BIOS 242 Week 7 Assignment; Lab 13 of 14 Onsite; Bacteria of Digestive System

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Institution	Chamberlain
Contributor	Nikki

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Lab 13: Bacteria of the Digestive System

Learning Objectives

Identify how selective and differential media can be used in the identification of bacteria that can inhabit the digestive tract.
Identify the growth of samples of presumed fecal bacteria from rest room swabs.

Introduction

The human intestinal tract is inhabited from birth by a variety of microorganisms acquired, at first, from the mother. Later, microbes are ingested in food and water, and from hands and objects placed in the mouth. Many of those do not survive the acidic pH of the stomach, which helps prevent infection from pathogens. However, those that do survive find the environment in the small intestine and colon more hospitable. Generally, our normal intestinal microbiota are harmless and are beneficial in two ways: they outcompete potential pathogens for space and nutrients; and produce vitamins, including vitamin B₁₂, folic acid, biotin, and vitamin K, that we cannot synthesize ourselves.

Most of the intestinal bacteria are anaerobic. However, when feces are cultured, facultative anaerobic bacterial species can be observed. These normal inhabitants of the intestinal tract are Gram-negative, non-spore forming bacilli. They grow rapidly and well under aerobic conditions on most nutrient media. The use of selective and differential culture media allows their isolation and identification.

In this experiment three bacteria that are often part of the normal intestinal microbiota, Escherichia coli, Enterobacter aerogenes, and Serratia marcescens will be investigated using selective and differential media, and carbohydrate fermentation.

Selective media typically contain substances that either favor the growth of certain microorganisms or inhibits the growth of others. Differential media are formulated so that the growth of particular organisms will result in a change in the color of the media or a change in the color of the bacterial colonies.

MaConkey agar (MAC) is both a selective and differential medium. Crystal violet and bile salts inhibit the growth of Gram-positive microorganisms, so only Gram-negative bacteria will grow. This medium contains lactose as its sugar source. Lactose fermenters will metabolize this sugar and secrete lactic acid as a waste product of lactose fermentation. This causes a localized drop in the pH around the colony, which, in turn, causes a pH indicator, neutral red, in the agar to turn bright pink. Colonies also appear pink. Non-lactose fermenters cannot utilize lactose and therefore do not cause a change in the pH. These colonies appear white on MacConkey agar, and the agar plate background turns yellow from the basic waste products normally produced by cells. The colonies of weak lactose fermenters may be pink, but the agar is yellow.

Eosin Methylene Blue agar (EMB) is also a selective and differential medium. It is typically used to isolate fecal bacteria. The Eosin and Methylene Blue in EMB inhibit the growth of Gram-positive bacteria. Strong fermenters of sucrose or lactose will produce large amounts of acid, turning the microorganisms dark purple or black. Colonies of E. coli, an active fermenter, often appear to have a green metallic sheen. Slow or weak fermenters will produce pink colonies. Non-fermenters will either be colorless or have their normal color.

In a previous laboratory Carbohydrate Fermentation of glucose (dextrose) and sucrose by S. epidermidis,

E. coli, S. marcescens, and B. subtilis was investigated. In review, the broth contains a single carbohydrate (such as lactose, glucose, and sucrose). A pH indicator, phenol red, is in the broth to detect the presence of acids as an end product of carbohydrate fermentation. When acid is produced, the pH of the broth is lowered, turning the pH indicator from red to yellow. In addition, each tube contains a small inverted tube (Durham tube). The purpose of this tube is to catch gas bubbles that may be produced during the fermentation process. The presence of a bubble in the top of the Durham tube is positive for gas production. The ability of the microorganisms to ferment lactose or glucose will be investigated. The glucose fermentation by the bacteria previously studied will be confirmed, with new data for lactose and glucose fermentation by E. aerogenes.

Environmental Sampling (MUST be set-up as a demonstration by the instructor): Swabs from a toilet rim will be cultured on EMB and MAC agar and grown at 37^◦C for 24-48 hours. Students will observe the resulting growth.

Note: Use personal protective equipment (PPE) at all times. Follow instructions at all times. Do not open the plates containing toilet swab sample.