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Bacteria in non-fat dried milk (NDM) were enumerated by a method involving preliminary solubilization of the milk proteins in 0.015 N NaOH followed by centrifuging, washing in the NaOH, and microscopically examining stained smears. The method was used to enumerate bacteria in samples of NDM obtained from government surplus stocks or from local retail sources. Bacterial counts from surplus NDM ranged from 4.64 × 105 to 2.83 × 106/g (the mean and median were, respectively, 6.23 and 2.84 × 106/g). Counts from retail samples ranged from 4.48 × 105 to 2.42 × 107/g (mean and median were 5.57 and 2.85 × 106/g). The predominant bacteria in some samples were paired streptococci; other samples contained rod-shaped bacteria, some with identifiable spores. Comparison of this method with the Levowitz-Weber method indicated that it produced fewer artifacts, was applicable to NDM samples containing a wider range of bacteria, and did not require the use of the potentially carcinogenic tetrachloroethane.

Samples of skim milk and nonfat dry milk (NDM) made from it were collected, paired and tested for pyruvate concentration, [P], and Direct Microscopic count (DMC). The skim milk was tested for Standard Plate Count (SPC) and Psychrotrophic Plate Count (PPC). The geometric average DMC of skim milk was more than three times higher than that of the paired NDM samples. However, [P] of NDM was not significantly different from that of the skim milk. Although [P] of skim milk was poorly correlated with SPC and PPC, r = .31 and .26, respectively, it was relatively well correlated with DMC, r = .64. Data were widely dispersed around the regression line when [P] was ≤ 4.0 mg/L. However, [P] increased rapidly when DMCs were > 106/ml. A limit of 10 mg/L of [P] in NDM reconstituted 1:9 was chosen to represent the current U.S. Department of Agriculture Standard for DMC in NDM. This limit failed to classify about 10% of the samples correctly, assuming that each geometric mean DMC was correct. However, the probability that...

In this work, we exploit a general flow cytometry technique involved in the differentiation of live and dead yeast cells for two applications in winemaking. The discrimination of yeast populations is achieved using two fluorescent dyes that measure the metabolic activity and membrane integrity of the yeast. This analytical approach is first applied for quality control of active dry yeast. Results are discussed in comparison with the Codex Oenologique International (International Oenological Codex) of the International Organisation of Vine and Wine (OIV), demonstrating that analysis using flow cytometry is a valuable alternative, given the ease of execution and the high quality of results obtained in terms of reproducibility, repeatability, and confidence interval. In the second case, we apply flow cytometry as a technique for monitoring the production of sparkling wines using the “Champenoise” method, and describe the evolution of yeast through the production process. In this case, results are directly compared with those obtained with the two methods (plate counts and direct microscopic count) listed in the OIV standards, in order to ensure a thorough understanding of the improvements related to the use of flow cytometry.

Summary The objective of this study was to develop a rapid quantitative detection method by triphosphate (ATP)-bioluminescence and determine the feasibility to assay total bacterial counts (TBC) in probiotic products. A useful pretreatment technique to eliminate the interference to the ATP-bioluminescence method was developed, resulting in a 10–100-fold improvement in the rapid quantitative detection method by ATP-bioluminescence. The TBC obtained by the ATP-bioluminescence rapid detection method was tested against the direct microscopic count method or the plate count method and validated on three ATP Assay Systems. The results generated by the ATP methods and the plate count method were comparable to each other when the interference due to non-microbial cell ATP and matter such as pigment were removed. This study reports the optimisation of an ATP-bioluminescence assay using a pretreatment technique to eliminate interference in order to quickly determine the viable counts of bacteria in solid and liquid probiotic products.

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.

