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Part 1: Outline how you would differentiate the bacterial strains listed below using selective media and microscopy.

Details of the following selective media and stains: check the protocol log book as this contains info that will help you.
Bacterial strains
Bacillus subtilis, Enterococcus faecalis, Staphylococcus epidermidis, Proteus mirabilis, Enterobacter cloacae, Eschericha coli
Growth on TSA plates, stabs, broths and slope cultures, SIM stab culture including use of Kovak’s reagent, EMB plates, PEA plates, and fermentation assays testing for acid and gas production when using glucose, lactose and sucrose as carbon sources. Gram staining, endospore staining and testing for catalase activity.
You have an unknown bacteria with the following characteristics: Gram negative short rod, H2S producer and ferments sucrose to give acid and gas.
Which bacteria do you conclude this unknown to be? Outline your reasons and explain what experiments you would do to confirm your identification giving details of results you would expect.

Part 2: You have a pear showing signs of brown rot caused by a bacterial pathogen. Outline the stages and techniques used to carry out a Koch’s postulate to identify the causal agent of the bacterial rot on the pear. Which bacterial pathogen is causes this disease?

Bacillus subtilis

The bacterial strains which will be used for differentiation through the selective media and microscopy are: Bacillus subtilis, Enterococcus faecalis, Staphylococcus epidermidis, Proteus mirabilis, Enterobacter cloacae, Escherichia coli

Bacillus subtilis- It is a catalase positive and gram positive bacteria which is found within the gastrointestinal tract of human and ruminants and also found in soil. The various identification tests which gives positive result are: Acid from glucose, acid from sucrose, catalase activity, endospore staining, gram stain. Bacillus subtilis grows in TSA plates and broth cultures.

Under the light microscope, the flagella are clearly viewable with whip like log tails. Bacillus subtilis has peritrichous flagella. This means that the cell is entirely covered with small tails. The tails are visible under a light microscope by staining with a specialized stain (Sciencedirect.com, 2018).

Enterococcus faecalis-  It is a gram positive bacterium that inhabits in the gastrointestinal tract of the mammals and other humans. The various identification test which gives a positive result are: gram staining, fermentation assays of glucose, sucrose, lactose are positive. Enterococcus faecalis grows in TSA plates, EMB plates, PEA plates, broth and slope cultures.

This bacterium is closely related to the streptococci and under the microscope appear to be cocci or spheres. It divides through binary fission and form chains of bacteria (Lebreton, Willems and Gilmore 2014).  

Staphylococcus epidermidis- It is a gram positive bacteria, which is a typically found on the human skin flora and is not pathogenic. However, if a person has a weak immunity then there is an increased chance of infection development. The various identification test which gives a positive result are: gram staining, hydrogen sulphide, fermentation assay of glucose, sucrose and lactose are positive. Staphylococcus epidermidis grows in TSA plate and broth, EMB plate, PEA plate.

The colonies of Staphylococcus epidermidis under the microscopic view shows grape like clusters, the colonies are cohesive and raised and measure about 1 to 2 mm in diameter (Namvar et al. 2014).

Proteus mirabilis- It is an anaerobic bacterium that exhibits in soil and water. This bacterium is responsible for the majority of infection in humans. The various identification test which gives a positive result are: hydrogen sulphide test, fermentation assay of glucose is positive. Proteus mirabilis grows in TSA plates and broth, PEA plate, EMB plate, SIM stab culture including use of Kovak’s reagent.

The bacterium under the microscopic view is rod shaped with elongated flagella (Armbruster and Mobley2012).

Enterococcus faecalis

Enterobacter cloacae- The various identification test which gives a positive result are: fermentation assay of glucose and sucrose is positive. Enterobacter cloacae grows in TSA plates, SIM stab culture including use of Kovak’s reagent, broth and slant cultures.

The microscopic view of the Enterobacter cloacae reveal straight rods, the colonies range from 1.2 to 3 micrometer, have 4 to 6 peritrichous flagella (Davin-Regli and Pagès 2015).

Escherichia coli- The various identification test which gives a positive result are:  fermentation assay of glucose and lactose is positive and is variable is sucrose. Enterobacter cloacae grows in TSA plate and broth, PEA plate, EMB plate, SIM stab culture including use of Kovak’s reagent.The microscopic view of the Escherichia coli reveal rod shape, 2 to 6 micrometers, occur in several strains and are found to be in capsules, either occur in pairs or in single (Canada.ca, 2018).

The unknown bacteria showing following characteristics: gram negative, short rod, hydrogen sulphide producer and ferments sucrose to give acid and gas.

