15: Oxygen Requirements

Learning Outcomes

By the end of this lab period, you will be able to

• Classify organisms by their oxygen requirements based on their growth in anaerobic chambers and BHI Shake Tubes
• Determine if the conditions within an anaerobic chamber are truly anaerobic

Introduction

When Earth was formed 4.8 billion years ago, there was no oxygen gas (O₂) in the atmosphere. The first organisms to appear on Earth did not use oxygen and were in fact, poisoned by it. Oxygen gas is a very reactive molecule, prone to forming free radicals, singlet oxygen, and other reactive oxygen species (ROS). These ROS can react with biological molecules and damage them.

When the levels of oxygen gas in the air began to increase as a result of early photosynthesis, most of the organisms on earth, all of them obligate anaerobes, became extinct. This was the first great extinction event on Earth (although it wasn’t the last). But evolution is a powerful force and life persisted. Some organisms adapted to this new environment by producing enzymes such as superoxide dismutase and catalase that are capable of detoxification of ROS, allowing organisms to survive. Many of these organisms evolved the ability to use oxygen gas to generate substantially more energy from metabolic pathways such as glycolysis. These organisms survived and thrived in the new atmosphere. There are, however, environments on Earth that remained oxygen-free, and obligate anaerobes continue to live there - think of deep-sea hydrothermal vents, swamps, deep lakes, etc. And, inside larger multicellular organisms such as humans, the bacteria that thrive in our digestive systems are facultative anaerobes, capable of living without oxygen.

In today’s lab, we will explore the oxygen requirements of several common microorganisms and use two methodologies to determine whether they are strict anaerobes, strict aerobes, facultative anaerobes, or aerotolerant organisms.

Anaerobic Chambers

One way to determine whether or not an organism can survive in the presence or absence of oxygen is to attempt to grow the organism in an oxygenated or oxygen-free environment. In our lab, our incubators are exposed to normal atmospheric conditions, and thus microorganisms that grow in them can either tolerate, prefer or require oxygen. To create environments that exhibit an absence of oxygen, we place petri plates in a sealable chamber with a "gas pack" that will react with oxygen thus removing it from the chamber. The presence or absence of oxygen is determined by a "test strip" which turns blue when oxygen is present and white when it is not.

Figure \(\PageIndex{1}\): A. Anaerobic Chamber for incubating Petri plates in the absence of oxygen. B. Sealed "Gas Pak" for removing oxygen from the chamber. C. Test strips are to be placed in an anaerobic chamber to detect the presence or absence of oxygen. A blue indicator strip means that oxygen is present. When the indicator strip turns white, oxygen has successfully been removed from the container.

Shake Tubes

Brain-heart infusion (BHI) agar deeps can also be used to evaluate the oxygen requirements of an organism. The deeps are first melted, and then the temperature is equilibrated to 60^oC or less (warm enough to remain melted, but not so hot that bacteria will be quickly killed). Several drops of liquid culture are dropped into the molten agar and the tube is mixed well (“shake”) to evenly distribute the organisms into the media. The tube is then cooled so that the agar solidifies. This process creates an oxygen gradient, where there will be atmospheric concentrations of oxygen at the top of the tube and very little oxygen present at the bottom of the tube. The pattern of growth in the tube can be used to determine what levels of environmental oxygen the organism is adapted to.

Figure \(\PageIndex{2}\): Diagram of growth patterns in BHI Shake Tubes of organisms with different oxygen requirements

Figure \(\PageIndex{3}\): Photograph of BHI shake tubes. Note how growth is only observed at the top of the tube inoculated with an obligate aerobe. The aerotolerant organisms demonstrates uniform growth from top to bottom. For the obligate anaerobe, note the absence of growth at the very top of the tube where oxygen is present. We also typically see large cracks in the agar as a result of gas production by the anaerobic microorganism. Facultative anaerobes grow best in the presence of oxygen as evidenced by the thick layer of growth at the top of the tube, however, they can also grow (more slowly) in the absence of oxygen. Gas can also be produced in these organisms as a result of fermentation.

Materials

• 6 molten tubes of BHI agar equilibrated to 60^oC in a dry bath.
• Inoculating loop
• Bunsen burner
• 2 TSA plates
• Anaerobic jar
• Gas packs
• Oxygen indicator strips
• Broth cultures of:
  • E. coli (facultative anaerobe)
  • Staphylococcus aureus (facultative anaerobe)
  • Pseudomonas aeruginosa (obligate aerobe)
  • Clostridium sporogenes (obligate anaerobe)
  • Enterococcus faecalis (facultative anaerobe)
  • Lactococcus lactis (facultative anaerobe)

Experiment

Day 1

BHI Shake Tubes

1. Retrieve 6 molten BHI agar tubes from the dry bath one at a time as you inoculate them. The agar will solidify if you take it out of the dry bath and allow it to cool down for too long.
2. Using a sterile transfer pipette, inoculate each tube with 3 drops of one of the six organisms below. Be sure to resuspend the organisms in the broth cultures thoroughly prior to inoculating. After aseptic transfer, mix the bacteria into the molten culture thoroughly (but without inverting the tube!).
   1. E. coli (facultative anaerobe)
   2. Staphylococcus aureus (facultative anaerobe)
   3. Pseudomonas aeruginosa (obligate aerobe)
   4. Clostridium sporogenes (obligate anaerobe)
   5. Enterococcus faecalis (facultative anaerobe)
   6. Lactococcus lactis (facultative anaerobe)
3. Cool the tube in ice (or by incubating it on the lab bench after inoculation) to solidify the agar.
4. Incubate tubes at 37 C for 48 hours or at 30^oC for 5 days.

Anaerobic Chambers

1. For each group of two students, you need two TSA plates. Divide each plate into 6 sections and perform a line inoculation with one organism in each section. After inoculation, you should have two, identical plates (Figure \(\PageIndex{4}\))

Figure \(\PageIndex{4}\)

1. E. coli (facultative anaerobe)
2. Staphylococcus aureus (facultative anaerobe)
3. Pseudomonas aeruginosa (obligate aerobe)
4. Clostridium sporogenes (obligate anaerobe)
5. Enterococcus faecalis (facultative anaerobe)
6. Lactococcus lactis (facultative anaerobe)
7. Incubate one plate at 37 ^oC for 48 hours or at 30 ^oC for 5 days.

Incubate the other plate anaerobically at 37 ^oC for 48 hours or at 30 ^oC for 5 days in an anaerobic jar with a palladium catalyst. Use the blue indicator strip to determine if the oxygen is removed from the jar. The blue strip will turn white under anaerobic conditions.

Data

Compare the growth on the plates to the growth in the BHI Shake Tubes. Is the pattern of growth for each organism consistent between the two methodologies? Compile your results into the table below.

Data Table:

Table 1: Student Data Table

Organism	Location of growth in BHI tube	Gas Production?	Growth on Aerobic Plate	Growth on Anaerobic Plate	Aerotolerance category
E. coli
P. aeruginosa
S. aureus
C. sporogenes
L. lactis
E. faecalis

Questions

1. Determine the aerotolerance category for each of the six organisms we looked at based on the data we collected. Now, look each up online or in the Gideon Guide. Do the categories match what you observed?
2. Where in/on the human body would you expect to find an obligate aerobe? What about a facultative anaerobe? Obligate anaerobe?
3. Which one of these two techniques did you find most useful for identifying aerotolerance categories? If you could only use one, which one would you choose?