Identification and detection of Bacillus sporothermodurans spores in 1, 10, and 100 milliliters of raw milk by PCR.

A PCR method was developed to detect spores of Bacillus sporothermodurans in 1, 10, and 100 ml of raw milk. Two primers were derived from a unique sequence after subtractive hybridization of B. sporothermodurans DNA with DNA of MB 397, a not yet identified spore-forming bacterium isolated from raw milk, closely related to B. sporothermodurans. Specific identification was proven on a large collection of Bacillus strains and on strains from relevant taxa. The detection of B. sporothermodurans in raw milk is based on activation, germination, and outgrowth of the spores, followed by PCR identification. Spores from 10 and 100 ml were concentrated by centrifugation after chemical extraction of the milk components. The total test takes 28 h. The detection limits are 9, 0.4, and 0.22 CFU/ml for 1, 10, and 100 ml, respectively.

A direct PCR detection method for Clostridium tyrobutyricum spores in up to 100 milliliters of raw milk.

A direct detection method for Clostridium tyrobutyricum spores in up to 100 ml of raw milk is presented. The bacterial spores are concentrated by centrifugation after chemical extraction of the milk components. The vegetative cells are selectively lysed, and their DNA is digested and washed away. Afterwards, the DNA is liberated from the spores by microwave treatment. For the identification of the C. tyrobutyricum DNA, a two-step PCR method with two nested pairs of primers is used. The primers were derived from the 16S-23S rRNA spacer region of C. tyrobutyricum, and the specificity of each of them for C. tyrobutyricum is demonstrated. The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk.

Impedance Measurements to Detect Bacteriophage Problems in Cheddar Cheesemaking.

A simple method is described to detect, within 2 h, complete failure of the starter due to bacteriophages in the manufacture of Cheddar cheese. This method is based on the observation that about 105 disturbing bacteriophages per ml, which cause complete failure of the starter, inhibit the normal impedance decrease brought about by growth of lactic starter bacteria, as recorded in the Bactometer 32 Microbial Monitoring System.

Impedance Measurements to Detect Post-pasteurization Contamination of Pasteurized Milk.

Investigated was whether, or to what extent, impedance measurements are suitable to detect post-pasteurization contamination of pasteurized milk by gram-negative bacteria. Results were compared with those obtained with the benzalkon-crystal violet-ATP method (BC-ATP method). When 1-L portions of milk, containing agents selective for gram-negative bacteria (0.06% benzalkon and 0.002% crystal violet), were incubated at 30°C, a detection time of less than 24 h was found for post-pasteurization contaminated milk. When post-pasteurization contaminating gram-negative bacteria were absent in pasteurized milk, the detection time varied between 26 and 52 h. In total, 83 samples were investigated. This study shows that impedance measurements are useful to trace post-pasteurization contamination of pasteurized milk. The detection time obtained as such for 1-L portions does, however, not give sufficiently reliable information on the degree of contamination. Suitable information could be obtained by investigating different milk portions, as in the BC-ATP method.

Usefulness of the Benzalkon-Crystal Violet-ATP Method for Predicting the Keeping Quality of Pasteurized Milk.

Using the benzalkon-crystal violet-ATP method (BC-ATP method), post-pasteurization contamination of pasteurized milk caused by gram-negative bacteria can be determined within 24 h. This study determined to what extent the keeping quality of pasteurized milk can be predicted by applying this BC-ATP method. Results obtained with the BC-ATP method for 100 samples of pasteurized milk were compared with those recorded in the shelf-life test (total bacterial count after 10 d of storage of samples at 7°C; standard: 106 bacteria per ml) and the Moseley test (bacterial count after 5 d of storage at 7°C; standard: 105 bacteria per ml). Using the shelf life test and the Moseley test, 14 and 8% of the results, respectively, did not correspond with those obtained with the BC-ATP method. From the results obtained, it was obvious that the greater the post-pasteurization contamination of the pasteurized milk, the lesser is the keeping quality. A quantitative estimation of the degree of post-pasteurization contamination can be obtained satisfactorily by applying the BC-ATP method to 1000-, 100-, 10- and 1-ml portions. The Moseley test takes too much time to achieve a good coordination between the factory laboratory and the pasteurization and filling sections of the dairy factory. By substituting the BC-ATP method applied to 100-ml portions of pasteurized milk for the Moseley test (5 d at 7°C; standard: 100,000 bacteria per ml) almost the same information is obtained within 24 h.