I have to collect honeybees for gut microbial study through a culture dependent method. I have planned to collect in 95% ethanol. If the bee samples remain in 95% ethanol for many days, will all the gut microbes survive and be culturable on selective media?
If not, then please suggest any suitable method for collection and preservation of gut microbes.
Conservation of honeybees in 95% ethanol for few days will for sure kill all the vegetative bacteria, to my point of view it is not a good way to preserve the microbiota if you want to make a culture of it. However it seems to be a good way to analyse the bacterial diversity by pyrosequencing.
By culturing the remaining gut microbiota, you may obtain the growth of some spore forming bacteria, which can resist to ethanol treatment.
If you need to preserve them for many days, I would say that mixing them in glycerol 15 to 20 % solution and conserved them in liquid nitrogen is the best solution (but you'll still kill some sensitive bacteria).
Regards
Dear Cyril Laconelli,
Thanks a lot for useful information.
Actually, I have to collect bee samples from about 900 KM away from my research station and i need the gut microbiota to survive and able to culture after 3 or 4 days . Will they survive in 15 to 20 % solution with out liquid nitrogen?
If you have some literature please send me .
with warm regards
Khalid
Two parts to your question
1) You do realise there are a limited number of microbiota you will be able to culture and a good percentage of those are anaerobic, so the best bet is do a literature survey and identify the population you can realistically culture, once you do that make some stab agar, in the field take out the gut and stab it in the slants and stick it in a jar with a gas pack to create anaerobic condition. Or alternatively you can try using anaerobic transport media that is used for anaerobic bacteria in clinical situations like dental plaques or biopsies.
2) if you just need to survey the microbes then 95%EtOh is good, but make sure you follow sterile procedures while extracting the gut, else you will end up getting surface bacteria as contamination.
Thanks to Cyril Iaconelli and Jay Siddharth for your answers so far! these have been very helpful. Keep it coming please!
Jay Siddarth, you should note that due to the small size of insects, passive diffusion of oxygen from the trachea keeps much of the body tissue oxygenated. In a study of a beetle, "no obligate anaerobic bacteria were identified" (Rizzi et al 2013, http://dx.doi.org/10.1155/2013/420287). This is one case, but if one survey's the literature, there are many more facultative aerobic bacteria than you would expect from vertebrate systems.
Dear Clement, thanks for including the link, here is what i could glean from the publication,
1) They have not really mentioned using anaerobic culture techniques
2) The Paper is about beetles, fair enough though it belongs in the Arthropods same as for bees,
3) The total sequences for all samples pooled is less than 500 sequences including isolates(aerobic) dgge and clone library, which hardly scratches the surface to understand the microbial community esp when one does not handle samples to preserve obligate anaerobes, during collection, transportation and isolation.
4) I do understand your point about oxygenation using trachea, but we must understand that its a primitive respiratory system and despite that, examples like Termites(Insecta) harbour obligate anaerobes in their gut which are essential to their survival. Humans even with advanced respiratory systems still harbour obligate anaerobes in good numbers.
Finally I would like to point to some specific studies performed on bees which might be more relevant to the question presented here and have done a more rigorous scientific investigation.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0072106
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0095056
as one can observe there are indeed anaerobes that are seen in samples.
here is one isolated only in anaerobic conditions specifically from honeybees
http://www.ncbi.nlm.nih.gov/pubmed/23606484
Points well taken. However, I think the main point is that many insects harbor a mix of species, some of which are aerobic or aerotolerant. The first paper you cite was done on larvae, and the results may not generalize to adults. From that paper: "Acetobacteraceae (Alpha 2.2) is highly aerotolerant (Anderson unpublished data), and is found at low frequency in the adult midgut, but does not occur in the increasingly anoxic adult hindgut [14]." So I guess the question for Khalid Ali Khan might be, do you plan to look for all species, whether anaerobe or aerobe? Would it be worth dissecting workers in the field and preserving hindgut and midgut samples with different techniques?
The second reference notes that crop bacteria were cultured in microaerophilic or aerobic conditions: "Each crop was homogenized in 1 ml of physiological saline and 100 µl was plated onto deMan-Rosaga-Sharp (MRS) and Tryptic-Soy agar (TSA) plates and placed in aerobic or microaerophilic (5% CO2) conditions at 35°C for 24 h. Counts of the number of colony forming units (CFUs) per plate were made and, where appropriate, the log number of CFUs per gram of crop was calculated to compare to other studies [47], [57]. Comparisons were made between CFUs growing in aerobic versus microaerophilic conditions for each media type or between the CFUs growing on MRS versus TSA plates incubated in similar environmental conditions." ... and ... "Overall, foragers contained a crop microbiota that flourished in both ambient and microaerophilic atmosphere on both MRS and TSA media"
So, Khalid, it seems Jay's advice about anaerobic storage may be good if you are interested in hindgut bacteria, while other storage techniques may suffice if you are interested in crop or midgut bacteria.
Dear Dr. Clement F. Kent,
Thanks a lot for such a detailed answer. Actually, I am not only interested in hindgut but my aim is to characterize the bacteria from the entire gut (foregut, midgut and hindgut) of worker bees (Apis mellifera jemenitica).
Sorry, I do not think so. Because we found that it was difficults to detect viruses in honey bee midgut when they were in 95% ethanol.
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