INDICATOR BACTERIA TOTAL AND FECAL COLIFORMS, E. COLI

ENVR431: TECHNIQUES IN ENVIRONMENTAL HEALTH SCIENCES, FALL 2008 INDICATOR BACTERIA TOTAL AND FECAL COLIFORMS, E. COLI Multiple Fermentation Tube (MFT) or Most Probable Number (MPN) Meods and Membrane Filter (MF) Meods Aug 22, Sept 3, 2008 INTRODUCTION AND BACKGROUND Traditionally, indicator bacteria have been used to determine e possible presence of fecal contamination and to estimate e amount of contamination in water, foods, and oer samples. The detection of indicator bacteria is preferred over direct paogen detection because e former are considered to be normal, non-paogenic intestinal inhabitants at are present in feces and wastewater in much higher numbers an are paogenic microorganisms and because ey are technically easier to detect and quantitate an paogens. Present standards for e sanitary quality of water, foods and oer materials, wi respect to fecal contamination, are based on concentrations of indicator bacteria. The most widely used indicator bacteria in e U.S. are e so-called total and fecal coliforms. Alough taxonomically meaningless, e word coliform has been used to describe various genera of e family Enterobacteriaceae at ferment lactose. It should be emphasized at coliforms are actually defined in operational terms at are based upon e media and incubation conditions used for eir isolation and quantitation. None of ese operational definitions detects all members of e family Enterobacteriaceae and some include members of oer families. Historically, e term coliform was meant to include all of e lactose-fermenting species of e family Enterobacteriaceae which are commonly found in e feces of man and e higher animals and to exclude genera of nonor slowly-lactose-fermenting bacteria, some of which are enteric paogens in man and animals and some of which exist naturally in e environment. Thus, lactose fermentation is a characteristic of considerable diagnostic importance in distinguishing among e various groups of enteric bacteria. The ability to ferment lactose depends upon e possession of e enzyme beta-galactosidase as well as a galactoside permease which facilitates lactose entry into e cell. 1

Two major operational definitions of coliforms have been distinguished: total coliforms and fecal coliforms or ermotolerant coliforms. The latter group (fecal or ermotolerant coliforms) are considered a more specific indicator of fecal contamination because e former group may include bacteria of non-fecal origin. Fecal or ermotolerant coliforms are able to grow at higher incubation temperatures and are considered more likely to have come from e intestinal tract of a warm-blooded animal. Total Coliforms Total coliforms are operationally defined in two different ways in e 17 edition of Standard Meods for e Examination of Water and Wastewater (1). The Multiple-Tube Fermentation Technique defines coliforms as all aerobic and facultative anaerobic, gram-negative, non-sporeforming, rod-shaped bacteria at ferment lactose wi gas formation wiin 48 hr. at 35ºC. In is meod e coliform bacteria are detected and quantitated by eir ability to grow and produce gas in lactose-containing liquid medium under specified incubation conditions. This technique actually consists of ree successive steps or tests: Presumptive, Confirmed, and Completed. For e Presumptive Test, dilutions of e sample are inoculated into fermentation tubes of lactose or lauryl tryptose bro and incubated at 35ºC for 48 hr. For e Confirmed Test, organisms from all positive fermentation tubes (ose wi grow plus gas) of e Presumptive Test are transferred to fermentation tubes of brilliant green lactose bile bro and incubated at 35ºC for 48 hr. Tubes showing bo grow and gas are considered positive Confirmed tubes. For e Completed Test, organisms from positive Confirmed tubes are isolated in pure culture on agar plates of differential/selective media (Endo or eosin meylene blue agar) and en tested for: (1) grow and gas production in fermentation tubes of lactose or lauryl tryptose bro incubated at 35ºC for 48 hr.; and a negative reaction in e Gram stain. For a positive Completed Test, e organisms must show grow plus gas production in e fermentation tubes and be Gram negative. (2) The Membrane Filter Technique defines total coliforms as all organisms at produce a colony wi a golden-green metallic sheen wiin 24 hr. of incubation at 35ºC on Endo-type medium containing lactose and are aerobic or facultative anaerobic, gram-negative, non-spore-forming rods. In is meod, measured volumes of sample or sample dilution are vacuum-filtered rough 47mm diameter, microporous cellulose membrane filters at retain bacteria. The membranes are transferred to petri dishes containing endo-type lactose medium and incubated under specified conditions for e development of bacteria colonies. Colonies having e characteristic appearance of coliforms are counted and e coliform bacteria density is computed. Alough e total coliform group as presently operationally defined includes bacteria usually found in e feces of humans and oer warm-blooded animals, some of e bacteria detected by ese procedures are sometimes also found in soil (Citrobacter, Enterobacter and Kleibsiella) on various plants, including grains and trees (Klebsiella and Enterobacter) and in certain industrial wastes. Furermore, some coliform bacteria can also be paogenic for humans and animals when present eier in e gut (enteropaogenic E. coli) or in oer parts of e body (Klebsiella pneumonia in e respiratory tract). In addition, organisms at are not members of e family 2

