Koch’s Postulates

Koch’s Postulates

Introduction

Koch created four guidelines to determine the causal agents of disease in humans, animals, and plants. Koch proved that a disease-causing agent could be transferred from one organism to another and create the same illness. Isolation of pure cultures and the introduction of the disease-causing agent to a healthy organism will transmit the disease and infect the inoculated organism. Koch’s four guidelines by which one must follow to transmit a disease from an infected organism to a healthy one are as followed:

1. The specific organism should be shown to be present in all cases of animals suffering from a specific disease but should not be found in healthy animals.

2. The specific microorganism should be isolated from the diseased animal and grown in pure culture on artificial laboratory media.

3. This freshly isolated microorganism, when inoculated into a healthy laboratory animal, should cause the same disease seen in the original animal.

4. The microorganism should be reisolated in pure culture from the experimental infection.

In this exercise, Penicillium was utilized, a common, safe, mold. Certain species of Penicillium will spoil fruits, vegetables, grains, and grasses. Other species will ripen various chesses. Still, other species are used in the production of antibiotics. The species of Penicillium, italicum is provided for the lab because of its pronounced hyphae. Penicillium italicum, along with Penicillium digitatum attack citrus fruits post-harvest. In this experiment, the effect of Penicillium italicum on two types of citrus fruits and one non-citrus fruits were tested.

Materials & Methods

1. Pick several appropriate fruits.

2. Gently was fruit in cool, soapy water, using a scrub brush on the citrus fruits, then rinse thoroughly with cool running tap water.

3. Place citrus in a beaker and cover with a 10% bleach solution. Let soak for 10 minutes.

4. Rinse thoroughly with cool running tap water for 10 minutes.

5. Flame sterilize a teasing needle, cool, then pierce skin of disinfected fruit.

6. Unscrew cap on Penicilium italicum culture tube with one hand and flame the mouth of the tube.

7. Using the first three fingers of your writing hand, obtain a sterile applicator stick and remove a small sample of the fungus and smear over the puncture wound of the fruit.

8. Flame tube and recap. Discard swabs appropriately.

9. Obtain a second sample with a sterile applicator stick and smear over an unpunctured section of the fruit.

10. Flame tube and recap. Discard swabs appropriately.

11. Maintain a control for the experiment.

12. Make observations periodically (make note of the fungus’ odor, shrinkage, discoloration or growth characteristics).

13.Make a slide of your inoculating organism.

14. Disinfect all work surfaces.

15. Partially unseal bag to allow quick but aseptic entrance for sample collection.

16. With sterile applicator stick collect a sample of spores (blue-green, moldy decay) from the infected fruit.

17. Streak a Potato Dextrose Agar plate and incubate at room temperature for approximately 5 days.

18. Observe growth of culture periodically with lid intact.

19. Growth may appear as circular, a white fuzzy mass of mycelium or white specks that gradually change color, from white to blue-green. Observe with a steromicroscope.

20. Repeat cleaning and washing procedure as in steps 1-4 with appropriate number and type of fruits.

21. Reinoculate your fruits (hosts) as in steps 5-11.

22. Stain a sample of the organism that you have reisolated and compare with starting culture.

23. Observe and record periodically.

24. Repeat procedures as in steps 14-19.

25. Observe periodically.

26. At the