Tetrazolium Agar Research Articles

Short communication: Coliform Petrifilm as an alternative method for detecting total gram-negative bacteria in fluid milk

Postpasteurization contamination (PPC) of fluid milk remains a challenge for some dairy processors. Pseudomonas is the most common contaminant of fluid milk after pasteurization, and therefore methods to detect PPC should be inclusive of Pseudomonas and other gram-negative contaminants (e.g., coliform bacteria). Our objective was to compare the ability of 3M (St. Paul, MN) coliform and Enterobacteriaceae (EB) Petrifilm to detect total gram-negative bacteria with that of the standard method, crystal violet tetrazolium agar. To that end, we evaluated coliform Petrifilm, EB Petrifilm, and crystal violet tetrazolium agar to detect gram-negative bacteria in naturally contaminated samples of fluid milk. A total of 92 observations derived from shelf-life testing of 33 milk samples from 5 different processing facilities were evaluated for (1) presence of coliforms on coliform Petrifilm at both 24 and 48 h of incubation; (2) presence of any growth, regardless of gas production, on coliform Petrifilm at both 24 and 48 h of incubation; (3) presence of EB on EB Petrifilm at both 24 and 48 h of incubation; (4) presence of any growth, regardless of gas or acid production, on EB Petrifilm at both 24 and 48 h of incubation; and (5) presence of gram-negative bacteria on crystal violet tetrazolium agar after 48 h of incubation. Sensitivity and specificity analysis of results indicated that compared with the standard method (i.e., crystal violet tetrazolium agar), the method that performed the best, based on balanced accuracy (i.e., the average of sensitivity and specificity), was coliform Petrifilm evaluated for the presence of any growth after 48 h of incubation (sensitivity = 0.787; specificity = 0.839). This method can be easily adopted by the dairy industry as many processing facilities already test for coliforms using coliform Petrifilm. Improving the ability of processors to detect PPC will improve the quality of the fluid milk supply.

Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life

Postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk by gram-negative (GN) bacteria continues to be an issue for processors. To improve PPC control, a better understanding of PPC patterns in dairy processing facilities over time and across equipment is needed. We thus collected samples from 10 fluid milk processing facilities to (1) detect and characterize PPC patterns over time, (2) determine the efficacy of different media to detect PPC, and (3) characterize sensory defects associated with PPC. Specifically, we collected 280 samples of high temperature, short time-pasteurized milk representing different products (2%, skim, and chocolate) and different fillers over 4 samplings performed over 11 mo at each of the 10 facilities. Standard plate count (SPC) as well as total GN, coliform, and Enterobacteriaceae (EB) counts were performed upon receipt and after 7, 10, 14, 17, and 21 d of storage at 6°C. We used 16S rDNA sequencing to characterize representative bacterial isolates from (1) test days with SPC >20,000 cfu/mL and (2) all samples with presumptive GN, coliforms, or EB. Day-21 samples were also evaluated by a trained defect judging panel. By d 21, 226 samples had SPC >20,000 cfu/mL on at least 1 d of shelf life; GN bacteria were found in 132 of these 226 samples, indicating PPC. Crystal violet tetrazolium agar detected PPC with the greatest sensitivity. Spoilage due to PPC was predominantly associated with Pseudomonas (isolated from 101 of the 132 samples with PPC); coliforms and EB were found in 27 and 37 samples with spoilage due to PPC, respectively. Detection of Pseudomonas and Acinetobacter was associated with lower flavor scores; coagulated, fruity fermented, and unclean defects were more prevalent in d-21 samples with PPC. Repeat isolation of Pseudomonas fluorescens group strains with identical partial 16S rDNA sequence types was observed in 8 facilities. In several facilities, specific lines, products, or processing days were linked to repeat product contamination with Pseudomonas with identical sequence types. Our data show that PPC due to Pseudomonas remains a major challenge for fluid milk processors; the inability of coliform and EB tests to detect Pseudomonas may contribute to this. Our data also provide important initial insights into PPC patterns (e.g., line-specific contamination), supporting the importance of molecular subtyping methods for identification of PPC sources.

Spathaspora passalidarum selected for resistance to AFEX hydrolysate shows decreased cell yield.

