BACTERIAL CONTAMINATION OF MEAT
POUR PLATE ASSAY

David B. Fankhauser, Ph.D.
Professor of Biology and Chemistry
University of Cincinnati Clermont College
Batavia OH 45103.
weigh 1 g meat
to suspend

This page has been accessed Counter times since 3 August 2001. 
7/5/89, rvsd 28 July '93, 21 July '95, 21 July 1996, 18 Aug '97, 17 July 00
 adding melted agar 
to plate

RELATED PROTOCOLS:

Pour Plate Technique for Bacterial Enumeration
Dilutions
Bacterial Contamination of Milk, Pour Plate Assay
DISCUSSION:
Ground beef for human consumption may legally contain up to 50 million bacteria/gm. Since the number of bacteria may vary by several orders of magnitude, when samples of these foodstuffs are assayed for bacterial count, they must be diluted in order to achieve the desired range of colonies per plate (30-300). Typically, 1.0 mL of several dilutions from undiluted to 103 (or higher) should be plated (greater dilution is necessary for highly contaminated samples). The following procedure is for that purpose:

SUPPLIES:

Ground beef to be tested. Have date of origin, if possible. Calculate age of material.
Standard Plate count agar*
Sterile dH2O in 4 repipets
Clean sterile petri dishes
EQUIPMENT:
15 mL melted Plate Count Agar sterilized in capped 16 x 150mm test tubes
45o C  Hot Block (or water bath deep enough to equal agar depth.)
vortex
balance
stainless steel spatula in test tube with 95% EtOH
sterile 16x150mm test tubes
0.1, 1.0, 2.0 & 10 mL pipets, sterile
flame
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 initials, date, specimen, aliquot volume (1.0 mL each) and dilution factor (101, 102 or 103).
Prepare meat dilution blanks by labeling three 16x150mm sterile tubes with 1, 2 or 3 for dilution factors 101, 102 or 103.
Repipet 9.0 mL sterile dH2O into each of the three tubes.
  Weigh out 1.00 g ground beef sterilely into a fourth sterile capped 16x150mm test tube.  
 

Add 3.0 mL sterile dH2O 2, vortex with sterile spatula inserted to suspend well, add 7.0 mL more sterile dH2O, vortex to mix well again.
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.]:
 

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.)
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
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.
Use 1.0 mL pipet, deliver 1.0 mL of 103 dilution into marked empty plate
  Add 15 mL 45oC melted agar to each plate in turn, swirl to mix completely. Plunge the emptied tube immediately into warm water before agar solidifies to ease cleansing.
 
 		 When solid, invert plate and incubate 35oC for 48 hr.
Count the colonies on the plates and calculate CFU per mL1
 
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.