Enterobacteriaceae and not of strictly intestinal origin, such as e members of e genus Aeromonas, may be detected in some forms of e total coliform test. For ese reasons, anoer group, e fecal or ermoteolerant coliforms, has been established in an attempt to separate e total coliforms into ose of fecal and non-fecal origin. Fecal Coliforms The basis for is distinction is a higher incubation temperature of 44.5ºC, at which presumably coliforms of only fecal origin will grow. Coliforms from non-fecal, environmental sources are generally incapable of growing at is elevated temperature. Thus, fecal coliforms can be defined as gram-negative, non-spore-forming, rod-shaped bacteria which ferment lactose wi e production of gas at 44.5ºC wiin 24 hr. Alough e fecal coliform test is applicable to investigations of surface and ground water pollution, sewage treatment systems and general monitoring of natural waters for sanitary quality, including recreational and shellfish waters, it is so far not considered a substitute for e coliform test in e examination of potable waters. Coliform bacteria of any kind are not to be tolerated in a finished (treated) drinking water. There are actually two approved Multiple Tube Fermentation Techniques for fecal coliforms: a single-step and a two-step procedure. The latter is e traditional test at has been widely used for may years, ad e former is a more recent test at appears in e 17 edition of Standard Meods. In is single-step procedure dilutions of e sample are inoculated into fermentation tubes of A-1 medium (see Standard Meods 17 ed.). The tubes are first incubated for 3 hr at 35ºC and en transferred to a 44.5ºC water ba for an additional 21 hr of incubation. Tubes showing grow plus gas are considered positive for fecal coliforms. For e two-step Multiple Tube Fermentation Technique for fecal coliforms, positive tubes from e Presumptive (total) coliform test are inoculated into fermentation tubes of EC medium and incubated at 44.5ºC for 24 hr. Tubes showing grow wi gas production are considered confirmed positives. In e Membrane Filter procedure, samples are filtered onto membranes as in e total coliform test, e membranes are placed onto plates of m-fc medium, sealed in water-tight plastic bags, and submerged in a 44.5ºC water ba incubator for 24 hr. Colonies wi a characteristic fecal coliform appearance are en counted and fecal coliform density is computed. Escherichia coli (E.coli) In recent years, ere has been increased interest in detecting E. coli exclusively as e lactose-fermenting coliform at invariably indicates fecal contamination. An early approach to is effort was e use of four biochemical tests to separate E. coli from oer lactose-fermenting Enterobacteriaceae: Indole test detects indole production from tryptophane. E. coli is positive (+); many oer coliforms are negative. Meyl Red test detects acid production in e medium; intended to distinguish between type of fermentation reaction (mixed acid vs. butylenes glycol). E. coli is (+) and some oer 3

