Culture media

CULTURE MEDIA

A significant number of common microorganisms are capable of synthesizing all the materials they need for growth (e.g. amino acids, nucleotides and vitamins) from simple carbon and nitrogen sources and mineral salts. Such organisms can grow on truly synthetic (chemically defined) media, but many organisms do not have this capability and need a medium that already contains these biochemicals. Such media are far more commonly used than synthetic ones, and several terms have been used to describe them, e.g. routine laboratory media, general purpose media and complex media. They are complex in the sense that their precise chemical composition is unknown and likely to vary slightly from batch to batch. In general, they are aqueous solutions of animal or plant extracts that contain hydrolysed proteins, B group vitamins and carbohydrates.

Readily available and relatively inexpensive sources of protein include meat extracts (from those parts of animal carcasses that are not used for human or domestic animal consumption), milk and soya. The protein is hydrolysed to varying degrees to give peptones (by definition not coagulable by heat or ammonium sulphate) or amino acids. Trypsin or other proteolytic enzymes are preferred to acids as a means of hydrolysis because acids cause more amino acid destruction; the term ‘tryptic’ denotes the use of the enzyme. Many microorganisms require B-group vitamins (but not the other water or fat-soluble vitamins required by mammals) and this requirement is satisfied by yeast extract. Carbohydrates are used in the form of starch or sugars, but glucose (dextrose) is the only sugar regularly employed as a nutrient.

Microorganisms differ in terms of their ability to ferment various sugars, and their fermentation patterns may be used as an aid in identification. Thus, other sugars included in culture media are normally present for these diagnostic purposes rather than as carbon and energy sources. Sodium chloride may be incorporated in culture media to adjust osmotic pressure, and occasionally buffers are added to neutralize acids that result from sugar metabolism. Routine culture media may be enriched by the addition of materials like milk, blood or serum, and organisms that need such supplements in order to grow are described as ‘exacting’ in their nutritional requirements.

Culture media may be either liquid or solid; the latter term describes liquid media that have been gelled by the addition of agar, which is a carbohydrate extracted from certain seaweeds. Agar at a concentration of about 1–1.5% w/v will provide a firm gel that cannot be liquefied by the enzymes normally produced during bacterial growth (which is one reason it is used in preference to gelatin). Agar is unusual in that the melting and setting temperatures for its gels are quite dissimilar. Fluid agar solutions set at approximately 40 °C, but do not reliquefy on heating until the temperature is in excess of 90 °C. Thus agar forms a firm gel at 37 °C which is the normal incubation temperature for many pathogenic organisms (whereas gelatin does not) and when used as a liquid at 45 °C is at a sufficiently low temperature to avoid killing microorganisms—this property is important in pour plate counting methods (see section 5).

In contrast to medium ingredients designed to support microbial growth, there are many materials commonly added to selective or diagnostic media whose function is to restrict the growth of certain types of microorganism while permitting or enhancing the growth of others. Examples include antibacterial antibiotics added to fungal media to suppress bacterial contaminants, and bile to suppress organisms from anatomical sites other than the gastrointestinal tract. Many such additives are used in media for organism identification purposes, and these are considered further in subsequent chapters. The term enrichment sometimes causes confusion in this context. It is occasionally used in the sense of making a medium nutritionally richer to achieve more rapid or profuse growth. Alternatively, and more commonly, an enrichment medium is one designed to permit a particular type of organism to grow while restricting others, so the one that grows increases in relative numbers and is ‘enriched’ in a mixed culture.

Solid media designed for the growth of anaerobic organisms usually contain nontoxic reducing agents, e.g. sodium thioglycollate or sulphur-containing amino acids; these compounds create redox potentials of −200 mV or less and so diminish or eliminate the inhibitory effects of oxygen or oxidizing molecules on anaerobic growth. The inclusion of such compounds is less important in liquid media where a sufficiently low redox potential may be achieved simply by boiling; this expels dissolved oxygen, which in unstirred liquids only slowly re-saturates the upper few millimetres of liquid. Redox indicators like methylene blue or resazurin may be incorporated in anaerobic media to confirm that a sufficiently low redox potential has been achieved.

Media for yeasts and moulds often have a lower pH (5.5–6.0) than bacterial culture media (7.0–7.4). Lactic acid may be used to impart a low pH because it is not, itself, inhibitory to fungi at the concentrations used. Some fungal media that are intended for use with specimens that may also contain bacteria may be supplemented with antibacterial antibiotics, e.g. chloramphenicol or tetracyclines.