CIO SCIENCE I SPECTRUM 1216 STATE ST RE ET POSTOFFICE BOX 3003 -SANTA BARBARA, CALI FOR NIA 93105 T E L E: P H 0 N E cio (80 5) 963-8605 A NEW TECHNIQUE FOR STUDYING MICROORGANISMS 2 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 2 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 ena e bacterial presence and concentration to be determined easily. The 2 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 an@ question regarding the study or results reported in the Application Notes, pl2ease let us know. Sincerely yours, SCIENCE SPECTRUM, INC. James E. Hawes Vice President, Marketing Enclosures Approved for Releasib 13 C) 2 7 F P'P, ', r, 7,1. ,I)ate .-WS C I EN CE I SPECTRUM 121 6 S TAT E S T R E ET POST OFFICE BOX 30.03 SANTA BARBAMA, CALIFORNIA 931C T E L E P H 0 N (805) 963-86C Jo SEMINARS 2 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, 1971 W2ashington, D. C. - Exhibit September 17, 1971 Washington, D. C. - Seminar September 21, 1971 Raleigh, North Carolina - Seminar September 24, 1971 New York, New York - Seminar September 27, 1971 Boston, Massachusetts - Semtlnar October 5, 1971 Los Angeles, California - Seminar 2 October 7, 1971 San Francisco, Calif. - Seminar (tentative) October 15, 1 971 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. 2 ------------------------------------------------------------------------ I will attend your seminar to be held on at (city) Please send my prograrr to: NAME TITLE INSTITUTION STREET CITY, STATE, ZIP TELEPHONE( 8 EXTENSION T -,,- - - @,4 -@ -,- - --' -,I - - Atlas of Light Scattering Curves. I n t r o d u c t i o n VERTICRL The interpretation of light scattering data has R 500 nm long been an obstacle to the widespread use of this powerful analytical tool. Whil2e 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 2make this computer-generated U9 159 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 2 9--- data measured by the Differential / and // light scat- .,. m TERaI.w-@t(xia tering photometers. Semitransparent vellum paper has A sample page from the Atlas showing the light scattering been used for the Atlas so that accurate comparison curves for homogeneous spheri2cal particles of 500 nm radius and four different refractive indices. of theory and experiment is achieved by merely over- laying the two sheets. Tables of normalization con- the scattering curves of spheres with radius between stants for absolute scattering power are also provided. 0.05 and 1 micrometer in steps of 02.05 micrometer, for refractive indices ranging from 1.33 to 1.59. The S i n g I e S p h e r i c a I P a r t i c I es i n A i r refractive index of the surrounding medium is that A wide variety of processes produce homogen- of air (n - 1.0). Curves for both linear polarizations aous spherical particles of 2 approximately one microm- are given. Inspection of the scattering atlas for spheri- ?ter in diameter. For example, photochemical aerosol cal particles shows that particle size can easily be )r "smog" droplets and colloidal particles like those specified to within 0.1 micrometer diameter. A de- n latex paints are spherical. Such small particles may termination of refractive index to well within 2ten per- !asily be suspended in air by nebulizing a liquid sus- cent accuracy is achieved for spheres by simply exam- )ension and their individual scattering patterns are ining the relative intensities at peak amplitudes. eadily measured with the Differential // scattering Supplements )hotometer. Periodically, additi4onal scattering curves are pub- This important class of scattering objects is corn- lished as supplement sections to the Atlas. Owners of letely described by two parameters: radius and re- the Science Spectrum Scattering Atlas will receive all -active index. The first section of the Atlas displays supplements issued within two years of the date of purchase without charge. Subjects selected for early diameter at a constant refractive index can be supplements include: the effect of size distribution vided for studies of colloidal size distributions. E6N upon scattering from suspensions of spheres; the effect this means sizing accuracy of + 10 nm diameter can upon scattering of size and size distribution changes be obtained as reported 2 by Phillips etal in the J. in model bacteria and mitochondria; scattering from Colloid Int. Sci. 34 (1970), p. 159. Scattering curves conductive particles; scattering from absorbing parti- on absorbing spheres can also be computed as needed. cles; and scattering from airborne bacteria. Measured Curves on -any spherically symmetric structure with scattering curves from known non-spheri2cal particles varying complex refractive index can be generated. may also be provided. Specific applications for specialized shell structures include bacterizf, bacterial spores, microencapsula- Computation of special scattering curve sets for tion particles, compound aerosol particles with large a wide variety of o2bjects will be done at moderate nuclei, etc. Even more varied shapes can be computed cost using proprietary Science Spectrum compu-ter exactly when the particle nearly matches the refrac- codes. The scattering from spheres of different diam- tive index of the medium in which it is immersed eters or refractive index can be computed on order. as in the case of bacteria in water. For example, curves for smal2l variations in particle 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 s2hipping 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: Purc5hasers of a Differential / or Differential photometer receive with the instrument two copies of the Atlas and all supplements for two years. Al-17-061(2) Physiological Monitoring of Bacteria and Mitochondria Introduction servatives such as phenol, formatin, or alcohols can be measured precisely4. Subtle changes in response to elevated Optical 2methods ranging from microscopy to turbidi- temperatures5 or pressures are easily determined. Size metry have long been used to monitor bacterial growth and modification and cellular damage occuring in phage-infected division. However, the optical microscope is unable to re- bacteria can be measured. The process of spore germina2tion solve features smaller than a few wavelengths of light in size. can be monitored as it proceeds. The response of chloro- Turbidimetric measurements are subject to large errors plasts to various processes including photophosphorylation because the attenuation of light is a function of the product have been followed by light scattering6-8. The sus2ceptibility of particle scattering cross section and particle density. of bacteria to various antibiotics can be measured within Since the particle scattering cross section is not in general minutes of contact9. Changes in mean cell size during syn- the same as the particle's geometrical cross section, signifi- chronous growth can be monitored with an a2ccuracy of cant interpretive problems arise. A given value of transmit- ± 20 nm. The effect of different growth media on the size tance will often correspond to several different products of distribution of cells can be seen clearly via light scattering particle density and particle size. On the other hand, differ- patterns. These measurements can be made wi2thout dis- ential light scattering measurements (i.e., recording the turbing the growth of the culture. Some details of such pattern of light scattered by such particles as a function of studies are discussed below. angle relative to the direction of the illuminating beam) are unambiguous, often yielding size and shape information of 2 much hig'her precision than obtainable with a microscope. LASER CUVETTE Under optimum conditions, cell size determinations of ± 2% 0 accuracy are achievable with differential light scattering E::@ measurements. 2 Figure 2 R The Differential I Photometer The Differential / light scattering photometer 2 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 2 Fig. 2. In use, a cuvette containing the suspension is placed in the instru- ment and illuminated by the intense monochromatic beam Figure 1 of an argon-ion laser. A spec2ially designed scanning detector system records, as a function of the scattering angle 0 relative to the beam direction, the intensity of light scat- A variety of biologically important processes can be tered by the cells. This2 differential light scattering pattern accurately studied by differential light scattering. Physical embodies a wealth of information about the cell ensemble, changes in mitochondria subjected to various enzymes, pH such as cell size, shape, structure, size distributionio,i 1, variations, and osmotic stresses can be directly moni- and 0 even structural details such as cell wall thickness and toredl,3.