Experiment 2: Characterizing Bacteria Isolated from Soil

Bacteria can be identified by differences in staining (e.g., Gram positive versus Gram negative) and by biochemical assays by virtue of different enzymes produced. In this experiment, you will characterize some of the bacteria you isolated from soil samples using two staining techniques and one biochemical assay for the enzyme catalase. Some species of bacteria produce catalase to inactive the toxic metabolic byproduct hydrogen peroxide according to the following reaction:

2 H₂O₂ → 2 H₂O + O2

The oxygen produced forms bubbles over the bacterial colony if the bacteria are catalase positive. The catalase assay is used along with other biochemical assays to characterize not only Bacillus and Clostridium in soil samples, but also to differentiate catalase positive Micrococcaceae from catalase negative Streptococcaceae.

Procedure

Experiment 2a - Staining:

Use the wax pencil to make three circles on four different microscope slides.

1. Use the wax pencil to label two slides as “Normal” and two slides as “Heated”.
2. Use a pipette to transfer one drop of sterile saline into each circle.
3. Select one of your Normal plates from Experiment 1; try to select a plate with well isolated colonies.
4. Use a clean inoculating loop to transfer a small amount of one of the bacterial colonies to a circle on the slide. Mix gently.
5. Repeat Step 5 for five more colonies (using the remaining 5 circles that you drew on the slides). Allow the samples to air dry.
6. Select one of your Heated plates from Experiment 1; this plate should have well isolated colonies.
7. Use a clean inoculating loop to transfer a small amount of one of the bacterial colonies to a circle with saline and mix gently.
8. Repeat Step 8 for 5 more colonies (using the remaining 5 circles that you drew on the slides). Be sure to use a clean inoculating loop for every transfer. Allow the samples to air dry.
9. Sterilize the forceps with your candle. To do this...
1. Pour isopropyl alcohol into a 100 mL beaker until you have a depth of 2-4 cm. Place the cap back on the bottle and position it clearly out of the way
2. Light your tea-candle and set it aside. Be very cautious that your tea-candle is safely positioned away from the beaker with the isopropyl alcohol.
3. Dip the forceps into the isopropyl alcohol for 10 seconds.
4. Without touching the forceps to anything, carefully pass the end of the forceps through the flame several times.
10. Pick up one of your bacterial slides with the forceps and flame it by passing the slide, smear side up, through the candle flame 4-5 times. Repeat this process until all of the slides have been flamed. Extinguish your flame when complete.

Gram Staining:

*Use one Normal and one Heated slide to perform the Gram staining procedure.*

12. Over a sink or disposable plastic container, saturate the sample with crystal violet and wait 1 minute.
13. Rinse the slide with deionized water for 30 seconds.
14. Saturate the sample with Gram iodine and wait 1 minute.
15. Rinse the slide with deionized water for 30 seconds.
16. Cover the sample with the decolorizer for 5 seconds. Do not let the decolorizer remain on the sample for more than 5 seconds as this will completely destain even a Gram positive organism.
17. Rinse the slide with deionized water for 30 seconds.
18. Saturate the sample with safranin and wait 1 minute.
19. Rinse the slide with deionized water for 30 seconds.
20. Use the bibulous paper to blot the excess water from the slide. Take caution not to disturb the sample. Repeat Steps 12 - 19 for the remaining slide

Endospore Staining:

*Use one Normal and one Heated slide to perform the Endospore staining procedure.*

21. Put a small piece of bibulous paper over the smear.
22. Saturate the paper with the malachite green solution.
23. Re-sterilize the forceps with your candle. To do this...
1. Re-light your tea-candle and set it aside. Be very cautious that your tea-candle is safely positioned away from the beaker with the isopropyl alcohol.
2. Dip the forceps into the isopropyl alcohol for 10 seconds.
3. Without touching the forceps to anything, carefully pass the end of the forceps through the flame several times.
24. Pick up a slide with the forceps and hold the slide above the candle for five minutes Use caution when using an open flame.
    Note: It is important to ensure that the malachite green does not dry out while the slide is heating. Add more malachite green solution to the bibulous paper if it is drying out. The slide should be steaming during the heating procedure. Add more malachite green if the slide stops steaming.
25. Remove the slide from over the flame. Remove the bibulous paper and dispose of it in the trash.
26. Repeat Steps 21-25 with the remaining slide.
27. Gently rinse the slides with deionized water. Carefully blot the excess water from the slide with bibulous paper.
28. Counterstain by placing a drop of safranin over each smear and incubating them for two minutes.
29. Rinse them gently with deionized water. Carefully blot the excess water from the slides with bibulous paper. Endospores will stain green while vegetative cells will stain pink/red.
30. Record any differences you noticed between the Gram stain and endospore stain procedures in Table 1. Be sure to acknowledge the differences which may have been required in the normal vs. the heated samples, and indicate why you believe these differences were needed.

Figure 3: Gram-positive Clostridium subterminale bacteria. This is an anaerobic, spore-forming bacteria.strong> Image courtesy of the CDC: Public Health Image Library.	Figure 4: The image above shows the bacteria Clostridium botulinum. Endospores are in green, while vegetative cells are stained red. Image courtesy of the CDC: Public Health Image Library.

Experiment 2b: Catalase Slide Assay

1. Use a wax pencil to label two new microscope slides as Normal and as Heated.
2. Use the wax pencil to make three circles on each of the slides.
3. Place each slide in the bottom of the large petri dish and place the petri dish on a dark surface, such as a piece of black construction paper or dark table/countertop.
4. Transfer individual bacterial colonies from the Normal plate to each of the circles on the slide labeled Normal. Repeat this process for the Heated plate and slide.
5. Place one drop of 3% H₂O₂ on the samples in each circle. Immediately place the covers on the petri dishes to limit bacterial aerosols. Observe the samples immediately for the formation of bubbles (effervescence). If both treatment conditions produce the same number of catalase positive samples, repeat with three more colonies from each plate.
6. When you are finished with your experiment, flood the plates with 10% bleach, incubate it for 20 minutes, pour the bleach down the sink with running water, and carefully dispose of the slides in the trash.