(ESTIMATED PUB DATE) NEWSLETTER WITH VARIOUS ARTICLES AND MAILINGS FROM SCIENCE SPECTRUM, INC. RE LIGHT SCATTERING, PHYSIOLOGICAL MONITORING OF BACTERIA, MICROBIOLOGY, & GALVANIC SKIN RESPONSE

• Document Type: SPECIALCOLLECTION
• Collection: General CIA Records
• Document Number (FOIA) /ESDN (CREST): 00173514
• Release Decision: RIPPUB
• Original Classification: U
• Document Page Count: 20
• Document Creation Date: January 21, 2025
• Document Release Date: January 15, 1983
• Publication Date: January 1, 1971

Coo
SCIEiNCE
SPECTRUM
Jo
1216 STATE STREET
POST OFFICE BOX 3003 SANTA BARBARA, CALIFORNIA 93105
TELEPHONE
[phone]
A NEW TECHNIQUE FOR STUDYING MICROORGANISMS
With differential light scattering you can learn more in minutes about
microbial morphology and physiology than others have learned in years.
Some examples are given in the accompanying Application Notes.
This technique - new to the study of microorganisms - shares some
capabilities of optical and electron microscopy. Sample preparation
is simple and rapid; submicron physical details can be studied; a wide
variety of samples can be examined in many different environments
and states, and physical changes monitored.
The differential light scattering measurements detailed in the accompanying
Application Notes were made with our DIFFERENTIAL I photometer. In
addition to determining structural parameters, such measurements enable
bacterial presence and concentration to be determined easily. The
scattering data can be analyzed rapidly, easily and quite accurately using
the Atlas of Light Scattering Curves described in the enclosed flyer.
For more information on these or other applications, or on our products,
simply fill out the enclosed prepaid card. A copy of our current seminar
schedule also is enclosed. If you have arir question regarding the study or
results reported in the Application Notes, please let us know.
Sincerely yours,
SCIENCE SPECTRUM, INC.
9f/aiwk,
James E. Hawes
Vice President, Marketing
Enclosures
Approved for Releaso -
pate 2 7 FEP, 1071
=a Aftnu, 3.,�11
r"
_,-SCIENCE
SPECTRUM
Jo
1216 STATE STREET
POST OFFICE BOX 3003 � SANTA BARBARA, CALIFORNIA 93IC
TELEPHON
(8 0 6) 963- 86 C
SEMINARS
Seminars with demonstrations explaining light scattering theory and its many
applications are offered by Science Spectrum periodically at various central
locations throughout the United States, free of charge. The DIFFERENTIAL I
and DIFFERENTIAL II instruments are also exhibited at selected professional
meetings. The seminars and exhibits currently scheduled are:
September 14 - 16, 1 971
September 17, 1 971
September 21, 1 971
September 24, 1 971
September 27, 1 971
October 5, 1 971
October 7, 1 971
October 15, 1 971
Washington, D. C. - Exhibit
Washington, D. C. - Seminar
Raleigh, North Carolina - Seminar
New York, New York - Seminar
Boston, Massachusetts - Seminar
Los Angeles, California - Seminar
San Francisco, Calif. - Seminar (tentative)
Chicago, Illinois - Seminar (tentative)
If you wish to attend one of the seminars listed above, please complete the
form below and return it to the Company. A program will be sent to you about
two weeks before the scheduled meeting, together with a confirmation of your
reservation.
I will attend your seminar to be held on
at (city) . Please send my prograrr
to: NAME TITLE
INSTITUTION
STREET
CITY, STATE, ZIP
TELEPHONE ( ) EXTENSION
Atlas of Light Scattering Curves.
Introduction
The interpretation of light scattering data has
long been an obstacle to the widespread use of this
powerful analytical tool. While some scientists de-
voted their careers to the theoretical understanding
of light scattering, their results were not readily adapt-
ed for use by workers in other fields. High speed
digital computers can now be used economically to
generate scattering data for a variety of model particles.
The purpose of the Science Spectrum Light
Scattering Atlas is to make this computer-generated
information available in a convenient form for a wide
range of light scattering applications involving small
particles. Computer-generated light scattering patterns
are plotted on the same scale as the experimental
data measured by the Differential I and // light scat-
tering photometers. Semitransparent vellum paper has
been used for the Atlas so that accurate comparison
of theory and experiment is achieved by merely over-
laying the two sheets. Tables of normalization con-
stants for absolute scattering power are also provided.
Single Spherical Particles in Air
A wide variety of processes produce homogen-
ous spherical particles of approximately one microm-
3ter in diameter. For example, photochemical aerosol
)r "smog" droplets and colloidal particles like those
n latex paints are spherical. Such small particles may
?asily be suspended in air by nebulizing a liquid sus-
')ension and their individual scattering patterns are
eadily measured with the Differential II scattering
Moto meter.
This important class of scattering objects is corn-
lately described by two parameters: radius and re-
-active index. The first section of the Atlas displays
VERTICRL
R = 500 nm
"grATTERga Amc,tilna Tr�
A sample page from the Atlas showing the light scattering
curves for homogeneous spherical particles of 500 nm radius
and four different refractive indices.
the scattering curves of spheres with radius between
0.05 and 1 micrometer in steps of 0.05 micrometer,
for refractive indices ranging from 1.33 to 1.59. The
refractive index of the surrounding medium is that
of air (n 1.0). Curves for both linear polarizations
are given. Inspection of the scattering atlas for spheri-
cal particles shows that particle size can easily be
specified to within 0.1 micrometer diameter. A de-
termination of refractive index to well within ten per-
cent accuracy is achieved for spheres by simply exam-
ining the relative intensities at peak amplitudes.
Supplements
Periodically, additional scattering curves are pub-
lished as supplement sections to the Atlas. Owners of
the Science Spectrum Scattering Atlas will receive all
supplements issued within two years of the date of
purchase without charge. Subjects selected for early
supplements include: the effect of size distribution
upon scattering from suspensions of spheres; the effect
upon scattering of size and size distribution changes
in model bacteria and mitochondria; scattering from
conductive particles; scattering from absorbing parti-
cles; and scattering from airborne bacteria. Measured
scattering curves from known non-spherical particles
may also be provided.
Computation of special scattering curve sets for
a wide variety of objects will be done at moderate
cost using proprietary Science Spectrum computer
codes. The scattering from spheres of different diam-
eters or refractive index can be computed on order.
For example, curves for small variations in particle
diameter at a constant refractive index can be
vided for studies of colloidal size distributions. .)1\
this means sizing accuracy of �- 10 nm diameter can
be obtained as reported by Phillips et al in the J.
Colloid Int. Sci. 34 (1970), p. 159. Scattering curves
on absorbing spheres can also be computed as needed.
Curves on any spherically symmetric structure with
varying complex refractive index can be generated.
Specific applications for specialized shell structures
include bacteria, bacterial spores, microencapsula-
tion particles, compound aerosol particles with large
nuclei, etc. Even more varied shapes can be computed
exactly when the particle nearly matches the refrac-
tive index of the medium in which it is immersed �
as in the case of bacteria in water.
Please send me____copies* of the Science Spectrum Scattering Atlas at $25.00 each, plus
$1.25 sales tax if delivered in California.
I understand that I will receive, without further charge, all supplemental sections to the Atlas
published in the next two years. Also, if payment is enclosed with my order, Science Spectrum
will pay shipping costs.
If I am not satisfied with the Atlas, I may return it postpaid within 10 days for a full refund.
Name
Title
Department
Organization
Street
City, State, and Zip
My main interest in light scattering is:
The supplement I am most interested in is:
* Purchasers of a Differential I or Differential II photometer receive with the instrument two
copies of the Atlas and all supplements for two years.
A1-17-081 (2)
Physiological Monitoring of Bacteria and Mitochondria
Introduction
Optical methods ranging from microscopy to turbidi-
metry have long been used to monitor bacterial growth and
division. However, the optical microscope is unable to re-
solve features smaller than a few wavelengths of light in size.
Turbidimetric measurements are subject to large errors
because the attenuation of light is a function of the product
of particle scattering cross section and particle density.
