A modified direct epifluorescent filter technique for the detection and enumeration of yeast in yoghurt

A modified direct epifluorescent filter technique (DEFT) for the detection and enumeration of visible yeast in fruit-flavoured yoghurt is described. The method involves an initial enrichment in oxytetracycline glucose yeast peptone broth (OGYP, 30C/24, 48 h), prefiltration prior to DEFT and use of the vital stain Viablue 1. Additional yoghurt samples were subjected to prolonged incubation at 12C or spiked with Kluyveromyces fragilis . When DEFT was compared with the plate count, regression and correlation coefficients of 1.12 and 0.85 respectively were obtained for values above the sensitivity threshold of DEFT (10³ cells/ml). The use of an enrichment stage (OGYP, 30C/24 h) enabled, by calculation, a theoretical minimum yeast contamination level of 7 yeast cells/g in the original yoghurt to be detected assuming the cells exhibit no lag phase of growth.     

Enzymatic sorting of bacterial colonies on filter paper replicas: detection of labile activities.

To utilize autoradiographic colony-sorting techniques (C. R. H. Raetz, Proc. Natl. Acad. Sci. U.S.A. 72:2274-2278, 1975) for the isolation of mutants with unstable enzymes, we report a new desiccation-induced lysis method, compatible with low temperatures. Furthermore, a general, two-step protocol is presented for clonal detection of hydrolytic reactions. The advantages of these critical modifications are demonstrated with the membrane enzymes glycerol 3-phosphate acyltransferase and cytidine 5'-diphosphate-diglyceride hydrolase.     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

Separation of micro-organisms from meat and their rapid enumeration using a membrane filtration-epifluorescent microscopy technique

A new method of separating bacteria from beef mince has been developed, in which an alkaline protease, Alcalase 0.6 L, was used to degrade the meat proteins, leaving micro-organisms in suspension. The organisms were then counted, using a membrane filtration-epifluorescent microscopy technique. A correlation coefficient of 0.97 was obtained between this method and the standard plate count, indicating its suitability for use in quality control.     

Improvement of the membrane filter technique for enumeration of enterococci in water

The membrane filter technique with AC agar medium supplemented with 0.04% NaN3 and 0.00015% 2,3,5-triphenyltetrazolium chloride for enumeration of enterococci in water is described. An appropriate volume of a water sample was filtered through the membrane filter. The membrane filter was put on an AC agar plate (designated as the AC.MF technique), which was incubated at 37 C for 18 hr and further at 45 C for 24 hr. By this AC.MF technique, all the colonies grown on the membrane filters were identified as enterococci, and the count of enterococci obtained by the AC.MF technique was similar to that by the AC.MPN technique. The AC.MF technique may be useful for accurate and rapid enumeration of enterococci in water and serve as a simple method for determining the sanitary quality of water.     

Use of the direct epifluorescent filter technique for the enumeration of bacterial spores

Heat treatment at 80°C for 10 min effectively destroyed all vegetative cells (except for Gram-positive cocci) and made easier the counting of bacterial spores, which stained orange, green or rarely transparent/black with a dull green halo, in the direct epifluorescent filter technique. The numbers of both orange- or green-staining spores were lower than the plate count. A variety of physiological conditions were used to investigate the relationship of the different staining patterns with germination status. It was concluded that orange-staining spores had germinated and their number agreed with the plate count after incubation in yeast glucose broth at 30°C for 4 h. This observation was unreliable, however, but it was found that a total spore count in the DEFT gave a good agreement with the plate count.     

Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms

AIMS: A method for rapid and simultaneous detection, identification and enumeration of specific micro-organisms using Peptide Nucleic Acid (PNA) probes is presented. METHODS AND RESULTS: The method is based on a membrane filtration technique. The membrane filter was incubated for a short period of time. The microcolonies were analysed by in situ hybridization, using peroxidase-labelled PNA probes targeting a species-specific rRNA sequence, and visualized by a chemiluminescent reaction. Microcolonies were observed as small spots of light on film, thereby providing simultaneous detection, identification and enumeration. The method showed 95-100% correlation to standard plate counts along with definitive identification due to the specificity of the probe. CONCLUSION: Using the same protocol, results were generated approximately three times faster than culture methods for Gram-positive and -negative bacterial species and yeast species. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is an improvement on the current membrane filtration technique, providing rapid determination of the level of specific pathogens, spoilage or indicator micro-organisms.     

Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT→GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10⁷ normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10¹⁰ normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.     