- Systematic distortions of bacterial cells by pre- the refractive indices of the cell wall and cytoplasml2. A final example of considerable interest concerns the radius of 432 ± 1 Onn which decreased to 403 ± 1 effects of heat killing on cell size and size distribution. In 30 minutes heating. Theaverage cell wall thicknessrem@,,-- preparing autologous staphylococcus vaccines, many labora- 2 nearly constant at 108 ± 20nm despite the heating, but th, tories use heat as a sterilization procedure. Such a treatment breadth of the size distribution increased by 15% after' supposedly does not destroy the immunogenic properties of heating. 2 vaccines and would be expected, therefore, to have little or no effect on cell walls. Figure 7 shows the changes in the References differential light scattering patterns as a function of heating times for S. epidermidis broth suspensions at 600C. ithe curves have 2 been broken at 650 and displaced relative to I . G. S. Gotterer, T. E. Thompson, and A. L. Lehninger, '.Angular light-scattering studies on isolated mitochondria," each other for visual clarity.) A subsequent analysis5 of this J. Biophysical and Biochemical Cytology 10, 15 (1961). data showed that the un-heat treated cells had an average 2. L. Packer, "Metabolic and structural states of mitochondria," 2 J. BioL Chem. 235, 242 (1960). 3. L. Packer and k. H. Golder, "Correlation of structural and metabolic changes accompanying the addition of carb2ohy- drates to Ehrlich ascites tumor cells," J. Biol. Chem, 235, 1234 (1960). 2 4. R. M. Berkman, "The effects of formaldehyde, phenol, and other alcohols on bacterial structure deduced from light scattering," Am. Soc. for Microbiol. Proceedings, May 1971. S. R. M. Berkman and P. J. Wyatt, "Differential light scattering measurements of heat treated bacteria," Appi. Microbiology 2 20, 510 (1970). 6. L. Packer, P. A. Siegenthaler, and P. S. Nobel, "Light induced - 2 high amplitude swelling of spinach chloroplasts," Biochem., 30 m' n-, 30 min., Siophys. Research Communications 18, 474 (1965). -io 7. L. Packer, "Structural 2 changes correlated vv;th photochemical 10 m@n 450,C -60 C phosphorylation in chloroplast membranes," Biochimica et 2 Biophysica Acta 75, 12 (1963). 8. L. Packer, R. H. Marchant, and Y. Mukohata, "Structural changes related to p2hotosynthetic activity in cells and 10 min.. chicroplasts," Biocl?imica et Siophysica Acta 75, 23 (1963). 60'C 9. R. M. Berkman, P. J. Wyatt, and 0. T. 2 Phillips, "Rapid detection of penicillin sensitivity in Staphylocaccusauteus," Nature 228, 458 (1970). 2 10. A. L. Koch, "Theory of the angular dependence of light Control scattered by bacteria and similar size(J biological objects," 2 J. Theoret. Siol. 18, 133 (1968). 3 min., 60'C 11. P. J. Wyatt, "Differential light scattering: a physical method 2 for identifying living bacterial cells," Applied Optics 7, 1879 (1968). 12. P. J. Wyatt, "Cell wall thickness, size distribution, refractiv2e Control index ratio, and dry weight content of living bacteria," Nature 226, 277 (1970). 2 13. Atlas of Light Scattering Curves, (Science Spectrum, Inc., Santa Barbara, California, 1971). 14. T. P. Wallace and J. P. Kratahvit, "Particle size analysis2 of polymer latices by light scattering", J. Polymer Sci. C, 25, 89 (1968). 2 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), 40 60 80 100i 120 140 16. P. J. Wyatt, "Light scattering in the microbial world," On the SCAT-TERING ANGLE Occasion of the Centennial of R,,yi2eigh Scattering Theory, Am. Chem. Soc., Sept. 1971 (to be published in J. Colloid and Interface Science). 2 17. R. J. Fiel, "Small angle scattering of bioparticles," Experi- Figure 7 mental Cell Research 59, 413 (1970). For further information Call or write the Director o2f Advanced Technology, Science Spectrum, Inc., 1216 State Street, Santa Barbara, California 93105; telephone (805) 963-8605, Ml-17-081 Rapid Assay of Bacteria in Urine I n t r o d u c t i o n LASER CUVETTE The detection of threshold concentrations of bacteria 0 in specimen solutions such as urine presents an important E2:: 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/ml (0.1 critical level) l, it would expedite enormously what is now a very time-consuming procedure. A testament to the urgency of this need is the recent work 2 D ECTOR at NASA2, whose luciferase - ATP assay to detect life on other planets is being considered for detecting bacteria Figure 2 in urine. A more direct bacterial counting capability, one which is simple, effective and rapid, is available via the 2Differential light scattering patterns can be analyzed technique of laser light scattering using a commercially theoretically using computer software already developed, available table-top instrument, the DIFFERENTIAL 1. or simply compared to previously compiled "known" scat- tering2 curves in a pattern recognition approach, analogous to fingerprint identification. An Atlas of Light Scattering DIFFERENTIAL I Photometer Curves5 is available which permits even those not previously 2 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 2 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 2number of these applications have already been carried out using the DIFFERENTIAL I instrument4,6,7,8. Urine Specimen Assays The simple task of determining concentrat2ions of bacteria does not need to utilize these analytical aids how- ever. In studies of bacterial suspensions using the Figure 1 DIFFERENTIAL I thedetection of bacterial concen2trations of 105/mi is routine. Indeed, in applications such as anti- biotic susceptibility testing, solutions are prepared at about The DIFFERENTIAL I laser light scattering photom- this concentration for optimal results. At these 2 concentra- eter is a highly versatile semi-automatic instrument designed tions and lower, the intensity of the scattered light at any to study liquid suspensions of cells with minimum altera- angle relative to the background from the liquid system-is tion of their normal environments. The instrument, shown approximately proportional to the number and densi2ty of in Fig. 1, records the intensity variation with angle, 0, of the cells, especially when the cell size distribution is narrow. scattered light which results when a cuvette of the solution Thus, calibration of the light scattering patterns in terms of under study is illuminated by a laser beam. The operation is cell concentration is straightforward. 2 shown schematically in Fig. 2. Figure 3 shows a set of light scattering recordings The variation with angle of scattered light intensity is taken on the DIFFERENTIAL I for pure distilled water detected by a speciaily-designed scanning system which and with several bacterial concentrations as indicated. The 2 records the output on a strip chart or x-y recorder, or on a detectability of these levels can be clearly seen. digital data card punch unit. In specimen solutions such as urine, appreciable back- When the size and internal structure of the illumi- ground light may be scattered from various materials other nated p2articles have dimensions approximating the wave- than the bacteria, materials such as tissue cells, granules, length of the incident light, as do bacteria, the scattered cel I debris, leukocytes, erythrocytes and various crystals. To light pattern is particularly sensitive to these particle param- gauge the magnitude of this background scattering, bacteria eters. The2 features (amplitude and angular positions of in known concentrations were added to unprocessed urine maxima and minima) in the scattering pattern give a precise and the samples examined in the DIFFERENTIAL 1. Some measure of the size, shaoe, structure, and size distrit)@ition of thP Tvni(-?] 1,;rRt'rPrinn ni8ttOIr'f ?rP il r-;' 4. It Figure 4 Figure 3 Bacteria added to urine S.aureus in water 2 2 5lo,lcc 2.3 10 'CC 2 205ec 2 @.GR D z 130 150 > 30 50 -G LE. 2 Figure 5 Clinical urine samples 7 . 