Microbiology Laboratory

Sterilization and Direct Plate Counting Technique

Written by MicroDok

Sterilization techniques is used to make microbial culture media, reagents and other objects or apparatuses in the microbiology laboratory sterile. To make an object sterile simply means to make it germ-free. Several sterilization methods are used in the microbiology laboratory to decontaminate materials in the microbiology laboratory. The autoclave is one of the most important equipment used in the microbiology laboratory to achieve sterilization; and it uses moist heat under pressure to achieve this purpose.

Another piece of equipment used for sterilization in the laboratory is hot-air oven, which uses dry heat to sterilize materials especially glass wares and other heat-stable materials. Sterilization can also be achieved through heating, filtration, radiation and by other chemical and physical means; but the type of sterilization method used is largely dependent on the nature of the material to be sterilized and the level of sterilization to be achieved. This technique is also applied in the food and pharmaceutical industry to ensure the safety and sterility of manufactured goods or products. Check section on “equipment and instrument used in Microbiology” for detailed explanation of the working principles and care of the autoclave. The autoclave¬†is one of the most important equipment used in the microbiology laboratory to achieve sterilization.

DIRECT PLATE COUNTING

Direct plate counting method is a microbiological technique used to evaluate the actual bacterial content of a product or specimen; and it gives an estimate of viable or living cells present in a sample. It is usually carried out by plating an aliquot of the sample on solid agar medium that supports the growth of the bacteria being sought for. The number of viable cells in the sample of interest is assessed from the number of colonies which develop on the solid agar medium after incubation. The colony counts of bacteria may range from < 1000 to > 106 colonies per ml (m-1) of the diluted sample or product. And the counted organisms or colonies on the solid agar plate is usually expressed as colony forming units per ml (cfu/ml). Colony forming units (cfu) is used to express a single cell of bacteria that is capable of producing a single colony because it is generally assumed that each colony on the agar plate developed from a single bacterial cell.

CFU is the microbiological expression of the number of bacteria or fungi in a microbial population (which is usually seen as distinct colonies on solid agar plates); and it is a measure of the viable cells (bacteria and fungi inclusive). While direct microscopy of cells under the microscope may give an estimate of both viable cells and dead cells; CFU (which is obtained via direct plating on solid culture media) measures only viable or living bacterial cells, and the theory behind it is based on the fact that a single bacterium can grow on agar medium and become a colony especially through binary fission. Direct plate count can also be used for counting fungi and other microbial cells excluding viruses. Electron microscopy is used for viral counts, and the counted viral particles are expressed as plaque forming units (pfu).

Spread plating and pour plating are the two methods of direct plate counting techniques for microorganisms (with the exception of viruses). But the pour plate method is more preferable to the spread plate technique when counting microbes because the later (in which the diluted sample is spread over an agar plate) usually produces colonies that only form on the surface of the agar medium. In the pour plate technique, colonies do not just form on the surface of the agar but throughout the agar medium (i.e., in the substratum of the agar medium).

The pour plate technique involves the dispensing of an aliquot of the test sample (usually in a diluted form in the range of 0.1 – 1.0 ml) on a clean Petri dish; and then the sterile molten agar is poured into the plate and the Petri dish is swirled in order to ensure an even mixture of the medium and the inoculum and/or diluted sample. The plate is allowed to set or dry prior to incubation at the appropriate temperature conditions. Other techniques available for the counting of microbes in the microbiology laboratory include microscopy and turbidometric method.

REFERENCES

Atlas R.M (2010). Handbook of Microbiological Media. Fourth edition. American Society of Microbiology Press, USA.

Balows A, Hausler W, Herrmann K.L, Isenberg H.D and Shadomy H.J (1991). Manual of clinical microbiology. 5th ed. American Society of Microbiology Press, USA.

Basic laboratory procedures in clinical bacteriology. World Health Organization (WHO), 1991. Available from WHO publications, 1211 Geneva, 27-Switzerland.

Black, J.G. (2008). Microbiology:  Principles and Explorations (7th ed.). Hoboken, NJ: J. Wiley & Sons.

Garcia L.S (2010). Clinical Microbiology Procedures Handbook. Third edition. American Society of Microbiology Press, USA.

Garcia L.S (2014). Clinical Laboratory Management. First edition. American Society of Microbiology Press, USA.

Ira R (1995). Bacteriology, Standard Operative procedure manual for microbiology laboratories, National Institute of Biologicals.  Pp. 73-97.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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