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.     

Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurized milk within 24 hours

The keeping quality (KQ) of pasteurized milk stored at 5°C and 11°C was predicted within 24 h by pre-incubating samples and counting bacteria by the Direct Epifluorescent Filter Technique (DEFT). For samples from 5°C storage, 0.03% (w/v) benzalkonium chloride and 0.002% (w/v) crystal violet (final concentration) were added to inhibit the growth of Gram positive bacteria during pre-incubation. The samples from milk stored at 11°C were pre-incubated without the addition of inhibitors. After pre-incubation there was a satisfactory relationship between the DEFT count and the KQ of milks at both 5°C and 11°C. The DEFT count following pre-incubation correctly classified > 80% of pasteurized milks on the basis of KQ.     

Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurized milk within 24 hours

The keeping quality (KQ) of pasteurized milk stored at 5 degrees C and 11 degrees C was predicted within 24 h by pre-incubating samples and counting bacteria by the Direct Epifluorescent Filter Technique (DEFT). For samples from 5 degrees C storage, 0.03% (w/v) benzalkonium chloride and 0.002% (w/v) crystal violet (final concentration) were added to inhibit the growth of Gram positive bacteria during pre-incubation. The samples from milk stored at 11 degrees C were pre-incubated without the addition of inhibitors. After pre-incubation there was a satisfactory relationship between the DEFT count and the KQ of milks at both 5 degrees C and 11 degrees C. The DEFT count following pre-incubation correctly classified greater than 80% of pasteurized milks on the basis of KQ.     

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.     

Detection of low numbers of osmophilic yeasts in creme fondant within 25 h using a pre-incubated DEFT count

The results presented show that the direct epifluorescent filter technique (DEFT) can be used to detect low numbers of osmophilic yeasts in creme fondant after pre-incubation, or high numbers without pre-incubation, and within 30 min. Pretreatment with trypsin and Triton X-100 was necessary to achieve efficient filtration of the fondant. The detection limits for osmophilic yeasts in the pre-incubated DEFT count, as extrapolated from the initial plate count, were approximately 1/g after 25 h and 1/10 g after 49 h.     

Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua.

The ability of the divalent cations magnesium, iron, calcium and manganese; yeast extract; pyruvate; catalase; and the carbohydrates glucose, lactose, sucrose, esculin, fructose, galactose, maltose, and mannose to facilitate repair of heat-injured Listeria monocytogenes and Listeria innocua was evaluated. Listeria populations were injured by heating at 56 degrees C for 50 min. To determine the effects on repair, Trypticase soy broth (TSB) was supplemented with each medium component to be evaluated. Repair occurred to various degrees within 5 h in TSB supplemented with glucose, lactose, sucrose, yeast extract, pyruvate, or catalase. Chelex-exchanged TSB was supplemented with divalent cations; magnesium and iron cations were found to have a role in repair. Listeria repair broth (LRB) was formulated by utilizing the components that had the greatest impact upon repair. When incubated in LRB, heat-injured Listeria cells completed repair in 5 h. After the repair, acriflavin, nalidixic acid, and cycloheximide were added to LRB to yield final concentrations identical to those of the selective enrichment broths used in the procedures of the Food and Drug Administration and the U.S. Department of Agriculture. The efficacy of LRB in promoting repair and enrichment of heat-injured Listeria cells was compared with that of existing selective enrichment broths. Repair was not observed in the Food and Drug Administration enrichment broth, Listeria enrichment broth, or University of Vermont enrichment broth. The final Listeria populations after 24 h of incubation in selective enrichment media were 1.7 x 10(8) to 9.1 x 10(8) CFU/ml; populations in LRB consistently averaged 2.5 x 10(11) to 8.2 x 10(11) CFU/ml.     

RAPID DETECTION OF MICROBIAL CONTAMINATION IN TRICLOSAN AND HIGH FLUORIDE DENTIFRICES USING AN ATP BIOLUMINESCENCE ASSAY

Abstract The Celsis ATP Bioluminescence method was optimized and validated to detect the presence of microbial contamination in High Fluoride and Triclosan dentifrice formulations. Several enrichment broths were evaluated by using a 24–27 h incubation period. The ATP concentrations of the enrichment broths were found to a range from 0.012 to 0.040 nM. None of the tested enrichment broths were found to exhibit any sample inhibition/enhancement effects on the ATP Bioluminescence reaction. Dentifrice suspensions were inoculated with bacteria, yeast, and mold. All test microorganisms (ca. 1–15 CFU/g) were detected within a 24–27 h incubation period by using TAT Broth Base enrichment broths containing different concentrations of the following ingredients: Tween 20, Neopeptone, Dextrose, Triton X-100, Thiosulfate, Sodium Dibasic Phosphate, and Glycine. Negative ATP response after 24–27 h of incubation at 35C indicates the absence of contamination from these products.     

