so i wrote a lot of this.. like 21 of them.. i never knew i had the drive to write bullshits like this... makes me nauseated.. these are my explanations for every one of the experiment i made for that class..
gelatin talls is an experiment designed to test the ability of an organism to produce an enzyme called gelatinase to help hydrolyze gelatin. A gelatin is a protein derived from connective tissue or protein produced by the hydrolysis of collagen which is solid at the temperature of 32 degrees celsius and liquid above 32 degrees celsius. The enzyme gelatinase gives the organism strength to break down gelatin into polypeptides, peptides, and amino acid that will allow it to cross the cell membrane in able for the organism to use. A positive result of this experiment displays a liquified gelatin where the organism has grown. My organism however, didn't liquify the gelatin, but remained solid, meaning that it didn't have the capabilities of producing the enzyme gelatinase to help hydrolyze the protein gelatin. This medium is differential because it distinguishes organisms that can produce the enzyme gelatinase, that will help hydrolyze gelatin. Gelatinase test is differential because it test the ability of organism to hydrolyze gelatin by producing the enzyme called Gelatinase in which breaks down gelatin into smaller polypeptides, peptides, and amino acids.
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Sim Medium is one of the combination differential medial that is designed to indicate a bacteria's ability of sulfur reduction, indole production and motility.
The sulfur part of the test is to differentiate enteric organisms. Organisms that produce the enzyme thiosulfate reductase has the ability to reduce sulfur to hydrogen sulfide gas. Sodium thiosulfate is the source of sulfur in the medium, so when hydrogen sulfide gas is produced, it will react with ferrous sulfate, giving the medium a black color or precipitate, indicating that sulfur has been reduce(positive result). My unknown organism however, didn't produce a black precipitate in the medium indicating that it didn't produce the enzyme thiosulfate reductase responsible for reducing sulfur to hydrogen sulfide gas. (Negative result)
The indole is part of the IMViC series of test that test if an organism produces the enzyme thyptophanase, which will break down tryptophan into indole, pyruvate and ammonia by way of deamination. To test if an organism is able to produce the enzyme tryptophanase, the agar must be inoculated with a needle and incubate it for 24-48 hours. After the incubation period, a few drops Kovac's reagent which contains (DMABA) and and hydrochloric acid is added in the medium. Any indole present in the medium will react with one of the ingredients of Kovac's reagent (DMABA) and producing a color red. My organism is indole positive because it was able to produce this result, meaning that it was able to produce the enzyme tryptophanase that hydrolyzes tryptophan to indole and pyruvate. A negative result display no color change in the Reagent, meaning the organism didn't have the capabilities of producing trytophanase to hydrolyze tryptophan.
The Motility part of the test is to distinguish if an organism is motile by having a growth spreading outward from the stab line in the medium, whereas organisms which are not motile wont have a growth spreading out from the initial stab line.
These test are differential because each test is uniquely designed to distinguish which reacts and bring out a specific biochemical reaction. The Indole test is used to differentiate enteric bacterias. The sulfur part of the SIM medium is used to distinguish enterics that can reduce sulfur. But as a whole, the SIM is recommended or primarily used for differentiating between Salmonella and Shigella.
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TSI or Triple Iron SUgar is a differential medium made up of a small amount of glucose,sucrose and lactose peptone, ferrous sulfate and Phenol Red as a pH indicator. It is designed to differentiate bacteria that can ferment these carbohydrates (glucose,lactose,sucrose) and also the ability to reduce sulfur. In able to get the results of fermentation and sulfur reduction, we have to wait up 18-24 hours for the fermentation of the three carbohydrates and 48 hours for the reduction of sulfur. If an organism can ferment any of the three carbohydrates present (glucose and lactose and/or sucrose) in the medium, then the result would be yellow slant/yellow butt. When the medium have Red slant/ yellow butt, interprets that glucose is fermented with acid production. If there is no change in the slant/ butt of the medium, interprets that the organism is growing in a slow phase or not at all. My unknown organism however, had a result of red slant/ red butt indicating that there is fermentation of the carbohydrates. Another results is when the medium has black precipitate in the agar, giving it the interpretation that sulfur has been reduced. Cracks inside the agar indicates gas production. TSI is solely designed to distinguish enteric bacteria from other gram negative rods such as Alcaligenes or Pseudomanas.
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The BGLB , Brilliant Green Lactose Bile broth and Escherichia coli broth coliform contains lactose and 2% of bile salts that helps inhibits bacteria that are noncoliforms but also use to determine the presence of a coliform. The BGLB broth contain a 10mL that has a Durham tubes inside to indicate if the organism produced gas product.My unknown organisms didn't turn turbid nor produce gas production, confirming that it is not a coliform bacteria. this experiment it selective since it contains bile salts and lactose that selects for E.coli
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Right after I received my unknown bacteria, I did a simple and negative stain to find out if my organism is gram + or -, and also its morphology. I soon found out that i have Gram - bacillus. As a beginner, i didn't quite know what experiments to do first so i could narrow it down into the group it belongs to in the Bergeys manual. Half-way through the experiments, I slowly started to gain an understanding. After finding out that my bacteria is noncoliform, i knew that my options are not broad.
After all my required experiments are done, I decided to go to my lab manual to check which flowchart my unknown bacteria belongs to. By having a positive sign for Indole and Methyl-Red and a negative sign for both Voges-Proskauer and Citrate test, the list of commonly isolated Enterobacteriaceae table lead me to Figure 7-15, which is accurate of my test results, and that my unknown organism is Shigella flexneri. To make sure it is my unknown organism is Shigella flexneri, i checked the Bergeys manual just to make sure. By doing so, i found out that it mostly match its subspecies Shigella sonnei, but the difference is that S. sonnei is positive Orinthine decarboxylation, which my unknown organism is not and also neither is S.flexneri. I also had to find out another organism that is very similar to my unknown and the other bacteria that i can compare it with. This leads me Klebsiella pneumonia subsp. rhinoscleromatis, although most of its test results match my unknown organism, i found out that it is a probable coliform because it is a lactose fermenter in MacConkey agar test, indicating a positive result for a probable coliform. After that I conclude that my bacteria is Shigella flexneri.
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