(SANITIZED)UNCLASSIFIED FOREIGN-LANGUAGE BROCHURE ON INSTITUTE OF BIOLOGY(SANITIZED)

• Document Type: CREST
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): CIA-RDP80T00246A002900500021-2
• Release Decision: RIFPUB
• Original Classification: K
• Document Page Count: 71
• Document Creation Date: December 21, 2016
• Sequence Number: 21
• Content Type: REPORT

Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA (PRAHA)
Me~trayrlapoatloe usaat~ue aicg~~~a~2o~ Ceslcoslovenskcc biologie ra Ceskosloverzska
milcrobiologie
I' 0 ~ a IC I[ li 0 II II a fI If 0 JI JI C P K ft
AK8J~0MIIK ~. MiAJICK ~1'JIBBIIbIi[ 11e]~aICTOp~, ~. BhIIIAIICKnIt, >tI, I'aIDelt, YJL-KOl)p. ~C11I~
(~. I'e1N111 IC, afGaA0 MI3K O. IIp O13e Is, IO. ~I.AI[~'pH, aIC &~ie MLIK ~.. IIh aT, li. YOCll1j lCI111 (UO KI), I)eJ~.
xo~nerirx), JI. 'icptmlii, }I. Lll~repusb.
IIepex;oui,I IIa pycexllli sxaial:: uoq. ;~-p II[IIpoIIa, IIa ancsniicrstlii srar,ul: R-p PII;~ecolla, IIa ue-
~Iegleuii nahu:: ~-p ~aKr~a
Ila~ae?rcll 131IO~IOrIl~ieculrnl nncrxrr~--ronl rlexoc~Imlalll~oiiAuaue~xnll Ilayx I; Ilai~a7~e~i~cTite xlC~ll[.
13i.ISOJSIrr 6 h,Ia I; ro;S. IIo;~,uucnasl Itel[a na 1 ro;S Ii+rc 60.-, IIeIIa o;~uoro Ilonlep:I I{+~c i0.-.
A,Spec peA:n.11~In: Iiuoalorn~ICCxuCI I3xcTnTyT LiCAlI, IIa Ip3II~~xmTII 2, llpara XIX. 3altaeia:
Ap?rxltix. Cnle~ucla 30, Ilpara II, rlexocno~saxi~rl.
FOLI A BIOLOGICA (PRAHA)
International 1+,'dit-ion of the Jou~?rcals Ceslcoslovenslca biolog~ae and Ceskoslovenslca
milcrobiologie
Academician I. iVlalek (Chief I+:ditor), L. ferny, M. Hasek, Corresponding Member of the Czeohoslovali
Academy of Scienco F. Hercik, Academician O. Jirovec, J. Macura, Academician S. Prat, B. Rosieky
(Editorial Sccretar,y), J. ~terzl, V. Vrsansky.
Trtmslations into Russian: llr Schierova, into Lnglish: Dr Ridesovxz, into German: Dr Feigcl,
Issued by Biologicky ixstav ~eskoslovenske akadexnie ved at Nakladatelstvi ~s. akademio ved. Annual
subscription (G numbers) Kcs 00. Single number I~cs 10. Address: Biologicky iistav t/'SAV, Na cvicisti 2,
Praha XIX. Agonts: Artia, Smecky 30, Praha II, Czechoslovakia.
FOLIA BIOLOGICA (PRAHA)
Internatio7~.ale Ausgabe der Zeitschriftera ~'eslcoslovensleci biologie u7zd Ceskoslovenskci
mikrobiologie
Reclaktionsrat:
Akademiemitglied I. Malelc (leitendor Redakteur), L. ferny, M, Hasek, korresp. Mitgl. d. ~s. Akadomie
d. Wiss. 1+'. Hercik, Akademiemitglied O. Jirovec, J. Macura, Akademiemitglied S. Prat,
B. Rosicky (Redaktions-Sekretiir), J. ~terzl, V. Vrsansky.
Die IJbersetzungen besorgt Doz. Dr A. Schierova fiir die russischen, Dr A. Ridesova fur die
englischen and Dr T. Feigel fiir die deutschen Artikel.
Heraxxsgeber: Biologicky xistav ~eskoslovenske akademie ved lurch Vermittlung des Nakladatelstvi ~s.
akademie ved. 6 Lieferungen jahrlich. Abonnementpreis 60 I~cs, Preis der Linzelmxmmer 10 Kcs.
Anschrift der Redalction: Biologicky ustav LSAV, Na cvicisti 2, Praha XIX. Zu beziehen lurch: Artia,
Smecky 30, Praha II, ~eskoslovensko.
Fol. biol. (Praha) pp. 129-192 Praha, 27. VI. 1957
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
The Adaptive Period for Foreign Antigens in Ontogenesis in Ducks
V. HA~KOVt~
Institute of Biology, Czechoslovak Academy of Science, Department of Experimental Biology
and Genetics, Praha
In previous work, specific suppression on the formation of heteroagglutinins was
found following long-term, repeated postembryonic injections of foreign blood
in ducks (Ha~kova and Pokorny 1956). The most important factor in the origin
of this non-reactivity of ducks to hen erythrocytes is adaption of the organism
in the immediate postnatal period. A further analysis of the origin of this non-
reactivity led to findings on the character, duration and significance of this period
(i. e. the adaptive period), when the administration of foreign blood results in inhibi-
tion of the formation of heteroagglutinins in fully grown birds.
Peking ducks, which had hatched out on the same day and had been reared under the same conditions,
were used for the experiments. These were given 1-15 intravenous injections of fresh hen or goose blood
(one part 3.8% citrate to nine parts blood) on alternate days, in amounts of 0.3- I ml., according to the
scheme given in the tables.
Immunisation was carried out in the experimental and control birds at exactly eight weeks, with four
doses of 1.5 ml. blood on alternate days; blood was collected on the fifth day after immunisation had been
completed. Re-immunisation was carried out at 13 weeks, with four doses of 1.5 ml. hen blood or 4 ml.
goose blood, and blood was again collected five days after completion of the immunisation series. The
control ducks received their first injections at eight weeks.
In order to determine normal reactivity of ducks to a foreign erythrocyte antigen, some of the ducks
were immunised with four doses of 1.5 ml. citrated turkey blood; blood was again collected on the fifth
day after completion of the immunisation series.
Agglutination was carried out by taking two drops of serum which had been stored for 24 hours
at -20? C and one small drop of a 50% suspension of erythrocytes washed three times in physiological
saline and incubating at room temperature. The results were read off after 10 and 60 minutes. The titres
given in the tables are the inverted values of the final dilution of the serum which gave agglutination?
visible to the naked eye after 60 minutes.
Incomplete antibodies were determined by agglutination in protein medium and by the Coombs test
(Dunsford and Bowley 1955). Antiglobulin serum was prepared according to the method suggested
by Milgrom et al. (1956), by immunising a cockerel with its own erythrocytes, agglutinated by immune
duck serum.
Skin grafting: When applying homografts, the skin was always exchanged from the middle of the back,
between ducks of the same age. After turning the akin by 180?, the graft was sutured by eight stitches
and lightly covered with collodion. Evalution of a permanent take was based on growth of the feathers
in the reverse direction. In heterografts in adult ducks, a piece of skin measuring approximately 2 X 2 cm.
was taken from the leg of a goose, sutured into position and lightly covered with collodion.
It was found that long-term, repeated injections of hen blood in ducks, if com-
menced within six days of hatching, lead to complete inhibition of the formation
of heteroagglutinins in fully grown birds. If the series of injections is commenced
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Table 1. The Influence of a Series of Postembryonic Injections of Hen Blood Commenced on Various
Daps after Hatching, on the Formation of Heteroagglutinins in Ducks.
-
Day after
hatching on
which injections
commenced
-
No. of
injections
I
i
i
----
Titre
Total amount --- -
injected after first after second
(ml.) immunisation imrnunisa~tion
series series
Titre of
incoxnplcte
antibodies
3
15
6.3
1
2
I
~ 0
3
15 ~
6.3
2
1
0
6
15
6.6
2
2
0
6
15
6.6
0
1
0
8
15 ~
6.9
8
2
8
15 I
6.9
16
16
15
15
7.5
2
4
15
15
7.:i
4
8
18
15 ~
8.1
1
2
IS
15
8.1
256 ;
64
23
15
8.7
64
16
23
lb
8.7
32
16
-
-
-
64
32
-
-
-
16
32
-
-
-
32
32
-
-
-
64
32
between the 8th and the 18th day, some ducks form immune heteroagglutinins
to the same titre as the controls, while others do not form them to a titre higher
than that for natural heteroagglutinins (Haskova and Pokorna 1956). If the series
is commenced on the 23rd day, the ducks react in the same way as the control birds
not given injections before immunisation (tab. 1, fig. 1). Since the period when
the first injections are administered is the decisive factor, the authors are of the
opinion that formation of immunological tolerance similar to that resulting from
embryonal parabiosis or intraembryonal injections (Hayek 1953, Billingham, Brent
and Medawar 1953) is involved. It confirmed the conclusion of the preceding com-
munication (Haskova and Pokorna 1956).
For postembryonal injections in ducks, goose blood was also used, and it was
found that the same inhibition of formation of heteroagglutinins occurred in adult
life following a series of 15 postembryonal injections in doses of 0.3-0.7 ml.
.on alternate days, five injections of 0.3 ml. or two injections of 0.3 ml. on alternate
days, or even after a single injection of 1 ml. goose blood, i. e. if a sufficient amount '
of antigen was administered up to the 10th-13th day after hatching (tab. 2, fig. 2).
In non-reacting ducks, the presence of incomplete antibodies was not demonstrated
either by agglutination in protein medium, or by the Coombs test. From all the above
results it is concluded that the period in which ducks are capable of adaptation
to a foreign antigen (i. e. the adaptive period) extends for several days into post-
embryogenesis. The length of the adaptive period depends on the nature of the
antigen. For goose blood it lasts, in all ducks, up to about 10-13 days and for hen
blood cells up to six days after hatching. This period varies considerably however,
among individual birds. Some ducks are capable of adaptation to hen or goose blood
as late as 18 days after hatching; others do not develop tolerance when injected with
antigen on the 8th day after hatching.
The origin of non-reactivity depends, without any doubt, on the dose of antigen
administered during the adaptive period. When a single dose of 0.3 ml. goose blood
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
was injected during the adaptive period, it had no effect on the formation of anti-
bodies at eight weeks. When the same amount was split up into three doses and
administered on the first, third and fifth days, or when a single dose of 1 ml. was
administered, the same inhibition of antibody formation was found as following
Fig. 1. Fig. 2.
Fig. 1. Titres of heteroagglutinins against hen erythrocytes after immunisation at the age of eight weeks
in ducks that had been given a aeries of 15 injections of hen blood, starting on varying days after
hatching. x: day after hatching when the ducks received the first injection. K denotes the titres in the
control ducks. y: last dilution of serum which gave visible agglutination.
Fig. 2. Titres of heteroagglutinins against goose erythrocytes after immunisation at the age of eight weeks
in ducks which had been given one or several injections of goose blood starting on varying days after
hatching. x: day after hatching on which ducks received first injection .K denotes titres of control ducks.
y: last dilution of serum which gave visible agglutination. The mark ?'0.3 ml." denotes that the ducks
indicated were given only the border-line amount of 0.3 ml. in the adaptive stage and that therefore
their titres lie within the limits of the titres of the controls.
a aeries of injections (tab. 2). From this it is concluded that the important factor
is not so much the amount of antigen administered in a given dose, but that the
antigen should remain in the body for a certain time during the adaptive period,
in order to be able to influence the mechanism of antibody formation.
Strong suppression of the formation of antibodies, against soluble heterologous
proteins, however, was successfully obtained by Hanan and Oyama (1954) and
by Dixon and Maurer (1955), following a series of postembryonal injections. It is
evident that in these experiments also the organism was adapted to the antigen
primarily in the adaptive period, shortly after birth. In the experiments described
in the present communication, the formation of heteroagglutinins was completely
inhibited; this had previously been successful only after turkey-hen parabiosis
(Hraba 1956). Intraembryonal injections of foreign blood resulted at most in
a decrease in the formation of heteroagglutinins, but never in complete inhibition
(Hayek and Hraba 1955, Simonsen 1955, 1956).
Homografts were carried out in ducks on various days after hatching out, in order
to verify the duration of the adaptive period. In five-day-old ducks, the growth
of feathers in homografts was 100%, in seven-day-old birds 50%. This confirms
that the adaptive period in ducks extends into postembryonic life for several days.
Hraba and Hawk (1956) also obtained complete growth of feathers in homografts
on one-day-old ducks. The lower percentage of their successful homografts is prob-
ably due to the different technique used (tab. 3).
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Table 2. The Influence of Injections of Goose Blood on the Formation of Heteroagglutinins in Ducks.
Day when
Titres
first
Dosage scheme
\o
- --
in ection
1
m ections iven on
( ~ g
.
of birds
after first after second
administered
alternate da s
y^)
immunisation immunisation
--
series i series
1st
0.3 ml?
2
128, 4
256, 32
1 ml.
2
0, 2
0.1+0.1-~0.1
3
0, 0, 1G
U.3 -}- U.3
2
Q U
0, 0
0.3-F0.3~-0.3-{-0.3-x-0.3
2
2, 0
64, 0
U.3 to 0.6 in 15 injs.
2
0, 0
U, 0
3rd
0.3
2
32, 2
128, 16
0.3 -}- 0.3
1 ~
2
0
0.3~-0.3+0.3 }-0.3
1 ~
1
G4
5th
U.3 to 0.6 in 15 injs.
1
~
~~
0.3 {- 0.3
2
2, 2
1, 2
0.3+0.3+0.3 fi 0.3-~ 0.4
1
1
8
9th
LO
2
1. 1
0.3+0.4-{-0.4
2
0, 0
10th
0.3 -} U.3
2
4, 2, 0
32
0.3~-0.4-1-0.4-X0.4-{-0.4
2
2, 4
32
13th i
0.4 + 0.4
2
4. 1
U.4 }- 0.4 -{- 0.4 ~-- 0.6 -} 0.5
2
] , 2
20th
0.4 ~ 0.5
2
16, 8 I
0.4 + 0.5 ~ 0.5 ~- 0.5 {- 0.6
3
1, 32, 256
~
0.4 to 0.7 in 15 injs.
1
128
Control birds of same age, injocted at age
o
16, 128, 64, :i12,
of eight weeks
16, 32, 64, ~ 128, 2.5fi
64, 16, 64,
16 ~
Table 3. The Proportion of Feathered Homografts and Others Carried out in Ducks on Various Days
after Hatching
I1~1s 5.~ 2~4 ohs o~4 0/4
(Hraba and
Hasek 1956)
Skin grafts from the legs of geese were also made in ten-week-old ducks in which
the formation of heteroagglutinins had been inhibited. These heterografts did not,
however, survive longer than 15 days, either in ducks which did not form agglutinins
on immunisation, or in the controls.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
It is known that an injection of blood from another bird of the same species, when
administered during the adaptive period, can result in permanent survival of a skin
homograft, as the leucocytes and the skin have the same antigens in common.
Injections of blood from a different species, although causing complete inhibition
of the formation of heteroagglutinins did not, in these experiments, lead to prolonged
survival of heterografts in adult birds in a single case. The question of heterografts
is discussed elsewhere (Ha~kova and Hasek 1957).
Summary
1. Complete inhibition of the formation of heteroagglutinins in adult life was
obtained in ducks by means of postembryonal injections of goose or hen blood.
The experiments constituted a further demonstration of immunological tolerance.
2. It was found that the adaptive period for foreign erythrocytic antigens in ducks
extended for 6-13 days into postembryogenesis.
3. The duration of the adaptive period depends not only on the species of the
experimental birds, but also on the nature and degree of heterogenesity of the
antigen, and even displays individual variations.
4. The important factor in the development of adaptation to a foreign antigen
is not only the amount of antigen administered during the adaptive period, but
rather the length of time over which it can act during this period. A single dose
of 0.3 ml. administered during the adaptive stage did not affect antibody formation,
but the same amount administered in three separate doses resulted in inhibition
of the formation of heteroagglutinins in maturity.
5. Inhibition of the formation of immune heteroagglutinins did not lead to the
development of incomplete antibodies.
6. The administration of blood during the adaptive period did not result in pro-
longation of the survival of heterografts.
7. Homografts carried out in ducks from 5-7 days after hatching out took
permanently.
References
B i 1 1 i n g h a m, R. E., Brent, L., Medawar, P. B.: Actively Acquired Tolerance
of Foreign Cells. Nature 172:603, 1953.
Dixon, F. J., Maurer, P. H.: Immunologic Unresponsiveness Induced by Protein
Antigens. J. Exp. Med. 101:245, 1955.
D u rr s f o r d, I., Bow 1 e y, C. C.: Techniques in Blood Grouping. Edinburgh, London
1955.
H a n a n, R., O y a m a, J.: Inhibition of Antibody Formation in Mature Rabbits by Con-
tact with Antigen at an Early Age. J. Immunol. 73:49, 1954.
H as e k, M.: Vegetativni hybrrdisace iivoLichu spojenim krevnich obehu v embryonalnim
v~voji. G`s. bio ogie 2:265, 1953.
H as a k, M., H r a b a, T.: Immunological Effects of Experimental Parabiosis. Nature
175:763, 1955.
H as k o v a, V., H as e k, M.: Increased Tolerance of Heterografts in Newborn Birds.
Fol. biol. (Praha) 3:49, 1957.
H as k o v a, V., P o k o r n a, Z.: The Influence of Intraembryonal and Repeated Post-
embryonal Injections on the Formation of Heteroagglutinins. Fol. biol. (Praha) 2:249, 1956.
H r a b a, T.: Immunological Behaviour of Embryonal Parabiosis between Turkey and Hen.
Fol. biol. (Praha) 2:165, 1956.
H r a b a, T., H as e k, M.: Kozni homotransplantace u jednodennich ku~at, kachen a krut.
~`s. biologie 5:89, 1956.
M i l g r o m, F., L u c z y n s k i, T., D u b i s k i, S.: Preparation of Antiglobulin Sera.
Nature 177:329, 1956.
Simonsen, M.: Induced Tolerance to Heterologous Cells and Induced Susceptibility
to Virus. Nature 175:763, 1955.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Simonsen, M.: Actively Acquired Tolerance to Heterologous Antigens. Acta pathol.
microbiol. scand. 39:21, 1.956.
I'a m e x, M.: BereTaTxsxax rx6pxAxsauxx xtxsoTxblx xyTeM coeAxxexxx xposo6pau;exxx
s Te*~exxe aM6pxoxanbxoro paasxTxx. LIc.x. Sxo,norxR 2:267, 1953.
I' p a 6 a, T., F a uI e x, M.: PoMOTpaxcn.naxTauxx xoxcx y oAxoAxesxblx ublx.nxT, yTxT x xx-
AloulaT. Fol. biol. (Praha) 2:61, 1956.
AAaHTI~IBHbIIi HO OTHOIileHI~IIO ,If iIy}IfepOAHbIM aHTYIPOHaM 3TaH
B OHTOP@H08e yTOK
B. PAIIIKOBA
MbI ycTaxoBK.nK, aTO c HoMOII~bIO noBTOpxblx BHpblcxl~saxr~H xypi3xol=l HpoBr~
MO?IfHO y BapOCJIbIX yTOH HOJIHOCTbIO rlOJjaBI3Tb OGpa30BaHKe POTepOaPPJIIOTIIHI3HOB
IIOCJie LIMMyHLIaaI[LII3 IIpI3 yCJIOBLIYI, tITO yISOJIbI 6bl.nx HaLIaTbI He noaxce 6-ro Axx
noc~Ie BblxseBblBaxHH (cps. Taxxce PaHlxosa, IIoxopxax 1956). ECJII3 cepKH yxo~IOB
6bIJIa HaLIaTa Ha c4-1g-bIYI J~eHb, y HeISOTOpbIX yTOIf YIMMyHHble aPPJIIOTLIHLiHbI
O~paayIOTCH B TaIfOM xce TI3Tpe, xax I3 B HOHTpOJIe, a y ApyPI3X - B TLITpe He BbIILie
TI3Tpa eCT0CTB8HHbIX P0Tep0aPPJIIOTYIHYIHOB. ECJII3 cepKH yxo.aoB 6bl.aa xa~IaTa xa
23-xI~ AeHb HOCJIe BbIIfJIeBbIBaHi3H, nOJ~OnbITHble yTKK peaPr3pyloT Tax axe, Kax r3 HOH-
TpOJIbHble, 6ea yHOJIOB IIepeA HMMyHI3aaI[i3eI3 (Ta6JI. 1, rpaI~I3It 1). Ilpr~ aTr~x onblTax
J~eJIO LIJ~eT OHHTb OC IIMMyHOJIOPI3LIeCHOH To.nepaxur~t~I (xax B peay.nbTaTe aM6pxo-
HaJIbHOPO IIapa6YI03a IIJIII BHyTpI33apOJ~bIITIeBbIX BHpbICK~IBaHYILI - I'aHIeH 1953,
Billingham, Brent t3 Medawar 1953). J~.xx snpblcxxBaxr3Fl Hoc.ne Bblx~IesblBaxHx
MbI I~ICIIOJIb30BaJIi3 TaIC?ICe PyCIIHyIO KpOBb I~i y6eRr3.nr3Cb, tITO TaI{OPO axe nOAaB.xeHxx
OC)pa30BaHIIR PBTepOaPPJIIOTYIHLIHOB BO B3pOCJIOM COCTOHHI3I3, xax HOCJIe cepHx
15 HOCT3MCpLIOHaJIbHbIX BrlpbiCxxBaHr3H OT O,3 Z[O O,7 MJI LIepe3 J~eHb, MO}IfHO ~[OCLITbCH
C HOMOH;bIO 2 yKOdIOB ri0 O,3 MJI ilepe3 JjeHb LIJII'I Aaxce OJ~HOpa30BOP0 BrtpbICxHBaHHx
1 MJI ryclaHO%I xpOBr3, T. e. npLI yCJIOBI3I3, iITO JjOCTaTOiIHOe ISOJIIIYeCTBO aHTYIrexa
6blao BBeRexo xe noaaxe 10-13-ro AHH rloc.ne sblx.aeBblBaxxx (Ta6~I. 2, pKC. 2).
IIepHOR, xorRa yTxr~ eH~e cnoco6xbl aRanTZ3posaTbcH x ~yxcepoAHbIM apr3TpouH-
TapHbIM aHTYIPeHaM (aJ;aIITI3BHbI~i aTaII) 3aTxPI~IBaeTCSI J;O 6-13-ro Rxx nocT-
aM6pI30P8Heaa. ,~JILITeJIbHOCTb aJ~anTNBHOPO aTaria 3aBLICI3T, nOBLIj~IIMOMy, He TOJIbIfO
OT BYIj~a 7I4YiBOTHOPO, HO H OT XapaxTepa YI CTerleHx *IyHSepOJ~HOCTI3 aHTLiPOHa, a Talcaxe
HpOHBJIHBT H I3HJjLIBI3J~yaJIbHble ISOJIe6aHyIH. ~,aJIbHeI~IiINe OIIbITbI IIOKa3aJIII, LITO J~JIH
B03HIIKHOBOHLIH aj[aIITaI~YIIi K LIySIfepOJ~HOMy aHTYIPOHy H0T HeOCXOj;LIMOCTI3 BO BBe-
J;eHLII3 aHTLIPBHa B TezIeHLIe aj[aIITIIBHOPO 3TaIIa TOJIbISO B KaILOM-HI36yJjb OHpeZ[eJIeHHOM
IfOJIIItIeCTBe, a CKOpee - B J~OCTaTOiIHO ROJIPOM ReHCTBI3II aHTI3P0Ha B TeLIeHI3e aAarl-
Ti3BHOP0 aTaHa. OJ[HOpa30B0e BBeJjeHLIe O,3 MJI B TeLIeHI~Ie a~aIITYIBHOPO nepHOAa
xe oxaablsa~IO s.nr~HHr~x xa o6paaosaxtae aHTKTe.a, BseAexi~e xce aTOro xo.nx~IecTBa
TpeMH J~03aMIi HO O,1 MJI BbI3BaJI0 IfOJ~aBJIeHi3e O~paaOBaHi3H reTepOaPPJIIOTIIHI3HOB
BO B3pOCJIOM COCTOFIHIII~I. HaJII3LIi3e HBIIOJIHbIX aHTYITeJI He rObIJIO HaMI~i J;OKaaaHO HLI
B OJ~HOM CJIyLIae. IIpOA.nHTb BpeMx BbI7IfI3BaHHH POTepOTpaHCnJIaHTaTOB nyTeM BBe-
~[eHHH IfpOBx J~OHOpa B TeileHIie aJ~aIITIIBHOPO nepr3oAa xe yAasa.nocb. PoNloTpaHC-
HJIaHTaTbI, IlepeCa?IfIIBaeMble yTKaM J;O 5-7-PO J~HH IIOCJIe BbIKJIeBbIBaHI3FI, yCTOii-
~r3BO npl~Iaxr~BaloT.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
Immunological Tolerance to Non-cellular Antigens
J. HORT and T. HRABA
Institute of Biology, Czechoslovak Academy of Science, Department of Experimental Biology
and Genetics, Praha
Not long after it had been found that tolerance to foreign cell antigens could be
produced (Hayek 1953, Billingham, Brent' and Medawar 1953, Ripley, cit. by
Owen 1954), it was demonstrated that a similar state could be induced using hetero-
genous proteins. In adult rabbits which had been injected during embryogenesis
and the immediate postnatal period with equine serum albumin, Hanan and Oyama
found absence of immunological response to this antigen. In rabbits which had
received from birth large, repeated doses of human plasma or equine serum albumin,
Dixon and Maurer (1955) found complete inhibition of the formation of precipitins
against the antigen concerned. By injecting newborn rabbits with human seralbumin,
Cinader and Dubert (1955) obtained complete suppression of antibody formation
in later life. In hens which had been embryonal parabionta with turkeys, Hraba
(1956) found depression, and in one case complete inhibition, of the formation
of precipitins against turkey serum proteins.
The aim of the present work was to ascertain whether immunological tolerance
to guinea-fowl serum proteins could be produced in chicks. The antigen was injected
intra-embryonally, but because only small amounts could be administered in this
way, larger doses were injected immediately after the chicks hatched out.
Injections were administered into the yolk sacs of 4 to 5-day-old embryos by drilling a small opening
in the shell in approximately the equatorial plane. The egg was illuminated and placed in such a way
that the extra-embryonic vascular field was situated below the opening. The antigen was injected below
the area vasculosa to a depth of about 0.5 em. (from the shell) in the direction of the embryo. After
completing the injection, the opening was closed with paraffin wax and the egg returned to the incubator.
About 15-20% of these embryos hatched out, the most frequent cause of death being rupture of the
yolk sac. Injections into the allanto-chorionic blood vessels of the embryo were carried out according
to a method described in previous communications (Hraba et al. 1956, Hayek 1958).
The experimental birds were immunized and blood collected by the intravenous route. Guinea-fowl
citrated plasma (1 part 3.S% sodium citrate to 9 parts blood) was used for all the injections and
immunization doses, and guinea-fowl and hen plasma were used for the reactions. Precipitins were
demonstrated by the ring reaction in miniature test-tubes; the titre is given according to the highest
dilution of the antigen which still produced a discernible positive reaction. The antiserum waa not diluted
for the reactions and unless stated otherwise, the antigen was diluted in the geometric series: 10 X , 20 X ,
40 X , etc., commencing with tenfold dilution.
Results
Guinea-fowl plasma was injected either intra-embryonally or into newly hatched
chicks. Intra-embryonal injections were administered to three groups of chick
embryos of different ages. For these injections a constant dose of 0.2 ml. was used.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Formation of Precipitins against Guinea-fowl Plasma in Experimental and Control Birds
Titres in blood collected on 7th day after immunization (in group 4 on 7th day after second
immunization dose). Antigen diluted in geometric series (1:10, 1 :20, 1 : 4Q etc.), commencing
with tenfold dilution
Groups
Method
Amt. of
Vo. of
Titre
of antibodies
Diff. between
exp. birds and
of birds
of administration
antigen
birds
7 days after
controls eval.
administration
by t-test
~
1. Exper.
Inj. in yolk sac in
0.2 ml.
4
160; 320; 640; 1280
t 0.45
' 4-5-day-old embryos
chick
Controls
-
-
6
160; 160; 160; 320;
~ P = 0.70
1280;].280
2. Exper.
Inj. in allanto-
0.2 ml.
12
70; 100; 100; 100;**
t = 0.68
chorionic blood vessels,
100; 100; 100;
11-13-da,y-old chick
100. 400; 400
embryos
Controls
-
-
10
100; 100; 100; 100;
P = 0.50
100; 100; 400; 400;
400; 400
3. Exper.
- -
Inj. in allanto-
-- ---
0.2 ml.
5
80; 160; 640; 640;
t 0.:"i
chorionic blood vessels
1280
in 18-day-old chick
~
embryos
Controls
-
-
7
160; 160; 160; 160;
0.7 P < O.G
fi40; 640; 640
4. Exper.
I. p. inj. 1st day after
1.0 ml.
8
160; 160; 320;
t = ].01
hatching
1280; 1280; 1280;
P = 0.30
1280;5120
~
Exper.
I. p. inj. 1st and 2nd
3.0 ml.