105 ML. 7 . 104 ML. > 2 I.. PURE H20 0 B z 40 SCATTERING ANGLE. d"lsft 120 WITH COLIMYCIN able, as expected; even at a lower concentration of 2.8 x W 2 104/mi the pattern is emerging from the background signal. These results for untreated urine are very encouraging. Simple techniql;es such as warming, mild acid treatment, and sedimentation all of which are compatible with rapid DN 1*1 processing, and should not affect the bacteria, can remove 2 most of the background-producing material, thereby reduc- ing the background scattering levels so that bacterial con- centrations appreciably lower than 105/mi can be measured. Qualitative Studies Figures 5A through 5D show light scattering patterns, 40 SCATTERING ANGLE, degm-m 100 t2aken with the DIFFERENTIAL 1, based on four patient urine samples9. The protocol for all four was: References * 1 loopfull of urine was placed in trypti case broth and 1. R. N. Barnett, "Conference on the Medical Usefulness of Micro- incubated for six hours; biology," Amer. J. of Clinical Pathology 54 Part 11, 521 (1970). 2. G. L. Picciolo, B. N. Kelbaugh, E. W. Chappelle, A. J. Fleig, "An * 0.5 ml of the incubated solution was placed in 13.5 Automatic Luciferase Assay in Bacteria in Urine," NASA ml of distilled water in a cuvette; Goddard Repor2t X-641-71-163 (Apr. 1971). * the cuvette was placed in the DIFFERENTIAL I and 3. A. L. Koch, "Theory of the Angular Dependence of Light scanned. Scattered by Bacteria and Similar-sized Biological Objects," J. Theoret. Hiol. 18, 2133 (1968). Samples A, C, and D showed no growth when incu- 4. P. J. Wyatt, "Cell Wall Thickness, Size Distribution, Refractive bated overnight on a nutrient plate. Sample B showed the Index Ratio, and Dry Weight Content of Living Bacteria presence of growth after incubation overnight on a plate, (Staphylococcus aureus)," Nature, 2262, 227 (1970). corroborating the clear indication of bacterial cells in the 5. Atlas of Light Scattering Curves (Science Spectrum, Inc., Santa light scattering pattern (specimen curve). The effect of Barbara, California, 197 1). A flyer (Al) describing the Atlas is treatment with an antibiotic (colimycin) was also deter- available on request. 2 6. R. M. Berkman and P. J. Wyatt, "Morphological Changes in Heat- mined. As shown in Fig. 5B, simply by adding it to sample treated Staphylococcus epidermidis as Derived from Light B and then rescanning its resulting light scattering pattern8, Scattering," Applied Microbiology, 20, 510 (1970). 2 susceptibility was clearly indicated by the dramatic change 7. P. J. Wyatt, D. T. Phillips, and R. M. Berkman, "Osmotic in the pattern. This illustrates the rapid antibiotic suscepti- Sensitivity in Staphylococcus aureus Induced by Streptomycin," bility test capability of the DIFFERENTIAL 1. When (submitted). coupled with the simple yet2 sensitive capability of the B. R. M. Berkman, P. J. Wyatt and D. T. Phillips, "Rapid Detection of Penicillin Sensitivity in Staphylococcus aureus," Nature 228, instrument to measure bacterial concentrations in urine, the 458 (1970). importance of differential light scattering as a clinical tool 9. 2 Recorded by Dr. S. Pantelick, Yale-New Haven Hospital is evident. (unpublished). For further information Call or write the Director of Customer Liaison, Science Spectrum, Inc., P. 0. Box 3003, 1216 State Street, Santa Barbara, California 931 05; telephone (805) 963-8605.1 standardization of Bacterial Culture Media and Suspensions Using the Differential I I n t r o d u c t i o n By comparing data obtained with bacteria grown on different media, one can immediately determine similari- 2 In the process of growing bacteria, it is often neces- ties or differences in the growth patterns. In addition, this sary to. maintain a strict uniformity of. the cultural con- simple comparison technique can easily be extended to ditions from one day to the next. The success of a long measuring the effects of toxins, antibiotics2, temperature 3 , and often costly experiment 2is directly dependent both on chemicaIS4, irradiation, and moisture on growing bacteria. the quality and on the uniformity of the growth media. In An Atlas of Light Scattering Curvess can be used to clinical laboratories, the lack of uniformity of culture determine quickly and accurately particle parameters such media can lead to grave consequences. For example, in 2as size, refractive index, and size distribution for various antibiotic susceptibility testing, errors not only in pre- species of bacteria and similar particles. 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 obtain2ing 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 2 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. Figure 1 2 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- Differential I ture of medium when plates are poured, ambient ternpera- ture and humidity, thickness of the medium, and agp of the2 plates affect subsequent growth. The DIFFERENTIAL laser light scattering photo- The difficulties in standardizing cultural conditions meter is a highly versatile semi-automatic instrument are quite apparent. However, Science Spectrum now pro- designed to study liquid suspensions of both viable and vides an instrument to monitor 2with high reproducibility nonviable cells. The instrument, shown in Fig. 1, records and precision, variability in culture. media and cultural the directional pattern of scattered light intensity from a conditions in general. The DIFFERENTIAL / measures the bacterial suspension illuminated by a laser beam. The physical and physiological state of growing bacteria, pro- operat7ion is shown schematically in Fig. 2. viding a means to quantify variability of the growth The pattern (i.e. the intensity of scattered light as a conditions. continuous function of angle relative to the incident beam LASER CUVETTE 0 Figure 3 S. aureus 2 2.0 x 10'lmi 1.5 x 101/mi 1.0 x 10'/ml Figure 2 5.20 x 101/mi direction) is recorded by an automatic scanning detector system, the output of which is designed to drive either a cn z chart recorder or a digital data card punch unit. If the average radius of the bacteria approximates the wavelength of the laser light, 2 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 / can be used to deduce average size, size distribution6 and even general st2ructure of the particles. Measuring Relative Cell C o n c e n t r a t i o n s 50 70 90 110 SCATTERING ANGLE (DEGREES) To determine cell numbers by differential light Monitoring of Solid Media for 2 scattering, a standard set of light scattering measurements Growth Potential for different known concentrations of bacteria is used to establish control patterns. In making subsequent light Difco Tryptic Soy Agar (TSA), Nutrient Agar (NA), scattering measurements, a quick comparison to the control and Heart Infusion Agar (HIA) were 2 evaluated using S. curves allows a very accurate determination of bacterial aureus (Seattle) and the DIFFERENTIAL /. Cells removed concentrations. Differences in cell concentration of less from each plate after incubation for 8 hours at 370C were than five percent are easily recognized by this method. resuspended in water to an optical density 2(OD) of 0.38 Figure 3 shows a typical standard set of curves which (N = 650 nm). For the differential light scattering measure- were used as control patterns. In this case, Staphylococcus mervts, the cells were then diluted 1/40, corresponding to aureus (Seattle) was the test organism. The cells were spread cell counts of approximately 8 x 2 106 bacteria per mi. In evenly on Heart Infusion Agar (HIA) and after incubation addition, a set of four Tryptic Soy Agar plates, freshly pre- at 37" C for 10 hours, an aqueous suspension of cells was prepared. The initial cell concentration was 2.0 x 107 cells Figure 4 per mi; by dilution concentrations of 1.5 x 2 107, 1.0 X 107, S. aureus from fresh 106 and 5.0 x bacteria per mi were also prepared. Note Tryptic Soy Agar plates from the figure that large differences in overall scattering were produced with cells differing by only 25 percent in concentration2. 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 0would 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 6 Effects of media on growth of S. atireus D Figure 5 SIZE DISTRIBUTION EFFECTS N SCATTERING FROM @ML BACTERIAL SUVENS.aft EM M@ETER 900 1.0 - . . . . . . . 120 - 100 - C Figure 5 shows a series of computer-plotted curves 2 for cells of varying size distribution. The loss in peak defl- nition is clearly increasing with widening of the size distri- bution. The Science Spectrum Atlas of Light Scattering 2 Curvess can be referred to for excellent approximations of size and size distribution. A 20 - The scattering curves shown in Fig. 6 were obtained when S. 2aureus was grown on various solid media for 8 hr 0 ' 0 at 370 C. The plates contained (A) TSA, stored at 4 C for a 20 N so 100 120 140 160 16 days, (8) TSA2, freshly prepared, (C) NA, stored for 6 SCATTERING ANGLE, d.W- wks at 40 C, (D) HIA, 6 wks at 40C, and (F) HIA, sealed 2 pared, served as a control for variability within a single mos at 40 C. At first glance the differences in scattering medium batch. The four contro2l curves are shown in Fig. 4. appear small; however, a composite of Curves A, C, and E, Note that while the relative heights of the peaks differ shown to the right, clearly reveals some significant quanti- slightly, the four curves are almost identical in shape. (The tative and qualitative differences in three of the cultures. differences in peak height suggest that the cell concen- 2 Such differences cannot be measured with other conven- tration of each sample differs by a small percentage, an tional particle sizing or monitoring instruments. Only by error resulting from lack of sensitivity of the spectrophoto- differential scattering measurements could it be ascertained meter used to prepare the suspensions.) that, for example, the cell conce2ntration is highest for 30' 30' 30' 30' 30, 30' D F Figure 7 Endogenous growth 5 of S. aureus SCATTERING ANGLE (in 10 degree increments) Curve A and lowest for Curve C. (The OD's for all three was barely detectable. Note also how cell shrinkage, readily ,suspensions were the same. The so-called OD as determined deduced from shifts of the primary peak to larger angie% with conventional spectrophotometers depends critically (A, 8, D, E, F) can be obse2rved to various degrees for each upon the average size of the particles and is not a mono- of the six preparations. Lastly, one can deduce that cell tonic function of this average size.) Furthermore, cells size distribution had narrowed significantly in some cases producing Curve C are smaller than the others, as evidenced (e.g., curves 8, 2D, E, and F) as is evident from the signifi- by the shift of the scattering peak to higher angles. Lastly, cant sharpening of the primary peaks. the cells producing Curves A and E are similar in size, but In a set of control determinations (not shown) inocula differ in that the size distribution among the latter cells on four plates made from a 2single batch of TSA were found (E) is narrower. to change uniformly when held in water for 8 hours. Of equal significance is the ability of the DIFFER- ENTIAL / to measure physiological differences in the cells taken from these six cultures. The method is described in 2 Summary the next section. Differential light scattering measurements provide a sensitive means to measure numbers, size, size distributions, refractive index, and refractive index dist2ribution of bac- terial cultures. With the DIFFERENTIAL I laser light Endotrophic Metabolism, a measure scattering photometer the most critical applications in- of cell quality volving standardizing of culture 2media are possible and practical. In addition to its ability to monitor quantity The cultures tested directly from agar media have and quality of bacterial growth, the instrument provides a characteristics which differ not only with respect to size sophisticated means to s2tudy physical and physiological or size distribution, but also with respect to nutritional changes in growing and resting cells. makeup. The DIFFERENTIAL I can also measure these nutritional or physiological differences simply by measuring changes in light scattering among cells reincubated for R e f e r e n c e s sever2al hours in liquids having no nutritional value, such as water or certain buffer solutions. 1. A.W. Bauer, W.M. Kirby, J.C. Sherris, and M. Turck, "Anti- biotic susceptibility testing by a standardized single disc Figure 7 shows the scattering cur2ves of the same cell method." Arner. J. Clit7. Pathol. 45, 493 @ 1966) suspensions as Fig. 6 taken after changes due to endotra- apid de- phic metabolism had occured. The data of Fig. 6 are in- 2. R.M. Berkman, P.J. Wyatt, and D.T. Phillips, "R 2 tection of penici)lin sensitivity in Staphylococcus aul-ems," cluded in Fig. 7 as broken lines for the purpose of com- lvature 228, 458 (1970) parison. The solid, unretouched curves show how the 3. R.M. Berkman and P.J. Wy2att, "Differential light scattering scattering signatures changed for each of the six cultures measurements of heat treated bacteria," Appl. Microbiology when they were allowed to stand in the cuvettes contain- 20, 510 (1970) ing water for an additional 1 0 hours at 250 C. The changes 4. R.M. Berkman, "The 2 effects of formaldehyde, phenol, and other alcohols on bacterial structure deduced from light observed consisted of changed in cell numbers, average cell scattering," Am. Soc. for Microbial. Proceedings, May 1971 size and size distribution. Endotrophic growth was appar- 5. 2 Atlas of Light Scattering Curves, (Science Spectrum, fnc., ently best among cells previously grown on fresh TSA Santa Barbara, California, 1971) (Curve 8) since the scattering curve was shifted upwards 6., P.J. Wyatt, "Cell wall thickness, size distribution, refractive significantly. On the other hand, the residual growth of 2 index ratio, and dry weight content of living bacteria:' Staphylococcus aureus taken from Nutrient Agar (Curve C) Nature 226, 277 (1970) For further information Call or write the Director of Technical Liaison, Science Spectrum, Inc., P.O. Box 3003 1216 State Street, Santa Barbara, California 953105; telephone (805) 963-8605. M4-17-091 New Laser Instruments f or M icrobio lozy Demonstrated With the advent of new laser light scattering instrum tation, on display by Science Spectrum at booths 812-813. 2 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 Ic 2 powered lasers, permitting their structural characteristics a their responses to various environments and processes to quantified accurately and rapidly. 2 One of the basic laser instruments for such studies is I DIFFERENTIAL TM light scattering photometer, shown the left. Applications of this new instrument, and this n 2 approach to microbiology, demonstrated by the Company its exhibit include quantifying the effects on microorganis of pesticides and germicides, antisera, 2 and various chemicz Antibiotic susceptibility determinations also will be dem( The Differential instrument strated. for studying microbial suspensions and molecular solutions. Another basic laser instrument being demonstrated 2 Science Spectrum is the DIFFERENTIAL /ITM photome Periodic Demonstrations Scheduled for studying single microorganisms. Using this unique inst Periodic demonstrations of each instrument will be given ment, the structure of single bacteria 2and spores can be det by Company personnel at its exhibit booths 812-813. mined while the individual particles are still viable and ir Every two hours, beginning at 9:30 a.m. various applica- natural environment. tions of the DIFFERENTIAL / will be presented. These appli- Another instrument, one very important to th2e clini cations include - laboratory, is the DIFFERENTIAL //ITM. Shown below, t • bacterial growth and morphology instrument enables antibiotic susceptibilities to be determir • the effects of various alcohols on bacterial suspensions automatically and rapi2dly. It will be demonstrated periodicz; • antibiotic susceptibilities by Company personnel for the duration of the exhibit usi • accurate measurement of bacterial concentrations in viable organisms and several antibiotics. Susceptibilities will suspension 2 computed by the instrument within two minutes of sample Many other applications will be discussed. Also, Application troduction. Samples of the data card printed with the compu@ Notes will be available for inspection and discussion with Com- susceptibilities will be distributed together with brochures pany personnel. 2 scribing the instrument and test. The DIFFERENTIAL /// instrument for the automatic determination of antibiotic susceptibilitieswill 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, 2 susceptibilities of viable bacterial isolates to several antibiotics will be automatically computed by the DIFFERENTIAL IIL 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 2 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 The D2ifferential I// instrument for automatically det -b- Monday afternoon. M. W. Wolfe and D. Amsterdam mentioned susceptibilities. 'Approved f some preliminary differential light scattering measurements re- lated to their study of the "Interactions of Bacteria of Medical Date 2 7 Importance with Plant Agglut2inins", paper M 107 of Session 69 The DIFFERENTIAL III I Tuesday afternoon. The new DIFFERENTIAL /// instrument, shown abc A survey of the potential of differential light scattering rapidly and automatically determines antibiotic susceptibili- was 2 discussed in a seminar, Session 25, held Monday afternoon of microbial specimens in an entirely different arxl nc and titled "Instrumental Approaches to the Rap@d Detection manner. It will be demonstrated periodically throughout and ChAr,-,-terizitior,. r@f Partqrii". h\, r),ivi(i Am@.rpr- c@pllr p@n-4 co@lp,;,[ ?ni dete-- Differential Light Scattering Papers cont. The DIFFERENTIAL /// TM Instrument cont. dam, Kingsbrook Jewish Medical Center, Brooklyn, the speakers ing susceptibilities in a few minutes. During each demonstra- were Donald A. Glaser, University of California, Berkeley; tion, first sample suspensions of2 the bacterial isolate are pre- Henry Lubatti, University of Washington, Seattle; Norman G. pared and exposed to different antibiotics. Then in the instru- Anderson, Oak Ridge National Laboratory; Philip J. Wyatt, ment, each sample is illuminated in succession with a laser Science Spectrum, Inc., Santa Barbara; and Henry D. 2 Isenberg, beam of low power. The illuminated bacteria scatter the inci- Long Island Jewish Medical Center. An abstract of Dr. Wyatt's dent radiation, producing characteristic'light scattering patterns presentation "Applications of Differential Light Scattering in which respond in a manner corresponding to the response of the C2linical Microbiology Laboratory" follows: the cells to the antibiotic. The patterns produced by suspen- Dr. Wyatt described a powerful new approach to many of the sions incorporating antibiotics are compared in succession to problems of clinical microbiology. Noting that the size of bacteria and the pattern produced by a control 2suspension without anti- the wavelength of visible light are about the same, Dr. Wyatt pointed biotics. The degree of susceptibility indicated by pattern out thatasa consequencea variety of unusual effects are observed when- changes then is automatically computed and printed on a data ever laser light is scattered from bacteria. By carefully measuring an2d card. interpreting the manner by which such microorganisms scatter laser light, numerous microbiological phenomena may be rapidly and accur- A DIFFERENTIAL /// placement and evaluation pro- ately characterized. These light scattering techniques have thus opened gram will begin shortly after the ASM meeting in several labora- 2 the way for extensive new instrumentation that promises dramatic tories throughout the nation. The results of this evaluation changes in near future for the clinical laboratory. Dr. Wyatt described program will be made available periodically to interested instrumentation and techniques currently available. including a new and clinical laboratories. 2 revolutionary automated system (DIFFERENTIAL 111) 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 Differential Light Scattering, Briefly identificati2on and susceptibility testing of clinical specimens without When particiesare illuminated by light,theywill ingeneral the requirement for initial isolation and incubation. All the techniques described appear to be equally applicable to aerobes and anaerobes, as scatter this light in all directions. The intensity of the scattered well as the more fastidious myco2bacteria. In this la"er regard, a high- light as a function of thedirection has been termed the differen- light of his talk was the preliminary repo" that Dr. Claude Reich of tial scattered light intensity. This is illustrated schematically in Johns Hopkins (Leonard Wood Memorial) had measured an antibiotic the figure on this page. The trace adjacent the schemati2c shows effect on a species of mvcobacteria in less than 15 minutes using the such a differential light scattering pattern (measured in a plane differential light scattering technique. with respect to the direction of the incident light) for a single 2 Bacillus sphaericus spore. Comparison of this pattern with swe theory permits the unique determination of the spore's diam- ARTICAL eter and refractive index of the spore's cortex and coat. For the 2 example shown, the radius of the spore was found to be 483 + 5nm, the coat thickness 80 + 10nm, the refractive index of tt@e cortex 1.56 + 0.02 and of th2e coat 1.48 + 0.03. Your Invitation A schematic of the differential To see the instruments, and to discuss the application of light scattering measurement particular interest to you, please drop 2 by booths 812-813 at and a sample of the resulting your convenience. Company scientists will be there, and litera- tt f ngle spore ture and data folios will be available for your inspection. Also, 2 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 2 publications, please call of write the Vice President, Marketing, Science Spectrum, Inc., 1216 State Street, Post Office Box 3003, Santa Barbara,2 California 93105, telephone T 2a' w a IV 120' la' law ior (805) 963-8605. SCAMRING ANGLL PUBLICATIONS AVAILABLE FROM SCIENCE SPECTRUM Reprints 2 Light Scattering in the Microbial World, P. J. Wyatt, J. of Colloid and Differential Light Scattering., A Physical Method for Identifying Living I nterf ace Science 40 (1972) in press. Bacterial Cells, P. J. Wye". Applied Optics 7, 1879 (1968). Dielectric Structure of Spores from Differential Light Scattering, P. J. identification of Bacteria2 by Differential Light Scattering, P. J. Wyatt, Wyatt, Spores V, (1972) in press. Nature 221, 1257 (1969). Bibliographies Call Wall Thickness, Size Distribution, Refractive Index Ratio, and Dry DIFFERENTIAL I bibliography - selected reference material including k*ight Content of Living Bacteria (Stap2hylococcus aureus), P. J. Wyatt, particle suspensions and molecular studies. Nature 226, 277 (1970) DIFFERENTIAL 11 bibliography - selected reference material including Morphological Changes in Hear-treated StaphVIococcus epidermidis as single particle measurements. Derived from Light Sca2ttering, R. M. Berkman and P. J. Wyatt, Appl. Application Notes: Microbiol. 20, 510 (1970). Physiological Monitoring of Bacteria and Mitochondria Measurement of the Lorenz-Mie Scaffering of a Single Particle. Polysty- Rapid Assay of Bacteria in Urine (currently being updated) rene Latex, P2.J. Wyatt, 0. T. Phillips and R. M.Berkman, J. of Colloid Standardization of Bacterial Culture Media and Suspensions Using the and I nterf ace Science 34, 159 (1970). DIFFERENTIAL I Rapid Detection of Penicillin Sensitivity in Staph ytococcus aureus' R. M. Size Measurements of Single Microparticles Berkman, P.2 J. Wyatt and 0. T. Phillips, Nature 228, 458 (1970). Characterization of Airborne Particulates Using the DIFFERENTIAL 11 Evolution of a Light Scattering Photometer, 0. T. Phillips, BioScience The Structure of Individual Microorganisms 21, 865 (1971). Measuring Antibiotic Susceptibilities and MI2C's by Differential Light Size Distribution of Bacterial Cells, V. R. Stull, J. Bacteriol. 109, 1301 Scattering (1972), Brochures describing - Osmotic Sensitivity in Staphylococcus aureus Induced by Strepromycin, DIFFERENTIAL I instrument for studying microparticle suspensions 2 P. J. Wyatt, R. M. Berkman and D. T. Phillips, J. Bacteriol. 110 (May and molecular solutions 1972) in press. DIFFERENTIAL 11 instrument for studying individual micropar-ticies A New Instrument for the Study of Individual Aerosol PartiCleSr F) J. DIFFERENTIAL III instrument for autom5ated antibiotic susceptibility 01@IGI.N OF Tlli-.