Hello all,
I am trying to attempt morphological identification of ticks and fleas collected in Wales on wild small rodents.
My samples have been frozen and after the morphological identification I need to extract DNA to attempt molecular identification and to check whether DNA from various pathogens is present.
My question is what is the best technique to treat the samples during morphological identification in order to identify the key morphological features and avoid DNA degradation?
I am using a dissecting microscope for big specimens and I used ethanol to fix them, but I am really struggling with tiny larvae. I thought about mounting slides, but I am not sure which medium to use.
Thank you very much,
flavia
You could try Dietrich's fluid, a mixture of equal parts of ethanol, glycerol and water. It preserves specimens, doesn't interfere with with nucleic acid extraction and doesn't dry out during manipulation or long-term storage. It is also ideal for transferring specimens into glycerol jelly for microscopic examination.
Thank you very much for replying.
I have some more questions, I hope you can give me advice.
Can I use it on the frozen samples? Is ethanol 96% ok to use it? How long the sample has to be put in ethanol before being safe to handle under the microscope for a while (these specimens are very difficult to identify for me).
Do I have to wash the samples afterwards? If so, what do you recommend?
Thank you so so much.
flavia
For frozen samples I'd add the specimen into the fluid while still frozen and let it thaw in there.
96% thanol is perfectly ok, after all, you add water anyway. In fact, I regularly use IMS, but then, I do not plan on DNA extraction.
Equilibration time depends of course on the size of the sample and a sample packed in chitin will take longer than something soft. I'd guess that you should be completely safe after overnight incubation, but you could try an hour or two on a non-critical sample (perhaps on a slowly rotating wheel as diffusion isn't very fast in glycerol).
For further processing, I found it safe to transfer samples directly from Dietrich's into water or 96% ethanol, depending on needs.
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