The detection of positive blood cultures in children using the acridine orange, gram, and methylene blue stains

The efficacy of the acridine orange (AO), gram (G), and methylene blue (MB) microscopic procedures was analyzed using positive blood cultures monitored by a radiometric procedure (Bactec) in children. Sixty-eight positive cultures included the following isolates: Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, Enterobacteriaceae, Staphylococcus aureus, Candida spp., and seven other pathogens. The MB stain yielded the highest detection rate, 99%, in comparison with 94 and 93% for the AO and G stains, respectively. Since the MB stain yielded comparable results to the AO procedure with no requirement for a fluorescent microscope, the former method is recommended for confirming the presence and initial characterization of microorganisms in blood cultures monitored by Bactec from children.     

Factors affecting the recovery of Campylobacter spp. from retail packs of raw, fresh chicken using ISO 10272-1:2006

Aims:  This study sought to determine the most effective protocol for the detection of Campylobacter spp. in retail packs of fresh, raw chicken based on ISO 10272-1:2006.
Methods and Results:  Three sample preparation protocols were studied; two based on excision and one combining excision with a rinse of the remaining sample. Enrichment cultures were incubated both in closed bottles and microaerobically, and sub-cultured at 24 and 48 h. Packs of chicken (110) were analysed and only two yielded no Campylobacter spp. Subculturing enrichment broths at 24 h gave the same prevalence as at 48 h, P  >   0·4 but microaerobic incubation yielded approximately 50% more positive samples than did incubation in closed bottles. Sampling based on excision plus rinsing gave the highest Campylobacter prevalence (92·7%).
Conclusions:  To isolate Campylobacter spp. from retail packs of chicken, enrichment cultures must be incubated in a microaerobic atmosphere and sub-cultured at 24 h and, possibly, 48 h. Sampling packs by excision plus rinsing maximized recoveries.
Significance and Impact of the Study:  ISO 10272-1:2006 permits the use of inefficient protocols which markedly underestimate the true prevalence of Campylobacter spp. in retail, fresh chicken. Equivalent results could be obtained 24 h earlier, with consequent savings. Its revision is essential.     

Comparison of Signal and Bactec NR-660 blood culture systems

M. STEVENS, M.B. PRENTICE, R.A. SWANN, C.J. MITCHELL AND A. DONKIN. 1993. The Signal blood culture system was compared with the Bactec NR-660. A total of 1617 blood culture sets yielded 143 (8.8%) significant isolates; 113 (79.0%) were from positive bottles in both the Bactec and Signal systems. Twelve organisms (8.4%) were detected and isolated from the Signal system only and another 18 (12.6%) from the Bactec system only. Of these 18, five were Signal-positive but the organism was not recovered and four organisms were isolated from negative Signal bottles on terminal subculture. The time taken to detection for each system was similar; the Signal system detected 68% and the Bactec 63% of significant positives within 24 h. At 48 h Bactec detected 91% and the Signal 85%. A significantly-reduced number of bottles which gave a positive signal but were negative by microscopical and cultural methods was found, compared with previous reports. The 1 h incubation period prior to the insertion of the Signal growth indicator device was considered to be the cause of this reduction in the proportion of false positives.
Fifty-five percent (42/77) of the Bactec false positives were due to delta growth value. This is when there is an increase in the growth index of ≥ 15 without the positive threshold level of 30 being attained. This occurred in the anaerobic bottle on day 2 with 42 bottles. Another 40% (31/77) of the false positives had a growth value between the positive threshold of 30 and a value of 35.
Eighty (4.9%) of Bactec and 65 (4.0%) of Signal sets yielded clinically non-significant isolates. Although the yield of significant isolates for the two systems was similar, Signal failed to detect a small number of medically important organisms without subculture.     

Evaluation of a new chromogenic agar for the detection of Listeria in food

AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.     

Comparison of Macroscopic Examination, Routine Gram Stains, and Routine Subcultures in the Initial Detection of Positive Blood Cultures

Blood was cultured in two vaccum bottles containing Columbia broth with sodium polyanethol sulfonate and CO₂. Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in CO₂) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture.     

