Culture media use in microbiology
- 1. Culture Media use in microbiology By:Assist.Prof.Chaital Kalamkar.S Arihant COP, Ahmadnagar
- 2. Content.... • Definition • Silent features • History • Classification
- 3. Microbiological Culture • Microbiological culture A microbiological culture, or microbial culture, is a method multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. • Culture media The base where microorganisms can grow with the help of nutrients and under the influence of physical growth parameters. All microorganisms cannot grow in a single culture medium . • Obligate parasite Some organisms cannot grow in artificial culture medium are known as obligate parasites. Examples: Mycobacterium leprae, Rickettsias, Chlamydias, Treponema pallidum
- 4. Silent feature of Bacterial culture... • Bacteria have to be grown (cultured) for them to be subsequent clinical diagnosis. • Culturing bacteria is also the initial step in studying its morphology and it’s identification. • By appropriate procedure they have to be grown separately (isolated)on culture media and obtained as pure for study. • Bacteria have to be cultured in order to obtain antigens from developing serological assay for vaccines. • Certain genetic studies and manipulation of the cells also need that bacteria be cultured in vitro. • Culturing on solid media is another convention way of separating bacteria in mixture.
- 5. History • Louis Pasteur used simple broths made up of urine or meat extract. • Robert Koch realized the importance of solid Media and used potato pieces to grow Bacteria • It was on the suggestion of Fannie Eilshemius,wife of Walther Hesse(who was an assistant to Robert Koch) that agar was used to solidify culture media. • Before the use of agar , attempt were made to use gelatin as solidify agent. • Gelatin had some inherent problem. • It existed as liquid at normal incubating temperature (35-37°C) • Digested by certain Bacteria
- 7. A.Culture media based on consistency • Solid media: Solid media contains agar (1.5-2.0%) as solidifying agent and suitable for isolation of bacteria and determination of colony characteristics. Examples: Nutrient agar, BAP etc. • Semi-solid media Semi-solid media also contains agar but the percentage content is less (0.5%). This media is useful for determination of motility of bacteria and cultivation of microaerophilic bacteria.
- 8. A.Culture media based on consistency • Liquid media This media is useful for determination of motility of bacteria and cultivation of microaerophilic bacteria. Liquid media that do not contains agar except for required amount of nutrients. This media is useful for propagation of large number of organisms, fermentation studies and various tests (Sugar fermentation test).
- 9. B. Culture media based on composition • Synthetic medium: • Chemically defined • Contain Pure organic and inorganic compounds • Exact formula (little variation) • Example: Peptone water(1%of Peptone and 0.5% Nacl in water) • Non synthetic • Complex Media • Containn at least one ingredients that is not chemically defined(extract from plant and animal) • No exact formula/tend to be general and grow wide variety of organisms. • Example: Blood agar,yeast extract broth,milk agar
- 10. C.Culture media based on applications 1. General purpose media/Basic media: Basal media are simple media that supports most non-fastidious bacteria and are used for the isolation of primary microorganisms. Examples:Peptone water, nutrient broth and nutrient agar are considered as basal medium. 2. Enriched medium: Addition of extra nutrients in the basal medium in form of blood, serum, egg yolk etc. to make the medium enriched. They are used to grow nutritionally exacting bacteria. Examples: Blood agar, chocolate agar etc.
- 11. C.Culture media based on applications 3.Selective media: • They are designed to inhibit unwanted contaminating bacteria and help to recover pathogen from a mixture of bacteria. • Selective media are agar based and enrichment media are liquid in consistency. • Addition of antibiotics, dyes, chemicals, alteration of pH etc. and makes a medium selective.
- 12. C.Culture media based on applications 3.Selective media • They are of various types like: 1. Thayer Martin Agar: It is used to recover Neisseria gonorrhoeae contains antibiotics; vancomycin, colistin and nystatin. 2. Mannitol salt agar and salt milk agar: They are used to recover S. aureus; contains 10% NaCl. 3. Potassium tellurite: This medium is used to recover C. diphtheriae; contains 0.04% potassium tellurite. 4. Mac Conkey’s Agar: It is used for Enterobacteriaceae members contains bile salt that inhibits commonly gram positive bacteria.
- 13. C.Culture media based on applications 3.Selective media 5. Pseudosel Agar or Cetrimide Agar: It is used to recover Pseudomonas aeruginosa contains cetrimide (antiseptic agent). 6. Crystal Violet Blood Agar: It is used to recover Streptococcus pyogenes contains 0.0002% crystal violet.
- 14. C.Culture media based on applications 3.Selective and enrichment media 7. Lowenstein Jensen: This medium is used to recover M. tuberculosis is made selective by incorporating malachite green. 8. Wilson and Blair’s Agar: It is used for recovering Salmonella typhi is rendered selective by the addition of dye brilliant green.
- 15. C.Culture media based on applications 4.Enrichment Culture Medium: • Enrichment medium, is liquid media, used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective medium. It is mainly used as broth medium. • Example: Selenite F broth, tetrathionate broth, alkaline peptone water are used to recover pathogens from fecal specimens.
- 16. C.Culture media based on applications 5.Differential/Indicator Medium: • In this medium dyes, metabolic substrates etc. are added so that the bacteria that utilize them appear as differently colored colonies. Such media are called differential media or indicator media. • Differential media allow the growth of more than one microorganism but with morphologically distinguishable colonies.
- 17. C.Culture media based on applications 5.Differential/Indicator Medium: • Examples: 1. Mannitol Salt agar(mannitol fermentation = Gram positive bacteria Staphylococcus aureus produces yellow colonies with yellow zones. 2. Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis). 3. Mac Conkey Agar (lactose fermenters, pink colonies whereas non-lactose fermenter produces pale or colourless colonies. 4. TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose).
- 18. C.Culture media based on applications 6.Transport media: • These media are used when specimen cannot be cultured soon after collection. This media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. • Example: Cary Blair medium, Amie’s medium, stuart’s medium etc. Stuart’s and Amie’s media are semi-solid media in consistency. • Addition of charcoal serves to neutralize inhibitory factors. Cary Blair transport medium is used to transport feces from suspected cholera patients. • Pike’s medium is used to transport streptococcii from throat specimens.
- 19. C.Culture media based on applications 7.Anaerobic media: • Anaerobic media is mainly supplemented with hemin and vitamin K where bacteria need Low oxygen content, reduced oxidation–reduction potential and extra nutrients for their growth. • The media is also contains 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings. • Before use the medium is to be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin. • Example: Robertson Cooked Meat (RCM) medium is commonly used to grow Clostridium spps. contains a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. • Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate.
- 20. C.Culture media based on applications 8.Assay Media: • These media is used for assay of amino acids, vitamins and other antibiotics. • Antibiotic assay media are used for determination of antibiotic potency (by microbial assay).