ENVR 421 - Environmental Health Microbiology - Spring, 2008
Total and Fecal Coliform and E. coli Analyses by Membrane Filter Methods
INTRODUCTION
As previously noted, E. coli and other coliform bacteria can be analyzed in water and other environmental samples by membrane filter (MF) as well as other methods. MF methods for total and fecal coliform analyses are well-known historically and are described in standard reference works (Section 9222, Standard Methods for the Analysis of Water and Wastewater). MF methods for E. coli were developed more recently (Mates and Shaffer, 1989) and have been standardized by the US EPA for routine use (Fed. Reg, 1991; US EPA, 2000; 2002; USEPA).
Initial E. coli MF methods required a confirmatory step in which membranes with presumptive E. coli colonies were transferred to a urea substrate to test for urease activity (E. coli is urease-negative). Newer developments in MF methods for E. coli are the transfer of TC and/or FC membrane filters with their colonies to nutrient agar containing the glucuronide substrate MUG (4-methylumbelliferyl -D-glucuronide). After 4 hours of incubation at 35oC, the colonies are examined under long wavelength UV light, and those colonies fluorescing blue are considered E. coli (Mates and Shaffer, 1989; Fed. Reg., 1991). More recently, glucuronide substrates for β-glucuronidase activity by E. coli, either fluorogenic ones like MUG or chromogenic ones, have been included in the primary culture medium. Hence, as bacteria grow and form colonies on the membrane, they produce the β-glucuronide hydrolysis product that either fluoresces under long wavelength UV light (as from MUG) or has a distinctive color (red or green, for example) (Grand and Baumgartner, 1996; Moini et al., 1996).
In this lab exercise several types of membrane filter methods will be used to detect total and fecal coliforms and E. coli in water and wastewater samples and the results from the different methods will be compared.
PURPOSE
To use the following membrane filter methods for analyses of total and fecal coliforms and E. coli in selected samples of water and wastewater.
Total coliforms on mEndo LES medium followed by blue fluorescence detection of E. coli on nutrient agar-MUG
Fecal coliforms on mFC medium followed by fluorescence detection of E. coli on nutrient agar- MUG
Total coliforms and E. coli on medium containing a TC chromogen and an E. coli fluorogen.
Total coliforms and E. coli on medium containing a TC chromogen and E. coli chromogen
MATERIALS
Water and wastewater samples - assigned by instructor
Membrane filter apparatus - consists of membrane filter holder, manifold assembly or vacuum flask and vacuum source
Membrane filters - mixed cellulose esters, 47 mm diameter, ~0.45 um pore size, "up" side has a grid pattern.
Flat blade forceps - for holding and transferring filters
50 and 60 mm diameter petri dishes of media:
mEndo LES for total coliforms
mFC for fecal coliforms
Nutrient agar containing 100 μg/ml MUG (to identify which fecal coliforms are E. coli based
on appearance of blue fluorescing colonies
TC and E. coli medium (TC chromogen and E. coli fluorogen) (Based on Manafi and
Kneifel, 1991.).
Bio-Rad TC and E. coli agar (TC and E. coli chromogens) (Based on Grand and
Baumgartner, 1996; Moini et al., 1996).
Diluent – Standard Methods buffer OR peptone water; in milk dilution bottles OR polypropylene round bottles, dispense 99 ml per bottle
Pipets - 10 ml
Washing (“squirt”) bottles - containing Standard Methods buffer OT peptone water; to rinse interior of filter funnel after filtering samples
Whirl-pac or zip-lock leak-proof plastic bags - for mFC plates
Water baths - set at 44.5oC
Air incubator - set at 35oC for mEndo, fluorogen-chromogen and chromogen-chromogen plates
Hand-held long wavelength UV light to detect fluorescence (from fluorogenic substrate hydrolysis product)
NOTE: Before making dilutions, make sure the diluent volumes in dilution bottles is 99.
Sample Dilution:
-mix the sample 25 times by shaking.
-then dilute the sample serially 10-fold as directed by the instructor. To make each dilution, pipet exactly 11 ml of sample (or previous dilution) into 99 ml of dilution water in a dilution bottle. Replace bottle cap and mix 25 times before making the next dilution.
Sample Filtration, Plating and Incubation
1. Set up a vacuum assembly and connect the lower section (base) of a filter unit to it. Be sure to handle the base using aseptic technique (avoid touching the top surface of the base on which the membranes are placed).
