The Methyl Red, Microbiology Laboratory Theory And Application

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

The Voges-Proskauer test to detect organisms that are able to ferment glucose, but convert the products to acetoin and 2,3-butanediol. This is deduced by the addition of Reagent A and Reagent B, and the observation of the color change thereafter. Reagent A is a solution of -naphthol and alcohol. Reagent A catalyzes the conversion of acetoin to diacetyl. Diacetyl thens react with guanidine-containing compounds from the peptone to form a red color in the presence of -naphthol. Reagent B is a solution of potassium hydroxide and water. It

To perform this test, a tube of broth rich with glucose is acquired. In this tube is phenol red, a pH indicator. Initially, the tube appeared pink in color, indicating a normal pH level. Next, a sample of unknown #44 is introduced into this medium using the aseptic technique, and this is allowed to sit for several days. If the organism is able to ferment glucose, the pH in the medium would decrease and cause the phenol red to exhibit a yellow color. In addition to the straw color, gas can also be produced and trapped inside the Durham tube placed in the medium. This production of acid and gas is a direct result of the fermentation of glucose, as seen with unknown

In the Voges-Proskauer test, I inoculated the tube with bacteria from my TSA plate and incubated the tube at 37 degrees Celsius for 3 days. After three days I placed some Barrits Reagent A and Barrits Reagent B in the test tube. The color change of red or pink indicates a positive reaction for acetoin which tells you that the organism is a butanediol fermentor.

Bacteria groups or species can be differentiated by the fermentation patterns. The end-product of carbohydrate fermentation is an acid or acid with gas production and is dependent on the organism involved in the fermentation process. The carbohydrate fermentation tests detect if an organism is able to utilize glucose, lactose and sucrose. Phenol red is used as a pH indicator because it can indicate a change in pH when acid products are formed. Bacteria can utilize certain sugars resulting in an alkaline by-product which changes the color of the carbohydrate broth from red to yellow. Bubbles trapped within the Durham tube indicate the production of gas. The Phenol red carbohydrate fermentation tests determine that my organism E. coli can utilize glucose, lactose and sometimes sucrose but can only produce gas in glucose and lactose. (Phenol red carbohydrate fermentation lab

The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were oxidase, catalase, lactose and sucrose fermentation, Kugler/iron agar, nitrate reduction, gelatin hydrolysis, starch hydrolysis, manitol salt, MR-VP, citrate, bile esculin,

Mannitol Salt Agar contains the carbohydrate mannitol, 7.5% sodium chloride (NaCl), and a pH indicator called phenol red. At pH levels below 6.9, the medium is a yellow color. In the neutral pH ranges (6.9 to 8.4) the color is red; while above pH 8.4, the color of phenol red is pink (Gerhardt P., 2011). Acid is produced when mannitol is fermented by bacteria, which lowers the pH and results in the formation of a yellow halo surrounding the isolated colony on MSA. Non-fermenting bacteria that withstand high salt concentrations will display a red to pink area due to peptone breakdown (Mahon C.R. et al.

Thus we inoculated lactose broth, glucose broth and tryptophone broth with our organism. At our final lab meeting, we observed that the lactose broth was still phenol red in color, glucose tube had turned yellow with a bubble present in Durham tube, and the oily ring in our Indole test was colorless. Taking all the results into consideration and rechecking all of our steps against the guidelines and steps to ensure all required tests had been performed, we concluded that our organism belonged to Group IX genre, Proteus, Providencia, and Morganella. We then proceed to consult with our instructor.

