Lab Work to Begin Soon!
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Welcome back! After a long summer of completely-unrelated field work (so I could keep a roof over my head), I'm back at school. This semester, I'm focusing all of my attention on field and lab work. The field work is related to the experiment you backed, but it focuses on the behavior of coyotes in the presence of free-roaming dogs. BUT! you want to hear about the lab work that you helped to fund.
I've been feverishly pushing paperwork, scheduling meetings, and cutting red tape over the past few weeks so that I can get some actual work started and get you some results. Some colleagues and I have worked out a protocol that ensures biological safety without sacrificing efficiency.
On Monday, we will conduct a trial run to ensure that we are doing the procedure correctly and not wasting supplies. This trial run will consist of using a single slide and small quantities of reagents. Each slide has 12 wells, which allow for positive and negative controls plus ten specimens. The amount of specimen used in each well is less than 1 µL, and we have at least 500 µL of each serum specimen. That way, even if we mess up the assay, we can try again without losing specimens.
All specimen preparation, incubation, and slide mounting will be done in a Biosafety Cabinet (BSC) similar to the one pictured above. A BSC is a specialized fume hood, fitted with a vacuum and sophisticated filters that capture biological agents such as bacteria. Ours is also equipped with ultraviolet light for sterilization of the BSC, pipettes, and other equipment. The BSC protects lab personnel from accidental infection due to splashing or inhalation of infectious agents.
Once the slide is mounted and fitted with a cover slip, the bottom and sides will be disinfected with 70% ethanol. At this point, it is safe to remove the slide from the BSC and put it under the microscope.
If antibodies for R. rickettsii are present in our specimens, they will bind to the formalin-fixed (inactivated) bacteria that have been attached to the slides by the manufacturer. When this bond is formed, a secondary (anti-canine) antibody that has been labeled with a fluorescent molecule called FITC binds to that complex. This second bond causes the FITC-labeled antibody to fluoresce. The fluorescence microscope allows us to see that fluorescence - if present - in each well.
Initial tests of each specimen will be of the recommended 1:50 dilution. Because the intensity of fluorescence depends on the concentration of antibody in the sample, this level of dilution will allow us to see even a small amount of antibody. Any specimens we find to be positive for R. rickettsii antibodies will be tested again with serial dilutions. In other words, we will test each positive sample again, this time at dilutions of 1:50, 1:500, and 1:1500. If a sample still fluoresces at the most-diluted ratio (1:1500), then we know it contains a higher concentration of antibody, which suggests that the infection was either very recent or especially severe.
The full procedures are outlined on Page 2 of the attached document (under "Recommended Staining Procedure for Indirect FA").
I know these procedures and concepts are very technical (for me too!), so hopefully I've explained them sufficiently. Feel free to drop me a line if you have any questions.
Eric
Image used without permission: labeled for non-commercial reuse. Source: http://uespak.com/project/tissue-culture-laboratory-air-conditioning/
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