6 July 2007
Chytrid Swab Protocol (2004-2007)
UC Berkeley Briggs et al. NIH Group
PDF version of this page.
This protocol was developed to allow field biologists to non-destructively sample amphibians in the field for the presence of Batrachochytrium dendrobatidis. This document was produced for the Briggs NIH research group based on Boyle et al 2004. Contact Vance Vredenburg (vancev AT berkeley.edu) or Cherie Briggs (cbriggs AT berkeley.edu) if you have questions.
Supplies:
Swabs- these can be ordered directly or from a distributor:
- Directly: Medical Wire and Equipment: go to http://mwe-usa.com/mwe/mwe.php
The product code is MW113.
- Distributor: Advantage Bundling SP. (catalog number MW113). Advantage Bundling can be reached either by phone, 1-866-Bundling or orders can also be placed through email, sales@advantagebundlingsp.com.
Vials-
Screw cap 1.5ml microcentrifuge tubes, available through Fisher (catalog number 05-669-12). These are standard and can be ordered through other companies, we just give one example. All microcentrifuge tubes used should be sterilized either by autoclaving before use or they can be purchased at a higher price as pre-sterilized (Fisher catalog number 05-669-17).
Marking pen-
When possible, use ethanol- proof black markers to label your vials as they tent to withstand the time best. Some people prefer to scratch the sample id on the vial because they cannot be washed off or erased by accident.
Procedure:
- Preferably, capture amphibians by hand. Wear gloves when swabbing animals and change gloves between animals. If you are using a dip net, be aware that B. dendrobatidis zoospores could be caught on the net and transferred between individuals, therefore, use different nets whenever possible, or disinfect the net as often as you can (there is no perfect solution to this problem).
- Swab the underside or ventrum of adult/metamorphs 30 times. Remember you are in effect scraping small amounts of tissue from the skin. Some pressure must be applied, but this does not mean that you must squash the animal. Areas to target are the drink patch, thighs and webbing between the toes.
- Air dry the swab for approximately 5 minutes, avoid direct sunlight if possible (if conditions are too humid to air dry then store in 95% EtOH)
- Break swab ~3cm from tip and drop into empty screw cap tube. The swab stick should not touch or bump against the top of the vial. Screw the cap on the vial and store in the shade.
- Samples can be kept a room temperature for a week or maybe longer, but it is best to keep the samples cool and placed as soon as possible in a 4 degree C freezer (the kind you have at home is fine). Avoid extreme high temperature and direct sunlight. Samples may be stored in a freezer for many months without problems.
- Analysis of swabs: We use the quantitative PCR methodology as described by Boyle et al (2004).
CLICK TO SEE INSTRUCTIONAL VIDEO: SWABBING VIDEO
Labeling:
Tubes should be labeled with the collectors first and last initials followed by an “S”, for swab, followed by a three-digit number, starting at 001. Example for Cherie Briggs first swab: should be labeled CBS001, etc… We are not assigning any other ID number to these vials so do not reuse numbers.
Other data should also be collected along with the swab reference # such as: Site ID, Site Name, Observer, Time, Species, Location (inlet, marsh or stream, pool), Life stage (larva, subadult, adult), Gosner Stage, Weight, SVL, Sex, pit tag number, and notes on animal condition (i.e., lethargic, righting reflex response, etc.).
Analysis of swabs:
Unfortunately, the Briggs Lab does not have the resources to run swabs for other research groups. Samples typically cost about $4-10 each not including labor costs. As quantitative PCR machines become more common, we believe costs will fall and the technique described by Boyle et al. (2004) will become more widely applied to measure and monitor the spread of chytridiomycosis.
Reference:
Boyle, D. G., D. B. Boyle, V. Olsen, J. A. T. Morgan, and A. D. Hyatt. 2004. Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Diseases of Aquatic Organisms 60:141-148.
Contacts:
Cherie Briggs
Associate Professor
Department of Integrative Biology
College of Letters and Sciences
University of California, Berkeley
email:cbriggs@socrates.berkeley.edu
office: 4014 VLSB, (510) 643-3889
lab: 4183 VLSB, (510) 643-3890
fax: (510) 643-6264
mailing address:
Dept. of Integrative Biology
University of California
Berkeley, CA 94720-3140
Vance T. Vredenburg
Postdoctoral Scholar
Museum of Vertebrate Zoology and
Department of Integrative Biology
3101 VLSB
University of California Berkeley
Berkeley, CA 94720
tel (510) 642-7960