Gram Negative Lab Report

INTRODUCTION
The Gram Negative lab had two parts; the aim of the first part is to determine the concentration of gram negative bacteria in a water sample collected from a creek near Providence Road, Strickling. Gram negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an additional outer membrane composed of phospholipids and lipopolysaccharides. Because gram negative bacteria have a relatively thin cell wall when compared to gram positive organisms, they are consequently unable to retain the color of the crystal violet dye during gram staining (Barron, 1996). Two enumeration techniques were used during this part: Direct Count Procedure and Viable Count Spread Plate Procedure.
The aim of the second part of the experiment was to isolate enteric coliform bacteria from the water sample and determine its identity using biochemical testing. Enteric bacteria, members of the Enterobacteriaceae family, reside in the guts of animals, such as humans, and are usually harmless. Some of them, on the other hand, are pathogenic and are responsible for infections, food poisoning and plaque. Coliforms are bacteria that are always present in the
environment, digestive tract of humans and other animals, and water sources (Garrity, 2005). The presence of coliform in water is a determinant that the water has been contaminated with fecal matter or sewage; when this water is ingested without proper treatment, gastrointestinal infections occur (Rinehart-Kim & Hynes, 2016). To avoid the contraction of such diseases, the EPA has set water quality standards, statements that describe water quality requirements for various uses.

MATERIALS AND METHODS
A water sample from a creek near Providence Road, Strickling was collected and two enumeration procedures were performed. During the Direct Count Procedure, a drop of the water sample was placed on the counting chamber and visualized under a microscope. The number of bacteria seen in 3 of squares was counted by 2 different people. The results were then averaged out. For the Viable Count Spread Pate, the water was diluted to the following factors: 10-1, 10-2and 10-3. Three tubes, containing 4.5mL of sterile water, were labelled with 10-1, 10-2, and 10-3. 0.5mL of the water sample was aseptically transferred into the 10-1 tube and thoroughly mixed. 0.5mL was then transferred from 10-1 to 10-2. The procedure was repeated from 10-2 to 10-3. Samples from each dilution were inoculated on three MacConkey Agar plates, and the plates were incubated at 37oC for 48 hours. After incubation, the colonies observed on the MacConkey Agar plates were counted and recorded.
After the enumeration was done, several biochemical tests were conducted to determine the identity of the bacteria. An isolated bacterial colony was then T-streaked on a Trypticase soy agar (TSA) plate and then incubated at 37oC for 48 hours. The isolated colonies observed on the plate was used for biochemical testing. For the Cytochrome Oxidase Test, 2-3 drops of oxidase test reagent was added to a filter paper and some bacteria was then added. It was allowed to stand for 30 seconds and then checked for color change. During the Indole Tryptophan test, a tryptophan broth was inoculated with bacteria and incubated at 37°C for two days. 15 drops of Kovac’s reagent was then added. A color change to cherry red was then checked for. A Simmons Citrate agar slant was lightly inoculated with bacteria and incubated at 37°C for 48 hours. It was then checked for color changes. For the Carbohydrate Fermentation Test: Three phenol tubes, each containing glucose, lactose and sucrose, were inoculated with the bacteria and incubated at 37°C for 48 hours. It was then checked for color change and production of gas. The Nitrate Test was subsequently done: A nitrate broth was inoculated with the bacteria and incubate for 48 hours at 37oC. The broth was then checked for gas formation, and 5 drops of solution A and B were added.
3ml of MR/VP broth medium was inoculated with the microorganism and incubated for 48 hours at 37oC.
5 drops of methyl pH red indicator was then added. During the Triple Sugar Iron Agar (TSI) test, A TSI slant was inoculated with bacteria at 37oC for 48 hours. Color changes and production of gas were then checked for. For the Phenyl Alanine Deaminase test, a phenylalanine slant was inoculated and incubated at 37oC for 48 hours. 5 drops of 10% Ferric Chloride was then added, and was then checked for color change. The Decarboxylase Test was then conducted: Three test tubes of decarboxylase base, one containing no amino acid, and the other two had Lysine and Ornithine. Each test tube was inoculated with the bacteria and then overlaid with a thin layer of mineral oil. The tubes were then incubated at 37oC for 48
hours.
For the Oxidation Fermentation Test, Two tubes of O/F were inoculated with a needle. One tube from each pair was then overlaid with 10mm of mineral oil. The tubes were then incubated for 48 hours at 37oC. During the Urease Test, The Urea agar slant was inoculated with the bacteria in a zig zag pattern and was then incubated at 37oC for 48 hours. A color change was then checked for. An Eosin Methylene Blue (EMB plate was sectioned and inoculated with bacteria. The plate was then incubated at 37oC for 48 hours. An MR/VP broth was inoculated with the bacteria and the incubated at 37oC for 48 hours. 30 drops of -naphthol was added and the solution was thoroughly mixed. 10 drops of KOH was then added. The tubes were incubated in a slanted position for one hour.