Biology Report: Experiment on Koch's Postulates and Disease Causation

This report details a biology experiment investigating Koch's postulates to determine the causative agents of disease in apples and beans. The methodology involved isolating suspected pathogens, sub-culturing them, and inoculating healthy hosts. Observations focused on visual characteristics, including sporulation and discoloration in apples, and necrotic appearances in beans. The results indicated that Sclerotinia fructigena caused brown rot in apples, while Pseudomonas syringae and Pseudomonas phaseolicola infected beans. The discussion highlighted the importance of Koch's postulates in identifying pathogens and the limitations encountered. The study was conducted in order to prove the pathogenicity of the identified organisms following Koch's postulate. The experiment confirmed the presence of Pseudomonas syringae and Pseudomonas phaseolicola. The report concludes with a discussion of the findings, limitations, and references to relevant literature.

Running head: EXPERIMENT ON KOCH’S POSTULATES
Experiment on Koch’s Postulates
Name of student:
Name of university:
Author Note:

1EXPERIMENT ON KOCH’S POSTULATES
Abstract
The following consists of a series of investigations to identify the causative agent of disease
in apples. The experiments were conducted using four criteria of Koch’s postulates. The
microscopic observations of visual characteristics were noted. A detailed discussion of the
experimental results are provided.

2EXPERIMENT ON KOCH’S POSTULATES
Introduction
The epidemics of dreadful diseases are associated with causative microorganisms and
this formed the basis of Koch’s postulates in microbiology. Koch postulates are based on a
set of four identifying criteria which can be used to determine the causative pathogen
responsible for a particular disease (Byrd and Segre2016). The following postulates are:
1. The suspected pathogen must have a regular association with the disease.
2. The pathogen must be isolated from its diseased host and grown in pure culture.
3. A pure culture of pathogen must produce the disease symptoms when introduced into
the susceptible host.
4. The pathogen must be re-isolated from the experimentally infected host and must
resemble with that of original pathogen.
Koch’s postulates have been applied to identify the causative agents of anthrax and
tuberculosis which are bacterial diseases caused by different bacterial species in cattle and
humans respectively (Cambau and Drancourt 2014).
Despite the importance of Koch’s postulates in microbiology, several obvious
limitations occur while considering viral diseases. Viruses cannot be replicated in culture
medium; a suitable animal model is required for their propagation (limitation of postulate 2
and 3) (Gradmann2014). Similar virus causes different diseases in the infected, whereas
different viruses can result in same diseases in the infected (limitation of postulate).Koch
postulates could not be fulfilled for identifying causal agents of cholera and
leprosy(Gradmann2014).This paper will illustrate the methodology of the experiment,
observation of the experiment and result and discussion of the experiment in following
paragraphs.

3EXPERIMENT ON KOCH’S POSTULATES
Methodology
Isolation of suspected pathogen
 The surface of infected apple was sterilized with 70% ethanol.
 An incision was made at the edge of lesion with a sterilised scalpel; two incised
pieces of tissue each measuring 0.5cms were removed to minimise the risk of
secondary infection.
 The tissue pieces were placed across each other in Potato Dextrose Agar (PDA)
medium, a general medium to allow propagation.
 The tissue samples were kept at room temperature (20°C).
 The infected beans were sterilized in the similar way and the edge of infected beans
tissue was smeared across in King B medium, a general medium to allow for bacterial
growth with the help of a sterilised loop.
 The tissue sample was incubated at room temperature (20°C).
Sub culturing of the associated pathogen
 A sterile procedure was used to perform sub culturing of potential pathogen from both
apple and beans tissues.
 The cleanest portion of the apple tissue that came out at the edge of the fungal growth
was isolated with the help of a sterile scalpel.
 Two pieces of apple tissue each measuring 0.5 cms were excised with sterile scalpel.
 The fungal colonies were placed into the PDA general medium and kept for
incubation at room temperature (20°C) for two weeks.
 For the beans tissue, a sterile method was used to obtain a pure culture by dilution.
 A single bacterial colony was picked from the culture using a sterile loopand the
colony was added on the King B general medium.

4EXPERIMENT ON KOCH’S POSTULATES
 The loop was flamed, cooled to room temperature and the colony was smeared across
in three streaks using the sterile loop.
 The loop was again sterilised by flaming.
 The sample was kept for incubation at room temperature (20°C) in presence of light.
Inoculation of the culture into susceptible host
 Sub culturing of the samples were done beforehand and a pure culture was obtained.
 A health apple was inoculated with fungal colony and healthy beans were inoculated
with bacterial colony.
Inoculation of healthy apple:
 The surface of healthy apple was sterilized with 70% ethanol.
 A small edge measuring 0.5cms was incised and flicked to create a flap.
 A loop of fungal culture was inserted through the little opening of the flap by a
sterile technique; the opening was sealed with a tape.
 Two inoculations were performed similarly for each apple and thereafter kept for
incubation at room temperature (20°C) in presence of light.
Inoculation of healthy beans:
 A sterile scalpel was used to inoculate each bean with pure single bacterial colony
by a stabbing method.
 The samples were incubated at room temperature (20°C) in presence of light.
Observations
 A careful observation was carried out to identify visual characteristics to link with the
disease with the help of a microscope.

