Direct Epifluorescent Filter Technique Count Research Articles

Application of the microbiological method DEFT/APC to detect minimally processed vegetables treated with gamma radiation

Marketing of minimally processed vegetables (MPV) are gaining impetus due to its convenience, freshness and apparent health effect. However, minimal processing does not reduce pathogenic microorganisms to safe levels. Food irradiation is used to extend the shelf life and to inactivate food-borne pathogens. In combination with minimal processing it could improve safety and quality of MPV. A microbiological screening method based on the use of direct epifluorescent filter technique (DEFT) and aerobic plate count (APC) has been established for the detection of irradiated foodstuffs. The aim of this study was to evaluate the applicability of this technique in detecting MPV irradiation. Samples from retail markets were irradiated with 0.5 and 1.0 kGy using a 60Co facility. In general, with a dose increment, DEFT counts remained similar independent of the irradiation while APC counts decreased gradually. The difference of the two counts gradually increased with dose increment in all samples. It could be suggested that a DEFT/APC difference over 2.0 log would be a criteria to judge if a MPV was treated by irradiation. The DEFT/APC method could be used satisfactorily as a screening method for indicating irradiation processing.

Semi-Automated Direct Epifluorescent Filter Technique for Total Bacterial Count in Raw Milk

The Direct Epifluorescent Filter Technique (DEFT) and the reference method of counting total bacterial colonies on Petri dishes were compared. IDF Standards 128 (1985) and 161A (1995) were applied. A total of 496 samples of milk were analyzed. Colony forming units per microL milk were transformed to decimal logarithmic units: log (cfu/microL). The repeatability standard deviation, Sr = 0.114, was typical for a routine microbiological method. To study the carryover at different levels of bacteria, 3 tests were performed on milk samples of approximately 100, 700, and >1000 cfu/microL. For the first 2 experiments, no carryover was detected; in the milk sample with >1000 cfu/microL, the carryover was <0.12%. When the DEFT counts were regressed versus the reference method, the values of the slope and intercept were 0.92 and 0.17, respectively; the correlation coefficient was r = 0.84; and the residual standard deviation was Syx = 0.287. The paired t-test showed that the reference method and DEFT do not give significantly different results (p = 0.05).

Direct enumeration of Escherichia coli and enteric bacteria in water, beverages and sprouts by 16S rRNA in situ hybridization

A fluorescent oligonucleotide probe complementary to a published 16S ribosomal RNA sequence ofEscherichia coli was used for in situ hybridization on membrane filters for direct enumeration of cells. The direct epifluorescent filter technique (DEFT) was modified by performing a 2-h oligonucleotide hybridization on intact cells collected on a membrane filter surface (‘oligo-DEFT’), instead of the traditional acridine orange staining. The filter was examined by epifluorescence microscopy, and digital image analysis was used for the cell enumeration. Oligo-DEFT counts were linear over the range of 103–106cells ml−1and correlated with DEFT counts (r=0·99). The oligo-DEFT detection limit was approximately 1 cell ml−1. Oligo-DEFT counts correlated with standard coliform counts by membrane filtration or plating on selective media for analysis of water and beverages artificially inoculated with E. coli. Heat inactivation of the cells did not destroy their reactivity in the oligo-DEFT. Natural coliform populations in aquarium water were countable in the oligo-DEFT after a 90-min incubation of the filter on nutrient agar before the hybridization step, which allowed the dormant cells to increase their rRNA content. Natural populations in bean sprouts were countable directly, without having to perform the incubation before hybridization.

Detection of irradiated deep‐frozen foodstuffs by comparison of DEFT and APC counts

The suitability of a microbiological method based on the comparison of an aerobic plate count (APC) with a count obtained with the direct epifluorescent filter technique (DEFT) for the detection of irradiation of deep‐frozen foodstuffs was evaluated. The study was carried out on parsley, mechanically deboned poultry meat (MDPM) and liquid egg white. The foodstuffs were stored at—18°C and examined after storage for 0, 4 and 12 months.The results showed that this method is discriminating during the whole storage period. Nevertheless, it has been observed that for non‐irradiated liquid egg white the difference between DEFT and APC tends to increase with the time of storage. This phenomenon which did not limit the interest of the test, was not observed for the other products tested.