A simple method for the detection of two types of thiaminase-producing colonies

Abstract A plate method which can detect two types of thiaminase-producing colonies was developed. After growing bacterial colonies on a plate with good thiaminase production for 2–4 days, soft agar was overlayed which contained the substrate thiamin, and substituting base (pyridoxine) for type I enzyme, and a buffer of appropriate pH for each type of enzyme. The plate was incubated at 37°C for 1–2 h for Bacillus thiaminolyticus and clostridia, and at 60°C for 2 h for Bacillus aneurinolyticus . Good results were obtained for laboratory strains of average thiaminase activity.     

A simple method for detection of imprinting effects based on case-parents trios

Using data from families in which marker genotypes are known for the father, the mother and the affected offspring, a simple statistic for testing for imprinting effects is developed. The statistic considers whether the expected number of families in which the father carries more copies of a particular marker allele than the mother is equal to the expected number of families in which the mother carries more copies of the allele than the father. The proposed parent-of-origin effects test statistic (POET) is shown to be normally distributed and can be employed to test for imprinting in situations where the marker locus need not be a disease susceptibility locus and where the female and male recombination fractions are sex-specific. A simulation study is conducted to characterize the power of the POET and other properties, and its results show that it is appropriate to employ the POET.     

Rapid method for selecting appropriate solid media for the enumeration of aerobic micro-organisms

A quick and cheap method for selecting appropriate solid culture media has been devised. It consists in the rapid picking of fragments of test colonies with the aid of a rubber strip in which pins are fixed in parallel, dispensing up to 8 colonies simultaneously in the wells of a Microtiter plate and streaking 4 strains at the same time on square Petri dishes containing the media under comparison. The approximate diameters of well-isolated colonies are measured with the aid of a series of calibrated spots. The results corresponded with those given by the spiral plate method used as a reference for colony count and diameter measure.     

Rapid method for selecting appropriate solid media for the enumeration of aerobic micro-organisms

A quick and cheap method for selecting appropriate solid culture media has been devised. It consists in the rapid picking of fragments of test colonies with the aid of a rubber strip in which pins are fixed in parallel, dispensing up to 8 colonies simultaneously in the wells of a Microtiter plate and streaking 4 strains at the same time on square Petri dishes containing the media under comparison. The approximate diameters of well-isolated colonies are measured with the aid of a series of calibrated spots. The results corresponded with those given by the spiral plate method used as a reference for colony count and diameter measure.     

Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique.

Filtration of "stomachered" food suspensions through nylon filters (pore size, 5 microns) removed most of the food debris without affecting the recovery of microorganisms. Two to ten milliliters of these prefiltered suspensions could be filtered in the direct epifluorescent filter technique (DEFT). The technique takes less than 30 min to complete and has a lower sensitivity of less than 60,000 microorganisms per g for all products examined. Vegetative bacterial cells, spores, fungal hyphae, and yeasts could be distinguished with the technique. For fresh meat and fish, the DEFT count of prefiltered suspensions agreed well with the plate count of unfiltered suspensions over the range of 10(4) to 10(10)/g (correlation coefficient of 0.91). For frozen meat and fish and frozen vegetables, the two counting methods had correlation coefficients of 0.87 and 0.66, respectively. The poor correlation for frozen vegetables was due to the inclusion in the DEFT count of nonviable bacteria killed by the blanching process used to inactivate enzymes. Good agreement was obtained between the prefiltered DEFT count and unfiltered plate count for cooked meats, cream doughnut, and whole peppers. Possible reasons for the poor agreement between the DEFT count and plate count for certain products are discussed.     

Development of a rapid detection and enumeration method for thermophilic bacilli in milk powders

Thermophilic strains of Geobacillus, Anoxybacillus and Bacillus that are able to grow at 55 degrees C and above are recognized as commonly occurring contaminants during the production of milk powders. In particular, Anoxybacillus flavithermus strain C and Bacillus licheniformis strain F are often the most prevalent. We describe here the development of a TaqMan-based real-time-PCR assay using a small amplicon of the ribosomal 16S rRNA gene for the selective and quantitative detection of thermophilic bacilli in milk powders. We further present an effective, rapid and inexpensive method for the isolation of total bacterial DNA from milk powder for quantitative PCR analysis within 20 min. With this method, the detection of thermophilic bacilli in milk powder can be accomplished within 1 h. The detection limit for reconstituted and inoculated milk was 8 vegetative cfu ml(-1) and 64 spores ml(-1), respectively.     

A quick and simple biostrip technique for detection of lactose

A quick, simple and economical biostrip technology was developed for estimation of lactose by immobilizing -galactosidase, galactose oxidase and horseradish peroxidase on to a polymeric support. The biostrip is dipped in milk or milk products and, from the colour that develops from an added chromogen, the concentration of lactose can be estimated from < 20 to 100+g l^–1. The biostrips may be used in dairy industries, hospitals and remote areas where expensive instruments are not available.