The bacteria identified from the given characteristic is Aeromonas hydrophila. Identification of this bacteria, involves the macroscopic tests, biochemistry test and gram staining. Microscopic tests are done through gram staining and observing the color and shape of the bacteria, including the biochemical tests. The biochemical test included the indol test, motile test and hydrogen sulphide test. The results of the identification tests revealed gram negative staining, form of short rod and motile. Whereas, the biochemical tests revealed the test results to be fermentative, negative indol, positive oxides and positive glucose. Thus, from the several tests conducted Aeromonas hydrophila can be identified to be exhibiting the similar characteristics (Kusdarwati, Kurniawan and Prayogi 2017).

In this part of the study, a pear is found to be showing signs of brown rot which is caused by an unknown bacterial pathogen. To identify this unknown pathogen, a procedure can be followed known as Koch’s postulate. Koch’s postulate is developed by a German physician named Robert Koch in the year 1890. This postulate is used to effectively judge a given bacterium which is responsible for the cause of the disease. This postulate brought the much needed clarity within the study of the plant pathology which was previously filled with confusions (Wingfield et al. 2012).

The stages that are used to carry out the Koch’s Postulates in order to identify the bacterial rot on pear are as follows:

  • Microorganisms are isolated from the infected pear.
  • Microorganisms are cultured in a pure culture
  • Microorganisms are identified
  • Microorganisms are injected into another fresh pear
  • The disease is reproduced in the fresh pear
  • Microorganisms are isolated and grown in a pure culture
  • Identification of the identical microorganisms

The various stages in the Koch’s postulates requires the usage of certain instrument will be required that will assist in carrying out of the experimental procedure. Culture media (agar plates), scalpel, 70% alcohol for sterilizing the scalpel and the table surface, candle flame.

  1. The agar culture and the and the infected pear are incubated at 20 to 25 degree Celsius.
  2. Just before the start of the process of inoculation, the surface upon which the experiment will be performed is sterilized using the 70% alcohol.
  3. The scalpel used for the isolation of the inoculum is heated over a flame to sterilize it.
  4. From the infected pear, a small incision is made with the scalpel and is isolated into an agar culture.
  5. The culture is then incubated for a week at 20 to 25 degree Celsius.
  6. In this step, from the culture of the unknown microorganism, a small amount of the inoculum is inserted into a fresh pear.
  7. The pear is then incubated for a week at 20 to 25 degree Celsius and the infection is cross checked with the first infected pear.
  8. When the comparison between the two infection in different pear is established, then it can be confirmed that the causal organism is similar (Barak and Schroeder 2012).

The pathogen which is causing the brown rot in pear is found to be a fungi called Monilinia laxa (Poniatowska, Michalecka and Bielenin 2013).

Reference

Armbruster, C.E. and Mobley, H.L., 2012. Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis. Nature Reviews Microbiology, 10(11), pp.743-754.

Barak, J.D. and Schroeder, B.K., 2012. Interrelationships of food safety and plant pathology: the life cycle of human pathogens on plants. Annual review of phytopathology, 50, pp.241-266.

Canada.ca, P., 2018. Pathogen Safety Data Sheets: Infectious Substances – Escherichia coli, enteropathogenic - Canada.ca. [online] Canada.ca. Available at: https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/escherichia-coli-enteropathogenic.html [Accessed 18 Jan. 2018].

Davin-Regli, A. and Pagès, J.M., 2015. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Frontiers in microbiology, 6.

Kusdarwati, R., Kurniawan, H. and Prayogi, Y.T., 2017, February. Isolation and Identification of Aeromonas hydrophila and Saprolegnia sp. on Catfish (Clarias gariepinus) in Floating cages in Bozem Moro Krembangan Surabaya. In IOP Conference Series: Earth and Environmental Science (Vol. 55, No. 1, p. 012038). IOP Publishing.

Lebreton, F., Willems, R.J. and Gilmore, M.S., 2014. Enterococcus diversity, origins in nature, and gut colonization.

Namvar, A.E., Bastarahang, S., Abbasi, N., Ghehi, G.S., Farhadbakhtiarian, S., Arezi, P., Hosseini, M., Baravati, S.Z., Jokar, Z. and Chermahin, S.G., 2014. Clinical characteristics of Staphylococcus epidermidis: a systematic review. GMS hygiene and infection control, 9(3).

Poniatowska, A., Michalecka, M. and Bielenin, A., 2013. Characteristic of Monilinia spp. fungi causing brown rot of pome and stone fruits in Poland. European journal of plant pathology, 135(4), pp.855-865.

Sciencedirect.com, 2018. Bacillus Subtilis - an overview | ScienceDirect Topics. [online] Sciencedirect.com. Available at: https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/bacillus-subtilis [Accessed 18 Jan. 2018].

Wingfield, M.J., De Beer, Z.W., Slippers, B., Wingfield, B.D., Groenewald, J.Z., Lombard, L. and Crous, P.W., 2012. One fungus, one name promotes progressive plant pathology. Molecular plant pathology, 13(6), pp.604-613.

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