Despite changes in taxonomy in recent years media for the isolation and enumeration of this group of organisms show little change. Pseudomonas spp. belong to a group of organisms of great importance as pathogens and as spoilage organisms. Methods described for isolation and enumeration are primarily selective, since nonselective pre-enrichment is not possible due to competing flora. The selective principles are based on antibiotics and the indicative principles on production of fluorescence, pigmentation and hydrolysis of casein. As with all selective media, the recovery of stressed cells is sometimes prevented and the competing flora is not always completely inhibited so that confirmatory tests need to be made on presumptive positive colonies.
The genera Aeromonas and Plesiomonas are included in the family Vibrionanceae and are primarily quatic inhabitants. Aeromonas taxonomy has changed very much in recent years and now at least 16 genospecies have been defined. Although identification to the species level of the most commonly found Aeromonas can be done on the basis of a limited number of biochemical tests, identification of some species is more difficult. Plesiomonas shigelloides is the only species of the genus Plesiomonas. A review of the culture media components used for Aeromonas and Plesiomonas isolation is made. This paper also reports on the media used for the isolation of Aeromonas and Plesiomonas from foods and comparative studies. In the case of Aeromonas spp. the most promising media are Starch Ampicillin Agar (SAA) and Bile Salts Irgasan Brilliant Green agar (BSIBG) as plating media and Alkaline Peptone Water (APW) or Trypticase Soy Broth Ampicillin (TSBA) as enrichment broths. With regard to P. shigelloides, it is more difficult to make recommendations, but enrichment in APW or tetrathionate broth without iodine and plating on Inositol Brilliant green Bile salts agar (IBB) and Plesiomonas agar (PL) can be useful.
Dilution plating techniques are designed to determine populations of viable fungal propagules per unit weight or volume of food. Direct plating techniques, on the other hand, are designed to assess the internal mycoflora of individual pieces of foods, e.g., seeds or dried fruits, and results are expressed as a percentage of infected pieces. Both techniques are used by industry and regulatory agencies to monitor fungal contamination at various stages of food handling, storing, processing and marketing. Peptone (0.1%) water is commonly used as a diluent for samples to be homogenized, pummelled or blended. Buffered diluents containing up to 30% glycerol, 40% glucose, or 60% sucrose are recommended for enumerating xerophiles. No one medium is satisfactory for detection or enumeration of all yeasts and moulds in all foods. Antibiotic-supplemented media are superior to acidified media for enumeration of yeasts and moulds. Dichloran 18% glycerol agar performs well for enumerating moderately xerophilic yeasts and moulds. Fastidious xerophiles require media containing high concentrations of sugars and/or sodium chloride. Media have been formulated to detect potentially aflatoxigenic aspergilli and mycotoxigenic strains of penicillia, fusaria and other moulds but increased selectivity and specificity of media for detecting mycotoxigenic moulds are needed. Heat-resistant mould ascospores often require heat treatment prior to plating in order to activate the germination process. The spread-plate technique is strongly preferred over the pour-plate technique for enumerating yeasts and moulds. The recommended incubation temperature is 25°C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of mycoflora to 4 weeks or more for fastidious xerophiles. There is a need for new and improved media for selectively isolating various groups, genera, species and/or strains of fungi capable of growing only under specific environmental conditions, e.g., low aw, low pH or, in the case of sublethally injured cells, under conditions which facilitate resuscitation. Improved media are needed which accurately detect moulds producing specific mycotoxins in a wide range of food types.
Media used for isolation of Shigella spp. are not very specific or sensitive and are also in use for isolation of other Enterobacteriaceae. Media for enrichment and isolation from foods are available and in use in reference methods developed by official organisations. In general these methods use one or two liquid enrichment media and up to three isolation media. These media, as well as some modifications, are reviewed in this paper.
In this review methods for the quality control of media were compared, taking the following questions as a guidelines. (i) Which methods are easy to use and give reliable results? (ii) Which experimental design should be used in order to obtain reliable data with a minimal input of resources (staff and materials)? These questions can be answered satisfactorily using statistical methods. This review shows that solid media can be assessed with acceptable accuracy using well established methods like the spread plate technique. In order to assure a minimum of statistical error, at least two plates with an average count of 100 colonies per plate seems to be the best design. This also applies to the ecometric streaking technique, a good alternative to the more quantitative methods. For an accurate assessment of liquid media, large numbers of tubes need to be tested. This is very expensive in terms of laboratory resources and therefore unlikely to be used routinely. Therefore it is proposed to use the serial dilution technique, in which the broths are tested in triplicate (Richard, N. (1982) Monitoring the quality of selective liquid media used in the official serial dilution technique for the bacteriological examination of food. In: J.E.L. Corry (Ed.). Quality Assurance and Quality Control of Microbiological Culture Media. Proceedings of the Symposium held on 6-7 September 1979, Callas de Mallorca, Spain, G.I.T.-Verlag Ernst Giebeler, Darmstadt. pp. 51-58). The recommendations in this review can be used together with the methods recommended by the International Committee for Food Microbiology and Hygiene, Working Party on Culture Media (ICFMH, WPCM: Baird et al., 1987) to assist laboratories setting up QC tests for culture media.
