Comparison of Media for Isolation of Salmonellae and Shigellae from Fecal Specimens

Five transport media, eight plating media, and three enrichment broth media for the isolation of salmonellae and shigellae were evaluated. Eight laboratories in widely separated regions of the United States participated in this evaluation by submitting 490 fecal specimens in the transport media provided. The results suggest that the newer transport media may not offer any advantage over the use of buffered glycerol-saline in the isolation of these enteric pathogens. Shigellae were best isolated by direct inoculation, whereas salmonellae were isolated in greater numbers after tetrathionate (without Brilliant Green) enrichment with subsequent culturing on the plating medium. The use of a variety of plating media is recommended for the recovery of a larger number of these enteric pathogens.     

Form of Selenium in Selenite Enrichment Media for Isolation of Salmonellae

Selenite-F and selenite-cystine media, commercially available for the routine isolation of salmonellae, were treated by anion exchange chromatography to separate the selenium from other components of the media. A chemical assay, based on an ascorbic acid reduction, showed that the selenium was all in the form of selenite.     

Studies on the Multiplication of Salmonellae in Various Enrichment Media at Different Incubation Temperatures

Several selected Salmonella strains did not multiply in tetrathionate brilliant green bile medium when the inoculum was small and the medium was incubated at 43° C. Gradual heating from 20° to 43° C and dilution of the medium with buffered peptone water (1:10) containing egg yolk did not decrease its inhibitory properties. It became less inhibitory, however, after the growth of other Enterobacteriaceae. These findings stress the advantage of using non-selective pre-enrichment since in that case the Muller-Kauffmann tetrathionate broth will be inoculated with a large number of salmonellae and other Enterobacteriaceae thus facilitating the isolation of the former.     

Evaluation of Culture Media for the Isolation of Salmonellae from Feces

Serial passage of the 64-2389 strain of type 3 parainfluenza virus in cercopithecus monkey kidney tissue cultures at low temperatures resulted in the selection of a variant which had a higher efficiency of plaque formation at 25 C than the parent line grown at 37 C. The cold variant, unlike the parent strain, plaqued readily at 25 C, and at 37 C it produced significantly larger plaques. Virus titers of the cold variant in hamster lungs were significantly lower and this was probably caused by the stimulation of interferon by the cold variant during the early phase of the infection. The cold variant, like the virus grown at 37 C, also induced the synthesis of interferon late in the infection. Hamsters responded to the intranasal inoculation of each virus line by the development of hemagglutinating-inhibiting antibodies in the sera.     

Isolation of Salmonellae from Sewage with a New Procedure of Enrichment

Forty samples of sewage on Moore's swabs were examined for the presence of salmonellae. They were first pre-enriched in buffered peptone water. From each pre-enrichment, three enrichments were made: (1) in a new, considerably modified, formula of Rappaport medium (R 10) incubated at 43 °C (R 10/43 °C), (2) in the usual formula (R25) of the same medium at 37 °C (R25/37 °C) and (3) in Muller-Kauffmann's tetrathionate broth at 43 °C (MK/43 °C). Practically the same numbers of swabs were found positive by the first two enrichment procedures, 38 and 39 respectively, while only 17 were found positive by the MK procedure. The R10/43 °C method was superior to the two other procedures; it yielded 103 strains of salmonellae as against 82 with the second Rappaport procedure, and only 25 with the MK/43 °C technique. A similar observation was made concerning the frequency of isolation of different serotypes by the three procedures; the number of the isolated serotypes was 24, 19 and 11, respectively. The new R 10/43 °C method of enrichment had also a much stronger inhibitory effect on the competing bacteria than the two other procedures of enrichment used.     

Isolation of Salmonellae by Selective Motility Systems

Salmonellae were isolated from naturally infected swine and human feces by means of selective migration through semisolid enrichment (SM) media in modified “U” tubes. Comparative studies showed that recovery of Salmonella by SM techniques was equal or superior to that of standard procedures employed in two routine diagnostic laboratories. Primary Salmonella isolations from SM enrichment were relatively free from normal fecal bacteria and often pure. The SM method required only 15% of the media and correspondingly less time and skill than the usual methods for isolating Salmonella.     

Evaluation of Media for Selective Isolation of Yeasts from Oral, Rectal, and Burn Wound Specimens

Six media were evaluated to determine their ability to isolate yeasts and inhibit bacteria. The media included the following: Snyder, Snyder tellurite, Sabouraud tellurite, Littman-gentamicin, molybdate, and Mycosel (BBL). Doses of mixed intestinal gram-negative bacilli and enterococci were most effectively inhibited by Snyder tellurite agar. Klebsiella pneumoniae was the most common bacterial contaminant of the other media. All six media were comparable in isolating yeasts while preventing the growth of the oral bacterial flora. The selection of a basal fungal growth medium for tellurite incorporation to inhibit bacteria but permit growth of yeasts was affected by pH. The bacteriostatic effect of tellurite was decreased with increasing pH of media while fungistatic action was increased. The arbitrary selection of Snyder and Littman agars to isolate yeast from burn wound cultures demonstrated the need to include a selective medium for these specimens. Blood, phenylethyl alcohol blood agar, and Columbia CN blood agar were all inadequate for isolating yeasts from burns. Growth of a variety of filamentous saprophytic and pathogenic dimorphic fungi grew adequately on four of five selective media tested.     

Selective Medium for the Isolation of Streptococci from Clinical Specimens

Incorporating neomycin and nalidixic acid into a blood-agar base resulted in a medium highly selective for beta-hemolytic streptococci under conditions in which detection of streptococcal colonies by conventional means would have been very difficult.     

