“Although a few bacteria are so morphologically remarkable as to make them identifiable without isolation, pure cultures are nearly always a necessity before one can attempt identification of an organism. It is important to realize that the single selection of a colony from a plate does not assure purity.”Bergey’s Manual of Systemic Bacteriology, vol. I Testing pure cultures is essential to the success of the any microbial identification system. Very often live but non-growing contaminants may be present in, or near a colony and can be sub-cultured along with the chosen organism. This is the reason that non-selective media are preferred for final isolation.

A big difficulty occurs with bacteria that form extracellular slime; many contaminants often become firmly embedded or entrapped in this slime/mucus and are very difficult to separate.

Mixed cultures that are processed as though they are pure most often produce poor results and cause much frustration and extra work. There are some key elements that can reduce the likelihood of using a mixed culture when processing a bacterial sample.

How can I be sure that I have a pure culture and species?
It has been said that a mistake in determination of shapes, Gram stain, colony morphology, pigment and many other characteristics of the species is the most frequent cause of mistaken identity of bacteria.

There are three preliminary actions to follow in order to aid in the classification of bacteria.
1: be sure that one colony has been selected when streaking on the appropriate media.
2: perform a Gram stain.
3: perform other tests required to classify the organism in the corresponding category.

Key #1: How do I proceed when cultures are not pure?
A. Be sure to select one colony and transfer it into 5 ml of sterile water or saline solution.
B. Mix well, place a very small drop of the suspension onto BUG+B media and proceed to streak the plate to obtain isolated colonies.
C. After twenty-four hours of incubation check again for purity. Never assume that the culture is pure. Repeat the procedure as many times as necessary until a pure culture has been obtained before processing the bacteria. This may seem like more work than it is necessary but it will save time and materials if tests do not have to be repeated.

Key #2: After you have a pure culture.
Select colonies from the last (third or fourth) quadrants of the agar plate. This will minimize the chance of a contaminant being carried over in the final step of inoculation. There will be times when bacteria from the second quadrant must be used but this should be done only when necessary (slow growing organisms).

Key #3: If you are attempting to identify environmental organisms, you may find several different species of bacteria in your initial sample.
Many bacteria utilize the nutrients of other bacteria to grow and synergistically stick to each other. When your sample is initially grown on rich media, numerous types of bacteria grow even if your plate appears to be pure. This is where Key #1 is extremely important because isolating one colony and sub-culturing it three times will drastically increase the odds for a pure culture and a correct identification.

Key #4: Check the plate for several different types of bacteria.
Frequently one finds that it is necessary to leave cultures for more than two days at room temperature in order to see differences in morphology. Look for different colonies using a colony magnifier lamp. Look for colonies growing within the colony in question, different colors, sizes, colony edges, and textures.

Key #5: Remember that bacteria are everywhere, including the bench top, doorknobs, hands, hair and air.
To reduce contamination, all aspects of processing a specimen should be done using aseptic technique. Everything that touches the bacteria should be sterile. Every time the top of the petri dish containing the sample is opened, contamination is possible. Try not to talk when the agar plate is opened, as respiratory isolates are often contaminants.

Using these keys will help to achieve a correct identification. While they may seem time-consuming and tedious, they greatly reduce the chance of having to repeat the test, which costs money as well as time.
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