Recently I established cultures of the large diatom Odontella sinensis by picking single cells from a field sample using a pipette, washing them individually in sterile culture medium (f/2) and incubating them at different salinities and temperatures.
After several weeks I noticed growth and started cell counts using a phase-contrast microscope. Unfortunately, I noticed that a small (cell ca. 2 µm) heterotrophic (no red fluorescence) flagellate (flagella ca 10 µm long) was present too. Most of them were attached to the outer cell wall of the diatoms, in all cultures.
Experience learnes that these flagellates will multiply rapidly and can slow down growth. Because they are attached to the diatom they cannot be easily washed out over a filter, e.g. a 50 µm gauze. What can I do to get rid of these flagellates?
He Louis,
Maybe germanium oxide might help? Diatoms can survive low concentrations, but I am not sure about the flagellates. However, vice versa, higher concentration of Germanium oxide are used to get rid of epiphytic diatoms from macro algae.
What about UV light. Some diatom are less sensitive (as I recall from previous study by Harry Peletier and possibly Anita Buma)
Engel
I have used the following technique, which is already explained elsewhere in RG.
Place the algal slurry in a polythene container upto70 % to 80% by volume & remove all the top air.Then add CO2 in the empty space through a plastic pipe, under slight pressure & close the container air tight. Place the bag/container over night in fridge. Take out the bag the next day, & all the flagellates would have been dead, whereas the plants would be alive.
The reason is CO2 doesn't effect plants, but kills the animals through this long period.
You may try to put your culture plates in a 4-degree refrigerator for different time, which may kill the flagellates while diatoms remain viable.
are you sure that they are not part of the reproductive cycle ' of the species ?(i.e. sexual stages?). Otherwise, you might try with good, old fashioned formalin or CuSO4 baths (between 10-5 and 10-6 usually work fine, or try your own range in decreasing strength starting from 10-4, 0.5x10-4 and so on). good luck
you can add some antibiotics into diatom culture mediums or you can purified the diatom in soild agar plates with antibiotics. you can try these methods.
Thank you all for this information; it seems a variety of methods might work. So its back to the lab! Louis
Eukaryote contaminants are particularly hard to get rid of. Streaking on plates is your best chance, in fact the most realistic one I can think of. You could try fungicides like nystatin but usually it does not work. Friends tell me some fungicides for treating seeds work well (from your local supermarket or garden store). Pharmaceuticals for treating Candida are worth a try. The suggestion of Trivedi above using CO2 sounds very interesting but I have never tried it. Of course you could always try saying "boo" at them.
I do agree with the answer of Ying Zhong Tang, refrigeration could be the right choice to get rid off flagellates as diatoms can survive very long on solidified plate during refrigeration and their revival is also easy from the plates or you can just try frequent transfer on solidified media, diatoms colonies are very much distinct (golden brown in color) on agar plates and you can see them by simple microscope by focusing the entire plate under light microscope, and can easily pic the colonies from the plate. Bacterial contaminant can be removed on subsequent plating.
re-isolate the culture from a single cell using the micropipette method - washing the cell in consecutive drops of distilled water.
try a freshwater or distilled water bath for increasing times, from 10-15 min to 2-3 hours. that should do the trick. you can also try a CuSO4 bath between 10-5 and 10-6 mol, again for increasing times. Transfer to new médium and let grow for 24 hours (48 if your diatom is slow grower ori f you don’s see any growth) Treatments should be repeated at least twice every 24 or 48 h.
local aquaculture technicians get rid of unwanted flagellates with commercial formalin (about 35% formaldehide) diluted between10-4 and 10-5 (depends on their experience with their strain), but I have never tried this and i am not sure of the outcome, while several times I have had good results using the previous two.
Good luck
Thanks all!
In the end I isolated fresh cells and started over.
The problem re-occurred last year in a photobioreactor with Tetraselmis.The problem is that these large-scale (200 L) reactors are difficult to maintain without contamination. A colleague did a web search and came up with a (Chinese) solution: decrease the pH to 3 for 1-2 hours (HCl), than get it back to normal (NaOH). This dramatically reduces the contaminating flagellates, but not Tetraselmis. We have not been able to retrieve the reference from China.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology