Hi Emma, For most freshwater protozoa, deionized, spring (not mineral!) or reverse osmosis water will do as a stand alone culture medium, or in the preparation of other culture media. For really fastidious taxa, you may want to filter some of the source water through a 0.2um membrane filter to remove any contaminants, and use this as the culture medium. I hand pick critters using a micropipetter with either 200 uL gel loading pipet tips or plain old 200 uL tips. Place the drop of sample on an empty sterile petri dish, and rinse gently by removing as much water as possible without disturbing the protozoan, and then add more sterile water so that you form a small pool around the cell. Continue to rinse the individual at least 5 or 6 times, and then transfer it to something like a 24 well tissue culture plate (or whatever type you have, just so long as it is sterile). Do a bunch of wells, and you should find at least one that is clear of other species and growing well. You can then add either additional water, or you can try some sterile Protozoa Pellet Medium or dilute (24%) Cerophyll medium. If you need the recipes, I can send them. I feed my cultures either E. coli or Klebsiella pneumoniae. Specifically, there is a Carolina strain of Ecoli that is used for feeding Dictyostelium - it is a non-mucoid strain. The Kleb should also be an attenuated strain. Make sure everything is sterile. Materials and media should be autoclaved or purchased as pre-sterilized. This should reduce any chance of extraneous bacteria or mold growth. As for contaminating protists, the tiny microflagellates are the hardest to eliminate (so are algae), so make sure you really rinse and rinse the individuals to remove any remaining contaminating cells. After you find a well that has good growth without obvious contamination, repeat the collection procedure once more when you start your stock cultures, i.e. isolate individuals from the good well, rinse them, and then transfer to wells on a fresh sterile tissue culture plate. Feed them, and once the population is ticking along, transfer to larger sterile containers (glass or plastic will do). Keep protozoa cultures below 80F at all times. I culture mine around 20C. You will have to determine the optimum time for setting up new cultures. It is very easy for a population to rapidly exceed K, and then crash - possibly to extinction. This is especially common for cultures growing in water without some organic resource (cerophyll etc) for the bacteria. These cultures feed on whatever Ecoli you provide, but since the bacteria are not growing (at least not very rapidly), the grazers run out of food and crash. The success of long term cultures will depend on making sure that the food resource and the medium is appropriate for the species. Most will do fine on bacteria, but some of the larger ones require other protists such as Chilomonas as their prey. If you have any other questions, feel free to contact me. One word of caution. Never use soap or detergent around protist cultures. The slightest trace of soap can kill sensitive cultures. Never use tap water or "mineral spring" water. Metal ions are also deadly. I wash all my glassware with non-iodized table salt and rinse with DI water. Hope this helps, Liane **************************************** D. Liane Cochran-Stafira, Ph.D. Associate Professor Department of Biological Sciences Saint Xavier University 3700 West 103rd Street Chicago, Illinois 60655
phone: 773-298-3514 fax: 773-298-3536 email: [EMAIL PROTECTED] http://faculty.sxu.edu/~cochran/ <http://faculty.sxu.edu/~cochran/> ________________________________ From: Ecological Society of America: grants, jobs, news on behalf of Emma Moran Sent: Wed 9/17/2008 6:12 PM To: [email protected] Subject: [ECOLOG-L] environmental protozoa isolations Our ecology lab is attempting to use protozoa collected from field samples to run a series of experiments which require isolating individual protozoa species that range in size from 4 to 60 microns. We have tried to isolate these species by diluting samples until we get individual drops of water with the desired species in it. However, most of these isolation attempts are unsuccessful either because the samples get "contaminated" with other species or because the cultures just never take off. We have also tried filters of various sizes, which strangely do not seem to work, as well as centrifugation (though we used a very old centrifuge and had no other media besides water). We do not want the protozoa to evolve to lab conditions, so the isolations also need to be done in a timely manner (within about a week). I was thus wondering if anyone had any suggestions or advice that could help us with this problem. Thanks in advance! Emma Moran