Fluorescent Antibodies Applied to Direct Epifluorescent Filter Technique for Microscopic Enumeration of Escherichia coli 0157:H7 in Milk and Juice

In a modification of the direct epifluorescent filter technique (DEFT), direct fluorescent antibody staining was used for the rapid (<1 h), specific enumeration of foodbome Escherichia coli 0157:H7 by epifluorescence microscopy. Cell counts obtained by this method (Ab-DEFT) correlated well with DEFT counts obtained by acridine orange staining and with viable plate counts ranging from 108 to 101 cells per ml for pure cultures in buffer. Ab-DEFT also was effective for counting E. coli 0157:H7 cells inoculated into milk and juice; the sensitivity limit was about 103 for milk. The highly specific nature of the technique was demonstrated by enumeration of E. coli 0157:H7 cells in the presence of large numbers of indigenous spoilage microorganisms in milk. This is the first known demonstration of the combination of DEFT and antibody probe technology for the specific enumeration of a microbe directly in food without a growth or enrichment step.

Rapid Detection of Psychrotrophic Bacteria In Manufacturing Grade Raw Milks

Methods to rapidly assess the bacteriological quality of raw milk were investigated. Whereas direct microscopic count, modified psychrotrophic plate count, and direct epifluorescent filter technique (DEFT) did not correlate well with initial psychrotrophic bacterial count of raw milk, improvements were obtained after preincubation of the milk samples. The best preincubation conditions were identified as 30°C for 6 h, 21°C for 10 h, 13°C for 15 h, 13°C for 20 h, or 7°C for 37 h.The “square root” equation was applied to the data, and a model was produced for predicting growth of the native microflora of raw milk. Using this equation, a DEFT count after preincubation of the milk at 21°C for 10 h could accurately predict the initial psychrotroph count and the count after storage of the milk at 6°C for 48 h.

Evaluation of a microbiological method for detection of irradiation of spices

The suitability of a microbiological method, a combination of the direct epifluorescent filter technique (DEFT) and the total aerobic plate count (APC), for the detection of irradiation of spices was evaluated. The evaluation was carried out using different kinds of spices including peppers, paprika, cardamom, cinnamon, ginger; herbs such as thyme, marjoram, basil, and oregano; and garlic powder. Ground spices were tested as well as cut and whole spice samples. Samples were analysed before and after gamma-irradiation mainly with doses of 5 and 10 kGy. Two different methods (centrifugation, filtration) for sample pretreatment were compared. Differences between the DEFT count and the APC with doses of 5 and 10 kGy varied between 3.9 and 6.8 and between 5.7 and 7.5 log units, respectively. The duration of storage after irradiation had no effect on the microbiological results. When applied to fumigated samples, the method might cause confusion in the absence of chemical analyses. The results of the analyses of samples treated by an unknown process indicated that the method could, in fact, detect irradiated samples, although it could not differentiate between irradiated and heat-treated samples.

Evaluation of a television image analyser as an aid to estimation of microbial numbers in food using the direct epifluorescent filter technique

Seventy food samples (23 samples of raw milk, 24 samples of minced meat and 23 samples of unsalted butter) were examined using the direct epifluorescent filter technique (DEFT). For each sample, micro-organisms captured on duplicate filters were counted both by eye and with the aid of Bio-Foss 40-10 image analyser. Each count was performed by two operators. The correlation coefficients for the image analyser counts against the manual DEFT count for the 3 products ranged from 0.94 to 0.98. An analysis of variance indicated that both counting methods were equivalent in their repeatability. Operator experience and the quality of the microscopic image were important factors in determining the accuracy of the automatic count.

The detection of irradiated foods using the Direct Epifluorescent Filter Technique.

A method was evaluated which has the potential to detect a food sample which has been irradiated. The technique will give an indication of the total number of viable micro-organisms present before irradiation. It is based on the comparison of an aerobic plate count (APC) with a count obtained using the Direct Epifluorescent Filter Technique (DEFT). When the APC of an irradiated sample was compared with the DEFT count on the same sample, the APC was considerably lower than that obtained by DEFT. The count of orange fluorescing cells after irradiation, however, correlated well with an APC of the same sample before irradiation. For the samples examined the DEFT count determined the viable microbial population in the sample before irradiation. The difference between the APC and the DEFT count gave the number of organisms rendered non-viable by the process.

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.

