ENVE 301: Environmental Microbiology Laboratory Indicator Organisms and Enumeration of Coliforms
Water is a transparent, odorless, tasteless liquid. It is derived from five sources,&emdash;rains, rivers, surface-water or shallow wells, deep wells, and springs. Water is never found pure in nature; it is nearly pure when gathered in an open field, after a heavy rainfall, or from springs. For town and city supply, surface-water is furnished by some adjacent pond or lake. Samples of such water are carefully and frequently analyzed, to make sure that it is not polluted with disease germs. - Farmer, Fannie Merritt. The Boston Cooking-School Cook Book. Boston: Little, Brown, 1918.
Upon completion of this laboratory you should:
1. Be able to state the characteristics of an ideal indicator organism.
2. Explain why coliforms are considered to be indicator organisms for potability of drinking water.
3. Explain the limitations of coliforms as indicator organisms.
4. Distinguish between total coliforms, fecal coliforms and E. coli
5. Know how to perform coliform analysis using the MPN, MF and PA methods.
The contamination of water supplies with fecal discharges from sewage, farm animals and wild animals may transport a variety of pathogens such as bacteria (Shigella, Salmonella, Vibrio, Escherichia, Campylobacter , etc.), viruses, and protozoans into drinking water. The World Health Organization estimates that water borne diseases account for 10 million deaths annually, worldwide.
Detection of all potential water borne pathogens is difficult, time-consuming, and expensive. Therefore, most water quality surveys use various indicators of fecal contamination such as total coliforms and fecal coliforms to indicate the potential presence of pathogens. These indicator organisms are normally not considered pathogens themselves (although ingesting E. coli can make you sick). Rather, they represent a group of organisms which can be easily detected and enumerated and which serve as indicators of the presence of sewage. The ideal indicator organism would have the following characteristics:
- Always present when pathogens are present (No False Negatives)
- Never present when pathogens are absent (No False Positives)
- Easy to culture
Unfortunately, there is no ideal indicator organism as of the present. Coliforms may give false negative and false positive results and many alternative indicators have been proposed and are currently under evaluation. In spite of the limitations of the coliform group as indicator organisms, empirical evidence and years of experience in the drinking water industry have shown these organisms to be extremely useful in protecting the public health and well-being. Therefore, the use of coliforms as indicators of the sanitary quality of water is likely to remain for the future.
The term coliform is actually a broad term encompassing several related groups of organisms. These are total coliforms, thermotolerant (fecal) coliforms and Escherichia coli (E. coli).
The total coliform (often simply called coliforms) group of microorganisms is composed of microorganisms which meet the following criteria. They are:
- aerobic and facultatsively anaerobic
- rod shaped bacteria that
- ferment lactose with gas and acid production in 24 to 48 hrs at 35oC
In addition, with the recent development of commercially available media and test kits, two criteria have been added:
- cytochrome oxidase negative
- B-galactosidase positive
Thermotolerant (Fecal) coliforms
These organisms are a subset of the total coliform group. In addition to the criteria defining the coliforms, thermotolerant coliforms are able to grow and ferment lactose with the production of acid and gas at 44.5oC. The presence of these organisms in water has been shown to have a generally excellent correlation with fecal contamination although some members of this group (e.g. Klebsiella) can be isolated from environmental samples where no fecal pollution can be found.
The use of E. coli as a specific indicator of fecal pollution has been used by the European Union (EU) for some time and recently has been incorporated into U.S. drinking water regulations. E. coli meets all the criteria used for the definition of both total coliforms and thermotolerant coliforms. In addition, the organism can be distinguished from other fecal coliforms by the lack of urease and the presence of B-glucuronidase enzymes.
Methods for the Determination of Coliforms in Water Samples
The techniques used for the detection and enumeration of indicator organisms for regulatory purposes are specified by the US EPA and described in detail in Standard Math Methods for the Examination of Water and Wastewater. Currently there are three basic techniques accepted for coliform analysis - the multiple-tube-fermentation or most-probable-number (MPN) method. The membrane filtration (MF) method, and the presence-absence (PA)/defined substrate method. The choice of which method to use depends on such factors as cost, characteristics of the water sample, and applicable regulations. In this laboratory, you will analyze a drinking water sample for total coliforms and E. coli using the MF and PA methods. In addition, you will analyze a stream sample for fecal coliforms using the MPN method.
Most Probable Number (MPN)
The traditional metnod for detecting coliforms in drinking water is the multiple tube fermentation most probable number (MPN) technique. In this procedure, replicate subsamples of a serial dilution of the sample are inoculated into an appropriate media (Table 1). After incubation, the replicates are scored as positive or negative on the basis of growth or some metabolic characteristics.