Of the four known species of the genus Alicyclobacillus only Alicyclobacillus acidoterrestris is of significance for the beverage industry. Alicyclobacillus acidoterrrestris is a Gram-positive, obligately aerobic rod-shaped bacterium which is able to multiply event at pH 2.2. The heat resistance of the spores is extremly high (D95°C 1.0–14.5 min, z-value=6.4–11.3°C). Alicyclobacillus acidoterrestris can be detected using various methods, including flow cytometry, PCR, detection of fatty acids, ribotyping and cultural methods with biochemical identification. For routine analysis a cultural method is recommended because it is more reliable and simple.
Conventional isolation of Salmonella, in food microbiology is accomplished by using cultural methods. Media for pre-enrichment, selective enrichment and isolation have been developed. Several official organisations for standardisation have developed reference methods for the isolation of Salmonella. In general these use one pre-enrichment medium, two different selective enrichment media and two or more isolation media. In this paper the main media used for conventional isolation of Salmonella are reviewed. Also some recent developments in culture media for Salmonella are described. Modified pre-enrichment media are sometimes required for specific cases, and can be accomplished by addition of supplements as ferrioxamine E or OxyraseTM to standard media or though the development of new media such as Universal Purpose Broth. For the selective enrichment procedure, motility enrichment in semi-solid media shows equal or better results than the use of the standard liquid selective media. Recently developed isolation media use different selective and diagnostic properties, such as glucuronate fermentation, acid formation from propylene glycol, fermentation of glycerol and addition of Tergitol 4 as selective agent. There seems to be a trend in the official Standards Organisations to follow the more recent developments but progress is slow.
This review deals with culture media for the detection, selective isolation and cultivation of different groups of lactic acid bacteria (LAB). A number of elective and semi-selective media are available and currently used for LAB. Most of them have been developed with the intention to isolate certain groups of LAB from a specific habitat such as meat or dairy products. These media can be rendered more selective by the addition of specific inhibitory agents or by reducing the pH. Members of the genera Lactobacillus, Leuconostoc, Pediococcus and Weissella (so-called LLPW group) share a number of physiological similarities and generally respond in the same way to conditions or compounds inhibitory to non lactics. Therefore, most culture media developed for the detection of Lactobacillus or Leuconostoc are not completely selective for the respective genus. Carnobacteria can easily be distinguished from the LLPW group by their non-aciduric nature. However, because of physiological similarities to the genus Enterococcus such as ability to grow at pH-values up to 9.5, media developed for the selective isolation of Carnobacterium do not suppress growth of enterococci often sharing the same habitat. A number of useful selective media is available for beer pediococci, Tetragenococcus and Oenococcus, organisms characterised by specific properties associated with their adaptation to special environments. Because of the growing interest in probiotic strains and the inhabitants of the intestine, an increasing number of media have been proposed in recent years for selective isolation of particular species or strains from those habitats typically containing mixed populations of different LAB.
Clostridia are the anaerobic bacteria most frequently associated with foods. They are widely spread in the environment and show an extensive diversity in metabolic activity and nutritional requirements. As they are a rather heterogeneous group, no medium is available which will allow the growth of all clostridia and at the same time exclude the growth of all competitive flora. Most clostridia reduce sulphite to sulphide and hence will produce rather large black haloes of iron sulphide, in iron-containing media under anaerobic conditions. Consequently, most isolation media include sulphite and an appropriate iron salt, so that blackening due to sulphite reduction can serve as a diagnostic test for clostridia. In addition to their diversity in metabolic activity and nutritional requirements, the enumeration of both vegetative cells and clostridial spores using one method is difficult. Many spores only germinate after heat activation, a process which kills the vegetative cells present in the sample. Thus the suitability of a medium depends on the objective of examination, the competitive flora in the sample and the Clostridium species under investigation. The newest media described in the literature are sulphite cycloserine azide medium (SCA) for samples which do not undergo heat treatment before analysis and differential clostridial agar (DCA) for spore counts. Several media for the detection and enumeration of C. perfringens are discussed. Choice will depend on the sample under investigation, the required detection limit and the reason for the test.
