N.49 – Simple tips to facilitate the phenotypic identification of aerobic microorganisms after collection and incubation in Petri dish.

TRIO

SCOPE OF IDENTIFICATION

The purpose of the bacterial air monitoring is not only to count the total number of microorganisms for cubic meter (CFU/cm3), but also to identify the microorganism.

STARTING PROTOCOL

A. The best phase to pick up a colony from the agar surface for identification is the “LOG” phase (not the “LAG” and “Stationary”), because they are behavior properly being at the top of the health.

B. THE “LAG” phase is conditioned by 3 different situations: a. Environmental condition (e.g. temperature, media, pH, salt, etc.); b. Kind of bacteria (e.g. slow or fast growing.); c. Stress. The stress of bacteria is the most important factor to consider and overcame.

C. The production and Q.C. people of the pharma or food company are interested to destroy the bacterial population and therefore consider the stress in positive way. The microbiologist that intend to identify the microorganism has a total different point of view. His goal is in fact to help the microorganism to grow well to facilitate the correct identification.

D. To help the bacterial population it is necessary to evaluate the condition of the colonies on the agar surface and note morphology, rate of growing, color, etc. In order to understand to whom  we are working with and consider the following condition of growth.

WRONG OR IMPOSSIBLE MICROBIAL IDENTIFICATION

When you select a colony from the agar surface of a Petri dish or Contact plate, two are the mainly reasons of identification problems: A. Stress; B. Mixing colonies. They represent almost  80% of the cases.

Some other reasons are: Bacterium is not in the Database or Bacterium mutations.

E. How to help the bacteria to recover from stress. If it is necessary a sub-culture, the passages should “not less than two, no more than five”. If the colonies are not very well separated, adopt a new medium or consider different streaking technique. Transfer the colony on the agar surface of a media like Blood Agar. TSA is not always the best medium for this purpose. The streaking of the colonies should be performed with a low amount of bacteria to avoid mixed colonies and that will fight for the “food” too producing further stress. If the separation is still not good, made a second streaking on another Blood Agar plate.

Do not waste the Petri dish with Blood Agar but save it for 2-3 days at room temperature (not in the refrigerator), just if you did not reach good identification.

In case you are thinking to have mix colonies, it is suggested to prepare two vials with 10 or 5 ml of peptone water.  Transfer a colony from the saved Petri dish in 10 ml of peptone water, vortex per 1 minute, transfer 1 or 0,5 ml from the first vial in the second vial, vortex per 1 minute. Spread on Blood Agar, incubate and repeat the test.

IDENTIFICATION

The specimen  is now ready for identification (e.g. Biolog). If the identification will not be possible, the possible reason could be the incomplete data base, mutant strains or the adopted procedures.

I suggest to evaluate also the quality of the Identification proposed from your system, sometime they are not correct.

Look for system are providing you as much Identification details as possible.

REMEMBER

Bacteria are living cells and they are doing not want the microbiologist want, but what they like to do! And Chemists sometime are not accepting that!

REFERENCE

Fabio Zenna lecture.