coliforms are (-). Voges-Proskauer test detects acetoin, an intermediate in e butylene glycol paway. Acetoin is oxidized to diacetyl under alkaline conditions in e presence of air, and when reacted wi creatine, it forms a pink color. E. coli is (-) and some oer coliforms are (+). Citrate utilization as sole carbon source. E. coli is (-) and many oer coliforms are (+). Therefore, in is series of four tests, called e IMViC tests, E. coli is typically ++-- and Enterobacter aerogenes is typically --++. Oer coliforms give different reaction patterns and are designated intermediates. However, e IMViC reactions have been found to be imperfect in speciating E. coli, and subsequently oer approaches to E. coli and coliform speciation have been developed. Two approaches are wor noting here. One is e use of rapid, commercial, biochemical test kits designed to carry out several biochemical tests simultaneously in incubation periods of 4 to 24 hours. The results of each test are scored as (+) or (-) and assigned a number based on e relative reliability of e test. These results constitute a code which is compared against a data base of accumulated reaction codes for most of e medically important Enterobacteriaceae. The code at is identical or closest to e organism tested is taken as its identity at e genus and species level. Anoer approach is e use of a single property or biochemical test in addition to lactose fermentation to definitively identify E. coli among oer coliforms. One such property is e presence of e enzyme beta-glucuronidase, which is found only in E. coli and some Salmonella and Shigella. A rapid test for is enzyme has been developed in which e beta-glucuronidase substrate MUG (4-meylumbelliferyl-beta-D-glucuronide) is incorporated into a coliform medium. If E. coli is present, e MUG is hydrolyzed to yield a fluorogenic product (4-meylumbelliferone) whose fluorescence can be readily seen by shining a long-waveleng UV light onto e culture. Membrane Filter Meods for Total and Fecal Coliforms and E. coli As noted above, E. coli and oer coliform bacteria can be analyzed in water and oer environmental samples by membrane filter (MF) as well as oer meods. The meods for total and fecal coliform analyses are well-known and are described in standard reference works (APHA, 1985; 1989). A membrane filter meod for E. coli was developed more recently (Dufour et al., 1981), and it is now standardized for routine use in recreational waters (U.S. EPA, 4

1985; APHA, 1989). A more recent development in MF meods for E. coli is e transfer of TC and FC membrane filters wi eir colonies to nutrient agar containing MUG. After 4 hours of incubation at 35ºC, e colonies are examined under long waveleng UV light, and ose colonies fluorescing blue are considered E. coli (Mates and Shaffer, 1989; Fed. Reg., 1991). TODAY S EXPERIMENT: PURPOSE: To estimate e total and fecal coliform and E. coli concentrations in samples of surface water and wastewater by Multiple-Tube Fermentation and Membrane Filtration Techniques. Work in pairs. MATERIALS (per group): Samples of water or wastewater for analysis. You will be assigned samples. Dilution bottles, each containing 99ml of peptone water (0.1%) diluent. Pipets: 1, 5, 10 and 20 ml volume, serological. Marking pen or grease pencil. Water ba: set at 44.5ºC for fecal coliforms and E. coli analysis. Air incubator: set at 35ºC for total coliform analysis. Multiple Fermentation Tube Meods Lauryl tryptose or lactose bro tubes containing inverted gas tubes (25 tubes per sample). Brilliant green lactose bile bro (BGLB) tubes containing inverted gas tubes (no more an 25 per sample). EC+MUG medium tubes containing inverted gas tubes (no more an 25 per sample). Long waveleng UV light, to shine on EC+MUG tubes when looking for blue fluorescence indicative of MUG hydrolysis (beto-glucuronidase activity). Wooden applicator sticks, sterile. Test tube racks, for fermentation tubes. Use Aseptic Technique for all operations; do not mou pipet. PROCEDURES Sample Dilution: Dilute e samples(s) serially 10-fold as directed by e instructor. Mix e sample by shaking 25 times before diluting. To make 10-fold dilutions, pipet 11 ml of sample into a 99 ml dilution blank. Seal e top of e dilution container and mix vigorously 25 times. 5

NOTE: Before making dilutions, adjust e diluent volumes in dilution bottles to 99ml as directed by e instructor. PROCEDURES: MULTIPLE FERMENTATION TUBE METHODS Two-Step-Tube Fermentation Technique for Total and Fecal Coliforms and E. coli (5 tubes per dilution): Day 0: Inoculation of tubes for presumptive test: Place 25 fermentation tubes in a test tube rack as groups of 5 dilutions, and mark e tubes as to eir dilutions. Using a 5 or 10 ml pipet, inoculate 1 ml volumes of each sample dilution to be tested into e 5 replicate tubes. Incubate e tubes in a 35ºC air incubator. After 24 and 48 hr: Reading of presumptive tubes and inoculation of tubes for Confimed Total and Fecal Coliform Tests and for E. coli: Gently shake e rack of tubes back and for several times to release gas in positive tubes. Examine all tubes for e presence of grow (turbidity or cloudiness) and gas (look in e small inverted tube), and score tubes showing bo as presumptive positive. Submit all lactose bro fermentation tubes at are Presumptive positive at about 24 hr and all additional Presumptive positive tubes at 48 ±3 hr to e confirmed tests. Insert a sterile wood applicator into e bro of e positive tubes to a dep of > 1 inch to wet e end. Transfer e organisms on e wetted end of e applicator to a fermentation tube of brilliant green lactose bile (BGLB) bro by briefly immersing e wet end of e applicator into e BGLB bro. Use e same applicator and same procedure to transfer material from e same Presumptive positive tube to a fermentation tube of EC medium containing MUG. Discard e applicator. For each positive Presumptive tube, use a fresh applicator stick. Incubate e BGLB bro tubes in a 35ºC incubator and e EC tubes in a 44.5ºC water ba. Reading Confirmed Tubes Examine e EC tubes at 24 ±2 hr for grow plus gas. Tubes showing bo are scored as Confirmed positive for fecal coliforms. Examine e positive EC tubes under a long waveleng UV light. Tubes showing bluish fluorescence are scored as E. coli. Examine e BGLB tubes at 48 ±3 hr for grow plus gas. Tubes showing bo are scored as Confirmed positive for total coliforms. 6

