The Long Awaited Answer to My Unknown

The Long Awaited Answer to my Unknown

Johnny Tran

August 2, 2006

BIOL 3444-002

Unknown #12

Introduction

Bacteria are microscopic unicellular prokaryotic organisms characterized by the lack of a membrane-bound nucleus and membrane-bound organelles. They are remarkably adaptable to diverse environmental conditions and are found in bodies of all living organisms and on all parts of the earth. The purpose of microbial biochemical tests is to identify the unique traits it yields and with that knowledge we can then categorize them in groups and specify them by scientific name. These experiments included the Triple-sugar iron agar (TSIA), Sulfur Indole Motility (SIM), Methyl Red (MR), Voges-Proskauer (VP), Citrate, Urease, Gelatin, and Oxidase Test. In order for these tests to produce reliable and credible results, the bacterium organism must be grown using strict and meticulous procedure to produce viable colonies of pure culture. Having pure culture is significant to ensure that a single type of bacteria is used for identification without contamination so tests can be run without complications or confusion. Once all these tests are performed, the unknown bacteria in this lab will be one of the following: Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Klebsiella pneumoniae, or Salmonella typhimurium. This report included the results and details to these experiments which are discussed further on.

Abstract

Gram negative bacteria Unknown #12 was run through an array of tests which produced positive and negative results. The results obtained from the various tests were used for the process of elimination with the use of our Gram Negative Unknown chart to conclude that unknown #12 was Proteus mirabilis. This report will include information regarding the methods and materials used as well as the discussion about the biochemical tests and the results.

Materials and Methods

The first step in the process of identifying the unknown bacteria was to transfer the bacteria from a random unknown broth medium test tube onto a TSA (Trypticase Soy Agar) plate using the T-streak method as well as aseptic methods to check for contamination and colony morphology. A TSA slant was also streaked at the top for back-up purposes incase the TSA plate became invalid due to reasons such as contamination of the TSA plate.

The second step was to inoculate the bacteria into each test. After inoculation, all media are placed in the hot room which is approximately 37ўЄC, and if placed it the cold room, the temperature is approximately 4ўЄC. Each test had a different technique of getting the end result and this part of the report identifies each of those methods as well as their inoculation times spent in their designated temperature rooms:

The TSIA Test: The triple-sugar iron agar media was used to indicate which sugar had been fermented by the organism depending on where the color change took place between the slant and the butt. This test also determined if the organism produced hydrogen sulfide and gas as by-products of fermentation. Phenol red was used as an indicator for this experiment and the bacterium is inoculated by the stab-streak method. The notation for stating whether the slant or the butt fermented are K for alkaline (red/pink), and A for acid (yellow). Notation of the change are K/K, K/A, and A/A with the first being the slant and the second being the butt respectively. Incubation will be checked after 6 hours and 24 hours of incubation for changes.

The SIM Test: The Sulfur Indole Motility media was used to conduct three simultaneous tests. The media was inoculated by stabbing the bacteria into the media. These tests were incubated for 24 hours in the hot room.

1) Sulfur reduction test: tested for hydrogen sulfide production. Black meant that the test was positive for hydrogen sulfide production.

2) Indole test: tested for indole production after several drops of KovakЎЇs reagent was added. The color on top of the media was the color used to determined test result. Red meant it tested positive and brown meant that it was negative.

3) Motility: this test was determined by the growth around the stab area and by cloudiness on top of the media which was positive for motility.

The MR Test (mixed acid fermentation test): The Methyl Red liquid media was inoculated using the inoculating loop and incubated for 24 hours for a color change. Red meant that the test was positive for mixed acid fermentation, and yellow meant that it was negative.

The VP Test (butanediol fermentation test): The Voges-Proskauer test tested for the presence of butanediol. The liquid media was simply inoculated using the inoculating loop and incubated for 4 days before 18 drops of BarrittЎЇs A and BarrittЎЇs B were added. A pink/red color change meant that the test was positive for production of butanediol.

The Citrate Test (Simmons Citrate agar): The citrate test was used to detect if organisms could utilize citrate as a sole carbon source. The slant was inoculated by streaking the surface and no reagents were added to this experiment. A color change from green to Prussian blue meant that it was positive for utilization of citrate.

The Urease Test: The urease test tested for the presence of the urea hydrolyzing enzyme urease. The liquid media was simply inoculated using an inoculating loop and incubated for up to 8 days. A color change to cerise meant that it was positive for the presence of urease.

The Gelatin Test: The gelatin tested for organisms that produced the metabolic digestive enzyme, gelatinase. After inoculation of the liquid media, it was incubated in the hot room for up to 8 days. If gelatinase was produced, it hydrolyzed gelatin and produced liquefaction, gelatin would not return to solid state when cooled. If the media returned to solid after cooling in the cold room for 30 minutes, the test was negative for gelatinase production.

The Oxidase: The oxidize test tested for the presence of cytochrome oxidase. The unknown bacteria is inoculated onto a piece of filter paper and droplets of 1% oxidase reagent are added. The test was positive if the culture turned from yellow to purple. If the culture remained the same, it was negative for cytochrome oxidase production.