A new selective and differential agar medium for Escherichia coli and coliform organisms

W right , R.C. 1984. A new selective and differential agar medium for Escherichia coli and coliform organisms. Journal of Applied Bacteriology 56 , 381–388.
An enriched lauryl sulphate-aniline blue agar medium which is selective for Escherichia coli and coliform organisms is described. From faecal samples, the medium gave higher counts of colonies producing acid from lactose than media containing bile salts. From contaminated water and food samples, the medium gave comparable or higher counts of colonies identified as E. coli than standard media. Colonies of E. coli were more readily differentiated from those of other coliform organisms.     

A new selective and differential agar medium for Escherichia coli and coliform organisms

An enriched lauryl sulphate-aniline blue agar medium which is selective for Escherichia coli and coliform organisms is described. From faecal samples, the medium gave higher counts of colonies producing acid from lactose than media containing bile salts. From contaminated water and food samples, the medium gave comparable or higher counts of colonies identified as E. coli than standard media. Colonies of E. coli were more readily differentiated from those of other coliform organisms.     

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

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

Evaluation of four membrane filter media in anaerobic-MF recovery of faecal coliforms from freshwater in Nigeria.

MacConkey (MC), membrane lauryl sulphate (MIS), membrane faecal coliform amended with rosolic acid (MFC + R) and without the acid (MFC - R) were evaluated in the anaerobic membrane filtration (anaerobic-MF) recovery of faecal coliform populations (FCs), genera and faecal coliform positive (FC-positive) strains isolated from various sources of freshwater, i.e., rivers, rural wells, unchlorinated distributive supplies and hand pumps. Mean counts (x 10(2)/100 ml) of presumptive (typical) FCs varied from 13.69 (MC) to 40.81 (MLS) in river samples, and from 2.0 (MC) to 4.19 (MFC + R) in wells. The proportion of FC-positive, typical FCs ranged from 48.66 (MIS) to 66-67% (MC) in rivers, and from 50 (MC) to 90.22% (MFC + R) in wells. More than 30% of the typical FCs from all sources on each medium was FC-negative. These usually formed small (ca 1.0 mm diam.) colonies on the test agar, and were prevalent in wells. Typical FCs and FC-positive strains were not recovered from piped supplies and hand pumps. In spite of anaerobic incubation, non-faecal coliforms (NFCs) were often higher than the FCs; the FC:NFC ratios for rivers ranged from 1.65 (MC) to 7.65 (MLS) and (MFC + R) but were < 1.0 for wells on each medium. Escherichia coli, Klebsiella and Enterobacter species were recovered on all media: approximately 35-64% of the strains confirmed as FCs were E. coli, 20-42% were Kl. pneumoniae. The FC counts on the media were variable, but the overall performance in recovering 'true' FCs was similar; < 70% of strains per medium were FC-positive. None could count E. coli exclusively.     

Comparison of fecal coliform agar and violet red bile lactose agar for fecal coliform enumeration in foods

A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 +/- 1 degrees C and transfer to 44 +/- 1 degrees C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.     

Evaluation of four membrane filter media in anaerobic-MF recovery of faecal coliforms from freshwater in Nigeria

M.T. OGAN. 1992. MacConkey (MC), membrane lauryl sulphate (MIS), membrane faecal coliform amended with rosolic acid (MFC + R) and without the acid (MFC — R) were evaluated in the anaerobic membrane filtration (anaerobic-MF) recovery of faecal coliform populations (FCs), genera and faecal coliform positive (FC-positive) strains isolated from various sources of freshwater, i.e. rivers, rural wells, unchlorinated distributive supplies and hand pumps. Mean counts (x 10²/100 ml) of presumptive (typical) FCs varied from 13.69 (MC) to 40.81 (MLS) in river samples, and from 2.0 (MC) to 4.19 (MFC + R) in wells. The proportion of FC-positive, typical FCs ranged from 48.66 (MIS) to 66.67% (MC) in rivers, and from 50 (MC) to 90.22% (MFC + R) in wells. More than 30% of the typical FCs from all sources on each medium was FC-negative. These usually formed small ( ca 1.0 mm diam.) colonies on the test agar, and were prevalent in wells. Typical FCs and FC-positive strains were not recovered from piped supplies and hand pumps. In spite of anaerobic incubation, non-faecal coliforms (NFCs) were often higher than the FCs; the FC: NFC ratios for rivers ranged from 1.65 (MC) to 7.65 (MLS) and (MFC + R) but were < 1.0 for wells on each medium. Escherichia coli, Klebsiella and Enterobacter species were recovered on all media: approximately 35–64% of the strains confirmed as FCs were E. coli, 20–42% were Kl. pneumoniae. The FC counts on the media were variable, but the overall performance in recovering 'true' FCs was similar; < 70% of strains per medium were FC-positive. None could count E. coli exclusively.     

