KOCH'S POSTULATES FOR PROOF OF PATHOGENICITY

Robert Koch (1843–1910) was a medical doctor and a bacteriologist. He was the first to
show, in 1876, that anthrax, a disease of sheep and other animals, including humans, was
caused by a bacterium that he called Bacillus anthracis. He subsequently discovered, in
1882, that tuberculosis and, in 1883, that cholera are each caused by a different
bacterium, which led to the general conclusion that each disease is caused by a specific
microbe. These experiments confirmed for the first time the germ theory of disease
proposed earlier by Louis Pasteur.
Before Koch’s experiments, and while Koch himself was carrying out the work on the
diseases mentioned earlier, there was confusion and uncertainty about the occurrence and
the cause of each disease. Much of the time when bacteria or fungi were isolated from
diseased or dead human, animal, or plant tissues, the isolated bacteria or fungi were
subsequently shown to be saprophytes, i.e., they coexisted with the microorganism that
caused the disease but could not by themselves cause the disease for which they were
being considered. Based on his experiences, in 1887, Koch set out the four steps or
criteria that must be satisfied before a microorganism isolated from a diseased human,
animal, or plant can be considered as the cause of the disease. These four steps, rules, or
criteria are known as “Koch’s postulates.”
1. The suspected causal agent (bacterium or other microorganism) must be present in
every diseased organism (e.g., a plant) examined.
2. The suspected causal agent (bacterium, etc.) must be isolated from the diseased host
organism (plant) and grown in pure culture.
3. When a pure culture of the suspected causal agent is inoculated into a healthy
susceptible host (plant), the host must reproduce the specific disease.
4. The same causal agent must be recovered again from the experimentally inoculated
and infected host, i.e., the recovered agent must have the same characteristics as the
organism in step 2.
Koch’s rules are possible to implement, although not always easy to carry out, with such
pathogens as fungi, bacteria, parasitic higher plants, nematodes, most viruses and viroids,
and the spiroplasmas. These organisms can be isolated and cultured, or can be purified,
and they can then be introduced into the plant to see if they cause the disease. With the
other pathogens, however, such as some viruses, phytoplasmas, fastidious phloem-
inhabiting bacteria, protozoa, and even some plant pathogenic fungi that are obligate
parasites of plants (such as the powdery mildew, downy mildew, and rust fungi), culture
or purification of the pathogen is not yet possible and the pathogen often cannot be
reintroduced into the plant to reproduce the disease. Thus, with these pathogens, Koch’s
rules cannot be carried out, and their acceptance as the actual pathogens of the diseases
with which they are associated is more or less tentative. In most cases, however, the
circumstantial evidence is overwhelming, and it is assumed that further improvement of
techniques of isolation, culture, and inoculation of pathogens will someday prove that
today’s assumptions are justified. However, in the absence of the proof demanded by
Koch’s rules and as a result of insufficient information, all plant diseases caused by
phytoplasmas (e.g., aster yellows) and fastidious vascular bacteria (e.g., Pierce’s disease
of grape) were for years thought to be caused by viruses.
Despite the difficulties of carrying out Koch’s postulates with some causal agents, they
have been and continue to be applied, sometimes with certain modifications, in all cases
of disease. They have had and continue to have a tremendous effect in deciding and in
convincing others that a particular microorganism is the cause of a specific disease. By
attempting to carry out Koch’s postulates in all newly discovered diseases, a great deal of
work with potential saprophytes has been avoided, while, at the same time, doubt and
criticism are reduced to a minimum while confidence in and use of the identification
increase greatly and quickly.