- (;ALVA-,IC 5KIN' I-,LSPONsrr. 559 were used to identify the cells which were pre- vi(le the active pht,,,ocytic population. 1).trin,,, for2 di%-isioii tiid colloi(lal !;accharated i. Kelly, L. S., I)ob-@on,, E. L.,- Finney, C. R., iron oxide was used to identify the active Hirsch, J. 1)., @t?;t. J. Phvsiol., 1960, %-19S, 1134. pha.,,,oc),tic cells. In livers of mice whose re- 2. Abercr4)iiit)ic, M., Harkness, R. D., Proc. Roy. tictilo-eii(lotlielial systeni was stit-nulated' I)y Soc., seri(,s 8, 1951, vl3S, S44. 3. Be.Lr(l, 1. W., Itous, P., 1. Exp. Alctt, 193.1, cstra(liol, it was established that the cells pi-e- parin-, for division and those which had re. v-9, -"D3- 4. linwat-d, J. G., 2Rowley, 1)., War(lltw, A. C.1 centlx, (li%,idc(l were actively pha-ocytic. In liol??:Ii?zology, 1958, VI, 181. livers of mice Nvhose reticulo-eild2othelial s@,s- 5. Bensch, K. G., Simbonis,' S., Hill, R.'B., King, teni liici been "blockaded" with saccharated D. %%'., Nal8tre, 19S9, vIS3, 47S. iron oxide, it %vis established that t2he cells 6. Kelly, L. S., Dobson,'E. L, Fed. Proc., 1961, which had phagocytized colloid were able to v2O, 265. divide in the process of recovery from "block- 7. Howard. J. G., J. Patig. Bact., 1959, v78, 465. ade." No evidence was found for a stem cell 2 8. l,ison, 1,., Smul(lers, J., Nature, 1948, vl62, 65. which proliferates and differentiates to pro- Acceived May 14, 1962. P.S.E.B.M., 1962, vIlO. Origin of the Galvanic Slcin Response.* (27579) BENJA'.1l.v A. SI-IAVER, JR.t SAUL W. BRUSILOW,- i%ND ROBRRT E. CooxE (Introduced by C. P. Richter) Departrite?tt of'Pediatrics, Johns -Hopkins U@tiversity School of Vedicijte and Ararriet Lane Monte, Johns Hopkins2 Hojpilai, Ballititore, Afd. If a metal plate electrode is placed on the skin reflex and the galvanic skin response, f sl-iii surface of the human palm, o2r on the wneii recor ed from te electrda-ds- foot or toe I)ads of the cat, and the body of Cnereatter referred to as 4'macroeiectro the subject -rounded by means 1.@'s suriace are of arotlier metal 2 plate electrode at some.point difier as would on the body at a distance from the first elec- ce in the path trode, a chaii-e in electrical potential will oc- length and conduction time of the nerve fi- cur betwee2n the 2 electrodes in response to a bers carryin- the stimulus to the skin(l). ,k stimulus transmitted to the skin by the s3,in- Various theories of ori-in of. the -alvanic There is a -simul- the -alvanic skin response 2 pathetic nervous system. skin reflex and ski taneous decrease in the n's resistance to the have been advanced at one time or another passage of ar. electric current. When the po- qince their discovery by Fere(2) and 2Tar- tential chan-e is elicited bv any p7a-i'hf -I,'- are u st r -@" chanoff (3), respectively, over 70 years ago. ing, or t reatening event in t e nvironment 1'era-uth(4), by implanting the grounded,2 h -6 or indifferent, electrode beneath-the skin sur- ot e txper,.menta subject, it is referred to rc ex. When the stiiiiu- as t e2 ,alvanic ski;,i@' face, demonstrated that the potential change f@@is applied peripherally as when a peril)h- ori-inated within the skin, rather than from eral sudomotor nerve, or the sympathetic muscle and other underlying tissues. The 2 tt:'uii@--@o the hin-d 'extr"e -mity is stimulated,. the controversy primarily centers around the pii(-Cn-tiai c@aii,,e is called the galvanic -skin cluest-.on of whether this phenomenon is rcspo;ii-c- The -%@ave 2 forms of the' galvanic caused by sweatin-, the widely accepted view. This project was supported in part by grant These theories and the experimental evidence from U. S. Public Health Service. suj)l)ortin- them have been reviewed by Wan- i Fellow of 2Nfaryland Heart Assn. Senior Research Fellow, U.S.P.H.S. Richter(S) pointed out that the skin 1)o- :ro 9 r Approve 'nato 560 ORIGIN OF THE GALVANIC SKIN Rnspo-,qsE tential tracings obtained from the palm of pipette was provided for by inclusion of a he human sub ect contain 2 components: a silver-silver chloride electrode as an inte-ral t 2 fast component, negative in direction, and a part of the electrode holder. slow component, positive in direction. He Indifferent, or ground return, electrodes of recorded the slow positive component from several kinds, placed in a variety of locations the skin of a patient with congenital absence 2 were used with uniform results. Electrode of the sweat (,lands. The fast negative corn- materials included platinum, steel, stainless ponent was absent in this patient. He con- steel, and zinc. Locations included the ab- cluded Liat the fast negative component was doininal cavity of the la2l),trotomi7ed animal,. related to sweating, but that the slow positive skin of the thigh, muscle of the thigh, the component was not, and had its or:,Iin in ton-ue, and the external car. The recorded capillary or epithelial cells of the skin. responses were not affec2ted bv the na,ure or Lloyd (6), studying the galvanic skin re. location of the indifferent electrode, unless sponse of the skin of the cat's paw pad, re- the latter was placed near the stimulatin", centlv discovered that Richter's observation electrode within the abdominal cavity. In 2 (5) OL' 2 components was valid in that species the latter case, as would be expected, consid- -ilso. crable stimulus artifact was introduced into However, Lloyd interpreted his data to indicate that the slow component was a the traci2ngs. @,weat -land "secretory, potentiaf.' that was The si- al from the recording micropipette n related to reabsorption, and to the amount of electrode was fed into a couplin- amplifier moisture in the sweat2 cland ducts. employing asin-le-encled CK-588 9 electrom- The present study was undertaken to de- eter tube. The measured electrometer tube lermine which of these conflictin- interi)reta- ,,rid current 2was 10-14 amperes. The signal tions is valid. Cats were employed as the ex- was furl,her amplified by Offner transistorized perimental iubjects. The use of micropipette voltage and power anplifiers. electrodes permitted direct measurement of The electrical resistance of the micropipette 2 the electrical potential from the lumen of in- electrodes used in these experiments ranmed dividual sweat -lands and from the epidermal from 2(> to 60 me-ohms. This resistance was and (lermal tissues surrounding them, Si. contintiouslv monitored by means of the pulse nitiltaneouslv, the galvanic skin response was injection technic, and did not sil-nificantly recorded from a macroelectrode covering the change durin.- the experiments reported here. --urface of a paw pad of the same extreiility. riie u,@ual precautions in selectin- niicropip- 2 Tticthods. In our experiments, the -alvanic t!tte electrodes with low tip potentials were skin response was studied by meastiriii- the obseev(-d. '\Te,-ative feedback was used to el,.@ctr;cal potential arisiner in the skin of the cincel out input capacitance. 2 C, paw pad of anesthetized mongrel cats in re- The microelectrode imt,-)Iantations were sponse to electrical stimulation of the Itimbar made tinder direct vision with the aid of a sviiipathetic trunk supplyin- the homolateral dissectin- microscope and a Zeiss micromani2- hin(i extremil@v. A nerve preparation similar ptilator. To obtain the -alvanic skin response to thit of Dale and Feldberg(7) was used. in the @ame animal, a small stainless steel cup The apparatus and technics employed are electrode, 1.5 cm in diameter, covered another 2 similar to those used in the microclectro(le im- toe pad of. the same paw. A layer of electro- palenient of single nerv@ fibers. Glass capil- lvte-coiitaining paste was interposed between larv tubes were rlranvn out into microl)ipettes the toe pad and the cul) electrode. The latter 2 with a pipette puller similar to the one de- electro(le was connected to an in(lependen', ,crii)c(l I)v Alexander and Na!;tuk(,I). The liirect-current differential amplifier. electrodes were filled with methanol I)v boil- Beciuse 'of its ready availabilitv and 2 con- iiig tinder v;icuum, and subsequentlv with 3- v(,nience, the electrolvte paste used in most Noriiial potissium chloride bv (lifftision re- of these experiments was that made bv tile 1)lacement of the methanol. Flectrical con- Stnborn Co. to b1e used between the qkiii ajid tact with the electrolvte solution within the aii electrocardio-raph electrode. To exclude ORIGIN OF TIIE GALVANIC SKIN RESPONSE 561 polarization phenomena at the skin Lnd in- IA) show this slow positive component, but ti different electrodes,2 the results obtaineti when the simultaneously recorded galvanic skin re- usin.