Bacterial Contamination of Milk: Pour Plate Assay

Bacterial Contamination of Milk: Pour Plate Assay

RELATED PROTOCOLS:
Pour Plate Technique for Bacterial Enumeration
Dilutions

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:

01_Milk_protocol_P7261201md

SUPPLIES:

    1. Milk to be tested. Have date of origin, if possible. Calculate age of material.
    2. Standard Plate count agar*
    3. Sterile dH2O in 4 repipets
    4. Clean sterile petri dishes

EQUIPMENT:
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)
vortex
paper towel
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

02_milk_aliquot_P7261206
dilute milk

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.

03_dilute_sample_P7261207

Dilute the milk: pipet 0.1 mL milk into above dilution tube (10^2 dilution), vortex to mix

04_sample_to_plate_P7261208
Diluted sample is added directly to empty plate

Add the aliquots: Using a 2.0 mL pipet, pipet 0.1 mL into first plate, 1.0 into the second.

05_hot_block_P7261209_md

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.)

06_add_melted_agar_P7261205

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.

11_meat_incubate_plates_P8081424

When the agar is solid, invert the plate and incubate 35C for 48 hr.

07_Milk_pour_plate_P7281229

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
wax pencils
16×150 mm sterile capped test tubes (5 per student)
test tube racks to hold 16×150 tubes (one per student)

Milk Dilution Station:
milk
sterile capped 16×150 mm test tubes
repipet with sterile dH2O, set for 9.9 mL
vortex
flame
sterile pipets:
0.1, one per student
2.0, one per student

Pour Plate Station:
hot block with styrofoam cage, 45 C
flame
wash tub with warm water in it.
16x150mm test tubes with 15 mL 45 C melted Standard Plate Count Agar (5 per student)
paper towel
Be sure to have enough tubes of SPCA (at least 3 tubes/person if they work in pairs on both experiments).

Accredited labs
advanced testing laboratories
4700 Smith
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
100,000-50,000,000/ gm

531-9222

Nutra Lab
Kempter
346-3567

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