The species Escherichia coli contains both diarrhoeagenic and non-diarrhoeagenic strains and it is very important to have methods available which can differentiate between them. Adequate culture methods have been developed for the isolation of enterohaemorrhagic E. coli (EHEC) of serogroup O157 from foods. However, at present no single isolation procedure is available for the recovery of all EHEC. Additionally, there are still no simple sensitive procedures available for the direct cultivation of strains of the other groups of diarrhoeagenic E. coli. The isolation of these organisms will best be accomplished by a combination of culture and molecular biological methods. In this paper some comparative studies of the media described for EHEC, especially EHEC O157, are noted and the difficulties associated with the isolation and enumeration of these organisms considered. Modified trypticase soya broth supplemented with novobiocin or modified E. coli broth supplemented with novobiocin and incubated at 41–42°C are the most appropriate selective enrichments. Injured EHEC O157 cells require pre-enrichment in a non-selective broth. Methods for the isolation of EHEC O157 should include sorbitol MacConkey agar supplemented with cefixime and potassium tellurite as the most effective isolation medium for typical sorbitol-non-fermenting EHEC O157 and a second isolation medium not based on the fermentation of sorbitol but, for instance, on β-D-glucuronidase activity. Where the background flora is low, washed sheep blood agar supplemented with calcium (“EHEC agar”) may be used.
Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods have been described. Use of many of these results in the isolation of non-pathogenic Yersinia strains. At present no single isolation procedure is available for the recovery of all pathogenic strains of Y. enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of Y. enterocolitica serotypes. Enrichment in irgasan ticarcillin chlorate (ITC) broth is the most efficient method for recovery of strains of serotype O:3, the most prevalent clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most frequently used plating media. For the recovery of serotype O:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN agar seems the most efficient procedure. Selection of the proper isolation procedure will depend on the bio/serogroups of Yersinia spp. sought and on the type of food to be examined. Use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Serotyping, biotyping and virulence testing is essential for differentiation between pathogenic and environmental Yersinia strains. The International Standard Organization method for the detection of presumptive pathogenic Y. enterocolitica includes parallel use of the following two isolation procedures: (1) Enrichment in peptone, sorbitol and bile salts (PSB) broth for 2–3 days at 22–25°C with agitation or 5 days without agitation; plating on CIN agar directly and after alkaline treatment and incubation for 24 h at 30°C. (2) Enrichment in ITC for 2 days at 24°C; plating on SSDC agar and incubation for 2 days at 30°C.
Conditions for the isolation of Gram-positive, catalase-positive, food-spoilage bacteria are discussed and media for the selective isolation of Micrococcaceae and Brochothrix thermosphacta are described. No selective media are available for the isolation of Microbacterium, Kurthia, Brevibaterium or Propionibacterium spp. An outline scheme for identification is proposed.
The first isolation methods for the detection of Listeria spp. were based on the direct culture of samples on simple agar media, but isolation of the pathogenic Listeria monocytogenes was difficult. In time, new media were developed based on a variety of selective and elective agents in enrichment and isolation enumeration media. The recovery of low numbers of L. monocytogenes from foods and environmental samples requires the use of enrichment cultures followed by selective plating. In this chapter the development of selective media, including so-called differential media on which L. monocytogenes can be distinguished from other, non-pathogenic, species, are discussed. Little attention is paid to pre-enrichment media, recommended for the recovery of injured organisms, because in our opinion the presence of listeriae in products that receive a listericidal treatment is mainly due to post-process contamination. The introduction of chromogenic media, such as agar Listeria according to Ottoviani and Agosti (ALOA) allows the recovery of Listeria and L. monocytogenes from a variety of foods with relative ease.
Recent developments in enrichment and selective media for the isolation and enumeration of “total” Enterobacteriaceae, coliforms, faecal coliforms and E. coli from foods are described and effects of time and temperature of incubation discussed. Coliforms and E. coli are both important indicators of food contamination; both therefore need to be detected in the same medium. Several attempts have been made to detect coliforms and E. coli simultaneously and novel methods have been introduced, based on the detection of β-d-galactosidase (β-D) and β-D-glucuronidase (GUD) using enzymatic methods. Factors to be considered in the selection of media for this group of organisms are noted, based on the results of comparative studies.
Publisher SummaryMannitol lysine crystal violet brilliant green (MLCB) agar is a selective and diagnostic agar for the isolation of salmonellae, other than Salmonella typhi and Salmonella paratyphi, from feces and foods. It is particularly useful when lactose fermenting strains of salmonellae are sought. The inhibitory properties of the medium are conferred by the inclusion of brilliant green and crystal violet. The recognition of Salmonella colonies depends on the fermentation of mannitol and the decarboxylation of lysine together with an indicator system for hydrogen sulfide detection. The medium is not suitable for the isolation of Salmonella typhi or Salmonella paratyphi, because of the inhibitory concentration of brilliant green, or for other brilliant green-sensitive strains. The medium is found to be excellent and with enhanced selectivity for the isolation of hydrogen sulfide positive salmonellae after enrichment in Rappaport–Vassiliadis broth containing soya peptone (RVS) in place of tryptone. Salmonellae grow as large purple-black colonies because of H2S production. Atypical salmonellae that produce little or no H2S grow as mauve-grey colonies and may develop a central black “bull's-eye”. Contaminating organisms grow as small colorless colonies, although some strains of Citrobacter species may mimic the appearance of Salmonella and some Proteus species may swarm.
