We often use 70-80% IPA or ethanol to wipe LAF and clean scissors and forceps... but how effective these cleanings are? How extant these IPA or Ethanol will help us to avoid contamination in plant tissue culture?
Dear @Badhepuri Mahesh Kumar Contaminants in tissue culture laboratory are most commonly of biological nature, and can include bacteria, fungi, viruses and parasites. For each category, a separate disinfectant is required. For example, the work surface should be decontaminated with antifungal agent (5% Trigene) followed by 70-80% ethanol as antibacterial agents.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149418/
Although ethanol solution (70 or 75 %) is used widely as a disinfectant in cell culture room, the experiment or should be careful on the quantity sprayed and where to spray it, and particularly to avoid its contact with experimental cells, since this will lead to radical influence on cells pathophysiological condition.
https://medcraveonline.com/JMEN/ethanol-in-cell-culture-disinfectant-or-contaminant.html
Please also check a similar discussion at RG; hope, it may throw important insight to this discussion too:
https://www.researchgate.net/post/How_can_one_reduce_contamination_in_the_tissue_culture_lab
Best wishes, AKC
The use of ethanol as an anti-microbial is fairly well established, for example https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection-methods/chemical.html. What has prompted your question exactly?
If you are concerned about your particular set-up, you could test it yourself by doing swab tests of the LAF bench before and after cleaning and culturing any bacteria/fungi/etc. If colonies develop on your plates, then you may have to increase the alcohol percentage in your spray, or consider a different anti-microbial treatment (UV light, bleach, etc).
We used 70% ethyl alcohol for years and now using 80% IPA for the last 3 years. The results are the same only and IPA is equally effective in the disinfection of LAF. IPA is even more cost-effective as compared with ethanol. Even the commercial units are using IPA only in India.
Arbind K. Choudhary Thank you so much sir for your valuable information
Matthew Wheal really ur words given me a new idea to test my LAF Sir.
and Actually, Why I have asked this question is, I am working on different medicinal plants, my cultures are responding good in the initiation step, but when I am subculturing them few of them are getting contamination (sometimes bacteria and some times fungal), that is really a great loss (even after wiping the bench and tools thoroughly with IPA and burning the tools under spirit lamp)
So that if I get any new ideas are the new way to avoid this contamination it will be a great discussion...!
I agree with above answers and I think cleaning with 70%ethanol or IPA is enough.
But I have other points to mention if still you get contamination during subculture it might be possible due to several reasons-
- Try to service ur LAF as HEPA filters might cause contamination
- similarly autoclave sterilisation is proper or not which you can check by autovlaving nutrient agar plate and simply keep in culture room and see if contamination occurs or not
- your plant also might contain endophytes, which we have observed during subculture, so if this is the case with you than you have to add antibiotic in media or treat culture at the time of subculture.
All the best
Thank you Ashutosh Pathak Sir
As of now our medium is pretty good sir. Because we will keep the medium in the same incubation conditions for few days depending on the use, there were no contamination observed yet.
As you told there might be a endophytic microbes may be making this contamination.
Here I would like to ask you small thing that, generally what kind of plants has this endophytes (as I have observed in Bamboo, Tectona grandis, Santalum album few others). is there any best way to avoide endophytic microbes?
thank you for your time Ashutosh Pathak
Yes so I think first you do small experiment using disc diffusion method. By this you can confirm which antibiotic is suitable for your samples. Companies like HiMedia supply readymade discs containing narrow and broad range antibiotics.
This will also reduce your time for optimisation of sterilization treatment.
Equally effective and much cheaper than ethanol. Most suitable for big PTC industries and has had no side effects till today (using it for the last 4 years).
Ethanol alcohol is has anti-microbial action mechanism by denaturation of dehydration of proteins and inhibition of metabolite production that indispensable for rapid cell division.
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