2. Use a sterile forceps to put (aseptically) a membrane filter, grid side up, on the surface of the filter base. Align and attach the upper, funnel section of the filter unit to the base.
3. Starting with the highest sample dilution to be tested, shake the sample 25 times, remove the bottle cap and pipet exactly 20 ml into the funnel. Then, turn on the vacuum to filter the 20 ml through the membrane. As soon as the sample has filtered, squirt sterile diluent water onto the inside of the filter funnel and allow it to filter through the membrane. Then, turn off the vacuum.
Aseptically remove the filter funnel from the filter base and set aside (turn upside down on a sterile surface).
4. Using a pair of flat-blade forceps, lift the membrane filter from the base by its outermost edge and place it grid side up onto the surface of a plate of mEndo LES. The filter is applied to the medium by "rolling" it onto the surface. Avoid trapping air bubbles under the filter.
Repeat the procedures 2, 3 and 4 above a total of three (3) more times, each with 20-ml volumes of the same sample dilution. Place each membrane on:
a plate of mFC medium for fecal coliforms
a plate of fluorogen-chromogen medium for TC and E. coli
a plate of chromogen-chromogen medium for TC and E. coli
Repeat these membrane filtration procedures for the next lowest sample dilutions in succession. Check with instructor for which dilutions of water, treated sewage effluent and raw sewage are to be filtered.
Incubate all plates inverted in a 35oC air incubator. After 2 hours of incubation remove fecal coliform (mFC) plates from the incubator, place them in watertight plastic bags and incubate submerged in a 44.5oC water bath.
Reading Results (after 22 +/- 2 hours of incubation, total).
Remove the plates from the water bath or the air incubator. Count the total coliform colonies on mEndo LES and the fecal coliform colonies on mFC. You may have to carefully remove the lids from the plates to clearly see the colonies on the filter surface. If possible, count plates having 20-80 TC colonies and 20-60 FC colonies. However, count all plates, even if they have fewer than 20 colonies per plates. Plates with >100 colonies are considered "too numerous to count"; do not count colonies in these plates.
Total coliform colonies on mEndo LES are pink to dark red and have a greenish-gold metallic sheen.
Fecal coliform colonies on mFC medium are any shade of blue
Bio-Rad TC/E. coli chromogenic medium: TC colonies are blue to blue-green (Gal+/Gluc-) and E. coli colonies are violet to pink colonies (Gal+/Gluc+), Figure 1 below.
Figure 1
Fluorogen-chromogen TC/E. coli medium:
TC: Blue-green colonies (due to the cleavage of the chromogenic substrate by b-galactosidase) under ordinary light but no blue fluorescence.
E. coli: Colonies fluoresce blue under long wavelength UV light due to the cleavage of the fluorogenic substrate by β-glucuronidase. See Figure 2 below.
Figure 2
Detection of E. coli on membrane filters for total (mEndo LES) and fecal coliforms (mFC) based on blue fluorescence of MUG hydrolysis
First count the total coliforms on m-Endo medium and the fecal coliforms colonies on mFC medium and mark them on the bottom of the plate using an indelible marker (“Sharpie”).
Then, transfer the membranes having countable colonies on m-Endo and mFC to nutrient agar-MUG plates.
Incubate 4 hours at 35oC. Then, observe the colonies under long wavelength UV light. Colonies having typical TC and FC appearance (at least initially) and fluorescing blue are E. coli.
Recording Data and Calculations
Record the numbers of TC, FC and E. coli colonies for each of the media tested above.
Calculate the total and fecal coliform and E. coli densities as described in Standard Methods and in the next section of the lab handout. Bring your data to the next laboratory class.
Computing Bacteria Concentrations
Using the individual colony counts of each replicate filter at the most countable dilution(s), calculate the total and fecal coliform and E. coli concentrations of each replicate sample per 100 ml. To do these calculations, you must first determine which sample dilution(s) is (are) most countable. For total coliforms and E. coli, the desired counting range is 20 to 80 colonies per
filter; for fecal coliforms it is 20 to 60 colonies per filter. If the number of colonies per filter are above the countable range at all dilutions at all dilutions tested, estimate the number of colonies by counts from plates having >80 (or >60) but <200 colonies per filter. Compute the estimated concentration per 100 ml and report as a "greater than (>)" value. If the membrane filter counts of all dilutions are below 20 per filter, sum the colony counts for all filters and the sample volumes filtered for all filters and then compute the number of colonies per 100 ml; report as an approximate count.
