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Colilert Rapid test was compared with the traditional methods (multiple tube fermentation (MTF) and Membrane filtration (MF)) to enumerate total and fecal coliform in marine water. Colilert method is based on specific indicator nutrients: ONPG and MUG for the target microbes, and chemically suppresses non-coliform bacterial growth. It is more rapid, providing results in as little as 18 h compared to the 24–96 h required for traditional methods. The results reported that Colilert method was more sensitive in detecting fecal coliform than the traditional methods (MTF and MF). No significance difference between it and MF in enumeration of total coliform. While, there were significant differences with both MF and MTF in enumeration of fecal coliform. It was more close to MF than MTF. The study recommended MF technique for detecting and enumerating coliform bacteria in marine water due to the high values of false positive results detected by Colilert method. INTRODUCTION The coastal water is an important economic and recreational resource that is influenced by human activities.(1) The hygienic quality of water is of utmost importance to society, and efficient bacteriological control of water is essential for implementing a good management of this vital resource.(2) The main criterion for assessing the potential health risk of waters is the density of indicator bacteria. Although indicator bacteria do not necessarily cause illness, they are abundant in human waste where pathogenic organisms, such as pathogenic bacteria, viruses, and parasites are also likely to exist.(3) The typical indicators used include total coliform, fecal coliform, Esherichia coli, and enterococci.(4) Total and fecal coliform were recommended by the US Environmental Protection Agency (EPA) in 1976.(5) Indicator bacteria have historically been measured using either membrane filtration (MF) or multiple tube fermentation (MTF), which both as a rule require 24 to 48 h for completion(6).The 78 Bull High Inst Public Health Vol.38 No.1 [2008] Colilert reagent, based on IDEXX’s defined substrate technology, is used for the simultaneous detection and confirmations of total coliforms and E. coli in water. It provides specific indicator nutrients: ONPG (O-Nitrophyenyl-B-D-Galactopyran-oside) and MUG (4-Methylum-belliferyl-B-D Hlucuronide) for the target microbes, and chemically suppresses non-coliform bacterial growth. This reagent system is specifically formulated to achieve optimum sensitivity and specificity in the simultaneous detection and identification of total coliform and E. coli. After 24 hours incubation at 35°C, if E. coli is present, the reagent should show yellow color and fluorescence when exposed to a long-wave (365-366 nm) UV lamp.(7) The allure of the Colilert test is that it is more rapid, providing results in as little as 18 h compared to the 24–96 h required for traditional methods. It has the additional advantage of being less expensive (3).Qatar is located half way along the western coast of Arabian Gulf. The total area of Qatar’s sea waters are approximately 35,000 sq. Km (approximately 15% of the Gulf). Qatar has an extremely shallow sea with an average depth of 30 m. The high evaporation of the water especially in summer, the very low rainfall, and the little fresh water inflow from the land are the main factors which produce very high sea water salinity. The salinity within Qatar's waters varies mainly between 39 ppt and 41 ppt at the surface. At the bottom, close to the offshore boundaries salinity tends to be 1 2 ppt higher than that at the surface.(8) So, two major objectives of the present study: are to compare the Colilert method with the standard MTF and MF in quantification of coliform bacteria, and to assess the efficient one used in high salinity water. MATERIAL AND METHODS Sea water samples, (n=32), were collected randomly from the Doha coastal region. Samples were collected aseptically Magda M. Abed El-Atty EL-Magharaby 79 in 250 ml sterile Pyrex glass bottles, placed on ice and transported immediately to the laboratory and analyzed within an hour. MTF was carried out by using lactose broth for presumptive test and Brilliant green bile broth for confirmed test to estimate the total coliform density and Eijkman medium at 44.5°C to estimate the fecal coliform density. MF total Coliform and fecal coliform analyses with m-Endo Agar LES (Fluka) and mFC Agar (Fluka) media respectively were used to estimate the total coliform and fecal coliform densities. The media used and analytical methods were performed in accordance with the standard procedures outlined in standard methods (6). The estimation of total coliform and E. coli by Colilert test was performed in accordance with Standard Operating Procedure for: Escherichia coli and Total Coliform using the IDEXX Quanti-Tray/2000 System with Colilert reagent (4010R01 E. coli IDEXX.doc).