Gram Negative Unknown for Microbiology

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James Arnold
December 4, 1999

Gram Negative Unknown

Upon being given a bacterial culture containing two bacteria, I was assigned the task of isolating the individual bacteria. The two bacteria of the mixture were one gram positive and one gram negative. The experiment has two parts and this is the part of isolating the gram-negative bacteria. The technique used was called streaking for isolation in which the bacteria were streaked thinly on nutrient agar in different directions in a pattern that left individual colonies on the final growth streak. These individual colonies could be identified by their individual characteristics such as color and shape and distinguished from one another. This was not easy with this culture because both bacteria grew in colonies that were small, white, and shaped similar. To separate these bacteria I had to first gram stain the bacterial mixture to see if I had both types of bacteria and so I used the following steps to perform the gram stain.

1. The bacteria are smeared on a slide and allowed to dry.
2. The bacteria is heat fixed to the slide
3. Crystal violet pigment is added to the smear for 1 minute
4. Wash the pigment off with distilled water
5. Apply Gramís Iodine for 1 minute
6. Wash iodine off with distilled water
7. Decolorize the smear with 95% ethyl alcohol drop by drop until it runs off clear
8. Wash off alcohol with distilled water
9. Counterstain with Safranin for 45 seconds
10. Wash off Safranin with distilled water
11. Blot dry with Bibulous paper

Following this test, I found that I had a lot more gram negative bacteria than the gram positive and so I could easily take one colony of my streaked culture and have a high chance of growing a pure culture from one of the isolated colonies. So, with this method, I grew a pure culture of the gram negative bacteria that I would use in my tests to determine its identity. I ran several tests on the bacteria staring with the Gram stain that proved that the bacteria were gram negative. From this test, I concluded that the bacteria were rods. According to the flow chart, the next test to be performed was the oxidase test which would tell if my bacteria were non-fermentative aerobes or if they were fermentative facultative anaerobes. I think they would be non-fermentative aerobes.
The oxidase test, as described in the lab manual on page 179, is used to determine if a bacteria has the ability to catalyze the reaction between reduced cytochrome and molecular oxygen using the enzyme Cytochrome oxidase to produce water or hydrogen peroxide. The test was conducted and the bacteria was found to be oxidase negative which means that it does not contain the enzyme cytochrome oxidase and means that it is a fermentative facultative anaerobe according to the flow chart. The next two tests conducted on the bacteria were the Hydrogen Sulfide test and the Citrate Utilization test.
From the Hydrogen Sulfide test, I can determine if the bacteria are motile and if they produce hydrogen sulfide as a product of peptone fermentation. The SIM medium contained ferrous ammonium sulfate as an indicator of hydrogen sulfide presence. When hydrogen sulfide reacts with the ferrous ammonium sulfate, an insoluble black ferrous sulfide precipitate forms. If the bacteria are nonmotile, the black precipitate will only exist along the line where the bacteria were stabbed into the media, but if the bacteria are motile, the black precipitate will be spread through out the media. The unknown bacteria were found to produce hydrogen sulfide and to be motile because the black precipitate was spread throughout the media. This test is illustrated on page 157 of the lab manual. The next test was the Citrate Utilization test that is illustrated on page 150.
The purpose of the Citrate Utilization test is to determine if a type of bacteria can use citrate as a carbon source in the absence of lactose and glucose. The enzyme citrate permease functions to transport citrate into the cell where the enzyme citrase acts on it to produce oxalacetic acid and acetate. These two chemicals are then enzymatically changed into pyruvic acid and carbon dioxide, which makes the medium alkaline. The alkaline condition of the media causes the bromothymol blue indicator in the media to turn Prussian green. For bacteria to be citrate positive, it has to have growth on the slant of the tube with blue coloration to the media. A citrate negative result is when there is no growth on the slant and the media remains green. The unknown bacteria grew on the slant and turned the media blue signifying that it is positive for the use of citrate as a carbon source in the absence of glucose and lactose. The next test was the Phenol Red Lactose test which determines whether bacteria release acidic byproducts after fermentation of lactose. At this point, I hoped my bacteria would be lactose fermentation positive.
The Phenol Red Lactose test is designed to determine if a bacteria releases acidic byproducts of fermentation of lactose. The media is phenol red broth that includes the pH indicator phenol red. The phenol red indicator turns yellow in the presence of pH levels below 7.0. The bacteria are inoculated into the media via the inoculating loop and left to grow for 24 hours. After this period, if the media is yellow, this means that the fermentation of lactose creates acidic products and is a positive reading. If the media remains red, the fermentation of lactose either didnít produce acidic products or lactose fermentation didnít occur and the reading is negative. The unknown bacteria turned the media yellow and produced a positive result on this test. This means that my unknown bacteria can either be a Citrobacter, Enterobacter, or Hafnia depending on the outcome of the methyl red test.
The methyl red test, as outlined on page 148 in the lab manual, is used to determine the ability of microorganisms to oxidize glucose with the production and stabilization of high concentrations of acid end products. A positive result on this test occurs when the methyl red indicator stays red. The media used is MR-VP broth that contains peptone, dextrose, and Potassium phosphate that are utilized by the bacteria for growth. The dextrose is the sugar used for fermentation. Following a 24 hour incubation period, the media is tested using methyl red indicator. This indicator behaves contrary to phenol red indicator because the phenol red indicator turns yellow in pH levels less than 7.0 while the methyl red indicator turns red. The result for the unknown bacteria was positive, meaning that the bacteria produced a high concentration of acidic products causing the indicator to remain red. This means that my unknown bacteria are of the Citrobacter genus. Through use of Bergeyís Manual, I found that my Citrobacteria were actually Citrobacter freundii. They resemble E. coli and produce hydrogen sulfide, which makes them different form all other Citrobacter.
Citrobacter freundii are not considered an enteric pathogen because they are of the normal intestinal flora. They have no medical interest, but have been suspected of diarrhea and possible extra-intestinal infections. Other members of the genus Citrobacter have been associated with pathogenic diseases such as meningitis caused by C. diversus.

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