0
40; 80; 80; 160;
t == 2.74
day after hatching
(1 + 1 + 1)
160;320;1280;
0.01 ~ P < 0,02~a
1280;5120
I
Controls
~
-
- ~
9
160; 320; 1280;
-
1280; 1280; 1280;
2560;10240
** Antigen diluted 1:10, 40, 70, 100, 400, 700, 1,000.
In the first group, guinea-fowl plasma was injected into the yolk sac of 4-5-day-old
embryos. Transmission of these foreign proteins into the embryonic blood stream was
demonstrated in embryos killed five days after the intravitelline injection had been
carried out. In the other two groups, guinea-fowl plasma was injected into the
allantochorionic blood vessel on the 11th-13th day and on the 1 Sth day of incubation.
All three groups of chicks which were given intra-embryonal injections of guinea-fowl
plasma were immunized at the age of six weeks with a single injection of 1 ml. of the
same antigen. All these birds formed antibodies and in no group was there any signi-
ficant difference in the titre of the precipitins formed as compared with the controls.
Newly hatched chicks were injected intraperitoneally with guinea-fowl plasma.
In the first group a single injection of 1 ml. was administered within 12 hours after
hatching. In the second group three doses of 1 ml. were administered, within 12,
j
r
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
24 and 48 hours respectively after hatching. These birds were immunized with two
injections of 1 ml. guinea-fowl plasma administered at the age of six and eight weeks
after hatching. All birds again formed antibodies, but those which had received
3 ml. guinea-fowl plasma after hatching, formed precipitins against the antigen
in a statistically significantly lower titre than the controls (P < 0.02) while in the
group which had received only 1 ml. guinea-fowl plasma the titres did not differ
significantly from those of the control birds.
Discussion
Hasek (1955) found that the injection of homologous blood led to the formation
of immunological tolerance in about 50% of newly hatched chicks to a skin graft
from the donor. These results indicate that the adaptive period, i. e. the period
in which exposure to a foreign antigen leads to the development of immunological
tolerance, does not end in chicks until after they hatch out, at least as far as homo-
logous cells are concerned. It would appear, however, that the adaptive period for
different antigens may end at different periods. Ha,~kova (1957), for example, suc-
ceeded in producing tolerance to homologous cells in ducks up to the tenth day
after hatching, whereas to hen erythrocytes the latest limit was the fifth day. It was
not certain, therefore, whether it would be possible to produce immunological toler-
ance in chicks to guinea-fowl plasma after hatching out.
The experiments demonstrated that it is possible, but that relatively large amounts
of antigen must be administered. This offers the most probable explanation for
failure to produce immunological tolerance by means of intra-embryonal injections,
in which only a small amount of antigen can be administered. The importance
of the amount administered is evident from a comparison of the results in the two
groups injected with guinea-fowl plasma after hatching out; a dose of 3 ml. led
to the development of tolerance, while a dose of 1 ml. was ineffective. A relationship
between immunological tolerance to turkey serum proteins and the amount of antigen
injected into the chick embryo is also seen from the results obtained in chicks
which were given intra-embryonal injections of whole turkey blood (Hraba et al.
1956); while tolerance to the serum proteins of the partner's species was found
in hens which had been embryonal parabionts with turkeys, no reduction in the
formation of precipitins against turkey serum was found in these other birds.
With reference to the different methods of administering the antigen, it would,
however, appear that apart from differences in the amount of foreign protein
administered, other factors might also participate. For instance, Ha~kova (1957)
found that in the case of postembryonal injection of hen blood in ducks, fractionation
of the same dose at given intervals is more effective for inducing tolerance than
administration of the whole amount in a single dose; for the induction of tolerance,
therefore, it appears necessary for the antigen to act at least over a minimum
given period.
Simonsen (1956) found that on the 17th-19th day of incubation of the chick
embryo, immunological tolerance could be induced by a dose of human blood cells
which at other periods would have no, or little effect.
Although injections ofguinea-fowl plasma were administered from the 4th-18th day
of incubation, no developmental period of increased sensitivity was found, in which
a relatively small amount of this antigen could produce tolerance. It is assumed
that these experiments do not demonstrate that no such sensitive stage to guinea-
fowl plasma exists, as the negative results were plainly due to the fact that only
sub-threshold amounts of antigen could be administered.
It will, however, be necessary to verify Simonsen's results apart from this, as when
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
chick embryos were injected with turkey blood from the 12th day of incubation up
to the first day after hatching out, no reduction in the formation of agglutinins
against turkey erythrocytes was found in any of the groups, although the same dose
of foreign blood was used for injecting the embryos as had been used in Simonsen's
experiments (Hraba et al. 1956).
On the other hand, the results in the group of newborn chicks and in chicks
injected with guinea-fowl plasma administered into the yolk during early embryo-
genesis, together with the results of embryonal parabiosis between turkey and hen,
are further evidence that the weak forms of tolerance induced in duck and hen
embryonal parabionts are not due to termination of the adaptive stage for the
partner species antigens before the time when the embryos are joined, i. e. the
11th-12th day of incubation, as assumed by Billingham et al. (1956).
The degree of immunological tolerance to guinea-fowl serum proteins obtained
in these experiments, and the degree of tolerance to turkey serum proteins obtained
in hen embryonal parabionts with turkeys, are lower than the degree of tolerance
to foreign proteins induced in rabbits. This is probably due to differences in
ability to acquire immunological tolerance in different species.
This relationship is particularly clear in duck-hen embryonal parabionts. On im-
munization at the same age, the duck parabionts form agglutinins against hen erythro-
cytes in lower titres than the controls, whereas in the hen parabionts the titres of
agglutinins against duck erythrocytes are the same as in the controls (Hasek and
Hraba 1955). A similar relationship can be seen in hens which received intra-
embryonal injections of turkey blood. Whereas parabiosis with this species produces
marked or even complete suppression of the formation of agglutinins against turkey
erythrocytes, the titres in the birds which only received injections of turkey blood
did not differ from those in the controls (Hraba et al. 1956). On the other hand,
in ducks injected with the same amount of goose blood as in the previous experi-
ment, areduction in the titre of antibodies against goose erythrocytes was found
(Ha.~ek 1956). In view of the fact that the acquisition of immunological tolerance
to foreign proteins depends on the amount of antigen administered, it might naturally
be possible that the difference between the reaction of hens and rabbits could be due
to differences in the amount of protein injected. That this is not the case is de-
mdnstrated by the finding that complete inhibition of the formation of precipitins
against the corresponding antigen was obtained in young rabbits by the injection
of 20 mg. human or equine seralbumin (Cinader 1955, Smith and Bridges 1956).
These are considerably smaller amounts of antigen than those required to produce
only partial depression of the formation of precipitins against guinea-fowl plasma
in chicks, showing that the rabbit belongs to the species in which immunological
tolerance is acquired more easily than in hens.
Summary
Chick embryos were injected with amounts of 0.2 ml. guinea-fowl plasma, 1. on the
4th- 5th day of incubation into the yolk sac, 2. on the 11th-13th and on the 18th day
of incubation, into the allantochorionic vein. No decrease in the formation of preci-
pitins was found in either of these groups following immunization with guinea-fowl
plasma as compared with the controls.
Newly-hatched chicks were injected intraperitoneally with 1 ml. or 3 ml. guinea-
fowl plasma. Following immunization, the chicks which had received 3 ml. guinea-
fowl plasma formed precipitins against this antigen in a significantly lower titre
than the controls (P < 0.02), while there was no significant difference between
the titres in the birds which had received only 1 ml. and those in the controls.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
B i l l i n g h a m, R. E., Brent, L., M e.d a w a r, P. B.: Actively Acquired Tolerance
of Foreign Cells. Nature 172:603, 1953.
B i 1 1 i n g h a m, R. E., Brent, L., Medawar, P. B.: Quantitative Studies on
Tissue Transplantation Immunity. III. Actively Acquired Tolerance. Phil. Trans. Royal
Soc. (B) 239:357, 1956.
C i n a d e r, B., ll u b e r t, J. M.: Acquired Immune Tolerance to Human Albumin and
the Response to Subsequent Injections of Diazohuman Albumin. Brit. J. Exp. Pathol. 36:515,
1955.
Dixon, F. J., Maurer, P. H.: Immunologic Unresponsiveness Induced by Protein
Antigens. J. Exp. Med. 101:245, 1955.
Hannan, R., O y a m a, J.: Inhibition of Antibody Formation in Mature .Rabbits by
Contact with the Antigen at an Early Age. J. Immunol. 73:49, 1954:
H a ~ e k, M.: Vegetativni hybridisace ~ivoLich$ spojenim krevnich obLh~ v embryonalnim
v~voji. Ls. biologie 2:265, 1953.
H a ~ e k, M.: ProblLm pf'ekonani nesluLivosti tkani pii homoplastickych pl?enosech. Las.
1Lkai:il Les. 94:41, 1955.
H a ~ e k, M.: The Influence of Intra-embryonal Injections of Foreign Blood on the Formation
of Antibodies. II. Observation of Reactivity in Ducks, Geese and Guinea-fowl. Fol. biol.
(Praha) 1:48, 1956.
Hayek, M., Hraba, T., Benesov'a, H., H1avaLkova, H.: Imunologicke
vztahy u fet'alnich parabios mezi kachnou a slepici. II. Ls. biologie 4:135, 1955.
H as k o v a, V.: The Adaptive Period for Foreign Antigens in Ontogenesis in Ducks. Fol.
biol. (Praha) 3:129, 1957.
Hraba, T.: Immunological Behaviour of Embryonal Parabionts between Turkey and Hen.
Fol. biol. (Praha) 3:165, 1956.
Hraba, T., Haskova, V., Lengerova, A., Vojtiskova, M.: The Influence
of Intra-embryonal Injections of Foreign Blood on the Formation of Antibodies. I. Observa-
tion of Reactivity in Hens. Fol. biol. (Praha) 1:43, 1956.
Owen, R. D.: Transp. Bull. 1 : 83, 1954.
Simonsen, M.: Actively Acquired Tolerance to Heterologous Antigens. Acta Pathol.
Microbiol. Scand. 39:1, 1956.
Smith, R. T., Bridges, R. A.: Response of Rabbits to Defined Antigens following
Neonatal Injection. Transp. Bull. 3 : 145, 1956.
I' a ui a x, M.: BereTaTxsxax rx6pxAxaauxx xcxsoTBxx nyTeM coeAxnexxx xposo6pau~exxA
B Tevenxe aM6pxoxa~cbHOro paasxTxx. LIcn. BxOJIOrxst 2:267, 1953.
I/IMMyHOJIOPIILIOCI{Oe CrOJIII]KeHI3e IIO OTHOIIleHI3I0 I{ H8I{JIOTOLIHbIM
c`tHTIII'OHaM
Bcxope noc.ne oTxpbiTHx xMMyxo.aorH~ecxoro c6~HxcexHx no oTxoIIiexHio x *~yaxe-
poAxbiM xneTO~xbiM axTxrexaM (I'aIIiex 1953, Billingham, Brent H Medawar 1953,
Ripley 1953, I[IIT. IIO Owen-y 1954) BblfICxI3JIOCb, iITO nOJ~OrOHOPO COCTOHHI3H MOxtHO
ROCYITbCx K HO OTHOIII0Hi3F0 x ~yxcepoAxblM 6e~xaM (Hanan H Oyama 1954, Dixon
H Maurer 1955, Cinander H Dubert 1955 - y xpo.aHxoB, Ppa6a 1956 - y xyp).
L~eabio xaCTOHii;e~3 pa6oTbI rObiJIO yCTBHOBHTb, MOaxHO JII3 ROCI3TbCx y ubin~RT
HMMyxo.aorH*~ecxoro c6.aHaxeHHA c uecapxaMx nyTeM BxyTpxaapoAbiiuesbix H nocT-
aM6pHOxa.abxbix BnpbicxuBaxH~ Hx cbiBOpoTO~xbix 6e.axoB. Mbi BnpbicxKBanK aapo-
J~bIILiaM IIO O,2 MJI naaaMbl IjecapxH: 1) Ha 4-5-bIl'I jjeHb HHxy6auKK B 1H8JITOiiHbi~i
McIIiox (nepexoA ~yaxepoAxbix 6esxoB s xposxxoe pyc.ao aapoAbiuia tibia HaMH
Aoxaaax); 2) Ha 11-12-bI~I H.ax 18-bIl'i Aexb Hxxy6auHx - B xopHOxa~naHTOHRxyio
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
sexy. IIoc.ae xMMyxHaauxx 1 MJI H.nasMbl uecapxx xa 6-oiI xeAe.ne Hoc.ne Bblx.aeBbl-
BaHHH Hx B OJ(HOII x3 BTHX PpyxH He HaOJIIOJ(aJIOCb xOHH7IfexxH CIIOCOrOHOCTLI K 06pa-
30BaHIdIO Hpel[LIHHTxHOB B CpaBHOHxII C ftOHTpOJIeM. HOMeRJIeHHO HOCJIe BbIKJIeBbI-
BaHxFi I(bIHJIRTaM BBOJ~xJIOCb B HOJIOCTb ~pIOHIHHbI HO 1 HJIx IIO 3 MJI HJIa3MbI ueCapxi~.
11OCJIe IIMMyHx3auI3II L~bIHJIFITa, HOJIytIxBHIIIe 3 MJI aHTLIPBHa, o6paaosa.ax HpeuH-
HI3THHbI B 3HaiixTeJIbHO ~OJIee HI33ifOM TI3Tpe, LIeM B iCOHTpOJIe (t = 2,74, P ~ Q,Q2),
Tr~TpbI I(bIHJIfIT, IfOTOpbIM HOCJIe BbIICJIeBbIBaHIIFi 6bIJI0 BBeJjeHO TOJIbKO HO 1 MJI
aHTIIPeHa, He OTJIIILIaJIxCb CKOJIbHO-Hx6yAb BHatiIITeJIbHO OT KOHTpOJIH.
HaHIr~ OHbITbI IIOIfa3aJIx, tiTO YIMMyHOJIOPYILIeCKOPO C6JIII}IfeHHFi y ubIHJIHT MO?I{HO
J~OCHTbCA H C HOMOH;bIO HOCT3M~pxOHaJIbHbIX BHpbICifLIBaHI3I%I CbIBOpOTOYHbIX ~eJIKOB
uecapxx. HO J[JIH ROCTxxceHi~x 3TOPO 8(~(~eICTa HOO6XOJ~xMO BBOCTH CpaBHHTeJIbHO
6OJIbHIOe I{OJIxtIeCTBO aHTI3PCHa, LIeM, HOBxJ~xMOMy, OGT>HCHFIeTCH HeyCIIeX IIOHbITOK
BbI3BaTb eP0 HyTOM BHyTpI33apOJ[bIHIOBbIX BHpbICKxBaHIILi. HeCMOTpFi Ha TO, LITO
BHpbICKHBaHxFI HJIa3MbI L[eCapKI3 HpOLI3BOJ~xJIxCb C 4-PO HO 18-bII3 j~eHb i~IHxy6aui~x,
HaM He yJ;aBa~IOCb OHpej[eJIxTb ISaIfOi3-Hx6y];b HepI3OJ~ 6OJIee LIyBCTBHTeJIbHbII~,
j(Jlfi riOJIyLIeHHA CCJIxSiteHIIH, HOJ~OCHO TOMy, xax ero xaHle~I Slmmonsen (1956) R.ax
apxTpouxTOB ~esosexa.
CTeHOHb I3MMyHOJIOPI3tieCKOPO CrOJIII?I{eHLIFi HO OTHOIileHxIO K CbIBOpOTOtIHbIM ~eJIKaM
uecapxH y ubIHJIFIT, KaK H C6JILI?ifeHxe HO OTHOHI0HxI0 K CbIBOpOTLIHbIM 6esxaM
HHj;e~IKH y xyp-aM6pHOHaJIbHbIX xaparOHOHTOB C HH~(eIIHOII, 6bIBaeT xxaice, ~eM
CTexeHb C~JILI?ifeHxfi x0 OTHOHIeHHIO K LIy}IfepOj[HbIM rOeJIifaM, J.(OCTHPHyTaFi y xp0-
JII~IISOB. HpxtiHHa 3TOLI pa3HI3I~bI ifOpeHIITCFi, BepOFiTHO, B BIIj[OBbIX pa3JIxiIHHX
B CHOCOrOHOCT$ K IIMMyHOJIOPHLIeCKOMy C6JIx?ICeHIiIO.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
Tom. III. (1957) - Fa$c. 3.
Ammonia Oxidation by Nitrosomonas Enzymes
A. A. IMSHENETSKY and E. L. RUBAN
Institute of Microbiology, Academy of Sciences of the USSR, Moscow
In studying the biochemical transformations caused by microorganisms two
stages have as a rule to be passed. First the peculiarities of the process induced by
living, multiplying microbes are established and only then an attempt is made
to reproduce the same process without the aid of cells, i. e. by culture filtrates
or enzyme preparations. In spite of the considerable progress made in the physiology
and biochemistry of microbes, many transformations of carbon, nitrogen, sulphur
and other elements have been followed up only in cultures of micro-organisms.
So far, the enzymatic nature of a number of processes has not been proved. Occasion-
ally new enzymes are described but the evidence is based upon experiments with
resting microbe cells only. This is hardly justified, since there is no fundamental
difference between experiments with microbe cultures and acute experiments with
resting cultures. It is hardly j ustifiable to speak of enzymatic action unless the chemical
process in question has been reproduced in a liquid free of microbes. The terms
"extracellular" fermentation or "extracellular" oxidation, so often used previously
do not meet the purpose as they emphasize that the chemical process occurs outside
the cell. The term "cell ?free" fermentation would therefore be preferable.
Six years have elapsed since Vinogradsky's (1952) brilliant studies on nitrifica-
tion. During this period our knowledge of the biology, occurrence and ecology
of the nitrifying bacteria has been greatly supplemented with new data. However
the chemistry of the nitrification process is still unclear. It is quite probable that
ammonia oxidation to nitrites is accomplished through the agency of several enzymes
but these are still unknown. The discovery of ammonia oxidation by the enzymes
contained in Nitrosomonas cells would however have opened vast prospects for the
study of the mechanism of nitrification. It is sufficient to mention the great role
of cell-free alcohol fermentation in elucidating the chemistry of individual stages
of this process.
Only one paper by Omeliansky (1953), has been published on nitrification in
a medium free of Nitrosomonas cells. These cells were triturated with washed sea-
sand, after which distilled water and ammonium sulphate were added to the sus-
pension. No ammonia decrease was noted in these experiments as due to Nitrosomonas
enzymes. Omeliansky therefore concluded that the chemical activity of these
microorganisms cannot apparently be dissociated from their vital activity. It should
be mentioned, however, that Omeliansky's procedure is not free from objections.
His principal experiments were carried out with very old (4-month) Nitrosomonas
cultures, the cells being dessicated prior to trituration over sulphuric acid, which
should have affected the activity of the enzymes.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
The present studies were carried out with a pure culture of Nitrosomonas europaea isolated from soil.
The methods of isolation and control of pure Nitrosomonas cultures have been described elsewhere-
(Imshenetsky and Ruban 1952, 1953). It will be noted that subculture on meat-peptone broth-a pro-
cedure commonly used for checking the purity of nitrifying bacteria-cannot be recommended sine+e
lack of growth does not demonstrate the purity of these cultures. A s a matter of fact, a number
of bacteria are known which, although present in the Nitrosomonas culture, do not multiply on meat-
peptone broth. These are mostly mycobacteria or myxobacteria. A very careful microscopic control
of culture purity is required as some bacterial satellites, e. g. mycobacteria, are incidentally very similar
in appearance to Nitrosomonas.
In the present study considerable amounts of nitrifying cultures were used, grown in large glass
bottles of 15- 201. capacity. Vinogradsky's medium was used, of the following composition: (NH4)zSO4
2.0 g.; K2HP04 - 1 g.; MgSO, - 0.5 g.; NaCI - 2.0 g.; FeSOq - 0.4 g.; a mixture o# microelements
(LiSO4, CuSO4, A12(S04)3, SnC12, MnC12, NiC12, CoSO4, TiC14, KBr) - 1 ml.; distilled water - 1000 ml.,
carefully ground chalk - 1.0 g.; pH of the medium 7.2-7.4. A pure 16-20 day Nitrosomonas culture
was used, grown in 20 ml. medium in Erlenmeyer flasks of 200 ml. capacity. After inoculation of the
bottles, glass tubes were introduced 3-4 cm. from the bottom; these were used for aeration of the
cultures with sterile air at a rate of 15-20 1. per minute. The air escaped through short glass tubes like-
wise passing through the corks and supplied with cotton filters.
The cultures were incubated 10-12 days at 22-24?. As a rule, the bacteria were cultivated in a set
of bottles. Subsequently, the culture was filtered through aSeitz-filter, 13.5 cm. in diameter, with
a membrane filter No. 3. The sediment was washed with sterile distilled water until the disappearance
of traces of ammonia and nitrite. Then it was carefully ground for 30 minutes with sterile glass powder
in a sterile agate mortar. Glass powder (Schott glass) in amounts of 0.25-0.40 g. were added per 10 g.
of sediment (consisting of calla and salt crystals, mostly phosphates). 15 ml. sterile water was added
per 10 g. of triturated sediment, the suspension agitated and incubated in a thermostat at 40? for
24 hours. During this period some autolyais of the nitrifying bacteria took place. The autolysate was
then filtered through a small Seitz filter (35 mm. diameter with an asbestos filter SF to free it from the
cells. Sterility of the filtrate was checked including for the nitrifying bacteria, by inoculating different
nutrient modia. The sterile autolysate was divided into two parts. In one of them, used as a control,
quantitative colorimetric determinations of nitrites were made with the Griss reagents, of ammonia
by distillation in vacuo, and of total nitrogen by the micro-Kjeldahl method. The second, experimental
portion of the filtrate was incubated for five days in a thermostat at 37? after which similar determina-
tions of nitrites, ammonia and nitrogen were carried out. A comparison of the results in the experimental
and control parts indicate the quantitative changes undergone by ammonia and nitrite in the cell-free
solutions.
As an additional control, filtrates of autolysates were used from cultures of heterotrophic organisms,
viz.Pseudomonas sp., Mycobacterixim rubrum and Saccharomyees eerevisiae. The filtrates were obtained
by the same methods as those from nitrifying bacteria.
Decrease of Ammonia Content in Autolysates Obtained
from Nitrosomonas Cells
Enzymatic oxidation of ammonia was studied on filtrates of autolysates of the
Nitrosomonas cells. These were shown to be free of Nitrosomonas cells as well
as of other bacteria. Quantitative ammonia determinations in the filtrates showed
that they always contain NH3. Hence no ammonium sulphate was added to the
filtrates. Instead, the degree of oxidation by Nitrosomonas enzymes of ammonia
already contained in the autolysates was ascertained. Ammonia and nitrite
determinations were carried out in duplicate (30 determinations 15 experiments)
(tab. 1). The data obtained may be summarized as follows: -
1. In the filtrates of autolysates, the nitrite content increases within five days
on an average from 0.94 to 1.48 mg. N per litre.
2. The ammonia content of the autolysates greatly decreases, from 26.4 to 7.4 mg N
per litre, i. e. by more than three and half times.
3. The amount of nitrites formed is much less than that of ammonia oxidized.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Table 1. Experiments with autolysate filtrates of Nitroaomonas calla without the addition of (NH4)zSO4.
(All figures are given in mg.~l. Duration of experiment 5 days.)
Amount of ammonia
Amount of NOZ
Decreased I
Formed
Exp.
initial
final
initial ~
final
NH4
N02
No.
I
-
NH3
N2
NH3
NZ
NOz
I
NZ
NO2
N2
in mg. N2
1
38.0
31.3
35.0
28.8
4.0
1.2
8.8
2.7
2.5
1.5
2
25.0
20.6
14.7
12.1
0.2
0.06
0.3
0.1
1.5
0.04
3
3.0
2.5
0.1
0.1
0.7
0.23
2.5
0.8
2.4
0.57
4
16.2
13.3
10.0
8.0
1.1
0.3
2.0
0.6
5.3
0.3
5
17.6
14.5
10.0
S.0
1.1
0.3
2.3
0.7
6.5
0.4
6
28.0
23.0
0.2
0.1
0.7
0.23
0.8
0.26
22.9
0.03
7
28.0
23.0
0.2
0.1
0.9
0.3
1.1
0.36
22.9
0.06
8
3.7
3.0
2.0
1.5
8.3
2.5
12.5
3.8
1.5
1.3
9
45.2
37.2
26.6
21.9
0.7
0.2
1.1
0.3
15.3
0.1
10
0.9
0.7
0.12
0.1
4.3
1.3
5.6
1.8
0.6
0.5
11
54.4
45.2
32.3
28.6
2.3
0.7
3.6
1.1
18.6
0.4
12
51.9
42.7
0.12
0.1
0.6
0.2
4.5
1.5
42.6
1.3
13
1.1
0.9
0.9
0.8
1.9
0.6
5.9
1.6
0.1
1.0
14
108.8
89.6
4.0
3.3
9.8
3.0
10.7
3.3
86.3
0.3
15
61.6
48.4
0.035
0.03
9.8
3.0
10.7
3.3
48.37
0.3
Aver-
age
32.3
26.39
9.05
7.43
3.09
0.94
4.82
1.48
18.49
0.54
Such are the principal conclusions. Let us discuss certain details. Attention is
drawn to the fact that the initial ammonia content of the autolysates varies greatly
i. e. from 0.9 to 108.8 mg. per litre. This is apparently due to a number of causes,
such as the degree of autolysis of Nitrosomonas cells, the initial ammonia content
of the cells, the cultivation conditions, etc. However, in all 15 experiments the de-
crease of the ammonia content was quite regular. Not in a single experiment did
ammonia increase above the control level or remain unchanged. The rate of ammonia
oxidation varies from one experiment to another. Sometimes almost all ammonia
is completely oxidized (experiments No. 3, 6, 7, 12 and 15). One can even speak
of complete oxidation of ammonia. Thus, for example in exp. No. 12 the initial
ammonia content decreased from 51.9 to 0.12 mg,~l, and in exp. No. 16 from 61.6
to 0.035 mg.~l. In some other experiments ammonia oxidation was much leas intense.
The question arose as to whether the decrease in ammonia content of the auto-
lysate is related to a change in the forms of nitrogen or to NH3 volatility. Determina-
tions were therefore made of total nitrogen of the filtrates of Nitrosomonas auto-
lysates and the heterotrophic Pseudomonas sp., immediately on preparation and
after five days incubation in a thermostat. The analyses were made according
to Kjeldahl in a modification used for nitrogen determination in the presence
of nitrites and nitrates. The figures obtained are summarized in table 2. It will
be seen that the total nitrogen content does not change during the experiment.
Thus, no ammonia is volatilized from the autolysates but oxidation of ammonia
takes place under the influence of enzymes contained in the cells of nitrifying
bacteria.
To prove the enzymatic nature of ammonia oxidation, the filtrates containing
the enzyme systems were inactivated by high temperature i. e. boiled for different
time intervals or autoclaved. A comparison of the ammonia content after five days
in heated and non-heated filtrates indicates the degree of thermostability of the
ammonia oxidizing systems. The results are summarized in table 3. It will be noted
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Table 2. Total N2 content of the autolysate filtrates of the cells of Nitrosomona,~ and Pseudomonas sp.
(N2 in mg.~l.).
Exp. No.
Table 3. Effect of thermal treatment of cell-free autolysates of Nitrosomonas europea on ammonia
oxidation.
that in all control experiments there occurs a regular drop in the ammonia content,
i. e. its oxidation through the agency of enzymes. A short, 5-10 minute boiling
of the filtrates does not cause complete inactivation of the ammonia oxidizing
enzymes. Boiling for 15-20 minutes causes a distinct inhibition of ammonia oxida-
tion while complete inhibition is induced by boiling for 30 minutes and autoclaving
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
85.6
86.6
109.2
111.6
103.9
102.69
108.49
109.6
84.56
85.8
98.4
96.9
63.7
64.0
136.9
135.7
137.2
138.1.
62.5
63.2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2 .
for 10 minutes at 0.5 atmosphere. In all experiments there invariably occurred
a regular decrease of the filtrate ammonia although no considerable accumulation
of nitrites took place. An inspection of tables 1 and 3 will show that the amount
of nitrites formed in the filtrates is insignificant as compared with that of ammonia
oxidized. It was suggested that a decrease occurs in the nitrite content of filtrates
incubated in the thermostat. To check this the course of nitrite accumulation was
investigated. The figures obtained are summarized in table 4. They confirm the
previous conclusion as to ammonia oxidation and also show that on the second day
the nitrite content of the filtrates is higher than on the fifth day. However, in these
experiments the nitrite maximum is also lower than was expected. This fact may
be accounted for in two ways: 1. Nitrite formation from ammonia is accomplished
in several stages through the agency of several enzymes, the experimental conditions
favouring the enzymes that oxidize ammonia but not those participating in further
oxidation. 2. The nitrites formed interact with the amides contained in the autolysate
filtrates and undergo a secondary reduction. Further enquiry is necessary to account
for the lack of the expected quantitative ratio between oxidized ammonia and
nitrites formed. It must be noted that such a correlation is likewise lacking in a pure
developing Nitrosomonas culture.
To check the assumption that hydroxylamine is the intermediate product formed
on oxidation of ammonia into nitrites, an attempt was made to oxidize hydroxyl-
amine by cell-free filtrates. These data are summarized in table 5.
~
Decrease of NH3
Formation of N02
I'xp'
days
Initial
days
No.
__ _
1 2 3 4 5
1
2 ~ 3 4 5
1
24
45
-
-
810
1.6
6.5
G.3
-
-
3.9
2
120
158
176
-
180
1.8
1.37
0.8
0.47
0.34
0.35
3
9
14
103
154
157
1
4.5
4.1
2.2
1.6
0.9
4
12.5
38
SO
110
123
2
3
2.3
1.8
1.8
-
5
82
132
132
137
2.2
1.8
1.7
L7
2.2
1.8
6
=
3.75
40
17
8.3
7
5.9
31
41.6
-
55
27.7
30.9
32.7
32
30
~ 8
3
5
7
-
8
8
15
8
S
-
5
9
+ 72
+ 98
-}- 148
-}- 165
+ 155
1
1.3 i
1.2
1
-1.5
1
~
Exp.
