Evaluation of Culture Media for the Isolation of Salmonellae from Feces

Transcription

1 APPuED MICROBIOLOGY, Oct. 1969, p Copyright 1969 American Society for Microbiology Vol. 18, No. 4 Printed in U.S.A. Evaluation of Culture Media for the Isolation of Salmonellae from Feces R. M. SHARMA1 AND R. A. PACKER Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa Received for publication 12 June 1969 A study conducted on 300 fecal samples from a cow and a pig, each artificially contaminated with approximately four Salmonella organisms revealed that, of the three enrichment broths used in conjunction with the three selective media, the maximum number of isolations were obtained with Brilliant Green MacConkey broth (BGMB), followed by those obtained with tetrathionate (TTB), and the least with selenite broth. The combination of BGMB with Brilliant Green-neutral red-lactose agar (BGNRLA), and lttb with desoxycholate citrate agar (DCA) gave an equal number of isolations. Of the three selective media used in conjunction with the three enrichment broths, the maximum number of recoveries were obtained on BGNRLA, followed by those on DCA, and least number of isolations on bismuth sulfite agar (BSA). The combination of selenite F broth-bsa appeared to be somewhat inhibitory for the growth of Salmonella organisms. Of the two selective media combinations, the DCA-BGNRLA combination yielded the highest number of isolations. The use of all three selective media gave still better results. Selenite and tetrathionate broths were found unsuitable for isolating Salmonella choleraesuis from feces. BGMB, containing 100 jsg of Brilliant Green per ml, proved to be a useful enrichment medium for the isolation of this organism from cow and pig fecal samples, each inoculated with 16 organisms. Observations recorded seemed to suggest that contaminant bacteria perhaps outgrow and mask S. choleraesuis. An incubation period of 24 to 30 hr was found optimum for the three enrichment broths. A longer period was detrimental in the case of TTFB but not with selenite broth and BGMB. The isolation of salmonellae from morbid material is often necessary to establish a diagnosis but it involves rather detailed laboratory technology, especially when isolation is attempted from contaminated materials, such as feces, sewage, etc. A number of enrichment and selective media have been used by various workers for the isolation of Salmonella organisms from organs and feces, and various claims have been made for the superiority of one or the other medium. In view of the diversity of opinion existing in the literature on the subject, it was considered desirable to carry out controlled trials with commonly employed enrichment broths and selective media to evaluate their comparative efficiency for the isolation of salmonellae from feces. Leifson (17) developed selenite broth and reported that, used in conjunction with desoxycholate citrate agar (DCA), it was very effective for isolation of typhoid bacilli from feces, sewage, I Permanent address: College of Veterinary Medicine, Punjab Agricultural University, Hissar, Haryana, India. or water. Edwards and Ewing (5) mentioned that tetrathionate (TFB) broth of Mueller (21) as modified by Kauffmann (13) was a useful enrichment medium for the isolation of salmonellae. The efficacy of this medium was confirmed by the results of Kauffmann (14). Knox, Gell, and Pollock (16) reported that a medium containing balanced tetrathionate provided optimal selectivity Ṁany persons prefer selenite broth for general use because Salmonella typhi usually is inhibited when tetrathionate broth is used in conjunction with Brilliant Green agar (5). Hobbs and Allison (12) and Cook, Friesby, and Jebb (2) found selenite superior to tetrathionate in the isolation of S. paratyphi B. Smith (23) reported that selenite broth and TTB were greatly superior to other enrichment media. He added that selenite medium was preferable to tetrathionate for examining cow and chicken feces but the reverse was true in the case of dog feces. Banwart and Ayres (1) found that, though TTB supported the growth of most of the species of Salmonella studied by them, it was 589

2 590 SHARMA AND PACKER APPL. MICROBIOL. definitely inhibitory to S. paratyphi. Selenite-F broth appeared to be one of the better broths; however, during the initial incubation period, inhibition and actual destruction of S. anatis was statistically significant. Smith (24) reported that it was necessary to use Brilliant Green-MacConkey broth (BGMB) for the isolation of S. choleraesuis since the organism failed to develop in selenite broth. Slavin (22) found Brilliant Green-neutral red-lactose agar quite useful to isolate Salmonella strains, mostly S. suipestifer on direct culture, from artificially infected pig feces. Mackie and McCartney (18) indicated that best results were obtained by employing two or three different methods simultaneously, since this gave a higher percentage of positive