Since the particle scattering cross section is not in general
the same as the particle's geometrical cross section, signifi-
cant interpretive problems arise. A given value of transmit-
tance will often correspond to several different products of
particle density and particle size. On the other hand, differ-
ential light scattering measurements (i.e., recording the
) pattern of light scattered by such particles as a function of
angle relative to the direction of the illuminating beam) are
unambiguous, often yielding size and shape information of
much higher precision than obtainable with a microscope.
Under optimum conditions, cell size determinations of � 2%
accuracy are achievable with differential light scattering
measurements.
A variety of biologically important processes can be
accurately studied by differential light scattering. Physical
changes in mitochondria subjected to various enzymes, pH
variations, and osmotic stresses can be directly moni-
tored1-3.- Systematic distortions of bacterial cells by pre-
servatives such as phenol, formalin, or alcohols can be
measured precisely& Subtle changes in response to elevated
temperatures5 or pressures are easily determined. Size
modification and cellular damage occuring in phage-infected
bacteria can be measured. The process of spore germination
can be monitored as it proceeds. The response of chloro-
plasts to various processes including photophosphorylation
have been followed by light scattering". The susceptibility
of bacteria to various antibiotics can be measured within
minutes of contact9. Changes in mean cell size during syn-
chronous growth can be monitored with an accuracy of
� 20 nm. The effect of different growth media on the size
distribution of cells can be seen clearly via light scattering
patterns. These measurements can be made without dis-
turbing the growth of the culture. Some details of such
studies are discussed below.
LASER
CUVETTE
0* 0
Figure 2
DETECTOR
The Differential I Photometer
The Differential I light scattering photometer is a
highly versatile instrument, uniquely suited to the study of
liquid suspensions of bacterial cells. It is shown in Fig. 1 and
its operation is represented schematically in Fig. 2. In use, a
cuvette containing the suspension is placed in the instru-
ment and illuminated by the intense monochromatic beam
of an argon-ion laser. A specially designed scanning detector
system records, as a function of the scattering angle
relative to the beam direction, the intensity of light scat-
tered by the cells. This differential light scattering pattern
embodies a wealth of information about the cell ensemble,
such as cell size, shape, structure, size distribution10,11,
and even structural details such as cell wall thickness and
the refractive indices of the cell wall and cytoplasm12.
A final example of considerable interest concerns the
effects of heat killing on cell size and size distribution. In
preparing autologous staphylococcus vaccines, many labora-
tories use heat as a sterilization procedure. Such a treatment
supposedly does not destroy the immunogenic properties of
vaccines and would be expected, therefore, to have little or
no effect on cell walls. Figure 7 shows the changes in the
differential light scattering patterns as a function of heating
times for S. epidermidis broth suspensions at 60�C. (The
curves have been broken at 65� and displaced relative to
each other for visual clarity.) A subsequent analysis5 of this
data showed that the un-heat treated cells had an average
RELAIIVE IN If NSITY
30 min..
60 C
10 mm..
60 C
3 min
60 C
Control
E
60 80 100
SCATTERING ANGLE
Figure 7
30 mm.,
60-0
10 mm..
60�C
3 min.,
60'C
Control
120 140
For further information
radius of 432 � 10nrn which decreased to 403 �
30 minutes heating. The average cell wall thickness rem
nearly constant at 108 � 20nm despite the heating, but th,
breadth of the size distribution increased by 15% after
heating.
References
1. G. S. Gotterer, T. E. Thompson, and A. L. Lehninger,
"Angular light-scattering studies on isolated mitochondria,"
J. Biophysical and Biochemical Cytology 10, 15 (19611.
2. L. Packer, "Metabolic and structural states of mitochondria,"
J. Biol. Chem. 235, 242 (1960).
3. L. Packer and R. H. Golder, "Correlation of structural and
metabolic changes accompanying the addition of carbohy-
drates to Ehrlich ascites tumor cells," J. Biol. Chem, 235,
1234 (1960).
4. R. M. Berkman, "The effects of formaldehyde, phenol, and
other alcohols on bacterial structure deduced from light
scattering," Am. Soc. for Microbic!. Proceedings, May 1971.
5. R. M. Berkman and P. J. Wyatt, "Differential light scattering
measurements of heat treated bacteria," App!. Microbiology
20, 510 (1970).
6. L. Packer, P. A. Siegenthaler, and P. S. Nobel, "Light induced
high amplitude swelling of spinach chloroplasts," Biochem.,
Biophys. Research Communications 18, 474 (1965).
7. L. Packer, "Structural changes correlated with photochemical
phosphorylation in chloroplast membranes," Biochimica et
Biophysica Acta 75, 12 (1963).
8. L. Packer, R. H. Marchant, and Y. Mukohata, "Structural
changes related to photosynthetic activity in cells and
chlorop/asts," Biochimica et Biophysica Acta 75, 23 11963).
9. R. M. Berkman, P. J. Wyatt, and D. T. Phillips, "Rapid
detection of penicillin sensitivity in Staphylococcus aureus,"
Nature 228, 458 (1970).
10. A. L. Koch, "Theory of the angular dependence of light
scattered by bacteria and similar sized biological obiects,"
J. Theoret. Biol. 18, 133 (1968).
11. P. J. Wyatt, "Differential light scattering: a physical method
for identifying living bacterial cells," Applied Optics 7, 1879
(1968).
12. P. J. Wyatt, "Cell wall thickness, size distribution, refractive
index ratio, and dry weight content of living bacteria,"
Nature 226, 277 (1970).
13. Atlas of Light Scattering Curves, (Science Spectrum, Inc.,
Santa Barbara, California, 1971).
14. T. P. Wallace and J. P. Kratohvil, "Particle size analysis of
polymer latices by light scattering", J. Polymer Sci. C, 25, 89
(1968).
15. T. P. Wallace and J. P. Kratohvil, "Size distribution analysis
of polymer latex systems by use of extrema in the angular
scattering intensity," J. Polymer Sci. A-2, 8, 1425 (1970).
16. P. J. Wyatt, "Light scattering in the microbial world," On the
Occasion of the Centennial of Rayleigh Scattering Theory,
Am. Chem. Soc., Sept. 1971 (to be published in .1. Colloid
and Interface Science).
17. R. J. Fiel, "Small angle scattering of bioparticles,'' Experi-
mental Cell Research 59, 413 (1970).
Call or write the Director of Advanced Technology, Science Spectrum, Inc.,
1216 State Street, Santa Barbara, California 93105; telephone [phone],
M1-17-081
Rapid Assay of Bacteria in Urine
Introduction
The detection of threshold concentrations of bacteria
in specimen solutions such as urine presents an important
medical challenge. If it were possible to make a rapid deter-
mination of whether the bacterial count in urine is greater
or less than 104/m1 (0.1 critical level)1, it would expedite
enormously what is now a very time-consuming procedure.
A testament to the urgency of this need is the recent work
at NASA2, whose luciferase - ATP assay to detect life
on other planets is being considered for detecting bacteria
in urine.
A more direct bacterial counting capability, one
which is simple, effective and rapid, is available via the
technique of laser light scattering using a commercially
available table-top instrument, the DIFFERENTIAL I.
DIFFERENTIAL I Photometer
The DIFFERENTIAL I laser light scattering photom-
eter is a highly versatile semi-automatic instrument designed
to study liquid suspensions of cells with minimum altera-
tion of their normal environments. The instrument, shown
in Fig. 1, records the intensity variation with angle, 0, of the
scattered light which results when a cuvette of the solution
under study is illuminated by a laser beam. The operation is
shown schematically in Fig. 2.
The variation with angle of scattered light intensity is
detected by a specially-designed scanning system which
records the output on a strip chart or x-y recorder, or on a
digital data card punch unit.
When the size and internal structure of the illumi-
nated particles have dimensions approximating the wave-
length of the incident light, as do bacteria, the scattered
light pattern is particularly sensitive to these particle param-
eters. The features (amplitude and angular positions of
maxima and minima) in the scattering pattern give a precise
measure of the size, shaoe, structure, and size distribution
LASER
Figure 2
Differential light scattering patterns can be analyzed
theoretically using computer software already developed,
or simply compared to previously compiled "known" scat-
tering curves in a pattern recognition approach, analogous
to fingerprint identification. An Atlas of Light Scattering
Curves5 is available which permits even those not previously
familiar with differential light scattering to quickly and
accurately determine many of the important physical para-
meters of cells in suspension. In addition, measured changes
in the light scattering pattern can be employed to monitor
the effects of variation of conditions (heat, nutrient
changes, drug treatment, etc.) on bacterial suspensions. A
number of these applications have already been carried out
using the DIFFERENTIAL I instrument4,6,7,8.