This study employed cell recycling, batch adaptation, cell mating and high-throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability. The most promising candidate YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and an ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Rapid detection and characterization of postpasteurization contaminants in pasteurized fluid milk

Microbial spoilage of pasteurized fluid milk is typically due to either (1) postpasteurization contamination (PPC) with psychrotolerant gram-negative bacteria (predominantly Pseudomonas) or (2) growth of psychrotolerant sporeformers (e.g., Paenibacillus) that have the ability to survive pasteurization when present as spores in raw milk, and to subsequently grow at refrigeration temperatures. While fluid milk quality has improved over the last several decades, continued reduction of PPC is hampered by the lack of rapid, sensitive, and specific methods that allow for detection of PPC in fluid milk, with fluid milk processors still often using time-consuming methods (e.g., Moseley keeping quality test). The goal of this project was to utilize a set of commercial fluid milk samples that are characterized by a mixture of samples with PPC due to psychrotolerant gram-negative bacteria and samples with presence and growth of psychrotolerant sporeforming bacteria to evaluate different approaches for rapid detection of PPC. Comprehensive microbiological shelf-life characterization of 105 pasteurized fluid milk samples obtained from 20 dairy processing plants showed that 60/105 samples reached bacterial counts >20,000 cfu/mL over the shelf-life due to PPC with gram-negative bacteria. Among these 60 samples with evidence of gram-negative PPC spoilage over the shelf-life, 100% (60/60) showed evidence of contamination with noncoliform, non-Enterobacteriaceae (EB) gram-negative bacteria (e.g., Pseudomonas), 20% (12/60) showed evidence of contamination with coliforms, and 7% (4/60) showed evidence of contamination with noncoliform EB. Among the remaining 45 samples, 28 showed levels of gram-positive bacteria above 20,000 cfu/mL and the remaining 17 samples did not exceed 20,000 cfu/mL over the shelf-life. Evaluation of the same set of 105 samples using 6 different approaches {all possible combinations of 2 different enrichment protocols (13°C or 21°C for 18 h) and 3 different plating media [crystal violet tetrazolium agar, EB Petrifilm (3M, St. Paul, MN), and Coliform Petrifilm]} showed that enrichment at 21°C for 18 h, followed by plating on crystal violet tetrazolium agar provided for the most sensitive, accelerated detection of samples that reached >20,000 cfu/mL due to PPC with psychrotolerant gram-negatives (70% sensitivity). These results show that tests still required and traditionally used in the dairy industry (e.g., coliform testing) are not suitable for monitoring for PPC. Rather, approaches that allow for detection of all gram-negative bacteria are essential for improved detection of PPC in fluid milk.

Survival and detection of coliforms, Enterobacteriaceae, and gram-negative bacteria in Greek yogurt

Despite the widespread use of coliforms as indicator bacteria, increasing evidence suggests that the Enterobacteriaceae (EB) and total gram-negative groups more accurately reflect the hygienic status of high-temperature, short-time pasteurized milk and processing environments. If introduced into milk as postpasteurization contamination, these bacteria may grow to high levels and produce a wide range of sensory-related defects. However, limited information is available on the use and survival of bacterial hygiene indicators in dairy products outside of pasteurized fluid milk and cheese. The goal of this study was to (1) provide information on the survival of a diverse set of bacterial hygiene indicators in the low pH environment of Greek yogurt, (2) compare traditional and alternative detection methods for their ability to detect bacterial hygiene indicators in Greek yogurt, and (3) offer insight into optimal hygiene indicator groups for use in low-pH fermented dairy products. To this end, we screened 64 bacterial isolates, representing 24 dairy-relevant genera, for survival and detection in Greek yogurt using 5 testing methods. Before testing, isolates were inoculated into plain, 0% fat Greek yogurt (pH 4.35 to 4.65), followed by a 12-h hold period at 4 ± 1°C. Yogurts were subsequently tested using Coliform Petrifilm (3M, St. Paul, MN) to detect coliforms; Enterobacteriaceae Petrifilm (3M), violet red bile glucose agar and the D-Count (bioMérieux, Marcy-l'Étoile, France) to detect EB; and crystal violet tetrazolium agar (CVTA) to detect total gram-negative bacteria. Overall, the non-EB gram-negative isolates showed significantly larger log reductions 12 h after inoculation into Greek yogurt (based on bacterial numbers recovered on CVTA) compared with the coliform and noncoliform EB isolates tested. The methods evaluated varied in their ability to detect different microbial hygiene indicators in Greek yogurt. Crystal violet tetrazolium agar detected the highest portion of coliforms, whereas EB Petrifilm detected the highest portion of EB, as well as highest portion of total gram-negative bacteria. Additionally, the D-Count method allowed for faster detection of EB in yogurt by generating results in approximately 13 h rather than the 24 h required when using EB Petrifilm and violet red bile glucose agar. Results from this study indicate that the coliform and EB groups encompass a broad range of dairy-relevant gram-negative bacteria with the ability to survive in Greek yogurt, supporting their use as microbial hygiene indicator groups in low-pH fermented dairy products.