results than when one method only was used. Brilliant Green agar is recommended as a useful medium for the isolation of salmonellae. Banwart and Ayres (1) reported that Brilliant Green agar supported more luxuriant growth of various species of Salmonella than did the other media tested. Bismuth sulfite agar was found to be significantly inhibitory to four of the six species of Salmonella used in their study. Thomson (25) mentioned that Brilliant Green-MacConkey agar was the best for the isolation of S. paratyphi B. Harvey (9) reported that Brilliant Green-Mac- Conkey agar was found superior to DCA and Tabet's modification, irrespective of the serotype of Salmonella present, with the exception of S. typhi. Hoag and Rogers (11) reported that S. typhimurium in mice could be isolated more frequently by tetrathionate enrichment used in conjunction with Brilliant Green agar than on Brilliant Green agar alone. Guinee and Kampelmacher (8) found that Mueller-Kauffman's medium and selenite Brilliant Green broth detected equal numbers of Salmonella in infected porcine feces and skin scrapings; the number of positive findings was increased by using both media simultaneously. Heidrich (10) reported that there was no appreciable difference between TTB and selenite broth with regard to the isolation of salmonellae from fecal and organ samples. MATERIALS AND METHODS Enrichment broths. The following enrichment broths were used: (i) selenite F broth, Leifson (17); (ii) TTB, Mueller (21) and Kauffmann (14); and (iii) BGMB. BGMB consisted of peptone, 20 g, mannitol 10 g, Oxgall, 5 g; bromocresol purple, 0.01 g, Brilliant Green (0.1%), 100 ml; and water, 1,000 ml. The above ingredients were mixed thoroughly in water and heated to boiling (100 C). The medium was dispensed in 12- ml quantities in tubes, and autoclaved at 121 C for 12 min. Selective media. The following selective media were used: (i) DCA (Difco), (ii) Bismuth sulfite agar (BSA, Difco), (iii) Brilliant Green-neutral red-lactose agar (BGNRLA). BGNRLA consisted of Proteose Peptone No. 3, 10 g; beef extract, 3 g, lactose, 10 g; sodium chloride, 5 g; saccharose, 5 g; agar 20 g, neutral red; 0.1 g; Brilliant Green (0.1%), 33.3 ml; and water, 1,000 ml. The above ingredients were added to water and mixed thoroughly to obtain a uniform suspension. The mixture was heated to boiling (100 C), autoclaved for 15 min at 121 C, and poured into plates. The culture media were stored in the refrigerator and were used within 48 hr of their preparation. Salmonella cultures. Stock cultures of S. anatum, S. typhimurium, S. newport, and S. choleraesuis, maintained in the Department of Veterinary Microbiology and Preventive Medicine of the University, were used in the study. They had been typed at the Animal Health Division, National Animal Disease Laboratory, Ames, Iowa. Actively motile 6-hr tryptose broth cultures of the above organisms were used. Estimation of number of Salmonella. Serial 10-fold dilutions of the broth cultures were made in sterile distilled water containing 0.1% bovine albumin, and bacterial counts were made on nutrient agar by the method of Miles and Misra (19). Bovine albumin was used, as it was found by Smith (23) to be the best for maintaining small numbers of salmonellae without any apparent decrease in the viable count. The cultures were diluted, if necessary, to contain the requisite number of organisms needed for adding into the fecal samples. The counts were made on them every time they were used for inoculating the fecal samples. Method of testing. Specimens of feces were obtained from a healthy cow and a healthy pig maintained in the animal units of the University. The feces of these animals were examined for Salmonella infection before including them in the study. The fecal samples were collected from the same animals throughout the study to avoid individual differences. The feces were diluted with sufficient sterile distilled water to impart a liquid consistency to them. Feces (1 ml volume) was added to each of the three enrichment broth tubes. Four fecal samples were examined on each occasion. Each specimen was inoculated separately with each of the four Salmonella serotypes. A dilution of the broth culture that contained the desired number of Salmonella organisms was added to each tube. The enrichment broths were incubated for 24 hr at 37 C and then streaked onto plates of selective media, with a 7-mm platinum loop. The plates were incubated for 24 hr at 37 C. Those plates which did not show growth of salmonellae at the end of 24 hr were incubated for a further period of 24 hr. Colonies resembling Salmonella were tested by slide agglutination with anti-o-serum of the particular species of Salmonella with which the enrichment broth had been inoculated. Approximately four salmonellae were added to each tube of enrichment broth. In case of S. choleraesuis, the number had to be raised to 16 because no growth occurred with 4 and 8 organisms added to the tubes.

