Matthew Mouck

Lab Partner: Taylor Bickle

MICRO 203 Section 003

2 March 2018

Koch’s Postulate Observed Through Infected Pepper

Hypothesis:

The infected pepper (pepper #29) is infected by a microbe.

Prediction:

If the pepper is infected by a microbe, then all four of Koch’s postulates will be fulfilled.

Postulate #1. A consistent association between the disease condition, which is the symptoms

observed on the infected pepper, and the presence of a specific microbe will be observed.

Postulate #2. The microbe will be isolated and will grow in pure culture outside of the original

host.

Postulate #3. A healthy, susceptible host pepper will be inoculated with the pure culture and

disease symptoms that are the same as those in the original host pepper will be observed.

Postulate #4. The microbe from the inoculated host pepper will be isolated and it will be

demonstrated that it is the same as the microbe from the original diseased pepper.

In short, a microbe isolated from the original infected pepper will infect a healthy pepper and

cause the same symptoms.

Treatments and Controls:

Experimental Group: The microbe on the infected pepper is isolated by streaking on both T. Soy

and PDA agar. These are incubated at 28°C for 2 days. This is done so that the microbe can be

examined and then reintroduced onto a healthy pepper.

Negative Control: The healthy pepper was streaked on T. Soy and PDA agar as well. However,

there is no growth to be expected since it is a healthy pepper. The purpose of having this

negative control is to control for the source of infection. If the healthy pepper possesses the same

microbes as the infected pepper, then the infection is likely not due to microbes.

Experimental Group: A colony from the PDA or T. Soy agar (wherever the microbe grew

better—in this case the T. Soy) is used and introduced onto a healthy host pepper. This is

completed to observe if the healthy pepper expresses similar symptoms to the original infected

pepper.

Negative Control: A healthy pepper was introduced to an inoculating loop with no microbes on

it. It is expected to remain healthy and not express similar symptoms that the original infected

pepper expressed since there is no microbes exposed to it. This negative control can control for

the effects of the procedure of using the inoculating loop to introduce the bacteria. If this pepper

expressed similar symptoms to the original host pepper or pepper infected by the microbe, then

the experiment would be nullified as there something has gone amiss.

Both of these peppers were stored at room temperature for 5 days.

The healthy pepper from the previous step that was exposed to the microbe (The experimental

group) was streaked onto T. Soy agar. This was stored for 28°C for 2 days.
 Results:

Figure 1: T. Soy Original Pepper Figure 2: PDA Original Pepper

The above figures are the results of the original host pepper being streaked onto T. Soy

agar and PDA agar. There was substantial growth on the T. soy agar. The PDA agar also

experienced growth, but it is not quite as much as the T. Soy agar. The size of the colonies is

approximately the same diameter. The T. Soy agar is a more mustard yellow, but this is likely

because of the agar itself causing a unique tint.

Figure 3: T. Soy Healthy Pepper Figure 4: PDA Healthy Pepper

The above figures illustrate the results of the healthy pepper streaked onto T. Soy and

PDA agar. It was not expected for there to be growth. The growth is very similar to the infected

pepper growth. This means that the size of the colonies is very similar to the infected pepper

colonies. Also, just as with the experimental group, there was more growth on T. Soy for these

healthy peppers as opposed to the PDA agar. It is worth noting that there is, however,

significantly less growth on the PDA agar from the healthy pepper as opposed to from the

infected pepper on the PDA. Furthermore, there are different sized colonies, with some being

large and some others being rather small.

 Figure 5: Infected 40x Power

The image above is the result of bacteria being taken from the T. Soy agar that was streaked with

the original infected pepper. It is on 40x power under the microscope. A simple stain was used to

view the bacteria using methylene blue. This shape of the bacteria can be seen in the middle of

the photograph. They are slightly tinted blue. They are clustered together and they appear

elongated in shape.
Figure 6: Host Pepper with Figure 7: Negative Control Host
Microbe

The host pepper with the microbe introduced to it that was isolated from the original

infected pepper became sleek and glossy in texture. It also appears that there is slime on the

pepper and that it has lost rigidity on the sides and top. The pepper is easier to penetrate with a

loop and the red color has almost completely disappeared. When looking at the negative control

host pepper, there is an obvious red section at the end of the pepper and it is bright. Furthermore,

the pepper is still rigid and still retains a rigid structure. Compared to the microbial pepper, there

is no definitely no shiny color and it has a darker green color. The microbial pepper has a green

color that appears to have lost a true green color and instead is a faded green. The microbial host

pepper displays characteristics that are similar to the original infected pepper. Both were slimy

and had lost the bright red color in the pepper.

 Figure 8: Microbial Host 40x

Using a gram stain, the above results were formed. The resulting microbe is purple and appears

to be clustered together. Furthermore, its shape appears to be elongated, which is similar to the

shape that was observed in Figure 5.

 Figure 10: Separate Group #2
Figure 9: Separate Group #1

The figures on above are from two different groups. Figure 9 is from Hailee and Neely Colvin.

Figure 10 is from Jared Burk and Andrew Ward. The microorganisms in both are red, fairly

separated, and also appear elongated in shape which is similar to our microorganism, as observed

in Figure 5 and 8.

Discussion:

The results of the experiment support the hypothesis that the pepper was infected by a microbe,

specifically a bacterium. This is evidenced by the fulfillment of the four Koch’s postulates. The

first postulate states that there is an association between the disease condition and a microbe.

This is evidenced by Figures 1 and 2, as it was possible for the microbe to isolated. Furthermore,

the original host pepper was clearly infected due to its glossy color and loss of red.

The second postulate is simply to isolate the microbe and grow it on a culture. This was

completed on two separate agars: PDA and T. Soy. Although microbes grew on both agars, it

appears that there was significantly more growth on the T. Soy agar as compared to the PDA

agar as seen in Figures 1 and 2. This therefore suggests that the isolated microbe is a bacterium
since bacteria experience better growth on T. Soy as compared to PDA, whereas fungi grow

better on PDA as compared to T. Soy. It also appears to be rod-shaped as observed in Figure 5

due to the elongated shape.

For the third postulate, a healthy pepper was introduced to the microbe that was isolated

from the original infected pepper. This pepper then expressed sickness that was akin to the

original pepper, thus fulfilling the third postulate. There was an evident slime that covered the

pepper and the host pepper lost the red color that is so prominent in the control pepper.

Therefore, it appears that the disease the host pepper has is the same as the original infected

pepper, which also contained a green slime and a clear lack of red color.

For the fourth postulate, microbes from the now infected host pepper were isolated and

then compared to the microbes on the original infected pepper to ensure it is the same microbe.

Based on Figure 5, which was completed through a simple stain, the microbe is a rod-shaped.

After the microbe grew better on the T. Soy, a gram stain was performed since it was likely a

bacterium that infected the pepper, which is seen in Figure 8. The gram stain reveals that the

microbe is gram-positive since it is purple and, although it is less evident than the simple stain

results, is likely rod-shaped. Because the bacteria are rod-shaped, it is likely that this second

isolated bacteria is the same as the first isolated bacteria, thus fulfilling Koch’s fourth postulate.

Because all of Koch’s postulates were fulfilled, there is strong support that the infection on the

pepper was due to a microbe, specifically a gram-positive and rod-shaped bacterium.

The results of some of the other members of the class have also been collected. Figure 10

comes from Jared B. and Andrew W, which depicts a gram-negative and rod-shaped bacterium.

Haliee W. and Neely C’s bacteria (results are depicted in Figure 9) likewise depict a gram-

negative and rod-shaped bacterium. Our gram-positive bacteria would clearly not be of the same
species; however, pictures of everyone’s else microbes could not be reproduced here because no

pictures were provided. Therefore, it is possible that the gram-stain procedure was not performed

correctly and thus led us to believe it was gram-positive when it is actually gram-negative as

seen by the two previous examples.

It is important to take notice of Figures 2 and 3. The healthy pepper should not have

produced a microbe because it is healthy. However, it is highly likely that there was cross-

contamination and therefore the microbe was introduced onto the healthy pepper that was then

inoculated. There is no definitive method to know for sure, so the results of the experiment must

be taken lightly. If a healthy pepper possesses the same microbes as the infected pepper, then the

infection would likely not be due to the microbe because the healthy pepper possesses it but is

not infected.

In the future, further tests to specifically identify what bacteria has infected the pepper

should be conducted. This will help to understand how the bacteria can be controlled or

eliminated. This can be done by reconducting a gram stain and testing various other attributions,

such as fermentation of lactose, survivability in certain temperatures, and survivability in certain

salt concentrations. Furthermore, it would be worthwhile to study if this microbe infects other

types of produce, which will allow an assessment on the microbe’s potential threat to crop

output.

Key Question: What if the disease-causing pathogen does not grow outside of the host? How

might this have changed Koch's understanding of infectious agents? Can you think of any

examples?
 If the pathogen does not grow outside of the host, then it wouldn’t have been able to be

isolated on its own. There would just be no growth. This would then prevent the ability to

reintroduce the pathogen onto a healthy host. Therefore, it would essentially be vastly more

difficult to prove that the disease is caused by a pathogen because it Koch’s postulates could not

be fulfilled. Essentially, one could not demonstrate that this microbe caused the same symptoms

on the original infected organisms by infecting a healthy host. This would have changed Koch’s

understanding of infectious agents since he might have been inclined to believe pathogen

exclusively form parasitic relationships and could only grow when there is a host. Furthermore,

he may have been inclined to believe that the issue was genetic rather than microbial because it

would appear that there are no microbes to even cause the disease. A possible example would be

the virus HIV that causes AIDs. Although it can “survive” (viruses do not really live) in certain

conditions, it would be difficult to isolate without knowing in what conditions it can persist.