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

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

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.     

A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater

S ummary . Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published evidence could be found to judge the specificity of the method to assess faecal contamination in either fresh or saline waters. The method is used in our laboratory to monitor the extent and degree of sewage pollution in coastal areas, but there is need for information on what proportion of lactose-fermenting colonies from seawater, developing at 44° on a 4% enriched Teepol medium, are E. coli type I. A total of 1352 colonies from seawater was tested for production of indole and for gas from lactose at 44°. In addition, 46% of the colonies were screened by the IMVEC series of tests. The proportion of colonies tested ranged from 10–100%, depending on the number of colonies on the membrane. Many of the colonies (81.9%) to which IMVEC tests were applied were E. coli type I; a further 10.9% were Irregular type I. The practical implications of these findings are discussed.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

Evaluation of the Anderson Baird-Parker direct plating method for enumerating Escherichia coli in water

The direct plating (DP) method for enumerating Escherichia coli in food was adapted for water analysis by membrane filtration and a standardized protocol was described. The DP method was found to give equal or better recoveries of E. coli than a membrane filtration method using 0·1% sodium lauryl sulphate agar; the repeatability of the DP method was markedly better. The necessity to transfer membranes from the non-selective medium tryptone soy agar (TSA) to the selective medium tryptone bile agar (TBA) after pre-incubation for 4 h was considered disadvantageous for practical purposes. A double-layer method, where the membrane filter is placed on a layer of TSA poured over TBA, with incubation in an incubator that automatically switches from 37° to 44°C after 4 h, was found to be an acceptable alternative. Recovery of E. coli and inhibition of competitive flora were equal or only slightly less than for the standard DP method.     

A rapid fluorogenic method for the detection of Escherichia coli by the production of β-glucuronidase

A medium containing the fluorogenic substrate 4-methylumbelliferyl-β-d-glucuronide was developed for the isolation and identification of Escherichia coli within 7·5 h and was based on the detection of β-glucuronidase. Optimum conditions for the rapid development of fluorescent colonies were determined. The optimum temperature was 41·5°C. Development of fluorescence was delayed when carbohydrates were incorporated into the medium. Water samples were used to evaluate the medium by surface plating and membrane filtration. The frequency of false-negative results was 6·1% and false-positives were 3·7% for freshwater samples. The false-positive organisms were identified as Klebsiella spp. and Shigella sonnei. The potential applications of the medium are discussed.     

Enumeration of intestinal enterococci and interfering organisms with Slanetz-Bartley agar, KF streptococcus agar and the MUST method

The recovery of intestinal species of enterococci and streptococci and potentially interfering nonfaecal species was measured on KF streptococcus agar, Slanetz-Bartley agar and in a medium based on 4-methylumbelliferyl-β- d -glucoside (MUST method) using pure cultures. Both of the solid media yielded high recoveries of the target species. Their selectivity was better at elevated incubation temperature but nonfaecal Enterococcus and Staphylococcus species were not eliminated even at the elevated temperature. The MUST method tended to give slightly lower recoveries than the agar cultivation methods with some target species at 44°C but recoveries were better at 41°C.     

Comparison of selective agars for the isolation and identification of Klebsiella oxytoca and Escherichia coli from environmental drinking water samples

Various selective media were assessed for their ability to detect and differentiate Klebsiella oxytoca and Escherichia coli in environmental water samples. Only two, Membrane Lauryl Sulphate agar and Deoxycholate Agar, could differentiate the two coliforms from each other and from the 'background' heterotrophs in water and this was a consequence of E. coli's ability to grow at 44°C and 37°C whereas Kl. oxytoca could only grow at 37°C. Modified M-FC medium effectively differentiated Kl. oxytoca but not E. coli in environmental samples. Other media characterized the different coliforms in pure culture but failed to do likewise in environmental samples. For example, pure cultures of E. coli fluoresced when MUG was added to the medium but single colonies on a mixed species plate failed to do so. MT7 agar distinguished the two coliforms from water heterotrophs but not from each other.     