Minimum inhibitory concentration (MIC) isobolograms of fresh garlic extract (FGE) and NaCl against Staphylococcus aureus test strains ATCC 25923 and ATCC 6538 in Brain Heart Infusion broth (BHIB) and Pork Sausage broth (PSB) were constructed. Additive and synergistic relationships were observed in BHIB and PSB against the test strains, respectively. Action of indirect antimicrobic ingredients in PSB was used to explain the synergistic relationship of FGE and NaCl established. The Fractional Inhibitory Concentration (FIC) indices of the agents validated the antimicrobic relationships established in the MIC isobolograms. An Iodonitrotetrazolium-Direct Microscopic Count (INT-DMC) method was used to measure staphylococcal cell count reductions in both broth systems containing concentrations of FGE and NaCl typically used in sausage production. Garlic incorporation of ≥1.20 (v/v) together with minimum levels of NaCl (

The automated test for pyruvate concentration, [P], was evaluated as a substitute for the direct microscopic count (DMC) in determining quality of manufacturing grade milk. Samples of manufacturing grade milk from producers and tank trucks as well as skim milk were collected at a large milk drying plant. Each sample was tested immediately for standard plate count (SPC), psychrotrophic plate count (PPC) and DMC. Portions of milk were heated to 85°C for 10 min to stabilize [P] before being returned to the laboratory for analysis of initial pyruvate concentration, [IP]. Unheated samples stored at 4°C were analyzed for [P] and DMC daily for up to 120 h. [IP] was a good indicator of PPC in milk from individual producers (r=0.81). However, [IP] was not highly correlated (r=0.26) with PPC in skim milk samples which were characteristically high in SPC and homogeneous in PPC. With skim milk, [IP] was more highly and significantly correlated with initial DMC (r=0.61) than with initial SPC (r=0.31) or initial PPC (r=0.26). [IP] was a good indicator of [P] in stored fluid samples until counts exceeded about 10

Summary Acetomicrobium flavidum gen. nov., sp. nov. was isolated from a thermophilic, anaerobic sewage sludge digester operated at 60 °c. The Gram-negative, highly motile, non-sporeforming rods form 2 mol acetic acid, 2 mol CO 2 and 4 mol H 2 from 1 mol hexose. They represent at least 25% of the total population (microscopic count) of the fermenting sludge.

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Summary The standard plate count (SPC-32 C) and the direct microscopic count (DMC) of samples of commercially pasteurized milk inoculated with pure cultures of actively growing (18–24 hr growth) bacteria commonly found in milk were compared. Four staining procedures for DMC were used: (a) Levowitz-Weber's methylene blue stain; (b) a modified Levowitz-Weber stain incorporating basic fuchsin; (c) alcoholic-acetic acid fixation followed by periodic acid-bisulfite treatment and staining with pH4 toluidine blue; and (d) alcoholic-acetic acid fixation and staining with pH4 toluidine blue. Counting was standardized by the use of a geometrical pattern. Correlations between SPC and each DMC procedure, or among the DMC procedures were little influenced by the number of microscopic fields counted, their location on the smear or the definition of “clumping” used. Correlations were influenced by the type of bacterial culture inoculated in milk and by the staining procedure. Precision of DMC was shown to be independent from the staining procedure, but varied directly with the number of cells per field and inversely with the square root of the number of fields counted.

The bacteriological quality of manufacturing-grade milk is very similar to that marketed a decade earlier when bulk tanks first came into general use. Milk grading programs usually relied on reduction tests. These tests indicated that most milk supplies were good quality. Based on the Standard Plate Count, data is presented that show approximately one-third of the samples tested, in 1969–70 and in 1957–59, exhibit counts Dairy farmers learned they could substitute cooling for cleaning because psychrotrophic bacteria predominated the microflora of most high count bulk milk. These bacteria do not readily reduce resazurin and methylene blue. Psychrotrophs also tend to grow in clumps preventing an accurate evaluation of milk quality using the Direct Microscopic Count (DMC). USDA uses the DMC to test check manufacturing plant's milk supplies. Laboratories are recognizing the value of plating procedures, including the Plate Loop Count, to determine milk quality. Manufacturing-grade milk must be evaluated with a plating procedure before progress can be made in milk quality improvement. One grade of milk is far from being a reality if present levels of manufacturing-grade milk quality are considered.

During recent years the methylene blue reductase test has been advocated as a practical method to use for determining the approximate number of bacteria in milk. The test has been used quite extensively in some European countries and recently to some extent in this country at dairy manufacturing plants and milk receiving stations. The well established plate count method and the more recent direct microscopic count method have been adopted as official by the American Public Health Association. These tests are more expensive and complicated and demand more care and skill than is required in operating the reductase test. But the latter test has not been used in this country to such an extent that its place in the dairy industry has been definitely established. During the months of July, August, and September, 1924, a number of dairy manufacturing plants and milk receiving stations were visited by the writer and a sample of the milk delivered by each producer was tested for bacteria by both the methylene blue reductase test and the direct microscopic method. The obiect of the work was first, to determine the grade (A, B or C) of each producer's milk on the day it was tested and, second, to learn how the results obtained by the reductase test checked with those obtained by the direct microscopic count method.

