Module 10
Coliform Bacteria: What Do They Tell Us?

Figure 1. Colonies of coliform bacteria in a Coliscan Easygel® Petri dish.

 

Background and Overview

 

Coliform bacteria are a common group of bacteria that occur in soils, on plants, and inside animals. Among the general group of coliforms are the fecal coliforms, which live in the intestinal tracts of endothermic (“warm-blooded”) animals and are released into terrestrial and aquatic ecosystems whenever animals defecate. Although we expect to find fecal coliforms in our aquatic ecosystems, we expect them to be there in low levels. When they occur in higher levels, we get concerned because they could be accompanied by other intestinal microbes that can cause disease.

 

Last week you collected coliform bacteria samples and began incubating them in a Coliscan Easygel®. Since that time colonies of bacteria have grown in your Petri dishes. This week you will determine which of these colonies are general coliform bacteria, which are Escherichia coli (one particular species of coliform), and which are “other” kinds of bacteria. You can do this because different kinds of bacterial colonies will take on different colors in the Coliscan gels. Sugars in the gel are linked to a dye. When the sugar-linked dye is broken down by different enzymes made by the different kinds of bacteria, different colors are revealed.

 

After you make the counts of the different colonies, you will do some calculations to come up with the number of colonies present per volume of water sampled. After everyone’s data have been collated, you will write a report that presents and interprets the data, with the goal of answering the questions from Module 9:

  1. How many fecal coliform bacteria are present in the lake?
  2. What are the likely source(s) of the bacteria?

 

Objectives

After completing this work, you will be able to:

 

1.     “Read” a Coliscan Easygel® (i.e., distinguish and count different kinds of bacterial colonies), and

2.     Explain how the number of colonies in a Petri dish is related to the number of fecal coliforms per volume of lake water.

 

Part 1:  Determining the Amount of Coliform Bacteria Present in Samples

 

Procedures

Counting the Colonies

Š      Find your Petri dishes from last week (be sure you get your control dish).

 

Š      Look at the color photos and Petri dish examples of the different kinds of bacterial colonies.

 

The deep-blue to purple ones represent what?

 

The pinkish ones represent what?

 

The light-blue to blue-green ones represent what?

 

The whitish to colorless ones represent what?

 

Š      count all the deep-blue to purple colonies (do not count light blue, blue-green, white or colorless colonies) in each Petri dish and record counts as E. coli in Table 1.

 

Š      Next, count all pink colonies (do not count light blue, blue-green, white or colorless colonies), and record counts as “other coliforms” in Table 1.

 

Š      In Table 1, calculate the total coliforms in your samples.

 

  Table 1. Number of colonies counted in Coliscan Easygel® Petri dishes.

Sample identification E. coli (fecal coliforms) Other coliforms Total coliforms
       
       
       
       

 

 

Determining the relationship between counts of colonies (in Table 1) and bacteria per volume of sample water.

 

The unit of final measurement that you are trying to get to is bacteria per 100 ml of water (= no./100 ml).

 

Each colony represents the growth from one bacterium (at least that is the assumption). So, in Table 1 above, the number of colonies of “other coliforms” in your first sample represents how many bacteria that you put into the Coliscan® bottle? ______________________

 

That number of bacteria was contained in what volume of water that you added to the Coliscan® bottle? __________________   This is the volume of your inoculum.

 

Example

Just as an example, let’s say the amount of water you added to the Coliscan bottle was 5 ml (so your inoculum volume is 5 ml). How many inoculum volumes would there be in 100 ml (the volume of your final measurement)?  You could withdraw 5 ml from 100 ml twenty times before all of the water would be gone, right?  100 ml ÷ 5 ml per inoculum volume = 20 inoculum volumes

 

Now let’s assume your E. coli count was 4. That’s the count from one inoculum volume. But 20 inoculum volumes were possible for 100 ml of water. So, if every inoculum volume gave us 4 E. coli counts, we’d have 4 X 20 = 80 E. coli bacteria per 100 ml. So, in the category of E. coli our final measurement would be 80/100 ml.

 

Make Your Calculations

 

Step 1: Calculate the number of inoculum volumes in 100 ml for each of your samples

 

 

 

 

 

Step 2: Calculate the number of inoculum volumes in 1 ml for each of your samples

 

 

 

 

 

Step 3: Fill in Table 2

 

 


Table 2. Number of bacteria per volume of sample water.

Sample identification

E. coli/fecal coliform
(no/100 ml)

Total coliforms (no./ml)
     
     
     
     

 

 

 

Part 2: Writing a Lab Report on Fecal Coliform Studies

Purpose

 

In a nutshell, the purpose of this report is to: 1) tell the reader why you did what you did (= Introduction), 2) what you did (= Methods), 3) what you found (= Results), and 4) what the results mean (= Discussion). You will also need to provide citations for any written references that you used to help you make sense of your work (= Literature Cited).

 

What To Do

 

Š      Gather all the materials you have for this lab together: this module, module 9, your field/lab notes, any calculations that you did, and the guide for writing lab reports.

 

Introduction

Š      Carefully read about what is included in an Introduction.

Š      Get it all in there: background about coliforms, purpose of this study, hypotheses (you may have hypothesized about sources of bacteria), etc.

Š      For this report, you need to find out more about coliform bacteria, and, in particular, what levels of coliforms are considered unsafe in Minnesota. Go to the library; search on line—but find out about these critters and standards set by state and local governments. (Hint: here’s a place to get started: http://www.health.state.mn.us/divs/eh/beaches/howsafe.html).

 

Methods

Š      Be sure that a high school student from Buttermilk, Kansas, could read your methods, understand them, and repeat everything that you did. This includes field collection, laboratory processing, and data analysis.

Š      Remember to write in your own words. DON’T PLAGIARIZE. IT AIN’T WORTH IT!

 

Results

Š      Carefully read about what is included in a Results section.

Š      For this report, you are required to report the colony counts for all samples from your section, as well as the number of E. coli and total coliforms per volume of water.

Š      How you present the data is up to you, but it must all be there and there must be some text that introduces any tables or figures that you might have.

Š      Tables must be numbered and have titles.

Š      Figures must be numbered and have captions.

 

Discussion

Š      Carefully read about what is included in a Discussion section.

Š      Be sure for this report that you clearly indicate whether your results indicate water contamination problems or not.

Š      What do your samples tell you, if anything, about the sources of coliform bacteria?

Š      Is there any further speculation about sources that you would like to make?

Š      Overall, what conclusions to you draw and how confident are you about them?

Š      What are your sources of error? This would include measurement error (e.g., how you sampled, when, how you processed the samples, etc).

 

Literature Cited

Š      Use CBE citation style (see “Lab Report Guide: Literature Cited”).

Š      Here is where you list all the references you cited in the Introduction and Discussion. List in alphabetical order by first author’s last name.

Š      You must have at least two reference sources for this report. They can be print media or on line.

 

What To Turn In and When (35 Pts)

 

A printout of your full laboratory report containing Title page (1pt), Introduction (4pts), Methods (6pts), Results [all data as noted above] (10pts), Discussion (10pts), and Literature Cited (4pts):

 

Keep an electronic copy of your work AND HAVE A BACK-UP. If your work turns up missing and you have no back-up, you may be out of luck (and points).

**Submit your work to your laboratory instructor at the beginning of the class or laboratory period indicated by your instructor. Have your work printed out and ready to submit—there will be no opportunity to print after you arrive in the classroom or lab.