Lab Report

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 Methyl Red test is a differential test for bacterial respiration used to differentiate strains of coliform bacteria capable of performing mixed acid fermentation that will lower the pH despite the phosphate buffer (http://faculty.deanza.fhda.edu). Mixed acid fermentation is confirmed by using methyl red as an indicator. It is red ant pH 4.4 and below, yellow at pH 6.2 and above, and orange in between. Red is a positive result reported as (+), yellow is a negative result reported as (-), and orange is negative or inconclusive.

The Gram stain was used to determine if the bacteria was gram positive or negative. A negative test shows a pink color and a positive test is a purple color. When a bacterium is negative it is because it has an outer membrane and a thin layer of peptidoglycan that is much harder to stain. A positive bacterium has a thick layer of peptidoglycan and no outer membrane that can be penetrated by crystal violet.

The next step of the project included preparing a Gram stain to discover the cell shape, arrangement, and if the bacteria is gram positive or

This test is used to detect if the bacteria contains any deoxyribonuclease activity. Because no color change was observed from blue to clear my unknown bacteria displayed a negative result.

This experiment was conducted to find the genus and species of an unknown bacteria prescribed by the lab teacher, which was unknown bacteria GA3 in my case. Identification of unknown bacteria techniques are used on an every day basis to figure out what type of bacteria it is and to find the best method of how to treat a patient with this bacteria (1). All five “I’s” of Microbiology were used in the testing for the unknown culture. Inoculation was used several times to put the unknown culture into agar plates or into biochemical test tubes. After Inoculation of these tubes or plates, they always were placed into the incubator for further growth and development. Isolation was used to make sure we got the correct bacteria we were testing for. After each further isolation, we gram stained the culture and inspected the culture under a microscope to further help in the identification process of the unknown bacteria. Multiple tests were done on the unknown culture to make sure we were confident in what kind of bacteria the unknown was.

The IMViC test is a series of different tests that differentiate between enterics. One is the Indole test. This test tells whether the bacterium possesses tryptophanase which is the enzyme that breaks down tryptophan into indole. The agar contains tryptic soy broth so if the bacterium contains tryptophanase, indole is produced. This production of indole is seen by adding Kovac’s reagent which causes a red ring to be seen at the top of the tube. The citrate test is also used to see which kind of products the bacteria make. It uses a green agar slant that contains sodium and ammonium phosphate. Bromythymol blue dye is late added as an indicator. Inoculation of the slant with a needle using a zig-zag then stab technique was used with the gram positive bacterium. Conversion of the medium to blue is a positive citrate result. All plates, slants, and broths were incubated at 37°C for 24‐48 hours.

The purpose of this lab was to identify unknown bacteria cultures using various differential tests, and my unknown bacteria is #17. 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 Gram stain, Catalase, Mannitol Salt Agar (MSA), Blood Agar, Novobiocin, Coagulase, and DNAse (Alachi, 2007).

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.

This experiment was given to us to utilized previous knowledge learned throughout the semester to identify a gram negative unknown bacterium. We had to first learn the difference between a gram negative and a gram positive organism. We started off with doing gram stains to determine whether it was positive or negative. Based on the gram stains, a gram positive stains purple and a gram negative stains pink. A gram positive stains purple because the cell walls is made of a thick peptidoglycan layer and doesn’t

Gram staining is a technique used to determine if the bacteria is Gram positive or Gram- negative. Gram staining procedure uses crystal violet stain, iodine moderator, alcohol decolorizer and safarin counter stain. In Gram- negative bacteria the primary stain will be washed out with the decolorizer and it will be stained with the counterstain. Whereas in Gram-positive bacteria the primary stain will not leave the cell wall. This difference comes from difference in the structure of the cell wall that retains the stain.

The SFB test showed no change at all, shown in Figure 8. Positive test results are indicated by turbidity and yellow pigmentation (Stout et al, 2017). The turbidity would be caused by the organism’s ability to thrive and grow in the media, while the change in color would be attributed to the bromcresol purple, which is a pH indicator and turns yellow under acidic conditions (Stout et al, 2017).

The decolorized Gram negative cells are stained pink. With the results from the Gram stain I was able to follow the “Unknown Identification Flowchart” to the next step, which was to prepare for the Starch Hydrolysis Test by inoculating a starch plate.

Determination of the bacteria being a lactose fermenter or non fermenter is done through the growth on the MacConkey agar. Knowing this allow for the student to perform the necessary tests to determine which lactose fermenter was present in the sample. The Indole test allows for the determination of whether the unknown bacteria is Escherichia coli because this genus and speices is the only lactose fermenter that will produce a positive result here. Moving onto the Methyl red test this indicates glucose fermentation, more specifically microbes that produce mixture of acids as a result of fermentation. The Voges-proskauer tests for glucose fermentation, specifically organisms whose acid is converted to acetoin. The Citrate test differentiates an organism’s ability to use citrate as its only carbon source. The urea broth culture detects the enzyme urease, which allows break down of urea producing acid, which causes a noticeable change in color. The final test is the motility test to determine if the bacteria has the capacity of movement beyond the point of

Durham tubes differentiate a microorganism’s ability to ferment sugar (mannitol, dextrose, and lactose) that may produce a gas, but acidic byproducts will turn solution yellow. Unknown 6 was yellow in all three test tubes suggesting acidic conditions (fermentation occurred), however lactose and mannitol were cloudy, dextrose remained clear. There was no gas present in any of the Durham tube. Urease broth determines if a organism can hydrolyze urea with urease. It contains urea, nutrients, pH buffers, phenol red (indicator). Unknown 6 tested negative because it remained yellow and clear, therefore the pH did not rise, because no acid was produced from hydrolyzing urea. Sulfur Indole Motility Media (SIM deep) differentiates microorganisms that reduce sulfur, produce indole, and are motile. H2S is reduced by cysteine catabolism, or thiosulfate, and HCl, p-dimethylaminobenzaldehyde, n-amyl alcohol (Kovac’s reagent). SIM deep contains nutrients, peptone (tryptophan), iron, sodium thiosulfate. Unknown 6 tested negative to sulfur reduction and indole production, and remained yellow (after Kovac’s reagent was added), and it is non-motile since the agar remained clear. Kliger’s Iron Agar (KIA) differentiates glucose, and lactose fermentation to acids and gasses, specifically sulfur reducers. It contains small amounts of glucose (to be exhausted), phenol red (indicate acid), lactose (secondary sugar). Fissures are the result of gas byproducts. Unknown 6 fermented glucose, it failed to ferment lactose. It remained red with a yellow butt, the surface was pink. It tested negative for H2S, or gas formation.Simmons Citrate Agar tests an organism use of citrate as the only carbon source. Citrase hydrolyzes citrate into oxaloacetic and acetic acid. Oxaloacetic acid is hydrolyzed into pyruvic acid and carbon dioxide. It contains sodium citrate (carbon source), ammonium dihydrogen phosphate (nitrogen source), nutrients, and