Evaluation of the Microbial Quality of Raw Milk 1.

Commercial evaluation of the microbial quality of raw milk presents a major challenge, and new methods are burdened by being compared to imprecise presently used standard methods. Extensive comparisons in commercial and research laboratory environments were made using a method that involved direct enumeration of single cells in comparison to colony forming units. The correlations were from 0.50 to 0.99 depending on treatment of the data. Repetition of all tests on milk from individual farms indicated that inherent variation in quality at the farm, sampling, testing, and evaluating the results showed the extreme inadequacy of the presently established methods of grading raw milk. More frequent tests with appropriate averaging would improve the likelihood of correct decisions on quality grade.

Simple method to demonstrate radiation-inducible radiation resistance in microbial cells.

A simple method for detection of radiation-inducible radiation resistance was developed by irradiating aliquots (0.01 ml) of cell suspension on agar plates. Part of each experimental plate was subjected to an induction treatment, and subsequent radiation resistance was compared with that of untreated cells on the same plate. The UV radiation resistance of a Micrococcus sp. was increased approximately 1.6 times by an induction treatment. This simple procedure of irradiating cells in a "fixed" position on agar avoided washing, centrifugation, and cell enumeration required in traditional methods.

Effect of physiological age on radiation resistance of some bacteria that are highly radiation resistant.

Physiological age-dependent variation in radiation resistance was studied for three bacteria that are highly radiation resistant: Micrococcus radiodurans, Micrococcus sp. isolate C-3, and Moraxella sp. isolate 4. Stationary-phase cultures of M. radiodurans and isolate C-3 were much more resistant to gamma radiation than were log-phase cultures. This pattern of relative resistance was reversed for isolate 4. Resistance of isolate 4 to UV light was also greater during log phase, although heat resistance and NaCl tolerance after heat stress were greater during stationary phase. Radiation-induced injury of isolate 4 compared with injury of Escherichia coli B suggested that the injury process, as well as the lethal process, was affected by growth phase. The hypothesis that growth rate affects radiation resistance was tested, and results were interpreted in light of the probable confounding effect of methods used to alter growth rates of bacteria. These results indicate that dose-response experiments should be designed to measure survival during the most resistant growth phase of the organism under study. This timing is particularly important when extrapolations of survival results might be made to potential irradiation processes for foods.

Paper replication method for isolation of radiation-sensitive mutants.

A filter paper replication system particularly useful for isolation of radiation-sensitive mutants of pigmented bacteria was devised. The fidelity of replication was high. Adhesion between a paper disk and a properly dried master plate provided adequate contact pressure. The replicas arising from this technique constitute a convenient apparatus for general application in isolation of clones sensitive to a discriminating treatment.

Heterogeneity of Samples as a Problem in Shelf-Life Prediction.

A study of the common assumption that a single package of milk represents the production lot was made. Sensory and microbial observations on shelf-life were of sample sets of sequentially produced cartons of milk from four different commercial operations. Neither spoilage rate nor nature of spoilage was uniform for a typical sample set of ten units. A single package, therefore, provided low probability for predicting behavior of the entire production lot. Observations on individual colonies from standard plate counts at the time of sensory spoilage indicated the microflora to be a pure culture in each spoiled unit. The extreme differences in spoilage rates of individual units within sample sets indicated sensory evaluation of multiple samples to be the most logical, simple criterion for evaluating shelf-life. The size of the sample set to be observed and frequency of sampling awaits further observations and application of statistical techniques to establish the accuracy of estimates desired.

Colony-Forming Unit Enumeration by a Plate-MPN Method.

Concepts of the standard surface plate method and the most probable number method (MPN) were combined to provide a new enumeration technique (plate-MPN). Three discrete 0.01-ml samples of an appropriate decimal dilution were inoculated onto each quadrant of a pre-dried petri plate. The discrete spots from the inoculum were then observed for growth after incubation. Results were interpreted analogous to a 3-tube MPN test using presently available tables. Application of the test to pure cultures and mixed flora provided no evidence to indicate the plate-MPN technique to be any less accurate than the standard technique for microbial counts. The plate-MPN technique was less precise than the standard technique. However, the plate-MPN technique has many advantages over traditional methods.

UV light-induced survival response in a highly radiation-resistant isolate of the Moraxella-Acinetobacter group.

