Growth kinetics of mixed culture in salmonella enrichment media

A technique for investigating the kinetics of salmonella enrichment is reported. Its use with four enrichment media (Rappaport's medium, Muller-Kauffmann tetrathionate broth (MKT) tetrathionate broth and selenite F) is described and the effect of elevated temperature on the growth kinetics shown. Rappaport's medium at 37 degrees C and MKT at either 37 degrees C or 42 degrees C were far superior to selenite F and tetrathionate broth in their selective properties and, with the exception of Rappaport's medium, the use of elevated temperature increased the selectivity of the media.     

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.     

Improved Salmonella recovery from moderate to highly polluted waters

A new enrichment procedure for the recovery of salmonellas from aquatic environments is proposed. It has been tested in a eutrophic lake showing moderate to high faecal contamination levels (the Albufera lake near Valencia, Spain), and in effluents coming into a wastewater treatment plant. The new method consists of the addition of sodium novobiocin to a modification of Rappaport's medium (R10/43°C). The new medium (NR10/43°C) allows a better recovery of salmonellas from water than selenite broth.     

A note on the performance of Rappaport's medium, compared with Rappaport-Vassiliadis broth, in the isolation of salmonellas from meat products, after pre-enrichment

Salmonellas were isolated from meat products using a slightly modified Rappaport's enrichment medium (R25), Rappaport-Vassiliadis procedure (Rappaport's broth containing 10 ml instead of 30 ml of Malachite Green solution and incubated at 43^oC instead of 37^oC), and Muller-Kauffmann's tetrathionate broth. From 255 samples, 89 were found positive with the Rappaport-Vassiliadis procedure, 83 with the R25 broth, whereas only 43 were positive with Muller-Kauffmann broth. It is concluded that the R25 medium may be used as an alternative to the more effective Rappaport-Vassiliadis broth when the only available incubation temperature is 37^oC.     

Evaluation of the Growth of Salmonellae in Rappaport's Broth and in Muller-Kauffmann's Tetrathionate Broth

S ummary . The behaviour of 70 strains of salmonellae belonging to 44 serotypes in Rappaport's broth and in Muller-Kauffmann's tetrathionate broth was examined. With an inoculum of 5–25 cells, 5 strains did not grow in Rappaport's medium, 2 multiplied slowly and 63 grew strongly in 24 h. With an inoculum of 100–500 organisms all but one strain grew readily in 24 h. In Muller–Kauffmann's tetrathionate broth inoculated with pure cultures of salmonellae, growth of many strains was markedly inhibited, in the absence of added faeces, at 37° and 43°. This inhibition was more severe with light inocula at 43°. The addition of 0.05% (w/v) of salmonella-free human faeces to Muller–Kauffmann's tetrathionate broth, did not stimulate growth of salmonellae. In contrast, the addition of 5% (w/v) of human stools to this medium resulted in a heavy growth of the added salmonellae, especially at 43°.     

Isolation of Yersinia enterocolitica and related species from porcine samples obtained from an abattoir

H armon , M.C., S waminathan , B. & F orrest , J.C. 1984. Isolation of Yersinia enterocolitica and related species from porcine samples obtained from an abattoir. Journal of Applied Bacteriology 56 , 421–427.
Swabs of swine carcasses and samples of porcine tongue and trim obtained from an abattoir were examined for the presence of Yersinia enterocolitica and related species ( Y. intermedia, Y. kristensenii and Y. frederiksenii) . Three enrichment media (phosphate buffered saline, sorbitol-bile salts-phosphate buffered saline, and a modified Rappaport's broth) were compared at 4C for their efficiency of recovery of Y. enterocolitica and related species. Two secondary enrichment procedures (post-enrichment in modified Rappaport's broth for 2 d at 25C and treatment with 0.5% KOH in 0.5% NaCl) also were evaluated. The porcine isolates were characterised by biochemical and serological examination, speciation, and biotyping. Eight of 43 samples were positive for Y. enterocolitica and related species. The combination of incubation in sorbitol-bile salts-phosphate buffered saline for 21 d at 4C followed by post-enrichment in modified Rappaport's broth yielded maximum number of isolates. All isolates, except one, were avirulent as determined by auto-agglutination, calcium dependence at 37C, and HeLa cell invasiveness tests.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

F ricker , C.R. 1984. A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium. Journal of Applied Bacteriology 56 , 305–309.
The efficiency of Rappaport's broth (RB10) and Rappaport's broth containing novobiocin (NRB10) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used; no significant difference in their ability to recover salmonellae was demonstrated.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

The efficiency of Rappaport's broth ( RB10 ) and Rappaport's broth containing novobiocin ( NRB10 ) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used: no significant difference in their ability to recover salmonellae was demonstrated.     

