Plating procedure for the enumeration of coliforms from dairy products.

A "repair-detection" procedure consisting of pour plating of food samples with Trypticase soy agar, followed by 1-h repair incubation at room temperature and subsequent overlay with violet red bile agar, was found to be an effective method for the detection of injuried and uninjuried coliforms from dairy products. This method was relatively less effective for the detection of coliforms in many semipreserved foods as compared with dairy products, but more effective than the most-probable-number method.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.     

Enumeration of Escherichia coli and coliforms in surface water by multiple tube fermentation and membrane filter methods

The current investigation was carried out in order to compare directly the multiple tube fermentation method (MTF), using standard procedures (lactose broth, LB) and the Colilert reagent, with the membrane filter method (MF) using Les Endo agar (LEA), m-faecal coliform agar (mFCA) and chromogenic coliform agar (CCA), for recovery of coliforms and Escherichia coli in 80 surface water samples. Total coliforms were isolated from 100% of samples by all methodologies. Faecal coliforms/E. coli were detected in 100% of samples by MTF methods, but only in 75.5% by MF-mFCA and in 86.2% by MF-CCA. Even if MTF-LB counts were consistently higher, the Colilert reagent accurately determined total coliforms and E. coli levels within 24 h with no additional confirmatory tests. Therefore, it could be a powerful tool for rapidly assessing possible faecal contamination and a suitable alternative to the traditional MTF and MF techniques utilized for coliform detection.     

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Evaluation of m-T7 agar as a fecal coliform medium

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.     

Comparison of the recoveries of Escherichia coli and total coliforms from drinking water by the MI agar method and the U.S. Environmental Protection Agency-approved membrane filter method.

Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. coli requires two media, an MF transfer, and a total incubation time of 28 h. A newly developed MF method, the MI agar method, containing indoxyl-beta-D-glucuronide and 4-methylumbelliferyl-beta-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, respectively, by means of their specific enzyme reactions, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level, 0.05) showed that the new medium recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar and nutrient agar plus MUG (4-methylumbelliferyl-beta-D-glucuronide), respectively, and the background counts were significantly lower than those of mEndo agar (< 5%). Generally, the tap water source, overall chlorine level, wastewater source, granular activated carbon treatment of the tap water, and method of grouping data by E. coli count for statistical analysis did not affect the performance of the new medium.     

沈阳市售乳制品中β-内酰胺酶残留状况初探

目的 调查沈阳市售乳制品中添加生鲜牛乳抗生素分解剂β-内酰胺酶的情况.方法 按照卫生部颁发的“乳及乳制品中舒巴坦敏感β-内酰胺酶类药物检验方法——杯碟法”检测.结果 七个品牌的乳制品样品50份中共有四个品牌19份样品中检出β-内酰胺酶,检出率达到38％.结论 沈阳市售乳制品中不仅检测出β-内酰胺酶,且添加率较高.希望引起政府相关部门的重视,建议修订相关国家标准,改进检测方法,加大监管力度.     

Repair and Enumeration of Injured Coliforms by a Plating Procedure

Surface plating of coliforms on Trypticase soy agar, followed by 1 to 2 h of incubation at 25 C and subsequent overlay with violet red bile agar, was found to be a useful method for the repair and enumeration of coliforms injured by freezing.     

Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media

AIMS: To make a preliminary assessment of the incidence of Salmonella in Egyptian dairy products, and to investigate the effectiveness of various protocols for the detection of the pathogen in these products. METHODS AND RESULTS: Samples of milk and related dairy products were randomly collected from local markets and examined for the presence of Salmonella. While most samples were free of the organism, isolates of Salmonella enterica subsp. enterica serovar Typhimurium PT 8 could be recovered from 'matared' cream specimens. These isolates were susceptible to antibiotics usually used to challenge infections caused by Salmonella. A combination of buffered peptone water, Muller-Kauffman tetrathionate broth, and brilliant green phenol red agar gave the best results for the detection of the pathogen. Selenite-cystine broth and Hektoen enteric agar were ineffective as an enrichment and a plating medium, respectively, in the isolation of Salmonella. A modified identification strategy that reduces the burden of serological testing of presumptive isolates is proposed. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: 'Matared' cream could be a vehicle for transmitting Salmonella. Using the above combination of media, beside the suggested modified confirmatory procedure, should increase the effectiveness and ease of the detection of Salmonella in milk and dairy products.     

