Pour Plate Technique for Bacterial Enumeration
RELATED PROTOCOLS: Bacterial Contamination of Milk and Meat , Yeast Plate Count
The pour plate technique can be used to determine the number of microbes/mL or microbes/gram in a specimen. It has the advantage of not requiring previously prepared plates, and is often used to assay bacterial contamination of foodstuffs. The principle steps are to
1) prepare/dilute the sample
2) place an aliquot of the diluted sample in an empty sterile plate
3) pour in 15 mL of melted agar which has been cooled to 45C, swirl to mix well
4) let cool undisturbed to solidify on a flat table top
5) invert and incubate to develop colonies.
Each colony represents a “colony forming unit” (CFU). For optimum accuracy of a count, the preferred range for total CFU/plate is between 30 to 300 colonies/plate.
One disadvantage of pour plates is that embedded colonies will be much smaller than those which happen to be on the surface, and must be carefully scored so that none are overlooked. Also, obligate aerobes may grow poorly if deeply imbedded in the agar.
15 mL sterile Plate Count Agar (PCA)*, in capped 16 x 150 mm test tubes, melted and cooled to 45C
Hot Block, 45C (or water bath), 3″ deep to equal agar depth
sterile capped 16 x 150 mm test tubes
0.1, 1.0 and 2.0 mL pipets, sterile
petri dishes, empty and sterile
colony counter with magnifying glass
POUR PLATE TECHNIQUE:
1. Write out details of preparing and plating your specimen(s):
Construct a table in your notebook with a line for each plate: • the identity/source of the specimen (notebook entries should be detailed).
• the dilution of the specimen expected to contain between 30-300 CFU/0.1-1.0 mL and how you will prepare it
• the volume of diluted specimen you will plate (usually 0.1 to 1.0 mL)
Label the bottom of the empty, sterile plates your initials, seat number, date and the above data.
2. Dilute specimen to yield approximately 30 to 300 CFU per aliquot to be plated (from 1).
3. Inoculate labeled empty petri dish with the aliquot of diluted specimen (from 1)
4. Pour 15 mL of melted Plate Count Agar (45C) into the inoculated petri dish.
5. Cover and mix thoroughly by gentle tilting and swirling the dish. Do not slop the agar over the edge of the petri dish.
6. Place on a flat surface undistrubed for about 10 minutes to allow the agar to completely gel. In this illustration, the agar is completely gelled and the surface is “smooth as glass.”
7. Invert and incubate at 37 C for 24-48 hours.
8. Count, record, calculate:
Count all colonies (note that the embedded colonies will be much smaller than those which happen to form on the surface). A magnifying colony counter can aid in counting small embedded colonies. Record the data. Calculate CFU/mL or CFU/g. Enter results in your table.
CFU/ mL = CFU/plate x dilution factor x 1/aliquot
On the plate shown, milk was diluted 1 to 100 (10 2), 1.0 mL of the dilution was plated and 40 colonies formed. Therefore the count per mL in the milk was:
40 colonies x 10^2 x 1/1 = 4 x 10^3/mL
* For 600 mL of NA + 1% glu: 9 g agar, 4.8 g nutrient broth, 6 g dextrose. Dissolve ingredients at 95C, repipet into 16 x 150 mm tubes, cap, autoclave, 15 lb, 15 min. Cool to 45C before using. Plate Count Agar may also be used.