Lab Exercise 12: Effects of oxygen on growth OBJECTIVES

1. To differentiate between oxygen requirements using anaerobic culture media.

INTRODUCTION Microbes can be classified into 5 groups based on their requirement for air that contains 20% oxygen: 1. Obligate aerobes: Require oxygen for growth via respiratory metabolism in which oxygen is the terminal electron acceptor. 2. Obligate anaerobes: Can not survive in the presence of oxygen due to lack of enzymes superoxide and catalase or peroxidase, which detoxify damaging oxygen by- products such as peroxides and superoxides. Use fermentation or anaerobic respiration. 3. Facultative anaerobes: Can grow in oxygen or without. Use oxidative respiration and fermentation/ anaerobic respiration. 4. Microaerophiles: Require small amounts of oxygen (5-10%) for respiration but can not survive in atmospheric oxygen due to limited ability to carry out respiration or presence of oxygen-sensitive enzymes. 5. Aerotolerant anaerobes: Can survive in oxygen due to presence of superoxide dismutase but do not use oxygen as a terminal electron acceptor. Use fermentation or anaerobic respiration. The oxygen requirements of a given organism can be easily determined in the microbiology laboratory by the use of both solid and liquid media. When grown on solid media, the use of an Anaerobic Jar is required. Plates are incubated within the airtight container and the trapped oxygen is converted to H2O by the addition of a GasPak. This GasPak contains chemicals which, when combined with water, will reduce atmospheric O2 to H2O. This reaction is catalyzed by palladium pellets in the Anaerobic Jar. Additionally, an anaerobic indicator strip containing methylene blue which becomes colorless in the absence of O2, is added to confirm the anaerobic environment (see figure below).

Growth in liquid media is accomplished using a differential medium called FTM (fluid thioglycollate medium). FTM is considered because, although most all bacteria will grow in it, the oxygen requirements of each class of organism can be easily discerned when incubation is complete. Along with the nutrients necessary for growth, FTM contains the reducing agent, sodium thioglycollate, which effectively removes the O2 in the medium. In addition, FTM contains a redox indicator, resazurin, which is red in its oxidized form and colorless under anaerobic conditions. A minimal amount of agar is also added which prevents the rapid uptake of oxygen and helps to maintain the stratification of organisms growing in different oxygen-containing layers of the medium: an aerobic environment on top, a microaerophilic environment just below the surface of the agar, and an anaerobic environment toward the bottom. Organisms can be classified based on the localities of their growth (see figure below).

LAB EXERCISES I. Fluid thioglycollate medium Team supplies Individual supplies Culture of Clostridium sporogenes 3 tubes of fluid thioglycollate medium (FTM) Culture of Staphylococcus aureus Wire loop Culture of Escherichia coli, Culture of Bacillus subtilis Culture of Micrococcus luteus Protocol 1. Make sure that the FTMs are fresh. Freshness is indicated by the absence of a pink color in the upper one-third of the medium.

2. Aseptically transfer one loopful of each test organism (strict aerobe, facultative anaerobe, and strict anaerobe) to each of the 3 FTMs. Each student should pick one option from each category in the list below, but make sure that all organisms are covered within each team for comparative purposes.

Aerobic requirements Test organisms Strict aerobes B. subtilis

M. luteus Facultative anaerobes S. aureus

E. coli Strict anaerobe C. sporogenes II. Anaerobic jar

Class supplies Team supplies Anaerobic jar Same cultures as above 4 agar plates Wire loop

Protocol: 1. You will work in teams to inoculate 4 test plates: 2 plates with B. subtilis, S. aureus, and C. sporogenes and two plates with M. luteus, E. coli, and C. sporogenes. Use a straight inoculation line as seen in the example below:

2. One plate from each set of inoculations will be placed in the class anaerobic jar, the other from each set will be incubated at the appropriate temperature in atmospheric oxygen (i.e. in the incubator).

Strict aerobe

Facultative anaerobe

Strict anaerobe

DATA AND OBSERVATIONS 1. Draw and describe the growth of organisms in the FTM.

Strict aerobe Facultative anaerobe Strict anaerobe Descriptions of growth in FTM: Strict Aerobe: Facultative anaerobe: Strict anaerobe: 2. Draw and compare growth on TSA plates grown aerobically and anaerobically.

Aerobic Anaerobic

DISCUSSION 1. Why can media such as thioglycollate be used for cultivation of anaerobes? 2. What are the purposes of the indicator strip and gas generator in the GasPak anaerobic jar? 3. What is the role of oxygen in cellular respiration? 4. What type of metabolism occurs in the absence of oxygen? 5. Name two enzymes that are present in obligate aerobes but lacking in oglibate anaerobes. What is the function of each enzyme? 6. Differentiate between a microaerophile and an aerotolerant organsm.