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Syllabus

• Agar slant tubes
• Aseptic technique
• Assays
• Assays Illustrated
• Colony morphology
• Gram stain
• Isolating colonies
• Making serial dilutions
• Culture media
• Preparing spread plates
• Relationship to oxygen
• Viewing Gram stains
• Viewing living bacteria

• Characterization writeup
• Lab responsibilities
• Project proposal guidelines
• Using Bergey's

Supplemental pdfs

• Inoculating sticks
• Microscopy procedures

Relationship to Oxygen

Types of bacteria based upon their relationship to oxygen

• Obligate aerobes rely on aerobic metabolism for growth and cannot grow in the absence of oxygen


• Facultative anaerobes employ aerobic metabolism when oxygen is available but are capable of inducing alternative anaerobic pathways when oxygen is not available; they grow best in an oxygen atmosphere but demonstrate limited growth under anaerobic conditions


• Obligate anaerobes cannot grow in the presence of oxygen; they are poisoned by oxygen and are unlikely to turn up in our samples or be cultivable in our laboratory without using anerobic chambers


• Microaerophils rely on aerobic metabolism but their growth is suppressed in room air; they grow well when oxygen is present at relatively low partial pressures

Rationale behind the assay

Solutions. We incubate our cultures in sealed jars in which a chemical reaction removes oxygen, replacing it with carbon dioxide and water vapor. We incubate duplicate cultures aerobically to ensure that they were viable when placed in the jar. The aerobically grown cultures are our positive controls.

Applications. We use this assay as part of a plan to characterize bacterial species based upon phenotype. Analysis of the ribosomal RNA sequence is increasingly used to identify known species, however that method is most reliable for identifying well characterized species of clinical importance. It is less useful for characterizing isolates from environmental samples and even less useful for the study of newly discovered species.

A second valuable "application" is the opportunity to put the concept of an experimental control into practice.

Procedures

            
Fig. 1. (left) GasPak jars for anaerobic systems. (right) Bacterial growth patterns on three sets of duplicate plates incubated for 72h, one set grown aerobically (top row) and corresponding anaerobically incubated plates (bottom row). Note that in all cases anaerobic growth is sparse compared with aerobic growth.

Setting up the incubations

1. Mark off sections and label two sets of plates (test plates, to be incubated anaerobically and control, to be incubated aerobically), prepared with an agar medium that we know supports growth of the isolates to be tested. Two or more isolates can be streaked onto the same plate. Dilution streaking is not necessary.

Conduct the following procedures quickly and efficiently. Your cultures will begin to grow as long as they are in aerobic conditions, regardless of relationship to oxygen. Leaving the plates exposed to oxygen for too long may result in false positives for anaerobic growth.

2. Systematically prepare duplicate streak plates, taking care to ensure that each plate is streaked identically and labeled identically to ensure comparison of aerobically and anaerobically incubated cultures. To save time you may go directly from the first plate to the second without picking up additional inoculum. There will be plenty on the loop, however to avoid falsely identifying a facultative anaerobe as an obligate aerobe because the inoculum was lighter, you should streak the plate to be incubated anaerobically first, followed by the plate to be streaked aerobically.


3. Prepare the anaerobic jars


1. Place a drop of water on the blue part of an indicator strip and tape the strip on the inside of the jar where it is visible (blue part shows).
2. Place the test plates into the rack in an anaerobic jar, inverted. Try to fill up each jar to conserve on generator packets, which are expensive. Join one or more other teams if necessary. Small jars take up to 14 plates and require one generator packet. Large jars take up to 42 plates and require three generator packets.
3. Open a generator envelope and insert the packet into a rack.  Quickly place the rack into the jar, attach lid, tighten, and label the jar.
4. For fastest growth place test & control plates in the 30 degree incubator for at least 48-72 hours. Room temperature is okay if they grow vigorously at that temperature

Reading results

4. Open the jar, line up your test and control plates for quick comparison. Reject any test for which there was no aerobic growth. You will have to re-test that isolate if it is still viable.


5. Assess the presence or absence of anaerobic growth


1. Anaerobic growth is indicated by an obvious "lawn" covering the inoculated section of agar. Growth is always more sparse than the corresponding aerobic growth and pigmentation is usually absent, even for species that produce pigmented colonies aerobically.
2. If all or most of the agar is bare, you probably have an obligate aerobe. Report "anaerobic growth not observed." You may see some material at the start of the streak, where a large amount of colony material was placed on the agar. Lack of growth over the "tracks" made by the loop over the rest of the section supports the conclusion that you have an aerobe.
3. Occasionally you may see individual colonies pop up on the agar, but not a continuous lawn. This observation suggests that your isolate is an obligated aerobe, contaminated by a facultative anaerobe.


6. After making and recording your assessments discard the generator packets in the regular trash, rinse out the jars and place them back on the table to be used again.

Identifying a microaerophil

In a tube of liquid media we have aerobic conditions in the top half centimeter or so. If there is anything in the tube that consumes oxygen, then conditions become more and more anaerobic as we descend to the bottom. Starting about a half centimeter below the tube surface and ending perhaps a centimeter further down, conditions become ideal for microaerophilic growth. If you happen upon a broth tube that is clear except for a turbid region a bit below the surface, you may indeed have a microaerophil.