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Lecture Eleven

Isolation of Bacteria


Pure Culture Techniques
Attempts to identify bacteria or use them in nearly every application require a pure culture
"Last Drop Dilution Technique"
Louis Pasteur who was the first to show bacteria was the cause of food spoilage (wine spoilage). He used the "last drop dilution technique". This technique involves aseptic transfer of one drip of a culture to a sterile vile of broth. This diluted suspension then shaken and one drop transferred to a new sterile vile of medium. The serial dilution procedure is repeated may be 15 time
All the vials are incubated and examined for growth after 24 hrs. It is expected that the least diluted vials (one with most bacteria) will show growth as a cloudy suspension. The vile that was most dilute should show no growth. It is assumed that the most dilute tube that shows growth is a pure culture. This assumption is based on the idea that it was inoculated with a drop containing only one bacterium
After Incubation
The assumption may not be valid, as there may be a small number of cells. Any number greater than one may spoil the result
Agar Plates
Robert Koch developed a technique using agar plate to isolate in pure culture Bacillus anthracis and went on to prove it caused anthrax. He also discovered Mycobacterium tuberculosis the cause of TB. He invented the idea of using solid media after observing bacteria growing on a slice of potato.
The use of agar was suggested by his wife and has tremendous advantages. It does not melt until 85 C so it can be used to incubate cultures at temperatures higher than the 25 that was the limit when gelatine was used. He also was first to use petri dishes. This round pair of plates was suggested by a Mrs. Petri a lab technician working for Koch.
Streak Plates
Koch was able to obtain pure culture on agar plates by spreading a culture out on the surface of the plate so that the cells were far enough apart to grow into separate colonies. This was doe by passing a sterile wire loop through an inoculated area on the plate and streaking it over a sterile area of the plate.
This procedure is repeated so that the wire loop dilutes the number of cell each pass until they are far enough apart. This is called a Streak plate
First Steak Second streak Third streak
After incubation
Diagram taken from
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.

An example is the Eijkman test for coliforms, Which relies on the difference between the max temperature for growth of E. coli and Enterobacter aerogenes. The later occurs in the soil as well as in faeces and often gives a false presumptive coliform count. E. coli is the organism need to prove faecal contamination because it only occurs in faeces (ie not in soil) E. coli being adapted for a warm environment tolerates a higher temperature than the Enterobacter sp. The test is conducted by incubation at 44 C E. coli will grow but not. aerogenes.

The difference in temperature tolerance is very small and the temperature has to be regulated at 44 ± 0.5

Eijkman Test
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
Multi test Kits
There are kits available that includes 24 or more culture media in small wells on one plate.
These are all inoculated at the one time and 24 indications can be obtained in one incubation
Eg Microbact 24E for Coliforms and APT for staphylococci

The End

Prepared by Barry Brazier