Improved Isolation of Salmonellae from Naturally Contaminated Meat Products by Using Rappaport-Vassiliadis Enrichment Broth

A total of 454 specimens of meat products were examined for salmonellae by using five procedures of enrichment. The use of a selective motility medium, inoculated from enrichment in Muller-Kauffmann broth, resulted in an increase in the number of positive specimens. However, simple enrichment in Rappaport-Vassiliadis broth, after preenrichment, was more sensitive and specific for recovering salmonellae than the selective motility medium-Muller-Kauffmann broth method.     

Isolation of Salmonellae from Foods Samples: IV. Comparison of Methods of Enrichment

A comparison of various methods of enhancing frequency of Salmonella isolations revealed that inoculation of a second enrichment broth, with culture from the first, was no improvement over the single direct enrichment method. It was inferior to centrifugation.

Selenite was observed to produce more positive isolations at 48 hr than at 24. No change occurred in tetrathionate. Reconstitution of dried albumen with water produced a significant increase in isolations over direct inoculation of enrichment broth in the case of tetrathionate but not selenite broth.

Pre-enrichment in lactose broth before inoculation of enrichment media was vastly superior to reconstitution in water for both enrichment broths. A comparison of results obtained using dulcitol, mannitol, lactose and carbohydrate-free purple broths in pre-enrichment indicated that the carbohydrate added was immaterial.

     

Comparison of Media for Direct Isolation and Transport of Shigellae from Fecal Specimens

Xylose-lysine-deoxycholate (XLD) agar, SS agar, and MacConkey agar for isolating shigellae from fecal specimens were compared. XLD agar was superior to both SS agar and MacConkey agar for isolating Shigella sonnei, and both XLD and SS agar were superior to MacConkey agar for isolating S. flexneri. Direct plating of the fecal specimens in the field resulted in a greater yield of shigellae as compared to transporting specimens to the laboratory either in holding media or enrichment broth. Buffered glycerol saline was superior to other transport media evaluated, yielding 83% of shigella isolates when plated within 48 hr as compared to direct plating. The combination of XLD agar and SS agar is recommended for direct isolation of shigellae, and, whenever possible, these solid media should be taken to the bedside and inoculated directly.     

Isolation of Salmonellae from Pork Carcasses

Four hundred and twenty pork carcasses from four abattoirs were examined for the presence of salmonellae by use of swabbing-enrichment techniques and contact plate methods. Carcasses from only one abattoir were found to be contaminated by swabbing-enrichment (23.3%) and contact plate (17.9%) methods. The area of the skin side of the ham, near the anal opening, was determined to be the area to examine for isolating salmonellae from pork carcasses with the greatest frequency. The most frequently isolated species of salmonellae in this study were Salmonella derby, S. anatum, S. typhimurium, and S. indiana.     

A Comparison of Methods for the Enrichment of Salmonellae from Sheep Faeces

S ummary : A 10-tube MPN technique was used to test the efficiency of nutrient, tetrathionate, mannitol-selenite and mannitol-selenite-cystine broths as enrichment media for detecting salmonellae. Small numbers of broth grown salmonellae could be detected in all 4 media in the presence of 5% of sheep faeces. In naturally infected sheep faeces small numbers of salmonellae were not detected with either nutrient or tetrathionate broths. With mannitol-selenite the sensitivity of salmonella detection increased with both incubation temperature (37–43°) and the addition of faeces. The most sensitive and reliable medium for detecting salmonellae in naturally infected sheep faeces was mannitol-selenite-cystine broth. Neither incubation temperature (37–43°) nor the addition of faeces had a statistically significant effect on its sensitivity.     

Enrichment in Muller-Kauffmann's Broth and Rappaport's Broth from Buffered Peptone Water in the Isolation of Salmonellae from Minced Meat

Four hundred and ninetysix samples of minced meat were examined for the presence of salmonellae. The methods of isolation used in the study were: (a) the standardized direct technique of enrichment in Muller-Kauffmann's broth incubated at 43· for 24 and 48 h, described by Edel & Kampelmacher (1969), (b) secondary enrichment in Rappaport's broth from the former medium after 24 h incubation, and (c) pre-enrichments in buffered peptone water incubated at 37·, followed by enrichments in Muller-Kauffmann's and in Rappaport's broth. These last procedures resulted in the higher rates of isolation of salmonellae with a slight, but not statistically significant, superiority of the enrichment in Rappaport's medium.     

Evaluation of Three Media for Selective Isolation of Gram-Positive Bacteria from Burn Wounds

Three media, phenylethyl alcohol blood agar, esculin-mannitol agar, and Columbia CN blood agar, were studied for the selective isolation of gram-positive bacteria from swab cultures of burn wounds.     

Comparison of Two Plating Media for the Isolation of Erysipelothrix rhusiopathiae from Enrichment Broth Culture

Sodium azide-crystal violet-agar and a modified blood-azide (MBA)-agar were used to isolate Erysipelothrix rhusiopathiae from swine tissues. There was no significant difference in the number of isolations. However, 78% of the isolants from modified blood-azide medium required only 24 hr of incubation, whereas all of the isolants obtained on sodium azide-crystal violet medium required 48 hr.     

Comparison of Selective Media for Isolation of Presumptive Group D Streptococci from Human Feces

Pfizer Selective Enterococcus (PSE) agar, a medium containing bile, sodium azide, and esculin, was evaluated for its sensitivity and selectivity for detection and enumeration of presumptive group D streptococci in human feces. SF broth and SF broth plus agar (1.5%), representing selective media in common use, were studied simultaneously. Presumptive group D streptococci were recovered on PSE agar from the feces of all 25 subjects. No growth was observed in 8% of specimens in SF broth. No gram-negative organisms were recovered in any medium. PSE agar has the advantages of selecting out Streptococcus bovis, earlier appearance of distinctive reactions, and lack of requirement for special incubation temperature.     

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products

Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar.