Evaluation of the MicroFoss system for enumeration of total viable count, Escherichia coli and coliforms in ground beef

This study was conducted to evaluate the performance of the MicroFoss system (Biosys, Ann Arbor, MI) for enumeration of total viable organisms, Escherichia coli and coliforms in ground beef. The system performance was compared to that of the USDA Bacteriological Analytical Method (BAM) reference culture methods. The correlation coefficients for the regression lines comparing the MicroFoss system detection times to the results of plate count methods for the total viable counts, coliform counts and the most probable number (MPN) method for E. coli were -0.95, -0.96 and -0.97, respectively. Tests comparing the reproducibility of data generated independently by two technicians on the same batch of samples showed no significant differences (P>0.05) in the MicroFoss detection times and culture results. The plate count methods for the total viable counts and coliform counts, and the MPN method for E. coli required 10, 11 and 22 times, respectively, the amount of time to complete tests compared to the length of time required to perform these tests using the MicroFoss system. The MicroFoss system produced reproducible data and provided a rapid and cost-efficient alternative method for enumeration of TVC, coliforms and E. coli in ground beef.     

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.     

Evaluation of the MicroFoss system for the detection of Listeria species in environmental samples

The MicroFoss system was evaluated for its ability to detect Listeria species in environmental samples. The sensitivity and specificity of the MicroFoss were determined in relation to a standard culture method for Listeria detection. The sensitivities of both the MicroFoss and standard culture methods were similar (88.4%-MicroFoss, 90.7%-Culture) based on the total number of positive results obtained by both methods. The MicroFoss system detected Listeria spp. in 12 samples, which were not detected by culture, and the culture method detected Listeria spp. in 15 samples, which were not detected by the MicroFoss method. This was likely due to uneven distribution of low levels of Listeria organisms in the split sponge samples used to assess the performance of these test methods. The specificity value determined for the MicroFoss system was 92.7%. The majority of microbes causing false positive results in the MicroFoss system were Bacillus species, which were readily distinguishable from Listeria species by a simple Gram stain and morphological features. Listeria monocytogenes (89.4%-MicroFoss, 88.0%-Culture) and Listeria innocua (8.8%-MicroFoss, 7.7%-Culture) were the most common isolates of Listeria detected by the two test methods, with L. monocytogenes being the most predominant isolate detected. The highly comparable results and rapid nature of the MicroFoss system demonstrate its effectiveness as a detection system for species of Listeria in environmental samples. The fact that the sensitivity of the MicroFoss system was similar to that of the culture method and the Listeria results were obtained within 48 h of testing, support the use of the MicroFoss as an alternative rapid method for screening large numbers of environmental samples for Listeria spp.     

Comparison of TEMPO® EC and TBX medium for the enumeration of Escherichia coli in cheese

Aims: TEMPO^® EC (Escherichia coli) is based on glucuronidase activity and is a test for use with the TEMPO system for the automated 24 h enumeration of E. coli in food products. In this study, TEMPO EC was compared with TBX medium, the current standard plate method for the enumeration of E. coli in cheese. Methods and Results: For comparative purposes, both naturally (92) and artificially contaminated (31) cheese samples were studied. Pearson correlation coefficients were determined as 0.954 and 0.978 between the two methods for naturally and artificially contaminated samples, respectively. Regression analysis yielded the following equations: log₁₀ TEMPO EC = 0.340 + 0.889 log₁₀ TBX medium and log₁₀ TEMPO EC = 0.174 + 0.899 log₁₀ TBX medium for naturally and artificially contaminated samples, respectively. In general, absolute differences did not exceed one log between results obtained by the two methods. Conclusions: Statistical analysis of the results showed good agreement between the two enumeration methods. Significance and Impact of the Study: TEMPO EC is a practical and reliable alternative to the current standard plate method for the enumeration of E. coli in foods.     

EU Drinking Water Directive reference methods for enumeration of total coliforms and Escherichia coli compared with alternative methods

AIMS: The reference methods for enumeration of total coliforms and Escherichia coli as stated in the European Drinking Water Directive were compared with alternative methods. METHODS AND RESULTS: Laboratories used the reference method on Lactose TTC agar (LTTC), the Colilert/18 system, Laurysulphate Agar (LSA), Chromocult Coliform Agar and the E. coli Direct Plating (DP) method. They enumerated more total coliforms on LTTC than on LSA. CONCLUSIONS: LTTC is suitable for analysis of very clean water samples only, due to heavy background growth. Colilert/18 is a good alternative but it enumerates a broader group of total coliforms, resulting in higher counts. The DP method appeared to be the best choice for enumeration of E. coli because Colilert/18 produces lower counts and false-negative results. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the limitations of the EU reference method on LTTC due to lack of selectivity and suggests alternative methods for the enumeration of total coliforms and E. coli.     

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards

AIMS: To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. METHODS AND RESULTS: Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. CONCLUSIONS: The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. SIGNIFICANCE AND IMPACT OF THE STUDY: The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.     

