Lab 3:
Total Coliform Bacteria



Introduction

In this lab, you will test for total coliform bacteria at a water treatment plant.  The presence of coliform bacteria in the water indicates that the water may have been contaminated with sewage and is not safe to drink.  Regulations require that coliform bacteria be completely absent from 95% of the water samples at a water treatment plant.  If a sample is found to contain coliform bacteria, then the operator must test for fecal coliform, which is a separate test not outlined in this lab.  If fecal coliform are found to be present in the water, then both the state and the public must be notified. 



Equipment

All of the bottles, pipets, and graduated cylinders used in this lab must be made of sterilizable glass or plastic.  They must not release any substances which are toxic to bacteria during sterilization.  None of the equipment can have broken tips or mouths. 

The specific glassware needed for this procedure includes:

  • Sample bottles. 
  • Dilution bottles. 
  • Pipets and graduated cylinders.
  • Containers for culture medium.  (Erlenmeyer flasks with metal caps, metal foil covers, or screw caps are preferred.)
  • Culture dishes.  (These can be glass or plastic petri dishes of any appopriate size.)
  • 1-L filtering flask with side tube. 

In addition, the following equipment is needed:

  • Filtration units.  (This should include a seamless funnel fastened to a base by a locking device or by magnetic force.  Funnels with deep scratches on the inner surface or with chipped surfaces should be discarded.)
  • Vacuum line, electric vacuum pump, filter pump operating on water pressure, or hand aspirator.
  • Membrane filters.  (These filters must have a pore diameter which will retain all coliform bacteria.  The filters must also be non-toxic to bacteria and must not influence the pH.  The membranes should be grid-marked in a manner which neither inhibits nor stimulates bacterial growth along the grid-marks.) 
  • Absorbent pads.  (These disks of filter paper should not be toxic to bacteria and should not influence the pH.  Absorbent pads are only required if liquid medium is used instead of agar.) 
  • Forceps.  (These should be smooth and flat without corrugations on the inner sides of the tips.)
  • Incubators.  (The incubators must provide a temperature of 35+O.5°C and a humidity of 60%.)
  • Microscope and light source.  (The microscope should have a magnification of 10 to 15 diameters.  Binocular wide-field dissecting microscopes are preferred.  The light should be a cool white fluorescent.)
  • Tape.



Reagents


Laboratory Procedure

1. Sterilize all equipment using either alcohol or an autoclave. 

For pieces of equipment which do not seal closed (such as pipets, graduated cylinders, petri dishes, and filtration units), the openings should be covered with metal foil before sterilization.  This will prevent sterilized glassware from becoming contaminated before use.

To sterilize by autoclaving, partially loosen all caps or stoppers on the glassware to prevent pressure buildup inside the containers.  Then the equipment should be autoclaved at 121°C for 15 minutes.  After sterilization, be sure that the pressure has returned to zero before the autoclave is opened since residual pressure can cause injuries and loss of sterilized lids.  
 
2. Prepare petri dishes.

Following the manufacturer's directions, or the procedure in Standard Methods, fill sterilized petri dishes with media. 

3. Choose an appropriate sample size.

An ideal sample size will result in 20 to 80 coliform colonies per dish.  The table below gives suggested sample sizes for a variety of water sources. 

Water source
Sample size (mL)
Drinking water
100
Swimming pools
100
Wells and springs
10, 50, or 100
Lakes and reservoirs
10, 50, or 100
Water supply intake
0.1, 1, or 10
Bathing beaches
0.1, 1, or 10
River water
0.001, 0.01, 0.1, or 1
Chlorinated sewage
0.001, 0.01, 0.1, or 1
Raw sewage
0.0001, 0.001, 0.01, or 0.1

Once an appropriate sample size has been chosen, carefully collect a representative water sample and record the sample size in the Data section.  You should collect enough water from each source to run three separate samples through the filtration apparatus.

4. Set up the filtration apparatus as shown below:
 
First, place the membrane filter in the bottom piece of the filtration unit, as shown above.  The grid side of the filter should be facing up.  Sterile forceps should be used whenever you handle the membrane filter to prevent contamination and damage to the filter. 

Setup of filtration apparatus

Next, place bottom of the filtration unit in the mouth of the filtering flask.  Then place the top of the filtration unit onto the bottom.  The stopper should seal the bottom of the filtration unit into the flask and the magnet in the filtration apparatus should seal the top and bottom together. 

Finally, attach a hose to the side arm of the filtering flask.  Attach the other end of the hose to the vacuum pump.  The completed setup is shown below:

.

Filtration apparatus

5. Filter the sample water. 

Pour your water sample into the top of the filtration unit and turn on the vacuum pump.  All of the water should pass through the filter and into the flask.

Rinse the interior surface of the funnel by filtering three 20 or 30 mL portions of sterile distilled water through the unit.  Once the water has passed through the filter, turn off the vacuum pump.

6. Place the membrane filter in a petri dish. 

Take off the top of the filtraton apparatus, exposing the membrane filter.  Then, using sterile forceps, remove the membrane filter. 

Petri dish

Place the membrane filter on the medium in a petri dish using a rolling motion to avoid entrapment of air.  The grid side of the membrane filter should be up. 

Pour a small amount of sample water into the petri dish on top of the membrane filter.  The sample water will prevent the bacteria on the filter from going into shock. 

Place the lid back onto the petri dish.  Seal the dish by placing two pieces of tape around the dish.  The tape should go from the top of the dish to the bottom of the dish, like the ribbon on a present.  Placing the tape around the edge of the dish will prevent air flow into the dish and will kill the bacteria. 

You should repeat steps 4 through 6 until you have filtered three samples from the same source. 

7. Incubate the petri dishes. 

Invert each dish and place the dishes inside an incubator at 35+O.5°C for 24 hours.  This allows the bacteria captured by the filter to grow and form a visible colony. 

8. Count the number of colonies found on each filter. 

After the incubation period has been completed, take each petri dish out of the incubator and remove the lid from the dish.  The surface of the medium should have growths of both coliform and other bacteria present. 

Coliform bacteria

Chemicals present in the media will normally reduce the number of non-coliform colonies present to a minimum.  In addition, colonies of coliform bacteria will have turned a pink or dark red color with a metallic surface sheen.  You should count only bacteria with this coloring and sheen to ensure that you do not count other types of bacteria.  (Some commercial media cause the coliform bacteria to turn other colors, so you should always read the instructions before counting coliform colonies.)

Set the dissecting microscope to a 10 to 15x magnification and use the microscope to help count the number of colonies found in the petri dish.  The figure below illustrates one method of counting colonies which should insure that all areas of the filter are observed: 

.

Colony counting technique

Once you have counted the number of colonies found on the filter, record the number in the Data section.  Filters which show a growth over the entire surface of the filter with no individually identifiable colonies should be recorded as "confluent growth."  Filters which show a very high number of colonies (greater than 200) should be recorded as "too numerous to count." 

If the number of colonies counted is greater than 80 or less than 20 per filter, then an incorrect sample size was chosen.  You should choose a larger or smaller sample size and repeat the above procedure.  (When sampling drinking quality water, you can disregard the lower limit of 20 colonies per sample.)

9. Calculate the coliform density of each filter using the following formula and record the results in the Data section. 

Formula used to calculate coliform density.

10. Calculate the average coliform density from all three samples.

When calculating the average coliform density, operators usually use a geometric mean rather than an arithemetic mean.  A geometric mean, unlike an arithmetic mean, tends to dampen the effect of very high or very low values which might be the result of an improper procedure.  A geometric mean can be calculated using either of the two methods outlined below.  These methods are also often used to calculate the average coliform density over time. 

 

The nth Root Method

The general formula for calculating the Geometric Mean using the nth root method is: 

 

Formula used for calculating the nth root geometric mean

Where:

X1, X2, Xn are coliform densities
n is the number of densities being averaged

.

This formula means that you multiply all of the data points together and then take the nth root of this product.  To take the nth root, you will need a scientific calculator with a "yx" or "y^x" key. 

Let's consider an example to show you how to make these calculations.  The table below shows the coliform density found in four separate samples:

.
Sample
Coliform density 
(colonies/100 mL) 
1
5 col/100 mL 
2
7 col/100 mL 
3
90 col/100 mL 
4
1,000 col/100 mL 
.

The following calculations are used to calculate the mean:

 

Calculations


In the second step of the calculations above, you would insert the following values on your calculator in order to take the 4th root:

 
3,150,000
y^x
(1/4)
=

 You should attempt this calculation on your own calculator to ensure that you understand the procedure. 


Logarithmic Averaging Method

The logarithmic averaging method is another method that can be used to calculate the geometric mean.  In order to use this method, you will need a scientific calculator with a "log" and a "10x" key.  Using the same data that we used for the nth root method, we will calculate the logarithmic mean:

The first step is to calculate the logarithm of each data point.  To do so, type the coliform density value into your calculator and push the "log" button.  The logarithm of each data point is shown in the table below:

Fecal Coliform
(colonies/100 mL)
Logarithm
0.69897 
0.84510 
90 
1.95424 
1,000 
3.00000 

Total

6.49831 

.

Next, we have added up the logarithm values.  As the table above shows, the sum of the logarithm values is 6.49831. 

Finally, we calculate the logarithmic mean as follows:

 

Calculating the logarithmic mean.


Notice that we first divided the sum of the logarithms by the number of data points.  Then we took the antilogarithm of this value.  To take the antilogarithm of a value, first type the value into your calculator, then hit the "10x" key. 



Data



Water Source
Sample size (mL)
Number of coliform colonies Coliforms/100 mL
Sample 1




Sample 2




Sample 3





Average coliform density: ___________________________________________


Sources

American Public Health Association, American Water Works Association, and Water Environment Federation.  1998.  Standard Methods for the Examination of Water and Wastewater.  American Public Health Association, Washington, D.C.