A Rapid Method for the Pre‐Enrichment and Detection of Salmonella Typhimurium by Immunomagnetic Separation and Subsequent Fluorescence Microscopical Techniques

Detection of food‐borne pathogens is of great importance in order to minimize the risk of infection for customers. These analyses should be as fast as possible. Any detection method requires enrichment and quantitative analysis of the enriched microbes. Conventional enrichment methods, which take several days, need to be replaced by faster techniques such as immunomagnetic separation (IMS). This technique is based on the use of paramagnetic microspheres coated with antibodies as ligands that have specific affinity to the microbes that have to be detected. In the studies reported here, a rapid method for the detection of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium), combining IMS and Direct Epifluorescence Filter Technique (DEFT), was developed. It was focused on releasing the target cells from the magnetic beads after IMS, because this is a premise for combining IMS, as an alternative pre‐enrichment, with DEFT. Otherwise, the high number of beads form a layer on the filter membrane that makes the following microscopic analysis for the detection of the contaminants impossible. The CELLection^TM Dynabeads^® used in this study, are coated with recombinant streptavidin (rSA) via a DNA linker. The rSA binds biotinylated antibodies that are able to capture target cells. The DNA linker provides the cleavable site, so that the beads can be removed from the captured cells after isolation. In this study a releasing procedure was developed. This procedure allows for an average 74 % ± 4 % of the bead‐bound Salmonella Typhimurium cells to be released from the beads after IMS, so that the detection of the separated cells by DEFT will be possible.     

Influence of culture conditions on cell surface hydrophobicity of rods of genus Serratia

The cell surface hydrophobicity (CSH) is a non-specific adhesion factor that is important in the proliferation of microorganisms on solid surfaces. Serratia spp. is a bacterium that has been increasingly implicated as a primary pathogen in numerous human infections, particularly in urinary tract infections. CSH of 60 Serratia spp. strains isolated from clinical materials was evaluated using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed), enrichment agar with 5% human blood and medium composed of agar granulated (Becton Dickinson), neopeptone (Difco) and 1% (v/v) glycerol. CSH was estimated most frequently when the analyzed strains in enrichment broth were cultured. When grown in enrichment broth cells of Serratia spp. at room temperature were more hydrophobic (43% after 24 h and 47% after 48 h) than those at 37 degrees C (30% after 24 h and 33% after 48 h). CSH of the examined Serratia spp. strains were depended on the temperature, time of the culture of bacteria and the kind of media. The influence of the culture conditions on the changes in CSH of the analyzed bacteria may suggest significance of these properties in the pathogenesis of Serratia spp.     

An evaluation of possible alternative methods to the Howard Mould Count

The Direct Epifluorescent Filter Technique (DEFT) could be used as an alternative to the Howard Mould Count (HMC) to enumerate mould, bacteria and yeast in tomato concentrate. The DEFT counts of micro-organisms before and after thermal treatment were similar. ATP and chitin assays were also examined as possible alternatives.     

Semi-automated counting of bacteria and somatic cells in milk using epifluorescence microscopy and television image analysis

An epifluorescent microscope fitted with a 'Chalnicon' closed circuit television camera linked to an Optomax System III image analyser was used to count bacteria and somatic cells on membrane filters prepared from milk by the direct epifluorescent filter technique (DEFT). For both bacteria and somatic cells, the semi-automated DEFT count of low count milk exceeded the visual DEFT count but this difference became proportionately less as the count increased. There was close agreement between the semi-automated and visual DEFT counts over the range 10⁵-5 times 10⁶/ml for bacteria and 3 times 10⁵-5 times 10⁶/ml for somatic cells. The semi-automated DEFT count of bacteria and somatic cells correlated well with the plate count and Coulter count respectively, with correlation coefficients of 0.83 and 0.81.     

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.     

Evaluation of enrichment broths for their ability to recover heat-injured Listeria monocytogenes

J.R. PATEL AND L.R. BEUCHAT. 1995. Listeria selective enrichment broth (LEB), University of Vermont (UVM) broth, modified UVM (MUVM) broth and Fraser broth (FB) were compared for their ability to recover cells of L. monocytogenes from heated tryptose phosphate broth. Three strains of L. monocytogenes were heated at 54C for 30 min, inoculated into enrichment broths supplemented with 400 µg catalase ml⁻¹, and incubated for 8 h at 30°C. After incubation for 4 h, the total viable cell populations either decreased or did not change, whereas the number of healthy (non-injured) cells of all strains increased significantly in all broths except FB inoculated with the LCDC strain. With an increase in incubation time to 8 h, the number of healthy cells of all strains increased in all broths. At 8 h, the difference between populations of total (injured plus healthy cells) and healthy cells detected in LEB inoculated with two strains was not significant. Overall, recovery of heat-treated cells was significantly higher in LEB, followed by MUVM broth, UVM broth and FB. The addition of catalase to enrichment broths significantly enhanced recovery of heat-injured cells. A slight reduction of catalase activity of heated cells of all test strains in all enrichment broths except FB was observed by extending the incubation period from 4 to 8 h. A test strain that produces relatively higher catalase activity compared to the other strains exhibited the greatest resistance to exogenous hydrogen peroxide. Enumeration of viable L. monocytogenes cells in heated foods should be done using LEB supplemented with 400 µg catalase ml⁻¹ to maximize the recovery of injured cells.     

