Enumeration Of Fecal Coliforms Research Articles

A Treatment Design and Operation Effect of Medical Rehabilitation Hospital Wastewater

The process of “Classified pre-treatment + Regulating + AO treatment + Disinfection” was designed to treat the medical rehabilitation hospital wastewater, and the treatment scale was 800 m3/d. The engineering design process, main design parameters and engineering treatment effects are discussed. The stable operation results showed that when the influent concentrations of CODCr, NH3-N, SS and the enumeration of faecal coliforms were 185∼346 mg/L, 28∼55 mg/L, 55∼93 mg/L, 1.2×106∼1.4×108 MPN/L, the effluent concentrations were 22∼45 mg/L, 0.5∼3.4 mg/L, 22∼46 mg/L, 85∼220 MPN/L, which could meet the requirements of “Discharge Standard of Water Pollutants for Medical Organization in Shandong Province (DB37/596-2020)”. The direct operation cost of the project was 2.16 yuan/m3.

Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil.

The aim of this study was to evaluate the microbiological quality of 45 samples of corn-based farinaceous foods commercialized in Brazil. The bacteriological analysis performed were: detection of Salmonella and Cronobacter, and enumeration of faecal coliforms and Bacillus cereus. The Cronobacter isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene and MLST. No sample presented contamination by Salmonella or B. cereus (<102 UFC/g). Faecal coliforms were detected in two (4.4%) samples but in low concentration (≤23.0 MPN/g), and 20 samples (44.4%) contained Cronobacter. Twenty-nine unique Cronobacter isolates were identified as C. sakazakii (n=18), C. malonaticus (n=2); that presented 11 different fusA alleles, including new fusA 183. MLST analysis revealed 17 sequence types (STs), six of which were newly identified (ST687-690, 693, and 694). Resistance or intermediary resistance were found to ceftazidime (15.0%), aztreonam (15.0%), nalidixic acid (15.0%), nitrofurantoin (15.0%), cefepime (10.0%), gentamicin (5.0%), and tetracycline (5.0%). The presence of Cronobacter in corn-based farinaceous foods could be a significant risk to infants as these products are used as alternatives to commercially available infant formula. Strategies to manage the risk of Cronobacter infections due to the consumption of these alternative feeds need to be developed by the regulatory agencies.

New strategies for the enumeration of enteric pathogens in water.

Water quality standards for drinking water and recreational waters have long been based on the enumeration of faecal coliforms in the various water supplies, with 0 CFU Escherichia coli/100 ml for drinking water and <126 CFU generic E. coli/100 ml for recreational waters. Irrigation water will soon undergo the same scrutiny in the United States. For over 50 years the most probable number method has been used by laboratories to estimate the level of viable bacteria in a sample, but this method is labour intensive and slow, especially if large numbers of samples need to be tested. In this review, we describe some recent innovations in methods to enumerate enteric pathogens in water. These methods are based on different reasoning schemes that can be categorized as biosensors and nucleic acid-based methods. All the methods described here used natural water sources. Several were also used to survey the bacterial levels in naturally contaminated samples. The different methods vary in their limits of detection, ease of use, and potential portability. Some combine very good limits of detection with the ability to overcome technical challenges; however, there is considerable room for improvement, as none of the methods are without shortcomings.

A Comparison of Rapid Bacterial Methods to U.S. EPA‐Approved Methods for Biosolids

The U.S. Environmental Protection Agency (U.S. EPA) specifies methods for testing microorganisms in biosolids, including U.S. EPA Methods 1680 and 1682 for fecal coliforms and Salmonella sp., respectively. However, these methods require 3 to 5 days to complete. For research or nonregulatory process monitoring purposes, it would be desirable to have a more rapid, less labor-intensive method of enumerating these organisms. The purpose of this study was to compare a modified Colilert with Quanti-Tray/2000 method to U.S. EPA Method 1680 for enumeration of fecal coliforms and E. coli, and to compare spread plating on xylose-lysine deoxycholate (XLD) supplemented with 10 pg/L novobiocin to U.S. EPA Method 1682 for Salmonella sp. in biosolids. These alternative enumeration methods require only 1 day to complete, using fewer costly materials. This limited study indicated that the more rapid methods provide results that are statistically comparable to U.S. EPA methods under most conditions and would be acceptable for rapid, nonregulatory data collection.

Monitoring of active but non-culturable bacterial cells by flow cytometry

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.

Fate of physical, chemical, and microbial contaminants in domestic wastewater following treatment by small constructed wetlands

In order to evaluate the efficacy of constructed wetlands for treatment of domestic wastewater for small communities located in rural areas, small-scale wetland mesocosms (400 L each) containing two treatment designs (a mixture of Typha, Scirpus, and Juncus species; control without vegetation) were planted into two depths (45 or 60 cm) with pea gravel. Each mesocosm received 19 L/day of primary-treated domestic sewage. Mesocosms were monitored (inflow and outflow samples) on a monthly basis over a 2-year period for pH, total suspended solids (TSS), 5-day biochemical oxygen demand (BOD 5), total Kjeldahl nitrogen (TKN), dissolved oxygen (DO), and conductivity. Microbiological analyses included enumeration of fecal coliforms, enterococci, Salmonella, Shigella, Yersinia, and coliphage. Significant differences between influent and effluent water quality for the vegetated wetlands ( p<0.05) were observed in TSS, BOD 5, and TKN. Increased DO and reduction in fecal coliform, enterococcus, Salmonella, Shigella, Yersinia, and coliphage populations also were observed in vegetated wetlands. Greatest microbial reductions were observed in the planted mesocosms compared to those lacking vegetation. Despite marked reduction of several contaminants, wetland-treated effluents did not consistently meet final discharge limits for receiving bodies of water. Removal efficiencies for bacteria and several chemical parameters were more apparent during the initial year compared to the second year of operation, suggesting concern for long-term efficiency and stability of such wetlands.

Evaluation of Selected Membrane Filtration and Most Probable Number Methods for the Enumeration of Faecal Coliforms, Escherichia coli and Enterococci in Environmental Waters

Selected methods recommended in national and international water quality guidelines were compared in tests on environmental waters with different levels of faecal pollution. The following methods yielded no statistically significant differences in counts of faecal coliforms and Escherichia coli in raw sewage, semi-treated effluent, polluted urban run-off and stored potable water: Membrane filtration (MF) using MFc Agar or Chromocult Coliform Agar containing X-glucuronide, or a miniaturised microtitre-plate Most Probable Number (MPN) assay using a liquid growth medium containing chromogenic 4-methyl-umbelliferyl-β-D-glucuronide. Significant differences were, however, found between the Chromocult and the other methods for unpolluted river water. Counts of faecal enterococci in raw sewage, semi-treated effluent and polluted urban run-off, obtained by the following methods did not differ significantly: MF using M-Enterococcus Agar, Bile-Esculin Agar or Enterococcus Selective Agar, or a microtitre-plate MPN method with a liquid growth medium containing chromogenic 4-methyl-umbelliferyl-β-D-glucoside. Significant differences were, however, found between the MPN and the other methods for unpolluted river water and stored potable water. MF using Chromocult Coliform Agar had useful benefits for the simultaneous enumeration of coliforms and E coli. However, in view of cost and practical considerations, MF using MFc Agar or M-Enterococcus Agar proved the methods of choice for respectively enumerating faecal coliforms and E coli, or faecal enterococci, in analyses for general water quality surveillance purposes.

Quantitative determination of Escherichia coli in water using CHROMagar ®E. coli

A new medium, CHROMagar ® E. coli (CAEC), containing a combination of X-glucuronide and methyl-glucuronide for the detection of β-glucuronidase activity of Escherichia coli has been evaluated by the membrane filtration (MF) technique n fresh water samples. The CAEC agar was compared with conventional media, mFC agar and mLSB, for the enumeration of faecal coliforms. The variance analysis showed that CAEC was as sensitive as mFC agar and mLSB. A good correlation was found between E. coli versus those from faecal coliforms in the water sampling areas tested. Of 321 presumptively positive E. coli colonies (blue) and 154 presumptively negative E. coli colonies (white), only 8 (2.5%) false positive and 19 (12.4%) false negative colonies were found. Specificity of the CEAC agar in spanish samples was temperature dependent, false negative E. coli colonies occurred less frequently at 37°C (2.3%) than at 44.5°C (18.8%). The results of this study indicate that CAEC agar is efficient for the enumeration of E. coli from a wide range of environmental freshwater samples.

Comparison of a Rapid Plate Count and MPN Methods for Enumeration of Fecal Coliforms and Escherichia coli in Soft-Shell Clams

A direct elevated temperature plate count method utilizing modified fecal coliform agar with rosolic acid (ETPC/mFC) was compared to 5-tube and 3-tube most probable number (MPN) procedures for its accuracy in enumerating fecal coliforms and Escherichia coli in naturally and artificially contaminated soft-shell clams (Mya arenaria). The results indicated that the extent of overall recovery of fecal coliforms was similar in the two methods tested. Therefore, the ETPC/mFC method may be considered as a rapid procedure for fecal coliform screening during depuration of soft-shell clams.

