Experiment 1: Bio-Prospecting for Starch Degrading Bacteria

Ethanol derived from plant material is currently used as an alternative fuel source for petroleum-based products. Plant biomass is approximately 50% cellulose, which is composed of repeating linked subunits of glucose. Glucose is the primary carbohydrate used by most bacteria as an energy source. When joined together, glucose can also form starch, a great energy store. However, starch can be difficult to break down, and only a few types of identified bacteria are capable of doing so.

However, starch requires a specific enzyme to be broken down that not all bacteria have .The enzyme responsible for degrading starch is called amylase. In this experiment, you will select for bacteria that are capable of digesting starch by using starch agar petri plates and Gram iodine. Starch agar is very similar to nutrient agar, but it has 0.4% soluble starch added to the media. This alone is not enough to indicate if a microorganism possesses amylase. However, when Gram iodine is added to the plate the iodine in the solution reacts with the starch in the agar and creates a blue coloration on the plate. Blue coloring indicates that the starch is still present, and was not degraded by amylase. Clear coloring indicates that the starch has been degraded, which can be interpreted to mean that amylase is present.

Procedure

Prepare Agar Plates:

1. Loosen or remove the cap on the nutrient agar bottle.
2. Place the bottle in the microwave (if you do not have a microwave, place the bottle in a heat-safe bowl and pour boiling water around the bottle) and heat until the entire agar bottle is liquefied. You will need to remove the bottle from the microwave and swirl it every 10 seconds to distribute the heat.
   Note: If you notice the agar boiling over, STOP the microwave and let the bottle cool down before handling. Hot agar can violently explode out of the bottle if heated too quickly and/or shaken. After boiling has stopped, use a hot pad to remove the bottle from the microwave. Remember, the bottle will be HOT!
3. Gently swirl the bottle to mix the solution.
4. Turn four of the petri dishes over and use the permanent marker to label them as “Nutrient”. Slowly pour the liquefied nutrient agar into the bottom half of four petri dishes so that it covers the entire bottom of the dish. It is important that the entire bottom is coated and that the agar is given time to spread out over the dish.
5. Place the lids onto the dishes and allow the agar to gel undisturbed. If you will not be using the dishes immediately, store them upside down in the refrigerator after they have fully gelled. Remove from the refrigerator and allow them to sit at room temperature for at least one hour prior to use.
6. Repeat Steps 1 - 5 using starch agar instead of nutrient agar, to prepare four more petri plates.

Prepare Stock Solution:

7. Place 10 mL PBS into one 15 mL conical tube; add the cow manure sample to it. Tightly screw on the cap and invert 10 times to mix thoroughly. Label this tube “Stock Solution”.
8. Place 9 mL of PBS into three 15 mL conical tubes (each tube should receive 9 mL).
9. Label one tube “10% Solution”, the second tube “1% Solution”, and the third tube “0.1% Solution”.
10. Perform serial dilutions of stock solution:
    1. Using a pipette, transfer 1 mL from the “Stock Solution” tube to the “10% Solution” tube. Tightly screw on the cap and invert 10 times to mix thoroughly.
    2. Using a pipette, transfer 1 mL from the “10% Solution” tube to the “1% Solution” tube. Tightly screw on the cap and invert 10 times to mix thoroughly.
    3. Using a pipette, transfer 1 mL from the “1% Solution” tube to the “0.1% Solution” tube. Tightly screw on the cap and invert 10 times to mix thoroughly.
11. Label one nutrient plate “Stock”, one nutrient plate “10%”, one nutrient plate “1%”, and one nutrient plate “0.1%”.
12. Using a clean pipette for each tube to transfer approximately 4 drops from each serial dilution tube to the corresponding nutrient plate. You may need to re-invert the tubes the ensure that the manure remains in the solution.
13. Spread the diluted manure solution evenly over the plates with a sterile spreader. Remember to use a new spreader for each plate!
14. Allow to air dry then place tops on petri dishes.
15. Seal the plated with Parafilm™ and incubate them in a warm location (not to exceed 37.7 °C or 100 °F) for 1 - 2 days (until well defined bacterial colonies appear).
16. Label the starch plates as Stock, 10%, 1%, and 0.1%.
17. Choose one colony from the “Stock” nutrient agar plate and use an inoculating loop to individually streak the colony in an X shape onto the center of the “Stock” starch plate. It may be useful to draw a circle on the bottom of the plate and streak an X onto this area. See Figure 4.
    

    Figure 4

18. Repeat Step 17 for the 10%, 1%, and 0.1% plates.
19. Seal the plates with Parafilm™ and incubate them in a warm location (not to exceed 37.7 °C or 100 °F) for 2 - 3 days or until well-defined, isolated bacterial colonies appear (this may take up to 1 week if incubated at room temperature).
20. Flood the nutrient agar plates with a 10% bleach solution, incubate for 20 minutes, and pour the bleach down the drain with running water. Wrap Parafilm™ around the plates and dispose of them in the trash.

Perform the Gram Iodine Staining:

21. Record the growth on the starch agar plates in Table 3.
22. Cover each plate with Gram iodine (this should take approximately 1 mL of fluid).
23. Immediately examine the plate for any cleared areas surrounding the bacteria. The iodine will not stain the bacterial growth itself, so look for a halo-like effect of “no-staining” near the edges of the growth. Cleared areas indicate starch hydrolysis.
24. When you are finished with the experiment, flood the starch plates with a 10% bleach solution, incubate for 20 minutes, and pour the bleach down the drain with running water. Wrap Parafilm™ around the plates and dispose of them in the trash.