5EXPERIMENT ON KOCH’S POSTULATES
The apples showed evidences of sporulation and discoloration; the spores appeared
circular in morphology with brownish grey on the outside and dark brown on the
inside.
 The fruit developed a soft texture giving out a fermentation smell.
 Dissection of the fruit revealed an entire brown colour; development of whitish or
grey hyphae with the formation of mycelia was observed at the core of the fruit.
 In order to identify the bacteria an experiment was performed where isolated single
colony was dipped in 3% of the KOH.
Result:
The result of inoculation of fungi in the healthy apple:
After conducting the inoculation procedure, it was observed that the apple was
covered with the sporulation zone and sporulation texture that was found initially
resembles the sporulation texture found in healthy apple after inoculation of the fungi.
After dissecting the apple, it exhibited soft texture that further give rise to the
fermentation smells. However, microscopic observation did not exhibit conidia spores;
only exhibit a spore during initial observation. The initial hype suggested that in order to
start the sporulation, more daylight was required. The study failed to detect the structure
of the mycelium. However, on the basis of the conducted research it can be concluded
that the sporulation by Sclerotiniafructigena resulted in the brown rot in healthy apple.
The result of inoculation of fungi in the healthy bean:

6EXPERIMENT ON KOCH’S POSTULATES
After conducting the inoculation procedure, it was observed that the healthy bean
was infected with the bacteria rather than fungi. The appearance of the bean was necrotic.
An experiment was conducted for the bacteria obtained from the beans in order to
determine the type of the bacteria that infected the healthy bean. The result of the
experiment showed the colony was bright yellow in color and other bacteria present in the
colony was creamy and the texture was gloopy. on the basis of the conducted research, it
was confirmed the bacteria that infected the healthy bean was Pseudomonas phaseolicola
and Pseudomonas syringe. These two microbes further subjected to the UV light at
366nm. It was observed that bright yellow bacteria did not show the fluorescent
characteristics but bacteria with creamy texture showed the fluorescent characteristics.
Discussion:
The study was conducted for proving the pathogenicity of the identified organism
following Koch's postulate. The Koch postulate is a crucial part of the microbiological lab
experiments that aid identifying the accurate information about the pathogenecity of the
microorganism that causes disease. In the conducted experiment, the study found out the
Sclerotiniafructigena that causes the rotten brown texture in the apple. According to
Lagier et al. (2018), Sclerotiniafructigena is a plant pathogen belonged to rosaceous that
gives rise to the rotten texture in fruits such as apples, pears, plums, and peaches. It is the
most common and generally distributed microbes that attack the plant and causes disease.
In the conducted study, the fungi were isolated from the infected apple and inoculated in
the healthy apple in order to observe the ability of the microbes to cause the infection
which is the first criteria of Koch's postulate. According to Koch et al. (2017), in order
to identify the individual fungi, the structure of the mycelia is crucial but the study unable

7EXPERIMENT ON KOCH’S POSTULATES
to detect the mycelia, only hyphae was observed. Therefore, according to the criteria of
the Koch’s postulate, the pathogen that was isolated from the infected apple had the
ability to cause infection to the healthy apple (Hornef 2015).
On the other hand, the study conducted on the bean showed a similar result.
The pathogenic microbes that were isolated from the infected bean were inoculated in the
healthy bean and for confirming the gram character of the bacteria an experiment was
conducted with KOH. The experiment confirmed the presence of Pseudomonas syringae
and Pseudomonas phaseolicola. Since Pseudomonas syringae has a thick cell wall, KOH
did not lyse the cell and gave a bright yellow color (Chen et al. 2018). On the other hand,
phaseolicola has a thin cell wall that was lysed by KOH and gave rise to the creamy
texture and further exposure to the UV light gave fluorescent characteristics (Raoult et al.
2016). Therefore, according to the criteria of the Koch’s postulate, the pathogen that was
isolated from the infected apple had the ability to cause infection to the healthy bean.
However, the limitation of the study is that the study failed to compare the
isolated pathogen from that was inoculated in the healthy apple with the original pathogen
found in the infected fruits due to adequate time.

8EXPERIMENT ON KOCH’S POSTULATES
References:
Byrd, A.L. and Segre, J.A., 2016. Adapting Koch's postulates. Science, 351(6270), pp.224-
226.
Cambau, E. and Drancourt, M., 2014. Steps towards the discovery of Mycobacterium
tuberculosis by Robert Koch, 1882. Clinical Microbiology and Infection, 20(3), pp.196-201.
Chen, J., Ma, M., Uzal, F.A. and McClane, B.A., 2014. Host cell-induced signaling causes
Clostridium perfringens to upregulate production of toxins important for intestinal
infections. Gut Microbes, 5(1), pp.96-107.
Gradmann, C., 2014. A spirit of scientific rigour: Koch's postulates in twentieth-century
medicine. Microbes and infection, 16(11), pp.885-892.
Hornef, M., 2015. Pathogens, commensal symbionts, and pathobionts: discovery and
functional effects on the host. ILAR journal, 56(2), pp.159-162.
Koch, H., Beesley, H., Formby, C. and Fraser, F., 2017. Civil Claimant Embitterment: Five
Case Studies Exploring Clinical Presentation and Management. Med Case Rep, 3(3).
Lagier, J.C., Dubourg, G., Amrane, S. and Raoult, D., 2018. Koch Postulate: Why do we
Should Grow Bacteria?. Archives of medical research.
Raoult, D., 2016. The return of microbes. Clinical Microbiology and Infection, 22(10),
pp.822-823.