PMO 2007: Appendix G - Chemical and Bacteriological Tests.

Grade "A" Pasteurized Milk Ordinance (2007 Revision)

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Reference: Section 7, Items 8r, 19r, 7p and 17p.

Application: To private water supplies, used by dairy farms, milk plants, receiving stations, transfer stations and milk tank truck cleaning facilities, and to recirculated cooling water, used in milk plants, receiving stations and dairy farms.

Frequency: Initially; after repair, modification or disinfection of the private water supplies of dairy farms, milk plants, receiving stations, transfer stations and milk tank truck cleaning facilities, and thereafter; semiannually for all milk plants, receiving stations, transfer stations and milk tank truck cleaning facilities water supplies and at least every three (3) years on dairy farms. Recirculated cooling water in milk plants, receiving stations and on dairy farms shall be tested semiannually.

Criteria: A Most Probable Number (MPN) of coliform organisms of less than 1.1 per 100 mL, when ten (10) replicate tubes containing 10 mL, or when five (5) replicate tubes containing 20 mL are tested using the multiple tube fermentation technique, or less than 1 per 100 mL by the membrane filter technique, or less than 1.1 per 100 mL when using an MMO-MUG and XGAL-MUG technique. (The MMO-MUG and XGAL-MUG technique is not acceptable for recirculated cooling water). 100 ± 2.5 ml water will be used for this analysis. Any sample producing a bacteriological result of Too Numerous To Count (TNTC) - greater than two hundred (200) total bacteriological colonies per 100 mL by the membrane filter technique; or confluent growth by the multiple tube fermentation, MPN technique, without coliform present, shall have a subsequent heterotrophic plate count of less than five hundred (500) colonies per mL in order to be deemed satisfactory. Findings shall be reported as present or less than 1 per 100 mL, absent for coliform organisms.

Apparatus, Method, and Procedure: Tests performed shall conform with the current edition of SMEWW or with FDA approved, EPA promulgated methods for the examination of water and waste water.

Corrective Action: When the laboratory report on the sample is unsatisfactory, the water supply in question shall again be physically inspected and necessary corrections made until subsequent samples are bacteriologically satisfactory.


Reference: Section 6.

Frequency: When any laboratory phosphatase test is positive, or any doubt arises as to the adequacy of pasteurization due to noncompliance with equipment, or requirements of Item 16p.

Criteria: Less than 350 mU/L by an electronic phosphatase procedure. (Refer to the SMEDP)

Apparatus: Fluorophos (Advanced Instruments) and Paslite (Charm Science), approved/validated standards and accessories.

Methods: The test is based on the detection of the phosphatase enzyme, a constituent that is inactivated by pasteurization at 63°C (145°F) for thirty (30) minutes or 72°C (161°F) for fifteen (15) seconds. When pasteurization is faulty, some phosphatase remains and is detected through its action on phosphoricphenyl esters, releasing phenol, which is measured quantitatively by the addition of dibromo-or dichloro-quinonechlorimide to form an indophenol blue color.

Procedure: Refer to the SMEDP for details on phosphatase tests.

Corrective Action: Whenever a phosphatase test is positive, the cause shall be determined. Where the cause is improper pasteurization, it shall be corrected and any milk or milk products involved shall not be offered for sale.


The presence of an appreciable quantity of phosphatase in milk and cream after heat treatment has been traditionally regarded as evidence of inadequate pasteurization. However, with the advent of modern HTST methods, evidence has been accumulating that under certain conditions, the relationship between inadequate pasteurization and the presence of phosphatase does not hold.

A number of investigators who have studied HTST pasteurizing methods have concluded that while a negative test can be obtained immediately after pasteurization, the same sample may yield a positive test after a short period of storage, particularly if the product is not continuously or adequately refrigerated. This phenomenon has come to be known as reactivation.

Reactivation may occur in HTST pasteurized products, after storage at temperatures as low as 10°C (50°F), although 34°C (93°F) is optimum. Products of high fat content generally produce relatively more reactivable phosphatase.

Reactivation is greatest in products pasteurized at about 110°C (230°F) but may occur in products pasteurized at much higher temperatures and as low as 73°C (163°F).

It has been noted that an increase in holding time during pasteurization will reduce reactivation.

