There are various reasons why bacteria have to be grown (cultured) in the laboratory on artificial culture media. One of the most important reasons being its utility in diagnosing infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is an indication of its role in the disease process. Indeed, isolating an organism from the clinical specimen is the first step in proving its role as an etiologic agent. Culturing bacteria is also the initial step in studying its morphology and its identification. Bacteria have to be cultured in order to obtain antigens from developing serological assays or vaccines. Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro. Culturing bacteria also provide a reliable way estimating their numbers (viable count). Culturing on solid media is another convenient way of separating bacteria in mixtures.
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 & energy, source of nitrogen, trace elements and some growth factors. Besides these, optimum pH, oxygen tension and osmolarity too have to be taken into consideration.
Ingredients
Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. While tap water is suitable for culture media, it must not be used if it contains high amount of minerals. In such situations, distilled or demineralised water should be used. Peptone is a byproduct of protein (plant or animal) digestion. Proteins are often obtained from heart muscle, casein, fibrin or soya flour and is digested using proteolytic enzymes such as pepsin, trypsin or papain. The final product contains peptones, proteoses and amino acids besides a variety of inorganic salts including phosphates, potassium and magnesium. Casein hydrolysate is obtained from hydrolysis of milk protein casein using HCl or trypsin. Meat extract is obtained by hot water extraction of lean beef and then concentrated by evaporation. Meat extract contains gelatin, albumoses, peptrones, proteoses, amino acids, creatinine, purines, and accessory growth factors. Yeast extract is prepared from washed cells of bakers’ yeast and contains wide range of amino acids, growth factors and inorganic salts. Malt extract is prepared by extracting soluble materials from sprouted barley in water at 55oC and concentrated by evaporation. It contains maltose, starch, dextrin, glucose and small amounts of protein and protein breakdown products and growth factors.
Brief history
Initially, culture media were very simple; Louis Pasteur used simple broths made up of urine or meat extracts. 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, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems; it existed as liquid at normal incubating temperatures (35-37oC) and was digested by certain bacteria.
Classification
can be classified in at least three ways; Based on consistency, based on nutritional component and based on its functional use.
Classification based on consistency:
Culture media are liquid, semi-solid or solid. Liquid media are sometimes referred as “broths” (e.g nutrient broth).
Liquid media are available for use in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits but with a clear medium. Culturing bacteria in liquid media has some drawbacks. Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. Liquid media tend to be used when a large number of bacteria have to be grown. Culture media are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods).
Solid media:
Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. The word "agar" comes from the Malay word agar agar (meaning jelly). It is also known as kanten, China grass, or Japanese isinglass. Agar is chiefly used as an ingredient in desserts throughout Japan. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute any nutritive property, it is not hydrolysed 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 concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders.
For preparing agar in Petri plates, 3% agar (by weight) is added to the broth and autoclaved, when the medium is at
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