Experiences with the Coulter Counter in Bacteriology1

Experiences with the Coulter Counter in Bacteriology1 ELLEN M. SWANTON, WILLIAM A. CTJRBY, AND HOWARD E. LIND Sias Laboratories, Brooks Hospital, Brookline, Massachusetts Received for publication May 24, 1962 ABSTRACT SWANTON, ELLEN M. (Brooks Hospital, Brookline, Mass.), WILLIAM A. CURBY, AND HOWARD E. LIND. Experiences with the Coulter Counter in bacteriology. Appl. Microbiol. 10:480-485. 1962-Viable and killed suspensions of Staphylococcus aureus SM, Escherichia coli, and Serratia marcescens, as well as polystyrene spheres, 0.81 and 2.85 A in diameter, were counted electronically with a model A Coulter Counter. Simultaneous counts by standard bacteriological methods and microscopy were done for purposes of control and comparison with the data from the Coulter Counter. Results indicated: (i) electrical characteristics of different bacterial populations are different; (ii) electronic counts were consistent for species used; (iii) live S. aureus exhibits a denser pattern of thick bright pulses on the cathode-ray tube than does live E. coli; (iv) killed bacteria resemble inert particles in pulse pattern; and (v) some viable bacteria do not react independently of current flow, as do inert particles and killed bacteria. The object of this study was to determine the reliability as well as the limitations of an electronic counting system, the Coulter Counter, in enumerating bacteria in suspension in a fluid medium. Previous studies by Kubitschek (1958) and Lark and Lark (1960) cited the utility of the Coulter Counter in measuring the volumes of bacterial cells and volume ratios of bacterial populations and mixtures of bacteria and spores. As yet, reports with data on actual numbers of bacteria in a given population have not appeared in the literature. (During the completion of this study, such a work was published: Toennies, G., L. Iszard, N. B. Rogers, and G. D. Shockman. 1961. Cell multiplication studied with an electronic counter. J. Bacteriol. 82:857-866.) The assembled Coulter Counter includes an aspirator pump, a mercury manometer stand with aperture tube and microscope, and a unit containing the electronic components: the pulse-amplifying system, the oscilloscope, and the decade counter. Particles suspended in a 0.900% solution of sodium chloride (equivalent conductance, 97.5 mhos/cm at 22 C) are counted and sized while flowing through an aperture immersed in the sus- 1 Presented before the Laboratory Section of the American Public Health Association on Wednesday November 15, 1961, at Detroit, Mich. pension. As a particle passes through the aperture, it changes the resistance between two electrodes located on either side of the aperture. The resulting voltage pulse, proportional to the size of the particle, is amplified and counted. These pulses are visible on an oscilloscope screen. An adjustable threshold circuit permits counting of particles which reach and exceed the threshold level chosen. Counts taken at various levels provide data for plotting the range of sizes of particles in suspension. For this study, a model A Coulter Counter calibrated to count the particles in a 0.05-ml volume and equipped with an aperture tube 30 A in diameter was used. MATERIALS AND METHODS Preparation of inert material. Polystyrene spheres, 2.85 and 0.81,u in diameter, were used. They were supplied by J. W. Vanderhoff of Dow Chemical Co., Midland, Mich. and Difco Laboratories, Inc., Detroit, Mich. Prior to diluting the spheres, stock samples were placed in a shaker for 10 to 20 min to enhance even distribution and as an aid in breaking clumps. Appropriate dilutions were made in commercially prepared 0.900% saline (Abbott Laboratories, North Chicago, Ill.). Coulter Counter dilutions were made in 100-ml amounts. Simultaneously, dilutions were made in sterile water for visual counts. Before counting, all samples were shaken mechanically for at least 10 min. Preparation of organisms. Staphylococcus aureus SM, Serratia marcescens, and Escherichia coli were selected for counting on the basis of size, shape, safety, and ease of handling. Initial inocula into Brain Heart Infusion Broth were from Brain Heart Infusion Agar slants. Cultures were incubated at 37 C for varying lengths of time during the growth period. To prepare the organisms for counting, the broth cultures were centrifuged at 1,500 X g for 25 min, and the supernatant fluid was discarded. This procedure was repeated for two 0.900 % saline washes and the organisms were suspended in 10 ml of saline. Serial dilutions from 102 to 106 were made for standard plate counts and from 102 to 104 in duplicate 100-ml amounts for electronic counting. That dilution was selected which would yield counts ranging between 6 X 103 and 6 X 104 particles per ml per sec. One set of each dilution was incubated in a 56 C water bath for up to 2 hr to kill the bacteria. Killing was checked by plating in NIH Agar Medium. Specimens were shaken in the same way as the latex spheres. 480