The addition of magnesium chloride to HTST processed milk or cream, after pasteurization but before storage, accelerates reactivation. The difference in activity between an adequately pasteurized sample, stored with and without magnesium, and an inadequately pasteurized sample, stored with and without magnesium, forms the basis of a test for differentiating reactivated from residual, inadequately pasteurized, phosphatase.

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Any Regulatory Agency that has adopted this Ordinance should operate under a control program that will insure that milk supplies are free from pesticide contamination, in conformance with Section 2.

Pesticide compounds gain access to milk by various routes. Insecticide contamination may result from any of the following:

  1. Application to the lactating animals;
  2. Inhalation of toxic vapors, by the animals, following application of insecticides to their environment;
  3. Ingestion of residues in feed and water; and
  4. Accidental contamination of milk, feed and utensils. Herbicide contamination may result from residues on the lactating animals feed and in their water supply and/or rodenticides may be present in milk as a result of accidental contamination.

At the present time, chlorinated hydrocarbon pesticides are the chief concern. While there are other pest control compounds that are more toxic than the chlorinated hydrocarbons, many of the agents in this latter group tend to accumulate in the body fat of both lactating animals and human beings, and are secreted in the milk of contaminated lactating animals. The accumulation of these toxic agents in persons continually consuming contaminated milk may reach hazardous concentrations.

Advances in residue analysis have resulted in a radical decrease in the use of paper chromatographic screening procedures for milk, because of its rather limited sensitivity. Regulatory Agencies can now routinely detect residues as low as 0.01 ppm of many of the chlorinated organic pesticides. Satisfactory screening procedures should, therefore, attain this level of sensitivity, which usually necessitates the use of gas chromatography or thin layer chromatography.

General screening procedures of the latter two (2) types are described and discussed in Volume 1 of the Pesticide Analytical Manual (PAM) published by FDA.

The need for closer scrutiny of milk supplies for pesticide residues has stimulated considerable research in detection technology. The Regulatory Agency entering upon a surveillance program should carefully check the available equipment in relation to its adaptability to the indicated need.

While a schedule of testing comparable to that for microorganisms, four (4) tests of individual producer's milk during any consecutive six (6) months, would be desirable, broad-spectrum procedures are too time consuming to render such a schedule feasible. As a more practical approach, the following procedure is suggested:

  1. Test one (1) load of milk from each milk tank truck route, every six (6) months, by a broad spectrum method and trace positive samples; or
  2. Test each producer's milk four (4) times every six (6) months for the most common chlorinated hydrocarbon pesticides, by available instrumental methodology.

NOTE: The above testing disciplines may be applied conveniently to can milk supplies. Where Procedure 1 is used, samples of commingled milk from known sources are drawn from receiving station storage tanks. Sampling for Procedure 2 may be done directly from the weigh tank.

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The problem of drug residues in milk is associated with their use in the treatment of mastitis and other diseases. Failure to withhold milk from the market for a sufficient length of time after treatment may result in the presence of drug residues in milk. Such milk is undesirable for two (2) reasons:

  1. It comes from an unhealthy lactating animal; and
  2. It is adulterated.

The allergenic properties of certain drugs in common use make their presence in milk potentially hazardous to consumers. Also, substantial losses of byproducts may be sustained by the milk industry each year because of the inhibitory effects of drug residues on the culturing process. Drug residues should be tested for, using tests provided for in Section 6 of this Ordinance. These tests are specified in memoranda from the FDA. (Refer to the latest edition of M-a-85, M-a-86, and the 2400 series forms for each specific test method.)

NOTE: Bacillus stearothermopilus disk assay analysis performed to fulfill the provisions of Section 7 of this Ordinance must be capable of detecting at least four (4) of six (6) Beta lactam drugs at or below FDA reference levels. A zone equal to or greater than 16mm will be considered positive when the Bacillus stearothermophilus disk assay is used, provided the 5ppb Beta lactam control zone is 16-20mm. (Refer to the most recent FDA 2400 Series Form(s) for details related to this analysis.)


Reference: Section 6.

Frequency: Annually for each product type, or when any doubt arises as to the adequacy of vitamin fortification. (Refer to Appendix O.)

Methods: Vitamin testing shall be performed using test methods acceptable to FDA and other official methodologies that give statistically equivalent results to the FDA methods.


Official Methods of Analysis of AOAC INTERNATIONAL, 17th Edition, 2000.

Pesticide Analytical Manual, (PAM) available from the U. S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, HFS-335, 5100 Paint Branch Parkway, College Park, MD 20740-3835.

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