Calculations of Most Probable Number Calculate e total and fecal coliforms and E. coli densities of your sample(s) from e number of positive and negative tubes of ree sample dilutions according to e procedures described in Standard Meods and below. Bring ese results to e next scheduled laboratory period. MATERIALS: MEMBRANE FILTER METHODS Water or wastewater sample assigned by instructor. Membrane filter apparatus consists of membrane filter holder, vacuum flask and vacuum manifold assembly. Membrane filter mixed cellulose esters, 47 mm diameter, 0.45 ìm pore size, gridded ( up side has a grid pattern). Flat blade forceps for holding petri dishes of media. 50-60 mm diameter petri dishes of media: mendo agar for total coliforms mfc agar for fecal coliforms Nutrient agar containing 100 ìg/ml MUG for E.coli. Washing bottles containing sterile peptone water; to rise interior of filter funnel after filtering samples. PROCEDURES: MEMBRANE FILTER METHOD Starting wi e highest sample dilution to be tested, shake e sample 25 times, remove e bottle cap and pipet exactly 20 ml into e funnel. Then turn on e vacuum to filter e 20 ml rough e membrane. For each sample dilution, filter a 20 ml volume per medium (2 filtrations). Rinse e interior walls of e funnel by filtering about 20 ml of dilution water dispensed from a squeeze bottle. After filtration, turn off e vacuum. Each filter holder section of e vacuum manifold has its own shut-off valve. Carefully remove e funnel from e base. Use a sterile forceps to remove e filter, and apply it, grid side up, to e surface of a 50 or 60 mm diameter petri dish containing eier m-endo (total coliform) or m-fc (fecal coliform) agar. The filter is applied to e agar by rolling it onto e surface. Avoid trapping air bubbles under e filter. Apply new filters to e filter base and repeat e sample filtration procedure so at individual plates are prepared for total and fecal coliform/e.coli tests. 7

Repeat is membrane filtration procedure for e next ree lowest sample dilutions (1:1,000, 1:100, and 1:10; check wi instructor for raw sewage dilutions to be filtered). Incubate all plates inverted in a 35ºC air incubator. After 2 hours of incubation remove fecal coliform (mfc) plates from e 35ºC incubator, and place em in a 44.5ºC incubatorwater ba. Reading Results (after 22 ±2 hours of incubation, total). Remove e plates from e air incubator. Count e total and fecal coliform colonies. You may have to carefully remove e lids from e plates to clearly see e colonies on e filter surface. If possible, count plates having 20-80 colonies. However, count all plates, even if ey have fewer an 20 colonies per plate. Plates wi >100 colonies are considered too numerous to count. Total colifom colonies are pink to dark red and some (but not all) have a greenish gold metallic sheen. Fecal coliform colonies are blue. Detection of E. coli on Fecal Coliform Membrane Filters (mfc medium) (Procedure of Mates and Shaffer) Transfer membranes having countable colonies on mfc to nutrient agar-mug plates. Incubate 4 hr at 35ºC. Then, observe e colonies under long waveleng UV light. Colonies having typical FC appearance and fluorescing blue are E. coli. Calculate e total and fecal coliform and E.coli densities as described in Standard Meods and in e next section of e lab handout. Bring your data to e next laboratory class. RESULTS AND DATA TABULATION AND ANALYSIS Mutltiple Fermentation Tube Meod Compute and record e Most Probable Number of total and fecal coliforms and E. coli per 100 ml using e data from e confirmed tubes (BGLB and EC/MUG). Also record e upper and lower 95% confidence limits for ese MPN values. The MPNs and confidence limits are found in e table for ree dilutions and five tubes per dilution in Standard Meods for e Examination of Water and Wastewater, 17 edition. Note at e MPN table in Standard Meods is set up for a dilution series of 10, 1, and.01 ml sample volumes. The sample volumes examined in your samples are smaller (1, 0.1, and 0.01 or even less), and erefore, e MPN values in e table have to be multiplied by factors of 10 (or more for some samples) to correct for e difference in sample volumes analyzed. Computing Bacteria Concentrations for Membrane Filter Results 8

