DIRECT PLATE COUNTING

SOIL MICROBIOLOGY

BIOL/CSES 4684




This webpage was created by Allen Coleman


1. OVERVIEW OF DIRECT PLATE COUNTING METHODOLOGY

When one decides to count the number of cells in a sample, the issues of viable numbers, and the total numbers should come to mind.  There are many methods used for counting cell numbers, some of which count total cell numbers (live cells + dead cells), and others that count only viable, or live cells.  Direct plate counting is a method used to count the number of viable cells in a sample.  By the very nature of the procedure, the dead cells are unable to be included in the count.

Once the cells to be counted have been isolated, they are to be diluted due to the fact that too many cells will cause the Petri plate to be so densely populated with colonies, that they would be impossible to count.  After the cells have been diluted, they are incubated on an agar medium until colonies form.  It is at this time that the cells may be counted. The image on the left shows a soil dilution plate containing bacteria and fungi. This plate is countable, but on the difficult side.


2. OVERVIEW OF DIRECT PLATE COUNTING PROCEDURES
There are two main methods of direct plate counting: spread plate method and pour plate method.

The spread plate method consists of evenly spreading the diluted sample over an agar plate.  When using this method, a volume of 0.1 ml of the diluted sample should not be used since the agar will not be able to absorb the excess.  Using this method yields colonies that form on the surface of the agar.


Picture obtained from Biology of Microorganisms by Madigan, Martinko, and Parker

When using the pour plate method, a diluted sample is pipetted into a sterile Petri plate, then melted agar is poured in and mixed with the sample.  Using this method allows for a larger volume of the diluted sample.  Usually in the range of 0.1 - 1.0 ml.  This method yields colonies that form colonies throughout the agar, not just on the surface.  Caution must be taken with this method to ensure that the organism to be counted can withstand the temperatures associated with the melted agar.


Picture obtained from Biology of Microorganisms by Madigan, Martinko, and Parker


3. PROS AND CONS
Direct plate counting is a good method of counting cells.  A couple of advantages are that it works well for cells that separate in a short amount of time after they divide, and it counts only viable numbers.  However, direct plate counting is not without its flaws.  It does not work so well for those cells that stick together after cell division.  Another problem that may arise is the very specific nutritional needs of some organisms.  This is a special consideration when dealing with mixed samples such as those from a soil, because one cannot be sure the media has sufficient nutritional value for all of the cell types present.  Sampling error is another possible problem.  Sampling error refers to the uneven distribution of the sample on the agar surface.  Direct plate counting has been described as the best available method for determining viable numbers, despite the large chance of the occurrence of an error (175, Claus).


4. ADDITIONAL SOURCES OF INFORMATION
Claus, G. W.  1989.  Understanding Microbes.   W. H. Freeman and Company, New York, NY.  pp. 175-185.

Heggers, J. P., and Robson, M. C.  1991.  Quantitative bacteriology: its role in the armamentarium of the surgeon.  CRC Press, Boca Raton, FL.

Madigan, M. T., Martinko, J. M., and Parker, J.  1997.  Biology of Microorganisms.  Prentice Hall, Upper Saddle River, NJ.  p. 156.


5. LINKS TO OTHER SITES ON THE INTERNET
Counting Microbes This is a link to a microbiology course outline at Ohio State University.

Biotech This is another link to more counting of microbes.


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