Estimating 95% confidence intervals. Using the RAW data for all countable filters (all filter counts used in the calculation of concentration per 100 ml), compute the 95% confidence interval from the relationship between the variance and the mean of the Poisson distribution:
S2 = X,
where S2 = the variance and X = the mean count (or total count in this lab)
Then compute S, the square root of the mean:
S = +/- (X1/2)
We will take the upper and lower 95% confidence limits as +/- 2S.
After computing +/- 2S values, apply the appropriate dilution factors to obtain the upper and lower 95% confidence limits per 100 ml, as done for the mean value of counts.
For the membrane filter results, answer the same questions posed for the results of the Multiple Fermentation Tube (MPN) analysis.
In addition, compare the counts for TC, FC and E. coli from the different media used. Are the counts from the different media approximately the same? That is, does the mean count from one medium fall within the 95% CI of the count from the other medium OR do the 95% CIs of the counts overlap?
REFERENCES AND BIBLIOGRAPHY
(Note: recent US EPA microbiology methods documents are available on-line at: http://www.epa.gov/nerlcwww/, the EPA Microbiology Homepage
American Public Health Association (2005) Sections 9222, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, D.C.
Cochran, W.G. (1950) Estimation of Bacterial Densities by Means of the “Most Probable Number”. Biometrics, 6(2):105-116.
Grand, M. and A. Baumgartner. (1996) Enumeration of Escherichia coli by means of selective chromogenic agars – Comparison with the official method. Trav. Chim. Alim. Hyg. 87: 623-630.
Moini, R., M. Grimaldi, P. Zorzi, A. Mani A. and P. Bordin (1996) Comparison between selective media « RAPID’E. coli » and « VRBA with sodium malonate » for E. coli investigation. Industrie alimentari. 35: 793 - 796.
Manafi, M. and Kneifel W., (1991), Fluorogenic and Chromogenic Substrates - A Promising Tool in Microbiology. Acta Microbiologica Hungarica 33(3-4):293-304.
Mates, A. and M. Shaffer (1989) Membrane filtration differentiation of E. coli from coliforms in the examination of water. J. Appl. Bacteriol., 67:343-346.
Oblinger, J.L. and J. A. Koburger. 1975. "Understanding and Teaching the Most Probable Number Technique." J. Milk Food Technol. 38(9): 540-545.
U.S. Environmental Protection Agency (1978) Microbiological Methods for
Monitoring the Environment. Water and Wastes. EPA-600/8-78-017, pp. 108-
114 and pp. 225-230, U.S. EPA, Cincinnati, Ohio.
US EPA (1985) Test Methods for Escherichia coli and Enterococci in Water by
the Membrane Filter Procedure, EPA-600/4-85/076, U.S. EPA, Cincinnati, OH.
Federal Register. Vol. 56, No. 5, pp. 636-643, Jan. 8, 1991.
US EPA (2000) Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli. Office of Science and Technology. EPA/821/R-97/004. Washington, DC.
US EPA (2002a) Method 1103.1: Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC). Office of Water. EPA 821-R-02-020, Washington, DC
US EPA (2002b) Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar(Modified mTEC). EPA 821-R-02-023. Office of Water, Washington, DC.
US EPA (2002c) Method 1604: Total Coliforms and Escherichia coli in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium). EPA 821-R-02-024. Office of Water Environmental Protection Agency Washington DC
U.S. Food & Drug Administration, Center for Food Safety & Applied Nutrition, Bacteriological Analytical Manual Online, September 2002. Chapter 4 Enumeration of Escherichia coli and the Coliform Bacteria http://www.cfsan.fda.gov/~ebam/bam-4.html
World Health Organization (WHO). Water Quality: Guidelines, Standards and Health. Lorna Fewtrell and Jamie Bartram, eds. Published by IWA Publishing, London, UK. ISBN: 1 900222 28 0. Ashbolt, N.J., W.O.K. Grabow and M. Snozzi (2001) Chapter 13. Indicators of Microbial Water Quality. (http://www.who.int/water_sanitation_health/dwq/iwachap13.pdf)
World Health Organization and the Organisation for Economic Co-operation and Development. (2003) Published by: IWA Publishing London. Assessing Microbial Safety of Drinking Water: Improving Approaches and Methods. A. Dufour, M. Snozzi, W. Koster, J. Bartram, E. Ronchi and L. Fewtrell, eds. Chapter 2. Introducing Parameters for the Assessment of Drinking Water Quality. P. Payment, M. Waite and A. Dufour (http://www.who.int/water_sanitation_health/dwq/en/9241546301_chap2.pdf).