DIAGNOSIS OF DISEASE AND PROOF OF PATHOGENICITY

To demonstrate Koch’s postulates using fruit infected with the fungus

To determine which microbe caused that fuzzy stuff on your fruit you must:
1. Describe the symptoms you see on the infected fruit and isolate the suspected
fungus pathogen responsible.
2. Isolate the fungus in pure culture (this means grow the fungus on its own, away
from the host plant (fruit) and without any contaminating microorganisms).
3. Use the fungus that you isolated in pure culture to inoculate a healthy fruit.
 4. Record the symptoms that develop on the healthy fruit following infection with
the cultured fungus. Are your observations the same as recorded previously?
5. Re-isolate the fungus and check that it is the same as observed initially.

Materials needed
1. Fungus infected fruit
2. Slides and coverslips
3. Cotton blue stain
4. Stereo & compound microscope
5. Imersion oil
6. Sterile scalpels
7. Potato dextrose agar (PDA) plus antibiotic
8. Fresh fruits

Session 1:

You are provided with:

• A fruit infected with the fungus
• Three plates of Potato dextrose agar (PDA) supplemented with antibiotic (to
control bacteria);
• Microscopes, slides, coverslips and cotton blue stain
Method:
1. Carefully record the symptoms of the disease. Examine the fruit externally. What
do you see?
2. Slice through the infected fruit. Are there any colour and texture changes in the
infected fruit? Observe the symptoms using a microscope and compare them with
a healthy fruit (control).
3. Using a sterile scalpel blade, carefully remove a pustule (containing reproductive
structures of the fungus) from the infected fruit and place it on a microscope slide.
Place a drop of cotton blue stain onto the pustule and cover with a coverslip.
Lightly press down the cover slip and use a circular motion to squash the pustule.
 This is best done using the end of a pencil. Take care not to crack the coverslip.
Record your observations.
4. Using a sterile scalpel blade isolate small pieces of tissue from the edge of the
infected fruit and place one piece of tissue at the centre of each plate of PDA.
The plates will be incubated at 25oC and returned to you in session 2.

Session 2:

You are provided with:

• Isolation plates from session 1;
• Healthy fruits;
• Sterile scalpel;
• Microscopes, slides, cover slips, cotton blue stain.
Method:
1. Carefully examine the plate cultures of the micro-organism isolated from the
infected fruit.
2. Inoculate a fresh fruit with the micro-organism that you have isolated. Sterilize
the healthy fruit surface by wiping with 70 % ethanol, remove a piece of fungal
colony with a sterile scalpel and insert it into the healthy fruit.
3. If time permits, remove a small amount of the fungal culture (using a sterile
scalpel) and place on a microscope slide. Stain with cotton blue and observe under
the microscope following the same method as described previously. Look
particularly for the characteristic conidia (asexual spores) of fungus.
The fuits will be incubated at 25oC and returned in session 3.

Session 3:

You are provided with:

• The fruits which were inoculated in session two with the micro- organism isolated
on PDA plates from the rotted fruits supplied in session one;
• Sterile scalpel;
• Microscopes, slides, cover slips, cotton blue stain;
 • 3 plates of potato dextrose agar (PDA) supplemented with an antibiotic (to control
bacteria).
1. Compare the symptoms of disease in the inoculated fruits with those recorded in
session 1. Are they identical, or are there differences?
2. You must now re-isolate the micro-organism from the fruits inoculated in session
2, to show that it is identical to the organism originally isolated.
The plates will be incubated at 25oC and returned in session 4.

Session 4:

You are provided with:

• The re-isolation plates inoculated in session 3.
1. Carefully examine the cultures.
2. Use the skills that you have learnt in the previous sessions to examine the cultures
using microscopy.
3. Look for the characteristic spore bearing structures.

Assessment:

In the assessment, students address the following questions:

1. Decide whether you have proven Koch’s postulates for this disease.
2. What additional experiments would you wish to perform in order to further
substantiate your conclusions?

Safety Notes

• No eating, drinking or chewing in the laboratory

• Wear a lab coat or apron
• Keep lids on cultures when not in use
• Wear disposable gloves when using cotton blue stain.
• Wash hands before and after the practical work.

It is advisable to wear a face mask when dealing with sporulating fungal cultures.