17.3.02 (e) Col ony coun ter.
—Stan dard ap pa ra tus, Que bec model
AOAC Official Method 986.33 preferred, or one providing equivalent magnification and visibility.
Bacterial and Coliform Counts in Milk
C. Analysis
Dry Rehydratable Film Methods
(Petrifilm Aerobic Count Plate (a) Bacterial colony count.—Use Petrifilm Aerobic Count Plates
and Petrifilm Coliform Count Plate) Methods or equivalent. Place plate on flat surface. Lift top film and inoculate
First Action 1986 1 mL test portion onto center of film base. Carefully roll top film
Final Action 1988 down onto inoculum. Distribute test portion over prescribed growth
A. Principle area with downward pressure on center of plastic spreader device
(recessed side down). Leave plate undisturbed 1 min to permit gel to
Method uses bacterial culture plates of dry medium and cold
H2O-soluble gel. Undiluted or diluted test portions are added solidify. Incubate plates 48 ± 3 h at 32° ± 1°C.
directly to plates at a rate of 1.0 mL per plate. Pressure, when applied In incubator, place plates in horizontal position, clear side up, in
to plastic spreader placed on overlay film, spreads test suspension stacks not exceeding 10 units. Count plates promptly after incubation
over ca 20 sq cm growth area. Gelling agent is allowed to solidify period. After incubation is complete, plates may be stored frozen
and plates are incubated and then counted. Either pipet or plate loop (≤–15°C) up to 7 days. This should be avoided as a routine practice.
continuous pipetting syringe can be used for test portion addition for Use stan dard col ony coun ter for count ing pur poses.
bacterial count analyses. Magnifier-illuminator may also be used to facilitate counting. Colonies
B. Apparatus stain in various shades of red. Count all colonies in countable range
(30–300 colonies).
(a) Petrifilm Aerobic Count Plates.—Plates contain standard
meth ods me dia nu tri ents, 940.36A(g) (see 17.1.02), cold To compute bacterial count, multiply total number of colonies per
H2O-soluble gelling agent coated onto film base, overlay film plate (or average number of colonies per plate if counting duplicate
coated with gelling agent, and 2,3,5-triphenyltetrazolium chloride plates of same dilution) by reciprocal of dilution used. When
indicator. Circular growth area of single plate contains ca twenty counting colonies on duplicate plates of consecutive dilutions,
1 cm squares outlined on film base. Petrifilm Aerobic Count Plates compute mean number of col onies for each di lution before
(Microbiology Products, 3M Center, Bldg 275-5W-05, St. Paul, MN determining average bacterial count. Estimated counts can be made
55144, USA) or equivalent meet these specifications. on plates with >300 colonies and should be reported as estimated
(b) Petrifilm Coliform Count Plates.—Plates contain violet counts. In making such counts, circular growth area can be
red bile nutrients conforming to APHA standards as given in considered to contain ca twenty 1 cm squares. To isolate colonies for
Compendium of Methods for the Microbiological Examination of further identification, lift top film and pick colony from gel.
Foods (1990) 3rd Ed., American Public Health Association, (b) Coliform count.—Use Petrifilm Coliform Count Plates or
Washington, DC, USA, cold H2O soluble gelling agent, and equivalent. Proceed as in (a), but distribute test portion over plate by
2,3,5-triphenyltetrazolium chloride. Petrifilm Coliform Count using plastic spreader, flat side down. Incubate plates 24 ± 2 h at
Plates (Microbiology Products, 3M Center), or equivalent meet 32° ± 1°C. Count as in (a), but count only red colonies that have one
these specifications. or more gas bubbles associated (within one colony diameter) with
(c) Plastic spreader.—Provided with Petrifilm plates, consists of them. Count all colonies in countable range (15–150 colonies). Red
concave side and smooth flat side, designed to spread milk test colonies without gas bubbles are not counted as coliform organisms.
portion evenly over plate growth area.
Reference: JAOAC 69, 527(1986).
(d) Pipets.—Calibrated for bacteriological use of plate loop
continuous pipetting syringe to deliver 1.0 mL. Revised: March 2002
2005 AOAC INTERNATIONAL
