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 Bacteria in Heat-treated Milk and Milk Products Using a Membrane Filtration-Epifluorescent Microscopy Technique

A rapid direct epifluorescent filter technique (DEFT), taking ca. 20 min to complete, was used to enumerate bacteria in heat-treated milk and milk-products. The DEFT count could detect as few as 5750 bacteria/ml in pasteurized and skim milk, pasteurized cream, whey and sweet cream butter and was in agreement with the standard plate count. The technique was also used to determine the sterility of cartoned ultra heat-treated milk after incubation at 37°C. The agreement between the DEFT and plate count was poor for evaporated and condensed milks, some reconstituted skim milk powders, pasteurized whey and ripened cream butter. Possible reasons for this are discussed.     

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 bacteria 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 > 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.     

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.     

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.     

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.     

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.     

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.     

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.     

Rapid physicochemical detachment, separation and concentration of bacteria from beef surfaces

A simple, rapid, physicochemical treatment for the removal of viable bacteria from the surface of raw and cooked beef is described. The detachment method was linked to a differential centrifugation step which removed large amounts of particulate food matter and concentrated the detached bacteria. The method increased the numbers of bacteria released from beef surfaces and increased the numbers detected by at least one and a half orders of magnitude, when compared to the traditional 'stomaching' technique. This 1-h separation and concentration method produced cleaner suspensions of bacteria and improved the sensitivity of detection by DEFT and direct plate count.     

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.     

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.     

Microbiological quality and safety of zoo food

Two types of commercial products for feeding zoo animals (a frozen meat product, referred to as zoo food, and a dry product, referred to as dry food) were microbiologically examined for spoilage organisms (aerobic, psychrotrophic, coliform, Escherichia coli, mold, and yeasts) and pathogens (Salmonella spp., Listeria monocytogenes, and Campylobacter jejuni). Levels of microorganisms in frozen ground zoo food were compared with those in frozen ground beef and frozen ground turkey meat. The level of microbial contaminants in frozen ground zoo meat was found to be similar to that in frozen ground beef and higher than that in frozen ground turkey meat. Sixty percent of the frozen zoo meat samples were Salmonella positive, and all of the samples were L. monocytogenes positive. Dry zoo food was documented to have microbial levels lower than those in frozen zoo meat; the pathogen levels were less than 1/25 g of food. Defrosting zoo meat at 10, 25, and 37 degrees C for 24 h showed that 10 degrees C is the best temperature for defrosting frozen ground zoo meat loaves (length, 9 in. [22.8 cm]; radius, 2 in. [5.1 cm]) without affecting the microbiological quality or safety of the product.     

Microbiological quality and safety of zoo food.

Two types of commercial products for feeding zoo animals (a frozen meat product, referred to as zoo food, and a dry product, referred to as dry food) were microbiologically examined for spoilage organisms (aerobic, psychrotrophic, coliform, Escherichia coli, mold, and yeasts) and pathogens (Salmonella spp., Listeria monocytogenes, and Campylobacter jejuni). Levels of microorganisms in frozen ground zoo food were compared with those in frozen ground beef and frozen ground turkey meat. The level of microbial contaminants in frozen ground zoo meat was found to be similar to that in frozen ground beef and higher than that in frozen ground turkey meat. Sixty percent of the frozen zoo meat samples were Salmonella positive, and all of the samples were L. monocytogenes positive. Dry zoo food was documented to have microbial levels lower than those in frozen zoo meat; the pathogen levels were less than 1/25 g of food. Defrosting zoo meat at 10, 25, and 37 degrees C for 24 h showed that 10 degrees C is the best temperature for defrosting frozen ground zoo meat loaves (length, 9 in. [22.8 cm]; radius, 2 in. [5.1 cm]) without affecting the microbiological quality or safety of the product.     

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.     

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.     

Evaluation of a commercial fluorochromic system for the rapid detection and estimation of wine lactic acid bacteria by DEFT

A commercial fluorochromic system was evaluated for the rapid detection of lactic acid bacteria in fortified wines by the epifluorescent filter technique (DEFT). The viability test used, employing the fluorescence dyes SYTO 9 and propidium iodide, was able to detect and clearly differentiate viable from non-viable cells (killed with a 50% v/v ethanol solution). A good overall agreement ( r ₌ 0·92) was obtained between the DEFT count and the plate count in the range studied (5 × 10²–4 × 10⁹ cells ml⁻¹). Wine components which might otherwise interfere with the method could be removed by including simple wash steps in the protocol. This measure proved critical to the success of the procedure. For practical purposes, the rapid method studied seems to be a good alternative to the traditional cultural methods as part of quality control programmes in wine making. It may also be useful when studying the efficacy of certain treatments in the elimination of wine bacterial contaminants.     

Incidence and identification of mesophilic Aeromonas spp. from retail foods

Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively. Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps. Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1). GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level. Aeromonas strains belonging to the Aer. caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish. In addition, three strains of the virulent taxon Aer. veronii biovar sobria HG8 were isolated from poultry and minced meat. All members of the Aer. hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3. Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.     

TEMPO/TVC法与国标方法测定食品中菌落总数的比较研究

【目的】确证TEMPO/TVC计数食品中菌落总数的检测性能。【方法】使用TEMPO/TVC法和国标方法GB4789.2对熟肉制品、方便食品、速冻食品、膨化食品、糖果、糕点、调味品7类食品进行菌落总数测定。【结果】TEMPO/TVC法和国标方法检测食品中菌落总数的检测结果一致性较好(符合率95.4%);且两种方法检测7类食品样品的检测结果P值均大于0.05,无显著性差异。【结论】TEMPO/TVC法具有操作简便、快速、高效和人为误差小的特点,在日常检测中值得推广应用。