Lab Report

Microbiology-2460

Lab-003

March 31,2008

Lab Report-Escherichia coli

Abstract

The purpose for this lab report was to identify and inform of an unknown bacteria that has been causing a patient to have lower abdominal and pelvic pain. To obtain the identification of this unknown bacterium, several biochemical tests needed to be performed in order to prescribe the correct medication to treat and cure the symptoms.

Introduction

In a lab today, I am to identify an unknown bacterium that is causing a patient lower abdominal pain. There are six different organisms that the unknown bacteria could be. They are Eschericha coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aerogenosa, and Salmonella typhimurium. I will be using several different types of biochemical testing. I am using this type of testing because it differentiate and sometimes identify microorganisms based on specific biochemical characteristics.

A gram stain preparation was done first to confirm that the unknown bacterium was gram negative. From here a TSA slant and a TSA plate (for backup) was inoculated with the bacteria and then incubated at 37Ð'Ñ"C between 24-48 hrs. Afterwards, they were observed for growth and used to perform a series of eight different tests to identify the unknown.

Material and Methods

Eight different prepared types of media were provided to use to identify the unknown bacterium. They were a Gelatin Tube, MR Tube(Methyl Red), VP Tube(Voges-Proskauer) Urea Tube, SIM Tube(Sulfur Indole Motility), Citrate Slant, TSIA Slant (Triple Sugar Iron Agar) and Oxidase. Each one of the tubes and slants were inoculated using either the streaking or stabbing technique with one of the colonies from the growth of the unknown bacterium.

The Gelatin tube was incubated for up to a 48 hrs at 37Ð'Ñ"C. It was afterwards place in 27Ð'Ñ"C for about 1 hr to see if it solidifies.

The MR Tube was incubated for 48 hrs at 37Ð'Ñ"C. Afterwards 3 drops of Methyl Red reagent was place into the tube to observe for color change. Based on the result of a color change or not would determine if it was positive=mixed acid fermentation occurred or negative= no mixed fermentation occurred.

The VP Tube was incubated for 48 hrs at 37Ð'Ñ"C. Afterwards 15 drops (0.6 ml) Reagent A and 5 drops (0.2ml) Reagent B was added to tube to observe for color change in an hour. If a color change occurred the results would be positive=2,3-butanediol fermentation (acetoin produced) or negative=no 2,3-butanediol fermentation (acetoin is not produced).

The Urea Tube was incubated for 24 hrs at 37Ð'Ñ"C. Afterwards it was observed for color change. A positive result would have a color change of pink=rapid urea hydrolysis, strong urease production and orange or yellow=no urea hydrolysis, organism does not produce urease or cannot live in broth.

The SIM Tube was incubated for 24-48 hrs at 37Ð'Ñ"C. Afterwards it was observed for spreading from the stab line, and black precipitate in the medium. Next Kovacs Reagent was added to tube and after a minute observed for the formation of red color in the reagent layer. The results for Sulfur present would be positive if black in the medium=sulfur reduction (H2S Production), negative if no black in medium=Sulfur is not reduced. For Indole present the results would be positive if red in the alcohol layer of Kovac’s Reagent= tryptophan is broken down into indole and pyruvate, and negative if Reagent color is unchanged= Trytophan is not broken down into indole and pyruvate. The result for Motility would be positive if growth radiating outward from the stab line= motility, and negative if no radiating growth=nonmotile.

The Citrate Slant was incubated for 48 hrs at 37Ð'Ñ"C . Afterward, it was observed for color change and growth. A positive result would be a blue color change=citrate is utilized and no color change or growth would also indicate positive results. A negative result would be no color change no growth=citrate is not