3 VOL. 18, 1969 CULTURE MEDIA EVALUATION FOR SALMONELLAE 591 RESULTS Four Salmonella serotypes were included in the study. Observations recorded in respect to S. choleraesuis revealed that this organism could not be recovered through TTB and selenite F broth but was recovered by means of BGMB. In view of this variation and the difference in the number of organisms added, it is considered appropriate to present results in respect to S. anatum, S. typhimurium, and S. newport separately from S. choleraesuis to facilitate correct interpretation of results. The recovery rates of the three organisms from 100 fecal samples (76 of cow and 24 of pig), each ofwhich had been inoculated with four organisms, by means of ITTB, selenite F broth, and BGMB, are given in Table 1. Enrichment broths. With a view to studying the efficiency of the three enrichment broths used in conjunction with the three selective media, the positive results obtained for the three organisms were pooled (Table 2). It was observed that the maximum number of isolations were obtained by means of BGMB, 647 against 645 with TTB and 612 with selenite F broth. By using TTB with the three selective media, the maximum number of recoveries were obtained on DCA, followed by those on TABLE 1. Comparative efficiency of recovery of three Salmonella serotypes from feces through enrichment broths in conjunction with three selective media Serotype No. of samples examined Maximum number' positive by TTB SFBb BGMB S. anatum S. typhimurium S. newport a Statistical analysis of the data revealed that the above differences were not significant (Table 4). b Selenite F broth. BGNRLA, and the least with BSA, the three values being 223, 216, and 206, respectively. The corresponding values for selenite F broth were 214, 217, and 181. The combination of selenite F broth with BSA gave the least number of isolations. The three values with BGMB were 218, 223, and 206. The combinations of BGMB with BG- NRLA and TTB with DCA gave equal numbers (223) of isolations. Statistical analysis showed that the difference between broths was significant at the 5 % level (Table 4). Selective media. The data in Table 2, when analyzed to evaluate the efficiency of the individual selective media in relation to the three enrichment broths, revealed 656 isolations on BGNRLA, 655 on DCA, and 593 on BSA. The factor responsible for a decrease in the number of isolations on BSA was the selenite F broth-bsa combination, which isolated 181 cultures against an average of of that group. The differences between selective media, on statistical analysis, were found to be highly significant at the 1% level (Table 4). Selective media combinations. The effect of using two or more selective media on the rate of recovery of salmonellae from inoculated fecal samples was studied on 300 samples. Table 3 shows the number of isolations when two or more selective media were used in conjunction with one or more enrichment broths. The observations obtained indicated that of the three enrichment broths used in conjunction with three two-selective media combinations, TTB and BGMB gave almost equal numbers of isolations, the average being and , followed by an average of isolations obtained by means of selenite F broth. The data analyzed in relation to the selective media used in conjunction with the three enrichment broths revealed that, of the two-media combinations, DCA-BGNRLA yielded highest number of recoveries, the average being against for DCA-BSA, and for BSA- BGNRLA. The use of three selective media, however, gave still better results, the corresponding value being The inhibitory effect of SFB- TABLE 2. Total number of recoveries of S. anatum, S. typhimurium, and S. newport from artificially contaminated fecal samples Number positive by Species of animal No. of samplesttb examined SFB BGMB DCA BSA BG.NRLA DCA BSA BGNRLA DCA BSA BGNRLA Cow Pig Total