The use of lauryl tryptose broth containing 4-methylumbelliferyl-β-D-glucuronide (MUG) to enumerate Escherichia coli from freshwater sediment

Most-Probable-Number tests for Escherichia coli were performed on 45 sediment samples using Lauryl Tryptose broth (LTB) containing 4-methylumbelliferyl-β-D-glucuronide (MUG). Eighty-three percent of 493 LTB-MUG reactions agreed with results obtained by conventional faecal coliform analysis. Although false-negative reactions are suspected in about 9% of the LTB-MUG tubes, anaerogenic E. coli , that would not be enumerated by conventional faecal coliform tests were recovered using LTB-MUG. The high percentage of agreement between the two methods suggests that the MPN method employing LTB-MUG is adequate to enumerate E. coli from some freshwater sediments.     

Study of haemolytic activity of some Campylobacter spp. on blood agar plates

A total of 152 strains of Campylobacter jejuni, C. coli, C. laridis and C. fetus subsp. fetus were tested for haemolysis on blood agar plates. Distinct haemolysis was detected in 92.% (96/104) of strains of C. jejuni and 21.7% (5/23) of strains of C. coli on sheep blood heart infusion agar after incubation for 4 d microacrobically at 42°C. Haemolysis was also detected on horse blood heart infusion agar. Haemolysis was not detected at 37°C except with one of 50 strains of C. jejuni tested at this temperature, which was weakly positive. Campylobacter laridis was not haemolytic; C. fetus subsp. fetus , which does not grow at 42°C, showed no haemolysis at 37°C. Blood agar (Oxoid, BA Base No. 2) was not suitable for testing for haemolysis by these organisms. A microaerobic gas mixture containing hydrogen is better than that containing nitrogen because the medium has a brighter colour, making haemolysis casier to detect. There was no synergistic haemolysis with Staphylococcus aureus or Streptococcus agalactiae . The plate haemolysis test as described here may aid differentiation within the thermophilic campylobacters.     

Comparison of indole production and β-glucuronidase activity for the detection of Escherichia coli in a membrane filtration method

In a membrane filter method for the enumeration of Escherichia coli in water samples, the James' indole reagent has several advantages over the commonly used diaminobenzaldehyde (DAB) reagent. Results with James' reagent were easier to read because the red colour of positive colonies was more intensive and developed within a few minutes without exposure to UV light. DAB-coloured colonies were pale pink with a diffuse pink zone surrounding the colonies after 30 min of exposure to UV-source radiation. Incorporation of 4-methylumbelliferyl- β -D-glucuronide (MUG) into the selective medium to detect E. coli by means of β -glucuronidase-activity gave discouraging results. Fluorescence was difficult to read on membrane filters incubated on this medium and 14% of E. coli strains were β -glucuronidase-negative.     

Membrane filtration method for bacteriological testing of water: enhanced colony visualization and stability on purification of phenol red indicator

Commercially-available phenol red indicator, purified by adsorption chromatography was incorporated into lauryl sulphate broth (LSB) used in the membrane filtration method for the detection of Escherichia coli and other coliform bacteria. Relative to LSB containing the impure dye or its major contaminant, the purified phenol red provided clear visualization of discrete yellow colonies observed against a white background. The colonies remained stable for at least 24 h at 25°C under standard laboratory lighting conditions. This simple procedure will enhance the detection of coliforms in samples.     

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

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

Conjugative Plasmid in Corynebacterium flaccumfaciens subsp. oortii That Confers Resistance to Arsenite, Arsenate, and Antimony(III)

The membrane filter (MF) method for detection and enumeration of coliform bacteria in drinking water requires that the coliforms both grow and produce a green metallic sheen when the filter is incubated on modified Endo medium at 35 degrees C for 22 h. Large numbers of noncoliform bacteria, which are enumerated by the standard plate count (SPC) technique, can interfere with the detection of coliforms on MF. This paper presents quantitative evidence from laboratory experiments on the interference of specific SPC bacteria on coliform colony sheen production on MF. Pseudomonas aeruginosa and Aeromonas hydrophila caused significant reductions in Escherichia coli sheen colony counts when present at 3,000 and 220 per filter, respectively. The Flavobacterium sp. and Bacillus sp. selected for this study from SPC did not interfere with coliform colony sheen production. Excessive crowding of E. coli and Enterobacter cloacae colonies on MF also caused a reduction in the number of colonies that produced sheen. Even when there was no crowding (14 colonies per filter), only a fraction of the E. cloacae colonies produced sheen colonies on modified Endo medium.