Initial content
of the autolysate
-
Final N02 content
of the autolysate
NHZOH
oxidized in
Difference
No.
without
with
water
NH2OH
NOZ
NHQO
NHZOH
1
21.21
0.55
1.76
17.60
3.08
12.76
2
21.21
1.62
3.56
22.33
3.08
15.69
3
19.45
1.26
2.54
16.94
0.10
14.28
4
19.45
11.50
12.69
21.15
0.07
8.39
5
19.57
1.26
2.54
16.92
0.10
14.28
6
37.36
1.58
16.92
33.84
0.76
16.16
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Tabl. 6. Experiments with autolysate filtrates of cells of heterotrophic microorganisms.
(The figures are given in mg.~l. Duration of experiments 5 days.)
Amount of NH3
-
Amount of NOZ
In
Exp.
Microor
anis
s
initial final
initial final
-
creas-
~ No
g
m
NH3
-
N~
NH3
Nz
!
i~ ;Vpz
N2 ~ Np~ N2
i
NH3 I
~
1
Saccharomyces
cerevisiae
10.0
-
8.2
--
20.0
_
lfi.4
0.1
0.03
0.25
0.1 8.2
2
0.24
0.2
11.7
9.6
0.1
0.03
6.3
2.1 I 9.4
3
13.1
10.8
18.5
15.2
0.1
0.03
0.8
0.3 ~ 4.4
4
>,
30.9
25.4
36.7
30.l
0.93
0.3
l.ll
0.33 4.8
5
56.5
46.0
58.0
47.7
1.12 i 0.4
L5
0.5 ~ 1.7
6
0.24
0.2 ~
3.0
2.4
0.1 0.03
1.5
0.5 ~ 2.2
7
Pseudomonas sp.
0.24
0.2
10.2
8.4
0.1 0.03
0.6
0.2 ~ 8.2
8
5.4
4.4
6.0
5.0
0.1
i 0.03
0.1
0.03 0.6
9
?
50.19
41.3
54.31
44.7
1.88
O.fi3
2.19
i
0.7 3.4
10
~
62.5
51.5 ~
77.0
63.4
1.0
0.3
1.2
0.4 ~ 11.9
11
42.0
34.5
54.4
44.7
0.1 ~
0.03
0.93
0.3 10.2
12
94.3
77.7
108.8
89.6
0.25 ~
0.1
0.45
0.2 11.9
13
53.6
41.2
80.2
66.1
1.2
0.4
1.2
0.4 24.9
14
48.1
39.9
70.9
55.4
0.6
0.2
1.5
0.5 18.5
15
Mycobacterium
rubrum
10.0
j
8.2
32.0
26.2
O.b
0.16
1.31
0.4
18.0
16
Average
0.24
29.84
0.2
24.3 7
j
2.1
40.23 i
1.7
33.10
0.10
0.52
0.03
0.17
1.73
1.41
0.6
0.4 7
1.5
8.7 3
N Oz
form-
ed
0.07
2.07
(1.27
0.03
0.l
0.47
0.17
0.00
0.07
O.l
0.27
0.1
0.00
0.3
0.24
0.57
It appears that hydroxylamine oxidation by the enzymes proceeds at a fairly
high rate. It was additionally found that the capacity of cell-free Nitrosomonas
autolysatea to oxidize hydroxylamine is lost upon autoclaving-a fact which
aupports the suggestion as to the enzymatic character of oxidation. This capacity
is specific for Nitrosomonas since it is lacking in the autolysates of the moat diverse
heterotrophic bacteria, such as Mycobacterium rubrum, Myc. citreum, Sarcina lutea,
Ps. ~luorescens, Bac. mesentericus, Micrococcus rubi f aciens, Proteus vulgaris,
B. schiitzenbachi.
The method of comparative physiology not only enablea one to introduce some
corrections into the results obtained, but also to confirm the specifity of the bio-
chemical processes studied. The decrease in the ammonia content in the autolysates
of nitrifying bacteria made it necessary to show that such a decrease does not take
place in the autolysates of heterotrophic bacteria. The experiments were carried out
with the cells of Pseudomonas ap., Mycobacterium rubrum and Saccharomyces cere-
visiae. The methods of cultivation of heterotrophic microorganisms have been
described above. The filtrates of autolysates were obtained in the same way as those
of Nitrosomonas. The results obtained are presented in table 6.
The following conclusions are indicated by the figures of table 6: -
1. In the filtrates of autolysatea obtained from the cells of heterotrophic micro-
organisma an increase occurs in the ammonia content by approximately 30% in five
days. No decrease in ammonia, which is so characteristic for the autolysates of
nitrifying bacteria; was ever noted. Nor did ammonia content remain unchanged
in any of the experiments.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
2. The autolysates of heterotrophic bacteria always contain nitrites but in a lesser
concentration than those of nitrifying bacteria. '
3. Within five days the nitrite content of heterotrophic autolysates increases
by about two and a half times.
The above changes in the autolysates of heterotrophs are associated with a change
in the forms of nitrogen since the total N remains constant throughout the experi-
ment (table 3).
The above data show that there exists an enzyme which oxidizes ammonia.
In this connection it seems valuable to discuss the results obtained by Omeliansky
(1953) with Nitrosomonas cells. His attempts to oxidize ammonia from triturated
cells proved futile and this might have been due to several reasons. Thus, the nitri-
fying cells were dessicated prior to trituration, which might have affected the
enzyme activity. It will also be noted that the principal experiments were carried out
with a very old culture (4-month-old) in which Omeliansky himself noted degenerating
forms. The possibility is not excluded that Nitrosomonas cultures grown in flasks
in such small volumes, cannot yield, a number of cells which would suit these
purposes. It is also of importance that in these experiments a solution of ammonium
sulphate was added, whereas ground Nitrosomonas cells, even if washed free of am-
monia, are rather rich in NH3 which is first oxidized. Nevertheless, Omeliansky's
work was highly progressive, for at that time only a single oxidase lactase, was
known, and there was almost no information as to the chemistry of the oxidation
processes taking place in the bacterial cell. Experiments with heterotrophic micro-
organisms showed that on incubation of these autolysates in a thermostat, the
ammonia content does not only fail to diminish but regularly increases. This is exactly
what would be expected as in the autolysates desamination of amino acids occurs.
Not only did the experiments on heterotrophs suggest that the drop in ammonia
content of the Nitrosomonas autolysates is strictly specific but certain indicat-
ions were obtained to the effect that in these autolysates two processes occur
simultaneously. One of them is connected with ammonia oxidation resulting
in a drop in its content, while the other should be accompanied by the
accumulation of ammonia, as there are no grounds for negating the possibility
of a parallel process of desamination. It is probable that proteins, peptides and
amino acids do not in these autolysates undergo the same changes as those observed
in the heterotrophic autolysates. If this be so, oxidation involves, not only ammonia
contained in the autolysates at the beginning of the experiment, but also that
formed in preserved autolysates. Hence it seems natural to conclude that the enzyme
responsible for ammonia oxidation is sufficiently active (as is the case in some experi-
ments), to oxidize all the ammonia almost completely. This assumption will probably
be confirmed by determinations of various forms of nitrogen in the autolysates.
It appears quite plausible therefore that nitrification is an oxidation process
consisting of several phases. At least two of them can already be indicated. The first
consists in ammonia oxidation resulting in the formation of intermediate products
which are similar to hydroxylamine, oximes and the like. These products are
accumulated in the cells in appreciable amounts as evidenced by the capacity
of nitrifying bacteria for endogenous nitrification, i. e. for the formation of nitrites
in a medium devoid of ammonium salts. It is this initial enzymatic nitrification
process that is particularly intense in the autolysates, i. e. transformation of ammonia
into an intermediate oxidation product. The amount of ammonia oxidized is signi-
ficant and hence a considerable amount of the intermediate product accumulates.
The toxicity of hydroxylamine does not favour the suggestion that this substance
is the intermediate oxidation product. Without foreshadowing the character of the
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
compounds formed on the oxidation of ammonia, the enzyme responsible for this
oxidation may be spoken off as "ammon oxidase". The activity of this enzyme can
readily be demonstrated by the above methods. Much less active is the second stage
of nitrification, consisting in the oxidation of the intermediate product to nitrites.
Cell-free nitrification results in insignificant nitrite formation as compared with the
reduction of ammonia. It should however be pointed out that in liquid cultures
of nitrifying microbe cells, the amount of nitrites formed is likewise much less than
that of oxidized ammonia. The possibility is not excluded that the enzyme responsible
for the second stage of oxidation requires different conditions for its activity than
those required by ammon oxidase. It may also be suggested that the formation
of nitrites is caused by an enzyme which is present not only in nitrifying bacteria
but also in some other bacteria. This is indicated by the formation of nitrites in the
autolysates of heterotrophic bacteria. These problems cannot be settled until
further studies have been made.
Summary
1. The possibility of cell-free oxidation of ammonia by Nitrosomonas enzymes
has been proved. The filtrates of the autolysates obtained from the cells of nitrifying
bacteria contain both ammonia and nitrites. Within five days, the ammonia content
of the filtrates decreases four times, eventually almost completely disappearing.
The total nitrogen content of the filtrates does not change, hence the possibility
of ammonia volatilization is excluded.
2. The enzymatic character of ammonia oxidation is confirmed by the fact that
boiling for 30 minutes and autoclaving for 10 minutes inactivate the filtrates.
Appreciable thermostability of the oxidative enzymatic systems of Nitrosomonas
brings them close to peroxidases.
3. Oxidation of ammonia is accompanied by the formation of a rather insignificant
amount of nitrites.
4. Cell-free filtrates obtained from autolysates of Nitrosomonas cells oxidize
hydroxylamine to nitrites thereby confirming the theory according to which hydroxyl-
amine is an intermediate product of ammonia oxidation.
5. The capacity to induce enzymatic oxidation of ammonia is specific for Nitro-
somonas. Incubation of the filtrates of autolysates from cultures of various hetero-
trophic bacteria does not lead to a decrease, but rather to a considerable increase
their ammonia content.
6. Oxidation of ammonia is apparently accomplished in several stages. At first
ammonia is oxidized to intermediate products which in their turn are oxidized
to nitrites. In filtrates free of Nitrosomonas cells the first stage is particularly active.
V i n o g r a d s k y, S.: Soil Microbiology. Moscow 1952.
O m e 1 i a n s k y, V.: Short Notes on Nitrifying Microbes. III. Do these Microbes Liberate
on Oxidase Selected Works. Moscow 1953.
I m s h e n e t s k y, A., R u b a u, E.: On the Isolation of Pure Cultures of Nitrifying
Bacteria. C. R. Acad. Sci. USSR 86:176, 1952.
I m s h e n e t s k y, A., R u b a u, E.: Isolation of Pure Nitrosomonas Cultures. Micro-
biology 82:376, 1953.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
L-~iopM>~ 6axTepz3~
IV. B.aHxHHe TeMnepaTypbi Ha paaBHTxe L-uxx~a y Proteus vulgaris H er0 axa~exxe
J;JIH B08HI3KHOB0HHH yCTO~YI3BOCTI3 H HBHHI~LIJIJIHHy
M. HEPMYT
IZHCTHTyT 06Hje3X 6HOJIOPHH MejjHI~HHCHOrO l~laHyJIbTBTa yHHBepCHTOTa B bpHO
ICOHeLIHbIM aBOHOM L-LjHKJIa paaBHTHA 6aKTepH~ xB.nHOTCH B03BpaT K nepBOHaLIaJIb-
HO~ ~aKTepI3aJIbHO~f (~IOpMe. I~IHbIMi3 CJIOBaMYI, B paMKaX L-IjHKJIa gepe3 paaJIH4Hble
HepexoAxbte c~opMbt (Kpynxbte xpyr.nbie Te~bua - KKT, xpynxbte HpoRo~rosaTbie
TeJIbI~a - I{IIT, aJIeMBHTapHbie TeJIbI~a) BOCCTaHaBJIYi$aioTCH T. H. BTOpH~Hble
Hano~xH. IIpH Hay~exHK L-c~opM y Proteus vulgaris Hac HxTepecosano ABa Bonpoca:
1. ocyH~ecTB.aReTCx JIH L-I;HKJI npx TeMnepaTypax xHTKe 37? C K HpH xaxo3~ TeMIIepa-
Type eH~e ocy~ecTBxMa perexepauxH; 2. xe oxaabiBaioTCH JIH BTOpI3iIxble naJlogxK
6o.nee yCTO#iIHBbIMi3 K neHI3i[I3JIJIYIHy.
B,aHxxxe TeMnepaTypbt xa paaBHTxe xpynxbix xpyr.abix Te.neu P. vulgaris
noR Ae~cTBxeM nexxuxn~xHa
Mamepuan~ u .wemoBuKa
Mbt HOJIbaOBaJIHCb OjSHHM HITaMMOM Proteus vulgaris (Pz), a xexoTOpbie OHbITbi Z(OHOHHH7IH
CpaBHBHHeM C 3 HIT8MM3MH P. mlrab111S (Pg, Pq, Pa). ~JIH HOCOBOB HpHMeHHJIHCb 18-24-LIaCOBbie
HyJIbTypbl B 6yJIbOHe, T. e. yHfe B CTauHOHapHO# (~laae, iITO 6bIJI0 yJ{OGHO Z[JIH HaHIHX OHbITOB
(06paayeTCR MaKCHMyM I{KT).
MbI Ae~a7iH Hocesbi Ha 6noxH arapa, HOTOpbie Mbi HpHHpbIBaJIH CTepH7IbHbiM HOKpOHHbIM CT@K-
JIbiIIIKOM H aa~HBanH s Hapac~HH. IIHTaTeJIbHaH cpeAa coAepxca~ca 25% JIOIIIaAHHON CbIBOpOTKH
H HBHHI(HJIJIHH B KOHI;eHTpaI(HH 2, 6, 1O, 2O, 4O TbICHY ej[~MJI. I{OHTpOJIbHbie HpenapaTbi COJ(ep-
HfaJIHCb B TepMOCTaTe HpH 3~? C, OHbTTHbie -HpH KOMH2tTHON TeMHepaType (18-22? C) H B JIej[HHKe
HpH TOMHepaType OT O J(O -~ 6? C HpH CpeRHe~ TeMHepaType OKOJIO 4? C. TOMHepaTypa B JIeJ~HHHe
KOHTpOJIHpOBaJIaCb eH(eJ{H0BH0. TaK H8K Ha6JIIOJ;eHHH HpenapaTOB HpOHSBOJ(HJIHCb HpH HOMHBTHON
TBMHepaType, TO J~JIH HCCJIeJ~OBaHHGi HpH 4? C aaCOBaJIOCb HO MBHbHie~i Mepe 4 HpenapaTa, ABa
H3 HOTOpbIX OCMaTpKBaJIHCb eHieZ[H0BH0, TpeTH~i - Yepea ~ j[He$, a HOCneAHH4t - TOJIbKO B HepHOZ[
06paaOB1HHH BTOpHYHbIX HaJIO`ieK. ICI 7(e~CTBHTeJIbHO, MbI y6eJ{HJIHCb, tITO B peaynbTaTe Hpe6bi-
BaHHH HpeHapaTOB K3 JIeJ{HHKa B Te9eHHe HOCKOJIbHHX MHHyT eHteJ~HQBHO HpH KOMH3THOft TOMHepa-
Type o6paaosaxxe Ha~ovex y CKOpHJIOCb HpH6JIHBHTeJIbHO Ha 12-24 Baca. Mbi Ae~anH cxHMKH
Ha KHHOH.aeHKy FOHla-repr0 OrtI70 II, 06bIYH0 C HOMOH;bIO c~aaosoro KoHTpacTa (KpoMe pxc. 6).
Peay~camambz
PaCCMOTpLIM paaBI3THe Proteus npK Hcc.aeAyeMblx TeMnepaTypax H paaJll3~xbIx
KOHL~eHTpaI~LIFiX neHi3l[LIJIJIyIHa.
2000 e8. IIpH CHKaxeHH03~ TeMnepaType paaBHTHe I{I#T npoTexa.ao no cyWecTBy
Tax xce, KaK H B xoxTpo.abxbix npenapaTax (npH 37? C). P~aBHaH paaxHga aax~io-
~aJlaCb B aanaaJ~blBaHi3i3 paaBYITIiH, B 06H;eM IIpOROpI~I30HaJIbHOM CHI3?KeHI3I0 TBMIIe-
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
paTypbl (Ta6JI. 1). ~pyrHM OTJIHLIxTeJIbHbIM xpH3HaKOM ~blJia CIfJIOHHOCTb K O~Opa30-
BaHHIO L-I{OJIOHxLI (pxC. 6) B KOHTpOJIbHbIX xpexapaTaX, He Ha6JIIOj~aBxIaHCHxpx
HOMHaTHOii TeMxepaType. Hpx 4? C MbI OTMeTx.nH HBCI{OJIbICO Cx00060B 06paaOBaHxH
BTOpx~ixbix xa.ao~ex (pxc. 18):
a) Hax6o.nee ~acTO BcTpexaeTCH oTno~xosaxxe sTOpH~xbix xa.no~Iex xa oRHOM x.nH
Ha OGOLIX xOJIIOCBX KHT (pHC. 2). e~TO - CaMbIyl paCxpOCTpaHOHHbIII CHOCO~ H HpH
6o~Iee BbicoxHx TeMxepaTypax. Ox 6bl.a yaice xaMH oxHCax (HepMyT H He~ac 1954);
6) Mexee ~IacTO BcTpe~aeTCx xo.nHpxasl c~parMexTauHH (HepMyT H He~Iac 1955),
sax,nlo~aloH;aslcx B nocTexexxOM pacxaAe Te.aeu xa Bce 6o.aee H 6o.nee Me.nxHe c~par-
MOHTbI - BIIJIOTb J~O B03HHKHOBBHHH xOLITH xOpMaJIbHbIX xaao~ex;
B) xoc.aeAxHM cnocoooM HB.nHeTCH T. x. cerMexTauHH, xorAa Te.nbue cxa~Iasa pa3-
pacTaeTCH B A,aHxy (B HlHpxxy ono 6bIBaeT Han To.acTOe Bo.aoxxo H B Te~exxe pocTa
ex;e rOOJIbHIe yTOHiIaeTCfi), a HOTOM pa3jjeJIFIOTCFI xOiITH OJ;HOBpeMeHHO Ha HOCIfOJIbKxX
MOCTaX Ha r00JIee I{OpOTI{He OTpe3IfH, - HOBble IIaJIOYKH.
6000 e8. B xoxTposbxbix xpexapaTax 6poca.acH B rsaaa 6bicTpblil pacnaR 1#I{T
Ha THxHLIHble CKOxJIeHHH 3epHHCTOCTH (pxC. 3), H3 HOTOpbIX He pa3BHBaJIHCb HH xa-
JIOLIIiH~ HH L-IfOJIOHHH. 3aT0 xpH KOMHaT-
HOII TeMxepaType 3Z[eCb *iepe3 30 LIaCOB
HaLIHHaJIaCb pereHepaI~HA BTOpHiIHbIX
~ xaJIOtIeK xOJ~OCHO TOMy, xax B JIe~HHI{e,
~~a~ ~~
6 ~ ~O$oo~~,;yi
S~ `~
HO TOJIbKO TaM OHH xofIBJIHJIHCb LIepC3
6-7 AxeK. PacxaA KI{T ocyluecTB~IH.ncH
H xpH 4? C, HO BO MHOPO pa3 McJ~JIeHHee
H tIaCTO H8 xOJIHOCTbIO. BOKpVr HeHO-
Topbix IxapoB rObIBaJIH saMeTHbI Me~Ixr~e
xpyxxxxH, paaMepaMH oxo~IO 0,4-0,6,u
(pHC. 9). C.ny~a I perexepauHH 6auH.n-
.xHpxoil c~opMbl 6bisa.aH xe Tax Mxoro-
Pxc. 18. a) OTUlxyposxa nanovex xa xoxge I{IIT, ~IHC.nexxbi, xax y xpeAxleCTBylox~eil
b) nonxpxax c~parMexTagxx, c) cerMexTagxx. xoxuexTpauHH xexHux.a.aHxa; HcxoR-
(CxeMa.) xbIMH o6paaosaxHHMH jjJIH xxx 6bIJIH
HCMHOrO~IHCJIeHHble I{HT (pHC. 5).
10.000 ea. B xoxTpo.nbxbix xpexapaTax oxxTb xa6~IloAa.ncH 6bicTpblK pacnaR I#I~T.
To.abxo B oTAe~Ibxbix cslyaatix xacTyxarla elije perexepauHx xa~IO~ex. B xpexapaTax
xpH xoMxaTxoK TeMxepaType pacnaR I~1~T B rpaxy.nslpxbie Maccbl npoTexa.a ropasAo
yMepexxee H x 24-My *Iacy Bo Mxorxx c.ay~axx xacTyxana perexepauHH 6auHSI.nHpxoil
(~OpMbI H3 ICHT. y xITaMMOB P2 H Pq ilepe3 10 H 17 AHeiI BbIpaCTaJIH OTJ~eJIbxbie
L-IfOJIOHHH, H3 IfOTOpbIX xOCJIe HX xepeH8C0HHFi B CpeAy 6e3 xeHHL(HJIJIHHa B aHa3pOr0-
xbix yc~IOBHHx BosxHxa.nH na.no~IxH. Ha pHC. 17 noxaaaxbl Bo.aoxxa, BbipacTaloHtHe
H3 McJIHHX H KpyxHbIX xIapHKOB, B3HTbIX H3 L-KOJIOxHI3. LIepe3 6 ~IacoB H3 sTxx
xpOJ~OJIPOBBTbIX TeJIeL~ OrOpa30BaJIHCb MHOrOtIHCJIeHHble BTOpHLIHble xaJIOLIIiH.
HpH 4? C paCIIa~j KKT OCyIIjeCTBJIHJICH ilpe3BbILIaLIHO McJ~JIeHHO, H TOJIbKO LIepe3
7-9 RHeH O~pa30BaJixCb KHT, a H3 HHX xOTOM LIepe3 10-14 J~HeII - HOBble xaJIOLIKH
(TarOJI. 2). HepBax rCHepaL~HH BTOpHtIHbIX xaJIOLIeif OrObILIHO x0 c~opMe xecxo.abxo
OTJIHLIaeTCH OT HOpMaJIbHbIX 6axTepHH: OHH ~bIBaIOT 06bIiIHO TOJIII(e x C 6o.nee aa-
OCTpeHHbIMH IfOHL~aMx, LITO B HOpMaJIbHOI~I xy.nbType HHILOPJ~a He BCTpetiaeTCFI, XOTH
Proteus, xax H3BCCTHO, xpHHaj~JIe7ifHT K MHIfpO~aM, B 3Ha~IHTeJIbHOLI CTexeHI3 xOJIH-
MOp(~HbIM .
`0.000 C'J~. B HOHTpOJIbHbIX xpexapaTaX Ha~JIIOJ~aJICH BCerJ~a ~bICTpbII3 (B TeT3eHHe
6-8 ~iacoB) pacnaR KIST B IxapHxH paasHaxoH BeJIII~IHHbI. HH c L-EfoJIOxHHMH,
HH C pePCHepaL~Hei3 MbI He BCTpeLIaJIHCb. J1HHIb B OJ~HOM CJIyLIae H3 10 MbI JjO~OHJIHCb
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
perexepauxH xaxo~eK, Ho Tonbxo noc.ne xepecesa Ha cBexcyio nHTaTexbxyio cpeAy
x B axaapo6xbtx yc.aoBxHx.
IIpx KOMHaTxO~i TeMnepaType CIIOCO~HOCTb H pereHepauHH y BCex IiITaMMOB
COXpaHHJIaCb j~OJIPO. TaK, y iuTaMMa Pa pereHepauHx BTOpI3iiHbIX xaJIOLIeH HBCTyxaxa
BcerAa ~epea 3-4 Axx, y ixTaMMa P5 ilepea 10 AHe~ BbipaCTaJII3 L-KOJIOHI3I3, KOTOpbte
.xa cpeRe 6ea xexl3ljLIJIJIi3xa cxoBa Aasanx xaJlogKYi. Y ocTaxbxblx IiITaMMOB ~epea
17 Axei~ xaxo~xH Bbrpocxx xa cpeRe 6es xexHuunaxxa npx aapo6xbix ycxoBxxx.
I{yJIbTi3BauKx xpH axaapO6HbiX yCJI0BI3xX AaBana OTpI3gaTeJIbHbie peayxbT$TbI.
B XOJIOJ~I3JIbHI3Ke ~epea 12-14 Rxei~ o6paaosasxcb I{IIT, a xa HHx, xajIHxaR
c 19-23-ro AHH - xosbte xaxo~KH. I~Ia aTOro BKAxo, xaK Ao.aro coxpaxxeTCH xcxaxe-
CH0006HOCTb KJIeTOK i)aKTepi3~! (i3JII3 I3X (~1parMQHTOB) xpx xOHI3?KeHHO~ TOMxepaType.
SE~.~0~ Cll. IIpx TaKO~i BbICOKO# HOHI~exTpauxx MbI xpH BCeX TOMxepaTypax
xa6.aioAa~H pacxaA KKT B TKxKexbte MexKxe cxoxxexuR ixapKKOB, H.nx xee o6paao-
Baxxe aepxKCTbix uiapoB, xoTOpbie xocTexexxo pacxaAaaxcb. C~eRyeT xo~~epKHyTb,
iITO xpH aTO~I KOHL;eHTpagHx xpOL(eHT KKT 6bIBaeT aHaiILITeJIbHO McHblile (HepMyT
H He~ac 1956). IIoaTOMy B xpexapaTax xpeo6.naRaioT xaaogxH, xo 6o.abrue~ ~acTx
xyCTble, C OTileTJIIiBO BHJ~I3MO~i 3epHI3CTOCTbIO. e~TI3 xaJIOiIKI3 C TeLIeHI3eM BpeMeHI3
TaK?Ke paCxa~[aIOTCH. B xpI3MOKyJIbTypaX MbI HI3KOrAa He Ha~JIIO~[aJIi3 pereHepauxH.
B Oj(HOM CJIy~ae xpH KOMxaTHO~ T2MxepaType MbI HaxIJII3 y ixTaMMa Pb 4 L-KOJIOHI3i3
THxa 3 A, Ha KOTOpbIX TOJIbKO B aapO6H0~I Cy6KynbType BbipOCJIi3 HOJIOHI3H PTOtells.
XapaKTepxblM peayJIbTaTOM BbICOKi3X HOHI~exTpaI~I3I~ xeHHI~YIJIJII3Ha (HaLIxHaH
c 6000 K Bb~iue) xBnHeTCx pacxaA KKT, HOTOpbi~ Ha4I3HaeTCx OileHb CKOpO, B 000C)eH-
HOCTI3 B TexJle. C.aeAyeT xORtIepHHyTb, PTO OH xpOHBJIHeTCA HepaBHOMepHO, T. e. x0-
paxcaeT He BCe xiapbt OJ~HOBpeMeHHO x npH 37? C 6btBaeT CI3JIbHee H ~blCTpee, iieM npH
HOMHaTHO~ri TeMxepaType xJII3 B XOJIOJ~LIJIbHHKe. PYIC. 4 I3JIJIEOCTpxpyeT HepaBHOMep'
HOCTb paaBI3Ti3H KKT: HeKOTOpbie KKT xpeACTaBJIxIOTCH ex~e OAxOpOJ~HbiMLi,
Apyri3e - Baxyoxx3HpoBaxxbu-zx, a ocTaJIbxbie pacxaRaioTCH B Me.aKyIO K.ax Kpyxxo-
3epHI3CTyI0 MaCCy. HexoTOpbie xIapbi OKaabIBaIOTCH J~OBOJIbHO KpyHHIaIMI3 (OKOJIO
~,g ~C6) H, x0 HaxII3M Ha6JIIOj[eHYIHM, B TeLieHLIe J~aJIbHeI~xIerO paaBl3T~iH MOryT ex~e
6OJIbIIie yBe.nKgHBaTbCFI (i(O 1-2 ,u, pxc. 12) Hxx Baxyo.aHaKposaTbcx (Axx ~epe3
2-3 xoc~e pacxaRa KKT). Baxyo.aH Bxa4a.ae 6biBaioT cosceM He6o.abIIII3MI3, xoaRxee
axa~xTenbxo yBe.aK~xBaEOTCH (pxc. 13, 14). Tax xaK aepxKCTOCTb pacno~araeTCH
06bILIHO He6OJIbxII3MLI rpyxxaMH, TO iIaCTO I3 i33 BaKyOJII33HpOBaHHbIX xIapOB B03Hi3-
HaIOT rpOaj[beBI3J~Hble O~paaOBaHI3H, HaxOMI3HaEOH~YIe IrIHOPJ~a (I3 xO paaMepaM) CHJIbHO
BaKyOJI~I3I3pOBaHHbie KKT (pHC. 11). BaxyOJI~i O6bIKHO 6biBaioT OKpylKexbI OCTaTKaMH
xepBOHaiIaJIbHO# x.aaaMbI B BIIJ~e Ka~MbI I3JIH He6OxbxiO# HOtiKH (pxC. 15). AxaxorHio
BO3HI3KHOB0Hi3fi BaKyo.aeii B xiapax MOxcHO BS~I~[eTb B T. H. L-OpraHlriaMaX, Pie Mbi
xa6nioRanx xoRo6HOe xB.aexKe xoc.ne xepexecexxx axeMexTapxbix Te~eu xa xosyl0
cpeAy: y i33OJII3pOBaHHbIX Te.aeu OC)bI~IHO Ha~HHaJIaCb BaxyO.ax3auxH (pHC. 16).