Urine Specimen Assays
The simple task of determining concentrations of
bacteria does not need to utilize these analytical aids how-
ever. In studies of bacterial suspensions using the
DIFFERENTIAL I the detection of bacterial concentrations
of 105/m1 is routine. Indeed, in applications such as anti-
biotic susceptibility testing, solutions are prepared at about
this concentration for optimal results. At these concentra-
tions and lower, the intensity of the scattered light at any
angle relative to the background from the liquid systern�is
approximately proportional to the number and density of
cells, especially when the cell size distribution is narrow.
Thus, calibration of the light scattering patterns in terms of
cell concentration is straightforward.
Figure 3 shows a set of light scattering recordings
taken on the DIFFERENTIAL I for pure distilled water
and with several bacterial concentrations as indicated. The
detectability of these levels can be dearly seen.
In specimen solutions such as urine, appreciable back-
ground light may be scattered from various materials other
than the bacteria, materials such as tissue cells, granules,
cell debris, leukocytes, erythrocytes and various crystals. To
gauge the magnitude of this background scattering, bacteria
in known concentrations were added to unprocessed urine
and the samples examined in the DIFFERENTIAL I. Some
of the tvnical snatterinn nnttorns rechn�Arn in It rnn
RELATIVE INTENSITY
Figure 3
S.aureus in water
7 105 ML.
7 104 ML.
PURE H20
able, as expected; even at a lower concentration of 2.8 x
104/m1 the pattern is emerging from the background signal.
These results for untreated urine are very encouraging.
Simple techniques such as warming, mild acid treatment,
and sedimentation all of which are compatible with rapid
processing, and should not affect the bacteria, can remove
most of the background-producing material, thereby reduc-
ing the background scattering levels so that bacterial con-
centrations appreciably lower than 108/m1 can be measured.
Qualitative Studies
Figures 5A through 5D show light scattering patterns,
taken with the DIFFERENTIAL I, based on four patient
urine samples9. The protocol for all four was:
� 1 loopfull of urine was placed in trypti case broth and
incubated for six hours;
� 0.5 ml of the incubated solution was placed in 13.5
ml of distilled water in a cuvette;
� the cuvette was placed in the DIFFERENTIAL I and
scanned.
Samples A, C, and D showed no growth when incu-
bated overnight on a nutrient plate. Sample B showed the
presence of growth after incubation overnight on a plate,
corroborating the clear indication of bacterial cells in the
light scattering pattern (specimen curve). The effect of
treatment with an antibiotic (colimycin) was also deter-
mined. As shown in Fig. 5B, simply by adding it to sample
B and then rescanning its resulting light scattering pattern8,
susceptibility was clearly indicated by the dramatic change
in the pattern. This illustrates the rapid antibiotic suscepti-
bility test capability of the DIFFERENTIAL I. When
coupled with the simple yet sensitive capability of the
instrument to measure bacterial concentrations in urine, the
importance of differential light scattering as a clinical tool
is evident.
RELATIVE INTENSITY
RELATIVE INTENSITY
30
50
SCATTERING ANGLE. Amor.
Figure 4
Bacteria added to urine�
\
130
"I.-. 2 2 � 105,CC
150
Figure 5
Clinical urine samples
B WITH COLIMYCIN
100
References
1. R. N. Barnett, "Conference on the Medical Usefulness of Micro-
biology," Amer. J. of Clinical Pathology 54 Part II, 521 (1970).
2. G. L. Picciolo, B. N. Kelbaugh, E. W. Chappelle, A. J. Fleig, "An
Automatic Luciferase Assay in Bacteria in Urine," NASA
Goddard Report X-641-71-163 (Apr. 1971).
3. A. L. Koch, "Theory of the Angular Dependence of Light
Scattered by Bacteria and Similar-sized Biological Objects," J.
Theoret. Biol. 18, 133 (1968).
4. P. J. Wyatt, "Cell Wall Thickness, Size Distribution, Refractive
Index Ratio, and Dry Weight Content of Living Bacteria
(Staphylococcus aureus)," Nature, 226, 227 (1970).
5. Atlas of Light Scattering Curves (Science Spectrum, Inc., Santa
Barbara, California, 1971). A flyer (Al) describing the Atlas is
available on request.
6. R. M. Berkman and P. J. Wyatt, "Morphological Changes in Heat-
treated Staphylococcus epidermidis as Derived from Light
Scattering," Applied Microbiology, 20, 510 (1970).
7. P. J. Wyatt, D. T. Phillips, and R. M. Berkman, "Osmotic
Sensitivity in Staphylococcus aureus Induced by Streptomycin,"
(submitted).
8. Ft. M. Berkman, P. J. Wyatt and D. T. Phillips, "Rapid Detection
of Penicillin Sensitivity in Staphylococcus aureus," Nature 228,
458 (1970).
9. Recorded by Dr. S. Pantelick, Yale-New Haven Hospital
(unpublished).
For further information
Call or write the Director of Customer Liaison, Science Spectrum, Inc., P. O. Box 3003,
1216 State Street, Santa Barbara, California 93105; telephone [phone].
Standardization of Bacterial Culture Media
and Suspensions Using the Differential I
Introduction
In the process of growing bacteria, it is often neces-
sary to. maintain a strict uniformity of. the cultural con-
ditions from one day to the next. The success of a long
and often costly experiment is directly dependent both on
the quality and on the uniformity of the growth media. In
clinical laboratories, the lack of uniformity of culture
media can lead to grave consequences. For example, in
antibiotic susceptibility testing, errors not only in pre-
paration of the culture medium', but also variation between
batches of media supplied by the manufacturer can produce
erroneous results. In commercial laboratories (drug houses,
chemical manufacturers, etc.) a major obstacle to obtaining
optimum biological or biochemical yields arises from lack
of a precise means to measure and standardize growth
media.
The formulation printed on bottles of dehydrated
culture media, unfortunately, only approximates many of
the actual components. The chemical composition of com-
ponents such as peptones, tryptic hydrolysates and meat
extracts are known to vary greatly from one medium to
another. In addition, analyses or descriptions of amino
acid sequences, lengths of peptide chains, vitamin contents,
and contents of all other growth and inhibitory substances
which may be present in complex media are, of course,
not normally provided nor available.
In rehydrating and sterilizing a culture medium, the
chances of altering the medium to an unknown degree are
high. Temperature and duration of autoclaving, tempera-
ture of medium when plates are poured, ambient tempera-
ture and humidity, thickness of the medium, and age of the
plates affect subsequent growth.
The difficulties in standardizing cultural conditions
are quite apparent. However, Science Spectrum now pro-
vides an instrument to monitor with high reproducibility
and precision, variability in culture media and cultural
conditions in general. The DIFFERENTIAL I measures the
physical and physiological state of growing bacteria, pro-
viding a means to quantify variability of the growth
conditions.
By comparing data obtained with bacteria grown on
different media, one can immediately determine similari-
ties or differences in the growth patterns. In addition, this
simple comparison technique can easily be extended to
measuring the effects of toxins, antibiotics2, temperature3,
chemicals4, irradiation, and moisture on growing bacteria.
An Atlas of Light Scattering Curvess can be used to
determine quickly and accurately particle parameters such
as size, refractive index, and size distribution for various
species of bacteria and similar particles.
Figure 1
Differential I
The DIFFERENTIAL I laser light scattering photo-
meter is a highly versatile semi-automatic instrument
designed to study liquid suspensions of both viable and
nonviable cells. The instrument, shown in Fig. 1, records
the directional pattern of scattered light intensity from a
bacterial suspension illuminated by a laser beam. The
operation is shown schematically in Fig. 2.
The pattern (i.e. the intensity of scattered light as a
continuous function of angle relative to the incident beam
LASER
Figure 2
direction) is recorded by an automatic scanning detector
system, the output of which is designed to drive either a
chart recorder or a digital data card punch unit.