Underestimation of Fecal Coliform Counts in Shellfish-Growing Waters by the Spanish Official Method

We compared two tube fermentation methods for the enumeration of fecal coliforms in mussels: the APHA method and the Spanish official method (CP method). In the study area (Galicia, northwest Spain), the regional authorities have proposed that the CP method be adopted as standard. Results showed that the APHA method gave significantly higher counts (P < 0.01) than the CP method. The mean difference between APHA-method counts and CP-method counts was particularly high when only those samples containing less than 500 fecal coliforms per 100 g (as determined by either method) were considered. A significantly higher number of samples were classified as unacceptable (more than 300 fecal coliforms per 100 g) by the APHA method than by the CP method. These results suggest that the CP method is inappropriate for the enumeration of fecal coliforms in mussels.

Evaluation of C-EC-agar, a modified mFC-agar for the simultaneous enumeration of faecal coliforms and Escherichia coli in water samples

A new medium, C-EC-agar (Biolife, Milan, Italy), was evaluated for the simultaneous enumeration by membrane filtration of faecal coliforms and Escherichia coli in water. The medium is a modification of m-faecal coliform agar, from which the aniline blue and lactose have been omitted and 4-methylumbelliferyl-β-D-glucuronide, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside and isopropyl-β-d-thiogalactoside added. At 44°C E. coli gives blue-green colonies that fluoresce under u.v.-light (366 nm) and give a reddish-violet colour when Kovac's reagent is placed on the membrane. Under similar conditions, faecal coliform colonies do not fluoresce. To increase recovery on the medium, repair of sub-lethally injured cells by a 4-h incubation at 37°C on tryptic soy agar is recommended.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

Agar Medium for Enumeration of Fecal Coliforms

ABSTRACTSpread‐inoculated plates of fecal‐coliform agar (FCA) were pre‐incubated 2 hr at 35°C, and then transferred to 44.5°C for 22 hr. Fecal coliforms on FCA produce acids and CO2, which react with bromocresol purple and calcium lactate to form yellow to yellow‐green colonies surrounded by a pale yellow zone. Recovery of control or freeze‐stressed Escherichia coli on FCA was about 1 log cycle lower than that with nonselective media. The accuracy for FCA to enumerate fecal coliforms from 138 typical colonies of three food samples was 98.6%; while the false positive was 1.4%. When the method was compared with the standard 3‐tube MPN method for fecal coliforms, counts from two methods were not significantly different (p&lt;0.05).

Enumeration of faecal coliforms andescherichia coliin new Zealand receiving waters and effluents

Abstract A membrane filter method, mTEC, was evaluated under New Zealand conditions.This method can enumerate both faecal coliforms and Escherichia coli. The mTEC method was compared to MPN methods for faecal coliforms (the New Zealand standard reference method) and for E. coli (a British standard method). It was also compared to the mFC membrane filter method for faecal coliforms, recommended by the American Public Health Association. The mTEC method was found to be a satisfactory alternative to the MPN methods and the mFC method. Samples tested included freshwater, sewage and meat processing plant effluent

Coliform detection from river waters: Comparison between MPN and MF techniques

This paper compares three different methods used to determine fecal pollution in river water. Two of the methods are based on direct estimation using standard aerobic incubation of membrane filters (S-MF) and anaerobic incubation of membrane filters (A-MF); the third is based on statistical evaluation by the most probable number method (MPN). It was seen from regression analysis of the results obtained that the MPN, S-MF and A-MF techniques are equally valid as regards the enumeration of fecal coliforms, whereas the S-MF and A-MF techniques are more sensitive than the MPN procedure for the enumeration of total coliforms.

Fecal coliforms as indicators in tropical waters: A review

Nowhere is the importance of accurate determination of recent human fecal contamination greater than in the tropics. The diversity of waterborne diseases and their severity is greatest in tropical environments. Since most of the countries in tropical climates are underdeveloped, with large populations that are undernourished, ill‐housed, with poor medical services, waterborne diseases may have a much greater effect on public health in the tropics than in temperate areas. Universally, tropical areas accept water maximum contaminant levels developed by temperate nations, despite the obvious differences in tropical climates. High densities of total and fecal coliform bacteria have been detected in pristine streams and in groundwater samples collected from many tropical parts of the world, even in epiphytic vegetation 10 m above ground in the rain forest of Puerto Rico. Nucleic acid (DNA) analyses of Escherichia coli from pristine tropical environs has indicated that they are identical to clinical isolates of E. coli. Many tropical source waters have been shown to have enteric pathogens in the complete absence of coliforms. Diffusion chamber studies with E. coli at several tropical sites reveal that this bacterium can survive indefinitely in most freshwaters in Puerto Rico. An evaluation of methods for the enumeration of fecal coliforms showed that currently used media have poor reliability as a result of large numbers of false positive and false negative results when applied to tropical water samples. Total and fecal coliform bacteria are not reliable indicators of recent biological contamination of waters in tropical areas. Fecal streptococci and coliphages in tropical waters violate the same underlying assumptions of indicator assays as the coliforms. Anaerobic bacteria like Bifidobacterium spp. and Clostridium perfringens show some promise in terms of survival, but not in ease of enumeration and media specificity. The best course at present lies in using current techniques for direct enumeration of pathogens by fluorescent staining and nucleic acid analysis, and developing tropical maximum contaminant levels for certain resistant pathogens in tropical waters.