IIO~{BOJ~H I3TOPI3 OTJ~eJIbHbIX OxbITOB, MOHSHO CKaaaTb, i3T0 CHH1KeHHe TOMxepaTypbl
BbIabIBaeT xpeaKAe BC0P0 aaMeAneHLie paaBKTHH KKT, KOTOpOe xCHO BbipaxceHO xpx
4? C K MCHee BblpaaIITeJIbHO, paayMeeTCH, xpH KOMHaTHOLI TCMxeparype. c~aMeJjJIeHlie
OTpa}IfaeTCH B O6xjeM ~JIarOxpI3f3TH0 Ha pereHepauxH 6auH,a.axpHO~Y (~OpMbi: TaM, rJ~e
xpH TeMxepaType B 37? C KKT 6bICTpo pacnaRaaxcb, xe xa6xioAaxocb o6btexo Boc-
CTaHOBJIeHi3H BTOpLItIHbIX xaJIOLIeK, TOPRa KaK xpH TO~ri ?Ke KOHI[eHTpauxx xeHI3IjHJI-
JILIHa, HO 6OJIee HI33KIIX TeMxepaTypax - xacTynaaa perexepauHx. B xexoTOpbix
CJIyLIaxX MbI Ha~JIIOJ~aJILI (xpH KOMHaTHOLI TOMxepaType) BO3HIiKHOBOHLIe L-KOJIOHI3~
Raaxe LI xpx TaKxX BbICOKIdX KOHL~eHTpauxxX, KaK 20 H 40 TbICxLI eA/MJI, iiTO HI3KOr~(a
He Ha6JIIOJ~aJIOCb xpK 37? C. Moxcxo yTBepxcAaTb, iITO KOMHaTHax TOMxepaTypa
SIBJIHeTCx Hax6Oxee 6aiarOxpLIxTHO$I J.[dIH BOCCTaHOBJIeHI3H 6auxxxxpxoi~ (~opMbI
Proteus.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
JJ,pyrz~IM TIIHHLIHbIM peay.nbTaTOM HI33K~SX TeMHepaTyp HBJIHeTCH CKJIOHHOCTb
K pocTy B jj.;II3Hy. MbI HIiKOPJ[a He Ha6JIIOJ~aJIH CJIyzIaeB B03HYIKHOBeHHH BTOpI3tIHbIX
HaJIO~IeK H3 KKT HyTCM T. H. L~eHTpaJIbHO~i c~paPMexTauHn, TaK iITO HpaKTIIzIeCKLI
eAt3HCTB0HHbIM aJIeMBHTOM perexepauHr~I HpLI HOHI3aK0HHOLI TeMHepaType HB.RHIOTCH
I{IIT (KpoMe McJIKOI~I aepHnCTOCTI3 - 3JIeM0HTapHbIX TOCeI~, - HpeACTaB~IHIOH~KX
ocxoBy L-KO~IOxHiI).
MbI Hcc~IeAosa.nK paaBraTne Kpyxxblx Kpyr~Iblx Te~Ieu (I{I{T) Proteus vulgaris
H P. mirabilis Hpla TeMHepaType B 22? LI 4? C i3 npu BoaAe~ICTBUt3 paa.nt3~Ixblx
KoxuexTpauK~I nexHuH.a,ni3xa (2, 6, 10, 20 n 40 Tblc. e~/MJI).
1. IIpH KOMHaTHO~I TeMHepaType MbI Ha~JIIOZ[aJILI HOJIHbII~i H HBIIOJIHbI~i L-L~I3KJIbI.
IIpepsaxxbl~t uHK.a noxa xaM xe BcTpeaa~ICH.
2. IIpH 4? C xa6~IloAa~ICH To~Ibxo xenonxbl~i utaK~I.
3. IIpn HccsleAyeMblx TeMHepaTypax pacllaA I{I{T H perexepauHx BTOpH~xblx
HaJIOLIeK C IIOHIIaK0HH8M TOMHepaTypbI 3aMeZ(JIHIOTCFI.
4. CHOCOCHOCTb I{I~T (K.aK aloe Kpyxxblx HpoAo.arosaTblx Te~Ieu) o6paaosaTb Ha-
JIOLIKI3 npii HOHII7KeHH0I3 TeMHepaType COXpaxHETCH J~OJIbIIIe, TIeM HpYI 37? C, Aaaxe
npH 6o,nee BblcoKt3x KoxuexTpaut3xx nexHuH~I.nt3Ha.
c~HatieHI3e 1.-IjIIKJIa ~OaKTepI3i3 RJIH B03HIIKHOBeHLIH yCTOYiiIHBOCTLI K HCHHIjHJIJIIIHy
~JIH BbIHCHeHI3H BOnpOCa yCTOI~iIYiBOCTI~I BTOpI3LiHbIX Ha~IO~IeK B HOHLII~IdJIJIHHy MbI
IIOCT8BI3JII3 CJIeZjVIOH~Yie OHbITbI: 1. IIaCCaaKYi KyJIbTypbI Proteus vulgaris B aKt3AKxx
H Ha HJIOTHbIX CpeJ~aX C HeHIIL(IIJIJII~IHOM; 2. OHpe~eJIeHLIe yCTO~iYLIBOCT~I BTOpH~IHbIX'
Ha~IO~eK, BblpoclHHx rya oRxoro o6H;ero I~I~T; 3. onpeAe~IexHe coRepaxaxHH Hext~ux.a-
.nHxa B Ky.abType P. vulgaris s 6y~Iboxe.
OnpeAe.aexxe YyBCTBHTeJIbHOCTH nanoYex Proteus x HexHL[Hn~IHxy npoxasoAxnocb no McTOAy
Cepx~IHbIX pa3BeJ(eHH~f (H-YaCOBO~i HyJlbTypbI). MbI ZjeJIaJIH IIaCCaxtx x.nx B CbIBOp OTOYHOM 6yJIbOHe
(25?~?), x~IH xa 6.noxax arapa c 2000 eA~M~I (axaapo6xo). CoAepxcaxxe nexHgx~n~nHxa MbI onpeRe.nx.nx
I3JIx C HOMOH~bIO xOJ~OMOTpHH, x7II3 HO MBTOJ~y JIHHe~IHO~I Z[I3(~I(~Iy3HH HO IIeIHeKy (1952).
Peayrcbmamaa
C HOMOH~bIO HaCCaaKe~I B aKHj[KOII CpeRe He ~bIJILI HOJIytIeHbI OJ~H03HatIHble peayJib-
TaTbI. IIOCJIe 6 HaccaaxeH He HarOJIIOJ~aJIOCb CKOJIbKO-HI36yAb 3aM8THOP0 HOBbILIIeHHH
yCTO~ILII3BOCTI3 K HeHHI[HJIJIHHy.
IIpr3 C.ne~jyloH~HX OHbITaX MbI J~CJIaJII3 HaCCaaKH Proteus Ha IIJIOTHOII CpeAe C IIeHH-
I(LIJIJIAHOM x Hpn e?KeRHeBHOM MHKpOCKOIILItIeCKOM KOHTpOJIe OTMeiIaJIII HpOuexT
o6paaosaxr~H KIST ~epea 2 Baca Hoc.ne Hocesa, a TaKaxe BpeMH i~ cnoco6 perexepauHH
BTOpLILIHbIX HaJIOileK. MbI HCXOJ[YIJII3 YI3 TOPO HpeRHOJIO?KOHHfI, PTO y 6osee ycTOii-
~Ir~Bblx Hano~eK npouecc perexepaur~r~ xa KKT Ao~IaxeH npoTexaTb 6blcTpee H ~To
~epea onpeRe~Iexxoe BpeMx Hano~KH Boo6H~e Ao~Iaxxbl HepecTaTb o6paaosaTb I~KT.
MbI npoHase.nr~ B o6H~eM 27 Haccaaxe~I. OHr~IUeM caxaTO, KaK o6u~aH KapTHxa peaKuHH
xa Hexr~gH.n,aHH Mexx.aacb B Te~Iexue HaceaaxeH. Bxa~Ia~ne, npH HepsoM naccaaxe,
94% Ha.no~IeK pearr~posa.ao o6paaoBaxHeM I~I{T. PerexepauHx ocyruecTB.nH.nacb
o6bl~xblM cuoco60M ~Iepea 30 ~IacoB. IIpH 19-oM Haccaaxe oxo.no 40% Ha,no~eK o6pa-
aosaao I{I{T i~ perexepauHH Txxy.nacb 4 RxH. IIpr~ Hoc.neAxeM Haccaaxe I~I~T o6paao-
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Ba~HCb To.abxo B 5% c~y~aeB, a perexepauHA He 6bina oTMeaexa Hx Ha oAHOM Hpe-
napaTe. II08TOMy Mbi H H8 MOrJII3 yHte OnpeRe~xTb HO IIJIaHy yCTO~IiiI3BOCTb BTOpI34HIaiX
IfaJIO~IeK K HOHLSI~HJIJII3Hy HOCJie 3O-PO HaCCaxca. c~aIIaaj[biBaHI3e pePeHepauHx rpac~K-
4eCKi3 IlpeJjCTRBJIeHO Ha pxC.19. HaiiI3HaFi C 9-PO IIaCCa?Ka, HO~ITH Ha BCOX HperlapaTaX
BCTpeLIaJII3Cb L-HOJIUHi3i3 (TLIHa 3 S), ~IaCTO CJIy?KHBHII3e eAxHCTBexHr~M I3CTOKHHHOM
0 10 20
Pxc. 19. IIpoRnexxe nepxoAa perexepauxx
sTOpxvxbtx nanovett xa I{I{T s Tevexxe
naccaxce# xa n~corxo# cpeAe c nexxuxn-
nxxoM. Ocb X: xonxvecTBO naccaxce#, ocb
Y: spemR perexepauxx B Axxx.
0 1 2 3 6 5 6
Pxc. 20. B~xxxxe TeMnepaTypbt xa naAexxe
yposxR nexxgxnnxxa s c~xaxo~corx~eceoM pac-
Tsope. Ocb X: xonxvecTBO Axe#, ocb Y: xonx-
~ecTBO eA. nexxuxnnxxa s 1 Mn B Tbicxvax. I xpx-
BaR: 37? C, II xpxsax: 22? C, III xpxsaa: 4? C.
BTOpH*~Hbix na~o~eK. Ha*~HHaH c 21-ro naccaxca, L-xo.noxxH Ha xexoTOpbix npena-
paTax yaKe xe noHB.aR.nHCb, a perexepauxA eacTO xa6.aFOAa.aacb To.abxo xa 1 xa
3 npenapaTOS. Bce aTH xB.aer~Hx o~exb HanoMxHaaH xapTxHy, Ha6,aioRaBiHyiocA npx
soaAe#cTBHH ropaaRo 6o~ee BbicoKHx Roa neHxuH~~xxa (f~40 Tbic. eA~M~ -
HepMyT H He*~ac 1956). TaHI3M o6paaoM, K aTOT OIibIT H8 CBI3j~eTeJIbCTBOBaJI 0 HOBbI-
HIOHI~ILi yCTO~rIiII3BOCTi3 BTOpI34HbIX HaJIO~IeK HOT( BJII3HHI3eM MHOrOKpaTHOrO KOxTaKTa
C H8HI3I[I3JIJILIHOM.
nOaTOMy Mbi HpIdCTyHLIJIi3 K OIIpeJ~eJIeHI3I0 yCTO~ILII~IBOCTLI BTOpi3~IHbIX IIaJIO~IeK,
B03Hi3KHII3X Ha I{I{T HpH HpHMOM MHKpOCKOIII3ileCKOM KOHTpOJIe. YIa OGIIjerO KOJII-I-
iieCTBa Hpyi(JII3a~ITeJIbHO 1OO KaO.axuH# HaM 52 paaa yRa.nBaOCb IIOJIyYI3Tb BTOp13iIHble
nanoYKH Ka oRxoro I{I{T, K OHpeJ~eJIHTb y Hi3X LIyBCTBi3TeJIbHOCTb K IIBHLII[YIJIJII3Hy.
B 37 CJIy~aHX OHa COOTBeTCTBOBaJIa KOHTpOJIIO, B 3 c.ay~aAx 6bIJIa BbIIile (Ha 1 npo-
6Hpxy), a B 12 c~yeaRx - xxaxe. Bo Bcex c~yaaax HecooTBeTCTSxx xoxTpo,nio 6bi.na
OTMeaeHa HOKOTOpaH paaHHua B nOMyTHOHxIrI paaseAexHx Ky.nbTypbI B cpasxexxK
C KOHTpOJIeM. BHaLIaJIe MbI He npxAasanx aTOMy aHaiieHl3H, HO OIIbITbI HOKaaaJIH,~
LITO LIX peayJIbTaTbI B aHaiIYITeJIbHO~i CT8H8HI3 aaBI3CHT OT KOJII3LieCTBa KJIeTOK. YcTU~-
LILiBOCTb KOJIe6aJIaCb B rpaHI3IjaX OT 5OO J{O 2000 eR/MJI. OKaaaaocb, TaKI3M OGpaaOM,
LITO yCTO~i~ii3BOCTb BTOpI3LIHbIX HaJIOileK He IIOBbIIiIaeTCfi CKO.RbKO-HI36yRb CyH;e-
CTB0HH0. McTOR paaBeReHK# JjJIFI OnpeAe.aeHHA yCTO~IYI3BOCTi3 He I3CKJIIOYaeT B08MOxc-
HOCTI3 HOHOTOpOPO HOBbIHI8Hi3H yCT0~3YI3BOCTLI. HO, HaK YI3B0CTH0, HeaHa~IITCJIbHOe
HOBbITIIeHI3e He j[OJI?KHO 6bITb O~HaaTeJIbHO CHeI~I3(~1LI~IeCKI~IM, OHO MO1KeT BbIabIBATbCfi
HOTO~iHO OTMepeHHbiMI3 ROaaMI3 j[JIH IIOCBBa K T. H. MbI HOTOMy H npHAepaKHBa~HCb
aTOPO McTO~a, iITO OH, ~JIarOJ;apH CBOe# HBCJIYIHIKOM ~OJIbLiiO~i YyBCTBI3TeJIbHOCTI3,
I3CKJIIOLIaJI aTI3 HOCHeI~I~I(~IYILIeCKLIe OTKJIOHeHLIFi. H000XOj(I3M0 eHje IIOJ~YepKHyTb, ~iTO
Hpx BCOX I3CHOJIbaOBaHHbiX HaMId j[O CI3X nOp HOHI;eHTpauHxX IIeHHgH.n.aHHa (OT 7 J(O
4O TbIC. eA/MJI) MbI BCBr~(a BCTpeLIaJIYiCb C 06paaOBaHI3eM KKT H YTO HpK HOHI~eHTpa-
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
z;HHx B 2000 eR/MJI 13 BbIIIIe MbI xllxorAa xe xaxoAn~IH Ha~IO~ex, xoTOpble pasMHO-
alfaJII3Cb 6bI IIpHMO, He HpOXOJ~H L-I[I3KJIa. 110CIfOJIbEfy HeIfOTOpble IIaJIOLII{H He
O~p330BaaJIBI KKT, OHII OTMLIpaJII~I H paCIIaJjaJIIICb.
r'.CJILI BTOpyItlHble HaJIOtIHII He yCTOI3LILIBbt K HeHHI~HJIJIHHy, 3T0 03HailaeT, LITO
CO~[epaRaHl~ie HOHYII[RJIJII3Ha B HepIIO~[ pePCHepaI~YIYI AOJIaI{HO ~ObITb 3HatII3TeJIbHO HHaI{e,
LIeM B HaiIaJIe OHbITa. F.CJIH 8T0 Talc, TO HeOGXOJ~HMO yCTaHOBI3Tb HpIItIHHy ero yMCHb-
Pxc. 21. IIaAexxe yposxx nexxgxnnxxa Pxc. 22. IIaAexxe ypoxxH nexHgnn.nHxa B
s 6ynboxe npn 37? C. Ocb X: xonx*EecTSO xynbxype Proteus vulgaris npx 37? C. Ocb X:
Rxex, ocb Y: xo~IHgecTSO eAxxxu nexn~xn- BpeMx s gacax, ocb Y: xonxuecTBO eAxxxu
nnxa. nexngH.n.nxxa xa 1 Ms B TblcxYax. ICpxaax K:
HOHTp07Ib.
~i
IileHHH. H03TOMy MbI HCCJIeJ~OBaJIII CHaLIaJIa, xax Haj~aeT I{OJII3tleCTBO H0HI3I;IIJIJII3Ha
B axHRxoH cpeAe B cBHan c TeMHepaTypoH. IIpn HepsbEx axe onblTax oxasa.aocb, ~ITo
rlaAexlle coRepaxaxzlx Hexz~ull.n,nHxa npK pH 5,5-6,0 B 3Ha~HTe.abxoH cTerleH~I
3aBIICHT OT TCMHepaTypbI (pllc. 20): B TepMOCTaTe (HpII 37? C) THTp HeHHI;HJIJIIIHa
C HepBOHaLIaJIbHI,IX 16.000 eA/MJI yna.x B Te~exr~e 6 AHeH Ao 1800 eA/MJI, T. e. Ha
88,8%; HpK xoMxaTHOiI TeMHepaType xa6,nloAa,nocb HaAexlle c 16.200 eR/MJI xa
14.400 eA/MJI (xa 11,2%), a B .aeAHmxe (4-6? C) c 16.500 xa 15.200 eA/MJI (Ha 8%).
8TH HoxasaTesn 6bI.nH ycTaxoBSexbl HoAnMeTpr~~ecxnM HyTeM.
IIpH ApyroM oHblTe MbI c HoMOU;bIO RHC~c~yar~oxxoro McTORa onpeAe.xH.nH HaAeHI~e
coAepaxaxnH Hexnun.n.axxa B 6y.aboxe. 1Cax HoxasblsaeT pxc. 21, xapaxTep xpzaBO~I
sRecb npaxTll~ecxK oTBe~aeT xpr~BOK npH 37? C xa pHC. 20. yIa aTOro BblTexa~IO 6bI,
PTO B rlepnoR perexepaur~t~I Ha~IO~ex (*Iepea 24-30 mac.) coRepaxaHlle HexHuIl~I.nIIHa
BCe eHje ~bIJIO rObI OLIeHb BbICOIfIIM, LITO HpOTI3BOpeLIHJIO ~bI pe3yJIbTaTaM HaHILIX HpeA-
~HIeCTBOBaBHII~IX OHbITOB. II03TOMy MbI HpliHyal{J~eHbI rObIJIH HpOCJIeJ~I3Tb H3MOHeHyIH
ypoBxll neHIIuII.n,nIlHa B cpeAe, aacellxxoil 18-~IacoBOK xy,abTypoil P. vulgaris.
IIpK xecxo.abxxx HepsbEx oHblTax MbI onpeRe~ISI.ax nepsoxa~a.abxoe I3 I:oxe~Ixoe
coAepaxaxHe HeHHuH.n.nHxa, T. e. ero coAepaxaxHe B HepHOA, xorRa obl.na oTMeaexa
perexepaunx Ha.xo~ex. B HepIIOA perexepauHr~ ero coAepaxaHHe 6bIBaJiO BcerAa xllaxe,
~EeM 0,03 eA/M.n, T. e. Hpal(THLIOCICLI paBHHJIOCb HyJIIO, tIT06bI BbIHCHHTb XOA HpI3BOYi
HaJ~eHIIH coAepaxaxHx HOHLII~IIJIJIIIHa B I{yJIbType, MbI OTMeYaJILI COZjepal{aHLIe HBHH-
uII~I.aHxa ~epea xaaxRble 3 Baca BH.xoTb Ro 36-ro Baca (pllc. 22). 1#oxTpo.abxaH xpHBaH
sa Hcc~Ie~yeMblK oTpeaox BpeMexll HpaxTHaecxx coBCeM xe cxHaxaeTCH, TorAa xax
oHblTxaH xpHBax HaRaeT o~exb 6blcTpo (Tabs. 3). B aTOT HepxoR (c 3-ro Ho 12-bII3 ~Iac)
I{yJIbTypbl COCTOxJII3 HpeIIMyH;eCTBeHHO H3 HIapOB CpCRHHX pa3MepOB (OKOJIO 5-10 ~C4),
a oxoso 10% COCTaBJIxJIII HaJIOLIKLI IIJIH HX OCTaTItII. r1aJIOLIHII He 6bIBasK oRxo-
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
poRxbiMx x xe xpoHBSR.aH npHaxaxoB poc'ra H.aK paaMxoxceHUH. PerexepauHH
BTOpx~xbtx xa.ao~ex xaYHxaaacb Tonbxo c 24-ro *~aca H Honxoro paaBHTHx AocTHra~a
x 30-My racy, T. e. xa~xxanacb vepea 12-15 ~acoB Hoc.ae HaRexHA coRepaxaxxH
IIeHLIt~I3JIJII3xa J;O Cy66axTepKOCT3THaeCHHX Be.aI3tiHH. 'e~TI'I peay.nbTaTbI 04eHb BaxcHbI
K CBLIJ~eTeJIbCTByIOT 0 3Ha~IxTeJIbH01'`I CIIOCOCHOCTYI I{I{T paCII~eIIJIHTb xeHHuH.a~HH
Ha er0 I3HaIfTLIBHble COCTaBHble iIaCTlri.
Pea~o.~-tie
1. BTOpH~Hbie Ha.ao*~xH, perexepHpytou~xe Ha I{I{T Proteus vulgaris, He 6biBaFOT
6oJiee yCTO~IiII3BbIMI-i IS nexHIjI3JIJIi3xy, qeM HepBH~Hb~e (HCxoRxbte).
2. CoAepxcaxxe HexHux~xHHa B xtHRxo~ cpeRe B nepHOA perexepauHK npaxTx-
~ecxH pasxxeTCH xy~io. PerexepauKH Ha~HHaeTCH npHfi.aHaxTe~bxo *~epea 12 sac.
Hoc~e HaAexKR coAepaFCaxHA HexxuxanKxa Ro aTOro yposxx.
ABTOp no.aaraeT, qTO L-uxx~ 6axTepx# He xMeeT xaxoro-xx6yAb oco6oro gxa~eHxs
J{JIH BOaHI3xHOB8HxH IiITaMMOB, yCT01'I~IIdBbIX H HOHHLjI3JIJII3Hy.
(Ta6n. XIX, XX, XXI).
JIxTepaTypa
B n a m x o B x e, J~. c c o T p.: K npo6neMe c~xnbTpyiou{xxcx c~opM 6axTepx#. Fol. biol.
(Praha) 2 : 8, 1956.
H e p M y T, M., H e Y a c, O.: L-i~opM~ 6axTepx#. I. IdaMexexxx ~opM~ Proteus vulgaris,
Bvia~BaeM~e nexxuxnnxxoM. LIcn. Bxonorxa 3:370, 1954. II. Bnxxxxe axaapo6xatx ycnoBx#
Ha xaMexexxx c~opMS~ Proteus vulgaris noA Ae#cTBxeM nexxgx~[nxxa. Fol. biol. (Praha) 1:257,
1955. I I I . BnxAxxe xoBuexTpauxx nexxgBU~[xxa B cpeRe xa paaBxTxe L-o6paaosaxx# y Proteus
vulgaris .Fol. biol. (Praha) 2:36, 1956.
T p o x u x x #, B. JI., II e p m H H a, 3. P.: 1i{ypH. Mxxpo6xon., anxAe~xo~., HMMyxon. 1950.
Cit. M a 1 e k, I.: Promenlivost mikroorganismu v sovetske vedL. Thomayer. sbirka 293, 1951.
X o p o m e B H ~c, R.: BJILIHHHe XJIOpMi3LjeTnHa Ha H3M0HiII3BOCTb KI3BIe'IHO# nanOUKH H, B 'iaCT-
xocTx, xa o6paaoaaxxe L-~opM. Med. doswiad. i mikrobiol., 7 : 5, 1955.
Ili e M x x x B, M. M., X o x n o B, A. C.: XxMxx axTx6xoTx~ecxxx Beu~ecTB. Mocxaa 1953.
B l as k o v i L, D., R a u s, J.: K pproblemu filtrovatelnych foriem bakterii. II. Pokusne
podmienky pre vyvolanie L-cyklu Pfeiffrovho hemofila. Z`s. biologie 4:588, 1955.
C a n b a c k, T.: The Influence of Iodide Concentration in the Iodimetric Titration of Penicil-
lin. J. Pharm. Pharmacol. 2 : 364, 1950.
Dienes, L., Sharp, J. T.: The Role of High Electrolyte Concentration in the Produc-
tion and Growth of L-forms of Bacteria. J. Bact. 71:208, 1956.
Dienes, L., Weinberger, H. J.: The L-forms of Bacteria. Bact. Rev. 15:245, 1951.
G r a s s e t, E.: Observation comparees sur la production in vivo et in vitro des formes L
de Proteus vulgaris et Klebsiella pneumoniae sous 1'influence d'anticorps specifiques. Ann.
Inst. Past. 89:111, 1955.
G r a s s e t, E., B o n i f a s, V.: Modalites de transformation en formes L in vivo de Proteus
vulgaris et d'autres Enterobacteriaceae sous faction de la penicilline. Ann. Inst. Past.
88:651, 1955.
J o i r i s, E.: Etudes de certaines proprietes de la croissance bacterienne de Proteus mirabilis
apres passage par la phase L. Rev. belg. Path. 24:5, 1955.
L i e b e r m e i s t e r, K.: Untersuchungen fiber Bakterien-L-Formen. Zbl. Bakter. Orio. I.
160:250, 1953.
L i e b e r m e i s t e r, K., K e l l e n b e r g e r, E.: Studien zur L-Form der Bakterien.
I. Die Umwandlung d. bazilaren in die globulare Zellform bei Proteus unter Einfluss von
Penicillin. Z. Naturforsch. 11b :200, 1956.
N e r m u t, M., N e L a s, O.: L-formy bakterii. I. Tvarove zmeny Proteus vulgaris vyvolane
penicillinem. ~s. biologie 3:394, 1954. II. Vliv anaerobnich podminek na tvarove zmLny
P. vulgaris vyvolane penicillinem. ~`s. biologie 4:390, 1955. III. Vliv koncentrace penicillinu
v pude na vyvoj L-telisek u Proteus vulgaris. ~`s. biologie 5:20, 1956.
P es e k, J.: Stanoveni hladin~ penicillinu v krvi linearni diffusi. Lek. listy 7:517, 1952.
Report of the Analysts' Subcomittee of the Ministry of Health Conference on the Differential
Assay of Penicillin. Analyst 74:550, 1949.
S c h n a u d e r, G.: Die Formen-L and ihre Rolle bei der Entstehung der Penizillinresistenz
der Bakterien. Zbl. Bakter. Orig. I. 162:151, 1955.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
L-forms of Bacteria
IV. The Influence of Temperature on the Development of L-cycle of Proteus vulgaris
and its Significance for the Development of Resistance to Penicillin
Summary
The final link in the L-cycle of bacteria is their return to the original bacterial
form. In other words, ?~secondary" rods regenerate within the L-cycle from various
preceding forms (large bodies, long bodies, elementary bodies). When studying
L-forms of Proteus vulgaris, two questions were of particular interest: 1. Whether
the J`,-cycle would take place at temperatures lower than 37? C and at what other
temperatures could regeneration of secondary rods take place, and 2. Whether
these secondary rods were more resistant to penicillin than the original rods.
The Influence of Temperature on the Development of Large Bodies of Proteus vulgaris
Produced by Penicillin
Work was carried out with strains of P. vulgaris and P. mirabilis using the method of agar blocks
(anaerobic) at temperatures of 4-6? C, 22? C and 37? C. The concentration of penicillin was 2, 6, 10,
20 and 40 thousand units~l ml.; the medium contained 25% equine serum. Phase contrast photography
was carried out with a tine-film Foma repro ortho II.
2,000 units PE~ml.
Formation of LRB
Regeneration of
secondary rods
Remarks
37? C
2 hrs.
24-30 hrs.
22? C
4- 6 hrs.
24-48 hrs.
Occasionally after 15 hrs.
6?-8? C
8-24 hrs.
5- 7 days
Comm. 5th day,
ends 7th day
4 ?C
16-30 hrs.
8-10 days
Comm. 8th day,
ends 10th day
The results are summed up in part in tab. 1 and 2. A decrease in temperature
leads primarily to a slowing down in the development of large bodies. This is very
clearly expressed with a temperature of 4? C and is-understandably-less evident
at room temperature. Slowing down is seen to be propitious on the whole for rege-
neration of the bacillary form. In cases where the large round bodies rapidly
disintegrated at a temperature of 37? C, regeneration of secondary rods did not, as
a rule occur, whereas it occurred at lower temperatures, with the same concentration
of penicillin. In some cases the development of L-colonies (at room temperature)
was observed with concentrations of penicillin as high as 20 and 40 thousand
units~ml., whereas at 37? C this never occurred. It may therefore be said that room
temperature is the best for regeneration of the bacillary form of Proteus.
A further typical feature of lower temperatures is a tendency to elongated growth.
The development of secondary rods from large bodies by central fragmentation was
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Temp.
Conc.
PE in
thous.
Formation
of
LRB
Disintegration
of
LRB
Formation
of
LLB
Regenera-
tion of
rods
Formation
of
L-colonies
10
2 hrs.
8-24 hrs.
-
24 hrs.
-
37? C
20
2 - 4 hrs.