Comparison of automated culture systems with a CFR/USP-compliant method for sterility testing of cell-therapy products

BACKGROUND: Although widely used, commercially available automated culture methods are not US Food and Drug Administration-approved for sterility testing of cell-therapy products. For cell-therapy products regulated under Section 351 of the Public Health Service Act, sterility testing must be performed by the methods described in 21 CFR 610.12 and USP <71> (CFR/USP method), or by methods demonstrated to be equivalent. METHODS: Two automated methods, BacT/Alert (BTA; bioMerieux) and Bactec (Becton Dickinson), were compared with the CFR/USP method. Representative mononuclear cell (MNC) products were formulated using six different product media. MNC product aliquots containing 10-50 x 10(6) cells in a 0.5 mL volume were seeded with organisms, and cultured for 14 days in aerobic and anaerobic bottles of each system. Ten different organisms at target concentrations of 10 and 50 colony-forming units (CFU) per bottle were tested. RESULTS: Positives were detected in a mean (range) of 72% (7-100%) of cultures for CFR/USP, 82% (0-100%) for BTA, and 93% (57-100%) for Bactec. For nine of the 10 organisms tested, overall detection rates for BTA and Bactec were equivalent to or higher than CFR/USP. Of the six product media tested, detection of organisms was impaired only by the medium containing multiple antibiotics: this occurred in all three systems. Both BTA and Bactec had shorter times to detection than the CFR/USP method, with overall means (ranges) of 87 (24-264) h for CFR/USP, 24 (12-54) h for BTA, and 33 (12-80) h for Bactec. Detection occurred consistently within 7 days for both BTA and Bactec, but not for CFR/USP. DISCUSSION: Both BTA and Bactec are superior to the CFR/USP method for overall detection and time to detection of organisms in MNC products suspended in commonly used media. These data support general use of either BTA or Bactec for sterility testing of a variety of cell-therapy products, and suggest that a 7-day culture period is sufficient to detect clinically relevant organisms. These results confirm the need for bacteriostasis and fungistasis testing of antibiotic-containing products, even when antibiotic-binding substances are used.     

A comparison of different culture media for the membrane filter quantification of Aeromonas in water

A comparative assessment of culture media for the membrane filter enumeration of Aeromonas spp. in water was performed, testing the effects of different incubation conditions (aerobic and anaerobic), temperatures (30 and 37 degrees C) and times (24 and 48 h). Different water samples seeded with test suspensions of Aeromonas spp., fecal material or raw sewage were examined. Results indicate clearly that plates should be incubated aerobically at 30 degrees C for 24 h. If the bacterial contamination is likely to be low, the use of most sensitive culture media, such as SAA, mA, ADA or PADE Agar, is recommended. By contrast, samples with an expected high level of background microbial flora should be analysed through more selective media, such as MIX Agar. However, the low selectivity of all media tested and the high likelihood of false negatives based upon the macroscopic examination of colonies means that further research directed to the development of more efficient media is needed.     

Comparison of classical isolation protocols with a 24 h screen to detect viable salmonellas in faeces

A 24 h screen which detects three viable salmonella cells per g of faeces was compared with classical isolation procedures for their ability to identify salmonella-positive samples from a pig rearing unit. The screen involved an overnight enrichment in Muller-Kauffmann tetrathionate (MK) broth, subculture for 4 h in M broth containing 10 μg ml⁻¹ novobiocin, followed by detection of the presence of salmonellas by BacTrace and Salmonella-tek ELISAs. The classical protocols were: (1) an overnight and 48 h incubation in MK or selenite cysteine broth; or (2) overnight incubation in buffered peptone water and 24 h subculture in Rappaport-Vassiliadis broth (BPW-RV). Salmonellas were isolated from the broth cultures on xylose lysine deoxycholate and brilliant green agars. Thirty four of 100 samples were positive for salmonellas but no single isolation protocol identified all of them. The best of the classical isolation protocols, 48 h incubation in MK broth, identified 27 (79%) of the 34 positive samples whilst the screen identified 26 (76%) of the 34 positive samples. False-positive results were obtained from all isolation protocols except BPW-RV.     