4 U~~~~~~~~~~z 592 SHARMA AND PACKER APPL. MICROBIOL. TABLE 3. Rate of recovery of salmonellae from feces with three enrichment broths in conjunction with two or more selective media Number positive TTB Selenite broth BGMB io. of Serotype samples - examined 04 T O < O 4) C < O 4) CO. 4)f z mz z m X UU QA U Up C U UP n < ~~~~~~~~C S. anatum S. typhimurium S. newport TABLE 4. Statistical analysis of data presented in Tables I and 2 Analysis of variancevariates due to A-Days B-Organisms C-Enrichment broths D-Selective media Interactions BX C BX D CX D B X C X D Error Degrees of freedom Mean square F BSA combination on the growth of salmonellae adversely affected the results in the related combinations mentioned above. Isolation of S. choleraesuis from feces. The evaluation of the efficiency of the commonly used enrichment and selective media was initiated with four Salmonella serotypes, but it was observed that the addition of four and eight S. choleraesuis organisms did not promote growth in TTB and SFB. The number of organisms in the inoculum was increased to 16, 32, 64, 100, 1,000, 2,000, and 3,000, but still no growth could be obtained on any of the three selective media. Using BGMB containing a 200,gg/ml concentration of Brilliant Green, it was found the dye inhibited the growth of the organism even when 160 organisms had been added to the broth. The addition of 1,600 organisms to the BGMB, however, permitted recovery of S. choleraesuis. Trials were carried out to find a suitable concentration of Brilliant Green which could inhibit the growth of contaminants but promote growth of S. choleraesuis. It was observed that Brilliant Green used in a concentration of 100 Ag/ml in MacConkey broth did not TABLE 5. Rate of recovery of S. choleraesuis from artificially infected fecal samples through BGMB when plated on three selective media Species of animal samples No. positive by BGMB No. of DCA BSA BGNRLAa Cow Pig Total Use of BGNRLA gave the maximum number of isolations, 57 out of 72. inhibit the growth of S. choleraesuis when 16 organisms had been added to it. It was, therefore, decided to inoculate the feces with 16 S. choleraesuis instead of 4 organisms. Seventy-two fecal samples (48 cow and 24 pig), each of which had been inoculated with 16 organisms, were examined. TTB and selenite F broth did not promote the growth of the organism. The results obtained with BGMB used in conjunction with the three selective media are, therefore, reported (Table 5). Effect of autoclaving the feces before infection on rate of recovery. In this study, the feces were autoclaved for 15 min at 120 C before artificial infection with S. choleraesuis to study the rate of recovery of the organisms as compared to that obtained when the feces were not autoclaved. The 72 fecal samples (48 cow and 24 pig) were examined. TTB and selenite F broth did not promote the growth of the organism. The results obtained with BGMB are given in Table 6. It was observed that autoclaving the feces before infection helped to recover S. choleraesuis from 71 out of 72 samples. With nonautoclaved feces, the maximum recovery rate was 57 out of 72 specimens. Effect of duration of incubation of enrichment broths on the isolation of salmonellae from feces

5 VOL. 18, 1969 CULTURE MEDIA EVALUATION FOR SALMONELLAE 593 contaminated with small numbers of organisms. To study the effect of duration of incubation of enrichment broths inoculated with salmonella contaminated feces on the isolation of organisms from them, 10 tubes each of TTB, SFB, and BGMB were used. Of liquid feces from the cow, 1 ml was added to each of the 30 tubes of enrichment broths. They were then inoculated with 0.02 ml of suitably diluted Salmonella culture, containing approximately 15 S. anatum. Similarly, a second set of enrichment broth tubes containing feces were inoculated, each with approximately 15 S. typhimurium, and the third set of broth tubes were inoculated, each with approximately 15 S. newport organisms. The broths were incubated at 37 C and, after 0, 6, 9, 12, 15, 18, 24, 30, 36, and 48 hr, were subcultured on plates of DCA. Salmonellae were not recovered from any of the specimens before 12 hr. The optimal time of incubation in the case of TTB was between 24 and 30 hr, when Salmonellae were recovered from 28 of the 30 specimens. The efficiency of this medium then decreased. The 25 specimens proved positive at 36 hr, and Salmonellae