8 - 24 hrs.
-
-
-
40
6 -12 hrs.
12 - 24 hrs.
-
-
-
10
4 hrs.
1- 3 days
12 hrs.
24 hrs.
17 days
22? C
20
4 hrs.
1- 3 days
24 hrs.
3 - 4 days
10 days
40
8 hrs.
2-5 days
-
-
10-14 days
10
8-36 hrs.
3- 5 days
7-9 days
10-14 days
-
4 ?C
20
1- 4 days
6 -10 days
12 -14 days
19 - 23 days
-
40
1- 4 days
7 -14 days
-
-
-
never observed, so that at low temperatures the large long bodies are virtually the
only regenerative element (apart from the small granules, or elementary bodies,
which form the basis of the L-colonies).
What is the significance and position of the large long bodies in the individual
types of the L-cycle of the bacteria ? In the first place, what is their relationship
to the large round bodies ? It cannot be said that they are identical with the large
round bodies, for reasons of difference in form and function. The large round bodies
are for the most part spherical in shape, whereas the large long bodies are elongated,
usually bipolar and sometimes tripolar. Sometimes they are thin and very long,
sometimes thicker and shorter (figs. 1 and 2). They are a further stage in the deve-
lopment of the large round bodies, a stage capable of regeneration. The large major-
ity of large long bodies regenerate into rods. That cannot, however, be said of the
large round bodies. It is known, for example, that with higher concentrations of
penicillin the large round bodies disintegrate. On the other hand, development of
elementary bodies from large long bodies was never observed, only from large round
bodies. Large long bodies, therefore, are one of the possibly stages in the development
of the large round bodies and according to our experience develop more frequently
under aerobic conditions than under anaerobic conditions and at low temperatures
rather than at high temperatures (i.e. 37? C) and also in the presence of certain
growth substances (e.g. from yeast cells). They occur most frequently in an in-
complete L-cycle, less frequently in a complete cycle and never in an interrupted
cycle.
Conclusion
A study was made of the development of large round bodies of Proteus vulgaris
and P. mirabilis at temperatures of 22? C and 4? C under the influence of various
concentrations of penicillin (2, 6, 10, 20 and 40 thousand units~ml.).
1. At room temperature a complete and incomplete L-cycle were observed. An
interrupted cycle has so far not been observed.
2. At 4? C only an incomplete cycle was observed.
3. At the temperatures investigated disintegration of the large round bodies and
regeneration of the secondary rods takes place more slowly, in direct relationship
to the fall in the temperature.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
4. Ability of the large round bodies (or of the large long bodies) to produce rods
is maintai ned for a longer period, and with higher concentrations of penicillin, at
temperatures lower than 37? C.
The Significance of L-cycle of Bacteria for the Development of Resistance to
Penicillin
Investigation of the question of resistance of secondary rods which regenerate in
the course of a complete or incomplete L-cycle is important for comprehending the
biological significance of L-forms of bacteria. Troitsky and Pershina (1950, cited by
Malek 1951) are of the opinion that the new rods are more resistant to penicillin
than the original rods.
Other authors especially Dienes and Weinberger (1951), Joiris (1955) and
Schnauder (1955) take the view that the secondary rods are not more resistant to
penicillin than the primary rods. An attempt was made, on our own material, to
throw light on this question. The following experiments were carried out: 1. Deter-
mination of the resistance of secondary rods which regenerated from one large body,
2. Observation of the penicillin level in a broth culture of Proteus up to the time of
regeneration of the secondary rods.
Large bodies from a broth containing 1000 units of penicillin were isolated by the
Lindner drop method and regeneration of the rods was observed under the microscope.
The resistance of these rods to penicillin was then determined by the dilution method
in Lahelle's modification (1948). The results were read after 5-7 days and resistance
ranged from 500-2,000 units/ml. Most authors read results within 24 hours and that
is why their resistance values are so much lower. We, however, found that the
titration result is stabilised only after the third, or even fifth, day of incubation and
that reproducible results can only be obtained in this way. From a total number of
100 cells isolated, secondary rods were obtained in 52. In 37 cases resistance to
penicillin was the same as in the control culture, in three cases it was higher (by one
degree) and in 12 cases it was lower. In
Table 3. all the divergent cases a slight difference
in the inoculation dose had been recorded
Level of PE Level of PE
(technical failure).
Hours in culture of P. in bouillon
The level of penicillin in the broth was
vulgaris in (control)
units/ml. in units~ml.
determined iodometrically or by Pesek's
method of linear diffusion (1952). The
0 5,000 5,000
following considerations were taken into
3 2,000 5,000
account
6 1,500 5,000
If the secondary rods are not resistant
9 100 5,000
to penicillin, this must mean that in the
1`2 3 5,000
period of regeneration the level of peni-
l4 1 5,000
36 X0,03 5,000
cillin is considerably lower than at the
commencement of the experiment. If that
is the case, it is necessary to seek the
cause of its decrease. We therefore first studied the fall in the level of penicillin in the
fluid medium under the influence of temperature. In the first experiments it was
seen that the fall in the penicillin level was markedly dependent on temperature at
a pH of 5.5-6.0 (fig. 20). In the thermostat (37? C) the penicillin level dropped
in six days from the original value of 16,000 units~ml. to 1,800 ttnits~ml., i. e.
by 88.8%, whereas at room temperature it fell from 16,200 units to 14,400 units
(by 11.2%) and in the refrigerator (4?-7? C) from 16,500 units to 15,200 units
(by 8%). These values were determined iodometrically.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
It follows from these results that at the time of regeneration of the rods (24-30
hours) the penicillin level is still very high, but this contrasts with our previous
results. We therefore observed changes in the penicillin level in a medium inocul-
ated with an 18-hour-old culture of P. vulgaris. In the first few experiments the
initial level and the final level (i. e. when regeneration of the rods was found) were
determined. In the period of regeneration, the level was always lower than 0.03 units,
i. e. virtually zero. In order to obtain information on the decrease of penicillin in
the culture, the penicillin level was observed every three hours up to 36 hours
(fig. 22). During this period the control curve virtually does not fall at all, whereas
the experimental curve falls rapidly (tab. 3). During this time (3-12 hours), the
cultures were composed mainly of round bodies of medium size (about 5-10 ,u)
and about 10% consisted of rods or their residue. The rods were not homogenous and
showed no signs of growth or proliferation. Regeneration of secondary rods began
from 24 hours and was at its height at about 30 hours. It began, therefore,
12-15 hours after the penicillin level had dropped to subbacteriostatic values.
These results are very interesting and indicate that the large round bodies are in
large measure capable of breaking down penicillin into its inactive components.
1. Secondary rods which regenerate from large round bodies of Proteus vulgaris
are not more resistant to penicillin as compared with primary rods (i. e. the original
rods).
2. The level of penicillin in the fluid medium at the time of regeneration of the
secondary rods is practically zero. Regeneration commences approximately 12 hours
after the penicillin level has dropped to this value.
The author is of the opinion that the L-cycle of bacteria is not of especial sig-
nificance for the development of strains resistant to penicillin.
(Tables XIX, XX, XXI).
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
Induction of Bacteriophages by Ultraviolet Light in a Naturally
Polylysogenic Strain of Staphylococcus aureus
J. ~`MARDA
Institute of General Biology, Medical Faculty of the University, Brno
The effect of ultraviolet (UV) light on bacteria has already been the subject of
detailed analysis from many aspects. In recent years the induction of bacteriophage
formation by lysogenic bacteria, discovered by Lwoff, Siminovitch and Kjeldgaard
(1950) and elaborated for various species of bacteria, in particular by Lwoff
(1951 etc. ), Weigle and Delbriick (1951), Jacob (1950 etc.) and by other authors
leas come to form an independent branch of this subject. Induction in lysogenic
bacteria has been also studied by Rosenberg (1956), and this subject was dealt
with by Hercik (1953).
The increase in the spontaneous production of bacteriophages by lysogenic strains
{induction) by irradiation of cultures of these with UV light has therefore already
been investigated in detail. Only two authors, however, have so far dealt with the
influence of UV light on the production of phages by strains which produce two types
of phage simultaneously; these are Jacob (1952, 1954) and Welsch (1953). Both
these authors first of all attempted experimentally to find an answer to the question,
of whether every polylysogenic cell produced both phages when lysed, or whether
certain cells always produce only one type. Jacob, who used artificially cultured
double lysogenic strains of P. pyocyanea, arrived at precise, though not altogether
conclusive results. Welsch's work with a naturally double lysogenic strain of Micro-
coccus pyogenes aureus did not provide a clear answer to the problem.
An attempt was therefore made to resolve this problem along independent lines,
following on from the results obtained in earlier experiments with lysogenic staphylo-
cocci (Rosenberg and ~marda 1955). Staphylococcal strains are frequently naturally
polylysogenic, as described recently by Gorrill and Gray (1956). The work was
carried out with a naturally polylysogenic strain of Staphylococcus aureus haemo-
lyticus.
Previously described strains of the species Staphylococcus aureus haemolyticus (Rosenberg and
~marda 1955) were used in the experiments, in particular the lysogenic strain LS 2 and the sensitive
strains CS 1, CS 4, CS 10, CS 13 and CS 14. Before commencing the experiments, all the strains were
propagated at least five times from one colony and further cultivated in a similar manner at least once
a week. In the case of the strain LS 2, endogenous lysogeny was demonstrated serologically according
to the method of McKinley (1925).
The strain LS 2 now produces basically two types of bacteriophage, which differ both biologically
(in the lytic spectrum of the series of sensitive strains) and also serologically. These are phage F 4, for
which the specific indicator strain is CS 4 and F 13, the specific indicator strain of which is C 13. (This
means that F 4 does not lyse C 13 or vice versa.) F 4 is present in the filtrate of a 6-hour culture of LS 2
in an average titre of 104 and F 13 in an average titre of lOb. Phage 1 can also, however, be demonstrated
in filtrate of LS 2; this is identical with F 13, except that it also, to a low degree, lyses CS 4. A clone
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
of CS 4 which was sensitive only for F 4 and not for F 1 could not be isolated; every one of the seven
clones of CS 4 which were cultured out lost its sensitivity for F 4 at the same time as for F 1. It is probable
therefore, that a small number of phages in filtrates of LS 2 in these experiments formed plaques both
onCS4andonCS13.
The following media were used: meat-peptone broth enriched with Difco proteose-peptone and yeast
hydrolysate, and 2% peptone agar, also containing Difco proteose-peptone. The pH of both media
was 7.4.
The bouillon cultures of LS 2 were always irradiated at the commencement of the log phase of growth,
i. e. following 41/2 to five hours' incubation, in a cylindrical container of transparent quartz glass with
an internal diameter of 22 mm. and walls 1.5 mm. thick. The distance of the low-power Uv lamp;
the full spectrum of which was used, was 50 cm. from the slowly and regularly rotating container (about
45 r.p.m.). Unless otherwise stated, irradiation was carried out in the laboratory in daylight, or in
mixed day and electric light. After exposure lasting for 1'15", 2'30", 5', 7'30", 10', 12'30", 15' and
17'30", the same volume of culture waa always removed and stored under the same lighting and temper-
ature conditions until irradiation of the rest of the culture had been completed. All specimens were then
incubated together in the dark for 30 minutes at 34-37? C.
After incubation they were filtered through collodion ultrafiltera of medium porosity about 650 m?.
(The same batch of filters was used for all specimens in the same experiment.) The filtrates were diluted
serially with bouillon or with 0.6% physiological saline until a concentration was reached which was
suitable for counting the plaques, i. e. up to 10-'1. (These staphylococcal phages cause only very slight
clearing of the bouillon cultures and can be titrated only according to the production of plaques on agar.)
Small amounts (usually 0.1 ml.) of the filtrates were transferred by micropipette to agar plates freshly
inoculated with sensitive strains. When dry, the plates were incubated at 34-37? C. The results were
always read off the next day. The titres were calculated to a ,u of the titres of non-irradiated controls.
The experiments were based on the preliminary finding that a filtrate of LS 2
contains phages which consistently lyre the bacterial strains CS 1, 4, 10, 13 and 14.
Filtrates of specimens of an irradiated culture of LS 2, removed from the culture
at given intervals during exposure, were tested after diluting on one to three of the
above sensitive strains simultaneously. The results of 30 such
evaluated, i. e. an average of six experi-
ments per strain.
After plotting on a graph the percentual
increases in the titres of the phages car-
ried by the individual sensitive strains for
the various times of exposure, roughly
two types of curve were obtained (fig. 1).
The first gave approximately the develop-
ment of the titres of phages on CS 1 and 4,
and the other on strains CS 10, 13 and 14.
Both curves indicate a high degree of in-
duction of phage production by LS 2. At
the same time, however, the induction of
experiments
phages forming plaques on strains CS 1 and
4 is not in general so great as that shown
by the three other indicator strains. It
can, to a certain extent, be judged from the
curves that the titre of phages produced
spontaneously in higher quantities by the
Fig. 1. Arithmetical average of titres of phagep
forming plaques on CS 1 and 4 (curve I) and on
CS 10, 13 and 14 (curve II), in the course of
induction of LS 2, as compared with the controls.
x: time of exposure in minutes, y: titre as a % of
culture, will be increased by induction .
even more than the titre of phages, which are liberated spontaneously in lower
quantities. The second type of curve (CS 10, 13 and 14) shows a second peak after
exposure for 17'30", which is higher than the first one occurring after 10 minutes.
(In the case of CS 1 and 4, the filtrates were not tested after this period of exposure.)
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
The higher values of phage titres found after irradiation of a culture of LS 2
with UV light, as illustrated in fig. 1, cannot, however, be evaluated simply as the
result of induction. Under the given conditions, the titres of the phages are also
influenced by a whole series of other factors, including in particular the following:
inequality of the cells in the stock cultures of LS 2, from which a strain was taken
in these experiments (Malek 1955), duration of growth of the culture in the loo phase
during the experiment, and hence variability of the number of cells in the specimens,
the sublethal or lethal effect of UV light on the cells, adsorption of released phages
back on to the cells or debris, reactivation of induced cells by visible light and
inactivation of phages by UV light, together with a whole series of further factors
participating in filtration and also in testing the filtrates on the indicator strains.
(Of these factors, inactivation of liberated phages by UV rays can be excluded
to a certain extent, at least as far as exposures up to 12 minutes are concerned.
It is known that bouillon markedly absorbs UV light. Irradiations of filtrates
of a culture of LS 2 were carried out several times with exposure intervals up
to 12 minutes, under the swme conditions, as irradiations of the culture itself with
and without visible light, and no real decrease in the titre of any of the phages
was found. )
The effect of actual induction cannot be separated from the majority of factors
mentioned above. No further attention was therefore paid to evaluating it quantitat-
ively and statistically; we just confirmed the basic finding that the production
of phages by the strain LS 2 can be greatly induced by UV light. It was decided,
however, in the further work to study the interrelationship of the titres of the
individual types of phages liberated by LS 2 during irradiation. It was assumed
that the above factors which complicate the effect of induction on the absolute titre
of the phages would have approximately the same effect on all the phages in the
specimen and would not therefore fundamentally affect the relationship of the titres
of the individual phage types. The curves in fig. 1 indicated that this relationship
would not be constant in the course of induction.
In this part of the work, 30 irradiation experiments were carried out, using the
lysogenic strain LS 2 and the indicator strains CS 4 and CS 13. Irradiation was carried
out as described above, except that exposure was carried out for intervals of 3, 6 and
every further three minutes up to 30 minutes. After dilution, every specimen was
plated with the same micropipette on the two indicator strains. In these experiments
a modification of d'Herelle's titration method (1926) was used, based on the author's
own experience and consisting chiefly in greater accuracy of the count.
The filtrates were diluted by progressively adding 0.1 ml. filtrate to 0.9 ml.
0.6 % physiological saline. The cultures used in the experiment were not always
of the same concentration and the initial titre of the phages could not, therefore,
always be the same. Their relationship on a given medium, however, was constant.
The proportion of the titres of F 13 and F 4 in 41/Z-5-hour cultures of LS 2
was constant, varying only very slightly from the ratio of 3 : 1, regardless of the
absolute height of the titres, which were different in the individual experiments.
In the statistical evaluation, this ratio was seen to be statistically significant
(standard error of the difference of both averages = 1). In the course of induction
the titre of F 13 increased by as much as 400 times, the maximum being reached
after 15 and 27 minutes' exposure, while the titre of F 4 increased at most 12 times,
with the maximum again after 15 and 27 minutes' exposure. The development
of the ratio of the titre of F 13 to the titre of F 4 in the course of induction is best
seen if the value of the titre of F 4 in every specimen is taken to equal 1. This
relationship then reached the ratio of 367:1.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
As was expected, the development of the titres of the phages was not always
the same. The chronological course of the increase in the titres varied in the different
experiments. The maximum increase was therefore often obtained after different
doses, while the values of the increase in the individual experiments were also
different. In one experiment, the titre of F 4 actually showed no increase on irradia-
Fig. 2. Development of titre of F 4 (I) Fig. 3. Development of titre of F 4 (I) and F 13 (II) in
and F 13 (II) in the course of induction the course of induction of LS 2, in the presence of
of LS 2. Experiment carried out 16. 1. visible light (full lines) and without (interrupted lines).
1958. x: time of exposure in minutes, y: Experiment carried out 7. 6. 1955. x:' time of exposure
titre: 10~. in minutes, y: titre as a % of control: 100.
tion of LS 2, while the titre of F 13 rose 4.9 times (fig. 2). No attempt was therefore
made to give a statistical evaluation of the individual periods of exposure. In all
experiments without exception, however, and whatever the time of exposure,
the increase in the titre of F 13 was always far greater than in F 4, i. e. lysis of the
cells to F 13 was induced far more than lysis to F 4.
The first induction maximum after about 15 minutes' exposure was followed,
as a rule, by a somewhat steep drop in the titre of both phages, evidently as a result
of the relative preponderance of adsorption of liberated phages on to bacteria and
bacterial debris over further phage production by the cells. It is interesting to note
that in many experiments-as in the first part of the work-a further sharp increase
in the titres occurred after 20-30 minutes' exposure, which usually reached a peak
higher. than the first peak at 15 minutes. This phenomenon can be explained by
sudden lysis of many cells as a result of the large done of UV light, at a moment when
the increasing lethal action of the UV light reached a certain equilibrium with the
action of induction. (The possibility of such an interpretation is also borne out
by the conclusion of Franklin 1954, that lysogenic bacteria which have been inactiv-
ated by UV light can still liberate bacteriophage.)
A similar method was .used in four consecutive experiments in which the course
of induction during irradiation in the dark was studied. It is interesting that in these
experiments completely different results were obtained. The titre of F 13 attained
at most six times the value of the control, while the titre of F 4 increased as much
as 34 times. The most effective time of exposure for the induction of F 4 and F 13
was 5-6 minutes. An example is given in fig. 3, which shows the curves of the titres
of both phages in two experiments carried out consecutively under exactly the same
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
conditions, except that in one irradiation was carried out in the daylight and in
the other in the dark.
As compared with the experiments in the preceding series, the effect of induction
in the dark is seen to be almost always far less than in the light, and as far as the
proportional increase in the titres of F 4 and F 13 is concerned, a greater increase
was found in the titre of F 4 than in that of F 13.
After irradiating the lysogenic staphylococcal strain LS 2 with UV light, which
damages the normal course of nucleoprotein metabolism in its cells (in the presence
of visible light), a considerable increase occurred in the number of particles of both
phages produced by the strain. They did not increase proportionately, however,
but the titre of F 13 increased far more than that of F 4. It could, of course,
be assumed that the increase in the ratio of the titre of F 13 to F 4 was due to
production of both phages in changed proportion by every individual lysing cell.
Even if this could be assumed to be partially the case, the increase in the proportion
mentioned in all the experiments is so great that such an explanation alone is not
sufficient and the possibility must be admitted that as a result of induction many
cells lyse while liberating F 13, without simultaneous production of F 4.
This does not, of course, exclude the possibility that after induction a whole series
of different types of cell disintegration appear in the culture. Some cells may dis-
integrate without forming phage (Hradecna 1952, Her~ik 1953), others may
simultaneously produce both F 4 and F 13 in varying proportion and it is even
possible that some produce only F 4. The important fact, however, is that almost
certainly many produce only F 13. The proportion of titres of the two phages in an
irradiated culture, considered from this aspect, would then represent some kind
of section through this scale of forms of disintegration, among which lysis into F 13
predominates.
A study of the results of these experiments permits no definite conclusions as to
the normal, spontaneous manner of production of the two phages by a growing
culture of LS 2. It is possible that it is analogous to the one described above. At all
events, the ability to produce both phages in the proportion of 3 : 1 is genetically
bound to every cell of the strain, since a clone isolated from any cell again produces
the phages in this proportion. Nevertheless, it is very probable that during lysis
some cells produce only one phage.
In resolving the problem of whether one induced polylysogenic bacterium can
produce both types of phage, Welsch (1953) used the "single burst" technique,
i. e. the study of lysis of isolated induced bacteria. After making a statistical analysis
of the results, he came to the conclusion that the question could not be decided
by thin method.
Jacob (1952) states that weak doses of UV radiation result in the liberation
of one type of phage by one cell, while strong doses induce the simultaneous develop-
ment of both types of phage by one bacterium. The present author takes the view
that the experiments from which Jacob arrived, mathematically, at these con-
clusions, on the basis. of the conception of probacteriophage (Lwoff 1953, Jacob
1954), do not take sufficiently into account the whole dynamics of a lysogenic culture
subjected to irradiation during growth. Jacob's general view on this subject
is that in a culture of a strain which produces two phages, the development of which
can be induced, each phage, after induction, can be produced by given cells which
do not at the same time produce the other phage. The present author agrees
with Jacob.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
The results of the present experiments also concur with Jacob's finding that with
high doses of UV irradiation the effect of induction on the production of phage
decreases. It is assumed that this is a manifestation of the lethal effect of irradiation
on the cells themselves, of progressive sterilization of the culture and of a pre-
ponderance of adsorption of formed phage particles over production of further
phages by the surviving cells.
The differences in the results of irradiation in the presence of light and in the dark
are also explained in the same way. In the dark, the bacteria were evidently in-
activated far more by the same doses, induction was masked by inactivation and
the effect of induction was therefore displayed far more weakly in the titre of the
phages. (The somewhat lower production of F 13 than of F 4 indicates more rapid
inactivation of the cells producing them.) Induction was still displayed to some
extent with the lowest doses, after which there was no further increase in the number
of phages and the number of liberated mature phages decreased by adsorption,
the decrease being approximately the same in both types. In visible light, in which
photoreactivation evidently asserted itself through the interaction of low-energy
photons, induction was much more effective.
Summa-ry
1. The formation of both types of bacteriophage liberated by the lysogenie staphylo-
coccal strain LS 2 can be induced to a marked degree by UV light.
2. The constant quantitative proportion of 3 : 1 in which these two phages are
produced in all passages by a culture of LS 2, is disturbed as a result of induction,
production of one of the phages being increased by as much as 400 times, and of the
other, with the same dose, twelve times at the most. The one phage is therefore
probably liberated by cells which do not produce the other phage simultaneously,
although potentially every cell can produce both.
3. The quantitative effect of induction is displayed differently if the culture
is irradiated in the presence of visible light, which reactivates the cells, than if it is
irradiated in the dark.
Frank 1 i n, R.: The Action Spectrum for the Ultraviolet Induction of Lysis in Escherichia
coli K-12. Biochim. Biophys. Acts 13:137, 1954.
G o r r i 1 1, R. H., Gray, R. A.: The Induction of Bacteriophage in Staphylococci. J. Gen.
Microbiol. 14:167, 1956.
H e r e f k, F.: Problem bakteriofaga. Praha 1953.
H ~ r e 1 1 e, F. d': Le bacteriophage et son comportement. Paris, 1926.
H r a d e e n a, 7..: ULinek ultrafialoveho svetla na bakterie Escherichia coli. ~s. biologie
1:348, 1952.
Jacob, F.: Induction de la lyse et de la production de bacteriophages chez uu Pseudomonas
pyocyanea lysogene. Compt. Rend. Acad. Sci. 231:1585, 1950.
Jacob, F.: Developpement spontane et induit des bacteriophages chez des Pseudomonas
pyocyanea polylysogenes. Anrr. Inst. Pasteur $3:671, 1952.
Jacob, F.: Les bacteries lysogenes et la notion de provirus. Paris 1954.
L w o f f, A.: Conditions de 1'efficacite inductrice du rayonnement ultraviolet chez une bacterie
lysogene. Ann. Inst. Pasteur 81:370, 1951.
L w o f f, A.: Lysogeny. Bact. Rev. 17:269, 1953.
L w o f f, A., S i m i n o v i t c h, L., K j e l d g a a r d, N.: Induction de la production
de bacteriophages chez une bacterie lysogene. Ann. Inst. Pasteur 79:815, 1950.
M a 1 e k, I.: O mnozeni a pestovani mikroorganismu, zvlaste bakter~ii. Praha 1955.
M c K i n 1 e y, E. B.: Serum antilytique obtenu par immunisation contre urre bacterie
normale. Compt. Rend. Soc. Biol. 93:1050, 1925.
Rosenberg, M.: Dynamika rozpadu lysogennich bunek ozarenych ultrafialovym svetlem.
~`s. biologie 5:198, 1956.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Rosenberg, M., ~ m a r d a, J.: Staphylococcal Bacteriophages of Lysogenic Origin
and a Comparison of these with Bacteriophages Subjected to Passage. Fol. biol. (Praha)
1:339, 1955.
Weigle, J. J., ll e l b r ii c k, M.: Mutual Exclusion between an Infecting Phage and
a Carried Phage. J. Bact. 62:301, 1951.
W e 1 s c h, M.: Induction par irradiation ultraviolette d'un Staphylocoque polylysogene.
Comp. Rend. Soc. Biol. 147:931, 1953.
P o a e x 6 e p r, M.: J~xxamxxa pacxaAa ~rxaorexxbix xJleTOx xo~ Aer~cTxxeM y.nbTpac~xo.ne-
Tosbrx Jryve#. Fol. biol. (Praha) 2:206, 1956.
y~-I~IH}1(yI{L~IIFI CaRTepyIO(~aPOB y eCT8CTB0HH0 IIOJIYIJII~I30I`eHHOPO
IuTaMMa Staphylococcus aureus
IIoBbiHlexile HpoJ~yHr[HH JlHaorexxblMH IIITaMMaMH 6axTepHOC~raroB B pesyJibTaTe
OrOJIyLIeHIIFi HyJibTyp 3TLIX HITaMMOB yJIbTpa(~i30JIeTOBbIMH JIyiIaMH (y~), - T. e.
IlHjjyHI;tiIH, - HOJjpOOIiO HCCJIejjOBaJIOCb yxce LjeJIbIM pBjjOlVi aBTOpOB. 110 TOJIbKO ABa
H3 HHX 3aHHMaJIHCb Talf?Ite BOHpOCOM BJILIHHHH ~ ~-JIyLIeII Ha HpOJ;yKIjHiO c~aroB
IHTaMMaMt3, HoTOpbie npoHasoAHT oAxospeMexxo ABa THHa c~ara, Jacob (1952, 1954)
ICI WelSCh (1953). 06a OHH HOHbITaJIIICb 3i4CHepLIMeHTaJIbHO OTBBTHTb Ha BOHpOC
B03M0?iSHOCTII HpOj;yifI~HH Z[ByX pa3JIHLiHbIX (~aPOB O~[HO~I H TOI3 5If0 HOABeprHIel%ICH
I~iHj[yifI~HH HJIeTI{OI%I. JaCOb, KOTOpbII3 HOJIb30BaJICH HCHyCCTB0HH0 BbIpaHjeHHbIMH
~Ba?IfJ~bI JIH30PeHHbIMH HITaMMaMH P, pyOCyaneUS, HpHHIeJI B 3TOM BOHpOCe K OHpeAe-
JIeHHbIM, XOTH II HC BHOJIHe y6eJjHTeJIbHbIM pe3yJIbTaTaM. Pa60Ta xce Welsch-a
C eCTOCTBCHHO JjBa~iCJ;bI JIH30P0HHbIM HITaMMOM Staphylococcus aureus B aTOM Ha-
HpaBJIOHHH He HpHHeCJIa HCHOPO OTBOTa.
B CBOeLI par00TC MbI HOHbITaJIHCb HpIirOJIIi3HTbCH K per1I0HHI0 3TOLI HpO~JICMbI COC-
CTBeHHbIM HyT2M, HCXOjjfl H3 CBOHX HpeJ1;HICCTBOBaBHIHX OHbITOB C JIH30P0HHbIMH
CTa(~HJIOifOIfISaMH (P03eH6epP LI LTIMapAa 1955). MbI HOJIb80BaJIHCb COGCTBOHHbIM
HOJIHJIH30PeHHbIM HITaMMOM Staphylococcus aureus haemolyticus.
c'~TOT HITaMM (LS ~.,) HpOH3BOJ[HT, IIO CyHjeCTBy, 2 THHa 6axMepHOC~ara, oTJii~IYalo-
H;LIeCH ApyP OT Apyra xax B rOHOJIOPHTIeCI{OM, TaH H B CepOJIOrI~IYP,CI{OM OTHOIIICHLIHX:
(~1ara F 4, AJIfi IfOTOpOPO CIIeL~H(~1HLIeCiSII LIyBCTBHTeJIbHbIM HBJIHe1'CH HITaMM CS 4,
H ~raPa F 13, j[JIH I{OTOpOPO CHer~H(~HLIeCIfH LIyBCTBHTeJIbHbIM HB.TIHOTCH HITaMM CS 13.