The number of organisms in the initial sample is calculated using statistical methods based on the random dispersion of organisms in the original sample. To understand the MPN calculations it is essential that you remember thata bacterial cells are particles and thus the term concentration of bacteria in a sample is fundamentally different from the same term. When it is applied to disssolved chemicals.
Table 1: Analysis of Drinking Water, Surface Water and Treated Wastewater by the MPN technique.
Type of Analysis (organisms) Medium Incubation Positive Run
Lactose broth or lauryl tryptose broth
35oC; 24/48 hrs.
Gas and/or acid
44.5oC; 24/48 hours
Gas and/or acid
35oC; 3 hours then transfer to 44.5o for 21 hours
Consider a 100 ml sample which contains 1 bacterial cell. If I subsample 1 ml of this, I have a 1 in 100 chance of sampling in bacterial cell, but 99 out of 100 times I will get no cells. If I take 5 subsamples, the chances that I will get 1 cell increases to 5 out of 100 or 1 out of 20, and if I use 10 subsamples the likelihood of 1 cell increases to 1 in 10. For any given number of cells in the original sample and any given number of replicates, it is possible, therefore, to calculate the probability of getting at least 1 cell. The Most Probable Number procedure, therefore, is a statistiacal method of calculating the most likely number of cells in the initial sample which corresponds to a pattern of positive and negative results in a series of replicate analyses.
Membrane Filtration (MF) Method
The MF method is simple compared to the MPN procedure. It is based on the enterpment and concentration of bacterial cells by a membrane filter. The sample is filtered through a 0.45 um filter. Any particle with a size larger than the ole size will be retained on the surface of the filter. Given the size of a typical bacterial cell (>1um) the bacterial cells will be retained by the filter while viruses and dissolved substances will pass through. After filtration, the membrane filter is placed on the surface of the appropriate medium (Table 2) and incubated. Bacterial cells will replicate and form discrete colonies which can be counted.
Table 2: Analysis of Drinking Water, Surface Water,and Treated Wastewater using the MF Technique
Type of analysis Medium Incubation Positive run
LES Endo agar
35oC for 24 hours
Colonies w/metallic green sheen
44.5oC for 24 hours
pale to deep blue
Presence/Absence (PA) Methods.
Unlike the MPN or MF techniques which allow for the determination of the number of indicator organisms in a sample, PA methods simply test for the presence or absence of indicator organisms in a sample. Typically in these methods a 100 ml smaple is added to a sterile bottle containing a liquid medium which allows for the detection of lactose fermentation. Since federal regulations for drinking water have established a standard of < 1 total coliform/100 ml sample, any positive result indicates that the water does not meet the federal regulations.
It should be noted that the general procedures just described for the MPN, MF and PA tests reflect only the preliminary parts of the tests. In each of these cases, additional testing of positive smaples must be done to confirm that the organisms detected are really coliforms.
Recently, new characteristics have been added to the definition of coliforms. These definitions are based on the presence of certain characterist enzymes. Specifically the presence of b-galactosidase for total and fecal coliforms and b-glucuronidase for E. coli.
b-galactosidas catalyses the breakdown of lactose to galactose and glucose. It is an inducible enzyme (remeber the lac operon) found in coliforms. In addition to acting on its normal substrate (lactose) b-galactoside can also cleave [ortho-nitrophenyl-b-galactopyranoside] (ONPG). The reaction proceeds as follows:
ONPG ------------------> galactose + ortho - nitrophenyl (ONP)
(colorless) (to further metabolism) ( yellow)
The enzymatic cleavage of ONPG results in the generation of a yellow-colored substance (ONP). Thus, the use of ONPG as a substrate for b-galactosidase (instead of the normal lactose) allows for visual detection of b-galactosidase activity.
In addition to b-galactosidase, E. coli has the enzyme b-glucuronidase which is capable of hydroyzing the artificial substrate 4-methylumbelliferyl-b-D-glucuronide (MUG) in the follwoing reaction:
MUG -------------------------> glucuronide + methylumbelliferon (colorless) ( fluorescent blue/white)
There are several commercially available systems which use these two substrates (ONPG and MUG) to allow for the simultaneous detection of total coliforms and E. coli in water samples.
All of the techniques available for the enumeration of coliform organisms have limitations. In addition, the use of coliform organisms, and even of E. coli as an indicator organism is the subject of much debate and research. The review paper accompanying this lab exercise should provide you with references for further information on this topic.
ASM statement on drinking water monitoring
In this laboratory you will be enumerating coliform (total, fecal or E. coli ) using the MPN, membrane filtration, and defined substrate techniques.
Most Probable Number Technique (MPN): Fecal Coliforms
Supplies and Equipment Needed
- Water sample (stream water)
- 10 tubes concentrated LTB
- 10 tubes single-strength LTB
- 1 bottle (99 mL) sterile dilution water
- Waterbath set at 44.5o
- 15 tubes EC broth
- Strile wooden applicators
1. Using a sterile 10 mL pipette, transfer 10 mL of sample to each of 5 tubes of concentrated LTB. Label the tubes 10 mL.
2. Using a sterile 1 mL pipette, transfer 1 mL of sample to each of 5 tubes of single strength LTB. Label the tubes 1 Ll.
3. Using the same 1 mL pipette, transfer 1 mL of the sample to a dilution bottle containing 99 mL of EPC dilution water. This is Dilution A.
4. Using a new sterile 10 mL pipette, transfer 10 mL of Dilution A to each of 5 tybes of concentrated LTB. Label the tubes 0.1 mL.
5. Using a new sterile 1 mL pipette, transfer 1 mL of Dilution A into each of 5 tubes of single strength LTB. Label these tubes 0.01 Ll.
6. Place all of the tubes in the 35o incubator.
1. After the LTB tubes have incubated for 24 + 2 hours check the tubes for the presence of turbidity and gas.
- Positive tubes
Using a sterile wooden applicator (you will need one sterile applicator per positive tube) transfer growth from each positive tube to a tube of E.C. broth. Be sure to label the EC tubes to correspond to the LTB tubes from which the inculum was obtained.
Incubate the inoculated E.C. tubes in a water bath at 44.5 + 0.2oC for 24 + 2 hours.
- Negative tubes
Return to 35o incubator for an additional 24 hours incubation.
- E.C. tybes: Score as positive any EC tubes showing growth with gas production.
- LTB (Negative) tubes
- If now positive inoculate into EC, incubate and score.
- If still negative, record as negative.
- Record the results for the MPN technique in Table 3.
Table 3 MPN results
LTB 24 hours
LTB 48 hours
EC From 24 hours LTB
EC From 48 hours LTB
Calculation of MPN
1. Using the data reocrded in Table 3, enter the appropriate data into the MPN calculator - (http://members.ync.net/mcuriale/mpn/mpnscript3.htm). Note: remember that water has a density of 1 gram/mL
2. Record MPN per 100 mL
Membrane Filtration Technique
Supplies and Equipment Needed per student
- Water sample
- Sterile Filtration Apparatus
- Sterile 0.45µm filters
- Sterile dilution water
- Alcohol and flame
- Sterile petri dishes with absorbent pads
- Coliform media
coliforms form red/pink colonies with a metallic green sheen in the center
Coliforms form blue colonies
total coliforms and E. coli
Coliforms are red; E. coli blue
- Add the appropriate media to the absorbent pad in a sterile petri dish. Use the table above to determine the media to be used for the organisms you wish to enumerate.
- Attach the filter apparatus to the vacuum device. Be sure not to touch the inside of the filter apparatus
- Using alcohol flamed forceps, transfer a membrane filter to the platform base of the filter apparatus. Make sure not to tear or perforate the filter. Make sure not to transfer the colored separator paper with the membrane filter.
- Pour about 20 mL of sterile distilled water into the filter funnel
- Filter the appropriate volume of sample water by adding the sample to the funnel and turning on the vacuum.
- Drinking water: 100 mL
- Swimming pools: 100 mL
- Wells: 10-100 mL
- Lakes: 10-100 mL
- River water 0.001-1.0 mL
- Sewage: 0.0001-0.1 mL
- Turn off the vacuum and wash the walls of the filter apparatus with 20-40 mL of sterile dilution water. Apply the vacuum to filter.
- Using sterile forceps (alcohol flamed) transfer the filter to the saturated pad in the petri dish.
- Incubate the petri dishes at the appropriate temperature
- total coliforms: 24 hrs @ 35oC
- fecal coliforms: 24 hrs @ 44.5oC
- total and E. coli : 24 hrs @ 35oC
- After incubation count the number of colonies on the plate. You may have to use a dissecting microscope to aid in the counting.
- Calculate the number of indicator organisms per 100 mL in your sample.
Supplies and Equipment Needed per Student
1. Water Sample (Drinking Water)
2. P/A broth bottle.
1. Fill the P/A broth bottle to the line on the side (100 mL).
2. Incubate 24 +/- 2 hours at 35oC.
3. Check for color change
bluish/white under UV
4. Record your results as present or absent.
1. Summarize the results from the analysis of your samples in appropriate tabular form. For each sample/method include both the raw data and the final calculated concentration of indicator organisms.
2. Assuming that the samples were submitted by a client to your environmental laboratory write a brief explanation of the results and their significance appropriate for a layperson.