If the average radius of the bacteria approximates the
wavelength of the laser light, the interaction between the
cells and the light is strong. Hence, variation in intensity of
the scattered light is particularly sensitive to the size and
structure of the bacteria. Conversely, the features of the
light scattering pattern generated by and measured with a
DIFFERENTIAL I can be used to deduce average size,
size distribution6 and even general structure of the particles.
Measuring Relative Cell
Concentrations
To determine cell numbers by differential light
scattering, a standard set of light scattering measurements
for different known concentrations of bacteria is used to
establish control patterns. In making subsequent light
scattering measurements, a quick comparison to the control
curves allows a very accurate determination of bacterial
concentrations. Differences in cell concentration of less
than five percent are easily recognized by this method.
Figure 3 shows a typical standard set of curves which
were used as control patterns. In this case, Staphylococcus
aureus (Seattle) was the test organism. The cells were spread
evenly on Heart Infusion Agar (HIA) and after incubation
at 37� C for 10 hours, an aqueous suspension of cells was
prepared. The initial cell concentration was 2.0 x 107 cells
per ml; by dilution concentrations of 1.5 x 107, 1.0 x 107,
and 5.0 x 106 bacteria per ml were also prepared. Note
from the figure that large differences in overall scattering
were produced with cells differing by only 25 percent in
concentration. The position (scattering angle) of the maxi-
ma, i.e. the primary peak (arrow) provides a measure of the
average size of the bacteria. Note that dilution has not
altered the angular position of this peak, nor has it signi-
ficantly changed the overall appearance of the curves. If
the bacteria were of smaller average size, the position of
the peaks would have been shifted to larger angles (to the
right) or to smaller angles were the average size larger.
But the overall intensity level of the curves is essentially
only a function of cell concentrations.
Figure 3
S. aureus
LOG (INTENSITY
2.0x 10' /ml
1.5 x 10'/m1
1.0 x 10'/m1
5.0 x 10'/m1
50 70 90
SCATTERING ANGLE (DEGREES)
110
Monitoring of Solid Media for
Growth Potential
Difco Tryptic Soy Agar (TSA), Nutrient Agar (NA),
and Heart Infusion Agar (HIA) were evaluated using S.
aureus (Seattle) and the DIFFERENTIAL I. Cells removed
from each plate after incubation for 8 hours at 37�C were
resuspended in water to an optical density (OD) of 0.38
(X = 650 nm). For the differential light scattering measure-
ments, the cells were then diluted 1/40, corresponding to
cell counts of approximately 8 x 106 bacteria per ml. In
addition, a set of four Tryptic Soy Agar plates, freshly pre-
Figure 4
S. aureus from fresh
Tryptic Soy Agar plates
)
140 �
�
60 �
Figure 5
SIZE DISTRIBUTION
EFFECTS IN SCATTERING FROM
MODEL BACTERIAL SUSPENSION
MEAN DIAMETER 900 am
A 0%
20%
IF 30%
AO%
70%

20 40
60 80 100
SCATTERING ANGLE, degreel
120
140 160
pared, served as a control for variability within a single
medium batch. The four control curves are shown in Fig. 4.
Note that while the relative heights of the peaks differ
slightly, the four curves are almost identical in shape. (The
differences in peak height suggest that the cell concen-
tration of each sample differs by a small percentage, an
error resulting from lack of sensitivity of the spectrophoto-
meter used to prepare the suspensions.)
LOG (INTENSITY)
30�
Figure 6 Effects of media on growth of S. aureus
SCATTEAMG ANGLE 0,0 deqr�� rneelevonl
Figure 5 shows a series of computer-plotted curves
for cells of varying size distribution. The loss in peak defi-
nition is clearly increasing with widening of the size distri-
bution. The Science Spectrum Atlas of Light Scattering
Curves5 can be referred to for excellent approximations
of size and size distribution.
The scattering curves shown in Fig. 6 were obtained
when S. aureus was grown on various solid media for 8.hr
at 37�C. The plates contained (A) TSA, stored at 4� C for
16 days, (8) TSA, freshly prepared, (C) NA, stored for 6
wks at 4�C, (ID) HIA, 6 wks at 4�C, and (F) H IA, sealed 2
mos at 4� C. At first glance the differences in scattering
appear small; however, a composite of Curves A, C, and E,
shown to the right, clearly reveals some significant quanti-
tative and qualitative differences in three of the cultures.
Such differences cannot be measured with other conven-
tional particle sizing or monitoring instruments. Only by
differential scattering measurements could it be ascertained
that, for example, the cell concentration is highest for
30�
SCATTERING ANGLE (in 10 degree increments)
Figure 7
Endogenous growth
of S. aureus
Curve A and lowest for Curve C. (The OD's for all three
suspensions were the same. The so-called OD as determined
with conventional spectrophotometers depends critically
upon the average size of the particles and is not a mono-
tonic function of this average size.) Furthermore, cells
producing Curve C are smaller than the others, as evidenced
by the shift of the scattering peak to higher angles. Lastly,
the cells producing Curves A and E are similar in size, but
differ in that the size distribution among the latter cells
(E) is narrower.
Of equal significance is the ability of the DIFFER-
ENTIAL Ito measure physiological differences in the cells
taken from these six cultures. The method is described in
the next section.
Endotrophic Metabolism, a measure
of cell quality
The cultures tested directly from agar media have
characteristics which differ not only with respect to size
or size distribution, but also with respect to nutritional
makeup. The DIFFERENTIAL I can also measure these
nutritional or physiological differences simply by measuring
changes in light scattering among cells reincubated for
several hours in liquids having no nutritional value, such as
water or certain buffer solutions.
Figure 7 shows the scattering curves of the same cell
suspensions as Fig. 6 taken after changes due to endotro-
phic metabolism had occured. The data of Fig. 6 are in-
cluded in Fig. 7 as broken lines for the purpose of com-
parison. The solid, unretouched curves show how the
scattering signatures changed for each of the six cultures
when they were allowed to stand in the cuvettes contain-
ing water for an additional 10 hours at 25� C. The changes
observed consisted of changed in cell numbers, average cell
size and size distribution. Endotrophic growth was appar-
ently best among cells previously grown on fresh TSA
(Curve B) since the scattering curve was shifted upwards
significantly. On the other hand, the residual growth of
Staphylococcus aureus taken from Nutrient Agar (Curve C)
For further information
was barely detectable. Note also how cell shrinkage, readily
deduced from shifts of the primary peak to larger angles
(A, B, D, E, F) can be observed to various degrees for each
of the six preparations. Lastly, one can deduce that cell
size distribution had narrowed significantly in some cases
(e.g., curves 13, D, E, and F) as is evident from the signifi-
cant sharpening of the primary peaks.
In a set of control determinations (not shown) inocula
on four plates made from a single batch of TSA were found
to change uniformly when held in water for 8 hours.
Summary
Differential light scattering measurements provide a
sensitive means to measure numbers, size, size distributions,
refractive index, and refractive index distribution of bac-
terial cultures. With the DIFFERENTIAL I laser light
scattering photometer the most critical applications in-
volving standardizing of culture media are possible and
practical. In addition to its ability to monitor quantity
and quality of bacterial growth, the instrument provides a
sophisticated means to study physical and physiological
changes in growing and resting cells.
References
1. A.W. Bauer, W.M. Kirby, J.C. Sherris, and M. Turck, "Anti-
biotic susceptibility testing by a standardized single disc
method." Amer. J. Clin. Pathol. 45,493 (1966)
2. R.M. Berkman, P.J. Wyatt, and 0.1. Phillips, "Rapid de-
tection of penicillin sensitivity in Staphylococcus aureus,"
Nature 228,458 (1970)
3. R.M. Berkman and P.J. Wyatt, "Differential light scattering
measurements of heat treated bacteria," App/. Microbiology
20, 510 (1970)
4. R.M. Berkman, "The effects of formaldehyde, phenol, and
other alcohols on bacterial structure deduced from light
scattering," Am. Soc. for Microbial. Proceedings, May 1971
5. Atlas of Light Scattering Curves, (Science Spectrum, Inc.,
Santa Barbara, California, 1971)
6.� P.J. Wyatt, "Cell wall thickness, size distribution, refractive
index ratio, and dry weight content of living bacteria,"
Nature 226, 277 (1970)
Call or write the Director of Technical Liaison, Science Spectrum, Inc., P.O. Box 3003
1216 State Street, Santa Barbara, California 93105; telephone [phone].
M4-17-091
))
New Laser Instruments for Microbiology Demonstrated
The Differential I instrument
for studying microbial suspensions and molecular solutions.