Isolation of Salmonellae From Food Samples: VI. Comparison of Methods for the Isolation of Salmonella From Egg Products

The recovery of salmonellae from egg products was studied, by use of three different enrichment procedures: (i) selenite broth, (ii) selenite broth containing 10% sterile feces, and (iii) the lactose pre-enrichment procedure. Brilliant Green Agar was used throughout as the recovery medium. Although the lactose pre-enrichment methodology promoted Salmonella recovery from samples containing small numbers of dormant organisms, the efficiency of this enrichment method is adversely affected by unfavorable coliform-Salmonella ratios. Under such conditions, early subculture of lactose broth into selenite broth is indicated. Selenite broth containing 10% sterile feces was more efficient than the lactose pre-enrichment methodology in promoting the growth of “dormant” salmonellae. Albumen adversely affected recovery of salmonellae from selenite broth, whereas whole egg and egg yolk enhanced Salmonella recovery from this medium. The selenite-feces medium presents a solution to the major problems encountered in the detection of salmonellae in egg products and offers an approach to a single medium in which food-borne salmonellae will manifest themselves with a minimum of laboratory manipulation.     

An evaluation of selective broths based on the bi-selenite ion and on hypertonic strontium chloride in Salmonellae detection in egg products.

Of the 104 isolations of Salmonella sp. from egg pulp, 97 were obtained from strontium chloride M broth, 42 from strontium selenite broth and 57 from strontium selenite A broth. The results suggest that the first medium may be used more successfully than bi-selenite based media for enrichment and subsequent detection of salmonellae in egg products; however, the growth of S. pullorum was not satisfactory in strontium chloride M broth.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42°C) were higher even when MKT (43°C) performance was improved by the use of brilliant green sulphamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Comparison of Muller-Kauffmann tetrathionate broth with Rappaport-Vassiliadis (RV) medium for the isolation of salmonellas from sewage sludge

The Rappaport-Vassiliadis (RV, modification of Rappaport's) medium was superior to Muller-Kauffmann tetrathionate broth (MKT) for the enrichment and isolation of salmonellas from sewage sludge samples, either naturally contaminated or artificially inoculated with salmonella. Isolation rates for RV (42 degrees C) were higher even when MKT (43 degrees C) performance was improved by the use of brilliant green sul-phamandelate agar. Kinetic results show that the suppression of the competing flora was poorer in MKT than in RV. The use of high inoculum to medium volume ratios (e.g. 1:5000) increased the isolation rates from RV and kinetic data explain why this was so.     

Growth kinetics of mixed culture in salmonella enrichment media

R hodes , P., Q uesnel , L.B. & C ollard , P. 1985. Growth kinetics of mixed culture in salmonella enrichment media. Journal of Applied Bacteriology 59 , 231–237.
A technique for investigating the kinetics of salmonella enrichment is reported. Its use with four enrichment media (Rappaport' medium, Muller-Kauffmann tetra-thionate broth (MKT) tetrathionate broth and selenite F) is described and the effect of elevated temperature on the growth kinetics shown. Rappaport' medium at 37C and MKT at either 37C or 42C were far superior to selenite F and tetrathionate broth in their selective properties and, with the exception of Rappaport' medium, the use of elevated temperature increased the selectivity of the media.     

Laboratory study of several enrichment broths for the detection of Salmonella spp. particularly in relation to water samples

The selectivity and efficiency of several enrichment broths used for the detection of salmonellas were comparatively evaluated under laboratory and environmental conditions. Media with selenite were less efficient in their inhibition of the growth of Gram-positive micro-organisms. Salmonellas grew slowly in tetrathionate broth and in media containing brilliant green. These media inhibited the growth of Salmonella typhi, which grew only in media containing selenite. The results obtained in the experiments with stressed salmonellas indicate that the media selenite F, selenite F with novobiocin, selenite cystine and Rappaport-Vassiliadis (RV/43), in conjunction with the double agar layer technique, showed an optimal efficiency for the detection of stressed salmonellas. When natural samples (freshwater and seawater) were used to evaluate the media, however, those containing malachite green, whether or not supplemented with sodium novobiocin, enhanced the recovery of salmonellas.     

OBSERVATIONS ON THE ISOLATION OF SALMONELLAE FROM SELENITE BROTH

SUMMARY: Studies of growth curves of enterobacteria in selenite broth containing different carbohydrates showed that whereas mannitol and lactose brought about a steep fall in coli-aerogenes bacteria, mannitol improved the growth of Salm. typhi-murium . With mixed cultures of Cit. freundii I and Salm. typhi-murium the presence of lactose, utilizable by the former, adversely affected the viable count of the latter.
Comparative studies with routine faeces specimens showed that selenite broth was an efficient selective medium with MacConkey's agar; but much better results were obtained when it was combined with deoxycholate-citrate agar. Gentian violet introduced into selenite broth improved its selectivity for most Salmonella types when MacConkey's agar was used for final isolation.     

A comparison of strontium selenite broth and mannitol-selenite-cystine broth in the isolation of salmonellae from egg products.

Of 103 isolations of Salmonella sp from egg pulp and albumen, 92 were made using strontium selenite enrichment broth and 54 were made using mannitol-selenite-cystine enrichment broth. The results suggest that the former medium may be used successfully for enrichment and subsequent detection of salmonellae in egg products.     

Sensitivity and Specificity of Different Methods for the Isolation of Salmonella from Pigs

Three different selective enrichment media, Rappaport-Vassiliadis broth (RV), selenite broth (SB) and Müller-Kauffmann tetrathionate broth (MKTB), in combination with plating on modified brilliant green agar (BGA), were compared for the isolation of Salmonella from samples of pig feces. These conventional methods were also compared with a new ELISA kit in conjunction with RV and SB enrichment. Of the conventional methods, enrichment in RV had a higher sensitivity and selectivity than SB and MKTB. Recovery of S. typhimurium from MKTB was significantly poorer than recovery of other serotypes. The combination of RV enrichment and ELISA was as good as the conventional method involving RV enrichment, with a similar high sensitivity and specificity.     

Secondary selective enrichment of salmonellae from naturally contaminated specimens by using a selective motility system.

A selective motility medium was used as a secondary selective enrichment medium to examine specimens naturally contaminated with salmonellae. The medium, incubated at 37 degrees C, was inoculated from either selenite brilliant green sulfa enrichment broth or Müller-Kauffman tetrathionate broth, both of which had been incubated at 42 degrees C. The use of the selective motility medium resulted in an increase in the number of positive specimens from 65 and 74% to 80 and 82%, when inoculated at 24 and 48 h, respectively, from tetrathionate broth. Tetrathionate broth, when used singly, was significantly better than selenite brilliant green sulfa broth, which detected 55% of positive specimens at both 24 and 48 h. The use of the selective motility medium of Harper and Shortridge (J. Hyg. 67: 181--186, 1969) for the further examination of specimens culturally negative on primary selective enrichment is advocated.     

Enhancing Effect of Feces on Isolation of Salmonellae From Selenite Broth

The addition of feces to selenite broth significantly enhanced the ability of this medium to select for salmonellae in an environment initially containing overwhelming numbers of coliform bacteria. Either heat-sterilized or Seitz-filtered feces produced this effect. In most experiments, the selectivity of selenite broth was unaffected by unsterile feces. Human blood and plasma markedly reduced selenite efficiency. In a base medium supporting both coliform and Shigella growth, heat-sterilized feces imposed a measure of selectivity for Shigella.     

Secondary selective enrichment of salmonellae from naturally contaminated specimens by using a selective motility system.

A selective motility medium was used as a secondary selective enrichment medium to examine specimens naturally contaminated with salmonellae. The medium, incubated at 37 degrees C, was inoculated from either selenite brilliant green sulfa enrichment broth or Müller-Kauffman tetrathionate broth, both of which had been incubated at 42 degrees C. The use of the selective motility medium resulted in an increase in the number of positive specimens from 65 and 74% to 80 and 82%, when inoculated at 24 and 48 h, respectively, from tetrathionate broth. Tetrathionate broth, when used singly, was significantly better than selenite brilliant green sulfa broth, which detected 55% of positive specimens at both 24 and 48 h. The use of the selective motility medium of Harper and Shortridge (J. Hyg. 67: 181--186, 1969) for the further examination of specimens culturally negative on primary selective enrichment is advocated.