- the Sanborn paste and a st-.iinILss steel sponse (Fig. 1B) frequently did. All of tl-.e macroclectrode were compared with results microelectrode responses obtained from in- obtained when using a zi2nc macroelectro(le dividual sweat -lands were identical in wav(.-- covered witli a paste composed of kaolin and form to that shown in Fig. IA. The latent a saturated aqueous solution of zinc sulphate. period varietl from 0.6 to 0.8 second, the r. The zinc-zinc sulphate mac2roelectrode was mean and median values bein- 0.7 secon(l. used in Richter's experimcnts(5,10), and is The magnitude 6f the peak ne,,ative response a combination in which polarization over the varied with stimulus intensity, the niore elec- voltage range in these experi2ments is negli- trone,@.ative responses beinl- obtained with the -ible. Under the conditions of the experi- hi-,her volta-e stimuli. The positive Com- meiits reported here, the results obt,-Lined ponent of the -alvanic skin response (Fi". witii the 2 electrode and paste comb2inations IB) was not always present in the trzcings i were identical. obtained from the macroelectrode. It ap- The output si-nals from both the amplifier I)eired mcist consiste2ntly in response to hi,-her coni.ected to the microelectrode, and tl-,e am- voltage stimuli. Its presence or al).ience ap- plifier connected to the macroelectrode cov- peared to be related to the intensity of the crin,,, the toe pad, were fed into a 2-cliannel stimulus, discussed below. 2 Offner recordin- galvanometer by melns of Repetitive sqztarcwave stimulatioi;. In the which the 2 signals could be recorded ,imul- exi@)erimcnts performed with repetitive qtimu. tancotisly. litioll, a stimulatin.- frequency of 120 per sec- A Grass stimulator supplied the stimtilatinr oii(i was employed. In response to repetitive stimulation (Fio,. 2), the microelectrode with- pulses to a platinum boot electrode placed at laparotomv around the lumbar svmpatlieti2c in a sweat -,land duct lumen held a uniform trunk supplyiii- the hind extreniity of the negativity for the first half minute and there- animal. Squarewave pulses, varyin,, from after be,-an to decay toward the base line, thresliold, usually 2 to 3 volts, tr.) 7 to 10 N,olts and finally rose to l2umen positivity by the r@ were employed. Pulse duration was i -,o 5en(! of th,@- third minute of repetitive stit-nula- microseconds. tion, whereas the galvanic skin response re- Results. Single pulse stimulation. In corded from the niacroelect-rode cov2erinr a tracings recorded simultaneously from the toe pad became positive early in thi e first miti- ruacroelectrode coverin- a toe pad and fr6m ute of stimulation after an initial negative di!- a microelectrode with 2 the duct of a sinale. flection. sweat gland of the a@djacent toe pad, the ini- f§cct of stimulus voltage on fast a?zd slow sk es The tial fast ne-ative (downward) component of components of gaivayiic in r ponte. 2 the two curves ran an almost identical course. voltage threshold necessary to elicit the slow The exponential decay of the fast component positive component was hi-her than that re- in returning toward the base line proceeded quired to elicit the fast negative component.' more rapidly in 2the case of the recording In one experiment, the latter was obtained iiiade from the macroelectrode (galvanic skin from thq macroelectrode with a squarewave response) and became positive with resl)ect pulse of 2 to 3 volts in a fresh preparation. to the restinff potential, whereas the tracing Increasing the 2stimulus to. 4 volts, the fast from ,he luiren of a single sweat gland did negative and the slow positive components not (Fil-. 1). The electrical response from were obtained in the same preparation. The over a thousand individual sweat -lanes in volta-e threshold for the slow positive corn- 20 cats2 always returned to the base line after ponent was more variable than that required a decay period of from 4 to 6 secoiids follow- to elicit the fast negative response and became in- the initial ne-ative deflection. In. n(:i in- lower with the application of frequent stimuli. staiice did the tracin-, from a microelectrode If the preparation was rested, thi?. effect was in the lumen of a single swelt gland (Fia. reversed. 'ne same threshold effect was 562 ORIGIN OF Tiir GALVANIC SKIN REspoNsE +10 4, 2 A0 lo. I sec. 0. B 2 > -IOJ E 25 see. +5 MV 2 A Om E B _5 Stimuiu; ............................. 2 on -lo- C4) i ig 1 min. FIG. 1. (A) @Microclectrode rt-qpoiise from a qiiigte swetit gland. (B) Gilv,-tnic skin rc.41)oiise ieousl-,r witli (A). from t macroclectrode coveri2ng toe p.,kti of siine extr(-tility, recorded siniult.11 A singi.e iitotiopli@isic squireiv-,tve pulse stimulus w4is :tl)plicil it the irroxv. Negative (lefleetioti is (l2o%viiAv-.ir(I in this and succee(lill., 1-@'IG. 2. (A) Mieroelectrode re,;poiisi! from ltitni:ii of a siiigle-sireat glaiid. (B) G,@ilvtt)ic skill response from a -niaci-ociectro(le cq%@eriii- toe ).t(t of s;iiiie extreniity. Relictitive stiiiiiila- tioii, 10 per see., begins at -.Lrrow'.,-ill(l coi,.tiiities to ciiil @f tr:tciii@. (A) iiid (13) recorded siiii2iil- tliieougly. FIG. 3. Mieroelectrode reipoiise frotii lumeu of a 11-@V- t@oiqtld to iiitr-.L-,trteri.-tl iiii. of iiietliiclioliiie clil2oride. FIG. 4. (A) Microclectrode rcsl)oiise fimoin epi(lei-mal cell.4 of skin of cat's pt-,v pad obtained (luring the life of tljc aiiiiiial. A similir tr:iciiia eoiil(l be (@istlinc(l for lit,, 2 .trlv one tir post-Tnor- teiii, lon@r itfter swentiiig lia(l conscil, (f3) Ifieropleetroilo r"poiit4o fron-L dermis of skin of liviitg 2 -tilittiil. Itelictitive stiiiiul-.ttiott of 10 I)er see. Nvits u@ett iii ol)t:tiiiiiit@, I)otli trticiii gm2 noted in the experiments in which repetitive that electrical responses from the Itimen oi stimulation was used. Usin,, low voltage sti- the individual sweat lands and from the skin 2 to the elec- muli in a fresh, or rested, preparation, the surface are not artifacts related tracin- from the macroelectrode became ne,,,a- tric shocl-I stimulus. intites, as Loc-a2lizat tive ,in(I r,!maine(I so for several " ion of the slow co?izpotict.-! ?f gat- reported by Richter and Wheelan(10). At vanic skin response. The superficial co@-nified a sj,2,,-htly hi-her stimulus voltage, often a layer of the epidermis was dissected awav so differential of as little as I volt, the positive ibat the 1)apillarv layer of the epithelium was component ,tppeared (cf. Fig. 2A. and Llovd exposed. A microclectrode was touched 2to (6) ). Stimulus pulse duration over I to 2 5 the surface of the exposed epithelium, but not microseconds had no appreciable effect on the in proxii-nity to a sweat gland duct orifice, and res,,)onses obtained. 2 a single pulse was applied. No ne-ative de-- Ilhari@tacologic stimulation. The micro- ilection Nvas obtained with a stimulus which electrode tracin- from a sweat gland of a rea(lilv elicited the fast ne-ative component 2 in- cat!s toe pad in response to the intra-arterial %vhen'the microelectrode was'subsequently ' I injection of methacholine chloride (via the serted into the l2umen of a sweat -,land in the abdominal aorta) is shown in Fig. 3, which sime prepared area. With the microclectrode is included to show the similaritv of the re- in contzict with the epidermal laver of the sl)onse obtained by2 repetitive stimulation to sl.@iii I)ut not near a sweat -land, r(,I)ctitive .1 .!r th-,it I)ro(Itice(I by a 4 parasympathominictic stiziiulttion identical to that used in Fi,,. 2 i drug ((:(inipare FiL-1. 2 and 3), and to indicate was applied. There was observed a slow posi- i tt ORIGIN OF TIIE (;ALVANIC RESPO.NSE 563 A tive Component of the -alva-.iic skin resrio:ise able to maintain their lumen neg.