A 210-min solid phase cytometry test for the enumeration of Escherichia coli in drinking water

A 210-min-test for the enumeration of Escherichia coli in drinking water is described, based on solid phase cytometry (SPC) and a two-step enzymatic procedure for fluorescence labelling of single cells and small microcolonies. The test involves membrane filtration through a 25-mm black polyester filter, induction of beta-glucuronidase in the retained target cells, fluorescence labelling with fluorescein-di-beta-Dglucuronide as an enzyme substrate and laser scanning of the membrane filter. Scan results can be confirmed on-line by epifluorescence microscopy. Application to 149 naturally contaminated and uncontaminated well, tap, out-of-pump centre (distribution), surface and sewage-spiked water samples indicated > or =90% agreement and equivalence with plate count methods, including Chromocult Coliform agar and m FC agar. In 5.4% of all samples examined, SPC detected between 1 and 11 E. coli per 100 ml, while the two plate methods yielded negative results. Cases of a negative SPC result but a positive E. coli count on both reference media were not observed. This test would primarily be useful for 'emergency' monitoring of drinking water when rapid results are crucial.     

Enumeration of total coliforms and Escherichia coli from source water by the defined substrate technology

Many water utilities are required to monitor source water for the presence of total coliforms, fecal coliforms, or both. The Colilert system, an application of the defined substrate technology, simultaneously detects the presence of both total coliforms and Escherichia coli directly from a water sample. After incubation, the formula becomes yellow if total coliforms are present and fluorescent at 366 nm if E. coli is in the same sample. No confirmatory tests are required. The Colilert system was previously assessed with distribution water in a national evaluation in both most-probably-number and presence-absence formats and found to produce data equivalent to those obtained by using Standard Methods for the Examination of Water and Wastewater (Standard Methods). The Colilert system was now compared with Standard Methods multiple-tube fermentation (MTF) for the enumeration of total coliforms and E. coli from surface water. All MTF tubes were confirmed according to Standard Methods, and subcultures were made to identify isolates to the species level. The Colilert system was found equally sensitive to MTF testing by regression, t test, chi-square, and likelihood fraction analyses. Specificity of the Colilert system was shown by the isolation of a species of total coliform or E. coli after the appropriate color change. The Colilert test can be used for source water samples when enumeration is required, and the benefits previously described for distribution water testing--sensitivity, specificity, less labor, lower cost, faster results, no noncoliform heterotroph interference--are applicable to this type of water analysis.     

Enumeration of total coliforms and Escherichia coli from source water by the defined substrate technology.

Many water utilities are required to monitor source water for the presence of total coliforms, fecal coliforms, or both. The Colilert system, an application of the defined substrate technology, simultaneously detects the presence of both total coliforms and Escherichia coli directly from a water sample. After incubation, the formula becomes yellow if total coliforms are present and fluorescent at 366 nm if E. coli is in the same sample. No confirmatory tests are required. The Colilert system was previously assessed with distribution water in a national evaluation in both most-probably-number and presence-absence formats and found to produce data equivalent to those obtained by using Standard Methods for the Examination of Water and Wastewater (Standard Methods). The Colilert system was now compared with Standard Methods multiple-tube fermentation (MTF) for the enumeration of total coliforms and E. coli from surface water. All MTF tubes were confirmed according to Standard Methods, and subcultures were made to identify isolates to the species level. The Colilert system was found equally sensitive to MTF testing by regression, t test, chi-square, and likelihood fraction analyses. Specificity of the Colilert system was shown by the isolation of a species of total coliform or E. coli after the appropriate color change. The Colilert test can be used for source water samples when enumeration is required, and the benefits previously described for distribution water testing--sensitivity, specificity, less labor, lower cost, faster results, no noncoliform heterotroph interference--are applicable to this type of water analysis.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

盐度对稀释平板法研究红树林区土壤微生物数量的影响

在使用稀释平板法分离潮间带红树林及其对照光滩土壤微生物以及计数时,多数情况下使用陈海水制作培养基和稀释水,很少考虑培养基和稀释水的盐度对最终计数结果的影响.使用稀释平板法研究了盐度对福建九龙江口红树林区与深圳福田红树林保护区土壤微生物平板计数的影响,结果表明培养基与稀释水盐度对微生物数量有明显的影响.统计分析显示细菌的海水稀释效果优于淡水,而放线菌与真菌则刚好相反(P<0.05,一个例外).海水不适合配制红树林区土壤微生物平板计数的培养基,从0～35,高盐度的平板培养基会降低微生物的数量,尤其是放线菌的数量,尽管培养基的盐度对真菌影响无规律,但细菌数量在低盐度时比在高盐度和不加氯化钠时要多.根据盐度效应,提出了稀释平板技术应用于潮间带的红树林及其相应光滩时的优化方法,认为细菌应该用海水作无菌稀释水,而放线菌和真菌则应用淡水作稀释水;包括光滩在内的红树林区土壤微生物分离与计数的培养基宜控制较低盐度范围.     