APPLICATION OF POLYMERASE CHAIN REACTION-OLIGONUCLEOTIDE LIGATION ASSAY FOR THE DETECTION OF SALMONELLAE IN PROCESSED MEAT, POULTRY, FISH, AND PET FOODS

We have tested a rapid and sensitive DNA-based assay for the detection of Salmonella serovars in a number of different processed meat, fish, poultry, and pet food samples. This technique uses an enrichment broth cultivation followed by a Salmonella-specific polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to specifically detect amplified PCR products in an ELISA-based microtiter plate format. The combined cultivation and PCR-OLA techniques were compared with a conventional culture method and with DNA hybridizations of PCR products for the detection of Salmonella bacteria. Eighty-one different processed meat, poultry, and pet food samples were screened for the presence of Salmonella serovars after 24 h and 48 h of enrichment broth cultivation. After 24 h of incubation, one ground turkey sample was positive by both culture and PCR-OLA (100% sensitivity and 100% specificity). After 48 h of incubation, two additional samples (ground beef and a dog food sample) were positive by both culture and PCR-OLA (100% sensitivity and 100% specificity), and three other samples (two ground beef samples and one ground turkey) were positive only by PCR-OLA (96.1% specificity). All positive PCR-OLA results were confirmed in DNA hybridizations with an oligonucleotide specific for the amplified PCR product. When compared to conventional culture, the combined 48 h enrichment and PCR-OLA had a positive predictive value of 50% and a negative predictive value of 100%. We concluded that a combined cultivation and PCR-OLA could be used as a sensitive and specific presumptive screening method for detecting Salmonella serovars in processed meat, fish, poultry, and pet foods.     

Detection of Mycobacterium bovis in bovine tissue samples by the Abbott LCx Mycobacterium tuberculosis assay and comparison with culture methods.

A ligase chain reaction (LCR) DNA amplification method for the molecular diagnosis of Mycobacterium tuberculosis complex (Abbott LCx MTB) was evaluated in comparison with solid (Lowenstein Jensen), liquid (7H12, Bactec 460 system) phase culture and microscopic examination (ME) on 86 tissue samples collected from 86 intradermal tuberculin positive cattle and one pool from 4 guinea pigs experimentally infected with M. bovis. Overall, 48 samples (58.81%) were culturally positive for mycobacteria, and on the basis of biochemical characters, all the isolates were identified as M. bovis. Sensitivity was 83.92% for LCx, 53.57% for LJ, 85.71% for Bactec and 41.07% for ME. In 3 out of 25 "no visible lesion" tissue samples, M. bovis was detected only by LCx and Bactec but not by LJ and ME. The concordance in the determination of positives and negatives among the methods observed in pairs was calculated according to Cohen's K concordance coefficient and showed 81.1% of concordance of LCx vs Bactec, 68.8% LCx vs LJ, 72.2% LCx vs ME, 80.0% Bactec vs LJ, 66.7% Bactec vs ME, 85.5% LJ vs ME. Despite a certain variability in concordance rates, both Cohen's K concordance coefficients or standardized (Zk) values were statistically significant. Both LCx and Bactec appear not alternative but subsidiary to the other methods traditionally applied for direct diagnosis of bovine tuberculosis on tissue samples from cattle reacting to intradermal tuberculin test.     

Evaluation of Radiometric System for Detecting Bacteremia

An automated radiometric system (BACTEC, Johnston Laboratories) for detection of bacteremia was evaluated in parallel with a standard blood culture system in use in our laboratory. Of 1,445 blood cultures from 484 patients with possible bacteremia, 106 sets of cultures (excluding 39 presumed contaminated), representing 56 patients, were positive by both methods. The conventional system yielded 85 positive cultures from 48 patients, whereas the BACTEC system yielded 84 positive cultures from 43 patients. The BACTEC system failed to detect 22 cultures that were positive in the conventional system, and the conventional system failed to detect 21 cultures that were positive in the BACTEC system. The detection efficiency was generally equivalent in the two systems except for the lower detection rates of anaerobes and Enterobacter aerogenes by the BACTEC system and the lower detection rates of Torulopsis glabrata and, possibly, Pseudomonas sp. (group IVD) in the conventional system. The BACTEC system had a slight advantage over the conventional system in the time interval to detection of positivity. Approximately 20% of the positive cultures detected by the BACTEC system were detected on the first day of incubation compared with 7% by the conventional system. The recovery rates and detection times of anaerobes were less efficient by the BACTEC system than by the conventional system. It does not appear that the radiometric method has much advantage over available conventional methods.     

FLUORESCENCE CONCENTRATION IMMUNOASSAY FOR RAPID DETECTION OF CAMPYLOBACTER SPP. IN CHICKEN RINSE WATER

A total of 215 freshly processed post-chill whole chicken carcasses were assessed for Campylobacter spp. contamination by a fluorescence concentration immunoassay (FCIA) procedure. Whole chicken carcasses were sampled with low volume water rinses from which 5 ml portions were enriched with brucella enrichment broth with or without oxyrase supplement in a test tube enrichment system. After a 24h stationary incubation at 42C, each sample was assayed using a FCIA procedure for the presence or absence of campylobacters. The FCIA procedure indicated Campylobacter spp. contamination in 84% of carcasses using oxyrase supplemented enrichment, while only 47% of the chicken carcasses were positive from nonsupplemented enrichment. The corresponding incidence rates detected by culture method were 92% and 87% for oxyrase supplemented and unsupplemented samples, respectively. The FCIA procedure can be completed in less than 1 h with 48 samples including a positive and a negative control assayed on one plate. In summary, the test tube oxyrase-supplemented stationary enrichment system followed by the use of the FCIA procedure was found to be an effective, rapid method for the detection of Campylobacter spp. in chicken rinse water.     

养猪场空气中抗性基因和条件致病菌污染特征

养殖场空气中含有较高浓度的抗生素抗性基因和条件致病菌,对人畜健康具有潜在威胁.采用中流量TSP采样器在某养猪场的生活区、猪舍内和猪舍外3个地点分别采样24 h和48 h,并采集猪舍内的饲料、粪便和饮水添加剂样品.采用普通PCR检测样品中的3类抗生素抗性基因(大环内酯类、β-内酰胺类、四环素类各3个基因)和7种致病菌/条件致病菌基因(弯曲杆菌属、产气荚膜梭菌、肠球菌属、大肠杆菌、小肠结肠炎耶尔森菌、葡萄球菌属和猪链球菌);选取检出率较高的6种基因,采用荧光定量PCR对其浓度进行测定.结果表明: 空气中大环内酯类抗性基因检出了3个,四环素类检出了2个,肠球菌、大肠杆菌、小肠结肠炎耶尔森菌、葡萄球菌等4种条件致病菌在空气样品中和饮水添加剂中都被检测到.绝大部分目的基因的浓度均在10⁴ copies·m^-3以上,并且猪舍附近浓度远高于生活区;猪场内主要的抗生素抗性基因和条件致病菌的可能来源是猪粪便和饮水添加剂.在养猪场内采样24 h即可满足PCR检测要求;在生活区采样48 h的采样效率高于采样24 h,而在猪舍外和猪舍内采样24 h的效率高于采样48 h.     

RECOVERY and SPECIATION of LISTERIA FROM RAW and COOKED MEAT and POULTRY PRODUCTS

Recovery of Listeria from raw and cooked meat products was compared using Fraser broth (FB) enrichment incubated at 30 and 35C for 24 and 48 h. the Micro-ID Listeria test strip for biochemical characterization of Listeria was also compared with conventional tests. Listeria spp. were recovered from 33, 47, and 20 of the raw chicken, raw beef and cooked meat products, respectively. No false-negative reactions were observed and more total Listeria -positive samples were found using FB incubated for 48 h compared with 24 h. Samples incubated at 35C had fewer false negative tubes than those incubated at 30C. More false-positive FB tubes were observed after 48 h than after 24 h incubation. Over half of the cooked samples did not hydrolyze the esculin and turn the tubes black, and therefore did not have to be streaked onto selective plates. However, with raw chicken or beef because of the large number of false-positive FB tubes, almost all tubes had to be streaked onto selective plates and very little advantage was gained from using the FB. the Micro-ID Listeria test kit gave a 100% correlation with conventional biochemical reactions for pure cultures of Listeria isolated from the three categories of meat products in this study. When used in conjunction with hemolysis plates and CAMP reactions, this test identifies species of Listeria isolates within 24 h of visible colony formation.     

Changes in porcine,ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

The aim of this study was to determine if different methods of handling porcine, ovine, bovine and equine blood between collection and centrifugation influence measurable progesterone levels. A 2 × 2 × 5 factorial experiment was conducted for each species with heparin (with or without), temperature of incubation (4 and 22°C) and time of incubation (0, 6, 12, 24 and 48 h) as the main effects. Following centrifugation, plasma and serum samples were stored at ?20°C until progesterone concentrations were determined by radioimmunoassay. Method of handling porcine and equine blood between collection and centrifugation did not affect the levels of progesterone. However, heparinized blood held at 4°C resulted in the most consistent levels of progesterone over time. Progesterone levels were fairly consistent across time in the ovine blood by all methods of handling except heparinized blood incubated at 22°C. By 24 h after collection, plasma progesterone concentrations decreased by 50% for the ovine blood incubated at 22°C with heparin. Decreases were detected by all the methods of handling the bovine blood between collection and centrifugation. The rate of decline, however, was considerably faster for blood held at 22°C than blood held at 4°C. At 12–48 h after collection, the concentrations of progesterone averaged only 5% of the time 0 sample for blood incubated at 22°C. In contrast, at least 30% of the progesterone values in the time 0 sample were detected between 12 and 48 h of incubation for the blood held at 4°C.     

ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples

This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of > or =10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after > or =10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required < or =48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.     

Factors affecting the production of an antimicrobial agent, plantaricin F, by Lactobacillus plantarum BF001

Lactobacillus plantarum BF001 produced plantaricin F in MRS broth but it was detected only after ca a 50-fold concentration. Growth on MRS broth and appearance of plantaricin F were similar under aerobic and anaerobic conditions. No growth occurred at pH 3 or at 4°C. Plantaricin F appeared first at early stationary growth phase (24 h) and was stable thereafter (pH 2). Amounts found in liquid cultures were ca 2–3 times higher than those from solidified MRS medium, and specific activities were ca 6 times higher in liquid culture (48 h). Maximal amounts of plantaricin F were found (48 h) when medium had an initial pH of 4 and growth was at 30°C. Under these conditions, cell growth and fermentation were partially uncoupled. Plantaricin F was not produced endogenously, organic nutrients were necessary. A molecular weight range of 500–3500 Da was indicated. Plantaricin F appears to be a secondary metabolite.     

Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of Salmonella.

Monoclonal antibodies produced to Salmonella enteritidis outer membrane proteins were screened against 57 Salmonella serovars and several related enterobacteria. Those detecting all Salmonella serovars and none of the related enterobacteria were used in a microtitre plate antigen capture ELISA to screen clinical samples. Sixty-one of 2100 samples yielded salmonellas after incubation for 24 h in selective media by conventional culture. Of these 58 were detected by the ELISA. Sixty-five false positives by ELISA were found to be Enterobacter spp. The results show the potential of this ELISA to eliminate a large proportion of the salmonella-negative cultures at an early stage.     

Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes

A previous study showed that oleic acid was converted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid isomers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-[13C]trans-9 C18:1) elaidic acid were each added to four in vitro batch cultures with three cultures inoculated with mixed ruminal bacteria and one uninoculated culture. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectrometry. At 0 h of incubation, enrichment was detected only in elaidic acid. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid, 7% to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons six through 16, and 5% to 10% for cis-9 and cis-11 monoenes. After 48 h, 13C enrichment in the uninoculated cultures was only detected in the added elaidic acid. This study shows trans fatty acids exposed to active ruminal cultures are converted to stearic acid but also undergo enzymic isomerization yielding a multitude of positional and geometric isomers.     

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.