Sara F 4 HITaMM HpOH3BOJ~HT B CpeAHOM B TIITpe 104, a F 13 B CpeZ[H8M B THTpe 1~5.
OjjHalfO He HCIfJIIOLIeHO, LITO HeIfOTOpOe (HC60JIbHIOe) I{OJIHLIeCTBO (~aPOB B (~HJIb-
TpaTaX LS 2 B HaIIIHX OIIbITaX 06paaosaJIO HOPaTIIBHble ICOJIOHHH xaic Ha CS 4,
TaHHHaCS13.
MbI OrOJIytIaJIH KyJIbTypbI HITaMMa LS 2 B 6yJIbOHe B HaLIaJIe JlorapHC~MH~ecxoH
(~a3bI HX pOCTa B L~HJIHHJ~pHYCCISOM COCyj[HI{e H3 Hp03paLIHOPO HBapL~eBOPO CTeISJIa,
ILOTOpbILI Mej[JI0HH0 BpaHjaJICH. 1V1bI HOJIb30BaJIHCb HOJIHbIM CHeI4TpOM y~-JIaMIIbI
HH3KOP0 j[aBJIeHHH C paCCTOHHHH B 50 CM, HpH J~OCTyHe BHAHMhIX JIytIeYI. llOCJie
OTjjeJIbHbIX 3ISCH03HL~HLI (OT 1 RO 3O MIIH.) H3 COCyjjHHa ~paJICH BCOPJja OJ~HHaIfOBbIi~i
067>eM IfyJIbTypbI, ifOTOpbIi3 XpaHHJICH RO OI{OHLIaHHH OOJIyLIeHHFI HpH TaKiIX xce
ycJIOBHHx, npH xaxHx ocTaTOx xyJlbTypbl eH~e o6JIy~aJicH. IIoTOM Bce o6paaubl
OJ~HOBpeMeHHO LIHKyrOHpOBa~ZHCb B TeMHOTe HpH 34-37? C B TetleHHe 3~ MHH. rlOCJIe
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Idxxy6auxH Bce o6paaubi ~x~IbTposa~Ixcb ~Iepe3 KOJIJIOJ~i3~%IHble yJIbTpa(~HJIbTpbl
CO CpeJ~HI3M ~(xaMOTpOM rl0p OHOdIO 650 mu. C noMOIIjbIO 6y.nbOHa xJix c~xaxOJIOrx-
`Iecltoro pacTSOpa x3 (~1I3JIbTpaTa HpxPOTOBJIHJII3Cb Cepxi~Hbte paaBeAexi~x BOa-
paCTaIOILjePO pslAa (BnJIOTb J(O HOHuexTpauxi3 10-4), KOTOpble TI3TpOBaJILICb IIO KOJII3-
'IeCTBy HOraTi3BHbIX KOJIOHx~I Ha OTJ~eJIbHbIX TCCT-HyJIbTypaX Ha crape. HOJIy~ieHHble
TI3TpbI BbILIxCJIHJIHCb B % HO OTHOHIeHI3i0 K Ti3TpaM H006JIy~aBIIIxXCH HOHTpOJIbHbIX
IIiTaMMOB.
MbI xCXOjjxJIx xa IlpeJjBapI3TeJIbHO HOJIyLIeHHbIX JjaHxblX O TOM, TITO ~-x.abTpaT LS 2
coRepxcxT c~arK, xexaMexxo Bblabtsaloluxe ~Ixaxc HITaMMOB CS 1, 4, 10, 13 x 14.
Ha aTYIX 5 LIyBCTBi3~'eJIbHbIX IIiTaMMaX MbI xCIIbITbIBaJIx c~xJIbTpaTbl OTjjeJIbHbiX
O~paaI.~OB 06JIyLIaBIile~ICH HaMx HyJIbTypbI LS 2. ~iblJIa HpOx3BeJjeHa OueHKa B OGIueM
30 OIIbiTOB. HaHeCH Ha rpa~Irlx % rIOBbIIileHxx TI3TpOB (~larOB, KOTOpbI# IIOKaaaJIx
OTi;eJIbHble LIyBCTBI3TeJIbHble IIITBMMbI - B aaBi3CLIMOCTH OT IIpOJ~OJI3Ki'ITeJIbHOCTx
axcxoaxut3x, - MbI no.ny~x~Ix 2 Txna xpxsbix (rpaI~I3K 1). TI3TpbI c~IaroB xa CS 1
x 4 Mexx.axcb npx6~It3axTe~Ibxo Ho nepso# xpxso#, a c~aroB xa CS 10, 13 H 14 -
no BTOpo~. 06e xpi3Bble HoxaablBaloT Bblcoxylo cTerlexb r3xAyKuxx o6paaosaxxR
~IaroB IIITaMMOM LS 2. Ilpx aTOM oAxaxo xxAyKl[HH ~aroB, o6paaylouixx xeraTxsxbie
KOJIOHxx Ha xITaMM$X CS 1 H 4, B OGx;eM He J~OCTHraeT TaKO~i BbICOTbI, KBK I3HJjyKuHH
c~arOB, HORBJIFIIOIL(HXCH Ha 3 OCTaJIbHbIX ILiTaMMaX. Ha OCHOBaHxx KpxBbIX MOxcHO
TaK?Ke CyJ;i3Tb, LITO TI3Tp T0X (~IarOB, KOTOpbIX yxce Calla KyJIbTypa rlpOi3aB0]~xT
~OOJIbLtie, B03M0]KHO C IIOMOIubIO I3HJ~yKIjLII3 IIOBbICI3Tb 60JIbHIe, LIeM TI3Tp T@X (~arOB,
KOTOpbIX CaMOHpOLIaBOJIbHO 06paayeTCH McHbHIe. HHTepeCHO, LITO Ha BTOpO~i HpHBO~i
(CS 10, 13 I3 14) IIOCJIe aKCrlOaxui~x B TeLIeHHe 17 MI3H. 30 CeK. 06oaHaiIaeTCfi BTOpOfk
Maxct~IMyM, Blue 6o~Iee Bblcoxx~, =Tell nepBbli't (~epea 10 Mxx.).
He c.aeAyeT oAxaxo pacuexHBaTb npr3BeRexxoe xa rpac~z~Ke 1 Ilosblluexxe Ti3TpoB
(~arOB IIOCJIe y~-06JIy~IeHi3H KyJIbTypbl LS 2 KaK peay~IbTaT ORHO# TOJIbKO I3HJjyHuxx:
xa TLITp c~arOB Hpi3 AaHHbIX yCJIOBxHX OIIbITa BJIIrIHeT Blue ueJlbl~ pxR ~IaKTOpoB,
KaK HepaBHOL~eHHOCTb KJIeTOK B aaHaCHbIX KyJIbTypaX LS 2, OTKyRa rlpOxaBOJ{xJIHCb
nepecesbl (ManeK 1955); npoAo~Ixcalou~x~cx B Te*Iext3e onblTa pocT Ky~IbTypbl B ~IOra-
pi3~IMH~ecxo~ c~aae, a BMecTe c TeM x xaMexext3x xo.ax~ecTBa K~IeTOx B o6paauax;
cy6.aeTa~Ibxoe H Raxce .neTa.abxoe Re~cTBtae Y~-.ny~e# xa x~IeTxt3; o6paTxax aRcop6-
uxR BblRe.nReMblx ~aroB x~IeTxaMx I3JItI YIx TieTpI3TOM; peaKTraBauxx noABeprtxHxcx
I3HJ;yKuI3I3 KJIeTOK HOj~ Aei~CTBxeM BI3J(xMOPO CBBTa x xHAKTYIBauxx taros Hou Re~i-
cTBZ3eM Y~-~Iy~e~, - KpoMe pxAa Apyrxx taKTOpoB, npoxB.axloluxxcR npi3
tIIJIbTpaurar~I H npH TOCTaX C tI3JIbTpaTaMI3. (H30 BCBX BTLIX IIOCOLIHbIX BaIxxHxfY
MO?KHO I3CKJIIOiIxTb, - HO Kpa~IHe~f Mepe rtpl-I axcnoaHuxx Ro 12 MI3I3., - T07IbH0
y~-I-iHaKTYiBauxlo BbIJ~eJIfIeMbIX taroB, HOTOpbIX B ATOM c.ay~Iae B cosepluexcTBe
3aH(xH~aeT 6yJIbOH.) ~e~rICTBxe COGCTB8HH0 xHJ~yKuxx H0B08M0?Kx0 OTJ~eJIi3Tb OT
BJII3fIHYIFI ~O.RbHIxxCTBa aTLiX taKTOpOB. II08TOMy MbI He IIpOLIaBOJ~LIJIR TOtIHO~f
KOJILILIeCTBOHHO~i OI~eHKi3 ee J[e~ICTBI3R H yJ~OBJIeTBOpi3JILICb B ATOM OTHOHIOHHH
IIpxHIjYIIILIaJIbHbIM KOHCTdTxpOBaHxeM taKTa, LITO v~-JIy~II3 B BeCbMa CIiJIbHO~I
CTCIIeHI~I xHT,jyLjYIpyIOT npOj(yKIjxIO tarOB TIITaMMOM LS 2.
Aa.nee MbI LICC~IeAoBa~Ir3 BaaxMxoe oTxolIIext3e TYITpoB orAe.abxblx TLInoB taros,
BbIReJIHeMbIX B TeLIeHI~Ie 06JIyLIeHI3Fi HITaMMOM LS 2. MbI I3CXOJ~I3JII3 YI3 IIpeA~IOaKeHHx,
iITO BbIIIIeyHOMHHyTbie taKTOpbI, OCJIOHSHFIIOH[xe J~e~iCTBxe I3xJ~yHuxLi Ha TI3Tp
(~aroB, BJIi3HIOT HpH6~IHaxTeJIbHO OJ;xHaKOBO Ha BCOX tarOB B OGpaaue, T. e. He OKa-
3bIBaK1T CyH;eCTB@HHOrO BJIxfiHxfi Ha COOTHOIIIeHxe TI3TpOB OTJ~eJIbHbIX TxHOB (~larOB.
KpLiBble Ha rpatYlKe 1 xOKaabIBaIOT, LITO 8T0 COOTHOILieHLie B XOj[e YIHRyKuxi3 He x0-
CTOSiHHO. B aTO~I LIaCTYI paCOT MbI HpOLI3BeJIx OIIFITb 30 OIIbITOB 06JIytIeHi3H, AaIA
HOTOpbIX MbI IIOJIbaOBaJII3Cb JIII30reHHbIM HITaMMOM LS 2 x iIyBCTBI3TeJIbHbIMI3 HITaM-
MaMx CS 4 I3 CS 13. I{axcAoe paaseAexxe o6paaua HaHOCtIJIOCb BcerAa xa o6a
iIyBCTBYITeJIbHbIX HITaMMa.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
I{y.abTypbl, 6paBHlnecH J~JIH onblTa, xe Bcer~a 6blBa.nn oRHxaxoso rycTblMn,
n08TOMy H HCXO~[HbII%I THTp i~aPOB F 13 n F 4 He MOP 6bITb BCOr~a OJ~nH n TOT }IiC.
OAxaxo B Aaxxou cpeAe HX COOTHOIIIeHne 6bIBa~IO xOCTOHHHbIM n KOJIer0aJI0Cb
(HeaHaLIHTeJIbHO) OKOJIO BeJInLIxHbI 3 1 (HeaaBHCHMO OT a6COJIIOTxbIX 3HaLIeHHLI
TLITpOB). Bblsa j[OKaaaHa CTaTHCTHLIeCKaH 3HaLInMOCTb 3TOI'O COOTHOIIIeHnH. B XO~e
xHJ~yKI~IIn THTp F 13 nOBbIIiIaJICFi H B 400 paa, C MaKCHMyMOM HpH aKCHOanI(nH
B TetIeHl~Ie 15 n 27 MIiH., a THTp F 4 - He ~OJIbIIIe, LIeM B 12 paa, C MaKCHMyMOM TaK}Ke
npH 9KCHOanL~nn B TeileHne 15 Ii 27 MnH. PaaBHTne COOTHOIIIeHnH Me?I{~y THTpaMn
F 13 H F 4 B XOj[e HHj[yKI~nn CTc'1HOBHTCH HaI~IrOOJIee HaPJIHJ~HbIM, eCJIn IIpHHHTb
3HaLIeHne THTpa F 4 B Ka?KJ~OM 06paaL~e as e~(nHnL[y: aT0 COOTHOHIOHne J~OXOJ~n~TO
nAo367:1.
I~aK MbI x npeRHO~~arasl~I, paaBnTxe Tr~ITpoB c~aroB npoTexaso xe BcerRa oRHxaxoso:
KpnBaH xoBbllHexnH THTpoB B oTAe.abxblx onblTax npoxoAn.na no paaxoMy; MaxcH-
Ma.abxoe noBblHlexxe THTpa Ho.nyYa~IOCb noc.ae paa.nnYxbix boa o6.nyYeHxH H 6bIBaJIO
H00J~nHaKOBO B OT~(eJIbHbIX OnbITaX. IIpII OJ~xOM OnblTe THTp F 4 Z{a?Ke BOBCe ne
noBblHla.ncsl, TorAa KaK THTp F 13 yBe.nHYn.acH B 4,9 paa (rpai~nK 2). IIoaTOMy MbI
He nblTaeMCH J~aTb CTaTHCTnLIeCKyIO OI[eHKy J~eI3CTBnH OTJ[eJIbHbIX aKCHOaxL[H~i.
Ho xpn Bcex onblTax 6ea xcK~IloYexHH H npH .nlo6oil axcnoanuHn, npH Raxllbix
yc.aoBnxx oxblTa, noBbliuexne THTpa F 13 6bIBaJIO ropaaRo axaYHTe.nbxee, YeM
nosbluiexne THTpa F 4. TaKHM o6paaoM, pacxaR K.neTOK xa c~arH Tnna F 13 nHAy-
uHpoBa.acH ropaaRo nxTexcnsxee, YeM pacnaA xa darn THna F 4.
IIoc,ae nepsoro MaxcnMyMa nxRyxunn oxo.ao 15-oH Mnx. aKCnoaliunn cseRoBa~-lo
o6blYxo Z[OBOJIbHO peaxoe naAexne TnTpoB o6onx e~aroB, o6yc.aoB~Iexxoe, no-BHAn-
MoMy, oTxocnTe.abxblM npeo6.aaRaxHeM aAcop6ulln yaKe cc~opMnposaBHlnxcH c~aroB
xa 6aKTepnn n~IH nx AeTpnT - xaR Ra.nbxeHHleH npoAyKuneK c~aroB KJIeTKaMH.
~HTepeCHO, iITO IIpH MHOPOYHCJIeHHbIX OHbITaX, - Kali n B nepBOK tIaCTH pa~OTbI, -
nOCJie aKCHOanL(nH B TeLIeHne 20-30 MHH. Ha~JIIOJ~aJIOCb HOBOe peaKOe nOBbIILIeHne
THTpOB, OCbILIHO uaxce npeBbIHIaIOn[ee nepBbILI MaKCHMyM (OKOJIO 15-oii MHHyTbI).
PTO HBJIeHYIe MbI OC'bHCHHeM BHeaanlIbIM paCnaJ~OM 60JIbIII0r0 KOJInLIeCTBa KJIeTOK
nOJj BJInHHneM aHaLIHTeJIbHOI3 J[OabI y~-JIyLIeII B MOMeHT, KOrJja yCnJInBaIOlljeeCH
JIeTaJIbHOe j[eLICTBne 06JIyLIeHIIH Jj0 H3BeCTHOI~ CTeIIeHH ypaBHOBeIiInBaJ10 nHJ~yKI~HIO.
~JIH ouexKn s.nnslxnH ~ioTOpeaKTnBaunn xa HcxoR o6.nyYexnH MbI B 4 onblTax
ncc.neAosa.nn HxAyKunlo npH o6,nyYexHn B TeMxoTe. Iipn aTnx onblTax THTp F 13
npeBbllHa~I noxaaaTe.an KoxTpo.nH xe 6o.nee, YeM B 6 paa, TorAa xaK TnTp F 4 HoBbI-
xla~ICx H B 34 paaa. Han6o.aee ac~~ieKTnsxax j[JIH Hx~yKunn F 4 n F 13 npoAo.nxcn-
TeJIbHOCTb aKCHOax1[nx 6bIBaJIa 5-6 MHH. B KaLieCTBe HpnMepa npnBORnM Ha rpa-
(~IIIKe 3 KpnBble THT~10B OrOOHX i~larOB, HOJIyLIeHHble xpn 2 OnbITaX, nOCTaBJIeHHbIX
HeIIOCpeJ~CTB0HH0 OJ~nH 3a J~pyPHM npH COBepItIeHHO Oj~HHaKOBbIX yCJIOBHHX. IIpn
nepBOM n3 HLIX MbI npOnaBOj[HJIn O~JIy~IeHHH Hpn j(HEBHOM CBeTe, npH BTOpOM aloe -
B TeMHOTe. KaK BnJ[HO n3 OHbITOB, a(~(~eKTI3BHOCTb nHjjyKI~nn B TeMHOTe ~bIBaeT
MEHbliie, a LITO KaCaeTCH COOTHOIileHnH IIOBbiHIeHnH THTpOB F 4 H F 13, xa6rllo-
AaeTCH 6osee axaYnTe.nbxoe noBblluexne THTpa F 4, YeM F 13.
e~HaYHTeJIbHOe yBeJIH~IeHHe OTHOIIIeHnH THTpa F 13 K THTpy F 4 nOCJIe y~-nHJ~pK-
I~I3n npt3 Z[OCTyne Bn~[nMbIX JIyiIOYI Tpyj[HO OG'bHCHHTb KaK naMeHeHHe COOTHOIIIeHnH,
B KoTOpoM oTAe.abxbie pacnaAaloH~necH K.neTKn nponssoAslT o6onx c~aroB. IIoBbI-
IneHne aTOrO COOTHOIIIeHnH BO BCCX CJIyiIaFIX Hc`ICTOJIbKO 3HaTInTeJIbHO, LITO npn-
XOJ~nTCH J~OnyCTHTb, LITO MHOPne KJIeTKn xOA BJILIHHxeM nHJ[yKI;nn paCHaJ;aIOTCH
Ha F 13, He IIpOnaBOJjH OZ[HOBpeM8HH0 F 4. N1bI ~[OnyCKaeM, KOHeLIHO, LITO nOCJIe
nxAyKunn B Ky~IbType BcTpeYaeTCH ue.ablK pHR paa~InYxbix TnnoB pacnaAa K.neTOK.
HeicoTOpbie K.aeTxn MoryT pacnaAaTbcH Boo6H~e 6ea o6paaosaxnH c~ara (PpaReYxaH
1952, PepYnK 1953). B03MO7KH0, YTO caMOnponaBO.nbxoe o6paaoBaxne o6onx c~aroB
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
npoTexaeT axa.norr~axo. Bo BcxxoM c.ay~ae, cnoco6HOCTb o6paaosaTb o6a Tnna c~ara
B COOOTHIileHIdLI 3 1 P0H0TI3ileCKH CBFI3aHa c HaxcJ~O~i K.neTxO# IIITaMMa.
B BOnpOCe pa3BI3TI3Si B pe3yJIbTaTe I3HJ~yKI~I3I3 B JIH3OP8HH0~ KyJIbType J;ByX
TIInOB (~aPOB, KOTOpI~Ie BO3MOHSHO LSHJ~yL[YIpOBaTb, MbI npIdCOeJ~I3HSieMCH KO B3PJIH~[y
Jacob-a (1952), aTO nocae xHAyxuut3 xaxcAbl~ xa xHx MoaxeT npoHaBORHTbcsl onpe-
j{eJIeHHbIMi3 IfJIeTKaMI3, He npOI33BOj[HIIjIIMLi OAHOBpeMeHHO BTOpOPO (~aPa.
Pa3HHi~y pe3yJIbTaTOB O6~IyaeHr3A npl3 RocTyne BHAr~Mblx .nyxeK H B TeMxoTe
MbI OG'bFICHfIeM peaxTHBxpyloH~i3M Ae~ICTBi3eM BI3J~LIMhIX JIyLIe~i. ~OBIIJ~LIMOMy, B T0M-
HOTe rOalfTepnLI T0MI3 axe JjO3aMI3 O6.~ygeHr3x IIHaIfTI3BLIpOBaJIYicb POpa3JjO 8HaYId-
TeJlbHee, Li IIOBTOMy J[eiICTBI3e I3HRyxgz3z3 OTpaaxaJlOCb Ha TI3Tpe c~aroB ropasRo
c,na6ee, H To~Ibxo npz~ xal36o~Iee HII3ItLIX Roaax o6.ny~exHH.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
The ~ ew Antibiotic BU 271
V. ~`EV~fK, M. PODOJIL, M. KYSELOVA and A. VRTI~`KOVt~
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
The following large-molecular actinomycetic antibiotics have been described
in the literature: micromonosporin (Waksman et al. 1942), actinomycetin (Welsch
1937-1947), antibiotic 2377 (British patent 719.230) and thermomycin (Schone
1951). These antibiotics can easily be distinguished from the small-molecular anti-
biotics by means of dialysis through a cellophane membrane. Micromonosporin and
actinomycetin are of a protein character, antibiotic 2377 is not a protein, but gives
a positive anthrone reaction for carbohydrates, while in the case of thermomycin
no details are given as to the character of the molecule.
The new antibiotic, BU 271, was obtained from a strain of soil actinomycetes
listed under the number 271. On agar media, colonies of this actinomycete have
a whitish mycelium with yellowish spores. Underneath, the mycelium is rust colour.
Pigment is not diffused.
When cultured on a shaker apparatus, an isolated strain of the actinomycete
produced 43 streptomycin units/ml. The composition of the medium was as follows:
glucose 1 %, starch 1.5 %, corn steep 0.5 ?%, (NH4)zSO4 0.35 %, NaC10.5 %, CaC03 0.5 %, .
When inoculated on agar media, subcultures with a maximum production of
460 strep. units/ml. were obtained. On further reinoculation on to potato agar
slants, however, production of the antibiotic fell rapidly.
A second batch gave 20 subcultures, about half of which (9) did not produce
the antibiotic on the shaker, but of which about a quarter (5) gave very satisfactory
maximum production (about 1,000 s. u./ml.), which was not, however, very stable.
Considerable differences were observed between the original cultures and the sub-
cultures on individual days of submersion cultivation, and production of the anti-
bicticfluctuated considerably in the same strain when this was inoculated indifferent
weeks, the cause being probably poor stability of the strain.
On further distribution of a selected, highly productive strain (about 1,000 s. u./ml.
on cultivation on a shaker), 28 subcultures were obtained, all of which produced
the antibiotic on culturing on the shaker. About half the subcultures gave very
satisfactory production (1,000-2,000 s. u./ml.), but the latter still fluctuated quite
considerably, even when the strain gradually became stabilized. More stable strains
were obtained only after further inoculation, when evaluation on agar plates showed
no great differences in the size of the inhibition zones in the individual subcultures.
Antibiotic 271 is stable only at low temperatures. At 2? C it remains stable for
a number of weeks. At laboratory temperature it is rapidly destroyed in the presence
of an acid, neutral or basic pH, even after only 24 hours. On boiling for 15 minutes
it is destroyed in all pH values tested (2.0, 7.0, 9.0).
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
The antibiotic is more effective in the presence of an alkaline reaction (pH 7.5 to
8.0); with 50% blood serum its efficacy is reduced by half. It is destroyed by organic
solvents. Crude preparations of the antibiotic were obtained by lyophilization of
dialyzed filtrates of a culture of the actinomycete.
The antibiotic is not dialyzed through a cellophane membrane and its spot
on a paper electrophoregram stains with bromphenol blue, indicating that the
molecule is probably of a protein character.
Antibiotic BU 271 acts particularly on Gram-positive bacteria. It usually acts
on various species of Gram-positive bacteria in concentrations of 1- 8 units~ml.,
but does not act on species of Gram-negative bacteria even in concentrations of
100 units~ml. It has been found to act especially strongly on Mycobacterium phlei
and in a comparison with other bacteria its action on this species was greater than
that of streptomycin.
The antibacterial spectrum and large molecule of antibiotic BU 271 resemble
those of micromonosporin (Wakeman et al. 1942, Welsch 1947), but it differs from
this antibiotic by its stability, method of isolation (it is not precipitated on adding
b0-90% ethanol or acetone) and by its coloration. If a filtrate of a micromonosporin
culture is heated for 60 minutes to 100? C, about two thirds of the antibiotic are
destroyed, whereas on_ heating a solution of BU 271 to 100? C, the whole of the
antibiotic is destroyed in 15 minutes. Micromonosporin also has an orange pigment,
while BU 271 is colourless.
On intravenous administration of a preparation with a strength of 1,150 units~mg.,
LD50 in mice was 45 mg.~kg., corresponding to 1,035 units/mouse.
We are indebted to L. Novotny of the Institute of Chemistry of the Czechoslovak Academy of Science
for the crude preparations of the antibiotic.
British Patent No. 719,230, 1. 2. 1954.
S c h o n e, R.: An Antibiotic which Inhibits Corynebacterium diphtheriae Produced by the
S-form of Streptomyces thermophilus. Antib. Chemother. 1 : 176, 1951.
Waksman, S. A., Horning, E. S., Welsch, M., Woodruff, H. B.:
Distribution of Antagonistic Actinomycetes in Nature. Soil Sci. 54:281, 1942.
W e 1 s c h, M.: Influence de la nature du milieu de culture sur la production de lysines par les
Actinomyces. C. R. Soc. Biol. 126:244, 1937.
W e 1 s c h, M.: De quelques proprietes du principe bacteriolytique des Actinomyces. C. R. Soc.
Biol. 126:247, 1937.
W e 1 s c h, M.: Mise au point dune technique nephelometrique pour 1'etude de la mycolyse.
C. R. Soc. Biol. 128:795, 1938.
W e I s c h, M.: llosage nephelometrique du principe bacteriolytique des Actinomyces. C. R.Soc.
Biol. 128:1172, 1938.
W e 1 s c h, M.: Inactivation par la chaleur du principe bacteriolytique des Actinomyces.
C. R. Soc. Biol. 128:1175, 1938.
W e 1 s c h, M.: Mise au point dune technique nepbelometrique pour 1'etude de la mycolyse.
I I. Influence de la reaction du milieu sur le trouble des suspensions microbiennes. C. R. Soc.
Biol. 130:797, 1939a.
W e 1 s c h, M.: De 1'inactivation du principe bacteriolytique des Actinomyces par les rayons
ultra-violets. C. R. Soc. Biol. 131:1296, 1939b.
W e 1 s c h, M.: Phenomenes d'antibiose chez les Actinomycetes. Gembloux 1947.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
H OBbII%I 2HTI~ICHOTI~IIi BU X71
B. LIILBiII~II{, M. IIOuOLI~I, M. ICLICEJIOBA H A. P,YTLIllIICOBA
Pea~o.nce
AHTH6uoTHIC BU 271 6bl.a Ho.ay~eH H3 To~Hee HaMli He onpeAe.aHBUierocH BHRa
HOLIBOHHOPO JIyLIHCTOPO PpH6xa, 0~03Ha~IeHHOPO N~ 271. AHTHrOnOTHIf yCTOI%ILIHB
HpH 2~ C B T2Y0HHe HOCI{OJIbffHX Hej[eJIb, HO npH JIahOpaTOpHOII TeMHepaType ~bICTpO
paapyuiaeTCfl (yalce ~epea 24 Baca), HpH f{HHHLIeHHFi B Te~Iexue 15 M1Ix. paapyluaeTCfl
no,nHOCTbIO. AHTU6HOTHlc 6osee ac~c~efeTlisex HpH anlca.7lH~ecfcoH peafcuuu (pH 7,5 -
8,0). 50% cblBOpoTlca fcpoBH oc~Iab.uHeT ero Rei~cTBHe xa no.7IOBHHy. OpraHH~eclcne
paCTBOpHTeJIH eP0 paapyHIaIOT. AHTHOHOTn14 3T0 ~eCI~BeTHOe BeH~eCTBO, He J~HaJIH3n-
pylOll[ee LIepe3 H~IeHISy L~eJIOI~IaHa. HHTHO aHTH~lIOTlIffa Ha ~yMa?fSfIOLI 3JIeICTpO(~Ope-
PpaMMe Of{paHIHBaeTCFi 6pOM(~eHOJIOBbIM CHHHM. AHTH~nOTLIff J~eYICTByeT HpeHMyII~O-
CTBeHHO Ha PpaM-HOJI071CHTeJIbfIble rOaf{TepHH. Ha i~1yCO~JaCte1'lUiYl plllel OII JIe~ICTBCT
CHJIbHee, LIeM CTpeHTOMHL[HH. HO CB00My aHT1i~0af{TepHI%IHOMy CHefCTpy H f{pyHHOLI
MOJIefCyJIe OH 6JIH30ff ft MHfCpOMOHOCHOpIiHy, OT fCOTOpOPO OTJIHLIaeTCH Oj(HaIfO
HO CTeH0H1I yCTOYILIHBOCTH, HO CHOCO~Oy H30JIl~i[HH (OH He OCa?ff~aeTCH nOCJIe npn-
6aBJIeHLIH BTaHOJIa HJIH aL~eTOHa) fI HO OfLpaCffe. LDS, Z[JIH MbIHIeYI Hpn BBeJjeHHN
B BOHy 6blBana 45 MP/Kr.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
The New Antibiotic BU 306
V. ~`EV~fK, M. PODOJIL, M. KYSELOVt~ and A. VRTI~`KOV~
Institute of Biology, Czechoslovak Academy of Science, Department of Microbiology, Praha
Among the antibiotics of a protein character which are recovered from actino-
mycetes and act on Gram positive and Gram negative bacteria, only actinomycetin
has been described in the literature (Welsch 1937-1947).
