Growth of starved Escherichia coli O157 cells in selective and non-selective media.

Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter-sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non-selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non-selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non-starved cells from overnight cultures with an initial density of less than 10(3) colony-forming units (CFU)/ml grew to the density of over 10(7) CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a "resuscitation" of the cells with non-selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.     

Survival and detection of bacteria in an aquatic environment

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival and detection of bacteria in an aquatic environment.

A genetically engineered plasmid, pPSA131, was used as a DNA probe to detect homologous DNA in Escherichia coli HB101(pPSA131) after it was mixed with aquatic microorganisms from Lake Mead, Nevada, water samples. An isolate from the pLAFR1 chromosomal library of Pseudomonas syringae Cit 7 was used to detect parent P. syringae Cit 7 that had been mixed with Lake Mead water. E. coli(pPSA131) was kept in variously treated samples of lake water or buffer, and its survival was measured by viable cell counting on modified Luria-Bertani (LB) agar. Full-strength LB agar proved better than 0.1 x LB agar at recovering E. coli(pPSA131) after survival in low-nutrient environments. Survival of E. coli(pPSA131) remained high in filtered (0.22-micron pore size) lake water and salts buffer on both selective and nonselective agars but was lower in untreated lake water or lake water filtered with a 0.8-micron-pore-size membrane. Total recoverable colonies grown on LB agar were higher when lake water was filter treated (0.8-micron pore size) than when lake water was untreated. Microorganisms recovered from lake water alone grew rapidly on nonselective media, probably because of the "bottle effect." After being mixed with Lake Mead water, E. coli(pPSA131) and P. syringae were detected by colony blotting with non-radioactively labeled DNA probes. E. coli(pPSA131) were recovered at three times during 48 h from variously treated samples of lake water and from a mixture with Lake Mead water organisms. Colonies were supported on either nonselective or selective agar for comparison.(ABSTRACT TRUNCATED AT 250 WORDS)     

False-positive identification of Escherichia coli in treated municipal wastewater and wastewater-irrigated soils

The increasing use of treated wastewater for irrigation heightens the importance of accurate monitoring of water quality. Chromogenic media, because they are easy to use and provide rapid results, are often used for detection of Escherichia coli in environmental samples, but unique levels of organic and inorganic compounds alter the chemistry of treated wastewater, potentially hindering the accurate performance of chromogenic media. We used MI agar and molecular confirmatory methods to assess false-positive identification of E. coli in treated wastewater samples collected from municipal utilities, an irrigation holding pond, irrigated soils, and in samples collected from storm flows destined for groundwater recharge. False-positive rates in storm flows (4.0%) agreed closely with USEPA technical literature but were higher in samples from the pond, soils, and treatment facilities (33.3%, 38.0%, and 48.8%, respectively). Sequencing of false-positive isolates confirmed that most were, like E. coli, of the family Enterobacteriaceae, and many of the false-positive isolates were reported to produce the β-D-glucuronidase enzyme targeted by MI agar. False-positive identification rates were inversely related to air temperature, suggesting that seasonal variations in water quality influence E. coli identification. Knowledge of factors contributing to failure of chromogenic media will lead to manufacturer enhancements in media quality and performance and will ultimately increase the accuracy of future water quality monitoring programs.     

New medium for the simultaneous detection of total coliforms and Escherichia coli in water.

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.     

Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks

Fluorescence in situ hybridization (FISH) was used for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal, and Latvia, and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, polyvinyl chloride, or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after 1 to 6 months exposure to the drinking water. In order to increase the signal intensity, a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of beta-d-glucuronidase) methods. An additional verification was made by using PCR. Culture method indicated presence of E. coli in one of five pipes, whereas all pipes were positive with the FISH methods. E. coli was detected in 56% of the coupons using PNA FISH, but no E. coli was detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples that were negative according to culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low-nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but nonculturable state, unable to grow on agar media. E. coli contributed to ca. 0.001 to 0.1% of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g., temperature, chlorine, or biodegradable organic matter concentration). We show here that E. coli is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.     

