Milk Plate Count Agar (PCA) ISO

Description

Due to its lower agar concentration, it may be used for the pour plate method or the spread plate method.

Technique

To use, the contents of the bottle should be poured into plates. The melting of the culture medium should be carried out according to the manufacturer's instructions, either in a water bath (100 °C) or microwave oven. Never apply direct heat to melt a medium. The melting temperatures and times depend on the shape of the container, the volume of medium and the heat source. Before melting any medium, loosen the screw cap of the container to avoid breaking the container. The medium should be melted only once and used. Media with agar should not be melted repeatedly as their characteristics change with each remelting. Overheating should be avoided as much as prolonged heating, especially with regard to media with an acidic or alkaline pH. Once melted, pour the plates using aseptic techniques. To inoculate, follow standard laboratory methods or the applicable norms: spiral plate method, streak plating, ecometric methods, dilution banks, spread plating, etc.

Recommended technique of use

Prepare ten-fold serial dilutions of the sample and take 1 mL in duplicate aliquots from each dilution and put them in sterile Petri dishes. Pour approx. 20 mL of sterile cooled medium (around 45 °C) in each of the plates. Mix gently by swirling the plate in a figure 8. Leave the plates undisturbed to solidify and incubate in an inverted position. The incubation time and temperature depend on the type of microorganism under investigation. In general, for an aerobic count, incubate for 3 days at 30 °C, checking the plates at 24, 48 and 72 hours.

The plate count method proposed by the APHA consists of the pour plate method, i.e. pouring the molten agar at 50 °C on plates containing the diluted samples. The final count is carried out after 48 hours of incubation at 32-35 °C.

For microorganisms with other temperature requirements, the following incubations have been suggested: 2 days at 32-35 °C, 2-3 days at 45 °C, 2 days at 55 °C, 3-5 days at 20 °C, 7-10 days at 5-7 °C.

Sample dilutions are prepared with 1/4 Ringer's solution, Buffered Peptone Water or Maximum Recovery Diluent depending on their nature.

The poured plate count method is preferred to the surface inoculation method, since it gives higher counts, although the latter facilitates isolation and reseeding of the colonies.

Physical/chemical control

LC-5172, 125 mL bottles: physical/chemical control

Microbiological control

Melt medium - prepare plates - spiral spreading: practical range 100 ± 20 CFU; min. 50 CFU (productivity).

Microbiological control according to ISO 11133:2014/Amd 1:2018.

Aerobiosis. Incubation at 30 ± 1 °C, reading at 24-48-72 h.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• ATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc. London.
• BUCHBINDER, L., Y. BARIS & L. GOLDSTEIN (1953) Further studies on new milk-free media for the standard plate count of dairy products. Am. J. Public Health 43:869-872.
• CLESCERI, L.S., A.E. GREENBERG and A.D. EATON (1998) Standard Methods for the Examination of Water and Wastewater. 20th ed., APHA, AWWA & WPCF. Washington.
• DIN 10192 (1971) Prüfungsbestimmungen für Milch und Milcherzeugnisse. Deutsche Landwirtschaft, Fachbereit und Ernahrung.
• DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods. 4th ed., APHA, Washington.
• FIL/IDF Standards 3 (1958), 100, 101 (1981), 109 (1982) and 132 (2004).
• HORWITZ, W. (2000) Official Methods of Analysis of the A.O.A.C. AOAC International. Gaithersburg. Va.
• IFU Method No 6 (1996) Mesophilic, thermoduric and thermophilic bacteria: Spores Count. D-1 Mesophilic Aerobic Sporeforming bacteria: Spores count.
• ISO 4833 (2003) Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of microorganisms. Colony count technique at 30 °C.
• ISO 8552 (2004) Milk - Estimation of psychrotrophic microorganisms. Colony count technique at 21 °C (Rapid method).
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• ISO 17410 (2001) Horizontal method for the enumeration of psychrotrophic microorganisms.
• MARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products. 16th ed. APHA. Washington.
• PASCUAL ANDERSON, Mª R. (1992) Microbiología Alimentaria. Díaz de Santos, S.A. Madrid.