Periodic Demonstrations Scheduled
Periodic demonstrations of each instrument will be given
by Company personnel at its exhibit booths 812-813.
Every two hours, beginning at 9:30 a.m. various applica-
tions of the DIFFERENTIAL I will be presented. These appli-
cations include -
� bacterial growth and morphology
� the effects of various alcohols on bacterial suspensions
� antibiotic susceptibilities
� accurate measurement of bacterial concentrations in
suspension
Many other applications will be discussed. Also, Application
Notes will be available for inspection and discussion with Com-
pany personnel.
The DIFFERENTIAL III instrument for the automatic
determination of antibiotic susceptibilities will be demonstrated
each morning at 10:30 A.M. Afternoon demonstrations will
start at 12:30, 2:30, and 4:30 P.M. During each demonstration,
susceptibilities of viable bacterial isolates to several antibiotics
will be automatically computed by the DIFFERENTIAL III.
Samples of the data card printed with these computed suscepti-
bilities will be distributed, together with brochures describing
the instrument and test.
Differential Light Scattering Papers
Exciting results and potentials of differential light scatter-
ing investigations were discussed in papers presented during the
Annual Meeting. W. Khan et al described "Rapid Detection of
Bacteria and Antibiotic Sensitivity in Body Fluids by Differen-
tial Light Scattering", paper M45 presented in Session 24
Monday afternoon. M. W. Wolfe and D. Amsterdam mentioned
some preliminary differential light scattering measurements re-
lated to their study of the "Interactions of Bacteria of Medical
Importance with Plant Agglutinins", paper M107 of Session 69
Tuesday afternoon.
A survey of the potential of differential light scattering
was discussed in a seminar, Session 25, held Monday afternoon
and titled "Instrumental Approaches to the Rap'd Detection
anci Chlr2ct9rizatior rsf Pqr-hsrin" Cr,n�f.n.r4 Drivirf Arricrpr_
With the advent of new laser light scattering instrum
tation, on display by Science Spectrum at booths 812-813
is no longer necessary to wait for days to determine the resL
of an experiment involving microorganisms. Bacteria, spo
and other microorganisms now may be examined with lc
powered lasers, permitting their structural characteristics a
their responses to various environments and processes to
quantified accurately and rapidly.
One of the basic laser instruments for such studies is
DIFFERENTIAL /TM light scattering photometer, shown
the left. Applications of this new instrument, and this n
approach to microbiology, demonstrated by the Company
its exhibit include quantifying the effects on microorganis
of pesticides and germicides, antisera, and various chemicz
Antibiotic susceptibility determinations also will be dem(
strated.
Another basic laser instrument being demonstrated
Science Spectrum is the DIFFERENTIAL //TM photome
for studying single microorganisms. Using this unique inst
ment, the structure of single bacteria and spores can be det
mined while the individual particles are still viable and ir
natural environment.
Another instrument, one very im_portant to the clini
laboratory, is the DIFFERENTIAL IIII M. Shown below, t
instrument enables antibiotic susceptibilities to be determir
automatically and rapidly. It will be demonstrated periodicz
by Company personnel for the duration of the exhibit usi
viable organisms and several antibiotics. Susceptibilities will
computed by the instrument within two minutes of sample
troduction. Samples of the data card printed with the compu-
susceptibilities will be distributed together with brochures ,
scribing the instrument and test.
The Differential lll instrument for automatically det ntibic
susceptibilities. Approved for Ricas
Date 221r73 197q
The DIFFERENTIAL IIITM InstraThent
The new DIFFERENTIAL III instrument, shown abc
rapidly and automatically determines antibiotic susceptibili-
of microbial specimens in an entirely different and nc
manner. It will be demonstrated periodically throughout
exhih;t, celIF anri ce?�,Prpl Rniibrtir ?nri deter,-
Differential Light Scattering Papers cont.
dam, Kingsbrook Jewish Medical Center, Brooklyn, the speakers
were Donald A. Glaser, University of California, Berkeley;
Henry Lubatti, University of Washington, Seattle; Norman G.
Anderson, Oak Ridge National Laboratory; Philip J. Wyatt,
Science Spectrum, Inc., Santa Barbara; and Henry D. Isenberg,
Long Island Jewish Medical Center. An abstract of Dr. Wyatt's
presentation "Applications of Differential Light Scattering in
the Clinical Microbiology Laboratory" follows:
Dr. Wyatt described a powerful new approach to many of the
problems of clinical microbiology. Noting that the size of bacteria and
the wavelength of visible light are about the same, Dr. Wyatt pointed
out that as a consequence a variety of unusual effects are observed when-
ever laser light is scattered from bacteria. By carefully measuring and
interpreting the manner by which such microorganisms scatter laser
light, numerous microbiological phenomena may be rapidly and accur-
ately characterized. These light scattering techniques have thus opened
the way for extensive new instrumentation that promises dramatic
changes in near future for the clinical laboratory. Dr. Wyatt described
instrumentation and techniques currently available, including a new and
revolutionary automated system (DIFFERENTIAL III) that determines
the antibiotic susceptibilities of exponential phase isolates within 12
minutes. He also described other instrumentation to be developed within
the next decade using light scattering techniques that could permit rapid
identification and susceptibility testing of clinical specimens without
the requirement for initial isolation and incubation. All the techniques
described appear to be equally applicable to aerobes and anaerobes, as
well as the more fastidious mycobacteria. In this latter regard, a high-
light of his talk was the preliminary report that Dr. Claude Reich of
Johns Hopkins (Leonard Wood Memorial) had measured an antibiotic
effect on a species of mycobacteria in less than 15 minutes using the
differential light scattering technique.
20
10
B. sphaericus spore
VERT IC AL
=MON
A schematic of the differential
light scattering measurement
and a sample of the resulting
pattern for a single spore.
BACKGROUND
GC NT IDD" 120'
SGATOR INS ANGLE
140'
1NY 1ST
The DIFFERENTIAL III TM Instrument cont.
ing susceptibilities in a few minutes. During each demonstra-
tion, first sample suspensions of the bacterial isolate are pre-
pared and exposed to different antibiotics. Then in the instru-
ment, each sample is illuminated in succession with a laser
beam of low power. The illuminated bacteria scatter the inci-
dent radiation, producing characteristicAight scattering patterns
which respond in a manner corresponding to the response of
the cells to the antibiotic. The patterns produced by suspen-
sions incorporating antibiotics are compared in succession to
the pattern produced by a control suspension without anti-
biotics. The degree of susceptibility indicated by pattern
changes then is automatically computed and printed on a data
card.
A DIFFERENTIAL III placement and evaluation pro-
gram will begin shortly after the ASM meeting in several labora-
tories throughout the nation. The results of this evaluation
program will be made available periodically to interested
clinical laboratories.
Differential Light Scattering, Briefly
When particles are illuminated by light, they will in general
scatter this light in all directions. The intensity of the scattered
light as a function of the direction has been termed the differen-
tial scattered light intensity. This is illustrated schematically in
the figure on this page. The trace adjacent the schematic shows
such a differential light scattering pattern (measured in a plane
with respect to the direction of the incident light) for a single
Bacillus sphaericus spore. Comparison of this pattern with
theory permits the unique determination of the spore's diam-
eter and refractive index of the spore's cortex and coat. For the
example shown, the radius of the spore was found to be 483 +
5nm, the coat thickness 80 + lOnm, the refractive index of the
cortex 1.56 + 0.02 and of the coat 1.48 + 0.03.
Your Invitation
To see the instruments, and to discuss the application of
particular interest to you, please drop by booths 812-813 at
your convenience. Company scientists will be there, and litera-
ture and data folios will be available for your inspection. Also,
you may fill out a request form to receive- free of charge - any
Company publications you desire.