A new antibiotic, BU 306, was obtained from a strain of soil actinomycetes, listed
in our collection under the number 306. Colonies of the actinomycete, which are rusty
blown underneath, form yellowish spores on normal agar media; the pigment is not
produced into the medium.
In submerged cultivation on a shaker, on the medium described for antibiotic
BU 271 (~`evL~fk et al. 1956), the original strain of actinomycete produced 120 strepto-
mycin units/ml. On setting up cultures on agar media of the same composition,
generations with a maximum production of about 1,000 s. u.~ml. were obtained.
On progressive transfer to potato agar slants, production of the antibiotic decreased
from 1,000 to 300 s. u.~ml. Following fresh culture on an agar medium, 35 colonies
were obtained, most of which (27) produced the antibiotic, but on a smaller scale
(100-400 s. u.~ml.). These cultures were somewhat labile. The further transfer of
a culture producing 400 s. u.~ml. gave 32 colonies, in seven of which production
was much higher (1,000-1,600 s. u.~ml.).
Cultures of the actinomycete of BU 306 were set up on an agar medium containing
corn steep (0.5%), the amino acid content of which provides a suitable environment
on potato agar slants in a refrigerator at -}- 4? C.
A high yield of the antibiotic (1,500-2,000 s. u.~ml.) was obtained on culturing
on a reciprocal shaker on the medium containing corn steep described above. In some
cases a maximum production of 3, 500 s. u.~ml. was obtained, after 6-7 days' cultivation.
On fermenting in a glass laboratory tank with a capacity of 1,000 ml., a maximum
production of 1,150 s. u.~ml. was obtained after 120 hours' culturing. The laboratory
tank was inoculated with a 24-hour-old vegetative inoculum from the shaker
(2% of the medium volume). Mixing was carried out at 375 revs.~minute, aeration
one vol./minute.
At 2? C, the antibiotic remains stable for a number of weeks. It is destroyed
by boiling. It is also destroyed by organic solvents.
The antibiotic BU 306 does .not dialyze through a cellophane membrane. After
being separated from ballast substances on the paper electrophoregram, the spot
of antibiotic stains with bromphenol blue, indicating the possibility that the molecule
is of a protein character.
Unrefined samples of~ the antibiotic, with a strength of 2,000 units~mg. were
prepared by means of lyophilization of dialyzed filtrates of a culture of the actinomycete.
Using the diffusion method of microbiological titration, BU 306 is most effective
at a pH of 8.0. Its effectiveness decreases along with a fall in the pH. Its activity
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
is reduced by half by 50% blood serum. BU 306 acts on Gram positive and Gram
negative bacteria. It acts on the micro-organism Mycobacterium phlei in approxi-
mately the same concentrations as streptomycin.
BU 306 is not very toxic. A concentration of 1,000 units/ml. produced no toxic
effect on the protozoa Tetrahymena gelei and Euglena gracilis (culture filtrate).
Toxicity in mice depended on the amount of high-molecular ballast substances
present and not on the concentration (degree of effectiveness) of the antibiotic
itself. In intravenous injections, LD50 was 0.5 mg./20 g. in a lyophilized preparation,.
following dialysis, with a strength of 2,000 units/ml. In preparations with a strength
of 800 units/mg., LDSO was 0.7 mg./20 g., corresponding to 560 units/mouse, and in
a preparation with 120 units/mg., LD50 was 1 mg./20 g., corresponding to 120 units
for mouse.
BU 306 also had an effect on Ehrlich's ascitic tumour in mice. In a patch test,
in which the tumour cells were placed for three hours in a refrigerator together with
the antibiotic, the mice survived on the administration of doses of 125 units/mouse.
While the control mice, which were inoculated with the tumour, without the anti-
biotic, died in 12-15 days, the mice which were given the antibiotic survived
for over a month. For this experiment a preparation with a concentration of
1,000 units/mg. was used. Similar results were recorded in therapeutic tests with
the same concentration of the antibiotic, in which the latter was administered once
a day for a total of eight days. The antibiotic was injected intraperitoneally, the first
dose being administered three days after inoculation with the tumour.
Among the high-molecular antibiotics (of a protein character), which act on Gram
positive and Gram negative bacteria, only actinomycetin is known from among
the actinomycetes. BU 306, however, differs from actinomycetin by its stability
and by the method of its recovery. BU 306 is not precipitated either by acidification
to a pH of 3-4, using HCl, or by adding four volumes alcohol or acetone.
The authors wish to express their thanks to Dr. L. ~TOVOtny from the Institute of Chemistry of
the Czechoslovak Academy of Science for the crude preparations of BU 306.
References
~ev~ik, V., Podojil, M., Kyselova, M., Vrtiskova, A.: Nove anti-
biotikum BU 271. ~s. mikrobiologie 1:223, 1956.
W e 1 s c h, M.: Influence de la nature du milieu de culture sur la production de lysines par les
Actinomyces. C. R. Soc. Biol. 126 :244, 1937.
W e 1 s c h, M.: Actinomycetin. J. Bact. 53:101, 1947.
W e 1 s c h, M.: Phenomenes d'antibiose chez les Actinomycetes. Gembloux 1947.
HoBbIH aHTLIrOI~IOTIIIC BLT 306
B. llIEBLII~IK, M. IIOJ~OYIJI, M. ICI~ICEJIOBA x A. BPTLIiIII{OBA
Pea~o.~ce
AHTHCLIOTLIK BU 306 6bIJI HOJIyLIeH H3 TO~IHee xe OHpeJjeJIeHHOPO JIyT3I3CTOP0 rpH6Ka,
0603Ha`IeHHOrO HaMH N~ 306. B cblpoM Bi~Ae HperlapaT aHTH6HOTLIKa npej~CTaBJIHeT
?KeJITOBaTOe HeIITpaJIbHOe BeH~eCTBO, BepOaTHO 6e.nKOSOro xapaKTepa. AHTH6t~oTHK
7ieilcTByeT Ha rpaM-no.aoaKHTe,nbxble K rpaM-oTpt~Ir;aTe.nbHble 6aKTepraH t~ xa acui~THylo
OnyXOJIb e~pJIHXa y MbIIIIeT'I. TOKCHiIHOCTb aHTYI~LiOTI3ISa 3aB~ICIIT OT CTeHeHI3 LIHCTOTbI
HpenapaTa. Sr npeHapaTa C 3(~(~eKTLIBHOCTbIO 2000 eA. C./MP HpII BHyTpHBOHHbIX
BHpbICKHBaHIIHX LD50 6blsa 25 MP/Kr. IIO CB00My aHTH6aKTepLILIHOMy cneKTpy
ICI n0 pa3MepaM MOJIeKyJIbI aHTI3GLIOTIIK rOJIH30K K aHTI3HOMHIjeTIIHy, OT KOTOpOPO
OTJILILIaeTCH Oj~HaKO HO CBOeLI yCTOI%ILII3BOCTH~ HO CHOCOOy LI30JIHgIILI N n0 paCTBOpI~I-
MOCTI3 B OpraHHjIeCKHX paCTBOpI3TeJIFIX.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
Study of the Effect of Bacillus thuringiensis on Insects
J. VAI~KOVt~
Institute of Biology, Czechoslovak Academy of Science, Department of Insect Pathology, Praha
Received Semptember 19, 19.56
Some varieties of Bacillus cereua are known to be pathogenic for insects. In 1902, Ishiwata in Japan
(according to Steinhaus, 1947) isolated Bacillus sotto fromsilk-worms. Berlinor (1915) described aspore-
forming rod isolated from flour moths (Ephestia kiihniella) which he named Bacillus thuringiensis.
Metalnikov et al. (1928) described B. pyraustae and B. pyrenei, B. italicum and B. cazaubon (1930)
isolated from Pyrausta nubilalta and B. galeehuae (1932), isolated from Clalechaa gossypiella. Toumanoff
and Vago (1951) described B. alesti, isolated from Bombyx mori. It was assumed that all these strains
corresponded both morphologically and culturally to the saprophytic Bacillus cereua and that they
differed from this bacillus only in their pathogenicity for insects, chiefly Lepuloptera.
Because of their pathogenic properties, these strains, especially Bacillus cereus var. thuringieneis,
have been investigated in a number of laboratories in Hungary, Yugoslavia, France, the USA and
Canada, with reference to their possible utilization as insecticides. The communication of Hannay (1953)
drew attention to the formation of paracentral,cryatalline inclusions by Bacillus thuringiensis, which had
already been observed in this variety by Berliner in 1915 and by Mattes in 1927. Later, inclusions were
also found in the other varieties of Bacillus cereua pathogenic for insects (B. sotto, B. alesti and other,
unidentified varieties) and their relationship to pathogenicity for insects was therefore investigated.
Hannay (1953), when the composition of the inclusions was still unknown, assumed that they were either
of virus origin or that formation of the inclusions was a genetic characteristic of the organism in some
way related to the formation of a toxic substance producing septicaemia in insect larvae. Hannay and
Fitz-James (1955) found that the inclusions were of a protein nature and that they contained 17 amino
acids. Angus (1954) observed the action of inclusions of B. sotto dissolved in dilute alkali and came to the
conclusion that they were the toxic component of B. sotto and when taken orally caused paralysis in
silk-worms.
Toumanoff (1951), on the other hand, while attributing a toxic role to crystalline inclusions, regards the
enzyme lecithinase as the important toxic factor in these strains. Heimpel (1955) likewise emphasizes
the relationship between pathogenicity for insects and the ability to produce lecithinase in various species
of the Bacillus genus.
The question of the classification of the above-mentioned strains pathogenic for insects is still not
altogether clear. B. thuringiensis is not described either in the sixth edition of Bergey'a Manual or in
Krasilnikov's Manual; only Smith et al. (1948) mention B. thuringienais, as a variety of B. cereua.
According to Steinhaus and Jerrel (1954), B. thuringiensis will be included in the seventh edition of
Bergey's Manual as an independent species. The other varieties of B. cereua differ only slightly and it is
quite possible that some are synonymous (e. g. some authors regard B. sotto as the Japanese equivalent
of B. thuringiensis).
The aim of the present work was to investigate the pathogenicity of a Czech strain of B. thuringiensis
isolated by Dr. Weiser, to ascertain at what stage in its development the insecticidal substance is formed,
where it is localised, what its effect is and whether the production of this substance by strains forming
inclusions differs from its production by strains in which inclusions are not formed. It was further necessary
to elaborate a sensitive method for isolating the inclusions, as the authors who so far studied the question
of the pathogenicity of B. cereua var. thuringiensis (Huaz 1927, 1929, 1930, Metalnikov and Chorine
1929a, b, Metalni!tov et al. 1931, Steinhaus 1951, Toumanoff 1955, Heimpel 1955) always worked only
with aporulated cultures, i. e. with a mixture of spores, inclusions and the remains of vegetative cells,
while Angus (1954) dissolved inclusions of B. sotto in dilute alkalis; in our experiments this did not prove
satisfactory, as it resulted in loss of activity of the filtrate.
Materials and Method
The Czech virulent strain of B. cereua, var. thuringiensis, was isolated from severely affected larvao
of the sweet pepper moth (Plodia interpunctella) in 1952. It is characterised by greater viruleneo than
the strains obtained in Hungary and the USA (Weiser and Veber 1954, 'lofka 1955). Its diagnostic
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
characteristics coincide with those of B. cereus. Non-virulent strain: When maintained on ameat-peptone
agar slant at room temperature for half a year without subcultivation, this strain lost its ability to form
inclusions. Both strains were cultured on an agar sporulation medium in Roux bottles at 28? C.
Caterpillars of Lymantria dispar and Euproctis phaeorrhoea were selected for the experiments; these
pests damage deciduous trees, mainly oaks, hornbeams, willows and fruit trees. The caterpillars were
reared in large Petri dishes and infected by feeding on willow leaves (in early spring) and later plum
leaves smeared with a suspension of infected material. In every experiment, which was carried out in
duplicate, twenty caterpillars were used. Some experiments were further repeated using silk-worms
(Bombyx mori), which were fed on mulberry leaves.
Isolation of inclusions: This proved very difficult in cultures in which sporulation was complete and
which also contained spores and vegetative debris in addition to free inclusions. The specific weight of the
inclusions differs only slightly from that of the spores. Differential centrifugation (Behrens and Marti
1955) did not give satisfactory results, nor could it be used even in cases in which the spores in a mixture
containing inclusions were allowed to germinate beforehand, in order to increase their substance. As
regards time, germination of the spores was very uneven and the part which should have contained
inclusions still contained a large quantity of spores which had not yet germinated. Dissolving the inlu-
sions indilute alkalis, as described by Angus (1954), likewise proved unsatisfactory in our experiments as
the inclusions lost their effectiveness on being dissolved. The most satisfactory proved to be the density
gradient centrifugation technique (Schneider et al. 1953, l~iman 1955). Gradients of 1 ml. of 1.11M,
0.957M, 0.636M and 0.335M saccharose were layered into centrifuge tubes. A culture in which sporulation
was complete was washed and suspended in 0.25M saccharose and layered in amounts of 1 ml. into the
centrifuge tube over the fourth gradient. Centrifugation was carried out For 25 minutes at 300 G. A ring
containing most of the spores and inclusions then fell to the 0.636M gradient and in the upper gradients
slightly turbid saccharose remained, which contained almost only inclusions. By removing the upper
gradient and washing, a suspension of inclusions containing only very few spores was obtained (50 to
80:1).
In their latest communication, Hannay and Fitz-James (1955) describe the isolation of crystalline
inclusions by mechanical disruption of the spores and by the germination and autolysis of spore material.
This method of isolation was used for making a chemical analysis, however, and not for ascertaining the
biological effect of the inclusions. -
Isolation of the spores: Three methods were used. A culture in which sporulation was complete was
treated for a) 10 minutes at 112? C, at 0.5 atm, overpressure, b) 24 hours with O.1N NaOH, c) 24 hours
with 1% pepsin in a N~20 HCl medium. The protein inclusions were either destroyed or completely
dissolved. The germinating capacity of the spores was tested by inoculating on to meat-peptone agar.
The same concentration was always maintained in the suspensions, measured by the degree of turbidity.
Determination of antibiotic properties: This was carried out by the plate assay method, using agar blocks
(a few pieces of agar of 5 mm. diameter) taken from a fully developed culture of B. thuringiensis on
nutrient agar.
Influence of the Age of the Culture on Production of the
Insecticidal Substance of Bacillus thuringiensis
In these experiments a study was made of the stage at which the insecticidal.
substance develops.
Table 1 shows that the action of a young, S-hour-old culture containing only
vegetative cells is very weak. The first caterpillars did not die until the fourth day
and massive death did not occur until the eighth day. A post mortem examination
showed that the cells had proliferated in the weakened organisms sufficiently to cause
septicaemia. The most virulent were the cells of a fully developed 7-day-old culture
composed of free spores, inclusions and vegetative debris; this caused death in 50?j~
of the caterpillars within three days and in 100% within six days. The results
permit the assumption that the effective insecticidal substance develops during the
period of sporulation.
Comparison of the Effect of Spores and Inclusions of the Virulent Strain
of Bacillus thuringiensis
Since the experiments described above do not show whether the pathogenic agent
is the spores or the inclusions which are released at the same time as the spores,
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Table 1. Death Rate in the Third Instar of Caterpillars of Euproctia phaeorrhoea Fed with Cultures of
B. thuriragiensia of Varying Ages
Age of culture
8 hours
16 hours
48 hours
7 days
Vegetative
part of
Morphology
Vegetative
cells with
signs of
spores and
Free spores
and inclusions,
of culture
cells
spores and'
inclusions
vegetative debris
inclusions
released
Days
Mortality
1
0
0
0
5%
2
0
0
5%
10?,/0
3
0
5%
5%
50%
4
10%
25%
35%
80?/0
5
20%
65%
75%
90%
G
25%
85%
90%
100%
7
45%
95%
100%
8
90%
95%
Table 2. Death Rate in the Third Instar of Caterpillars of Euproctia phaeorrhoea Fed with Isolated
Spores and Isolated Inclusions
Spores
Inclusions
Inclusions
Inclusions
Inclusions
Isolated by
by density gradient
Days
denatured by
?
dissolved
dissolved
centrifugation
heating to 112
C
in O.1N NaOH
in 1% pepsin
technique
Mortality
1
0
0
0
10?%
2
0
0
0
50%
3
0
5%
5%
70%
4
15?%
10%
5%
85%
5
30%
20%
10%
100%
6
45%
35%
15%
7
55%
85?/0
25%
a further investigation was made to compare the effect of spores only and inclusions
only on the death of caterpillars.
Table 2 shows that the isolated spores do not have an immediate toxic effect.
The caterpillars do not die for 3-4 days, the cause of death being septicaemia follow-
ing proliferation of the bacilli which develop from the spores in the intestine. After
the ingestion of inclusions, however, some caterpillars died on the very first day.
By the second day 50?% of the caterpillars were dead and 100% died by the fifth
day. The toxic effect of the inclusions was evident only a few hours after ingestion.
The caterpillars stopped feeding and their movements decreased. In older, more
resistant caterpillars, damage to intestinal function was manifested by the intestine
remaining full and by cessation of peristalsis, while in younger, less resistant cater-
pillars, diarrhoea occurred and the anus became stuck to the underlying surface.
Susceptibility of the caterpillars to infection decreased with age. A loss of appetite
was observed when the leaves were smeared with a large dose of sporas or whole
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
culture, and the caterpillars fed with infectious material were therefore subjected
beforehand to starvation.
Caterpillars infected with spores died from septicaemia. In caterpillars infected
with inclusions, normal microflora appeared in the intestine in the first hours after
feeding; later, germination of the spores accompanying the inclusions in small
quantities occurred (proportion of inclusions to spores 50-80:1), while prior to
death vegetative forms even penetrated into the haemolymph. Paralysis in the first
hours of infection was not therefore accompanied by septicaemia.
Prior to death the caterpillars were soft in consistency and the epidermis acquired
a darker tinge. After death the internal organs rapidly disintegrated and the haemo-
lymph dried up.
Effect of a Strain of Bacillus thuringiensis not Forming Inclusions
The above results demonstrated that the inclusions constitute the toxic component
of the virulent strain. It was therefore of interest to ascertain the effect of a strain of
B. thuringiensis which had undergone degeneration and lost the ability to form
inclusions. Experiments with this strain showed that when given orally it is non-
virulent both in the form of young, vegetative cells (16 hours old) and also in the
Table 3. Death Rate in the Third Instar of Caterpillars of Euproctis plaaeorrhoea Fed with Inclusions
of the Virulent Strain and Spores of the Non-virulent Strain of B, thuringiensis
Inclusions of
virulent strain
Inclusions of viru-
lant strain -}- spores
of non-virulent
strain
Inclusions of
virulent strain
-~ spores of
virulent strain
Spores of
non-virulent
strain
Table 4. Death Rate in the Third Instar of Caterpillars of Bombyx mori Fed with Inclusions and Spores
of B. thuringiensis
Inclusions
of virulent
strain
Inclusions
{- spores of
virulent
strain
Inclusions of
virulent strain
+ spores of
non-virulent
strain
0
(20% paralysis)
50%
(20% paralysis)
100%
?%
(10% paralysis)
60?'
~o
(10% paralysis)
95?~
100%
0
(30% paralysis)
40%
(35% paralysis)
y ~o~
,e
100~~
Spores of
non-virulent
strain
0
0
0
0
U
of
.i io
Spores of
virulent
strain
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
form of spores from a fully developed 7-day-old culture, which pass through the
intestine in the excrements with their ability to germinate still intact, without
causing septicaemia (table 3, 4).
Infection with vegetative cells of the non-virulent strain did not cause death.
These experiments lend support to the view that inclusions of the pathogenic strain
merely induce germination of the spores in the intestine of the caterpillar which
results in septicaemia.
In experiments in which inclusions were added to the spores of the non-virulent
strain, the death rate among infected caterpillars of Euproctis phaeorrhoea did not
exceed the death rate among the control caterpillars to which only inclusions were
administered (table 3, 4).
Effect of Bacillus thuringiensis on Silk-worm (Bombyx mori)
Caterpillars in the third instar were very susceptible to infection with B. thurin-
giensis. Within only two hours after ingesting infected leaves they remained motion-
less on their sides and began to die. A microscopic examination of the haemolymph
and contents of the intestine confirmed that no germination of the spores had
occurred within this short period and that death was the result of intoxication
(table 4).
Antibiotic Properties of Bacillus thuringiensis
In the past few years it has been discovered that some strains of the species
B. cereus produce antibiotic substances. An attempt was therefore made to ascertain
whether the strain of B. thuringiensis used in these experiments produced an anti-
biotic, and if so, whether this was related to its toxic effect on caterpillars.
The plate assay method demonstrated that both strains-the strain forming
inclusions and virulent for caterpillars and the non-virulent strain without inclu-
sions-produce an antibiotic which acts on some gram-positive micro-organisms:
B. subtilis (15 mm. zone ), Sarcina lutes (18 mm.) and Staphylococcus aureus (12 mm. )
and not affecting the gram-negative micro-organisms Serratia marcescens and
Escherichia coli. Since the antibiotic was produced in the first hours of growth
(6-hour-old culture), before inclusions are formed, and was also produced by the
strain not forming inclusions, it may be assumed that the antibiotic is not identical
with the inclusions.
The present communication, dealing with the problem of ascertaining the effect of
spores and inclusions of B. thuringiensis on caterpillars demonstrates. for the first time
the use of a relatively very pure concentrated suspension of inclusions isolated
mechanically by a density gradient centrifugation technique. In agreement with
views expressed in the literature it was, found that ,the toxic substance does not
develop until during the sporulation period. The results given in tab. 2, however,
show a basic difference between the effect of isolated inclusions and spores on
mortality in caterpillars and show that the inclusions are the main toxic component.
The question thus remains of how far other factors (e. g. lecithinase) may participate
in the effect of the inclusions.
The observation that the inclusions are the main bearers of toxicity was also
confirmed in experiments with a strain of B. thuringiensis which had lost the ability
to form inclusions. This strain was non-virulent for caterpillars. Toumanoff (1955)
also described two strains of Bacillus sotto which differed in their virulence for the
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
silk-worm and observed inclusions only in the virulent strain. By passaging B. cereus,
var. alesti on alkaline agar with a pH of 9.0-9.5, Toumanoff also obtained a culture
in which inclusions were absent. This culture was less toxic than the initial culture.
On returning the culture to neutral agar, inclusions were again formed in some
cases. It may be assumed that in the alkaline medium the inclusions were dissolved
and thereby diluted, resulting in a decrease in their toxicity and perhaps a partial
degradation of their toxic agent. This observation is in agreement with our own
experiments in which the dissolving of inclusions in 0.1 N NaOH always led to the
loss of virulence of the filtrate. It differs, however, from the results of Angus (1954),
who in the case of Bacillus sotto succeeded in dissolving the inclusions in dilute
alkali and thus obtained an active, spore-free filtrate which produced paralysis in
silk-worms within 2-4 hours.
Summary
1. A method was elaborated for isolating inclusions from a mixture of spores,
inclusions and vegetative debris by means of a density gradient centrifugation
technique. The proportion of inclusions to spores was from 50 to SO : 1.
2. It was found that the crystalline inclusions formed in the cells of Bacillus thurin-
giensis during sporulation and released from the cells at the same time as the spores,
have a strong insecticidal action. They have a toxic effect on caterpillars within
a few hours after ingestion (immobilisation of intestinal function, reduced mobility)
and cause death in the third instar of caterpillars of Euproctis phaeorrhoea within
five days and in those of Bombyx mori within 24 hours.
3. Spores alone do not have an immediate toxic effect, but cause septicaemia
after a few days.
4. A strain of Bacillus thuringiensis which has undergone degeneration and has lost
the ability to form inclusions is not virulent for caterpillars of Euproctis phaeorrhoea,
Lymantria dispar and Bombyx mori when given by mouth. If, however, inclusions
from the virulent strain are substituted, infection with the degenerated strain has
the same effect as that with the virulent strain.
5. The strain of Bacillus thuringiensis investigated produces an antibiotic
substance which is not identical with the inclusions.
Angus, T. A.: A Bacterial Toxin Paralysing Silkworm Larvae. Nature 1i3 : 545, 1954.
B e r 1 i n e r, E.: tJber die Schlafsucht der Mehlmottenraupe (Ephestia kuhniella; Zell) and
ihren Erreger Bacillus thuringiensis n. sp. 7schr. Angew. Ent. 2 : 29, 1915.
Behrens, M., M a r L i, H. R.: Aufteilung der l.eukozyyten des Pferdeblutes Hach der
Grosse durch ein Analog der Craigschen Gegenstromverteilrur, arbeitendes Trennverfahren.
Naturwissenschaften 42:610, 1955.
Hannay, C. L.: Crystalline Inclusions in Aerobic Sporeforming Bacteria. Nature
172:1004, 1953.
Hannay, C. T.., Fitz -James, P.: The Protein Crystals oT BacillrLS thnringiensi~
Berliner. Cauad. J. Microbiol. 1 : 694, 1955.
H e i m p e 1, A. M.: Investigation of the Mode of Action of Strain of Bacillus cereus Fr. and
Fr. Pathogenic for the Larch Sawly, Pristiphora Erichsonii. Cauad. J. 'Cool. 33:311, 1955.
H u s z, B.: Bacillus thuringiensis Berl., a Bacterium Pathogenic Lo Gorn Borer Larvae.
Intern. Corn Borer Invest. Sci. Reports 1:191, 1927.
H u s z, B.: The Use of Bacillus thuringiensis B. in the Fight Against the Corn Borer. Intern.
Corn Borer Invest. Sci. Reports 2:99, 1929.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
H u s z, B.: Field Experiments on the Application of Bacillus thuringiensis Against the Corn
Borer. Intern. Corn Borer Invest. Sci. Reports 3:91, 1930.
Mattes, O.: Parasitare Krankbeiten der Mehlmottenlarven and Versuche fiber ihre Ver-
wendbarkeit als biologische Bekampfungsmittel. Gesel. Beford. Gesam. Natures. Sitzber.
62:381, 1927.
Meta 1 n i k o v, S., Chorine, V.: The Infectious Diseases of Pyrausta nubilalis Hb.
Intern. Corn Borer Invest. Sci. Reports 1:41, 1928.
Meta 1 n i k o v, S., Chorine, V.: Experiments on the Use of Bacteria to Destroy
the Corn Borer. Intern. Corn Borer Invest. Sci. Reports 2:54, 1929a.
Meta 1 n i k o v, S., Chorine, V.: On the Infection of the Gypsy Moth and Certain
Other Insects with Bacterium thuringiensis. Intern. Corn Borer Invest. Sci. Reports 2:60,
1929b.
Metalnikov, S., Ermolaev, J., Skobaltzyn, V.: New Bacteria Pathogenic
to the Larvae of Pyrausta nubilalis Hb. Intern. Corn Borer Invest: Sci. Reports 3:28, 1930.
Meta 1 rr i k o v, S., H e r g u l a, B., Miss S t r a i 1: Utilisation des microbes dans
la lutte contre la Pyrale du mail. Ann. Inst. Pasteur 46:320, 1931.
Meta 1 n i k o v, S., Meta 1 n i k o v, S. S., J r.: Maladies des vers du coton (Gelechia
gossypiella et Prodenia litura). C. R. Acad. Agr. France 18:203, 1932.
~t i m a n, J.: O postnatalni metabolicko-morfologicka organogenese krysi kostni d~en~. Diser.
prace. Praha 1955.
Schneider, W. C., Dalton, A. F., K u f f, E. L., Felix, M.: Isolation and
Biochemical Function of the Golgi Substance. Nature 172:161, 1953.
Smith, N. R., Gordon, R. E., C 1 a r k, F. E.: Aerobic Mesophilic Sporeforming
Bacteria. Washington 1946.
S t e i n h a u s, E. A.: Insect Microbiology. New York 1947.
S t e i n h a u s, E. A.: Possible Use of Bacillus thuringiensis B. as an Aid in the biological
Control .of the Alfalfa Caterpillar. Hilgardia 20:359, 1951.
S t e i n h a u s, E. A., Jerre 1, E. A.: Further Observation on Bacillus thuringiensis B.
and Other Sporeforming Bacteria. Hilgardia 23:1, 1954.
T o u m a n o f f, C.: Au srrjet de souches cristallophores entomophytes de Cereus. Observation
sur leurs inclusions cristallines. Ann. Inst. Pasteur 89:644, 1955.
T o u m a n o ff, C., V a g o, C.: L'agent pathogene de la flacherie des versa soie ende-
mique dans la region des CBvennes: Bacillus cereus var. alesti var. nov. C. R. Acad. Sci.
233:1504, 1951.
T o.u m a n o f f, C., V a g o, C.: Sur la virulence vis-a-vis du ver a soie de quelques Cereus
entomophytes en tant que tests de comparaison. Ann. Inst. Pasteur 88:388, 1955.
Weiser, J., Veber, J.: Moznosti biologickeho boje s pisastevniLkem americkym (Hyphan-
tria cunea Drury). Zool. a entom. listy 3:55, 1954.
'G o f k a, P.: Bakterialn} infekce hmyzu, vyvolane entomofytnimi kmeny B. cereus F. a F.
Diplomova prace. Praha 1955.