A highly radiation-resistant member of the Moraxella-Acinetobacter group, isolate 4, obtained from meat, was studied to determine the effect of preexposure to UV radiation on subsequent UV light resistance. Cultures that were preexposed to UV light and incubated for a short time in plate count both exhibited increased survival of a UV light challenge dose. This response was inhibited in the presence of chloramphenicol. Frequencies of mutation to streptomycin, trimethoprim, and sulfanilamide resistance remained the same after the induction of this survival response and were not altered by treatment with mutagens, with the exception of mutation to streptomycin resistance after gamma-irradiation or nitrosoguanidine or methyl methane sulfonate treatment. The results indicated that isolate 4 has a UV light-inducible UV light resistance mechanism which is not associated with increased mutagenesis. The characteristics of the radiation resistance response in this organism are similar to those of certain other common food contaminants. Therefore, considered as part of the total microflora of meat, isolate 4 and the other radiation-resistant Moraxella-Acinetobacter isolates should not pose unique problems in a proposed radappertization process.

Irradiation of Food for Public Health Protection 1.

Irradiation of food has a long history and has potential for improving public health protection, yet the technology is still not being used commercially. Public acceptance of the process has been hindered by fear and controversy, which involved erroneous definition of radiation as a food additive followed by some ill-conceived and ill-interpreted research. In general, all research has indicated radiation to be bactericidal with various degrees of effectiveness, depending on the specific bacteria being studied. The gram-negative psychrotrophic bacteria, as well as other Enterobacteriaceae , are quite sensitive to radiation and these bacteria are of particular interest to those persons responsible for protecting the quality of fresh red meat. Bacteria resisting less than sterilization doses in irradiation processes have been studied. Acquired increased resistance of bacteria through exposure survival, and subsequent growth of pure cultures can be obtained only through special laboratory manipulations. None of the presently available data indicate bacteria surviving irradiation are of special public health significance.

Fate of Microbial Contaminants in Lettuce Juice 1.

Lettuce is a common menu item that may involve bacteria of public health significance. The most favorable environment for bacterial growth is the exudate (juice) from cut or broken leaves. Bacteria from growing lettuce and those added during harvesting, transportation and storage are able to grow in the exudate. At 10 C gram-negative, psychrotrophic bacteria become the predominant elements of the microflora, while at 20 C the microflora resemble those commonly associated with lactic fermentation of vegetables. Increasing the concentration of solids in lettuce juice to simulate evaporation from droplets of exudate suppressed growth of some members of the natural flora but had little effect on added bacteria of public health significance. Vacuum packaging only decreased slightly the rate of growth of the normal flora in droplets of lettuce juice. All the results indicated lettuce juice may be important as a harborage for bacteria of public health significance.

Fate of Escherichia coli and Salmonella typhimurium in a Food Film on Stainless Steel at 5°C 1.

The fate of Escherichia coli and Salmonella typhimurium in a model system of food residue was determined. Bacteria were grown at 32°C in plate count broth or beef "serum", placed on stainless steel, then dried at 5°C under quiescent or forced air and held for 24 h. Survival was determined by enumeration on plate count agar, and injury was determined by failure of E. coli to grow on violet red bile agar or S. typhimurium to grow on brilliant green agar. The physiological age of a culture was a major determinant of survival and injury. At the most vulnerable age of bacteria in plate count broth, approximately 99.9% of the cells died during the test period and 90% of the survivors was injured. In beef serum there was less death and injury than in plate count broth. The forced air environment was less destructive than the quiescent environment. The model system indicated bacteria in a food film may be in an unfavorable environment, and the surviving bacteria may not be enumerated with commonly used selective media.

Surface Microenvironment and Penetration of Bacteria into Meat 1.

Surface contamination in the form of discrete colony-forming units is the main source of bacteria associated with meat spoilage. The fate of these bacteria is determined by the microenvironment at the meat-atmosphere interface, where the constraints determine the nature of a developing microflora. Nutrients, water availability and nutrient diffusion are prominent factors influencing microbial activity. While surface growth is most commonly recognized through enumeration studies based on removal of microorganisms, the less-studied phenomenon of movement of bacteria may be of considerable significance. In model systems, Serratia marcescens moves rapidly in intact meat as well as in compacted comminuted meat. The invasion process may depend on specific enzymes rather than the general class of collagenases. Need for more knowledge about factors that control surface microenvironment of meat is apparent.

Science of hemolytic activity of some radiation-resistant micrococci in food.

Micrococci resistant to 1 Mrad of gamma radiation were isolated from irradiated chicken. Three isolates were hemolytic on blood agar plates and were selected for further study. Two other radiation-resistant micrococci, Micrococcus radiodurans and Micrococcus radiophilus, were included in the study because there is only a very limited amount of information regarding hemolytic activity of these organisms and their potential role of public health importance. Tests to determine hemolytic patterns, hemolytic activity of extracellular substances, leukocytic activity, presence of enzymes commonly associated with pathogenicity (coagulase, deoxyribonuclease, phosphatase), and pathogenicity for laboratory animals all suggested that the organisms would not be of public health significance.

