commingling <300,000 cfu/mL after com-

Grade A pasteurized milk and

Total bacterial count

mingling <20,000 cfu/mL

milk products

Coliform count

<10 cfu/mL

Grade A aseptically processed milk and milk products

Total bacterial count

No growth by standard plate count or other comparable method

Source: U.S. Public Health Service, 1995.

Source: U.S. Public Health Service, 1995.

terms of milk quality and safety. Excessively high bacterial counts can overwhelm the bacterial thermal destruction capacity of a pasteurizer, resulting in pasteurized milk with high bacterial numbers that may be unsafe to consume and that may have reduced quality and shelf life. High bacterial counts in raw milk can also suggest the presence of bacterially produced enzymes that may adversely affect the quality of any fluid milk and processed product made from the raw milk.

For reasons noted above, analytical tests are routinely done to characterize the microbial population of raw milk samples. The TBC is typically determined by the standard plate count (SPC) or the Petrifilm (3M Company, St. Paul, MN) aerobic count (PAC). The SPC measures all bacteria able to form colonies on standard methods agar within 48 h under aerobic conditions at 32°C, whereas the PAC measures all bacteria able to form colonies on a nutrient medium embedded in a plastic film within 48 h at 32°C (Houghtby et al., 1992). Several alternative, but less commonly applied, techniques for estimating total bacterial numbers exist, including plate loop count, pectin gel plate count, spiral plate count, hy-drophobic grid membrane filter most probable number count, and impedance/ conductance method (Houghtby et al., 1992).

A notable new rapid method known as Bactoscan (Foss Food Technology Corp., Eden Prarie, MN) utilizes fluorescent staining to count individual bacterial cells. In this technique, somatic cells, fat globules, and casein particles are chemically degraded and then separated from bacterial cells by centrifugation in a sac-charose-glycerol gradient. Bacterial cells are then stained with acridine orange and channeled beneath the objective of an epifluorescence microscope. As they pass under the objective, the bacteria are irradiated with filtered blue light, which causes red light pulses to be emitted from live bacteria. A photodetector fitted to the objective detects these pulses, which are then counted as individual bacterial cells (IBCs) (Rodriguez-Otero et al., 1993). Differences in acridine orange intercalation into cell DNA cause dead cells to emit green light, whereas live cells emit red light, thus ensuring that Bactoscan only counts live bacteria (Sharpe and Peterkin, 1988). Calibration of the Bactoscan apparatus using reference standards allows IBC/mL values to be translated into colony-forming units per milliliter values. This calibration step facilitates comparison of Bactoscan results with other TBC techniques. The Bactoscan method is unique in that it counts individual bacterial cells rather than the colony-forming units measured by most other tests, leading to values of IBCs per milliliter which may be significantly higher than corresponding colony-forming units per milliliter values, particularly in the presence of organisms such as many Streptococcus spp. and Staphylococcus spp. that form, for example, clusters, chains, duplets, or triplets. Although widely applied in Europe to analyze raw milk quality, the Bactoscan method is not currently approved for regulatory use in the United States.

Although information provided by the TBC is useful for determining pre mium allocations and for satisfying PMO regulations, it is of less utility for identifying specific sources of high bacterial counts or for assessing risks to milk quality posed by a particular bacterial population. Selective and/or differential tests that detect and quantify a specific type or group of bacteria can prove to be more useful. By doing tests that distinguish among microbial groups, one can identify the dominant organism(s) in a given bacterial population. The identity of dominant organism(s) can often suggest a possible contamination source or route and thus aid in focusing future contamination prevention efforts. The identity of dominant organism(s) can also help assess bacterial threat(s) to milk quality and safety. Many spore formers and thermoduric organisms, which can survive pasteurization, can also grow in the processed product and diminish product quality and shelf life. Psychrotrophs, which grow under refrigeration conditions, can multiply while raw milk awaits pasteurization, creating off-odors and off-flavors and chemically degrading milk components. Many heat-stable enzymes produced by psychrotrophs can also survive pasteurization and degrade the finished product, decreasing the shelf life of fluid milk products and adversely affecting yield of cultured products (Cousin, 1982). Individual selective tests can also prove to be useful for monitoring elimination of a specific contamination source. For example, a selective test that detects S. agalactiae could be employed to gauge the effectiveness of an S. agalactiae eradication program.

Numerous selective and differential tests can be used to determine the presence or absence of specific types of bacteria in raw milk. The laboratory pasteurized count (LPC), in which milk samples are heated to 62.8°C for 30 min before plating onto standard methods agar, estimates the number of thermoduric bacteria that could survive a batch pasteurization-type process (Frank et al., 1992; Murphy, 1997). The preliminary incubation count (PIC), in which milk samples are held at 12.8°C for 18 h before doing an SPC, gauges the number of bacteria capable of growth at cooler temperatures. A significant increase in the SPC after preliminary incubation is considered to be indicative of unsanitary production practices. The coliform count, in which samples are plated on the selective and differential medium Violet Red Bile Agar and incubated for 24 h at 32°C, estimates the number of coliform organisms present (Christen et al., 1992). The presence of these organisms can also indicate unsanitary production and processing practices. The selective and differential Edwards Medium can be used to isolate streptococci, which can be indicative of mastitis in the herd (Atlas, 1993). To meet other specific diagnostic objectives, procedures have been established to detect and quantify thermophilic organisms, proteolytic organisms, li-polytic organisms, lactic acid bacteria, enterococci, aerobic bacterial spores, and yeast and molds (Frank et al., 1992).

Characterization of the bacterial population present in raw milk must always consider the limitation inherent in any analytical technique: No one test can detect all bacteria. Even nonselective tests designed to determine total bacterial numbers cannot detect fastidious organisms that require additional nutrients, slow-growing organisms that require more time to form visible colonies, or poor competitors that require selective media to ensure sufficient nutrient access. Furthermore, correlations are so low among results obtained from standard plate count, rapid psychrotrophic count, preliminary incubation count, aerobic spore count, and laboratory pasteurized count analyses from the same raw milk sample that one result cannot be used to estimate multiple different test results (Boor et al., 1998). Ultimately, no one test gives a complete picture of the microbial population; the picture must be pieced together using results from multiple different tests. Since doing all possible tests is neither economically nor logistically feasible, microbial analysis must involve deciding which tests will provide the most useful information about the microbial population of the particular product being examined. Additional information on the testing of milk and milk products can be found in Chapter 17.

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