1962] USE OF THE COULTER COUNTER 481 Analysis of counting procedures. The determination of total solids content (densitometric analysis) for the latex spheres (Difco) was made after appropriate dilution, as another check of the inert particle count. To examine visually and to count both latex spheres and the bacteria by microscopy, preparations of appropriate dilutions were used. Ten lambda samples were dropped and spread onto a 1-cm2 area and allowed to dry. The bacterial slides were heat-fixed and gram-stained. The latex spheres were examined unstained using phasecontrast microscopy. The dry-count method was not satisfactory for counting organisms in all phases of growth and was therefore discarded. Latex spheres were also counted in a hemacytometer. The lack of reliability of the hemacytometer counts concurs with that reported by Mattern, Brackett, and Olson (1957). Since differences were found to occur in the comparison of predicted counts with those obtained from the optical dry count, a counting method was developed which gave a measure of the population distribution of particles in the suspension to be evaluated by the electronic counter. The method is similar to that described in the optical dry method except that the counts are made of all particles in a field of a hanging drop. The assumption that, for motile organisms, the number leaving a random field will equal the number entering the field of count seems to be valid from consideration of data thus far obtained. The depth of focus for the microscope system used for counting particles in this study was 24 IA. Making the assumption that a hanging drop will assume a cross-sectional lower edge approximating a catenary, it was calculated that a 2-lambda drop having a diameter of 3 mm for its upper surface, using a Simpson's rule integration, would contain 844 field per drop. By use of drops of 2-lambda and optically measuring their upper diameter to be sure that drops 3-mm in diameter alone were counted, the standard error of the arithmetic mean was found to be i 7.7 particles about a field mean of 13.6 for 8 to 20 field samples. All electronic counts were made on a model A Coulter Counter set for maximal gain. Checks were made to determine the circuit characteristics of the counter following the procedures indicated by the manufacturer (Coulter Electronics Inc., Chicago, Ill.). For each specimen, the method of preparation for electronic counting was the same. Particles studied were less than 1,A in diameter and a 30-,u orifice was used with aperture current settings of 4, 5, and 6. Background noise was kept below 100 for all counts quoted, and threshold settings of less than three were not used. Counts were recorded from the lowest possible threshold above the noise level (threshold setting 4) and increased in increments of 1 unit to the threshold level where the counts dropped markedly (threshold setting 15). Any fall-off in count as a function of time was noted; however, none was seen in the case of particles less than 1,A diameter. The count distribution obtained with the Coulter Counter was compared with the results obtained by the other counting methods. Because of the size and shape of the particle studied, the data relating to the electronic counter are given with no corrections made for coincidence. Bacterial populations were also counted by standard plate methods. Depending upon the age of the culture, dilutions of from 103 to 106 were plated in NIH Agar Medium. After incubation at 37 C, the counts were made with the aid of a Quebec Colony Counter. Size and shape of organisms were determined with the aid of a calibrated ocular disc. RESULTS The results of our preliminary investigations are summed up in tables comparing counts from different procedures and TABLE 1. Comparison of counting methods and results from inert latex beads* Counting method Total Particle count per ml pgut Optical dry 3.18 X 10' 1.78 X 109 Optical wet 3.29 X 109 2.64 X 109 Densitometric 2.80-3.74 X 109 Coulter electronic (un- 2.75 X 109 corrected) * Latex spheres (0.81,u) follow a normal frequency distribution with respect to number of spheres in groups (1, 2, 3, 4, and more than 4) in the population. t For inert spheres, the pgu (pulse-generating units) count varies from the Coulter count by 4% for the same fluid medium. TABLE 2. Comparison of counting methods and results from Staphylococcus aureus SM* Counting method Particle count per ml Total pgut Optical wet 5.9 X 109 2.0 X 109 Standard plate 1.6 X 109 Coulter electronic (uncorrected) 0.9 X 109 * S. aureus (4-hr culture, 0.8,u) does not follow a normal frequency distribution with respect to grouping (1, 2, 3, and 4 organisms per grouip) in the population. t Pulse-generating units. TABLE 3. Comparison of counting methods and results from Serratia marcescens* Counting method Particle count per ml Total pgut Optical wet 1.8 X 109 1.6 X 109 Standard plate 2.2 X 109 Coulter electronic (uncorrected) 0.9 X 109 * S. marcescens (4-hr culture, 0.5 X 1.0 A) follows a normal frequency distribution with respect to the grouping (1, 2, 3, and 4 organisms per group) in the population. t Pulse-generating units.

