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Lecture Ten

Microbial Culture


Broth or Agar
Culture can be grown in either a liquid medium or n an agar gel. Both can be dispensed in a tube and the agar can be in a petri dish.
The advantage of an agar is that bacteria can form individual colonies on the agar and make it easier to obtain pure culture. It is easier to see if there are any contaminants growing in or on the agar.
A tube culture can be sealed easier than a petri dish (plate culture) and allows longer storage with out desiccation.
The manner in which a bacterium grown in either broth or plate can be used as an identification feature.
Agar in tubes can be prepared as deeps (ie with a horizontal upper surface) or as slants (ie with a sloping upper surface. The advantage of the slant is that is provides a larger area for aerobic growth.
A deep or slant can be used to observe growth characteristic on the surface and near the bottom of the tube (ie in the "deep")
Maintenance Culture:
Optimum Conditions
Generally when maintaining a culture we want it to retain its original character. (ie. not to mutate.) So the medium is produced with all conditions as close as possible to the optimum for the organism being stored.
Mutations can re reduced if the cell division can be restricted. This can be done by refrigeration. For longer storage refrigeration below 0 C can be obtained with out freezing by adding glycerine as an anti-freeze.
Silica Gel:
Maintaining culture over a longer time with out them drying out can be achieved by using silica gel instead of agar for plate cultures.
Freeze-dried Cultures
For really long time storage freeze-drying is used to stop all growth and the culture is stored in an evacuated capsule to stop oxidation.
Maintenance media
Maintenance media is media provided at optimum conditions for growth and may have extra ingredient added if the bacteria is fastidious
Selective Culture:

To isolate one bacterium from another we have to use the differences in their ranges of tolerance. And pick a set of conditions that allows one to grow but not another. There is no use in using optimum conditions for selection because even if one species grows faster than anther they may both grow and so no selection is obtained.

A selection procedure may exploit difference in minimum or maximum tolerance for temperature, pH,

nutrients, toxins and water activity. In all cases it is not using the optimum for any organism and will disadvantage all of them, just some less than others.

Enrichment media is media usually a broth that is used to select one type of bacteria and reduce the population of others so the community is enrich on regard to one population. The selective agent may be selenium, bismuth or a dye etc. If the organism to be selected is only present in small numbers it is possible that they will be killed of and a negative result will result. One solution is to incubate the sample to be examined in a non-selective medium. This is called Pre-selection. A simple nutrient broth may be used.

Indicative culture
The enzyme produced by bacteria make changes to the medium on which they grow. They may also produce product of metabolism. Some of these products and changes can be detected by including indicators in the medium or adding them after growth has occurred.
Eg. Enzymic effects looked at:
Starch hydrolysis detected with iodine
Fat hydrolysis detected with smell
Protein hydrolysis detected by visual appearance
Citric acid utilisation detected with pH indicators
Acid production detected with pH indicators
Gas production detected with inverted tubes
Indole production detected with indicators
Media used for these indicating reactions is called Indicating Media

Prepared by Barry Brazier