When checking the results, H2S was negative, there were no black precipitants in the tube, and the organism was motile. After recording those results, I went to the hood to add Kovoc’s reagent to the SIMs and waited for about 30 minutes before observing to get the results, and it came out to be negative for indole as well. At this point, only one organism matched every result that I received during all my tests, I decided to do my confirmation test. I did Methyl Red and Voges-Proskauer tests for my confirmation test. After inoculating the MRVP broth and incubating it at 37oC for 120 hours, I took my separated samples to the hood and added in the Methyl Red in one tube and reagent A then B in the other tube, I mixed it and checked in on both tubes after every 10 minutes. About 45 minutes later it was clear what the results were, negative for methyl red and positive for Voges-Proskauer. That confirmed that my unknown O17 was Enterobacter

Homoacetogens are strict anaerobic bacteria that produce acetate from CO2 as a diagnostic end-product of metabolism (Drake 1994, Muller 2003). They can grow on different substrates such as hydrogen, hexoses, formate, carbon monoxide, methoxylated compounds, and alcohols. Depending on what energy sources they are growing with, products in addition to acetate can be formed, and some can produce butyrate from CO2 using the same basic pathway as used for acetate formation. They are known as “homoacetogens” (organisms generating only acetate), “reductive acetogens”, “CO2-reducing acetogens”, or simply as “acetogens”. These different terminologies are confusing, and misleading, since they do not only produce acetate, and acetogens could form acetate by any pathway. In this thesis, the term “homoacetogen” will be used, because it is a simple one word descriptor for this taxonomically-diverse group of bacteria unified by using the Wood-Ljundahl pathway to form acetate from CO2. The term “homoacetogenesis” will be used for this metabolism, and “homobutyrogenesis” for the process in which butyrate is formed from CO2.

2. Introduction: Each student was given unknown bacteria and was instructed to perform a variety of experimental tests that would help to identify their bacteria. During the process of identification, the unknown bacteria was added to many different testing medias using aseptic technique. They are as follows: lactose fermentation on eosin methylene blue (EMB), TSI (Triple Sugar Iron agar), Phenol red sucrose, the SIM test, H2S by SIM, IMViC (indole, motility, voges-proskauer, and citrate), Urease (urea broth), PDase (Phenylalanine Deaminase), Lysine Decarboxylase, and Ornithine Decarboxylase. Colonial morphology on EMB was used to

How I determined that my bacteria were Pseudomonas fluorescens. The first test I began with was a gran stain to figure out if it was a gram positive or gram negative bacteria which removed half of the bacteria. With the knowledge that the bacteria were gram negative, I did the EMB test and the basic sugar tests glucose, lactose, and sucrose. The results for EMB on the bacteria was a non-fermenter, further limiting the choices. It was during the test on the sugars, that it was possible to zero in on the last couple bacteria. These test, which showed up all negative leaving me with only three bacteria, since the other all negative for sugars had to be a cocci and mine was a rod. Therefore, I was left with two test left to help, Urease and Nitrate.

Introduction: Through the conduction of numerous experiments, the identity of two bacterial isolates was determined. The tested specimen was an unknown sample of a mixed culture of two different species of bacteria. The first step that was taken was obtaining a pure culture of each species of bacteria by isolating one species from the other. Once isolation was complete, the isolated cultures were tested using procedures that had been performed during previous lab sessions. A gram stain was performed on the two isolates. The isolate which had tested gram negative was then tested for the presence of cytochrome C and lactose fermentation. For the gram positive isolate, cell shape was determined and a catalase test was performed.

The overall chemical reaction conducted by these bacteria may be represented as: C6H12O6 → 3CH3COOH. According to Jia et al., 2007 these acetogenic bacteria synthesize acetic acid from single-carbon mixtures, including methanol, carbon monoxide, or a blend of carbon dioxide and hydrogen: 2 CO2 + 4 H2 → CH3COOH + 2 H2O. It is also described in the figure below.

Low concentration of viologen can significantly alter the metabolic process of fermentation. By adding viologen in the fermentation broth, the yields of hydrogen, butyric acid, and acetic acid rapidly decrease, whereas those of butanol and ethanol increase. After the addition of viologen, the color of fermentation broth changes (if methyl viologen is added, the fermentation broth turns blue; if benzyl viologen added, the broth becomes purple), indicating the reduction of viologen by hydrogenase (oxidative viologen is colorless), and the reduced ferredoxin has to compete with viologen for