Isolation of radiation-resistant bacteria without exposure to irradiation.

Resistance to desiccation was utilized in the selection of highly radiation-resistant asporogenous bacteria from non-irradiated sources. A bacterial suspension in phosphate buffer was dried in a thin film at 25 degrees C and 33% relative humidity. Storage under these conditions for 15 days or more reduced the number of radiation-sensitive bacteria. Further selection for radiation-resistant bacteria was obtained by irradiation of bacteria on velveteen in the replication process, thereby avoiding the toxic effect of irradiated media. The similarity of radiation resistance and identifying characteristics in irradiated and non-irradiated isolates should allay some concerns that highly radiation-resistant bacteria have been permanently altered by radiation selection.

Patterns of cell division, DNA base compositions, and fine structures of some radiation-resistant vegetative bacteria found in food.

Representative highly radiation-resistant Moraxella-Acinetobacter (M-A), Pseudomonas radiora, Micrococcus radiodurans, and Micrococcus radiophilus exhibited a wide variety of division systems and cell wall characteristics. However, the most resistant M-A possessed unusually thick cell walls, indicating a possible role of the cell wall in radiation resistance in the M-A. Thick septation was present in most of the bacteria studied, but was absent in P. radiora, thus excluding this as a necessity for high resistance. Reliable determination of the number of division planes of the M-A for use as a taxonomic criterion was achieved by the direct observation of dividing cells. The highly resistant M-A were found to divide in multiple planes and had base compositions of 54.0 to 57.5%, unlike typical Moraxella and/or Acinetobacter species. The taxonomic position of most highly resistant bacteria remains unclear.

Temperatures of Water in Household Refrigerators 1.

Temperatures of water exemplifying liquid foods in household refrigerators were determined. The mean of a.m. and p.m. observations with 11 different refrigerators was 3.6 C. Only two refrigerators showed a significant difference between a.m. and p.m. temperatures. Both the variance and range were surprisingly low in view of the limited previous published data. These results indicated household refrigerators have received too much condemnation for inadequate protection of quality of foods.

A Test to Determine Microquantities of Milkfat in Cleaning Solutions by Surface Film Displacement 1.

Determining microquantities of soil is a major challenge in evaluating cleaning systems. A highly sensitive test was therefore developed to determine trace quantities of milkfat in cleaning solutions. It involved extraction of cleaning solution with organic solvents and subsequent determination of surface film displacement. The area displaced was directly related to the quantity of milkfat in the test solution with a correlation coefficient of 0.99. Usefulness of the test in evaluating cleaning systems has been shown in other research.

Lettuce Salad as a Carrier of Microorganisms of Public Health Significance 1.

Culture, distribution, and preparation of lettuce for salad offers opportunities for contamination with and growth of microorganisms. Protection and preservation methods, even when appropriate, may likewise be favorable for the contaminants. Fresh lettuce, as commercially available, was studied to determine the magnitude of contamination and the nature of representative contaminants. Specific contaminants of public health interest were added to test portions to determine their fate during storage of lettuce as a salad at room temperature. Storage of lettuce in bowls on ice resulted in very little cooling of most of the lettuce. Microbial plate counts on fresh lettuce commonly were over 105/g and the diversity of the microflora indicated a generally favorable microenvironment for many types of bacteria. Inocula of Salmonella typhimurium , Escherichia coli , and Staphylococcus aureus fared well on lettuce salad and were able to grow at room temperature storage. Commmercial "whitener" added to lettuce to preserve freshness reduced the total microflora and indicator organisms of public health significance.

Characterization of radiation-resistant vegetative bacteria in beef.

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized.

Changes in Escherichia coli associated with acquired tolerance for quaternary ammonium compounds.

Acquired tolerance for a quaternary ammonium compound produced a tolerance for a similar compound. Tolerance was associated with the structure and the extent of adsorption of the compound. Morphological changes and resistance to disruption by pressure and by sonic treatment accompanied the development of tolerance. An otherwise weakened culture evolved with the acquisition of tolerance. The maximum obtainable viable population density of tolerant cells in growth medium was approximately 5% of that obtained in the parent culture. Tolerant cultures died off more rapidly in the original growth medium as well as when washed cell suspensions were stored at 5 C. Since acquired tolerance was associated with an otherwise weakened culture, the occurrence of the tolerant cells to limit the efficacy of quaternary ammonium compounds in sanitation operations is highly unlikely.