Development of an internal control for evaluation and standardization of a quantitative PCR assay for detection of Helicobacter pylori in drinking water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

Comparison of media for enumeration of coliform bacteria and Escherichia coli in non-disinfected water

In this work alternative media for detection and enumeration of E. coli and coliform bacteria were compared to the reference method ISO 9308-1 (LTTC) using non-disinfected water samples with background flora. The alternative media included LES Endo agar medium (LES Endo), Colilert-18 with 51-well Quanti-tray (Colilert), Chromocult Coliform agar (CC), Harlequin E. coli/Coliform medium (HECM) and Chromogenic Escherichia coli/Coliform medium (CECM). A total of 110 samples of groundwater, bathing water and spiked water was used. Our results revealed that confirmation of coliform bacteria counts is necessary, not only on lactose-based LTTC and LES Endo media, but also on the chromogenic agar media tested, due to the growth of oxidase positive colonies. LTTC and CC media also allowed the growth of some morphologically typical coliform colonies containing gram-positive bacteria. The recovery of coliform bacteria was lower on LES Endo than on LTTC. In most cases Colilert, CC, HECM and CECM gave higher coliform counts than LTTC. The use of the LTTC medium led to higher E. coli counts than obtained with any of the alternative mediums. There are three explanations for this: (1) high sensitivity of LTTC, (2) false positives on LTTC or (3) false negatives especially with Colilert, but also with chromogenic agar media. Although LTTC was found to be a very sensitive medium, the high degree of background growth of non-disinfected waters disturbed substantially the use of it. In conclusion, our results suggest that Colilert, CC and CECM are potential alternative media for detection of coliform bacteria and E. coli from non-disinfected water.     

Methods for the isolation of water-borne Escherichia coli O157

AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments. METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E. coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples. The sensitivity of recovery of E. coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS. CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E. coli O157 culture from water and sediment samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E. coli O157 detection from water and sediments.     

Formation of nonculturable Escherichia coli in drinking water

AIMS: To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. METHODS AND RESULTS: Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp-tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20-60 days in nondisinfected drinking water resulted in detection of only 0.7-5% of the initial E. coli population using standard cultivation methods, whereas 1-20% could be resuscitated to a culturable state, and 17-49% could be clearly detected using cultivation-independent methods. CONCLUSIONS: Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10-20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. SIGNIFICANCE AND IMPACT OF THE STUDY: The existence of substrate-responsive but nonculturable cells should be considered when evaluating the survival potential of E. coli in nondisinfected drinking water.     

Effect of oxygen on survival of faecal pollution indicators in drinking water

AIMS: The aim of this study was to determine the effect of oxygen on the survival of faecal pollution indicators including Escherichia coli in nondisinfected drinking water. METHODS AND RESULTS: Aerobic and anaerobic drinking water microcosms were inoculated with E. coli ATCC 25922 or raw sewage. Survival of E. coli was monitored by membrane filtration combined with cultivation on standard media, and by in situ hybridization with 16S rRNA-targeted fluorescent oligonucleotide probes. Anaerobic conditions significantly increased the survival of E. coli in drinking water compared with aerobic conditions. Escherichia coli ATCC 25922 showed a biphasic decrease in survival under aerobic conditions with an initial first-order decay rate of -0.11 day(-1) followed by a more rapid rate of -0.35 day(-1). In contrast, the first-order decay rate under anaerobic conditions was only -0.02 day(-1). After 35 days, <0.01% of the initial E. coli ATCC 25922 population remained detectable in aerobic microcosms compared with 48% in anaerobic microcosms. A poor survival was observed under aerobic conditions regardless of whether E. coli ATCC 25922 or sewage-derived E. coli was examined, and regardless of the detection method used (CFU or fluorescent in situ hybridization). Aerobic conditions in drinking water also appeared to decrease the survival of faecal enterococci, somatic coliphages and coliforms other than E. coli. CONCLUSIONS: The results indicate that oxygen is a major regulator of the survival of E. coli in nondisinfected drinking water. The results also suggest that faecal pollution indicators other than E. coli may persist longer in drinking water under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of oxygen should be considered when evaluating the survival potential of enteric pathogens in oligotrophic environments.     