could be recovered from only 21 out of 30 specimens at the end of 48 hr of incubation. In the case of selenite F broth and BGMB, the maximum number of 28 positive isolations were obtained at 24 through 48 hr. TABLE 6. Rate of recovery of S. choleraesuis from autoclaved fecal samples, artificially infected, through BGMB when plated on three selective media Species of animal No. of samples Number positive by BGMB DCA BSA BGNRLA Cow Pig Total TABLE 7. Effect of duration of incubation of enrichment broths on the recovery of salmonellae from infected feces Duration of No. of fecal samples out of 30 positive by incubation in hours TTB Selenite F broth BGMB There was no falling off of efficiency at 36 or 48 hr, as was noticed in the case of TTB (Table 7). DISCUSSION This study indicated that, of the three enrichment broths used in conjunction with the three selective media, BGMB gave the maximal number of isolations of salmonellae, TTB was next in order of efficiency, and the least number of isolations were obtained by means of selenite F broth. The analysis of data on the efficiency of the three selective media in relation to the three enrichment broths revealed maximal isolations on BGNRLA, followed by those on DCA, and least on BSA. The comparatively low results obtained with SFB appeared to be due to the selenite F broth-bsa combination, which resulted in only 181 positives against an average of isolations obtained by means of selenite F broth used in conjunction with the other two selective media, and an average of of TTB, and of BGMB, each used in conjunction with the three selective media. The observation that selenite F broth-bsa combination exercised some inhibitory effect on the growth of salmonellae is in conformity with the findings of Smith (23), Hobbs and Allison (12) on S. typhi, Cook et al. (2) on S. paratyphi B., and Banwart and Ayres (1). The results obtained in this study with approximately four salmonella cells added to the enrichment broths revealed a lesser number of isolations by means of SFB on DCA, as compared to TTB and BGMB on DCA. It resulted in one more isolation on BGNRLA in comparison to TTB. The inoculation of approximately 15 organisms into the three enrichment broths revealed an equal number of isolations, when cultured on DCA, at the end of 24 hr of incubation. This seems to indicate that perhaps selenite F broth exercises some inhibitory effect when the feces are minimally contaminated. Banwart and Ayres (1) reported that SFB caused a decrease in numbers of viable cells during the initial period. They added that it was particularly undesirable in the case of samples with low counts of salmonellae since destruction during the lag phase might result in failure to isolate organisms of this genus. The selective inhibitory action of sodium selenite on S. thompson was considered by Weiss (Ph.D. Thesis, Iowa State University, Ames, Iowa, 1964) to be a function of the rate of selenite uptake. It was observed in the present study that selenite F broth seemed to score over TTB in that there was no falling off of efficiency during incubation for 36 and 48 hr, whereas a decrease in the efficiency of TTB was noticed beyond 30

6 594 SHARMA AND PACKER APPL. MICROBIOL. hr of incubation. This is consistent with the results obtained by Smith (23). The observations recorded in this study indicate that, of the two media combinations, DCA- BGNRLA yielded the highest number of recoveries, the average being out of 300 known positive samples. The use of all three selective media, however, gave still better results, the corresponding value being This is in conformity with the findings already reported (2, 5, 8, 15, 18). The significant result obtained is the isolation of S. choleraesuis from feces by means of enrichment through BGMB. Smith (23) reported that, for the isolation of S. choleraesuis, direct culture on Brilliant Green agar was superior to the use of enrichment media. He added that it was necessary to use an inoculum of 30,000 S. choleraesuis in TIB and liquid desoxycholate citrate medium to recover the organisms in 55 to 75% of the specimens. With an inoculum of 3,000 organisms, the recovery