What is the appropriate way to preserve fish intestine samples for one or two months for bacteriological studies ? Do I need to add Glycerol to the samples and keep them at -20 degrees C?
Freeze-thawing would rupture bacteria cell walls....So, you will loose many culturable bacteria (lot of VBNC bacteria might still survive). Considering the little fraction of culturable bacteria it will be a luxury to do so. If you cannot use fresh samples for culture-independent analysis, try using one of those methods mentioned by Mr. Andres.
I also think like Andres Arce's comment but in addition you may store the whole fish for few days maybe better than storing directly the fish intestines.
Andres answer is correct. If you are quantifying either a specific species or the general microbiota, then PBS at 4 for a day or so is fine. Given this is fish then there are unlikely to be be thermophiles like Campylobacter that don't like the cold. Ideally the sooner the better would be my experience from both avian and mammalian samples. It is also worth bearing in mind that any storage is likely to favour some bacterial species over others. Any culture-based system will not appreciate freezing. There are, as stated, plenty of bacterial-specific products now available that stabilise RNA/DNA in samples if this approach is to be used-though they all cost a fair amount.
I agree with all comments done. It depends on the study you need to do. Certainly, some bacteria are going to die with freezing, or adding "glycerol" or other preservative. Some species of strict anaerobes die easily in any case. So it depends in the specific bacteriological study you want to do.
Perhaps you can run a little experiment comparing different methods: by freezing (at -20 or -80º C) with or without preservative (ie glycerol or a buffer as PBS or simply SSF), by cooling at 4º with or without preservative. Other preservative could be a nutrient media in a semisolid agar, as the media used in transport swabs. Nevertheless, if you want to isolate, ie, species as Campylobacter or Clostridia, take into account the preservative culture media and the temperature.
If the comparative study I commented before is not possible, I recommend you to freeze all the samples directly, always in the same way (time, temperature, volumen). In any case, I think feces are the best preservative for gut/intestinal microbiota.
Other possibility that comes to mind is to lyophilize the samples.
Yes you can preserve the intestine samples not just from one or two months but for 6 months at a stretch. Rapidly freeze the samples to -50C (preferably to -80C) in a quick freezer. However, once you thaw it completely do not refreeze. You may cut portions of the samples in frozen conditions, so that you do not have to thaw and refereeze.
Thanks all. As I do not have possibility to do the sampling in two times, I will use freezing approach with different kind of preservatives and then I transport the samples. I will update this topic as soon as I find the differences. I am going to use DGGE and Pyrosequencing to determine the core microbiota of several valuable fish species.
Not enough information. Do you intend to isolate and identify by conventional microbiological culture and ID methods? If so then the best way is to plate them out fresh - do not store them in a frozen state at all. Chilling for a day or so in th fridge is acceptable. Freezing will result in bias as some are more freezing labile than others. If you must freeze then plate out later then what you have described is appropriate. Anywhere from 10 to 50% glycerol seems to work well. Just make sure it's well mixed in.
If you want to ID by shotgun sequencing/ next gen sequencing then preservation of the DNA/RNA is the key. I suggest the use of RNAlater preservation solution for either DNA or RNA.
I would recommend some of the specific products now available for bacteria-it's an approach we are using for RNA seq. That said snap freezing should preserve what you have pretty well-we are doing this for chicken microbiota analysis. DGGE may prove more difficult
Can you please suggest any suitable method for collection and preservation of gut microbiota of honeybees for culture dependent study?
Actually I have to collect honeybees for gut microbial study through a culture dependent method. I have planned to collect in 95% ethanol, will all the gut microbes survive or not for culture dependent isolation ?
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