Should you care for more information, or copies of our
publications, please call of write the Vice President, Marketing,
Science Spectrum, Inc., 1216 State Street, Post Office Box
3003, Santa Barbara, California 93105, telephone
[phone].
PUBLICATIONS AVAILABLE FROM SCIENCE SPECTRUM
Reprints
Differential Light Scattering: A Physical Method for Identifying Living
Bacterial Cells, P. J. Wyatt. Applied Optics 7, 1879 (1968).
Identification of Bacteria by Differential Light Scattering, P. J. Wyatt,
Nature 221, 1257 (1969).
Cell Wall Thickness, Size Distribution, Refractive Index Ratio, and Dry
Weight Content of Living Bacteria (Staphylococcus aureus), P. J. Wyatt,
Nature 226, 277 (1970)
Morphological Changes in Heat-treated Staphylococcus epidermidis as
Derived from Light Scattering, R. M. Berkman and P. J. Wyatt, Appl.
Microbiol. 20, 510 (1970).
Measurement of the Lorenz-Mie Scattering of a Single Particle: Polysty-
rene Latex, P.J. Wyatt, D. T. Phillips and R. M.Berkman, J. of Colloid
and Interface Science 34, 159 (1970).
Rapid Detection of Penicillin Sensitivity in Staphylococcus aureus, R. M.
Berkman, P. J. Wyatt and D. T. Phillips, Nature 228, 458 (1970).
Evolution of a Light Scattering Photometer, D. T. Phillips, BioScience
21, 865 (1971).
Size Distribution of Bacterial Cells, V. R. Stull, J. Bacteriol. 109, 1301
(1972).
Osmotic Sensitivity in Staphylococcus aureus Induced by Streptomycin,
P. J. WYatt, R. M. Berkman and D. T. Phillips, J. Bacteriol. 110 (May
1972) in press.
A New Instrument for the Study of Individual Aerosol Particles, P. J.
Light Scattering in the Microbial World, P. J. Wyatt, J. of Colloid and
Interface Science 40 (1972) in press.
Dielectric Structure of Spores from Differential Light Scattering, P. J.
Wyatt, Spores V, (1972) in press.
Bibliographies
DIFFERENTIAL I bibliography - selected reference material including
particle suspensions and molecular studies.
DIFFERENTIAL II bibliography - selected reference material including
single particle measurements.
Application Notes:
Physiological Monitoring of Bacteria and Mitochondria
Rapid Assay of Bacteria in Urine (currently being updated)
Standardization of Bacterial Culture Media and Suspensions Using the
DIFFERENTIAL I
Size Measurements of Single Microparticles
Characterization of Airborne Particulates Using the DIFFERENTIAL II
The Structure of Individual Microorganisms
Measuring Antibiotic Susceptibilities and MIC's by Differential Light
Scattering
Brochures describing -
DIFFERENTIAL I instrument for studying microparticle suspensions
and molecular solutions
DIFFERENTIAL II instrument for studying individual microparticles
DIFFERENTIAL III instrument for automated antibiotic susceptibility
ORIGIN OF THE GALVANIC SKIN RESPONSE 559
were used to identify the cells which were pre-
paring for division and colloidal saccharat ed
� iron oxide was used to identify the active
phagocytic cells. In livers of mice whose re-
ticulo-endothelial system, was stimulated by
estradiol, it was established that the cells pre-
paring for division and those which had re-
cently divided were actively phagocytic. In
livers of mice whose reticulo-endothelial sys-
tem had been "blockaded" with saccharated
iron oxide, it was established that the cells
which had phagocytized colloid were able to
divide in the process of recovery from "block-
ade." No evidence was found for a stem cell
which proliferates and differentiates to pro-
vide the active phagocytic population.
1. Kelly, L. S., Dobson,, E. L.; Finney, C. R.,
Hirsch, J. D., Am. I. Physiol., 1960, v19S, 1134.
2. Abercrombie, M., Harkness, R. D., Proc. Roy.
Soc., series /3, 1951, v13S, 544.
3. Beard, J. W., Rous, P., J. Exp. Med., 1934,
v.59, 593.
4. Howard, 5. G., Rowley, 1)., Wardlaw, A. C.,
Immunology, 1958, vi, 181.
5. Bcnsch, K. G., Simbonis; S., Hill, R. B., King,
D. W., Nature, 1959, v183, 475.
6. Kelly, L. S., Dobson, E. L., Fed. Prot., 1961,
v20, 265.
7. Howard. J. G., J. Path. Bact., 1959, v78, 465.
8. Lison, L., Smulders, J., Nature, 1948, v162, 65.
Received May 14, 1962. P.S.E.B.M., 1962, v110.
Origin of the Galvanic Skin Response.* (27579)
BENJAams A. SHAVER, JR.t SAUL W. BRUSILOW,: AND ROBERT E. COOKE
(Introduced by C. P. Richter)
Department of' Pediatrics, Johns -Hopkins University School of Medicine and Harriet Lane Home,
Johns Hopkins Hospital, Baltimore, Md.
If a metal plate electrode is placed on the
skin surface of the human palm, or on the
foot or toe pads of the cat, and the body of
the experimental subject grounded by means
of another metal plate electrode at some, point
on the body at a distance from the first elec-
trode, a change in electrical potential will oc-
cur between the 2 electrodes in response to a
stimulus transmitted to the skin by the sym-
pathetic nervous system. .There is a �simul-
i.
1
' 3
taneous decrease in the skin's resistance to the
passage of an electric current. When the po-
tential change is elicited by any p-giiifuT,-stare;
cir.* threatening event in the environment
orthe- experimental subject, it is referred to
as the galvanic skin reflex. When the stimu-
li lus is applied peripherally as when ,a periph-
eral sudomotor, nerve,.. or the sympathetic
titink-i'o the hind extremity is stimulated,, the
potential 'change is called the galvanic _skin
iespeo-iis-e7. The wave forms of the galvanic
* This project was supported in part by grant
from�U. S. Public Health Service.
I Fellow of Maryland Heart Assn.
; Senior Research Fellow, U.S.P.H.S.
v�,4414.-.- � t..c�w2b-
skin reflex and the galvanic skin response,
wnen recorded from metal plate electrocies
Csnereatter referred to as "macroetectrodes ')
avering a poruon of the skin's surtace, are
identical, Their latent perMr=s7v-oirci,
be expected from the difference in the path
length and conduction time of the nerve fi-
bers carrying the stimulus to the skin(1).
Various theories of origin of the galvanic
skin reflex and the galvanic skin response
have been advanced at one time or another
since their discovery by Fere(2) and Tar-
chanoff(3), respectively, over 70 years ago.
Veraguth(4), by implanting the grounded,
or indifferent, electrode beneath the skin sur-
face, demonstrated that the potential change
originated within the skin, rather than from
muscle and other underlying tissues. The
controversy primarily centers around the
question of whether this phenomenon is
caused by sweating, the widely accepted view.
These theories and the experimental evidence
supporting them have been reviewed by Wang
(1).
Richter(5) pointed out that the skin po-
4.0- �
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;
, Approved for. R31e
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to- 2, 7 Cr
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-
'4
560
ORIGIN OF THE GALVANIC SKIN � RESPONSE
tential tracings obtained from the palm of
the human subject contain 2 components: a
fast component, negative in direction, and a
slow component, positive in direction. He
recorded the slow positive component from
the skin of a patient with congenital absence
of the sweat glands. The fast negative com-
ponent was absent in this patient. He con-
cluded that the fast negative component was
related to sweating, but that the slow positive
component was not, and had its origin in
capillary or epithelial cells of the skin.
Lloyd(6), studying the galvanic skin re-
sponse of the skin of the cat's paw pad, re-
cently discovered that Richter's observation
(5) of 2 components was valid in that species
also. .However, Lloyd interpreted his data
to indicate that the slow component was a
sweat gland "..secretory, potentiar_ that was
related to reabsorption, and to the .amount of
inoisture in the sweat gland ducts.
The present study was undertaken to de-
termine which of these conflicting interpreta-
tions is valid. Cats were employed as the ex-
perimental subjects. The use of micropipette
electrodes permitted direct measurement of
the electrical potential from the lumen of in-
dividual sweat glands and from the epidermal
and dermal tissues surrounding them. Si-
multaneously, the galvanic skin response was
recorded from a macroelectrode covering the
surface of a paw pad of the same extremity.
