Bacterial Contamination of Milk: Pour Plate Assay
Pour Plate Technique for Bacterial Enumeration
Fresh food will typically have very low bacterial content, but as it is handled and stored, the bacterial concentration may increase dramatically. Pasteurized Grade A milk is required to have less than 20,000 bacteria/mL by standard plate count. Ground beef may contain up to 50 million bacteria/gm. Since the number of bacteria may vary by several orders of magnitude, samples of these foodstuffs must be diluted and several dilutions plated out in order to achieve the desired range of colonies per plate (50-500). Typically, for pour plate technique, 1.0 mL of dilutions ranging from undiluted to 103 (or higher) should be plated. (Greater dilution is necessary for highly contaminated samples.) The following procedure is for that purpose:
- Milk 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
15 mL melted Plate Count Agar in:
sterile capped 16x150mm test tubes
45o C bath (deep enough to = agar
depth. Hot Block, or water bath)
stainless steel spatula in test tube with 95% EtOH
sterile 16x150mm test tubes
0.1, 1.0, 2.0 & 10 mL pipets, sterile
colony counter with magnifying glass
DILUTIONS, AND ALIQUOT ADDITION TO PLATES:
Label two empty plates with your initials, the date, specimen (milk), aliquot volume (0.1 or 1.0 mL) and dilution factor (10^2).Prepare dilution blank: Add 9.9 mL sterile dH2O to a sterile 16x150mm test tube.
Dilute the milk: pipet 0.1 mL milk into above dilution tube (10^2 dilution), vortex to mix
Add the aliquots: Using a 2.0 mL pipet, pipet 0.1 mL into first plate, 1.0 into the second.
Pick up a tube of 15 mL melted plate count agar, cooled to 45C, (here in a hot block, a 45C water bath can be used.)
ADD MELTED AGAR TO MAKE POUR PLATE:
Add melted agar (dry off if maintained in hot water) to each plate in turn, swirl to mix completely. Plunge the emptied tube immediately into warm water before agar solidifies to ease cleansing.
When the agar is solid, invert the plate and incubate 35C for 48 hr.
Count colonies on the plates and calculate CFU per mL:
CFU on plate x dil’n factor (102) x aliquot factor (either 1/1.0 ml or 1/0.1 mL) = CFU/ mL milk
Example plate: 40 colonies on plate x 10^2 x 1/1.0 mL = 4000 CFU/mL of milk
Enter your results into the class table ( your initials, the milk manufacturer, its expiration date, CFU/mL)
* Standard Plate Count Agar: 5 g tryptone, 2.5 g yeast ext..1 g dextrose, 15 g agar, 1 L water
Suggested stations (at least two each? If enough repipets):
Empty plates station, wax pencil
sterile bags of empty plates
16×150 mm sterile capped test tubes (5 per student)
test tube racks to hold 16×150 tubes (one per student)
Milk Dilution Station:
sterile capped 16×150 mm test tubes
repipet with sterile dH2O, set for 9.9 mL
0.1, one per student
2.0, one per student
Pour Plate Station:
hot block with styrofoam cage, 45 C
wash tub with warm water in it.
16x150mm test tubes with 15 mL 45 C melted Standard Plate Count Agar (5 per student)
Be sure to have enough tubes of SPCA (at least 3 tubes/person if they work in pairs on both experiments).
advanced testing laboratories
Standard methods agar
Plate count agar:
5 g tryptone
2.5 g Yeast est
1 g dextrose
15 g agar
1 L water (pH 7
suggested limit total viable
basic food microbiology book, Banwart, OSU Microbiology