Comparison of four membrane filter methods for fecal coliform enumeration in tropical waters

Four membrane filter methods for the enumeration of fecal coliforms were compared for accuracy, specificity, and recovery. Water samples were taken several times from 13 marine, 1 estuarine, and 4 freshwater sites around Puerto Rico, from pristine waters and waters receiving treated and untreated sewage and effluent from a tuna cannery and a rum distillery. Differences of 1 to 3 orders of magnitude in the levels of fecal coliforms were observed in some samples by different recovery techniques. Marine water samples gave poorer results, in terms of specificity, selectivity, and comparability, than freshwater samples for all four fecal coliform methods used. The method using Difco m-FC agar with a resuscitation step gave the best overall results; however, even this method gave higher false-positive error, higher undetected-target error, lower selectivity, and higher recovery of nontarget organisms than the method using MacConkey membrane broth, the worst method for temperate waters. All methods tested were unacceptable for the enumeration of fecal coliforms in tropical fresh and marine waters. Thus, considering the high densities of fecal coliforms observed at most sites in Puerto Rico by all these methods, it would seem that these density estimates are, in many cases, grossly overestimating the degree of recent fecal contamination. Since Escherichia coli appears to be a normal inhabitant of tropical waters, fecal contamination may be indicated when none is present. Using fecal coliforms as an indicator is grossly inadequate for the detection of recent human fecal contamination and associated pathogens in both marine and fresh tropical waters.

Limitations of the A-IM Method for Fecal Coliform Enumeration in the Pacific Oyster (Crassostrea gigas)

Use of the A-1M method, which was originally devised for testing water samples, has recently been extended for enumeration of fecal coliforms and Escherichia coli in shellfish and other food products. Results of our study indicate that while this method is reliable for analysis of growing waters, the use of the A-1M method for testing Pacific oysters may be less reliable because bacteria not belonging to the coliform group but which are sometimes present in these animals also give a positive reaction.

Selective detection and enumeration of fecal coliforms in water by potentiometric measurement of lipoic acid reduction

Water samples of various origins were inoculated into a specific coliform-selective lactose broth provided with lipoic (thioctic) acid, and the time evolution of the redox potential of the cultures was monitored during incubation at 41 degrees C by use of gold versus reference electrodes. Positive potential-time responses, i.e., 100-mV potential shifts recorded within 20 h of inoculation, were related to the initial number of fecal coliforms in the broth determined by control enumeration techniques, and the organisms responsible were isolated and identified by conventional procedures. A total of 30 samples of wastewater, 38 of surface water, 553 of groundwater, and 110 of drinking water were tested successively. A total of 240 natural water samples, including 172 groundwater samples, and 1 drinking water sample were found to be positive in the potentiometric test. The majority (i.e., 92.5%) of the relevant potentiometric detection times were shorter than 15 h, and 96% of these could be attributed to Escherichia coli. Fifteen hours corresponded to the limit for detecting 1 E. coli cell per 100 ml of water. About 78% of the potentiometric responses occurring after 15 h were induced by fecal coliforms other than E. coli (Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter freundii). Calibration curves relating detection times shorter than 15 h to fecal coliform (i.e., E. coli) concentrations were constructed for the natural water samples tested. There were minor variations in the average growth rate of the organisms in the relation to the contamination level of the water tested. The number of false-positive samples in the potentiometric test was equivalent to that of false-negative samples (groundwater or drinking water).

Performance variability, ranking, and selection analysis of membrane filters for enumerating coliform bacteria in river water

Seven different kinds of membrane filters were evaluated for the enumeration of fecal coliforms in water samples from three rivers. Analysis of variance of filter performance indicated significant interaction between the filter and the river water. The ranking and selection of filters was performed by using two different statistical procedures. In this paper a newly developed statistical method—MCB (Multiple Comparisons with the Best)—is introduced. None of the filters examined were found to be perfectly suitable for enumeration of fecal coliforms or total coliforms, but based on relative merits, two filters were outstanding.