Kochs Postulates and experimental evidence: A lesson in

correlation vs. causation

Emily Cribas
Hypothesis. A specific disease is caused by a specific microbe in plant cells.

Prediction. When a healthy plant cell is infected with a microbe from a diseased
plant, it will exhibit the same symptoms and have the same disease (microbe) as
the original plant.

Treatment and Controls. Microbes were isolated using pure culture methods
from both an uninfected and infected pepper. These bacteria were streaked on
TSA and PDA plates to test whether the pathogen is a bacteria, which thrive in a
TSA media, or a fungus, which thrive in PDA media. Differences in both plates,
from the healthy and unhealthy strains were compared to make sure the microbe
present in both peppers was not the same.
The TSA and PDA colonies were then introduced into two healthy peppers,
respectively. After a week, the symptoms of the newly diseased peppers were observed, and microbes from the two were re-extracted again as a source of comparison to the original plates to make sure the same bacteria caused these symptoms.
The bacteria grown on the PDA plate were restreaked onto a TSA plate to compare growth and appearance under the same media at 28 C with the TSA strain.
The microbes from the TSA plates obtained from the old and newly diseased pepper were compared using gram staining and wet mounts.

Results. Bacteria did not grow as well in PDA media. PDA colonies were barely
visible and not distinguishable.

Original Infected Pepper

Healthy Pepper

Brownish on the sides

Cloudy yellowish liquid
Mushy/semi-solid texture
Olive green

Firm texture
Normal green

(b) Uninfected Pepper

(a) Infected Pepper

Original Samples

Bacteria did not grow as well in PDA media. PDA colonies were barely visible
and not distinguishable.

(a) TSA Colonies

(b) PDA Colonies

Bacteria from Original Infected Pepper

Both TSA and PDA infected peppers had dry, black edges and had a similar
appearance.

(b) PDA Infected

(a) TSA Infected

Newly Infected Samples

Both microbes obtained from the infected samples were rod-shaped (shown below) and came out pink after gram-staining.

Wet Mount Microbe

Conclusions. Initially, from the PDA and TSA streaking, it is clear that the microbe grew better in TSA media, indicating that the microbe is a bacteria. The
appearance of the newly infected peppers was at an early stage of the original
pepper and showed the same symptoms as the original, indicating that they both
had the same disease.
After comparing both TSA plates from the original and newly infected peppers,
they showed similar signs of growth with yellow circular and distingshable single
colonies. Upon further observation, the wet mount showed that both bacteria were
rod-shaped, and the gram-stain came out gram-negative for both. After consulting with my peers whose peppers showed signs of the same disease, we came to

the conclusion that one type of gram-negative bacteria was the cause of the disease.
Key Question. One of the key questions the results from this experiment answers is the following:
Why couldnt you simply grind up some of your initial plant tissue, spread
it on a second (healthy) host and see if the disease appeared? Assuming it did
appear, what would that show and what would that fail to show?
Grinding up plant tissue onto a healthy host could very likely lead to disease,
but it would be impossible to tell what caused it. Specifically, without streaking
the microbe onto a plate and comparing it to microbial growth of a healthy pet, it
would fail to show what bacteria actually caused the disease. It would also fail to
show that the same bacteria caused the same disease. Nothing could be deduced
by grinding it up except observing, at a macroscopic level, that the two peppers
developed the same disease.