Lab : Testing Water for Microbes

Introduction : Water testing is important for homes with wells. It is also important for environmental monitoring of streams, lakes and oceans where people are using the water. All natural water sources are likely to contain living microorganisms - something to keep in mind when hiking, fishing, swimming or any other instance of contact with water. Some microorganisms are harmless, other could cause serious medical problems.

Also, consider the water source when traveling outside of the United States.

A. Materials :

1. Students: Please provide a water sample from a pond, stream, lake, or pool.

2. Students: Please provide a water sample from your home water source. Be sure to label your containers.

3. Provided Lab materials : Other water samples, E.coli , Enterobacter aerogenes, Levine EMB agar, nutrient agar slants, lauryl tryptose broth, Gram-staining reagents, collection tubes, petri dishes, alcohol swabs

B. Procedures:

Prepare the Petri Dishes:

1. Melt the Levine EMB agar ( EMB = Eosin methyl blue)

2. Cool the agar to 50 ^oC

3. Pour the agar into a petri dish and cover it immediately, distributing the contents among five petri dishes.

4. Refrigerate until used.

Collect Water Sample :

1. Label the collection container. Samples should be tested soon after collection.

2. Fill the sample container and swab the top with an alcohol swab.

In LAB : Presumptive Test :

1. Shake the water sample well. Get one lauryl tryptose tube.

2. Invert the lauryl tryptose tubes and tap them lightly to eliminate any air from the vile.

3. Remove the caps from the lauryl tryptose broth tube and the water collection tube .

4. Use a sterile pipet; do not touch the tip to any surface; measure 1.0 mL of the water sample and transfer the water to the lauryl tryptose broth tube. Discard the pipet. Replace the cap to the tube.

5. Shake the tube gently.

6. ONE GROUP ,Make a control : Inoculate a new lauryl tryptose tube with a loop full of E.coli broth.

7. ONE Group, Make another control : Innoculate a second , new tube with a loop full of E.aerogenes broth.

8. Incubate the tubes at 37 ^oC for 24-48 hrs. At the end of 24 hrs , examine the tubes for the presence of gas in the inverted vial. The presence of gas indicates a positive test. Record results in a data table 1 on Classroom Google Doc. Save the positive tubes for the confirmed test. Begin the confirmed test on the same day that a positive presumptive test was observed.

9. Reincubate all negative tubes for another 24 hrs. At the end of 48 hours, reexamine any remaining tubes. Record the results in a data table. Save all positive tubes for the confirmed test. Discard any negative tubes.

Table 1: Presumptive Test :

In Lab : Confirmed Test :

1. Streak a petri dish of EMB agar with a loop of inuculum from a positive presumptive tube . Label the dish "Confirmed" and indicate the water source.

2. Repeat this for all positive tubes.

3. Incubate the plates for 24 hrs at 37^oC inverted.

4. After 24 hrs, examine the dishes for colonies . E.coli colonies will appear reddish purple with greenish metallic sheen.

E. aerogenes colonies are brownish pink, and tend to coalesce. Compare the growth of water samples to growth on the E.coli and E.aerogenes control plates. Record the results on Table 2.

Sample Results : Doc

5. If typical coliform colonies ( colonies described in step 4) are present on the EMB agar, the confirmed test is considered positive. The completed test is then performed on each isolated typical coliform colony as well as on all isolated atypical colonies that do not appear to be coliforms. Begin the complete test ASAP.

6. Discard all negative plates, those with no bacterial growth .

TABLE 2 : Confirmed Test :

In Lab : The Completed TEST

1. Inoculate a lauryl tryptose broth tube from each isolated colony by picking up a small amount of the colony with a loop and smearing it on the insides of the broth tube below the broth surface. Inoculate a nutrient agar slant from the same colony.

2. Label each broth tube "Coliform , Complete" or "Noncoliform , Complete" with the water source or species

3. Incubate at 37^oC for 24 hrs. Observe for gas formation. Record the results in Table 3. Discard all positive lauryl trytose tubes.

4. Reincubate any negative tubes. Record results.

5. Perform a Gram Stain on the slant cultures ( See Carolina Manual, BELOW) and examine the bacterial cells for endospores

,

Gram Negative

Gram Positive

Procedure for Gram Staining

1. Wear gloves...Place a rop of D.I. water on a clean slide.

2. Using a loop, remove a small amount of bacteria from the slant tube sample.

3. Mix the bacteria with the water on the slide, spreading it thinly.

4. Allow the smear to air-dry.

5. Heat the slide on an electric heater on low, smear side up This kills the bacteria and causes them to stick to the slide.

6. Allow the slide to cool.

7. Flood the slide with Hucker ammonium oxalate crystal violet for 60 seconds

8. Rinse with DI water.

9. Flood with Gram's iodine solution for 60 seconds.

10. Rinse with DI Water.

11. Decolorize with 95% ethanol. drip until almost clear run off.

12. Rinse with DI water.

13. Flood with safranin for 60 seconds.

14. Rinse with DI water and blot with a paper towel, be careful not to disturb the bacterial sample.

6. Based on the data , conclude if the original water samples contained coliforms. Record in Table 4.

Table 3 : Completed Test

Table 4 : Conclusions :

PART II: Chemical Testing of Water Samples

Directions: We will use a variety of tools available to test the water samples.