8: Plaque Assay and Biochemical Tests (Day 1)

Complete the Following Lab with Your Partner:

1. Obtain 14 microcentrifuge tubes and label 7 tubes 1-7 and 7 tubes E. coli 1-7.

2. Obtain 7 nutrient agar plates. Label them 1-7 and place them in the 37°C incubator to warm up until needed.

Dilution of Enterobacteria phage T4

3. Aseptically add 990 μL of sterile saline to tube #1.

4. Aseptically add 900 μL of sterile saline to tubes #2-7.

5. Finger vortex the E. coli broth. Then, aseptically add 300 μL to all seven E. coli microcentrifuge tubes.

6. Vortex the T4 bacteriophage suspension and then aseptically transfer 10 μL to tube #1. Close the lid and vortex. This is your 10-2 dilution.

7. Aseptically transfer 100 μL from tube #1 to tube #2, close the lid and vortex. This is your 10-3 dilution.

8. Aseptically transfer 100 μL from tube #2 to tube #3, close the lid and vortex. This is your 10-4 dilution.

9. Aseptically transfer 100 μL from tube #3 to tube #4, close the lid and vortex. This is your 10-5 dilution.

10. Aseptically transfer 100 μL from tube #4 to tube #5, close the lid and vortex. This is your 10-6 dilution.

11. Aseptically transfer 100 μL from tube #5 to tube #6, close the lid and vortex. This is your 10-7 dilution.

12. Aseptically transfer 100 μL from tube #6 to tube #7, close the lid and vortex. This is your 10-8 dilution.

Addition of diluted phage to E. coli

13. Aseptically transfer 100 μL of SALINE into tube E. coli #1, close the lid and vortex. This will become your negative control.

14. Aseptically transfer 100 μL of dilution tube #2 to E. coli #2, close the lid and vortex.

15. Aseptically transfer 100 μL of dilution tube #3 to E. coli #3, close the lid and vortex.

16. Aseptically transfer 100 μL of dilution tube #4 to E. coli #4, close the lid and vortex.

17. Aseptically transfer 100 μL of dilution tube #5 to E. coli #5, close the lid and vortex.

18. Aseptically transfer 100 μL of dilution tube #6 to E. coli #6, close the lid and vortex.

19. Aseptically transfer 100 μL of dilution tube #7 to E. coli #7, close the lid and vortex.

20. Let all seven tubes stand for 15 minutes. We are allowing time for the virus to infect the bacteria.

21. Remove one soft agar tube (containing 2.5 ml of agar) from the hot water bath and add the entire contents of E. coli #1 to it. Gently mix. 

22. Immediately pour the mixture onto plate A. Then, gently tilt the plate back and forth until the soft agar mixture is spread evenly across the solid medium.

23. Allow the agar to solidify completely with the lid on at your table (15-20 minutes). Then, invert and place in the 37°C incubator to grow overnight.

24. Repeat 21-23 for the remaining dilutions of bacteriophage. 

 Microbes are EVERYWHERE...so how can we isolate only the specimen we want?

I'm sure your first thought was, "Duh, we dilute the specimen by streak- or spread-plate techniques." BUT, what if the specimen you are looking for is only a small fraction of the total microbes in your sample? If we perform dilution techniques, we may lose sight of our desired microbe completely. Special media techniques take advantage of microbial nutritional and environmental requirements to enrich  your specimen of interest. Additionally,  special medias can be used to select or differentiate various microbes, thus allowing for identification. 

General Purpose Media

This is the media we most commonly use in lab. General purpose media will typically support the growth of a wide diversity of microorganisms. Examples of general purpose media include nutrient agar and tryptic soy agar (see table below). While this media is not special, it's important you understand how special media differs from general purpose media.

As you can see, both types of general purpose media contain similar components. Agar for solidification, various digests or extracts for nutrients, and sodium chloride (salt) to maintain appropriate tonicity. 

Enrichment Media

Enrichment media is used to culture fastidious microbes that grow slowly or are found in low abundance. The enrichment culture contains specific and/or essential nutrients which allow for increased growth of the microbe of interest. For example, some microbes may require blood or lysed red blood cells, while others require unique growth factors or vitamins. It is important to note that enrichment media DOES NOT inhibit growth of other microbes; it merely promotes growth of a specific microorganism. As a result, enrichment cultures do not always result in a pure culture. Instead, you may observe multiple colony types with your microbe of interest being more abundant. Common examples of enrichment media include sheep blood agar and chocolate agar. 

Selective Media

Selective media contains ingredients that prevent the growth of unwanted microbes without inhibiting the growth of the desired microorganism. Most commonly, selective media takes advantage of the microbes unique characteristics. For example, say your microbe of interest can grow at a low pH (acid). Most microbes prefer a neutral pH and will not grow on acidic media. Consequently, by using acidic media we select for our microbe of interest by inhibiting the growth of unwanted microbes.  An example of selective media is Mannitol Salt Agar (which is also differential). We will explore this media further in the next module and in today's lab assignment. Note that physical conditions can also be used for selection, including temperature. 

Mannitol Salt Agar

Differential Media 

Differential media contains ingredients that allow you to distinguish between organisms growing on the sample plate. The basis for differentiation is usually a biochemical process that reacts with specific indicators in the media, resulting in a color change. It's important to note that differential media alone does not select for any one organism, but instead allows for identification of different microbes. One example we just learned about was blood agar. In lecture we discussed different types of hemolysis or lysis of red blood cells. The resulting lysis (or absence of lysis) provides a visual indicator, allowing for differentiation of different microbes. Let's revisit blood agar:

Right away you should notice that there are three different microbes growing on this plate. We know this because we see three different types of hemolysis: beta hemolysis, alpha hemolysis, and gamma hemolysis.   Recall that  beta hemolysis is complete lysis (yellow appearance), alpha hemolysis is partial lysis (yellow-brown appearance), and gamma hemolysis is the absence of lysis (no color change). 

Summary

Cultivation of Media for Bacteria, by Cindy Arvidson

To further explore different types of special media, we will be exploring various  types of special media using resources created by Cindy Arvidson. This resource includes everything we just went over, as well as numerous examples of special media and how different microbes grow on special media. Please familiarize yourself with the composition and use of the special media listed in the provided resource. Additionally, familiarize yourself with how different microbes grow on special media by clicking the "Examples" link under each type of special media. You can even click on the individual example pictures to further observe colony morphology.

Cultivation Media for Bacteria, by Cindy Arvidson

Procedure

• Obtain a liquid culture of a bacterial culture and Gram stain the specimen, then inoculate the following forms of media:
  • starch hydrolysis
  • H₂S test
  • oxidase test
  • mannitol salt agar
  • lactose Fermentation
• NOTE: The above links should serve as a guide for inoculation of media. You will only be inoculating one microbe onto each type of media. The oxidase test will be performed on Day 2 of Biochemical Testing, as will any additional steps required for visualization of differential media. 
  • Today you will inoculate Starch Agar, SIM Agar, Mannitol Salt Agar, and EMB Agar.