Evaluation of the effectiveness of a commercially available defined substrate medium and enumeration system for measuring Escherichia coli numbers in faeces and soil samples

AIMS: To determine if a commercially available defined substrate medium and enumeration system could be utilized as an effective and accurate means of enumerating Escherichia coli in environmental samples containing faeces and soil. METHODS AND RESULTS: The samples tested were either inoculated with laboratory grown E. coli or natural E. coli populations in cow faeces. The number of E. coli recovered from faeces and soil samples using the defined substrate medium and enumeration system and a miniaturized MPN method (using traditional media) was compared by analysing the difference between the two methods in relation to the mean. For four of five groups of samples analysed there was no significant difference in the number of E. coli recovered by the two methods (P > 0.05). In one batch the difference was 0.30 log, which while being statistically significant (P < 0.01) was not considered to be biologically significant. CONCLUSION: The commercially available enumeration system was significantly more precise than the miniaturized MPN method (P < 0.001). SIGNIFICANCE AND IMPACT OF THE STUDY: We conclude that the commercially available defined substrate medium and enumeration system is a suitable method for the measurement of E. coli numbers in faeces and soil samples and should provide advantages of increased precision and a reduction in laboratory analysis time.     

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.     

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.     

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.     

Escherichia coli detection using mTEC agar and fluorescent antibody direct viable counting on coastal recreational water samples

Aims:  Escherichia coli is the faecal indicator species recommended by the US Environmental Protection Agency (USEPA) for monitoring fresh recreational water. Viable but nonculturable (VBNC) E. coli are living cells that are dormant and not culturable using standard microbiological cultivation methods. This study reports a comparison between the mTEC culture method recommended by USEPA for E. coli enumeration and a fluorescent antibody-direct viable count (FA-DVC) method to visualize living E. coli cells with a microscope.
Methods and Results:  Escherichia coli , faecal coliforms and Enterococcus were detected using standard methods recommended by the USEPA. VBNC E. coli was visualized with FA-DVC. Results were analysed with standard statistical methods (Pearson correlation; paired-sample t -test). Significantly higher numbers of E. coli were detected using the FA-DVC method than using the mTEC method. Escherichia coli results were also compared with faecal coliform (mFC broth) and Enterococcus (mEI agar) counts in the same samples.
Conclusions:  The results of this comparative study demonstrate that E. coli can be present in higher numbers than what are detected with standard culture methods.
Significance and Impact of the Study:  This study re-emphasizes the need for a rapid, accurate and precise method for detecting health risks to humans who use recreational waters.     

Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media

This study compared the performance of LMX(R) broth (LMX), Chromocult Coliform(R) agar (CC) and Chromocult Coliform agar plus cefsulodin (10 microg ml-1) (CC-CFS), with standard methods multiple tube fermentation (MTF), for the enumeration of total coliforms and Escherichia coli from marine recreational waters. LMX and CC are two media designed to concurrently detect total coliform (TC) bacteria and E. coli by the specific action of beta-galactosidase (total coliforms) and beta-glucuronidase (E. coli). Overall results for the TC test showed that LMX, CC and MTF recovered 2.63, 1.95 and 1.90 times as many TCs as CC-CFS, respectively. Data from the multiple range test showed significant differences (P < 0.05) between TC counts on CC-CFS and LMX. The traditional MTF was less sensitive for E. coli enumeration. However, there was no statistically significant differences between LMX, CC, CC-CFS and the MTF method for E. coli enumeration. Background interference was reduced on CC-CFS and the counts obtained reflected more accurately the number of TCs. Therefore, the contribution of beta-galactosidase positive, non coliform bacteria (Aeromonas spp. and Vibrio spp.) to TC counts should not be neglected.     

Comparison of Vogel-Johnson and Baird-Parker media for membrane filtration recovery of staphylococci in swimming pool water

Previous studies have indicated that the coagulase-positive Staphylococcus (Staphylococcus aureus) has potential as a useful indicator of the infection hazard associated with the use of swimming pools and other recreational waters. However, before this indicator system can be used effectively, a recovery system that is sufficiently selective, accurate, and reliable for the enumeration of S. aureus must be developed. In this study, Vogel-Johnson (VJ) and Baird-Parker (BP) agars were compared for efficacy in the primary isolation and recovery of S. aureus from swimming pool water. For equal sample volumes of pool water containing adequate free chlorine residual, VJ agar was found to be more selective for staphylococcal species and less inhibitory to general cell growth than was BP agar. However, neither medium was found to be sufficiently differential to permit the accurate identification of S. aureus. In contrast, water samples obtained from a swimming pool containing very low levels of chlorine (none of which was in the free form) showed abundant growth of staphylococci on both test media, with both VJ and BP agars showing increased sensitivity for the detection of S. aureus. Thus, VJ and BP agars show increased sensitivity for the detection of coagulase-positive staphylococci from unchlorinated versus chlorinated waters.     

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.