Microbiology Growth Media & Culture Techniques PDF

Enriched media contain complex media plus highly nutritious materials (e.g., serum or blood) used to culture fastidious (nutritionally demanding) microbes Selective media contain compounds that selectively inhibit growth of some microbes but not others Differential media...

Enriched media contain complex media plus highly nutritious materials (e.g., serum or blood) used to culture fastidious (nutritionally demanding) microbes Selective media contain compounds that selectively inhibit growth of some microbes but not others Differential media contain an indicator, usually a dye, that detects particular metabolic reactions during growth  Considerations for growth media For successful cultivation of a microbe, it is important to know the nutritional requirements and supply them in proper form and proportions in a culture medium. Cells can be grown in liquid or solid culture media. Solid media are prepared by addition of the gelling agent agar (Figure 5.11) to liquid media. When grown on solid media, cells form isolated masses (colonies).  Properties of bacteria to be utilized for differential media Example: Lactose utalization and anaerobic fermentation in E. coli Lactose being converted to glucose by β-galactosidase Lactate dehydrogenase Under anaerobic fermentative conditions, a β-galactosidase mixture of succinate, formate, acetate, lactate, and ethanol is produced. The products, including e.g. acetate and lactate, are acidic. 29 Front. Bioeng. Biotechnol., 23 May 2014  MacConkey agar A selective (bile salts inhibit most Gram-positive bacteria and crystal violet inhibits certain Gram-positive bacteria) and differential medium (lactose fermentation). Lactose-fermenting bacteria are those that consume lactose or other six-carbon sugars and metabolize them. Acids are produced in lactose-fermentation, reducing the pH thus turning the neutral red dye red. Peptone – 17 g Proteose peptone – 3 g Lactose – 10 g Bile salts – 1.5 g Sodium chloride – 5 g Neutral red – 0.03 g Crystal violet – 0.001 g Agar – 13.5 g Per litre; adjust pH to 7.1 http://www.microbiologyinfo.com/macconkey-agar-composition-principle-uses-preparation-and-colony-morphology/  CHROMagar™ enzyme-based chromogenic detection system Dr. Alain Rambach 1979 In an unrelated field, he pioneers, invents and patents a system for detecting E.coli bacteria by 'chromogenic differentiation' culture media. 1989 He patents a 'chromogenic' system for detection of the Salmonella pathogen, known as Rambach Agar. This is the first commercially available chromogenic culture medium. 1993 Dr. Rambach founds CHROMagar Microbiology. http://www.chromagar.com/  Typical Appearance of microrganisms VRE. faecalis/VRE. faecium → pink to mauve E. gallinarum/E. casseliflavus → blue or inhibited other bacteria → inhibited VRE: Vancomycin-Resistant Enterococci CHROMagarTM E.coli CHROMagarTM VRE β-galactosidase-based Most chromogenic mixes are proprietary. http://www.chromagar.com/  5.5 Growth Media and Laboratory Culture Microbes are everywhere. Sterilization of media is critical. To prevent contamination, aseptic technique should be followed. (Figure 5.12) Figure 5.12 5.6 Microscopic Counts of Microbial Cell Numbers Total cell count microscopic cell count: observing and enumerating cells present dried on slides or on liquid samples counting chambers with squares etched on a slide for liquid samples (Figure 5.13) Can be used on natural samples for studying microbial ecology Various staining methods can be applied (e.g., DNA dye, fluorescent stains, Phylogenetic stains )  Limitations of microscopic cell counts cannot distinguish between live and dead cells without special stains Precision is difficult to achieve small cells are often difficult to see - so difficult for small bacterial cells phase-contrast microscope required if a stain is not used cell suspensions of low density (< 106 cells/ml) hard to count Motile cells need to immobilized Debris in sample can be mistaken for cells