Selective Solid Media Research Articles

A COMPARISON BETWEEN TWO METHODS FOR THE RECOVERY OF LISTERIA MONOCYTOGENES IN MILK POWDER REFERENCE SAMPLES

ABSTRACTTwo methods (A and B) for the recovery of Listeria monocytogenes were compared using reference samples based on spray drying of milk containing this pathogen and competitive microflora. For method A, the sample was inoculated in a buffered liquid medium (same as IDF Pre‐enrichment Broth) and then passed to IDF Enrichment Broth plus phosphate buffer. For method B, the sample was inoculated in FDA Enrichment Broth (LEB) and then passed to LEB (subenrichment). The solid selective medium used in both cases was Listeria Selective Agar (Oxford formulation). The sensitivity and specificity of method B was 95.2% and 100% vs. 76.1% and 88.8% for method A, respectively.

Utilization of Low Molecular Weight Lignin-Related Aromatic Compounds for the Selective Isolation of Yeasts: Rhodotorula vanillica, a New Basidiomycetous Yeast Species

Summary Low molecular weight lignin-related aromatic compounds were used as sole sources of carbon and energy for the selective isolation of yeasts. A total of 227 yeast strains were isolated from terrestrial and aquatic habitats and ascribed to three groups: ascomycetous yeasts (17.6%), ascomycetous yeast-like microorganisms (5.3%) and basidiomycetous yeasts (77.1%). About half of the strains, representing the proportions of the various groups, were identified to the species level. Among the basidiomycetous yeasts, those affiliated to the Ustilaginales were present in higher number (75 strains) than the representatives of the Tremellales/Filobasidiales (16 strains). The ability of each identified isolate to grow in liquid medium with the same composition as the solid selective isolation medium was checked. Only 41% of the ascomycetous yeasts were able to grow, whereas all the ascomycetous yeast-like isolates and 90% of the basidiomycetous yeasts had that capacity. A novel basidiomycetous anamorphic yeast species was found among the strains collected and is described as Rhodotorula vanillica . Conspecificity with the phenotypically similar species Rhodotorula ingeniosa was excluded based on absence of DNA-DNA complementarity.

Microbiological‐Immunological Rapid Method to Detect Legionellaceae in Water

AbstractSpecific and sensitive immunological assays for the detection of bacteria are able to substitute the conventional microbiological control of drinking water. In cooperation with the Medical University of Hannover immunological assays were developed based on monoclonal antibodies against specific bacterial antigens. The immunological methods were combined with a shortened microbiological pre‐enrichment, because the sensitivity of the immunoassays was too low to fulfill the requirements of the drinking water ordinance. Legionella colonies grown on a solid selective medium are detectable within 2 h only by an immunological dot‐blot method using a monoclonal antibody against the legionella heat shock protein. The method proved its validity in first test series at the municipal water works Mainz and will be screened intensively in further studies. The intent is to find a standardization analogous to that of the immunological detection of enterobacteria [1].

Semisolid blood-free selective-motility medium for the isolation of campylobacters from stool specimens.

Isolation of Campylobacter jejuni and C. coli from stool specimens is done by growing campylobacter colonies on solid selective media with or without blood. However, recognition of these colonies can be difficult. Therefore, we decided to evaluate an isolation procedure based on the swarming of campylobacters through a semisolid medium. We developed a semisolid blood-free selective motility (SSM) medium which is composed of Mueller-Hinton broth with 0.4% agar and supplemented with cefoperazone (30 micrograms/ml) and trimethoprim (50 micrograms/ml). The SSM medium was compared with our previously described Butzler Medium Virion (Goossens et al., J. Clin. Microbiol. 24:840-843, 1986) and blood-free medium (Bolton and Coates, J. Appl. Bacteriol. 54:115-125, 1983) with cefoperazone (32 micrograms/ml) (Bolton et al., J. Clin. Pathol. 37:956-957, 1986). Of 1,890 routine stool specimens tested, 100 were found to be positive for campylobacters: 95 were recovered with the SSM medium, 94 with the Virion medium, and 90 with the blood-free medium. The SSM medium performed equally well whether it was incubated in the special incubator or the candle jar. Only 4.4 and 7.3% of the plates grew contaminating fecal flora when incubated in the special incubator and the candle jar, respectively. Clearly the SSM medium is easy, quick, cheap, sensitive, and more selective than any other medium which has been developed so far and does not require the addition of blood. We believe that this medium has a future in the routine microbiology laboratory in developed as well as in developing countries.

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique.

