Bacterial Contamination of Meat: Pour Plate Assay
Pour Plate Technique for Bacterial Enumeration
Bacterial Contamination of Milk, Pour Plate Assay
Ground beef for human consumption may legally contain up to (hard to believe) 50 million bacteria/gm. The number of bacteria in a given sample may vary by several orders of magnitude. Therefore, when samples of these foodstuffs are assayed for bacterial count, several dilutions must be prepared over several orders of magnitude in order to achieve the desired range of colonies per plate (30-300). Typically, 1.0 mL of several dilutions from each of the dilutios ( undiluted to 103, or higher) should be plated. Greater dilution is necessary for more highly contaminated samples. The following procedure is for that purpose:
Ground beef to be tested. Have date of origin, if possible. Calculate age of material.
Standard Plate Count Agar, sterile (SPCA) 15 mL in capped 16 x 150mm test tubes, melted, 45 C , three per student
Sterile dH2O in 4 repipets capable of adjustment to deliver 1 to 10 mL aliquots.
Clean sterile petri dishes, 3 per student.
sterile 16x150mm test tubes (four per student)
stainless steel spatula in test tube with 95% EtOH
45o C Hot Block (or water bath deep enough to equal agar depth.) One per15 students
2.0 mL pipets, sterile , 3 per student
1.0 mL pipet, 1 per student
colony counter with magnifying glass
EXPERIMENTAL PLAN FOR ASSAYING BACTERIAL CONTENT OF MEAT:
PREP OF MEAT SAMPLE, DILUTIONS, AND ALIQUOT ADDITION TO PLATES
Label the bottoms of three empty plates with:
aliquot volume (1.0 mL each)
dilution factor (101, 102 or 103)
Prepare meat dilution blanks by labeling three 16 x 150mm sterile tubes with your initials and the exponents 1, 2 or 3 (dil’n factors 101, 102 or 103.)
Prepare dilution blanks:
Repipet 9.0 mL sterile dH2O into each of the three tubes.
Prepare a 10% suspension of meat in sterile water:
Weigh out 1.00 g ground beef sterilely into a fourth sterile capped 16x150mm test tube. (Record actual amount weighted out.)
(If you only weigh out 0.8 g of meat, suspend it in 8 mL water in the next several steps. I.e., a 10% suspension.)
Suspend the weighed meat with a vortex and spatula:
Add 3.0 mL sterile dH2O 2, vortex with sterile spatula inserted to suspend well. Be sure to hold the tube near the top to prevent spraying…)
After thorough suspension, add an additional 7.0 mL more sterile dH2O. Vortex to mix well again.
In the following steps, you will prepare the serial dilutions:
Prepare serial dilutions of the specimen in steps of 101, vortex after each dilution to mix completely. [Greater dilution factors may be achieved by using 0.1 mL into 9.9 mL in one or more of the serial dilutions, or plating 0.1 mL into the final petri dish.]:
For the first dilution, because of chunks of meat in suspension, use an inverted 2 mL pipet as instructed:
1) Use an inverted 2.0 mL pipet to deliver 1.0 mL of sample into first dilution tube = 101. (Invert so that meat particles do not clog up the pipet. Draw fluid to the 1.0 mark, deliver to the 0.0 mark.)
2) Use a 2.0 mL pipet to deliver first 1.0 mL of 101 dilution into the appropriately marked empty plate, THEN deliver the rest (1.0 mL) into the 102 dilution tube, vortex
3) Use a fresh 2.0 mL pipet to deliver first 1.0 ml of 102 into the appropriately marked empty plate, then deliver the rest (1.0 mL) into 103 dilution tube, vortex.
4) Use a fresh 1.0 mL pipet, deliver 1.0 mL of the 103 dilution into the appropriately marked empty plate
Add 15 mL 45 C melted agar to each plate in turn, swirl well to mix completely. To ease cleaning, plunge the emptied agar tube immediately into warm water before agar solidifies
After the plate has completely solidified (be patient), invert plate and incubate 35 C for 48 hr.
Meanwhile, pour the contents of the initial meat suspension tube into the quart jar provided (do not pour down the stink, er…, I mean sink…)
The contents of the other dilution tubes may be washed down with hot soapy water.
Count the colonies on the plates and calculate CFU per mL.
Remember that colonies imbedded within the agar are quite small, while the colonies on the surface are quite large (see the image to the left).
Each counts as a colony when enumerating the total colonies formed.
Using which of the three dilution plates has between 30 and 300 colonies, calculate the CFU/g meat. (See formula below.)
Enter your results into the class table (your initials, the meat source, its expiration date, CFU /g
CFU/plate x meat suspension factor (10 mL/g) x dil’n factor x aliquot factor (1) = CFU/g meat
1 On the plate shown, 1 gram of meat was suspended in 10 mL, diluted 1 to 10, 1.0 mL of the suspension was plated and 293 colonies formed. Therefore the count per gram in the meat was 293 x 10mL/g x 10 D.F. = 2.93 x 103/gram
2 If testing for E coli H7:O157, substitute MacConkey Broth for sterile water in this step. After dilutions are completed, place suspension in 37oC.