Using e individual colony counts of each replicate filter at e most countable dilution(s), calculate e total and fecal coliform and E. coli concentrations of each replicate sample per 100 ml. To do ese calculations, you must first determine which sample dilution is most countable. For total coliforms and E. coli, e desired counting range is 20 to 80 colonies per filter; for fecal coliforms it is 20 to 60 colonies per filter. If e number of colonies per filter is above e countable range at all dilutions, estimate e number of colonies by counts form plates having >80 (or >60) but <200 colonies per filter. Compute e estimated concentration per 100 ml and report as a greater an (>) value. If e membrane filter counts of 11 dilutions are below 20 per filter, sum e colony counts for all filters and e sample volumes filtered for all filters and en compute e number of colonies per 100 ml; report as an approximate amount. Estimating 95% confidence intervals Using e raw data for all countable filters (all filter counts used in e calculation of concentration per 100 ml) for each organism, compute e 95% confidence interval from e relationship between e variance and e mean of e Poisson distribution: 2 2 s = S, where s = e variance and x = e mean count (or total count). Then compute s, e square root of e mean: s = ±(x) 1 / 2 We will take e upper and lower 95% confidence limits as ±2s. After computing ±2s values, apply e appropriate dilution factors to obtain e upper and lower 95% confidence limits per 100 ml, as done for e mean value counts. QUESTIONS How well do e results for e multiple fermentation tube (Most Probable Number) and membrane filtration meods compare? Are e estimated bacterial concentrations equivalent? Specifically, do e MPN values and MF values fall wiin e calculated 95% confidence limits of e oer meod? For each sample, compare e MPN and MF concentrations of total and fecal coliforms and E. coli. Which indicator concentration is e highest? Which is lowest? Are ese results consistent wi what you would expect, based upon e definition of each indicator and e expected relationships among em? Recall at e four samples analyzed were: raw sewage, treated sewage effluent, Morgan Creek (e receiving water) upstream of e sewage effluent discharge, and Morgan Creek downstream of e sewage effluent discharge. Compare e levels of e ree indicators in e four samples. Which sample had e highest levels of indicators? Which e lowest? How effective was e sewage treatment plant in reducing e concentrations of indicator bacteria in raw sewage? (Sewage treatment at is plant consists of (i) primary settling, (ii) secondary (biological) treatment and (iii) chlorine disinfection of e secondary-treated effluent.) Considering at e raw sewage was treated and en discharged to Morgan Creek, what is e impact of e sewage effluent discharge on e concentrations of indicator bacteria in Morgan Creek? 9

REFERENCES American Public Heal Asociation (1985) and (1989), Sections 908 and 909, Standard Meods for e Examination of Water and Wastewater, 16 and 17 editions, American Public Heal Association, Washington, D.C. Feng, P.C.S. and P.A. Hartman (1982), Fluorigenic assays for immediate confirmation of Escherichia coli. Appl. Environ. Microbiol., 43:1320-1329. Mates, A. and M. Shaffer (1989), Membrane filtration differentiation of E. coli from coliforms in e examination of water. J. Appl. Bacteriol., 67:343-346. U.S. Environmental Protection Agency (1978), Microbiological Meods for Monitoring e Environment. Water and Wastes. EPA-600/8-78-017, pp. 108-114 and pp. 225-230, U.S. EPA, Cincinnati, Ohio. Federal Register. Vol. 56, No. 5, pp. 636-643, Jan 8, 1991. Colonies of fecal coliform bacteria filtered from water samples and grown on mfc nutrient agar are indicators of fecal contamination of e water.(www.ars.usda.gov) 10