(9) The last ten samples were performed to intensive tests to recovery of coliforms and E. coli. Recovery of coliforms. Following incubation, the backing material of each Quantitray was disinfected by application of 70% ethanol with a sterile swab. After the residual ethanol evaporated, sterile pipette tips were used to pierce the backing material of all MUG-positive, ONPGpositive wells; all MUG-positive, ONPGnegative wells; all part florescent, two ONPG-positive wells and MUG-negative per tray. One tray was processed per each water sample. One hundred micro-liters of fluid were withdrawn from each well and added to a separate tube containing 5 ml of Ej broth with a Durham tube and incubated at 44.5°C. After 24 h, all of the tubes were examined for gas formation. At the same time the Ej tubes were inoculated, fluid from each well was used to inoculate selective-differential media. One drop (approximately 20 μl) of well content was 80 Bull High Inst Public Health Vol.38 No.1 [2008] streaked for isolation on MacConkey agar and EMB agar. Following incubation at 35.0°C for 24 h, colonies were examined for lactose utilization. Selected colonies isolated from each Colilert well were stained with gram stain and cultured on tryptone water and incubated at 35oC. After 48h, all were examined for indol formation. From the positive MTF fecal coliform tubes, full loop from each was streaked also on MacConkey and EMB agar and then stained and tested for indol formation. Selected one typical blue colony (fecal) from positive m-FC agar were stained and cultured on tryptone water and incubated at 35°C. After 48h, all were examined for indol formation. Statistics: Descriptive analysis, AOVA test , LSD, and Sensitivity tests were done on the results. RESULTS AND DISCUSSION Total coliform Results revealed that 12.5% from samples undetected of total coliform by MTF and 9% by MF while no samples undetected for total coliform by the Colilert method. MTF method recorded the highest value of total coliform in 9.4% of samples, MF method recorded the highest value in 37.5% of samples , and Colilert method recorded the highest value in 53.1% of samples. Two samples (6.25%) were coliform negative by MTF and MF procedure but coliform positive by the Colilert method. Covert et al.,(10) reported one sample only was coliform negative by MTF and MF procedure but coliform positive by the Colilert method. For the MTF method, the geometric mean value of total coliform was 1.8 MPN/100 ml with standard error of 0.290 MPN/100 ml. For the MF, the geometric mean value was 2.9 CFU/100 ml with standard error of 0.192 CFU/100 ml .For the Colilert method, the geometric mean value was 3.3 MPN/100 ml with standard Magda M. Abed El-Atty EL-Magharaby 81 error of 0.133 MPN/100 ml as presented in table (1). A number of environmental factors injure or kill coliform bacteria in seawater.(11) Furthermore, salinity has been shown to be detrimental to the survival of E. coli and other coliform bacteria, with aged seawater being more toxic than fresh water.(12) The lower recovery efficiencies of coliform may be due to the inhibition of biochemical reactions producing typical positive results in both MTF and MF assay methods. (13) Attributed to the MTF, the lower values detected may be due to that the enzyme formic hydrogenlyase (which produces hydrogen gas from formic acid) may be impaired or not induced in environmentally stressed cells.(14) An injury of this nature could explain the absence of gas production in the presumptive portion of the MTF test. This is the reason for giving false negative result and consequently lower count recorded by this method. Olson(14) stated in his study on seawater, a mean increase of 10.4% in the total coliform count occurred when incorporate of false negative into the coliform MPN method number. Total coliform results by Colilert about 1.8 and 1.1 higher than MTF and MF methods respectively. Noble et al.,(15) reported total coliform were 1.6 to 1.8 times by Colilert higher than those for MF and MTF. It was noticed that the highest geometric mean value of total coliform reported by the Colilert method, this may be indicated that this method was more sensitive than the standard methods or it may give false positive result due to the presence of interference of bacteria other than coliform bacteria. Previous study (16) indicated that various plant and algal extracts can significantly interfere with the Colilert system’s detection of both coliform and E. coli. In an earlier study, Palmer et al.,(17) also reported higher total coliform recovery in the marine environment when the original 82 Bull High Inst Public Health Vol.38 No.1 [2008] Colilert was used. They hypothesized that the Colilert medium did a better job of resuscitating coliforms stressed by exposure to highly saline bay water than does conventional recovery medium. Table (2), contributed to tota l coliform detection, 87.5% from samples concordance between Colilert and MTF while 90.6% samples were concordant between Colilert and MF. The present study reported

journal of high institute of public health

Comparison of Standard Methods, MTF, and MF, by the Colilert Rapid Test for the Enumeration of Coliform Bacteria in High Salinity Water, Arabian Gulf, Doha, Qatar