-.itivity. That Aroni the iiiztcroelectro(le. I,' the aii2imal NvaS the -,ite of this fatigue is the s%VLtt zlrci l,ille(l, visible swea.tina soon Iceased, all(i the not the su(ioniotor nerve,,i sti por q@(I fast cotill)oiietit of the -alvanic skiii resi)oiL-,e @2")eri@meiits @i-L@yhich pharmac%)Iogicstiiiiula-- could no longer be de tected. Ho Lion was tised_(I-@4% (See -Thaysen and V slow component could be obtained froiii.Ac LcLiNviiii(q)). e@7 )@i li7ii-iti@i-Nvit@h the@@iiero7el'e'ctrode for,nearly- We can account for the dzta presented in an l@jur I)os'@2-niorteni (Fig. 4A), Ion- after Fig. I and 2 if, as Richter(5) conten(led, the 75c'@s-%V'e7t ceased to function. Positive component is not reltted to the sweat To cxc'aude the possibility that the sloiv -lands, but ori.-inatei i;@7tRe-r ; -oln-str-uc-ttir'es. I)ositive component originates fron-, the sl@in Our d'a'ta substantiate Ricliter's view and Lre structures below the epidermis, a strip of epi- consistent with the following mechanism. dermis was dissected 2away leavin- tlie d(!rzilis With si@n-le shock stimulus, the f t.-r-egative as exr.osed. Sweat ,,lands in this 2art!a poured C29i@)onent ori-inates in i.he .sweat. 0 0 -glands. forth sweat when stimulated repetitively, I)ut With repetitive stimulation, the fast-com- the positive 2 conil-)onent was not recorded 1)otients fuse to become a sustained ne,,,,,itive e-ii7* "'- ol 'i'@ ' s been sho%i,n by Rieliter and wh ii@i-m-i'c-r'o-clectr'6de-wasli@n-2c .-taci-@@'L T@) t@eiitial as lia li@e exposed dermis (Fi-. 4 B). 'Whc,!ian(IO) and by Lloyd(6). 'I'his wou-Id bisclissI,on.' It Ls unlilely that curves such liso-appear to be 2 the case with a pharmacolo- as Fin-. 2A would be seen under ph-,rsiolo,,,ical aic stimulus of Ion.- duration of action as me- conditions. If a flat electrode is I)Iace(i on tliacholine chloride (Fig. 3). In resl.)onse to the skin surface of the palm, and 2aiiother on an enormous stress such as repetitive stiniula- the back of the hand of a human subject (the tion or with intra-arterial methacb-oline chlor- same obtains for the panv of the att), sl)on- ide, the sweat -land be,,ins to fatigue (after tan2cously arising ne-ative waves will b(- re- apl)rox-Imately 600 shocks in Fig. 2) and is corded which are identical to the iiiicro,.Iec- unable to maintain its lumen negativity. The trode responses from the lumen of a sin,,,Ie surface has simultaneously become 2 posi- sweat -,land of the cat. Thes@@-@esp@)nse!@-oQ- tive as the epidermal cells have become posi- cur in random fashion in the url@itiniulated tive (Fi-. 4A). "7hen the sweat aland is no subject and by their identity with the mici-o- 2 Ion- r able to maintain its lumen ne@,,,ativity electrode responses (Fig. IA), are I)resuiii- (@@@er approximately . 1400 shocks, . in - Fig. @ply the electrical potentials gener-a-t@ed by the' 2A), the rricroelectrode.- within. the. sweat ind2ividual sweat ,ands or groups of sweat -land duct lumen records the positive poten- ,I 0 I n * lal f the surrounding epidermal cells. c, ands covered by the skin electrode. I a t 0 2 restin,, patient with extreme hyperhydi-oiis 'Nve, -a --r,e-e with Lloyd (6) that the slow posi- t of the palms of the hands from whom ,;uch tive component 2 is obtained in response to a tracings were taken, the sweat gland pote;itial sitigle pulse stimulus only after a period of chan-es reci)rded from a skin electrode with rest following repetitive stimulation. The an area of 6 sq cm occurred a-s often as 100 slow positi2ve component in response to repe- per miiiute. These responses often were su- titive stimulation of a given intensitv reaches I)eriml)osed on each other because of their fi-e- a maximum or ceilina for that, stimulus quency, but in no case was fusion observed streii-th and frequency. Since the positive such as occurs with stron(r repetitive stimula- component runs an exceedingly long time tion of the luinbar sympathetic trunk t)r a course(6), subsequent stimulation, either sin- peripheral sudoiiiotor nerve(6,10). 2 On trac- -le shock or repetitive, would not elicit the in-S obtained from the resting, unstimulated slow positive component of the galvanic skin' cat or human subject, only the fast negative response until a sufficient period of time had component was seen. It appears that under elapsed for 2 the decay of the slow positive corn- the enormous stress upon the sweat -,Iinds ponent. During this period of rest, reabsorp- when repetitive stimulation is used (F!-. 2A), tion of .vater may be taking place in the the sweat glands become fatigued and are un- 7sweat gland ducts as Lloyd(6) has suggested. ORIGIN OF Tiir GALVANIC SXIN REspovsi, 564 ;ht However, th ot appear to (12,13,14) shoul(! be reevaluated 2in the lig Lg hypothesis does n be supported bv Lloyd's data, since, as we of our report. Siti?zm(irv. The galvanic skin response bive shown here, the slo v 2 r)ositive comnonent fror,-i a iii electrode coverin,, the toe pad is unrelated to sweat-(,land activity, If one acro 0 2 eouslv of the cit was compared with simultan must assi-n the term "secretory potential" to recorded potentials arising from individu@l either of the 2compone2 vanic nts of the gal skin response, it should be applied only to the sweat glands and cells of the surrounding epi- deriiiis and dermis of the toe pad skin of the 9 2 ini tial fast ne-Ittive component since the slow same animal. direct measurements em- positive component originates in the cells of 2 lov,in@ microelect;We-t ni@'cs-, thc"i@;@,neg- the epielermis. The physiological significance ative component of the ,,,alvanic skin re@,ponse of the.,.e potentials recorded from within the v to originate. in the sweat glands,-, 2- Itimeii of sin-le sweat gland ducts will be re- Che and to be related to sweat gland activitv.I ported later. SII)V.- positive component of the. galvanic skin-2- 4 In addition to the Emf gene ated,.b sweat Le@I)onse was shown to originate- in the cells bove, lylarius Ils described.a of the epide the, s in and is,,. 2 rmal lkver, of,,. k tv r an@)ther component of the Ivanic., skin re- pnrelated to sweat2ing.-.-. JAil 5 a r s lonse -is. the simultaneous fall in, skin resis- Tlic allLhors gratefully acknowled-.e the helpful tlnce. Althouch resistance 2 measurements comments and review of the mtnuscript by Dr. C. P. were not made during these experiments, tilis Richter, Johns Hopkins Univ. School of 'i%lcd., and report would not be complete without relat- te,:Iinical assistance of Miss Carmen Diaz and Miss in,- skin resistance to the present worl@. Ellen Hessler. Accordin,, to Lloyd(6), the slow phase of 1. Wang, G. I s. bfed., 19 7, ttie -alvanic skin response and the impedance 295 (Part 1) ;19SS, v37, 35 (Part 2). 2. Fere, C., Compt. Rend. Soc. Biol., 1888, VS, (alternatin,,- current resistance) chan- 2 M e in re- (ci,,,hth series), 217. sl-)onse to repetitive stimulation "show an 3. Tarchanoff, G. Pfietigers Arch. ges. Physiol., identica2l course and are, therefore, considered 1,1;90, v46, 46. as si,,iis of the same fundamental process at 4. Ver:iguth, O., Das psychogalvanishen Reflev work.-" Considerin- the data presented above, p.5enoine7z, Xarger, Berlin, 1909. it appears that the skin resistance changes re- 5. Richter, C. P., Brain, 1927, v5O, 216. corded by Lloyd ori,,inate in the epidermal 6. Lloyd, D. C. P., Proc. Nat.2 Acad. Sci., 1961, v47, cells and not in the sweat glands. Further 3@5i. evidence to support this view is -iven by 7. Dale. D. H., Feldberg, W., 1. Phvsiol.. 1(134, v82, Fdelberg(li) @@vho emplo ed a micrositrgi(7a2l 121. B. Aie%ander, J. T., Nastuk, W. L., Rev. Scient. technic to ,Sol irglig and electricai litsir., 1953, v24, 53S. 2 ermis from surrt)undinl, snat du-cts. Bv measurei 9. Thayscn, J. H., Schwartz, I. L., 1. Clin. Invest., ;ent-of resistance on the 2 195.i, v34, 1719. suriace @f such a preparation with micro- 10. Richter, C. P., Wh@clan, F., J. Neltrophysiol., electrodes, he demonstrated that the edider- 2 194-1, v6, 191. --octhe resistan mis contributes si-nificantfy -@p& iL. rd,lb,,g, R., Fed. Proc., 1961, v2O, 2 245. C -M 12. Lloyd, D. P. C., -Proc. Nat. Acad, Sci., 19S9, saunsi:@. ere ore, rpr tions relating v45, 410. 1 to the phvsiolo- of sweating which have2 been i.i. 1. Gen. Phvsiol., 1960, v43, 713. y based on the slow phase of the galvanic skin 14. Proc. Nat. Acad. Sci., 1961, v47, 3SS. respoiise(6) and on skin impedance chant,res Received May 11, 19692. P.S.E.B.M., 1962, vl iir