Lesson 10 Bacterial Growth 3

Some bacteria grow with or without the presence of oxygen, these are facultative. Bacteria that can be grown on solid medium (agar, a non nutrient polysaccharide gelling agent extracted from seaweed) will form colonies, the shape, colour, texture etc of these colonies will vary with temperature and the type of medium used, but within controlled conditions the nature of a colony on visual inspection, accompanied with simple colour reactions for oxidase and other enzymes, can give good preliminary clues as to what type of bacterium is present when combined with the performance of a Gram stain. Proper formation of colonies requires that few enough bacteria are applied to the medium so that one can be statistically certain that the colony grew from a single bacterium, if you don’t do this properly it is possible to get more than one type of bacterium forming a mixed colony in the same spot and this can lead to confused interpretation. Bacteria have specific nutritional requirements, and there is wide variation as to what any given bacterial species needs in its “diet”. I will discuss this more in class. The differences in the ability of bacteria to utilize given chemicals as nutrient forms the basis of diagnostic tests used to identify their species (taxonomic analysis). Many bacterial (and fungal) organisms cannot be grown on general laboratory culture media, some will not grow on any kind of medium. Some microbes are fastidious, they have special nutritional needs that are not met by standard growth media, some bacteria need high salt conditions to grow, these are called halophiles. Many bacteria will not grow on standard culture plates because they are strict anaerobes and have to be grown in an oxygen free atmosphere. Some bacteria are described as fastidious because they need a specialized growth medium, as with the bacteria that cause gonorrhea, they grow best on a blood based medium. The point here is that one must NOT assume that use of standard media will allow growth of the bacteria that may be responsible for a particular problem or infection, careful analysis of the situation has to take place so that other tests and growth conditions can be applied, if there is a suspicion that a causative organism may not show up using the faster and more convenient aerobic nutrient agar and other media. Bacteria can be grown in liquid medium, or more conveniently on the surface of a growth medium gelled with a seaweed polymer called agar. Agar is a polysaccharide purified from certain marine algae, and it has an unusual property that makes it ideal for use as a solid growth base. When agar powder is placed in water it is insoluble until the water is brought to boiling point, when it will melt and dissolve into solution (agar is very expensive, but you only need 1.5 to 2% in the medium). This molten agar based solution will NOT set solid until the temperature drops to below about 44 C. The great advantage of agar is that once it has set, it will NOT melt until it is heated all the way up to boiling point again. What this means is that there is a wide temperature range at which the agar based medium will remain solid and stable. This was not the case with other media used early on in microbiology, such as gelatin, which would form a gel base, but this would melt if the temperature was raised much more than a few degrees. Agar itself is NOT a nutrient in microbiological growth media, if it was, it would be no use because it would break down as colonies grow on it. It is the substances that are dissolved in solution along with the agar that are the nutrients, agar just provides a stable nutrient containing gel platform on which colonies can grow. Complex growth media contain rich organic materials such as beef extract, yeast extract, peptone (hydrolysed beef), malt extract etc, these are rich in the nutrients many bacteria need. They are called complex media because, while we know from experience that they are good substances for growth media, they contain many different compounds and we are not always sure of the exact composition of these media. Defined media consist of mixtures of known compounds (salts, glucose, minerals, vitamins etc) in known amounts. Defined media are used to test for the ability of an unknown bacterium to use known compounds, such as nitrogen or carbon compounds as sole sources of that element, and as such, defined media can be used to identify bacteria according to what sources they can and cannot use. Irrespective of whether a growth medium is complex or defined, it is also sometimes possible to compose a medium so that it excludes growth of a given bacterial type (selective medium), or so that it allows different bacteria to grow but differentiates between them in some way (differential medium). Selective media (which may be complex or defined) allow a particular type of bacterium to grow in preference to another, an example is certain media that contain dyes that will selectively allow one kind of bacterium to grow and not another - based on what their Gram type is (Rose Bengal agar for instance, which selects for the growth of yeasts and molds)). Differential media (which may be complex or defined) allow more than one type of bacterium to grow but identify one of the bacterial colonies - usually by means of a colour change caused by a chemical reaction that a particular bacterium can undertake but the others on the plate cannot, an example of this is EMB agar (Eosin-Methylene Blue) which specifically gives colonies of E. coli a metallic dark sheen, while other colonies of bacteria are darkish but do not have the metallic sheen. Strictly anaerobic organisms have to be grown in the absence of oxygen, I will discuss this further in class or tutorial