The growth patterns of microcolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0.89), pseudomonads (r = 0.93) and staphylococci (r = 0.92) after incubation at 30 degrees C for 3 or 6 h (staphylococci) at contamination levels of above 10(3) bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.

Discrepancies in the enumeration of Escherichia coli.

Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO(4).7H(2)O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods.

Discrepancies in the Enumeration of Escherichia coli1

Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO(4).7H(2)O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods.

Urease Activity of Enterobacteriaceae: Which Medium to Choose

Detection and intensity of urease activity in enterobacteriaceae greatly varies as a function of the media or techniques used, or both. A comparative investigation on several solid and liquid media led us to the following conclusions. (i) Detection of Proteus spp. can be adequately performed with the highly selective solid medium described by Cook (1948), as well as with the different liquid media described (Stuart standard and rapid media; Elek medium). (ii) Detection of Klebsiella should be based upon urease production on solid media with low buffer capacity (Christensen, 1946). (iii) For the identification of Yersinia, either the solid Christensen urea agar or the rapid Elek technique give optimal results.

Comparison of Methods for the Isolation of Salmonella from Bone Meal

The efficiency of some methods for the isolation of Salmonella from bone meal was studied. Grinding of the granulated, industrial product was found to increase Salmonella recovery. An improvement in the efficiency of the pre-enrichment method was obtained by adding deoxycholate (1%) to mannitol broth. Selective enrichment in selenite cystine medium gave better results than in Kauffmann-Müller medium, when small inocula were used. Experimental data showed that, in the presence of large numbers of coliforms and very small numbers of Salmonella, the Kauffman-Müller medium offers better results, but, in the presence of smaller numbers of coliforms, selenite cystine is preferable. As solid selective medium, Brilliant Green Agar gave, in our laboratory, better results than S S medium.

Rapid isolation of salmonellae from faeces.

Factors and techniques leading to the more rapid isolation of salmonellae from faeces that are negative by direct plating were examined. The aim was to increase the number of specimens on which positive reports might be given within 24 hours of receipt. Salmonellae were isolated from 279 of the specimens studied. Brilliant green MacConkey agar was found to be the most satisfactory solid selective medium both for direct plating and as a subculture medium, giving large characteristic colonies after 24 hours' incubation. Selenite F medium, inoculated with undiluted faeces, incubated at 43 degrees C., and subcultured after six hours' incubation on to brilliant green MacConkey agar, was the most successful rapid method of enrichment, though the results were considerably inferior to those obtained after 24 hours' incubation. Seventy-six of the positive specimens examined were negative by direct plating on three different selective media; 24 (31.6%) of these were positive on subculture from selenite F incubated for six hours at 43 degrees C. and 21 (27.6%) when incubation was at 37 degrees C. The use of six-hour subcultures from 43 degrees C. selenite F as a supplement to direct plating added significantly to the number of positive results. Other techniques that were tried but found to be of no practical value were dilution of the faeces before inoculation on solid and fluid media, and replica plating from one solid medium to another after a few hours' incubation.

A selective method for the isolation of Listeria monocytogenes from mixed bacterial populations.

Abstract A method and medium have been developed for the isolation of L. monocytogenes from mixed cultures as it commonly occurs in biologic materials. This consists of an enrichment broth in which the organisms are incubated for 18 hours, then streaked on a solid selective medium. L. monocytogenes produces gray colonies in 24 hours. Other organisms present could be easily differentiated by their appearance.

SELECTIVE MEDIA IN THE ENUMERATION OF BACTERIA IN PITCHING YEASTS

Examination of representative samples of top yeasts from British breweries revealed invariably the presence of strains of Flavobacterlum proteus, Lactobacillus and Acetobacter and the absence of significant numbers of Pediococcus and other bacteria. Three solid selective media have been developed which permit relatively rapid independent estimation of the numbers of each type present in pitching yeast. Flovobacterium proteus usually constituted 90% or more of the living bacterial population, cells of Lactobacillus and Acetobocter species in about equal proportions accounting for the remainder.

THE USE OF A SELECTIVE MEDIUM FOR THE ENUMERATION OF LACTOBACILLI IN CHEDDAR CHEESE

SUMMARY: The use of a selective solid medium for counting lactobacilli in Cheddar cheese is described. It has proved useful for this purpose, especially in the early period of ripening when large numbers of streptococci render other methods impracticable.The medium, in a semisolid form, is more sensitive for the detection of heterofermentation in lactobacilli than media previously available. The possibility of using it to count heterofermentative lactobacilli and leuconostocs is discussed.