Methods. In our experiments, the galvanic
skin response was studied by measuring the
electrical potential arising in the skin of the
paw pad of anesthetized mongrel cats in re-
sponse to electrical stimulation of the lumbar
sympathetic trunk supplying the homolateral
hind extremity. A nerve preparation similar
to that of Dale and Feldberg(7) was used.
The apparatus and technics employed are
similar to those used in the microelectrode im-
palement of single nerve fibers. Glass capil-
lary tubes were drawn out into micropipettes
with a pipette puller similar to the one de-
scribed by Alexander and Nastuk(A). The
electrodes were filled with methanol by boil-
ing under vacuum, and subsequently with 3-
Normal potassium chloride by diffusion re-
placement of the methanol. Electrical con-
tact with the electrolyte solution within the
pipette was provided for by inclusion of a
silver-silver chloride electrode as an integral
part Of the electrode holder.
Indifferent, or ground return, electrodes of
several kinds, placed in a variety of locations
were used with uniform results. Electrode
materials included platinum, steel, stainless
steel, and zinc. Locations included the ab-
dominal cavity of the laparotomized animal,.
skin of the thigh, muscle of the thigh, the
tongue, and the external ear. The recorded
responses were not affected by the nature or
location of the indifferent electrode, unless
the latter was placed near the stimulating
electrode within the abdominal cavity. In
the latter case, as would be expected, consid-
erable stimulus artifact was introduced into
the tracings.
The signal from the recording micropipette
electrode was fed into a coupling amplifier
employing a single-ended OK-5889 electrom-
eter tube. The measured electrometer tube
grid current was 10-14 amperes. The signal
was further amplified by Offner transistorized
voltage and power amplifiers.
The electrical resistance of the micropipette
electrodes used in these experiments ranged
from 20 to 60 megohms. This resistance was
continuously monitored by means of the pulse
injection technic, and did not significantly
change during the experiments reported here.
The usual precautions in selecting micropip-
ette electrodes with low tip potentials were
observed. Negative feedback was used to
cancel out input capacitance.
The microelectrode implantations were
made under direct vision with the aid of a
dissecting microscope and a Zeiss micromani-
pulator. To obtain the galvanic skin response
in the same animal, a small stainless steel cup
electrode, 1.5 cm in diameter, covered another
toe pad of. the same paw. A layer of electro-
lyte-containing paste was interposed between
the toe pad and the cup electrode. The latter
electrode was connected to an independent
direct-current differential amplifier.
Because .of its ready availability and con-
venience, the electrolyte paste used in most
of these experiments was that made by the
Sanborn Co. to be used between the skin and
an electrocardiograph electrode. To exclude
ORIGIN OF THE GALVANIC SKIN RESPONSE 561
polarization phenomena at the skin and in-
different electrodes, the results obtained when
using the Sanborn paste and a stainless steel
macroelectrode were compared .with � results
obtained when using a zinc macroelectrode
covered with a paste composed of kaolin and
a saturated aqueous solution of zinc sulphate.
The zinc-zinc sulphate macroelectrode was
used in Richter's experiments(5,10), and is
a combination in which polarization over the
voltage range in these experiments is negli-
gible. Under the conditions of the experi-
ments reported here, the results obtained
with the 2 electrode and paste combinations
were identical.
The output signals from both the amplifier
.conhected to the microelectrode, and the am-
plifier connected to the macroelectrode cov-
ering the toe pad, were fed into a 2-channel
Offner recording galvanometer by means of
which the 2 signals could be recorded simul-
taneously.
A Grass stimulator supplied the stimulating
pulses to a platinum boot electrode placed at
laparotoniy around the 'lumbar sympathetic
trunk supplying the hind extremity of the
animal. Squarewave pulses, varying from
threshold, usually 2 to 3 volts, to 7 to 10 volts
were employed. Pulse duration was I to 5
microseconds.
Results. Single pulse stimulation. In
tracings recorded simultaneously from the
ruacroelectrode covering a toe pad and from
a microelectrode with the duct of a single
sweat gland of the adjacent toe pad, the ini-
tial fast negative (downward) component of
the two curves ran an almost identical course.
The exponential decay of the fast component
in returning toward the base line proceeded
more rapidly in the case of the recording
made from the macroelectrode (galvanic skin
response) and became positive with respect
to the resting potential, whereas the tracing
from the lumen o'f a single sweat gland did
not (Fig. 1). The electrical response from
over a thousand individual sweat glands in
20 cats always returned to the base line after
a decay period of from 4 to 6 seconds follow-
ing the initial negative deflection. In. no in-
stance did the tracing from a microelectrode
in the lumen of a single sweat gland (Fig.
IA) show this slow positive component, but
the simultaneously recorded galvanic skin re-
sponse (Fig. 1B) frequently did. All of the
microelectrode responses obtained from in-
dividual sweat glands were identical in wave-
form to that shown in Fig. 1A. The latent
period varied from 0.6 to 0.8 second, the
mean and median values being 0.7 second.
The magnitude Of the peak negative response
varied with stimulus intensity, the more elec-
tronegative responses being obtained with the
higher voltage stimuli. The positive com-
ponent of the galvanic skin response (Fig.
1B) was not always present in the tracings
obtained from the macroelectrode. It ap-
peared most consistently in response to higher
voltage stimuli. Its presence or absence ap-
peared to be related to the intensity of the
stimulus, discussed below.
Repetitive squat-move stimulation. In the
experiments performed with repetitive stimu-
lation, a stimulating frequency of 10 per sec-
ond was employed. In response to repetitive
stimulation (Fig. 2), the microelectrode with-
in a sweat gland duct lumen held a uniform
negativity for the first half minute and there-
after began to decay toward the base line,
and finally rose to lumen positivity by the
end of the third minute of repetitive stimula-
tion, whereas the galvanic skin response re-
corded from the macroelectrode covering a
toe pad became positive early in the first min-,
ute of stimulation after an initial negative de-
flection.
Effect of stimulus voltage on fast and slow
components of galvanic skin response. The
voltage threshold necessary to elicit the slow
positive component was higher than that re-
quired to elicit the fast negative component:
In one experiment, the latter was obtained
from the macroelectrode with a squarewave
pulse of 2 to 3 volts in a fresh preparation.
Increasing the stimulus to. 4 volts, the fast
negative and the slow positive components
were obtained in the same preparation. The
voltage threshold for the slow positive com-
ponent was more variable than that required
to elicit the fast negative response and became
lower with the application of frequent stimuli.
If the preparation was rested, this effect was
reversed. The same threshold effect was
g
0
5- m-ct
> 0
-51
I Tifl.
0
ORIGIN OP THE GALVANIC SKIN RESPONSE
I--4
25 see.
A
stimuiui
on
+5 MV
min. �
FIG. 1. (A) Microelectrode response from a single sweat gland. (B) Galvanic skin response �
from a macroelectrode covering toe pad of same extremity, recorded simultaneously with (A).
A single monophasic squarewave pulse stimulus was applied at the arrow. Negative deflection
�� is downward in this and succeeding figur��s.
FIG. 2. (A) Microelectrode response from lumen of a single...sweat gland. (B) Galvanic
skin response from a i.nacroelectrode covering toe pail,of same .extremity. Repetitive stimula-
tion, 10 per sec., begins at arrow and continues to end of tracing. (A) and (B) recorded simul-
taneously.
FIG. 3. Mieroelectrode response from lumen of a single sweat land to intra-arterial inj.
of methacholine chloride.
FIG. 4. (A) Microelectrode response from epidermal cells of skin of cat's paw pad obtained.
during the life of the animal. A similar tracing could be obtained for nearly one hr post-mor-
tem, long after sweating had ceased. (11) Mieroelectroiln response front dermis of skin of living
animal. Repetitive stimulation of 10 per see. was used in Obt:thallg both tracings.
noted in the experiments in which repetitive
stimulation was used. Using low voltage sti-
muli in a fresh, or rested, preparation, the
tracing from the macroelectrode became nega-
tive and remained so for several minutes, as
reported by Richter and Wheelan(10). At
a slightly higher stimulus voltage, often a
differential of as little as 1 volt, the positive
component appeared (cf. Fig. 2A. and Lloyd
(6)). Stimulus pulse duration over 1 to 25
microseconds had no appreciable effect on the
responses obtained.