482 SWANTON, CURBY, AND LIND [VOL. 10 figures showing displayed pulses from inert particles and representative microorganisms. Table 1 shows results from counts of inert latex beads. Particle count (total) represents the total number of single spheres enumerated; pulse generating unit (pgu) count is the number of spheres counted either singly or as clumps. The latter count should agree with the Coulter count. This relationship between optical wet pgu and Coulter counts was observed consistently when using inert particles. For each counting procedure, results obtained from at least ten independent experiments varied within the following limits: the mean variation for the Coulter count was :1: 1 %; for the plate count, ±t5 %; for the optical wet count, 4t7 %. The number of spheres in groups of 1, 2, 3, 4, and greater than 4 was counted and plotted on normal probability paper as an index for complete mixing in the case of latex particles and to determine the degree of deviation from a chance distribution of particle aggregates existing in the actively growing microorganism populations. Table 2 shows a comparison of counting methods and results for S. aureus SM. A 4-hr culture was used for these data. The pgu count from the wet count does not agree with the Coulter count as closely as did the count with inert latex beads. The difference, however, is consistent with this organism. The differential is felt to be due to the effect of electric and surface characteristics peculiar to this organism. The closer agreement observed between standard plate count and Coulter count was encouraging. FIG. 1. Comparison of cathode-ray tube display obtained with inert latex particles (top), 1,u in diameter, and heat-killed (70 C, 1 hr) Staphylococcus aureus (bottom).

USE OF THE COULTER COUNTER 483 With S. marcescenrs (Table 3), one is not dealing with a sphere but a cylinder. Again, the variations between the Coulter count and the optical count, as well as the standard plate count, are consistent with this species. In addition to the counting characteristics, we observed that the pulses displayed on the cathode-ray tube of the counter were unique in some respects with regard to the condition, type, and viability of the particulate material being counted. After heat-killing (70 C for 1 to 1.25 hr), the pulse pattern displayed by any microorganism tested was seen to be similar to that obtained with inert particles of the same size and concentration. Further, viable populations, populations mixed with live and dead organisms, and nonviable populations display different pulse patterns. Figures 1 through 3 show representative patterns photographed using a camera synchronized to record one sweep of the electron beam on the Coulter Counter cathode-ray tube. Figure 1 compares the cathode-ray tube display obtained with inert latex particles and killed S. aureus. From Fig. 2, a comparison of oscilloscope tracings obtained from a 4-hr culture of S. aureus and E. coli may be made. Figure 3 demonstrates the oscilloscope pattern obtained with the same samples but incubated for 18 hr. For similar population densities, live S. aureus usually exhibited a denser pattern on the cathode-ray tube than did E. coli. Electrical properties, cellular elasticity, as well as the morphology of the organism, may be among the FIG. 2. Comparison of oscilloscope tracings from 4-hr cultures of Staphylococcus aureus (top) and Escherichia coli (bottom). In both cultures, organisms were 1,u in diameter in 1O4 dilutions.

484 SWANTON, CURBY, AND LIND [VOL. 10 factors contributing to this effect. At this time we have no further definitive information on this phenomenon. It seems that the patterns displayed are consistent for the species tested. Whether this will hold true among given species of each genus is still to be determined. DISCUSSION It appears that the use of the Coulter Counter in the analysis of dynamic bacteriology is appropriate and worthwhile. The necessity for the analysis of natural phenomena without changing the established equilibrium in a definite fluid environment suggests that an electronic counter may well be the instrument of choice. However, background noise arising from the circuitry, from the current generated at the aperture, and outside interference, must be taken into consideration. Because the electric and surface characteristics of individual species of organisms may be different, a profile must be made for each organism under study. Such data should be obtained before attempting to assay quantitatively from body fluids. Certain factors affect the motility of the organisms, their shape, electrical characteristics, conductive mass, and surface properties. We have seen changes in pulse pattern occur with changes in aperture-current setting when studying live S. aureus, but not with inert particles or with heat-killed S. aureus. This suggests that some viable bacteria do not react independently of current flow across the aperture of FIG. 3. Oscilloscope pattern from Staphylococcus aureus (top) and Escherichia coli (bottom) cultures of Fig. 2 after 18-hr incubation period. S. aureus, 1,u diameter, 104 dilution; E. coli, 1 X 0.6 u diameters, 104 dilution.

1962] USE OF THE COULTER COUNTER 485 an electronic counter, whereas inert latex particles and heat-killed cells show a reaction which is independent of current. This observation indicates that a technique may be developed to assess the viability of part of whole bacterial populations. These preliminary results show that: (i) the Coulter Counter is reliable within definite limits in the counting of a bacterial population, (ii) the Coulter Counter may indicate the degree of viability of a part of a whole bacterial population, and (iii) electrical characteristics of different bacterial populations are different. LITERATURE CITED KUBITSCHEK, H. W. 1958. Electronic counting and sizing of bacteria. Nature 182:234-235. LARK, K. G., AND C. LARK. 1960. Changes during the division cycle in bacterial cell wall synthesis, volume, and ability to concentrate amino acids. Biochim. et Biophys. Acta 43:520-530. MATTERN, C. F., F. S. BRACKETT, AND B. J. OLSON. 1957. Determination of number and size particles by electronic gating: Blood cells. J. Appl. Physiol. 10:56-70. COULTER ELECTRONICS COMPANY. Theory of the Coulter Counter. Chicago.