Do a lab summary of minimum 300 words.

Oxygen requirements

Depending upon the type of metabolic process or processes used for growth by a particular species of microorganism, that organism will display a characteristic dependency for, or sensitivity toward oxygen. By inoculating a tube of melted agar cooled to 50 degrees Celcius (ie. a “tempered” agar deep), and that is sufficiently deep enough to prevent oxygen from diffusing to the bottom of the tube, we can determine an organisms’ oxygen requirement. We use normal hard agar for this test (15 grams agar/liter as is used in plates or slants) rather than soft agar as was used for motility. The higher agar content helps to limit oxygen diffusion into the tempered deep. The result is an oxygen gradient with high oxygen at the top of the tube and decreasing to extinction toward the bottom. The goal is that agar in the top of the tube be highly aerobic and the agar below some point in the tube be anaerobic.

Organisms that require oxygen as terminal electron acceptor for the process of respiration, and have no other choices are called obligate aerobes (they are obliged to use oxygen). Examples of obligate aerobes that we will use this semester include Bacillus megaterium, Bacillus subtilus, Pseudomonas aeruginosa, Alcaligenes faecalis and Acinetobacter lwoffi. Most obligate aerobes prefer an oxygen concentration similar to that present in air. However, some others prefer a lower concentration of oxygen and cannot survive with the concentration present in air. These obligate aerobes are referred to as microaerophiles, and include the clinically significant genus Campylobacter.

Some bacterial species do not necessarily require oxygen for survival & growth. Some fermenters will grow in the presence of oxygen although they do not use oxygen in their metabolic processes. These species are referred to as aerotolerant anaerobes (they tolerate, but do not require oxygen). All species of the Genus Streptococcus are in this group. Streptococcus faecalis grows equally well in the presence or absence of oxygen. Streptococcus pneumonia will grow in the presence of oxygen but prefers an anaerobic environment. A few fermenting species, and species that use a very electronegative terminal electron acceptor for the process of anaerobic respiration (such as the Methanogens) cannot survive in the presence of oxygen. These species are referred to as obligate anaerobes. Anaerobic species can be cultured using media/methods which limit exposure of the organism to free oxygen. A commonly used method in medical labs is the addition of reducing agents such as sodium thioglycolate or cysteine to media. These compounds remove oxygen gas which is dissolved in liquid media by reducing that oxygen to water. Another method is to place the media inside an anaerobic chamber (glove bag) or anaerobe jar. These are air tight compartments containing chemicals which reduce oxygen in the air. Freshly autoclaved media is oxygen free, and if placed in one of these chambers, will remain anaerobic. Aerated media can be made anaerobic after-the-fact. As oxygen diffuses out of the media into the surrounding air, the oxygen is consumed. Eventually the media becomes anaerobic. Oxygen sensitive organisms such as obligate anaerobes can be handled in the anaerobic chamber to protect them from air.

Facultative organisms (often referred to as facultative anaerobes in an attempt to confuse microbiology students) are those that will respire aerobically in the presence of oxygen, but will ferment (or respire anaerobically, but we don’t generally deal with these in clinical micro) in the absence of oxygen. These organisms have choices. All the organisms that you will see in this lab other than those mentioned in the paragraphs above are facultative. That includes all of the enteric genera, Aeromonas, Staphylococcus, Bacillus cereus, Listeria and Corynebacterium.

You are going to inoculate agar deeps with organisms to determine in which of these groups they fit based upon oxygen requirements. The agar media in the deep limits the diffusion of oxygen resulting in a gradient of oxygen concentration: lots at the top & little-none at the bottom. Lets say that you inoculated 2 agar deeps as described below, one with an aerotolerant anaerobe and the other with a facultative organism. In both deeps, the organisms should grow from bottom to top. In other words, they will both grow throughout the entire range of oxygen concentrations in the tube. How can you tell one from the other? Respiration is a much more efficient form of metabolism than fermentation. The facultative organism is respiring aerobically at the top of the tube and fermenting at the bottom, therefore more growth should be present at the top than the bottom. Since the aerotolerant anaerobe is fermenting throughout the tube, there should be less growth overall than in the case of the facultative organism. Furthermore, since fermentation is occurring throughout the tube, the amount of growth should be equal throughout, or there should be more growth at the bottom than at the top. The latter is true of a somewhat oxygen sensitive fermentor such as Streptococcus pneumoniae. These rules may not work if you leave the tubes in the incubator too long, or if there is oxygen throughout the tube for some reason. Think about it. An aerotolerant anaerobe, although growing at a slower rate than the facultative, will eventually “catch up” to the facultative organism, and can be difficult to distinguish for this reason. But wait! There is another way to tell them apart. Look on the top of the agar in incubated tubes; not in the agar, but literally on the agar. Facultatives (and obligate aerobes) love to grow on top of the agar where the greatest amount of oxygen is present. Aerotolerant anaerobes will not be happy on top of the agar.

For this procedure we use tempered (agar that is held at 500C so it is molten) TSA agar deeps. Again, these are NOT soft agar, but agar that contains 15 grams agar/liter. You must inoculate the media while molten, so do not take the deep out of the water bath until you are ready to inoculate it. Collect a healthy size wad of inoculum on your loop. Mix the inoculum throughout the media (from top to bottom) being gentle so not to mix oxygen into the media. Allow the agar to cool and harden. Incubate the tube at 370C for 24hrs. Examine your deep for growth both throughout the agar as well as on top of the deep. If the results are inconclusive, incubate for another 24 hours. See the image “Oxygen requirements left to right: facultative, obligately aerobic (unclear in image), and aerotollerant anaerobe (fermenter).”

NOTES

* Oxygen requirement testing utilizes “tempered” agar deeps. These are NOT soft agar deeps. They are

regular hard agar deeps (15gm agar/liter) that have been melted and cooled to 50oC.

* Unlike motility tests in soft agar deeps, O2 requirement deeps should be checked after 24hrs

incubation, and maybe again at 48hrs. You CAN over incubate oxygen requirements tubes.

* Don’t simply look IN the agar, but also look ON the agar (ie. the top of the tube). Any organism

capable of aerobic respiration (obligate aerobes and facultatives) will love to grow on top of the agar.

* Organisms that produce gas within the agar (as many enterics will do) will cause the agar to separate

where gas pocket form. This is fine. You didn’t do anything wrong.

* Sometimes certain obligately aerobic organisms will not grow in oxygen requirement media,

presumably due to the sensitivity of their oxygen requirement. One such organism is Bacillus

megaterium.

* Just like with motility tubes, oxygen requirements results are more easily interpreted while the media

is warm (directly out of the incubator). Refrigeration causes the agar to become cloudy. This is a

problem.