rate was markedly reduced. Edwards, Bruner, and Moran (4) mentioned that the inability of S. choleraesuis to grow in the more modem enrichment media was a serious handicap to the study of the epidemiology of disease caused by this organism and that this might account for some of the observations that "healthy" fecal excretors of this organism were rare. Slavin (22) and Gitter (7) used BGNRLA to isolate Salmonella strains, mostly S. suipestifer, on direct culture from artificially infected pig feces. Slavin (22), however, remarked that in spite of the high efficiency of the Brilliant Green agar, it must be allowed that a direct method was inferior to a good enrichment method in view of the fact that in naturally infected feces salmonellae were probably present in most cases in small numbers. The results obtained in this study, with BGMB in conjunction with the three selective media, revealed that it gave the maximum number of isolations of S. choleraesuis on BGNRLA, 57 out of 72 specimens. The use of Brilliant Green in the enrichment and selective media for the isolation of salmonellae is often advocated (1, 3, 6, 9, 24, 25). The concentration of Brilliant Green suitable for the purpose was found to be 100 Ag/ml; the higher concentration of 200 Iug/ml was noticed to be inhibitory for the organism, since Brilliant Green did not permit growth in the broth tubes containing 160 organisms but did permit growth in the broth tubes with 1,600 salmonellae. A concentration of 30 pg/ml of Brilliant Green in the BGNRLA when used in conjunction with BGMB (Brilliant Green, 100 pg/ml) was found useful for the isolation of S. choleraesuis from feces. The higher concentration of 100,g/ml Brilliant Green in the selective medium inhibited the growth of organisms, since they had already been subjected to this higher concentration in the enrichment broth and a second exposure to the same concentration seemed to inhibit their growth. Gitter (7) recommended the use of 30 gg/ml Brilliant Green in the selective medium in preference to 100,g/ml. Slavin (22) used Brilliant Green in the selective media in 100,ug/ml and 50,g/ml concentrations as he found that its inhibitory effect varied with the source of the dye. Miller and Banwart (20) indicated that Brilliant Green proportionately lost its inhibitive effect on bacteria in the presence of increasing amounts of organic matter. Smith (23) mentioned that the reason for failure to recover S. choleraesuis from feces was not primarily that the feces contained bacteria which outgrew salmonellae but that many of the media used were too toxic to permit the growth of the organism. In the present study, the autoclaving of feces before inoculating them with S. choleraesuis resulted in a higher rate of isolations (70 out of 72) as compared with 57 out of 72 from nonautoclaved feces on the same selective medium. These observations appear to suggest that contaminant bacteria present in the feces perhaps outgrow and mask S. choleraesuis. It was observed that S. choleraesuis grew at a slower rate as compared to the other three Salmonella species and that the colony size was also comparatively smaller, given the same period of incubation. It may be for this reason that, before S. choleraesuis can grow in sufficient numbers, the contaminant bacteria multiply in large numbers to mask them and thus render their isolation difficult in naturally infected feces. Smith (24) reported that S. choleraesuis could be isolated from mesenteric lymph nodes of pigs by means of BGMB, but not from feces. Evidently it does not appear to be the toxicity of the enrichment broth for the organism that contributes towards failure to isolate it from feces but the possibility indicated above. The observations that 24 to 30 hr was the best incubation time for TTB and that there was no falling off of efficiency with selenite F broth from 30 to the end of 48 hr, as happened with TTB, were in conformity with the findings reported by Smith (23). Smith, however, mentioned that salmonellae were not recovered from any of the specimens before 15 hr, whereas we observed that the organisms could be isolated at the end of 12 hr of incubation of enrichment broths in 53 to 60% of the samples on DCA used in conjunction with the three enrichment broths. With the use of equal numbers of organisms for

