[Microbiology] Atlas of Aerotolerance

Aerotolerance, Atlas of Aerotolerance, A Photographic Atlas for the Microbiology Laboratory



Purpose
The two procedures discussed here—agar deep stabs and agar shakes—are good visual indicators of oxygen tolerance (aerotolerance) in microorganisms.

Principle
Most microorganisms can survive within a range of environmental conditions, but not surprisingly, tend to produce growth with the greatest density in the areas where conditions are most favorable. One important resource influencing microbial growth is oxygen. Some organisms require oxygen for their metabolic needs. Some other organisms are not affected by it at all. Still other organisms cannot even survive in its presence. This ability or inability to live in the presence of oxygen is called aerotolerance. 

Most growth media are sterilized in an autoclave during preparation. This process not only kills unwanted microbes, but also removes most of the free oxygen from the medium as well. After the medium is removed from the autoclave and allowed to cool, the oxygen begins to diffuse back in. In tubed media (both liquid and solid) this process creates a gradient of oxygen concentrations, ranging from aerobic at the top, nearest the source of oxygen, to anaerobic at the bottom. Because of microorganisms’ natural tendency to proliferate where the oxygen concentration best suits their metabolic needs, differing degrees of population density will develop in the medium over time that can be used to visually examine their aerotolerance. 

Obligate (strict) aerobes, organisms that require oxygen for respiration, grow at the top where oxygen is most plentiful. Facultative anaerobes grow in the presence or absence of oxygen. When oxygen is available, they respire aerobically. When oxygen is not available, they either respire anaerobically (reducing sulfur or nitrate instead of oxygen) or ferment an available substrate. Refer to the Appendix and Section 7 for more information on anaerobic respiration and fermentation. Where an oxygen gradient exists, facultative anaerobes grow throughout the medium but are more dense at the top. Aerotolerant anaerobes (or simply “aerotolerants”—not to be confused with “aerotolerance”), organisms that don’t require oxygen and are not adversely affected by it, live uniformly throughout the medium. Aerotolerant anaerobes ferment even in the presence of free oxygen. Microaerophiles, as the name suggests, survive only in environments containing lower than atmospheric levels of oxygen. Some microaerophiles called capnophiles can survive only if carbondioxide levels are elevated. Microaerophiles will be seen somewhere near the middle or upper middle region of the
medium. Finally, obligate (strict) anaerobes are organisms for which even small amounts of oxygen are lethal and, therefore, will be seen only in the lower regions of the medium, depending on how far into the medium the oxygen has diffused.

Agar deep stabs are prepared with Tryptic Soy Agar (TSA) enriched with yeast extract to promote growth of a broad range of organisms. Oxygen, which is removed from the medium during preparation and autoclaving, immediately begins to diffuse back in as the agar cools and solidifies. This process creates a gradient of oxygen concentrations in the medium, ranging from aerobic at the top to anaerobic at the bottom.

Agar deeps are stab-inoculated with an inoculating needle to introduce as little air as possible. The location of growth that develops indicates the organism’s aerotolerance (Figure 3-38).

Agar shakes are also prepared with enriched TSA, but differ from agar deep stabs in that, after autoclaving, they are cooled to 45°C and placed in a warm water bath until time for inoculation. Agar shakes are inoculated in liquid form, mixed gently to distribute the bacteria evenly throughout the medium, and allowed to solidify. Like agar deep stabs, the location of growth that develops in agar shakes indicates the aerotolerance of the organism (Figure 3-39). 

AGAR DEEP STAB TUBES
3-38 AGAR DEEP STAB TUBES
From left to right: Clostridium butyricum (strict
anaerobe), Staphylococcus aureus (facultative
anaerobe), and Pseudomonas aeruginosa (strict aerobe).  


AGAR SHAKE TUBES
3-39 AGAR SHAKE TUBES 
From left to right, Clostridium butyricum (strict anaerobe), 
Escherichia coli (facultative anaerobe), uninoculated control, and
Pseudomonas aeruginosa (strict aerobe)  

Suggested Reading
- Michael J. Leboffe & Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratory 4th edition 2011

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