Title: 6.1 Lab: Preparing for the Experiment
Course: ATEP - Biotechnology Module B > Section: Section 6 Test and Evaluate

6.1.1 Preparing to Measure Growth

Growth will be measured by growing cultures on an agar plate and counting colonies. For example, Lactobacillus-MRS Agar (LMRS AGAR) is an enriched selective medium intended for the isolation and cultivation of Lactobacillus found in clinical specimens and dairy and food products. It can be used for the cultivating of Lactobacillus from clinical, dairy and food samples. The medium consists of peptones, yeast extract and glucose.

It will be necessary to prepare dilutions of your culture samples before plating them on agar. If the culture is too concentrated, cells will appear as a lawn of growth as seen in the 1:10 dilution plate in the graphic.

Diluting the culture will allow for individual cell counts like the 1:10,000 plate in the graphic.

Visit the website at the bottom of the page to learn more about this procedure is carried out.

Using the website below, develop an SOP for:

1. How the plates will be made
2. How dilutions will be made
3. How growth will be measured

Find out more about counting colonies by visiting this site (this will open a new browser window).

6.1.2 Liquefying Gelatin

Protease activity will be tested by liquefying gelatin. Remember in lesson 2.2, you learned that protease will break down and can liquefy proteins by cutting the bonds between the amino acids. Gelatin is a protein, so when mixed with a bioreactor sample which contains protease secreted from the microorganism, it will liquefy. The greater the protease activity, the faster the gelatin will dissolve.

The gelatin solution must be prepared before growth occurs to allow time for it solidify. Prepare the gelatin solution now. Once you are done, return to this lesson.

The recipe is below:

Gelatin Solution:

1. Add 7.1 g of unflavored instant gelatin to a 1L flask.
2. Add 500 ml of deionized or distilled water.
3. Heat and stir the solution on a hot plate until gelatin dissolves, farming a clear solution.
4. Add 3 drops of red food coloring to the solution.
5. Pour the gelatin solution into six petri dishes.
6. Store in the refrigerator to prevent mold growth.

Protease solution (must be done the day the assay will be performed):

1. Dissolve 1.0 g of unseasoned meat tenderizer into 30 ml of distilled water in a 50-ml beaker.
2. Make a fresh solution for each assay.

Have you prepared your gelatin and stored it in the refrigerator labeled with your group’s name?

6.1.3 Testing for Protease

To test for protease:

PROTEASE ASSAY 

1. Remove a plug of gelatin from the petri dish using a straw. And add to first test tube.
2. Repeat for second test tube. 
3. Add 1mL of bioreactor sample to the first tube. 
4. Add 1mL of meat tenderizer solution to the second tube. 
5. Record the length of time required to dissolve the gelatin plug. 
6. Put the used tube in the cylinder of bleach.

Protease solution (must be done the day the assay will be performed):

1. Dissolve 1.0 g of unseasoned meat tenderizer into 30ml of distilled water in a 50 ml beaker.
2. Take a fresh solution for each assay.

Assignment

6.1.4 Preparing to Monitor Contamination

Contamination may be seen on your agar plates. You can also look for contamination by viewing culture samples under a microscope. There is no media to prepare, but you should gather your materials and make sure you know how to properly use the light microscope in your lab. Ask you instructor if you need to review procedures.

Gather the following materials:

1. Microscope slides and cover slips
2. Methylene blue
3. Light Microscope

To view a sample using the microscope:

1. Put 1-2 drops of culture on the microscope slide. Cover with a cover slip.
2. Put the used syringe in the cylinder of bleach.
3. Adjust the compound light microscope to low power.
4. Put your slide on the microscope stage with the sample over the hole in the stage. Draw and label  what you see.
5. Adjust the compound microscope to high power and draw and label what you see.
6. Put the used slide in the cylinder of bleach.

6.1.5 Preparing to Collect Data

You should now have SOPs developed for testing that will be done on cultures grown in the bioreactor. The last step before turning the bioreactor on is to prepare for all of the data you will be collecting. For each of the SOPs you have generated in this lesson, create a table that illustrates/explains:

1. What question are you investigating?
2. Which variable are you testing?
3. How will this variable be tested/monitored?
4. What is your control?
5. How many trials should be done?

In most cases, the independent variable (that which you purposefully change) is in the left column, the dependent variable (that which you measure) with the different trials is in the next columns, and the derived or calculated column (often average) is on the far right.