7 VOL. 18, 1969 CULTURE MEDIA EVALUATION FOR SALMONELLAE 595 inoculating the enrichment broths as Smith did, the difference may perhaps be due to variation in the nature and amount of the fecal matter added. Smith employed feces from various species of animals in contrast to our use of feces from only one animal (cow). ACKNOWLEDGMENTS This investigation was conducted during the tenure ofa scholarship awarded by the Rockefeller Foundation, New York, N. Y., to Radhey M. Sharma. We thank Roy Hickman, Department of Statistics, Iowa State University, for statistical analysis of the data. L1TERATURE CITED 1. Banwart, G. J., and J. C. Ayres Effect of various enrichment broths and selective agars upon the growth of species of Salmonella. Appl. Microbiol. 1: Cook, G. T., B. R. Friesby, and W. H. H. Jebb The routine use of selective and enrichment media for the isolation of Salmonellae. Gt. Brit. Min. Health Public Lab. Serv. Monthly Bull. 10: Dixon, J. M. S Rapid isolation of Salmonellae from feces. J. Clin. Pathol. 14: Edwards, P. R., D. W. Bruner, and A. B. Moran Further studies on the occurrence and distribution of Salmonella types in the United States. J. Infec. Dis. 83: Edwards, P. R., and W. H. Ewing Identification of Enterobacteriaceae. Burgess Publishing Co., Minneapolis. 6. Georgala, D. L., and M. Boothroyd A system for detecting Salmonellae in meat products. J. Appl. Bacteriol. 28& Gitter, M Isolation of Salmonella choleraesuis from post-mortem specimens. Vet. Rec Guinee, P. A. M., and E. H. Kampelmacher Influence of variations of the enrichment method for the detection of Salmonella. Antonie van Leeuwenhoek J. Microbiol. Serol. 28: Harvey, R. W. S Choice of a selective medium for the routine isolation of Salmonella group. Gt. Brit. Min. Health Public Lab. Serv. Monthly Bull. 15: Heidrich, D Comparative evaluation of tetrathionate broth and selenite broth for the demonstration of Salmonella in bovinefeces and in bacteriological meat inspection. Deut. Tieraerztl. Wochenschr. 70: Hoag, W. G., and J. Rogers Techniques for the isolation of Salmonella typhimurium from laboratory mice. J. Bacteriol. 82: Hobbs, B. C., and V. D. Allison Studies on the isolation of Bact. typhosum and Bact. paratyphosum B. Gt. Brit. Ministry of Health Public Lab. Serv. Monthly Bull. 4 (No.12): Kauffmann, F Die Technik der Typhenbestimmung in der Typhus-Paratyphus-Gruppe. Zentralbl. Bakteriol. Parasitenk. lnfektionskr. Hyg. Abt. Orig. 119: Kauffmann, F Weitere Erfahrungen mit dem Kombinierten Anreicherungsverfahren fur Salmonella-bacillen. Z. Hyg. Infektionskr. 117: Knox, R., P. G. H. Gell, and M. R. Pollock Selective media for organisms of the salmonella group. J. Pathol. Bacteriol. 54: Knox, R., P. G. H. Gell, and M. R. Pollock The selective action of tetrathionate in bacteriological media. J. Hyg. 43(No. 3): Leifson, E The effect of sodium selenite on the growth of bacteria and its use as the basis for a new enrichment medium for the isolation of typhoid bacilli from feces, water, milk, etc. J. Bacteriol. 31: Mackie, T. J., and J. E. McCartney Handbook of practical bacteriology, 9th ed. E. and S. Livingstone, Ltd., London. 19. Miles, A. A., and S. S. Misra The estimation of bactericidal power of the blood. J. Hyg. 38: Miller, V. R., and G. J. Banwart Effect of various concentrations of brilliant green and bile salts on Salmonellae and other microorganisms. Appl. Microbiol. 13: Mueller, L Milieu d'enrichment pour la recherche du Bacille typhique et des Paratyphiques. Compt. Rend. Soc. Biol. 89: Slavin, G Brilliant green-neutral red-lactose agar for the isolation of S. suipestifer from pig feces. J. Comp. Pathol. 53: Smith, H. W The evaluation of culture media for the isolation of Salmonellae from feces. J. Hyg. 50: Smith, H. W The isolation of Salmonellae from the mesenteric lymph nodes and feces of pigs, cattle, sheep, dogs, and cats and from other organs of poultry. J. Hyg. 57: Thomson, S The number of bacilli harbored by enteric carriers. J. Hyg. 52:67-70.