Enterobacter Aerogenes

Enterobacter Aerogenes

Unknown Lab Report #73

Joshua Odom

4/17/12

Professor Yakubovskaya

MCB2010L

Introduction

There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium.

MATERIALS AND METHODS

On March 21/2012, an unknown was given as #73 by Professor Yakubovskaya. The methods that have been learned thus far for identifying bacteria have been applied to this unknown. Procedures were directed by the professor, unless otherwise noted. The first procedure that needed to be done was to streak the unknown using the quadrant streak. This needed to be done to isolate a single colony of the bacteria. The unknown seemed not to be pigmented. The unknown seemed not to be pigmented. Using the isolated colony, three media were inoculated using aseptic techniques with an inoculating loop. A slant was inoculated along with two nutrient broths using aseptic techniques with an inoculating loop. The nutrient broths were used as working stock. The slant was used as reserve stock and was stored in the refrigerator. To determine the unknown's optimum temperature, one of the nutrient broths was stored at 22C and the other nutrient broth was stored at 37C. The slant and both broths were put away until evaluated next class. On the slant, there was white, kind of solid growth. In both of the nutrient broths, the bacterial growth was on the bottom of the tube and the solution was cloudy. The spectrophotometer was used to determine the optimum temperature of growth for the unknown. Blank solutions were placed inside of the spectrophotometer as a control followed by the tube containing the unknown placed in 22C and 37C. The turbidity of the bacterial growth at 22C was 1.314A and the turbidity of the bacterial growth at 37oC was 1.046A.The results showed, the optimum temperature for the unknown to be 37C. Then, the Fluid Thioglycollate liquid medium was inoculated using the unknown broth from the optimum temperature tube with the inoculating needle. The medium was then stored at 37C to be evaluated later.

The results of the Fluid Thioglycollate test were recorded. Using pictures from the lab manual, the unknown was closely compared to the aerotolerant anaerobe. Aerotolerant anaerobes are bacteria that are capable of fermentation and respiration and can grow in the presence or absence of oxygen. The tube was cloudy of the medium and kind of bubbly on the top. Then the catalase test was performed to determine whether the unknown had the ability to produce catalase. Using the slant, some of the unknown was inoculated aseptically onto a sterile microscope slide with the loop. Hydrogen peroxide was then added aseptically using a pipette. Two drops of hydrogen peroxide were placed on the bacteria and was observed for bubble formation. Bubbles were produced which shows the unknown is positive for catalase. Next, the Oxidase test was performed to determine whether cytochrome c oxidase is present in the unknown. A DrySlide from Becton Dickinson, Sparks, MD was given by the professor. Then a sterile cotton-swab was obtained and used to collect some of the unkown from the slant. The bacteria was then transferred to one square of the slide and observed. Negative results were observed for this test because a blue color change was not shown. Then working stock with the optimum temperature was obtained. Labels were then created for Glucose broth with PR, Mannitol broth, MR-VP broth, Lactose and Nitrate broth. The professor distributed five test tubes mediums for inoculation. Each medium was inoculated using inoculating loop and then put away in the refrigerator until later evaluation.

Next, the evaluation of the five test tube mediums were did. The Glucose Phenol Red Broth was inoculated from the pure culture using aseptic techniques. After incubation, the broth had a bright yellow color. The color indicated that there was fermentation with acid and gas products The Lactose broth was inoculated from the pure culture using aseptic techniques. After incubation the broth noticeably turned hot pink from light pink and there was a bubble present. This shows the bacteria to be an acid and a gas producer. The mannitol broth was also inoculated from the pure culture using aseptic techniques. After incubation, the mannitol broth turned a hot pink color. This Methyl Red Voges-Proskauer Test was performed by transferring 1.0mL of broth to two test tubes. In one test tube three drops of the indicator methyl red was added. The broth was observed immediately for a red color change. A color change did nwhich meant the unknown was negative for mixed acid fermentation. In the second test tube 15 drops of reagent A and 5 drops of reagent B were added. During the next hour the results were recorded every ten minutes. After one hour the color of the broth remained unchanged, therefore acetoin was not produced. The Nitrate test was negative for the unknown. Each agar plate was divided in half, labeled and used with a lab partner that had the same optimum temperature. The agar plates were inoculated aseptically using the inoculating loop. The urea broth