Evaluation of different methods to detect microbial hygiene indicators relevant in the dairy industry

It is estimated that 19% of the total food loss from retail, food service, and households comes from dairy products. A portion of this loss may be attributed to premature spoilage of products due to lapses in sanitation and postpasteurization contamination at the processing level. Bacterial groups including coliforms, Enterobacteriaceae (EB), and total gram-negative organisms represent indicators of poor sanitation or postpasteurization contamination in dairy products worldwide. Although Petrifilms (3M, St. Paul, MN) and traditional selective media are commonly used for the testing of these indicator organism groups throughout the US dairy industry, new rapid methods are also being developed. This project was designed to evaluate the ability of different methods to detect coliforms, EB, and other gram-negative organisms isolated from various dairy products and dairy processing environments. Using the Food Microbe Tracker database, a collection of 211 coliform, EB, and gram-negative bacterial isolates representing 25 genera associated with dairy products was assembled for this study. We tested the selected isolates in pure culture (at levels of approximately 15 to 300 cells/test) to evaluate the ability of 3M Coliform Petrifilm to detect coliforms, 3M Enterobacteriaceae Petrifilm, violet red bile glucose agar, and an alternative flow cytometry-based method (bioMérieux D-Count, Marcy-l’Étoile, France) to detect EB, and crystal violet tetrazolium agar to detect total gram-negative organisms. Of the 211 gram-negative isolates tested, 82% (174/211) had characteristic growth on crystal violet tetrazolium agar. Within this set of 211 gram-negative organisms, 175 isolates representing 19 EB genera were screened for detection using EB selective/differential testing methods. We observed positive results for 96% (168/175), 90% (158/175), and 86% (151/175) of EB isolates when tested on EB Petrifilm, violet red bile glucose agar, and D-Count, respectively; optimization of the cut-off thresholds for the D-Count may further improve its sensitivity and specificity, but will require additional data and may vary in food matrices. Additionally, 74% (129/175) of the EB isolates tested positive as coliforms. The data obtained from this study identify differences in detection between 5 microbial hygiene indicator tests and highlight the benefits of EB and total gram-negative testing methods.

Evaluation of various selective media for the detection of Pseudomonas species in pasteurized milk

Pseudomonas spp. are common gram-negative, post-pasteurization contaminants that contribute to spoilage of pasteurized dairy products. This study evaluated 5 common selective media for detecting Pseudomonas spp. in pasteurized milk. The performance of each selective medium for recovering 12 different Pseudomonas isolates (selected to represent a diversity of pasteurized milk isolates) was compared with that of standard plate count agar pour plates. Pseudomonas isolates showed varying abilities to produce colonies on different selective media. For 2 of 12 isolates, a 48-h incubation time was required for colony formation on any of the media tested. Violet red bile agar and coliform Petrifilm (3M, St. Paul, MN) were less effective than standard plate count agar pour plates at recovering Pseudomonas, regardless of incubation time, and MacConkey agar showed poor detection efficiency compared with SPCP after a 48-h incubation (R2=0.26). Therefore, the use of violet red bile agar, MacConkey agar, or coliform Petrifilm may not be sufficient for detecting common Pseudomonas spp. in milk. The methods showing the highest detection efficiencies were crystal violet tetrazolium agar (CVTA) pour plates (R2=0.95) and CVTA plates inoculated by spiral plating (R2=0.89) incubated at 32°C for 48h. Overall, plating milk samples on CVTA followed by a 48-h incubation at 32°C was the most effective selective method for recovering a diversity of Pseudomonas spp. from milk.