Pharmacologic stimuldtion. The micro-
electrode tracing from a sweat gland of a
cat's toe pad in response to the intra-arterial
injection of methacholine chloride (via the
abdominal aorta) is shown in Fig. 3, which
is included to show the similarity of the re-
sponse obtained by repetitive stimulation to
that produced by a parasympathomimetic
drug (compare Fig. 2 and 3), and to indicate
that electrical responses from the lumen of
the individual sweat glands and from the skin
surface are not artifacts related to the elec-
tric shock stimulus.
Localization of the slow corn pollen! of gal-
vanic skin response. The superficial cornified
layer of the epidermis was dissected away so
that the papillary layer of the epithelium was
exposed. A microelectrode was touched to
the surface of the exposed epithelium, but not
in proximity to a sweat gland duct orifice, and
a single pulse was applied. No negative de-.
tlection was obtained with a stimulus which
readily elicited the fast negative component
when the microelectrode was subsequently in-
serted into the lumen of a sweat gland in the
same prepared area. With the microelectrode
in contact with the epidermal layer of the
skin but not near a sweat gland, repetitive
stimulation identical to that used in Fig. 2
was applied. There was observed a slow posi-
Th
/4
a
ORIGIN OF TIIE GALVANIC SKIN RESPONSE
tive component of the galvanic skin response
from the macroelectrode. If the animal was
killed, visible sweating soon ceased, and the
fast component of the galvanic skin response
could no longer be detected. Howevs.the,
slow component could be obtained,frpni.the
\ V I tn tkie�..rtiicroelectrode rly.
an hour post-mortem, (Fig. 4A), long after
ItErrvgi i-4.13'n-r1sTha'd ceased to function.
To exclude the possibility that the slow
positive component originates from the skin
structures below the epidermis, a strip of epi-
dermis was dissected away leaving the derinis
exposed. Sweat glands in this area poured
forth sweat when stimulated repetitively, but
the, zositive component was, not recorded
when tie inieiroelectro-de-Wal in co:itact with
The exposed dermis (Fig. 4
Discussion: 'It is unlikely that curves such
as Fig. 2A would be seen under physiological
conditions. If a flat electrode is placed on
the skin surface of the palm, and another on
the back of the hand of a human subject (the
same obtains for the paw of the cat), spon-
taneously arising negative waves will be re-
corded which are identical to the microelec-
trode responses from the lumen of a single
sweat gland of the cat. These responses_ocr
cur in random fashion in the unstimulated
subject and by their identity with the micro-
electrode responses (Fig. IA), are. presum-
ably the electrical potentials generated by the
individual sweat glands or groups of sweat
glands covered by the skin electrode. In a
resting patient with extreme hyperhydrosis
of the palms of the hands from whom such
tracings were taken, the sweat gland potential
changes recorded from a skin electrode with
an area of 6 sq cm occurred as often as 100
per minute. These responses often were su-
perimposed on each other because of their fre-
quency, but in no case was fusion observed
such as occurs with strong repetitive stimula-
tion of the lumbar sympathetic trunk or a
peripheral sudomotor nerve(6,10). On trac-
ings obtained from the resting, unstimulated
cat or human subject, only the fast negative
component was seen. It appears that under
the enormous stress upon the sweat glands
when repetitive stimulation is used (Fig. 2A),
the sweat glands become fatigued and are un-
563
able to maintain their lumen negativity. That
the site of this fatigue is t.�1.1e_sw�eat and
not the suclomotor.�.nerye�is_supported by�..the
�ziziments in �which_pharmacologic,stimula-_
Lion was ise(Fig)See Tha sen and
"Schwartz-5)).
We can account for the data presented in
Fig. 1 and 2 if, as.Richter(5) contended, the
positive component is not related to the sweat
,,lands but originate 's in otrers1:7m� structures.
Our data substantiate Richter's view and are
consistent with the following mechanism.
With single shock stimulus the fast negative
component origirtate.s glands.
With repetitive stimulation, the. fast corn-
yonents fuse to become a negative
potential as has been shown by Richter and
11: heelan This wouca
also appear to be the case with a pharmacolo-
gic stimulus of long duration of action as me-
thacholine chloride (Fig. 3). In response to
an enormous stress such as repetitive stimula-
tion or with intra-arterial methacholine chlor-
ide, the sweat gland begins to fatigue (after
approximately 600 shocks in Fig. 2) and is
unable to maintain its lumen negativity. The
skin surface has simultaneously become posi-
tive as the epidermal cells have become posi-
tive (Fig. 4A). When the sweat gland is. no
longer able to maintain its lumen negativity
(after approximately 1400 shocks.. in Fig.
2A), the rnicroelectrode.. within the. sweat
gland duct lumen records the positive poten-
- -
tial of the surrounding epidermal_ cells.
We agree with Lloyd(6) that the slow posi-
tive component is obtained in response to a
single pulse stimulus only after a period of
rest following repetitive stimulation. The
slow positive component in response to repe-
titive stimulation of a given intensity reaches
a maximum or ceiling for that stimulus
strength and frequency. Since the positive
component runs an exceedingly long time
course(6), subsequent stimulation, either sin-
gle shock or repetitive, would not elicit the
slow positive component of the galvanic skin
response until a sufficient period of time had
elapsed for the decay of the slow positive com-
ponent. During this period of rest, reabsorp-
tion of water may be taking place in the
sweat gland ducts as Lloyd (6) has suggested.
i!
5
Po / r'epcnak
V
564 ORIGIN OF THE GALVANIC SKIN RESPONSE
However, this hypothesis does not appear to
be supported by Lloyd's data, since, as we
have shown here, the slow positive comoonent
is unrelated to sweat gland activity, If one
Must assign the term "secretory potential" to
either of the 2 components of the galvanic
skin response, it should be applied only to the
initial fast negative component since the slow
positive component originates in the cells of
the epidermis. The physiological significance
of these potentials recorded from within the
lumen of single sweat gland ducts will be re-
ported later.
In addition to the Emf generated. by,sweat
glands a�nr-,,epid-er-mal::cells described above,
4.4.4'1� -another component of the galvanic skin re-
, c suonse is the simultaneous fall in skin resis-
t
tance. Although resistance measurements
were not made during these experiments, this
report would not be complete without relat-
� ing skin resistance to the present work.
According to Lloyd(6), the slow phase of
the galvanic skin response and the impedance
(alternating current resistance) change in re-
sponse to repetitive stimulation "show an
identical course and are, therefore, considered
as signs of the same fundamental process at
work." Considering the data presented above,
it appears that the skin resistance changes re-
corded by Lloyd originate in the epidermal
cells and not in the sweat glands. Further
evidence to support this view is given by
delberg (11) who employed a micros' irgical
technic t-c7=15TivAirally and electricaily
a-773.17FreTaermis from surrounding sweat
By measurement of resistance on the
surface of such a preparation with micro-
electrodes, he demonstrated that the epider-
mis contributes significantly to the resistance
C n skin re-
sDatise. Therefore, interpretations relating
to the physiology of sweating which have been
based on the slow phase of the galvanic skin
response(6) and on skin impedance changes
(12,13,14) should be reevaluated in the light
of our report.
Summary. The galvanic skin response
from a macroelectrode covering the toe pad
of the cat was compared with simultaneously
recorded potentials arising from individual
sweat glands and cells of the surrounding epi-
dermis and dermis of the toe pad skin of the
same animal. 2,3,y direct measurernents em-
ploying microelectrode technics, the fasttneg-
ative component_ of the galvanic skin response
Vris�shown to originate in the sweat glands,- -
,and.to,be related to sweat gland activity. The--
slov.: positive component of the galvanic skin-
response _was shown to originate in the cells..
of the epidermal layer of the skin and is,.
tYler.e.fore unrelated to The authors authors gratefully acknowledge the helpful
comments and review of the manuscript by Dr. C. P.
Richter, Johns Hopkins Univ. School of Med., and
technical assistance of Miss Carmen Diaz and Miss
Ellen Hessler.
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Received May 11, 1962. P.S.E.B.M., 1962, v110.