Proportion of beta-D-glucuronidase-negative Escherichia coli in human fecal samples.

Convenient assays and reports that almost all clinical isolates of Escherichia coli produce beta-D-glucuronidase (GUR) have led to great interest in the use of the enzyme for the rapid detection of the bacterium in water, food, and environmental samples. In these materials, E. coli serves as an indicator of possible fecal contamination. Therefore, it was crucial to examine the proportion of GUR-negative E. coli in human fecal samples. The bacterium was isolated from 35 samples, and a mean of 34% and a median of 15% were found to be GUR negative in lauryl sulfate tryptose broth with 4-methylumbelliferyl-beta-D-glucuronide. E. coli from three samples were temperature dependent for GUR production: very weakly positive at 37 degrees C but strongly positive at 44.5 degrees C. These results remind us of differences between fecal and clinical E. coli populations, of diversity in GUR regulation and expression in natural populations of E. coli, and of the need for caution in using GUR for the detection of fecal E. coli.     

Proportion of beta-D-glucuronidase-negative Escherichia coli in human fecal samples

Convenient assays and reports that almost all clinical isolates of Escherichia coli produce beta-D-glucuronidase (GUR) have led to great interest in the use of the enzyme for the rapid detection of the bacterium in water, food, and environmental samples. In these materials, E. coli serves as an indicator of possible fecal contamination. Therefore, it was crucial to examine the proportion of GUR-negative E. coli in human fecal samples. The bacterium was isolated from 35 samples, and a mean of 34% and a median of 15% were found to be GUR negative in lauryl sulfate tryptose broth with 4-methylumbelliferyl-beta-D-glucuronide. E. coli from three samples were temperature dependent for GUR production: very weakly positive at 37 degrees C but strongly positive at 44.5 degrees C. These results remind us of differences between fecal and clinical E. coli populations, of diversity in GUR regulation and expression in natural populations of E. coli, and of the need for caution in using GUR for the detection of fecal E. coli.     

Estimation of viable Escherichia coli O157 in surface waters using enrichment in conjunction with immunological detection

The use of a minimal lactose enrichment broth (MLB) in conjunction with immunomagnetic electrochemiluminescence detection (IM-ECL) was evaluated for the estimation of viable Escherichia coli O157 populations in surface water samples. In principle, E. coli O157 populations (C(initial E. coli O157)) can be derived from enrichment data according to the equation: C(initial E. coli O157) = C(initial coliforms) x C(final E. coli O157)/C(final coliforms)), assuming that the growth rates and lag times of water-borne E. coli O157 and collective coliforms are sufficiently comparable, or at least consistent. We have previously described a protocol for determining C(final E. coli O157) in MLB-enriched water samples. In the present study, 80% of coliforms (red/pink colonies on MacConkey Agar) grew in MLB, indicating that this provides reasonably accurate estimates of C(initial coliforms). Estimates of C(final coliforms) were determined from turbidity data. Initial E. coli O157 populations (C(initial E. coli O157)) were calculated for 33 Baltimore watershed samples giving a positive IM-ECL response. The majority of samples contained E. coli O157 concentrations of < 1 cell per 100 ml. These data indicate that E. coli O157 are present in surface water samples but at very low levels. Growth rates for MLB-enriched coliforms were highly variable (k= 0.47 +/- 0.13 h(-1), n= 72). There was no correlation between growth rates and any measured water parameter, suggesting that coliform populations in water samples are spatially and temporally unique. Although variability in growth rates was expected to yield some low values, the fact that most E. coli O157 concentrations were < 1 suggests that other factor(s) were also responsible. Studies with E. coli O157:H7 and wild-type E. coli suggest that increased lag times due to starvation were at least partially responsible for the observed data. Based on estimates of C(initial coliforms) and k(coliforms), MLB was evaluated for sensitivity and quantitativeness. Simulated populations of E. coli O157:H7 at stationary phase varied from ca. 10(3) to 10(8) cells ml(-1) enrichment culture. Although not suitable for quantitation, MLB enrichment in conjunction with IM-ECL can detect as few as one viable water-borne E. coli O157 cell per 100 ml surface water. Experiments are in progress to evaluate alternative media for sensitivity and quantitative detection of enterohemorrhagic E. coli.     

