Bacterial Culture Media: Definition, Composition and Classification

Bacterial Culture Media

• Bacterial culture media are nutrient-rich substances designed to support the growth, proliferation, and maintenance of bacterial populations in a controlled laboratory environment.
• One of the most important reasons for culturing bacteria in vitro is its utility in diagnosing infectious diseases. Isolating a bacterium from sites in the body normally known to be sterile is an indication of its role in the disease process.
• Culturing bacteria is also the initial step in studying its morphology and its identification.
• Bacteria have to be cultured in order to obtain antigens for developing serological assays or vaccines.
• Certain genetic studies and manipulations of the cells also need bacteria to be cultured in vitro.
• Culturing bacteria also provides a reliable way for estimating their numbers (viable count).

Composition of Culture Media

• Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs.
  When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. Hence, an artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat.
• Most often, a culture medium contains water, a source of carbon and energy, a source of nitrogen, trace elements, and some growth factors. Besides these, the pH of the medium must be set accordingly.
• Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract, and malt extract.

Classification of Bacterial Culture Media

Bacterial culture media can be classified in at least three ways:

1. Based on consistency
2. Based on nutritional component
3. Based on its functional use

1) Classification based on consistency

• On the basis of consistency, culture media are liquid, semi-solid, solid, and biphasic.

a) Liquid media

• These are available for use in test tubes, bottles, or flasks.
• Liquid media are sometimes referred to as “broths” (e.g., nutrient broth).
• In liquid medium, bacteria grow uniformly, producing general turbidity.
• Liquid media tend to be used when a large number of bacteria have to be grown.
• Inoculating the bacteria in liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures.
• Culturing in liquid media can be used to obtain viable counts (dilution methods).
• Properties of bacteria are not visible in liquid media, and the presence of more than one type of bacteria cannot be detected.

b) Solid media

• Any liquid medium can be rendered solid by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae (of the genera Gelidium).
• Agar is composed of two long-chain polysaccharides (70% agarose and 30% agaropectin).
• Agar melts at 95°C (sol) and solidifies at 42°C (gel), doesn’t contribute any nutritive property, is not hydrolyzed by most bacteria, and is usually free from growth-promoting or growth-retarding substances. However, it may be a source of calcium & organic ions.
• Most commonly, it is used at a concentration of 1-3% to make a solid agar medium.

c) Semi-solid agar

• Reducing the amount of agar to 0.2-0.5% renders a medium semi-solid.
• Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (with the help of U-tube and Craigie’s tube).

d) Biphasic media

• Sometimes, a culture system comprises both liquid and solid medium in the same bottle. This is known as biphasic medium (e.g., Castaneda system for blood culture).
• The inoculum is added to the liquid medium, and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium.

2) Classification based on nutritional component

• On the basis of nutritional component, media can be classified as simple, complex, and synthetic (or defined).
• While most of the nutritional components are constant across various media, some bacteria need extra nutrients.
  • Bacteria that are able to grow with minimal requirements are said to be non-fastidious.
  • Those that require extra nutrients are said to be fastidious.
• Simple media such as peptone water and nutrient agar can support most non-fastidious bacteria.
• Complex media such as blood agar have ingredients whose exact components are difficult to estimate.
• Synthetic or defined media, such as Davis & Mingioli medium, are specially prepared media for research purposes where the composition of every component is well known.

3) Classification based on functional use or application

These include: basal media, enriched media, selective/enrichment media, indicator/differential media, transport media, and anaerobic media.

a) Basal media

• Basal media are basically simple media that support most non-fastidious bacteria.
• Peptone water, nutrient broth, and nutrient agar are considered basal media.

b) Enriched media

• Addition of extra nutrients in the form of blood, serum, egg yolk, etc., to basal medium makes them enriched media.
• Enriched media are used to grow nutritionally exacting (fastidious) bacteria.
• Examples include blood agar, chocolate agar, Loeffler’s serum slope, etc.

c) Selective and enrichment media

• Selective and enrichment 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 in consistency. 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. Various approaches to make a medium selective include the addition of antibiotics, dyes, chemicals, alteration of pH, or a combination of these.
• Examples: Selenite F broth, tetrathionate broth, and alkaline peptone water are examples of enrichment media used to recover pathogens from fecal specimens.

d) Differential media or 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 the 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.
• Examples: MacConkey’s agar, CLED agar, TCBS agar, XLD agar, etc.

e) Transport media

• Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media.
• Such media prevent drying (desiccation) of specimens, maintain the pathogen-to-commensal ratio, and inhibit overgrowth of unwanted bacteria.
• Examples:
  • Stuart’s & Amie’s medium,
  • Cary Blair medium,
  • Venkatraman Ramakrishnan medium,
  • Sachs buffered glycerol saline, etc.

f) Anaerobic media

• Anaerobic bacteria need special media for growth because they require:
  • 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.
• Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine, or Red-hot iron fillings can render a medium reduced.
• Before use, the medium must be boiled in a water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin.
• Methylene blue is an oxidation-reduction potential indicator that is incorporated in the thioglycollate medium. Under reduced conditions, methylene blue is colourless.