An improved tetrazolium agar medium for testing sugar fermentation in lactobacilli

An improved tetrazolium agar medium for testing sugar fermentation in lactobacilli is described. Basal medium 86 was essentially a modified MRS broth with the omission of glucose. The standard formula was 30 micrograms/ml of 2,3,5-triphenyltetrazolium chloride, 2% sugar to be tested, and 2% agar in medium 86. Plates were incubated anaerobically for 2 days at 37C or 5 days at 30C, depending on the strain. With three strains each of group II and III lactobacilli, colorless, fermentation-positive colonies were clearly differentiated from red, fermentation-negative colonies. For three strains of group I lactobacilli, this medium was not satisfactory because they grew poorly on it unless supplemented with a sugar.

Ineffectiveness of Crystal Violet Tetrazolium Agar for Determining Psychrotrophic Gram-Negative Bacteria

Gram-negative psychrotrophic and nonpsychrotrophic mesophiles grew equally well on crystal violet tetrazolium (CVT) agar at 30°C in 48 h, hence this medium is not suitable for enumerating or recognizing psychrotrophic bacteria.

Potato-Like Odor of Retail Beef Cuts Associated with Species of Pseudomonas

Retail cuts of beef and hamburger packages from a North Dakota meat processor were examined due to consumer complaints of a strong potato-like or musty odor associated with the meat. Examination for total numbers of aerobic bacteria on plate count agar and for gram-negative psychrotrophic bacteria on crystal violet tetrazolium agar revealed numbers in excess of 108 CFU/g. Numbers of coliform bacteria on violet red bile agar were in excess of 106 CFU/g. Gram-negative rods were isolated and identified. The isolates were characterized by a positive catalase reaction, oxidase production, an oxidative O/F reaction, nonutilization of lactose, liquefication of nutrient gelatin, slight motility, production of acid in litmus milk with decoloration and clotting, nonproduction of indole, and nonreduction of nitrate. The isolate was tentatively identified as a Pseudomonas of undetermined species, probably a variant of either Pseudomonas taetrolens or Pseudomonas perolens.

Characterization of Psychrotrophic Bacterial Contamination in Commercial Buttermilk

The extent to which fresh and stored commercial buttermilk was contaminated with psychrotrophic bacteria was determined with pour plates of crystal violet tetrazolium agar incubated at 7°C for 10 days. Fresh buttermilk samples were obtained from six dairy plants on 5 days during 3 mo. For these samples, psychrotrophic counts ranged from less than 10 to 86,000/ml. After samples were refrigerated for 14 days, counts ranged from less than 10 to 1,200/ml. Microbial isolates from the medium were identified, and their ability to reduce diacetyl was determined. The diacetyl content of fresh buttermilk ranged from undetectable to 8.6 µ/ml and declined after 7 and again after 14 days of refrigerated storage. The concentration of diacetyl in refrigerated buttermilk may be influenced by psychrotrophic contamination, but no consistent pattern was observed. Species of Pseudomonas, Enterobacter, Acineto-bacter, Escherichia, and Actinobacillus genera were isolated from the buttermilk. Isolates of these genera reduced diacetyl at 21°C in 24h 30 to 40%, 25 to 75%, 26%, 45%, and 48%. Yeast isolated from the buttermilk reduced 58% of the diacetyl under these conditions. Microbial contaminants of buttermilk have the potential to affect adversely flavor quality.

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.

Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12

Genetic recombination between a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1) in Escherichia coli K-12 has been examined. Since the deletions were nonoverlapping, rare lactose-fermenting (Lac+) recombinants occurred and were detected qualitatively on lactose tetrazolium agar indicator plates as white papillae growing on the surface of red colonies or quantitively on lactose minimal agar plates. Formation of Lac+ recombinants required the recA, recB, and recC gene products. Indirect suppression of recB21 by sbcB15 led to an increase in the frequency of Lac+ recombinants over wild-type levels. recF143 did not appreciably alter the number of Lac+ progeny, whereas recL152 and sbcB15 strains yielded increased numbers of Lac+ recombinants. The nature and formation of Lac+ recombinants was also examined. Respreading analysis indicated that formation of recombinants occurred primarily as the cells entered early stationary phase on the surface of the minimal agar plates and that over 90% of the recombinants contained a phi80dIIlac+ prophage. Time-of-entry experiments suggested that the region of deoxyribonucleic acid between the two operons was not inverted as a result of the recombinational event.