Bacteria and somatic cell counts made with the direct microscopic count and millipore membrane count on thirty fresh and on thirty milk samples preserved with one tablet of Lactab (12mg HgCl 2 and 52mg K 2 Cr 2 O 7 ), Thomson and Copper Ltd., Liverpool, England, per 50ml milk, were compared. The samples were classified in three groups of ten according to arbitrarily chosen somatic cell levels of 900,000 cells per ml. The lowest cell level yielded the greatest average count variance. The within somatic cell level count variance was not affected significantly by milk treatment and method. The average somatic cell counts were higher for the millipore membrane method. Although they reacted the same under both milk treatments, use of preservative resulted in higher counts with greater influence on the millipore membrane method. Therefore, the millipore membrane count is a better method for determining somatic cells; significantly lower bacterial counts were obtained with it. The preservative had a marked adverse effect upon the stainability of bacteria independent of the method.

When pasteurized whole eggs from breakers were examined by the Direct Microscopic Count (DMC) procedure, the bacterial count frequently appeared to be too low to correlate with the observed state of decomposition. The DMC of whole egg was found to decrease during pasteurization. To determine why, DMC's were done using the North Aniline Oil - Methylene Blue Stain and the Levowitz-Weber modification of the Newman-Lampert stain. Total bacterial counts also were made using the Petroff-Hausser counting chamber. Results indicated that the reduction in count resulted from lysis of some of the bacterial cells in egg rather than to loss of stainability. Crystalline lysozyme at the concentration found in egg and whole egg preparations produced similar reductions in the DMC of bacteria isolated from egg.

Five screening tests, Direct Microscopic Leucocyte Count, Wisconsin Mastitis Test, California Mastitis Test, Clemson Catalase Test, Wisconsin Catalase Test, used in estimating leucocyte concentrations were evaluated using 1,308 producer milk samples and 97 tank-delivered samples. These milk samples were representative of the South Carolina milk supply. The Wisconsin test was the most reliable method for estimating leucocytes in milk, using the microscopic count for comparison. This applied to both producer and tank-delivered samples. The Clemson and Wisconsin Catalase tests and the California test were not as reliable predictive tests of leucocyte concentration when compared with microscopic count. The leucocyte data were classified into three groups: less than 500,000, 500,000 to 1,000,000, and more than 1,000,000. The microscopic leucocyte count distributions of producer milk samples were 46.6, 42.4, and 11.0%, respectively, and for tank-delivered samples they were 47.4, 43.3, and 9.3%, respectively.

The usefulness of the Wisconsin Mastitis Test in an abnormal milk program is dependent, in large measure, on the degree of its correlation with the direct microscopic leucocyte count. This correlation is dependent to a large degree on the accuracy with which the microscopic count is performed. One of the most likely sources of error in the microscopic count is the criteria for recognizing leucocytes in milk. Wisconsin test results were compared with the microscopic method using counting criteria: whole leucocytes only, whole leucocytes plus damaged cells at least 50% intact, and whole leucocytes and all cell fragments. The results of 117 milk sample comparisons indicated that microscopic Procedures 1, 2, and 3 showed a good correlation with the Wisconsin test (r 1 = 0.88, r 2 = 0.82, r 3 = 0.79), but Procedure 1 was best for predicting Wisconsin test values.

Microbiological criteria in military and federal food specifications are presented. Approximately 10% of more than 600 specifications for food contain microbiological requirements which are applied in procurement of food for military and federal agencies. Foods were grouped into four categories which include dehydrated foods, milk and milk products, miscellaneous dairy products, and frozen foods. Microbiological requirements vary with the food item and include criteria for the aerobic plate count, coliforms, Escherichia coli, salmonellae, yeast and mold, proteolytic bacteria, lipolytic bacteria, psychrotrophs and direct microscopic count. Specifications may be obtained by writing to the U.S. Naval Publications and Forms Center, NPFC Code 1032, 5801 Tabor Avenue, Philadelphia, Pennsylvania 19130.