Discrepancies in the Enumeration of Escherichia coli

Stationary-phase cells of Escherichia coli were enumerated by the pour plate method on Trypticase soy agar containing 0.3% yeast extract (TSYA), violet red-bile agar, and desoxycholate-lactose agar, and by the most-probable-number method in Brilliant Green-bile broth and lauryl sulfate broth. Maximum counts were assumed to be those on TSYA. In general, numbers detected were lower with the selective solid media and higher with the selective liquid media. Inhibitory effects, especially on selective solid media varied with the strains of E. coli. The lower detection on selective solid media was partly due to the stress induced in some cells by the temperature of the melted media used in the pour plate method. These cells apparently failed to repair and form colonies in the selective media. Improved detection on the selective solid media was achieved by using 1% nonfat milk solids, 1% peptone, or 1% MgSO(4).7H(2)O in the dilution blanks. Higher detection on selective agar media was effected by surface plating or by surface-overlay plating of the cells. The surface-overlay method appeared to be superior for the direct enumeration of E. coli in foods.     

Quantitative determination of Escherichia coli from coliforms and faecal coliforms in sea water.

Escherichia coli concentration in sea water was determined by the MUG test after primary growth on membrane filters used to determine total coliforms or faecal coliforms. A good correlation (r = 0.86) was found between E. coli obtained from coliforms versus those from faecal coliforms. Verification procedures showed that all the MUG-positive colonies obtained on both media were E. coli. Evaluation of this data and the literature indicated that this technique for estimation of E. coli in sea water is a useful addition to laboratory procedures without generally increasing the time and the expense of the analysis of recreational water.     

Improved detection of acid mine water stressed coliform bacteria on media containing catalase and sodium pyruvate

Pure culture suspensions of two strains of exponential and stationary phase Escherichia coli exhibited significant reductions in catalase activity following exposure to acid mine water (AMW). The exogenous addition of catalase (500-2000 U) or sodium pyruvate (0.05-5%) to a nonselective recovery medium resulted in enhanced detection (12- to 465-fold) of AMW-stressed E. coli as compared with recovery on the medium lacking these supplements, whereas addition of 3,3'-thiodipropionic acid failed to improve recovery. Additional in vitro experiments utilizing selective M-FC, mT7, and M-Endo media containing 1000 U catalase or 1.0% pyruvate similarly resulted in improved detection of AMW-stressed cells, with the exception of M-Endo containing pyruvate. Appropriately modified media were then used to analyze an AMW-impacted stream by the membrane filtration technique. Addition of catalase, pyruvate, or a combination of both significantly improved recovery of fecal and total coliforms without promoting growth of noncoliforms. Supplementation of plate count agar with pyruvate and (or) catalase enhanced detection of total heterotrophs. These findings suggest that addition of catalase or pyruvate to standard recovery media may improve detection of coliform and total heterotrophic bacteria in AMW-impacted waters.     

Estimation of Escherichia coli in raw ground beef

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.     

Estimation of Escherichia coli in raw ground beef.

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.     

Fluorogenic assays for immediate confirmation of Escherichia coli.

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).