IiIayuexi~e AeHCTBi~x Bacillus thuringiensis xa xacexoMblx
YI. BAHbI{OBA
Peaw.atie
yIcc,neRosasacb cHOpoo6paayroruax 6axTepHx Bacillus thuringiensis, xax HasecTHO,
IIaTOPOHHaH J~JIH HaCeFfoMbIX. B COOTBOTCTBI3I3 C J;aHHbIMI~I JILITepaTypbl ~bIJIO yCTa-
HOBJIeHO, iITO TOKCYIYHOe AJIx HaCOHOMbIX BOH;eCTBO BOBH~iKaeT y B. thuringiensis
TOJIbI{O B nepHOA cnopoo6paaosaxKSi (Ta6~i. 1). 1{neTxH aTOro IIiTaMMa coAepxcaT
B 3TOT Hept3oA, xpoMe chop, Taxxce xpxcTaa.ai3~ecxxe Bicaiio~exHR (pHC. 1). Bbi~ia
paapa60T8Ha M@TO~jI3iSa BbIJ~eJIeHI3Fi BKJIIOLIeHI3I3 H3 CM0CI3 CHOP, BKJIIOLIeH}i)=f I3 OCTBTKOB
BBreTaTLiBHbIX K.aeTOx YICHOJIbayiOH;afi rpaJjl3eHTHyIO T0XHI3Ky IjeHTpH~yrHpOBaHHR.
Hat=iJ~eHHOe HaMI3 COOTHOHIBHLIe Me?IfZjy BKJIIOLI0HI3fIMH yI CHOpaMYI 6bi.n0 50-80:1.
A.ax BbiAe.aexHx criop Te~ibua Bx.nio~eHHi~ RexaTypHposaat3cb H.nH xarpesaxz3eM
(Ao 112 ?C), I3JIII HeHCYiHOM, LIJII3 axe 0,1 N-NaOH. I~Ia peay.nbTaTOS, Hpt3BeAexxbix
Ha Ta6~i. 2, OLIeBI3JjH0, ~ITO Cyu;eCTByeT nptiHuHnr3a.abHax paaHt3t;a Me?Kj(y TOKCI3-
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
~ecxHM ReilcTBneM xa ryceHnu oTAeJrbxo BxJIIO~eHIIH H oTReJlbxo cnop H PTO r.nasxoil
Toxcn~ecxoil cocTasxoil ~acTblo HBJIFIIOTCFI BxJIlo~exHH. Bx.nlo~eHr~IH oxa3blBaloT Ha
rycext~Iu Toxci~I~Iecxoe AeiICTBHe yaxe ~epe3 xecxoJlbxo ~acoB nocJle nx 3arJIaTblsaHi~x
(IIMMO6yIJIII3aL~I~IH xLIHIeLIHI~xa, HOHHTxeHLIe IIOJ~BPI?xHOCTI~I), BbI3bIBaIOT PI~16eJIb
3-eH cTaRr~n rycexi~u Euproctis phaeorrhoea Don. B Te~exr~e 5 RHeiI (Ta6Jt. 2),
a 3-eiI cTaRHr~ ryceHnu Bombyx mori - B Te~eHne 24 Tacos (Ta6JI. 4). I~I3oJIHpo-
Baxxble cnopbl He oxa3blBaloT HenIeAJlexxoro ToxcH~ecxoro AeHCTBnH, xo sbl3blsaloT
ceHTr~IxeMr3lo ~epe3 xecxoJlbxo AHeit.
HaCJIIOJ?~eHYIe, iITO I3HxJIIO3LIId HBJIHIOTCH OCHOBHbIM HOCIITeJIOM TOHCHLIHOCTH, ~bIJIO
nOJ~TBep?xJ[eH0 Tax?xe OIIbITBM.i CO HITaMMOM B, thUringlenSlS, xOTOpbILI yTpaTHJI
CHOCOF)HOCTb OrOpa3OBaTb TeJIblja BxJIIOTieHIIII. IIpH BBeJ~eHLILI per OS aTOT IIITc`1MM
xe 6bIJI BIIpyJIeHTxbIM AJIFI ryceHHu Lymantria dispar, Euproctis phaeorrhoea Don.
H Bombyx mori. EcJIn x cnopaM Hesi~pyJIeHTHOro uITaMMa rlpHrOaBJIHJIHCb TeJlblja
BxJIIOLIeHLiLI BI3pyJI0HTHOPO IiITaMMa, TO HOJIyLIeHHaH CMOCb Oxa3bIBaJIa TaxOe xce Aeil-
CTBI3e, xax n IIepBOHaiIaJIbHbII~ BIIpyJIOHTHbI~I HITaMM (Ta~JI. 4).
~ibIJIO yCTaHOBJI0H0, LITO HCCJIeJ~yeMbIII HITaMM B. thuringiensis o6pa3yeT aHT1~I-
CIIOTIILIeCIfOe BOH;eCTBO, xOTOpOe He TO?IteCTB@HHO C TeJIT~I~aMYI BxJIIOLIBHHLI.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
FOLIA BIOLOGICA
Darstellung von mit 131J markierten Eiweissstoffen
J. LIEBSTER, A. BABICK~', J. KOZEL, E. LISS, G. SYDOW
Biologiaches Inatitut, Tachechoslowakiache Akademie der Wissenachaften, Praha and Inatitut fur Medizin
and Biologie, Deutsche Akademie der Wissenachaften, Berlin-Buch
Mit iaiJ markierte Eiweissstoffe besitzen in Biologie, Biochemie and Medizin
grosse Bedeutung, vor allem fur Stoffwechseluntersuchungen. -Fur die meisten
Zwecke ist eine moglichst hohe Radioaktivitat des mittels 131J markierten Eiweiss-
stoffes bei minimalem Jodgehalt erforderlich, um die urspriinglichen Eigenschaften
des Eiweisses zu erhalten. Nach Francis verursacht ein Gehalt von 0,32 bis 0,87
Jod im Eiweiss keine Veranderung der Antigeneigenschaften (Francis and Mitarb.
1951). Bei der Jodierung ist es notwendig, immer moglichst schonende Bedingungen
zu wahlen and den Jodiiberschuss quantitativ sus der Eiweisslosung zu beseitigen,
ohne dass eine Strukturveranderung erfolgt.
Die Markierung von Eiweiss mittels 131J beruht auf der Jodierung des Tyrosin-
bzw. Histidin-Restes. Der mogliche Jodierungsgrad hangt vom Gehalt dieser zwei
Aminosauren im Eiweiasmolekul ab. Menschliches y-Globulin enthalt 6,7 % Tyrosin
and 2,6 %Histidin, menschliches Albumin 4,7 % Tyrosin and 3,55 %Histidin,
Pferde-Albumin 4,7 % Tyrosin and 4,02 %Histidin.
Bei den iiblichen Methoden der Eiweissjodierung wind Radiojod zusammen mit
dem Trager in der entsprechenden Menge von Kaliumjodid (Hughes 1950) nach
der Gleichung
J2 + KJ = KJ~
gelost. Aus dieser Gleichung folgt, dass eine moglichst geringe Tragermenge gewahlt
werden muss, falls Bute Jodierungsausbeuten erzielt werden sollen. Ein Nachteil
dieser Methode besteht darin, dass fur die Jodierung nur 33 % des Jodes ausgenutzt
werden konnen, wie sus der Gleichung
KJ3 -}- RH = KJ -{- HJ -}- RJ
folgt, wobei R den Phenylrest im Tyrosinmolekul bedeutet. Diese Jodierungsmethode
benutzte eine Reihe von Autoren unter Anwendung verschiedener Pufferlosungen.
In den meisten Fallen wurde eine Gesamtausbeute von 2-12 %bei der Jodierung
erreicht.
Francis and Mitarbeiter (1955) befassten sich mit der Methode der Markierung
von Eiweiasstoffen mittels Jod mit dem Ziel der Ausbeutesteigerung. Es gelang
den Autoren die Menge des Kaliumjodid-Tragers herabzusetzen, indem sie die
Oxydation des Kaliumjodides mittels Kaliumjodat nach der Gleichung
5 KJ -~ KJ03 ~- G HCl = 6 KCl + 3 H2O -}- 3 J2
ausfiihrten. Bei dieser Reaktion ist nur ein kleiner ~berschuss an Kaliumjodid
erforderlich, um das entstandene Jod in Losung zu erhalten. Die Autoren erzielten
bei der Jodierung von Eiweissstoffen eine Ausbeute von 35 %.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
~:
z
~
.~
~
w
~
~
~
o
o
~
x
~
z
~
o
~
x
~
h
~
~
~
~'
~
o
x
?
z
~
~
~
o
~
~
a
~.
~~
~m
~~
~y
~~
?.~
~
o
o
"'
o
x
0
~
~
~~
~~
wm
~~
1
1,0
8
8
6
1,5
1,50
3
10
20
-
50
9,15
2
1,0
8
8
6
1.5
0,06
3
10
4
0.3
50
0,74
3
1.0
8
8
6
1:5
0.06
3
20
20
0.3
50
! 0, 7 4
4
1.0
8
8
6
1.0
0,06
3
10
1
0,6
50
O,G6
5
1,0
3
3
~ 6
0,2
0,06
3
24
24
0,2
42
0,77
6
1,0
3
3
6
0,2
0,06
3
10
24
0,2
42
0,77
7
1,0
3
3
G
0,2
0,06
3
10
24
0,2
42
0,72
8
1,0
3,3
3,3
6
0,2
0,04
3
10
20
Q2
42
0.28 ~
9
1,0
3
3
4
0,2
0,03
3
10
20
0,2
42
0,21
10
1,0
3
2
4
0,2
0,03
3
10
20
0,2
42
0,18
11
1,0
3
3
6
0,2
0,02
3
10
20
-
42
0,12
12
1,0
3
3
6
0,2
0,03
3
l0
6
0,2
42
0,23
13
1,0
3
3
6
0,2
0,06
3
10
24
0,2
42
0,76
14
1,0
1
1
2
0,2
0,06 ~
1
10
24
0,2
25
0,61
15
1,0
0,3
0,3
0,9
0,2
0,12
0,45
10
b0
0,2
60
1,07
16
1,0
-
1
1
0,2
0,03
0,5
10
20
0,2
28
0,26
17
0,1
0,01
0,01
0,7
1,G
3,0
0,3
1
24
0,04
6,24
22,6
18
0,1
0,08
0,08
0,7
0,3
0,5
0,3
1
24
0,04
4,94
3,58
19
0,3
0,25
Q,25
2
1,85
3,0
1
3
24
0,1
11,5
23,3
20
1,0
3
3
6
0,5
0,5
3
10
24
Q2
78
1,7 i
21
1,0
3
3
6
0,5
0,5
3
10
24
0,2
73
1, 7 7
22 ~
~ 1,0
0,51
0,51
1,53
Q,8b
2,25
0,77
2,2
24
0,17
15,1
12,.5
23 ~
1,0
0,54
0,54
1,63
4,2
11,1 ~
0,9
4
24
0,1.8
21
57
:i
24
1,0
O,S3
0,83
1,6
2,35
5,0 ~
0,9
12,5 i
24
0,3G ~
37,5
,
39,6
25
1,0
0,75
0,75
2,25
3,25
10
1,13
3,25
24
0,25
28
79,5
26
0,1
0,09
0,09
0,75
4
10
0,4
10
24
0,2
31
79
27
0,1
0,16 i
0,16
1,6
20,4
16
Q,8
12
24
0,03
40
124
Versuche 1-19 wurden mit Rinder-y-Globulin auagefiihrt, Oxydationsdauer 1 Stunde.
Versuche 22-27 wurden 2 Stunden oxydiert.
Eine weitere Herabsetzung der Tragermengen wird durch die Extraktion des
Jodes mittels Ather aus dem Oxydationsgemisch ermoglicht (R.oche 1951), Die An-
wendung von Jod bei der Jodierung erhoht jedoch nicht nur die Gefahr der Jod-
sublimation beim Abdampfen des Athers, sondern such die Moglichkeit der Eiweiss-
denaturierung.
Nach einem anderen Verfahren kann Jod aus Jodid durch Oxydation mittels
Wasserstoffperoxyd in Freiheit gesetzt werden (McFarlane 1956). Die Jodierung
verlauft bei neutralem pH 7,5 in 0,9 % NaCl-Losung mit einer Ausbeute von 24 ?,~,.
Eine ahnliche Methode benutzte Gilmore (1954), welcher Natriumjodid mittels
Persulfat oxydierte and bei Jodierung von Serum-Albumin in Phosphatpuffer bei
Anwesenheit von Guanidin eine Ausbeute von 90 % der Theorie (bzw. 45 '~~ der
eingesetzten Jodmenge) erhielt. Fur die Oxydation kann ferner Natriumnitrit
in saurer Losung verwendet werden (Shulman and Tagnon 1950).
Fur die Abtrennung des Eiweiss von unverbrauchtem Jod and Salzen werden
Fallungsmethoden, z. B. mittels Essigsaure (Banks u. a. 1951), Dialyse (Melcher
and Masouredis 1951) event. auch in Anwesenheit von Ionenaustauschern (Stern-
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
~
~
.d e
W ~
?
y
m
A
~
~,
?m
a
y W
o .~
Anmerkung
1,98
22
0,44
Rinder-y-Globulin, 2 X mit Benzol ausgeschiittolt
0,22
29
0,58
dtto
0,39
53
1,06
dtto
0,24
49
0,5
dtto
0,41
52
0,36
dtto
0,39
51
0,36
Rinder-y-Globulin dialysiert
0,33
45
0,36
Rinder-y-Globulin 6 X mit Benzol ausgescliiittelt and voroxydiert
0,18
67
0,5
Globulin 5 X mit Benzol, 4 X mit CCl,~ ausgoschiittelt and voroxydiert
0,13
64
0,45
dtto
0,11
85
0,3
Globulin 10 Stunden dialysiert
0,04
30
0,4
Globulin 4 X mit Benzol ausgeschiittelt
0,07
31
0,3
dtto
0,35
47
0,37
Globulin 4 X mit Benzol ausgeschiittelt and voroxydiert
0,31
50
0,12
dtto
0,46
43
0,04
dtto
0,05
19
*)
dtto
11,0
49
0,1
Globulin mit Benzol extrahiert
1,74
45
0,09
dtto
13,3
67
0,11
dtto
0,33
19
0,15
Rinderalbumin mit Benzol extrahiert and voroxydiert
0,32
18
0,15
Rinderalbumin mit Benzol extrahiert
11,6
93
0,13
Pferdealbumin 24 Stunden dialysiert
50,0
87
0,13
dtto
13,4
20
0,06
Menschlichea Albumin, weniger rein, 24 Stunden dialysiert
51,5
65
0,12
Menschlichea Albumin rein, 24 Stunden dialysiert
3,6
4,6
*)
Tuberkulin 24 Stunden dialysiert
21,0
21,0
*)
BCG 1 24 Stunden dialysiert
Bei Vereuch 20 and 21 betrug die Oxydationsdauer 1 Stunde.
*) Der Jodgehalt im Eiweiss war Beringer als 0,01 %.
berg u. a. 1955) benutzt, wodurch der Dialysenverlauf beschleunigt wird. -Die
Dialysierrrlethoden arbeiten im allgemeinen achonender.
Da die in der Literatur angefiihrten Ergebnisse stark schwanken and die Arbeits-
methoden haufig nor ungeniigend reproduzierbar sind, befassten wir uns mit der
Methodik der Jodierung von Eiweissstoffen eingehender zwecks Ausarbeitung einer
verlasslichen Methods, welche hohe Ausbeuten and Erhaltung der urspriinglichen
Eigenschaften der Eiweissstoffe ermoglicht.
Fur die Versuche wards Rinder-y-Globulin, Ei-, Pferde-, Menschen- and Rinder-Albumin and zwei
Tuberkulinpraparate benutzt. Fiir die Jodierung warden folgende Losungen verwendet: 0,02 M NaJ;
0,02 M NaN02; 0,1 N HC1; 0,1 N NaOH; 0,2 M Phosphatpuffer pH 7,5; 20 % H2O2.
Die 10% Eiweisslosung wards vor der Jodierung entweder durch wiederholtes Ausschiitteln mit
0,5 Vol. Benzol zwecks Beseitigung event. vorhandener phenoliecher Stabilisatoren oder durch Dialyse
gegen 0,9 % NaCI unter Riihren bei 5? C wahrend 24 Stunden gereinigt. In manchen Fallen erfolgte
sine Voroxydation der Eiweisslosung, indem zu dem gereinigten Eiweias 0,2 ml 30% H2O2 zugeaetzt
and 2 Stunden bei 20? stehen gelassen words.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Fur die Jodierung wurde die in Abb. 1 dargestellte Apparatur benutzt.
In einon 20 ml-Erlenmeyer-Schliffkolben wurde 0,02 M NaJ-, 131J- and 0,02 M NaNOz-Losung pipet-
tiert (Tab. 1). Aus dem Scheidetrichter wurde 0,1 N HCl-Losung zugesetzt. Die Losung wurde 1 Stunde
bei Limmertemperatur mit elektromagnetischem Ruhrer geriihrt. Nach beendeter Oxydation wurde
aus dem Scheidetrichter 0,1 N NaOH-Losung zwecks Neutralisierung iiberschiissiger HCl zugesetzt.
Hierauf wurde die Eiweisslosung and der Phosphatpuffer in den Scheidetrichter pipettiert, diese Losung
bmnen emrgen Mmuten unter intensivem Ruhren zu der Jodierungs-
losung zulaufen gelassen and 30 % Wasserstoffperoxyd zugesetzt. Die
Jodierung verlief dann unter dauerndem Ruhren bei Raumtemperatur
wahrend einiger Stunden. Nach beendeter Jodierung wurde der Kolben-
inhalt in einen Dialysierschlauch iiberfuhrt and unter Ruhren bei 5? C
gegen 2 1 einer 0,9% NaCl-Losung dialysiert. Das Dialysat wurde 2 bis
3ma1 in Intervallen a 24 Stunden ausgetauscht.
Nach beendeter Jodierung wurde das Volumen der Losung gemessen
and in einem aliquoten Teil die Radioaktivitat bestimmt and auf das
Gesamtvolumen umgerechnet. Auf die gleiche Art wurde der Dialysen-
verlauf bei jedem Austausch des Dialysates verfolgt. Nach Beendung der
Dialyse wurde das Volumen erneut bestimmt and das Ergebnis der End-
analyse auf eine event. Volumenanderung korrigiert.
Die markierten Eiweissstoffe warden der Elektrophorese unterworfen.
Die Elektrophorese erfolgte in einer feuchten Kammer an gespannten
Filterpapierstreifen mit Veronal-Citrat-Puffer von pH 8,6 bei 250 V
wahrend 8 Stunden. Der Nachweis wurde durch Entwickeln des Elektro-
phoregrammes mittels Bromphenolblau in alkoholischer mit Quecksilber-2-
chlorid gesattigter Losung ausgefuhrt.
Die Radioaktivitat wurde mittels Geiger-Muller-Zahlrohr mit Glimmer-
Endfenster bestimmt. Die Proben warden unter gleichen geometrischen Be-
dingungen gemessen. Die Ergebnisse warden auf den Hintergrund and Zerfall
des Radioisotope korrigiert; Korrektion auf Eigenabsorption musste nicht
erfolgen, da die Masse der Proben zu vernachlassigen war.
Die Proben fur die Messung warden bereitet, indem wir 0,1 ml (nach
geeigneter Verdiinnung) auf ein Aluminiumschalchen von 2 cma Flache
pipettierten, 0,1 ml 0,1 N Natriumthiosulfatlosung zusetzten and unter
der Infrarotlampe trockneten.
Ergebnisse
Die Ergebnisse der Versuche, in welchen die zugesetzte Tragermenge, die Menge
an Radiojod, die Jodierungsdauer and die Menge zugesetzten Wasserstoffperoxyds
verandert wurde, Sind in Tab. 1 zusammengefasst. Aus der Tabelle folgt der Einfluss
der Reinheit des Eiweiss auf die Jodierungsausbeute, was am besten aus Versuch
Nr. 24 and 25 hervorgeht, in welchen zwei Albumine von verschiedener Reinheit
benutzt warden. Die besten Ergebnisse werden bei der Jodierung von durch Dialyse
gereinigten Eiweissstoffen erzielt. Die Tragermenge beeinflusst die Ausbeute in
weiten Grenzen nicht. Nur bei der Jodierung mit dem urspriinglichen Radiojod-
praparat, welches ohne Tragerzusatz oxydiert wurde, Sind die Ausbeuten besonders
niedrig. Die Oxydation von Radiojod and zugesetztem Trager erfordert fur den
quantitativen Verlauf mindestens 1 Stunde. Als optimale Dauer der eigentlichen
Jodierung erwiesen Bich 20 bis 24 Standen. Da bei der Jodierung eine dem an Eiweiss
gebundenen Jod aquivalente Jodmenge zu Jodid reduziert wird, ist es moglich, die
Ausbeuten durch dauernde Reoxydation des Jodids zu Jod zu erhohen, was durch
Zusatz einer kleinen Menge Wasserstoffperoxyd zu dem Jodierungsgemisch aus-
fiihrbar ist. Es gelang so, die Ausbeuten bei der Jodierung bis auf 90 ?,~ der eingesetz-
ten Menge an 131J zu erhohen. Wahrend des gesamten Versuches erfolgte keine
Veranderung des pH, welches rich dauernd auf einem Wert um 7,5 hielt. Fur die
Reinigung der markierten Eiweissstoffe von uberschussigem Jod eignet Bich
am besten die Reinigung mittels meist 2 bis 3ma1 wiederholter Dialyse, wodurch
mehr als 99 % des nicht gebundenen Jodes entfernt werden kann. Im Verlauf der
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Versuche erfolgte niemals Denaturierung des Eiweiss, wovon wir uns eineraeita
Burch Elektrophorese der Eiweissstoffe, andererseits Burch immunologischen Test
and Exkretionskurve iiberzeugten.*)
Die im Grossteil der Literatur beschriebenen, relativ niedrigen Jodierungsausbeuten
and deren betrachtliche Schwankungen besitzen mehrere Grunde. Dies ist in erster
Linie die Reinheit der Eiweissstoffe. Die fur die Jodierung bestimmten Eiweiss-
stoffe miissen vollkommen rein rein and diirfen vor allem keine Burch Jod oxydier-
baren Substanzen enthalten. Meist werden nach verschiedenen Verfahren gefallte
Eiweissstoffe benutzt, welche jedoch noch haufig Spuren von Verunreinigungen,
vor allem Stabilisatoren, enthalten. So enthielt z. B. ein von uns benutztea y-Glo-
bulinpraparat 0,3 % Trikresol. Fur die Erzielung hoherAusbeuten ist es vorteilhaft,
den Eiweissstoff vor der Jodierung einer Voroxydation mittels einer kleinen Menge
Wasserstoffperoxyd wahrend 2 Stunden zu unterwerfen, wodurch mittels Jod
oxydierbare Substanzen oxydiert werden, welche bei der Jodierung die fur die
eigentliche Jodierungsreaktion zur Verfiigung stehende Jodmenge herabaetzen
wiirden. Vorteilhafter ist ea, die Eiweissstoffe Burch Dialyse zu reinigen; die Vor-
oxydation entfallt Bann. Ein weiterer wichtiger Faktor ist die quantitative Oxyda-
tion des radioaktiven Jods and des zugesetzten Tragers, was Burch Wahl einer ge-
eigneten Reaktionsdauer erreicht wird. Die meisten Autoren vermischten die
Eiweisslosung im Puffer mit der frischbereiteten Jodierungslosung sofort; nach
unseren Erfahrungen erfordert die Oxydation von Jodid zu Jod mindestens 1 Stunde.
Bei den fur unsere Versuche benutzten Jodkonzentrationen bleibt Bas Jod nach
der Oxydation des Jodids in Losung, and es ist daher nicht notwendig, mit einem
Losungsmittel oder grosserer Tragermenge zu arbeiten. Es ist unter diesen Versuchs-
bedingungen nicht ausgeschlossen, Bass es zu oxydativen Veranderungen der Eiweiss-
stoffe kommt, diese beeinflussen jedoch nicht deren Verwendbarkeit fur immuno-
logische Zwecke.
Fiir die Erreichung hoher Ausbeuten eignet rich der Zusatz einer kleinen Wasser-
stoffperoxydmenge zu der Jodierungslosung, welcher Bas bei der Jodierung
entstehende Jodid erneut zu Jod oxydiert. Auf diese Art ist eine beinahe quantitative
Ausnutzung des gesamten Jods fur die Markierung von Eiweiss erreichbar. Fur die
Abtrennung von Salzen aus der Losung der jodierten Eiweissstoffe benutzen die
meisten Autoren Fallungsmethoden. Diese Methoden sind mit Verlusten verbunden
and gestatten es nicht, mehr als 99 % des ungebundenen Jods zu beseitigen. Es gelang
uns in unseren Versuchen such Burch 5ma1 wiederholtes Fallen nicht, diese Werte
zu erreichen. Wir fiihrten die Reinigung daher Burch Dialyse gegen 0,9 % NaCl-
Losung bei 5? C aus. Die Dialyse wurde fur gewohnlich 2 bis 3ma1 a 24 Stunden
wiederholt, bis der Jodgehalt in der Eiweisslosung wahrend 24 Stunden um nicht
mehr als 0,5 ?% sank. Auf diese Art gelang es, mehr als 99 % des ungebundenen Jods
zu beseitigen.
Zusammen f assung
Es wurde eine Methode der Jodierung von Eiweisastoffen ausgearbeitet, welche
die einfache Markierung mit 131J gestattet. Die Ergebnisse sind stabil and gut
reproduzierbar.
*) Wir darken Herrn Dr. 1$iha aus dem Biologischen Institut fur die freundliche Ausfiihrung der
immunologischen Teste and der Exkretionekurven.
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
 Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2
Die hohen Jodierungsausbeuten rind durch die Reinheit der zur Jodierung
benutzten Eiweissstoffe, den quantitativen Verlauf der Oxydation des Jodids
zu Jod and den Zusatz einer kleinen Wasserstoffperoxydmenge bei der Jodierung
bedingt.
Durch Einhalten der angefiihrten Bedingungen konnen Ausbeuten bis zu 90 ?,;, der
eingesetzten Gesamtmenge an Radiojod erzielt werden.
Fur die Beseitigung iiberschiissiger Salze eignet Bich die Reinigung durch Dialyse
wahrend 72 Stunden am besten.
Die Anwendung minimaler Tragermengen bei Einhaltung hoher Ausbeuten ge-
stattet die Darstellung von Eiweissstoffen mit einem derartigen Jodgehalt, dass
keine Veranderungen ihrer Antigeneigenschaften erfolgen.
Banks, T. E., Francis, G. E., Mulligan, W., Wormall, A.: (2uaatitative
Aspects of the Antibody-antigen Reaction. Biochem. J. 48:180, 1951.
Francis, G. E., Mulligan, W., W o r m a l 1, A.: T.abelling of Proteins with
Iodine-131, Sulphur-35 and Phosphorus-32. Nature 167:748, 1951.
Francis, G. E., Mulligan, W., W o r m a l 1, A.: Labelled Proteins and Protein
Antigens Containing both Iodine and Mustard Gas Sulphone Groups. Biochem. J. 60:118,
1955.
G i 1 m o r e, R. C.: Labelling of Bovine and Human Serum Albumin with J'a'. Nucleonics
12:65, 195xexxe no oTxoxrexxro x xercneTOexbrM axTxrexaM) 135
Imshenetsky, A. A., Ruban, E. L.: Ammonia Oxidation by Nitrosomonas Enzymes 141
HepmyT, M.: 1.-rj?opMbr 6axTepx#. IV. B~rxxxxe TeMxepaTypbr xa paasxTme L-uxxna y Pro-
teus vulgaris x ero sxaYexxe Rnx xoaxxxxosexxx ycmo#uxsocTx x nexxr~x~r.nxxy. (Ner-
mut, M.: .f.-forms of Bacteria. IV. The Influence of Temperature on the Development
of 1.-cycle of Proteus vulgaris and its Significance for the Development of Resistance
to Penicillin) 149
~marda, J.: Indiction of Bacteriophages by Ultraviolet Light in Naturally Polylysogenic
Strain of Staphylococcus aureus. (IIIMapua, R.: Y~-xxRyxuxx 6axTepnoc~arox y ecTe-
cTxexxo xosrxnxsorexxoro mTaMMa Staphylococcus aureus) 160
Sev~Yk, V., Podojil, M., Kyselova, M., Vrtiskova, A.: The New Antibiotic BU 271. (IITes-
~rxx, B., IIoAoxs, M., I{xcesosa, M., BpTKmxoxa, A.: Hoxbr# axTx6xoTxx BU `271) 170
5ev~}k, V., Podojil, M., Kyselova, M., Vrtiskova, A.: The New Antibiotik BU 306.
IlIerrvnrr, B., Ho~ox.n, M., I{xce~nosa, M., BpTxmxosa, A.: Honbr# axTx6xoTxx BU 306) 173
Vankova, .T.: Study of the Effect of Bacillus thuringiensis on Insects. (Baxbxoxa, H.:
hlayvexne Re#cTxxn Bacillus thuringiensis xa xacexoMbrx) 175
l,iebster, J., Bahicky, A., Kozel, J., hiss, E., Sydow, G.: llarstellung~ von rnit rarJ mar-
kierten Eiweissstoffen. (JIx6cTep, M., Ba6xgxx#, A., I{oae.n, H., JIxc, 3., CbrRox, r.:
HpxroTOn.nexne 6esxon Me~exbrx 13rJ) 183
Ghrastil, J., Petrtir, E.: The Physiology of the Formation of Anthocyanin in Carrot Root
Cultures (llaucus carota L.). (XpacTxn, ICI., lleTpy, 3.: ~xaxoarorxx o6paaosaxxx axTO-
uxaxorr s axcxsraxTaTax xopxx Mopxosx Daucus carota L.) 190
Approved For Release 2008/04/10 :CIA-RDP80T00246A002900500021-2