MICROBIAL COUNTS OF INDIVIDUAL PRODUCER AND COMMINGLED GRADE A RAW MILK

Microbial populations of Grade A raw milk samples from 105 individual producers and 74 bulk tank trucks (commingled) were enumerated by Standard Plate Count (SPC), psychrotrophic count (PBC), coliform count (CC), laboratory pasteurized count (LPC), thermophilic count (TBC), yeast and mold count (Y&M), and special penicillin (PEN) and crystal violet tetrazolium (CVT) agar count procedures. In addition, microbial populations were determined by the SPC, PBC, PEN, and CVT procedures after preliminary incubation (PI) of samples. Initial mean counts obtained on individual producer samples were generally lower than those for commingled samples. However, producer samples had higher mean counts after PI. Growth ratios were lower for commingled than for individual producer samples indicating slower growth during PI. Results obtained by the PBC, PEN, and CVT procedures were similar when viewed as correlation coefficients, distribution of samples according to microbial counts, mean counts, and growth ratios during PI. Before PI, the correlation between these three tests was poor and lacked statistical significance when the PBC was <50,000/ml. After PI, the tests were highly correlated (P<0.01) and the r values ranged from 0.8 to 0.9 for samples with PBC levels above 108/ml.

Transduction mechanisms of bacteriophage ε15III. A new class of mutations affecting the conversion ofSalmonella anatumby bacteriophage ε15

SUMMARYSalmonella anatumlysogenized by a doubly mutant strain of bacteriophage εγshows a markedly rough colonial phenotype on tetrazolium agar. Mutants that abolish the roughness are reported.

Detection and enumeration of fecal indicator organisms in frozen sea foods. II. Enterococci.

Consistently high recoveries of enterococci as compared to the low numbers of coliforms obtained from the same samples of frozen sea foods are indirect evidence that enterococci are better indicators of contamination in such foods. The use of azide dextrose broth, modified by the incorporation of bromthymol blue, and of ethyl violet azide broth as presumptive and confirmation tests, respectively, were found to be highly specific for the detection and enumeration of enterococci in these samples. Tetrazolium agar medium, when used as a third step after the confirmation test, provides a reliable differentiation of Streplococcus faecalis types from other group D streptococci. A simple procedure is described for further identification of S. faecalis varieties and other enterococcal species. Incidence of biotypes within certain species is noted and relationships of these subgroups to the organisms described by other workers is discussed. The striking resistance of all group D streptococci to dihydrostreptomycin and polymyxin B seems to offer promise for evolving a new selective medium for these organisms.

Ultraviolet Induction of Respiration-deficient Variants of Saccharomyces and Their Stability During Vegetative Growth

Ultraviolet radiation induces respiration-deficient variants with high frequency in both haploid and tetraploid yeasts. Variant colonies arising after irradiation, when overlaid with triphenyl tetrazolium chloride agar, can be classified as (1) “whole colony” and (2) “variegated or sectored.” The heterogeneity in respiratory phenotype of the latter type of colonies is not detectable by colony morphology prior to overlaying with tetrazolium agar. The variegated types may explain some cases of “reversion” of respirationdeficient cells to wild type. Whole colony variants can be grouped into two classes (vI and vII). The vI variants are similar to spontaneously occurring variants and fail to revert to wild type, whereas the vII variants generally revert to wild type at a low frequency. The vI type is produced most frequently and presumably results from nongenic radiation damage, while the less frequently occurring vI type results from nuclear damage.

METHODS FOR THE ISOLATION OF FAECAL STREPTOCOCCI (LANCEFIELD GROUP D) FROM BACON FACTORIES

SUMMARY: Two methods have been developed for isolating and enumerating group D faecal streptococci from localities such as bacon factories where they are heavily outnumbered by other organisms. The first depends on presumptive counts in a Lab‐Lemco‐peptone‐glucose broth (pH 6·0) containing 0·1% of thallous acetate with confirmation by streaking on tetrazolium agar. The other method involves direct plating on tetrazolium‐glucose agar (pH 6·0) containing 0·1% of thallous acetate. On the tetrazolium medium differentiation can be made between Streptococcus faecalis and its variants zymogenes and liquefaciens and the other group D organisms, Strep. faecium, Strep. durans and Strep. bovis.