The Microbiologics
blog has a useful back-to-basics article on good pour plate
practices. Included in the article is the following advice:
1. Keep
the molten agar in the water bath for no more than three to four hours. Don’t
pour the agar until it has cooled to <50°C (preferably 44°C to 46°C).
2.
Don’t re-melt the agar. Agar should only be melted one time.
3. Use
phosphate buffer pH 7.2 if necessary to dilute the suspension.
4.
Decrease the risk of contamination by pouring plates in a laminar-airflow
cabinet. When pouring multiple plates, flame the mouth of the flask before
moving on to the next plate to reduce the risk of contamination.
5. Fill
plates according regulators’ recommendations. The U.S. Food and Drug
Administration (FDA) Bacteriological Analytical Manual (BAM) recommends filling
plates with 12 ml to 15 ml of agar. The United States Pharmacopeia (USP)
recommends a fill of 15 ml to 20 ml of agar.
6. Some
microorganism species, such as obligate aerobes, may recover better on spread
plates than pour plates. When growing these strains, it is recommended to
verify counts with a spread plate.
7. Incubate most bacterial species for 48
to 72 hours. Note: Incubate Candida albicans and Aspergillus brasiliensis for three to five days.
8. Count small microorganism colonies, such
as Pseudomonas aeruginosa, with the aid of an illuminated
colony counter or magnifying glass.
9.
Keep in mind recovery will be lower on selective agar. If selective agar is
used, test non-selective agar in parallel using the same microorganism
suspension. A higher CFU concentration for the selective agar may be necessary.
10.
Don’t be surprised if the value obtained when performing tests differs from the
mean assay value. Note: Microbiologics uses non-selective Tryptic Soy Agar when
testing most microorganisms.
Posted by Dr. Tim Sandle, Pharmaceutical Microbiology