Rapid Estimation of Microbial Numbers on Meat and Poultry by the Direct Epifluorescent Filter Technique

The direct epifluorescent filter technique (DEFT) for rapid estimation of microbial numbers was evaluated by comparison with the plate count on a variety of uncooked red meat and poultry samples. Good agreement [correlation coefficient (r) = 0.95–0.96] was obtained from samples with plate counts of 5 × 103/g or /cm2 and above from red meat carcasses (surface swabbed), aerobic or vacuum packed chill-stored joints (surface sampled - stomachered) and frozen beef (thawed stomachered). For stored and unstored raw poultry sampled by skin scraping or stomachering of muscle and skin good overall correlation (r = 0.88–0.89) was obtained between the DEFT count and the plate count in the ranges 1.1 × 103 to 1.3 × 107/cm2 (skin scraping) and 1 × 104 to 9.5 × 106/g (muscle and skin) even though the DEFT always overestimated counts on samples on which no growth had occurred (plate count <7×104/cm2 or <1×105/g). However, good linearity between DEFT and plate counts allowed use of the regression equation to obtain a good estimate of the plate count on these samples. The DEFT was unsuitable for application to poultry neck skin sampled by shaking because particulate material interfered with counting. This was also a problem with Mechanically Recovered Meat although the DEFT gave a fair estimate (r = 0.72) of the plate count on certain types (beef and veal) of this product. The DEFT was capable of providing counts within 35 to 45 min and its applicability to the rapid estimation of bacterial numbers in meat and poultry is discussed.

A comparison of DEFT clump counts obtained in eight dairy laboratories receiving replicate samples of preserved raw milk

In an interlaboratory trial, the Direct Epifluorescent Filter Technique (DEFT) for estimating the hygienic quality of raw milk was compared with the colony count method. The results, from replicate samples of 60 chemically preserved milks examined at eight dairy laboratories, showed linear correlation coefficients ranging from + 0.19 to + 0.90. DEFT results depended on the skill of the operator and agreed less well with colony counts made before preservation than with colony counts made at the same time as DEFT counts. The combined DEFT counts from all laboratories compared with colony counts made after preservation showed a linear correlation coefficient of + 0.87; the equation for the regression line was y = 1.00x + 0.04, indicating that the DEFT can provide results comparable to those of the colony count.

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 direct epifluorescent filter technique, cytochrome c oxidase test and plate count method for predicting the keeping quality of pasteurized cream

The ability of the direct epifluorescent filter technique (DEFT) to predict the keeping quality (KQ) of pasteurized cream, after an 18-h preincubation stage, was poor unless (particularly with samples that had a corresponding long KQ) undiluted cream was filtered. The DEFT then gave as good, if not better, prediction of the KQ as the plate count. The cytochrome c oxidase test (Kroll 1985, J. Appl. Bacteriol. 59 , 137–141) could also be used successfully to predict the KQ of pasteurized cream but its predictive value was not as good as the DEFT or plate count method. However, because of its simplicity, rapidity and low cost, the method could be of use to milk processors to monitor the potential KQ of their products.

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.

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.

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.

Effect of Agitation on Bacterial Aggregates in Pure Cultures and Raw Milk

A comparison of the effects of various mechanical agitation treatments on bacterial aggregates was performed on 8 pure cultures and 27 raw milk samples. Although both syringing and blending produced significant increases in total counts and psychrotroph counts, blending for 2 min gave the greatest increase in count. Use of the direct epifluorescent filter technique (DEFT) confirmed that syringing and blending reduced bacterial clump size to approximately 2 cells. These agitation treatments markedly improved the correlation between DEFT counts and plate counts.

Collaborative trial of the direct epifluorescent filter technique (DEFT), a rapid method for counting bacteria in milk.

The direct epifluorescent filter technique (DEFT) is a new rapid method which uses membrane filtration and epifluorescent microscopy for counting in milk. A collaborative trial of the DEFT was conducted between six laboratories. Each laboratory obtained a highly significant relationship between the DEFT count and plate count with a correlation coefficient generally greater than 0.9 but there were significant differences between these relationships. The repeatability of the DEFT, although ca 1.5 times worse than that of the plate count, was of a level acceptable in practice. Reproducibility of the DEFT was ca 3 times that of the plate count. This poor reproducibility was probably mainly due to counting errors. Possible reasons for this and ways of reducing counting errors are discussed.

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.