Koch Postulates & Fungal Disease

Koch’s Postulates

 

Introduction: 

    In the late nineteenth century, German scientist Robert Koch established a set of procedures to isolate and identify the causative agent of a particular microbial disease. The following four steps, which are still used today, are known as Koch’s Postulates.

1. A specific organism must be always be observed in association with the disease.
2.  The organism must be isolated from an infected host and grown in pure culture in the laboratory.
3. When organisms from the pure culture are inoculated into a susceptible host organism, it must cause the disease.
4. The infectious organism must be re-isolated from the diseased organism and grown in pure culture.

Objective:

In this investigation, your group will demonstrate Koch’s Postulates by using oranges as the host organisms. The infectious agent will be Penicillium notatum, a mold. You will isolate the culture on petri dishes of Potato Dextrose Agar.

Materials:

Penicillium notatum mold,  3 oranges, incubator, 10% bleach solution, apron, gloves, paper towels, detergent, small scrub brush, wide-mouth jar, portable burner, dissecting needle, large Ziplock bags, permanent marker, petri dish, potato dextrose agar, sterile swab

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Procedure – Part A:     

Postulate 1.  A specific organism must be always be observed in association with the disease.

1. Disinfect the work area.

2. Obtain an orange and wash it thoroughly in cool, soapy water, scrubbing with a scrub brush. Rinse well.

3. Place the orange in a jar and cover with a 10% bleach solution. Let it stand for 10 minutes.

4. Rinse the orange for 10 minutes.

5. Flame a dissecting needle and allow it to cool. Then pierce the skin of the orange three or four times with the needle.

6. Flame the mouth of the tube of fungus and, using a sterile swab, aseptically remove a small sample and smear it over the puncture wounds in the orange.

7. Place the orange in a Ziploc bag. Label with your group number and date. The bag will be allowed to remain at room temperature or in an incubator at 25oC for about a week.

7. Prepare a data chart (Figure 1) to record daily observations. The chart should have places for the date, room temperature or incubator temperature, description of changes in the orange, and sketches.

8. Each day, record in a data chart your observations of the orange and the progress of the infection.

FIGURE 1:

Procedure – Part B  

Postulate 2. The organism must be isolated from an infected host and grown in pure culture in the laboratory.

During the week or so of incubation, you should see a white powdery spore mass on the orange that soon changes to a greenish color. When the green appears, it is time to isolate the pathogen.

1. Disinfect work area.

2. Obtain a petri dish of Potato Dextrose Agar. Label the bottom of the plate with your group number and the date.

3. With a sterile swab, aseptically transfer some of the spore mass to the plate of Potato Dextrose Agar. Streak across the plate in parallel lines.

4. Incubate the plates upside down at room temperature or in an incubator at 25oC for 5 – 7 days until the mold produces spores.

5. Make another data chart (Figure 2) to record observations of the growth on the petri dish.

6. Each day, record in a data chart your observations of the growth in the petri dish. (Do not remove the cover of the dish when making observations.)

FIGURE 2:

Procedure – Part C:  

Postulate 3. When organisms from the pure culture are inoculated into a susceptible host organism, it must cause the disease.

Once the culture in the petri dish has produced spores, you can inoculate susceptible organisms.

1. Disinfect work area.

2. Obtain two oranges and scrub them thoroughly in cool, soapy water. Rinse well.

3. Place the oranges in a jar and cover with a 10% bleach solution. Let stand for 10 minutes.

4. Rinse the oranges for 10 minutes.

5. Flame a dissecting needle and allow it to cool. Then pierce the skin of each orange three or four times with the needle.

6. Using a sterile swab, aseptically remove a small sample of mold spores from the petri dish. Smear it over the puncture wounds in one of the oranges.

7. Place the oranges in separate Ziploc bags. Label with your group number and date. Label the orange that is NOT inoculated, “CONTROL.” The bags will be allowed to remain at room temperature or in an incubator at 25oC for about a week.

8. Prepare a data chart (Figure 3) to record daily observations.

9. Each day, record in the data chart your observations of the oranges and the progress of the infection.

FIGURE 3:

Procedure – Part D:  

Postulate 4. The infectious organism must be re-isolated from the diseased organism and grown in pure culture.

When the spore mass appears on the inoculated orange, it is time to re-isolate the culture.

1. Disinfect work area.

2. Aseptically transfer a sample of the spores from the inoculated orange from Procedure 3 to a petri dish of Potato Dextrose Agar. Label the plate.

3. Incubate for the same length of time that you incubated in Procedure 2.

4. Make a data chart (Figure 4) to record your observations.

6. Each day, record in the data chart your observations of the growth in the petri dish.

FIGURE 4:

Analysis:

1. What is the importance of Koch’s Postulates?

2. Why have Koch’s Postulates remained unchanged for over a century?

3. Why were oranges and a mold used in this investigation?

4. Why were you instructed to scrub the oranges with a brush?

5. What was the reason you punctured the control orange?

6. What led you to the conclusion that the same organism caused the infection each time? Be sure that your data sheets support your answer.

7. Other than observations of appearance, what further investigations might have been done to prove that the organism that grew on the plates in Procedure 4 was the same one that you started with in Procedure 1?