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 byproducts 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 O 2 to H 2O. 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 O 2, 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
Culture of Clostridium sporogenes
Culture of Staphylococcus aureus
Culture of Escherichia coli,
Culture of Bacillus subtilis
Culture of Micrococcus luteus
Individual supplies
3 tubes of fluid thioglycollate medium (FTM)
Wire loop
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
Strict aerobes
Test organisms
B. subtilis
M. luteus
S. aureus
E. coli
C. sporogenes
Facultative anaerobes
Strict anaerobe
II. Anaerobic jar
Class supplies
Anaerobic jar
Team supplies
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:
Strict aerobe
Facultative anaerobe
Strict anaerobe
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).
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.