In this lecture we shall be looking into the aspects of culturing microorganisms, the different types of culture media, isolation of pure cultures.
In the laboratory, bacteria are normally grown or cultured in either liquid medium, in flasks, bottles or large culture vessels called fermenters or solid medium in Petri dishes which are round, normally plastic or glass dishes. Introduction of microbes into or onto these media is called inoculation. The nature of the medium depends on the microorganism's natural environment because its nutrient requirements reflect its natural surroundings. The culture medium must contain all the nutrients that the microorganism requires for growth like water, a source of energy, carbon, nitrogen, essential inorganic ions and a number of trace elements. Bacteria that can synthesize all they require from the basic ingredients are called Prototrophs – and most microorganisms that survive in the outside environment can do this. Microbes that have become adapted to life in a situation rich with nutrients such as human body may require other growth factors like vitamins, amino acids or nitrogenous bases to be provided. These are called auxotrophs.
The media that are used in microbiology laboratories to culture bacteria are referred to as artificial media or synthetic media, because they do not occur naturally; rather they are prepared in the laboratory. There are number of ways of categorizing the media, one way is to classify based on whether the exact contents of the media are known or not.
Chemically defined media : Is one in which all the ingredients are known; and was prepared in the laboratory by adding a certain number of grams of each of the components (Carbohydrates, amino acids, salts etc). Particularly photolithoautotrophs such as Cyanobacteria and eukaryotic algae can be grown on relatively simple media containing CO2 as a carbon source (often added as sodium carbonate or bicarbonate), nitrate or ammonia as a nitrogen source, sulfate, phosphate and a variety of minerals. Chemoorganoheterotrophs can be grown in a defined media with glucose as a carbon source and an ammonium salt as a nitrogen source.
Complex media : contains undefined ingredients and the exact contents are not known. Complex medium may be sufficiently rich and complete to meet the nutritional requirements of many different microorganisms. They contain undefined components like peptones, meat extract and yeast extract. Peptones – are proteins hydrolysates prepared by partial proteolytic digestion of meat, casein, soyameal, gelatin and other protein sources (Carbon, energy and nitrogen). Beef extract – aqueous extracts from lean beef and contain amino acids, peptides, nucleotides, organic acids and minerals and vitamins.Yeast extract – from brewer's yeast and contain an excellent source of B vitamins, nitrogen and carbon compounds. Three commonly used complex media are
1. Nutrient broth – Peptone (5.0g/L), Beef Extract (3.0g/L)
2. Tryptic soya broth – Tryptone (enzyme digest of casein), Peptone (enzyme digest of soybean meal), glucose, NaCl and K2HPO4
3. MacConkey Agar – Pancreatic digest of gelatin, casein, peptic digest of animal tissue, bile salts mixture, NaCl, neutral red, crystal red and agar.
These media are routinely used for cultivation of bacteria in the laboratory and particularly useful for cultivation of bacteria whose growth requirements have not been defined.
Culture media can also be categorized as liquid or solid. Liquid media (also called broths) are contained in tubes, flasks and fermenters. Solid media are prepared by adding agar to liquid medium and then pouring the media into tubes or Petridishes where the media will solidify. Agar is a complex polysaccharide that is obtained from red marine algae. Other solidifying agents are gelatin, silica. A 1% or 2% agar can be used to solidify, but 1.5% is the most commonly used.
Enriched Medium : Is a broth or solid medium containing a rich supply of special nutrients that promotes the growth of fastidious organisms (that have complex nutrition and environmental requirements). It is usually prepared by adding extra nutrients to a medium called nutrient agar. Blood agar (nutrient agar + 5% sheep red blood cells): It is bright red in color and distinguishes between the hemolytic and non-hemolytic bacteria ( Streptococci and other pathogens)
Chocolate agar (nutrient agar + powdered hemoglobin): It is brown in color and is considered more enriched than blood agar as hemoglobin is more readily accessible in chocolate agar. Pathogens like Neisseria gonorrhoeae and Haemophillus influenza, which will not grow on blood agar, can be cultured.