Bacterial Culture Media: Classification, Types, Uses
Culture media contains nutrients and physical growth parameters necessary for microbial growth. If a culture medium meets a bacterial cell’s growth requirements, then that cell will multiply to sufficient numbers to allow visualization by the unaided eye.
All microorganisms cannot grow in a single culture medium and in fact, many can’t grow in any known culture medium. Organisms that cannot grow in the artificial culture medium are known as obligate parasites. Mycobacterium leprae, Rickettsia, Chlamydia, and Treponema pallidum are obligate parasites.
Classification of Bacterial Culture Media
Bacterial culture media can be classified on the basis of composition, consistency, and purpose.
On the Basis of Consistency
Solid culture media contain agar at a concentration of 1.5-2.0% or some other mostly inert solidifying agent. Solid medium has a physical structure and allows bacteria to grow in physically informative or useful ways (e.g. as colonies or in streaks). MacConkey agar, chocolate agar, nutrient agar, blood agar, etc are some examples of solid culture media.
Uses of solid culture media
- For isolating bacteria from various types of specimen
- For determining the colony characteristics of the isolate (such as colony morphology, hemolysis, pigment production, etc.
- For performing antimicrobial susceptibility testing using Kirby Bauer disc diffusion method
Semisolid culture media are prepared with agar at concentrations of 0.5% or less. Semisolid medium has a soft custard-like consistency and is useful for the cultivation of microaerophilic bacteria or for the determination of bacterial motility. Motility test medium, Stuart’s and Amies transport media, etc are semisolid media.
Liquid (Broth) Medium
These media contain specific amounts of nutrients but don’t have a trace of gelling agents such as gelatin or agar. Commonly used liquid media in the lab are; nutrient broth, glucose broth, brain-heart infusion broth, alkaline peptone water, and selenite F broth. Broth medium serves various purposes such as propagation of a large number of organisms, fermentation studies, and various other tests.
Uses of liquid culture media
- To grow bacteria for motility testing
- To grow bacteria for inoculum production for antibiogram testing
- To revive bacteria from lyophilized or stock culture
- To study metabolism, toxin, and enzyme production
- To enrich and/or transport clinical material
On the Basis of Functional Use
Many special-purpose media are needed to facilitate the recognition, enumeration, and isolation of certain types of bacteria. To meet these needs, numerous media are available.
Bacteria with complex nutritional requirements are called fastidious bacteria. Special nutritional supplements must be added to culture media to grow such bacteria. Bacteria having relatively basic and straightforward nutritional requirements are easy to grow in a lab, they are known as nonfastidious bacteria.
Basal media also called general-purpose media are basically simple media that support the growth of most non-fastidious bacteria. Peptone water, nutrient broth, and nutrient agar (NA) are considered basal media. These media are generally used for the primary isolation of microorganisms.
Addition of extra nutrients in the form of blood, serum, egg yolk, etc, to the basal medium makes an enriched medium. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope, etc are a few examples of enriched media. Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar.
Selective and Enrichment Media
These media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogens from a mixture of bacteria. While selective media are agar-based, enrichment media are liquid. Both these media serve the same purpose. Any agar media can be made selective by the addition of certain inhibitory agents that don’t affect the pathogen of interest. Various approaches to making a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH, or a combination of these.
Principle: Differential growth suppression
Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others. Selective medium is an agar-based (solid) medium so that individual colonies may be isolated.
Examples of selective media include
- Thayer Martin Agar used to recover Neisseria gonorrhoeae contains antibiotics; vancomycin, colistin, and nystatin.
- Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
- Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium tellurite.
- MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits most gram-positive bacteria.
- Pseudosel Agar (cetrimide agar) used to recover Pseudomonas aeruginosa contains cetrimide (antiseptic agent).
- Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
- Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating malachite green.
- Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye brilliant green.
- Selective media such as TCBS Agar used for isolating Vibrio cholerae from fecal specimens have elevated pH (8.5-8.6), which inhibits most other bacteria.
Enrichment medium is used to increase the relative concentration of certain microorganisms in the culture prior to plating on a solid selective medium. Unlike selective media, enrichment culture is typically used as a broth medium. Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth, and alkaline peptone water (APW) are used to recover pathogens from fecal specimens.
Differential/ Indicator Media
Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony color. Various approaches include incorporation of dyes, metabolic substrates, etc, so that those 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 of interest but with morphologically distinguishable colonies.
Examples of differential media include:
- Mannitol salts agar (mannitol fermentation = yellow)
- Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
- MacConkey agar (lactose fermenters, pink colonies whereas non-lactose fermenter produces pale or colorless colonies.
- TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals and maintain the viability of the potential pathogens. This can be achieved by using transport media. Such media prevent drying (desiccation) of a specimen, maintain the pathogen to commensal ratio, and inhibit the overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid. The addition of charcoal serves to neutralize inhibitory factors.
- Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to transport feces from suspected cholera patients.
- Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery.
- Pike’s medium is used to transport streptococci from throat specimens.
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation-reduction potential, and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. The addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine, or red hot iron filings can render a medium reduced. Before using, the medium must be boiled in a water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin.
Robertson Cooked Meat (RCM) medium that is commonly used to grow Clostridium spp 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.
Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated into the medium. Under the reduced conditions, methylene blue is colorless.
These media are used for the assay of vitamins, amino acids, and antibiotics. For e.g., antibiotic assay media are used for determining antibiotic potency by the microbiological assay technique.
Other types of medium include;
- Media for enumeration of bacteria,
- Media for characterization of bacteria,
- Maintenance media etc.