In order to determine the amount of E. coli bacteria in our water sample, we are actually going to help it grow! A common way of determining the amount of E. coli in water is to place a pre-determined amount of sample water on a special plate. The plate contains a nutritious food source that helps the bacteria grow into colonies (visible “clumps” of bacteria). It also contains special dyes, so that as the bacteria “eat” the food, they are also stained a bright color that helps us see and count the colonies. Other bacteria may grow on the plate, but the dye will only stain the type of bacteria the plate is designed for.
All types of bacteria have an ideal temperature at which they survive and reproduce. Some bacteria live at extreme temperatures, much too hot or too cold for any other type of life to survive! The ideal temperature for E. coli is much closer to our own body temperature. We will store the E. coli test close to its ideal temperature to give it the perfect environment to grow. It takes a couple of days for the E. coli bacteria to develop into visible colonies, so don’t forget to read your results a couple of days from now!
The plates we are using will test for both E.coli, and the larger Total coliform group. Remember from the introductory material that Total coliforms are found naturally in soil, and that E.coli is part of the larger fecal coliform group, which is part of the larger Total coliform group.
Standard 3, Benchmark 4
9-12 Benchmark 4: Learners identify and evaluate environmental issues from multiple points of view.
By the end of the twelfth grade, the students:
1. Water sample
2. 3M Petrifilm EC Count Plates
3. Sterile 1-milliliter (mL) pipettes
4. Distilled water
5. Incubator or warm place to store samples
1. If you have more than one water sample, use a separate plate for each sample and be sure to label them clearly with the sample site, the date and the time at which you process the sample. Also prepare one plate for your “blank” sample, which will be explained in the next steps.
2. Make sure you have a sterile 1 mL pipette for each water sample, including your blank. You should use a new pipette with each sample - this helps us make sure we’re only growing bacteria from one sample on each plate. When you remove the pipette from its sterile covering, open the plastic wrapper at the bulb end. Be sure not to touch the tip or any part of the pipette that will go into the sample.
3. Let’s begin with the blank. We run a blank sample with distilled water to help us make sure there aren’t any problems with our equipment or our methods. Distilled water has no bacteria, or any other contaminants associated with natural water. When we place distilled water on a plate and incubate it with the rest of the samples, it should have no bacterial growth after incubation. If bacteria grow, it tells us that our equipment may have had bacteria on it, or we need to be more careful with our methods.
For your blank sample, carefully fill your pipette to the 1 mL mark with distilled water. Lift up the cover sheet on the plate, being careful not to touch anything but the edge. Gently squeeze the water into the center of the pink circle. Then slowly roll the top cover back down onto the sample, which will help the water spread over the circle. Gently pick up and tilt the plate from side to side to distribute the water evenly. Discard the used pipette.
4. Repeat the same process with your water sample(s). If your sample has settled, you may gently shake or roll it to mix it up. Carefully fill your pipette to the 1 mL mark with your sample water. Lift up the cover sheet on the plate, being careful not to touch anything but the edge. Gently squeeze the water into the center of the pink circle. Then slowly roll the top cover back down onto the sample, which will help the water spread over the circle. Gently pick up and tilt the plate from side to side to distribute the water evenly. Discard the used pipette.
5. Repeat until you have all of your samples processed.
6. The ideal incubation temperature for this test is 35C (93F). At this temperature, test plates can be counted in 48 hours (two days). If you don’t have an incubator, you can place them in a warm spot – on top of your refrigerator for example. Be careful not to place them near a heating vent or any place where they might reach temperatures higher than 95F, and do not expose them to sunlight. You can place the samples in a sealed plastic bag to keep them from drying out. If you don’t have a place to incubate the plates at 93F, you can incubate them at 80F and read them in three days, or at 70F and read them in four days. Always note what you did on your field sheet – where you placed them to incubate, what temperature, and for how many days.
7. After the incubation time, you can count the plates in a well-lit area. The pink colonies are fecal coliform colonies and represent several different types of bacteria that are normal and natural in nature. Count the number of pink dots. Count them at least twice to get an accurate number. If the entire plate is covered with colonies, you may write “tmtc” (too many to count) on your data sheet.
8. Now count the blue dots or colonies. These are the E.coli colonies that could be harmful to human health if swallowed. You can follow the same procedure as above for counting.
9. Be sure to record all of your counts on your data sheet. The units for these numbers are colony forming units (cfu) per 1 mL. Most water quality standards use units of cfu/100 mL, so if you would like to compare to these standards, simply multiply your counts by 100.
Record your Total coliform and E.coli ratings on your field sheet according to the charts below:
Total Coliform Rating
When you consider your results, first determine the use of the water you tested. If it is water that will be used for drinking or household use, the only acceptable level for both total coliform and E.coli is 0 cfu/1 mL. This includes groundwater (well water) used for drinking. If bacteria, especially fecal coliform bacteria, are present in well water, it indicates the groundwater has been contaminated by surface runoff.
If you are testing surface water, some total coliforms are to be
expected, and simply indicate soil or sediment in the water. However, E.coli
should be 0 or close to 0. The State of
Kansas sets standards for surface water based on what the water is used
for. “Contact recreation” includes all
activities in which you might swallow some water, such as swimming. “Non-contact recreation” includes activities
in which you wouldn’t expect to swallow any water, such as boating or
fishing. You can determine from the E.coli rating chart whether your water
sample is currently safe for contact recreation, non-contact recreation, or
Store unopened Petrifilm plate pouches at temperatures below 46º F. Allow pouches to come to room temperature before opening. Return unused plates to the pouch. Prevent exposure to moisture after opening pouches. Store resealed pouches in a cool dry place for no longer than one month. Exposure of plates to temperatures above 77º F and/or humidities above 50 percent can affect performance of the plates. Do not use plates that show orange or brown discoloration. Expiration date and lot number are noted on each package of Petrifilm plates. After use, Petrifilm EC plates will contain viable bacteria. Put all used Petrifilm in a plastic bag, inside another plastic bag, and take to sanitary landfill or transfer station. Alternatively, mix a mild chlorine bleach solution (10 percent) and put one dropper full on each exposed plate to kill the bacteria before disposal.