The survival of Staphylococcus aureus during the fermentation and storage of yoghurt

The effect of yoghurt culture R _x on the survival of Staphylococcus aureus CCM 5984 added to milk in various concentrations was observed during the fermentation and storage of yoghurt. The end of the fermentation process (3.5 h) was only accompanied by a slight reduction. During the storage of yoghurt at 4°C a 1-2 log reduction was observed. No Staph. aureus was detected in yoghurt produced from milk contaminated by 10³ Staph. aureus cells 1^-1 after 48 h of cold storage. When a concentration of 10² Staph. aureus cells was used for milk contamination, the pathogen was not recovered from yoghurt during the fermentation and storage. The fermentation and storage of yoghurt was accompanied by increases in lactic acid and titrimetric acidity, as well as by a decrease in pH value.     

Protein enrichment of potato processing waste through yeast fermentation

Potato starch obtained from waste waters of chips manufacturing was used as a fermentation substrate for yeast protein enrichment. Among 18 yeast strains, 6 strains were screened according to their biomass yield and protein content after fermentation for 16 h at 30 degrees C in an aerated glucose-based liquid media (4.5 Ls). Using concentrated media (25% solids) made from potato starch pre-hydrolyzed with malt flour and batch-fermented for 20 h at 26 degrees C under aerobic conditions, Candida utilis ATCC 9256 was the most efficient protein-forming strain. Scaled-up at the 100 Ls level, the aerobic batch process was improved under fed-batch conditions with molasses supplementation. After drying, fermented starch contained 11-12% protein, including 7-8% yeast protein.     

Role of the yeast ABC transporter Yor1p in cadmium detoxification

Growth of yeast strains, either deleted for the vacuolar ABC transporter Ycf1 or deleted for the plasma membrane ABC transporter Yor1p or overexpressing Yor1p, were compared for their sensitivity to cadmium. On solid medium cell death (or growth inhibition) was observed at cadmium concentrations higher than 100 microM when yeasts were grown at 30 degrees C for 24 h. However, for all tested strains cell death (or growth inhibition) was already observed at 40 microM cadmium when incubated at 23 degrees C for 60 h. Thus cadmium is more toxic to yeast at 23 degrees C than at 30 degrees C. At 23 degrees C, the Deltayor1 strain grew more slowly than the wild-type strain and the double Deltayor1, Deltaycf1 deleted strain was much more sensitive to cadmium than each single Deltayor1 or Deltaycf1 deletant. Overexpression of Yor1p in a Deltaycf1 strain restores full growth. Cadmium uptake measurements show that Deltaycf1 yeast strains expressing or overexpressing Yor1p store less cadmium than the corresponding Deltaycf1, Deltayor1 strain. The strains expressing Yor1p display an energy-dependent efflux of cadmium estimated for the yeast overexpressing Yor1p to be about 0.02 nmol 109Cd/mg protein/min. Yeast cells loaded with radiolabeled glutathione and then with radioactive cadmium displayed a twice-higher efflux of glutathione than that of cadmium suggesting that Yor1p transports both compounds as a bis-glutathionato-cadmium complex. All together, these results suggest that in addition to being accumulated in the yeast vacuole by Ycf1p, cadmium is also effluxed out of the cell by Yor1p.     

UV-B诱导的酵母凋亡现象及调节机制的作用

研究UV-B诱导的酵母凋亡现象及调节机制的作用。通过高密度细胞培养, UV-B能够抑制酵母细胞生长和诱导细胞凋亡。然而, 将UV-B已照射96 h活酵母细胞重新进行UV-B照射时发现, 培养12 d照射过细胞的存活率仍有10% (P<0.05), 而未照射细胞已经基本死亡。同时, 经0.01 mol/L和0.1 mol/L H2O2处理, UV-B照射24 h活细胞的存活率分别是对照的3.0倍和5.2倍; 而经30 min和60 min 55°C热处理, UV-B照射24 h活细胞的存活率分别是对照的3.5倍和9.0倍。     

A bacteriostatic mixture for milk samples and its effect on bacteriological, cytological and chemical compositional analysis

Of the bacteriostats tested, that comprising boric acid, glycerol, potassium sorbate and nystatin was the most suitable in preventing the multiplication of bacteria, yeasts and moulds in milk whilst causing least change in the Direct Epifluorescent Filter Technique (DEFT) count of bacteria and somatic cells. The DEFT and plate count of bacteria and the DEFT count of somatic cells were reduced after preservation and storage. The relationships between the plate count of fresh milk samples and the DEFT count and plate count of the same samples after preservation and storage for 3 d at room temperature (20–22°C) had correlation coefficients ( r ) of 0.80 and 0.85, respectively. The relationship between the Coulter count of somatic cells in fresh milk and the DEFT count of somatic cells in stored preserved milk had a correlation coefficient of 0.90. The addition of the bacteriostatic mixture to milk affected the determination of protein by the Milkoscan and lactose by the Infra Red Milk Analyser. It did not affect the estimation of the other milk components (fat, protein or lactose) by either instrument.     

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.     

The differential fluorescence of bacteria stained with acridine orange and the effects of heat.

Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence.