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Fecal Coliform

Bacteria are single-celled organisms that can only be seen with the aid of a very powerful microscope. Bacteria can be found everywhere- in air, water, soil, even in and on your own body. They can benefit us by recycling wastes, fixing nitrogen helping plants to grow, and by making certain types of food. They may harm us by causing diseases and food spoilage.

Bacteria reproduce rapidly if conditions are right for growth. Most bacteria grow best in dark, warm, moist environments with food. Some bacteria form colonies as they multiply which may grow large enough to be seen. By growing and counting colonies of fecal coliform bacteria from a sample of stream water, we can determine approximately how many bacteria were originally present.

There are many types of coliform bacteria. Coliform bacteria naturally occur in the human digestive tract and aid in the digestion or breakdown of food. Fecal coliform bacteria are found in the feces of humans and other warm-blooded animals. Fecal coliform bacteria can enter rivers through direct discharge of waste from mammals and birds, from agricultural and storm runoff, and from untreated human sewage.

Fecal coliform bacteria are indicator organisms. This means that fecal coliform does not cause disease in humans but it may indicate the presence of other pathogenic bacteria. Fecal coliform bacteria are a type of E. coli bacteria that is not pathogenic; they will NOT make you sick. However, some coliform are pathogenic. Water is tested for fecal coliform because it is a safe, inexpensive way to determine if other, harmful bacteria could be present. If fecal coliform counts are high (over 200 colonies/100 ml of water sample), it is very likely that pathogenic organisms are also present. Diseases and illnesses such as typhoid fever, hepatitis, gastroenteritis, dysentery, and ear infections may result from contact with water having such pathogenic organisms.

Fecal coliform like other bacteria can usually be killed by boiling water or by treating it with chlorine. Washing thoroughly with soap after contact with contaminated water can also help prevent infections. Gloves should always be worn when testing for fecal coliform to protect against infection by some of it's disease causing friends.

How to do a Fecal Coliform Test

There are several ways to test for fecal coliform. Some tests only indicate the presence or absence of the bacteria. Others indicate the relative amount of bacteria present in a sample. Two methods are described below for determining the relative amount of fecal bacteria present in a sample.

METHOD I: Using the Coliscan EasyGel (ReadyGel) Method

Collecting the Sample:

Use a sterile pipet to collect a 2 mL sample from below the water surface.

Using Redigel as a Pour Plate:

Remove the cap of a Redigel vial and add the 2 mL amount of sample. Swirl gently to distribute the sample.
Tilt the lid of a pretreated petri dish and pour the Redigel/sample mixture into the dish bottom. Replace the lid and gently swirl until the bottom is covered.
Carefully tape the lids to the bottom of the petri dish. Dishes should not be opened once prepared.
Allow 45 minutes for the gel to solidify. Invert and incubate as desired. It is best to incubate at a temperature range between 85-99 degrees F. They can be incubated at room temperature but will take longer to grow.

Coliscan pour plate key.
After 24 - 48 hours, count the number of purple colonies and colonies with a purple halo. Multiply the number by 50 to determine the approximate number of fecal coliform colonies in a 100 mL sample.
When finished, sterilize the plates before disposal by submerging them in a mixture containing one cup of bleach and three cups of water for twenty minutes.

NOTE - Use only the provided pretreated petri dishes. Plain petri dishes will not solidify.

CAUTION - The bottle medium and petri dishes are both sterile. Do not contaminate when opening and using the product.

WARNING - DO NOT OPEN PETRI DISHES TO COUNT THE COLONIES. WASH HANDS THOROUGHLY AFTER HANDLING THE SAMPLES.

METHOD II: Using Hach Millipore Filters

Collecting the Sample:

Remove the cap of a sterilized bottle just before sampling. Avoid touching the inside of the cap. Use gloves and hold the bottle near its bottom. Plunge it (opening downward) below the water surface, then turn the bottle underwater into the current and away from you. Do not sample the water surface or bottom sediments because they often contain greater numbers of fecal coliform bacteria. Allow some empty space in the bottle to allow mixing. Replace the cap.

Ideally, all samples should be tested within one hour of collection. If this is not possible, the sample should be placed on ice and tested within six hours.

Test Procedure: Read all directions and end notes carefully before beginning.

Label the bottom of a petri dish (side with the numbers) with the date, name of the group, test site location, and percent of dilution.
Use the blue plastic ampoule breaker to break the top off of a F-C broth ampoule. Pour the contents of the ampoule onto the white absorbent pad in the dish. Be careful not to touch or breathe on the inside of the petri dish or its lid. Throw the glass ampoule away.
Set up the sterilized filtration system.
Unscrew the top half of the filtration system. Carefully tear open a sterile filter membrane. With sterilized tweezers, carefully pull the white filter membrane from between the blue cover papers. Place the filter membrane, grid side up, on the center of the filtration system. Be careful not to touch the membrane or filter system. Be sure the filter membrane lies completely flat with no wrinkles.
Screw the top half of the filtration system to the bottom half making sure the filter membrane is centered. (Some filtering apparatus use magnets to hold the two halves together.)
Use a clean graduated cylinder to measure out 100 mL of sample water.
Unscrew and remove the lid on the filtration system. Pour the 100 mL water sample into the top of the filtration system and replace the lid.
Pump the sample through the membrane.
Carefully remove the lid from the petri dish that was set aside earlier.
Unscrew the top half of the filtration system. Use sterilized tweezers to remove the filter membrane and place it grid side up into the petri dish. Be sure not to touch anything.
Make sure that the membrane lies flat on the absorbent pad. Place the lid back on the petri dish.
Place the petri dish in a ziplock plastic bag or some other type of waterproof container.
Check to see that the temperature in the waterbath incubator is set at 44.5oC. Place the ziplock bag into the waterbath incubator and incubate for 24 hours.
Clean and put away the test equipment.
After 24 hours, remove the sample from the incubator. DO NOT TAKE the lid off of the petri dish. Use a 10x magnifying glass to count and record the number of blue dots that have appeared on the filter membrane. Each blue dot is one colony of fecal coliform bacteria. Cream or gray colored colonies are non-fecal coliform bacteria. Colonies should be counted within 20 minutes of removal from the incubator to be sure color changes do not occur.
Dispose the petri dish by placing it in a solution of one part bleach and three parts water for at least twenty minutes. Then the dishes may be safely disposed by placing them in plastic bag and putting it into the trash.

NOTE - a negative control sample using pure uncontaminated water should be run at first to be sure the system is clean. A positive control sample using water that has fecal coliform bacteria in it should be run last to prove that the system is working.

NOTE - If an unknown sample is being tested it is recommended that (in addition to the normal 100 mL sample) a 10% and 50% dilution sample be run. A sample should have between 20 and 80 colonies. If more than 80 or less than 20 colonies are present the accuracy of the results are questionable.

NOTE - Do NOT use deionized water for dilutions. It is best to use pure bottled spring water.

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