Membrane filtration differentiation of E. coli from coliforms in the examination of water

A membrane filter-Endo agar method for enumerating Escherichia coli as distinct from other coliforms in drinking water was developed. Membranes containing coliform colonies are transferred to nutrient agar containing 4-methyl umbelliferyl-beta-D-glucuronide (MUG) and incubated at 35 degrees C for 4 h. The MUG is hydrolyzed by the glucuronidase of E. coli and the fluorogenic product is visualized. The method recovered 98% of E. coli without false positives and is proposed as an additional test in routine water examination for the detection of pollution.     

Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects

The purpose of this study was to examine the use of Chromocult agar medium for isolation and enumeration of Enterobacteriaceae from human faecal samples, to compare it to MacConkey agar and to evaluate its usefulness as a possible alternative selective medium in human faecal studies. The medium was shown to be effective in identifying Escherichia coli and coliforms in faeces without the need for extensive accompanying biochemical tests for confirmation of identity. A positive correlation (r=0.86) was found between the recovery of Enterobacteriaceae on the two media, and no significant difference (P>0.05) between overall mean bacterial counts for the whole study group or at different intervals of faecal collection were observed. Chromocult agar is an effective replacement for MacConkey agar in human faecal studies and has the advantage of differentiating E. coli from other coliforms.     

Membrane filtration differentiation of E. coli from coliforms in the examination of water

A membrane filter-Endo agar method for enumerating Escherichia coli as distinct from other coliforms in drinking water was developed. Membranes containing coli-form colonies are transferred to nutrient agar containing 4-methyl umbelliferyl-β-d-glucuronide (MUG) and incubated at 35°C for 4 h. The MUG is hydrolyzed by the glucuronidase of E. coli and the fluorogenic product is visualized. The method recovered 98% of E. coli without false positives and is proposed as an additional test in routine water examination for the detection of pollution.     

Injured coliforms in drinking water

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

New medium for improved recovery of coliform bacteria from drinking water.

A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.     

Evaluation of new chromogenic substrates for the detection of coliforms

Aims: To evaluate a new range of chromogenic substrates for the detection of β‐galactosidase activity in coliforms and to compare their performance in agar media and broths. Methods and Results: Sixteen novel galactoside substrates were prepared and incorporated into agar and broth. Their performance was compared using Escherichia coli (five strains), Salmonella (two strains), Enterobacter (two strains), Klebsiella, Pseudomonas, Listeria, Serratia, Shigella, Citrobacter, Proteus and Staphylococcus as well as pathological urine samples. The six substrates out of the initial 16 that showed the greatest sensitivity were VQE‐gal, VQM‐gal, VLPr‐gal, VLE‐gal, VLM‐gal and VBzTM‐gal, whose released chromophores were red, brown or purple. VQE‐gal and VLPr‐gal were studied in greater detail and were incorporated into agar medium. Coliform colonies appeared red and brown respectively, following incubation at 37°C for 24 h; however, positive results were obtained within a working day. The VQE‐gal medium was compared with some commercially available media. Conclusions: The range of substrates described can be used in broths as well as in agars. The VQE agar allows the detection of coliforms within a working day. VQE‐gal medium proved to be more sensitive when compared to other available chromogenic media and allows the unambiguous detection of coliforms.     

A simple and cost-effective method for the quantification of total coliforms and Escherichia coli in potable water

In this study, a new simple and cost-effective method for the study of total coliforms and Escherichia coli in potable water, combining the use of lactose TTC agar and TBX agar, was developed and compared with methods using Chromocult agar and coli ID. The statistical analysis showed no significant difference and a good correlation (R(2)) between the three methods.     

AN ELECTRICAL TESTING METHOD FOR THE DETECTION OF LISTERIA SPECIES IN CONFECTIONERY PRODUCTS

An electrical testing method for the detection of Listeria spp. in confectionery products and associated raw materials was developed in which samples can be negatively screened within 48h. The method involves a 24h resuscitation in Listeria enrichment broth followed by a 24h test in a Bactometer M128 using a broth (Claremont broth) developed from Oxford agar. The comparative study involved analysis of 511 samples (chocolate, dairy, cereal, nut and fruit products) tested by the Interim Australian Standard method (AS) and the Bactometer method (BM). The sensitivity and specificity of the BM for samples §1 Listeria/g was 100% and 99.8%, respectively, when compared to the AS method. When samples containing < 1 Listeria/g sample were added to the analysis, the sensitivity and specificity marginally dropped (98.3% and 99.6%). The electrical capacitance method is rapid and easy to perform, with a negative result being available within 48h making it a viable tool in a positive release QA program in food manufacturing factories.