Microbes are considered invisible drivers of both soil health as well as plant health safeguard. A microbial species once established as an effective inoculant either as biofertilizer or as antagonist against variety of diseases as biofungicide, has to be maintained for its purity through number of sub-culturing process. It is still a matter of debatable issue , how can we maintain the purity of such microbial culture for mass multiplication and commercial use . But , it has been vehemently observed many a times , many of microbial bio-inoculants loose their potential after few cycles of sub-culturing, with the result , the agronomic response of crops to inoculation loose its consistency over time and space. With this background , i have few obvious querries :
* What is the best course of action to retain extended vitality of microbial inoculants from one sub-culture cycle to another cycle?
* Is there any difference in period of vitality for sub-culturing with regard to bacterial versus fungal inoculants?
* What shall we adopt that a microbial inoculants dont loose their vitality after sub-culturing?
* Can we procure the same strain of microbial inoculant after inoculation into the soil using sequencing technique ?
* And if so, whether the vitality of regained microbial inoculant will be the same to that of earlier inoculant ?
Thank you for your responses......
It all depends on how it was stored before sub-culturing. If it was undergoing rapid metabolism, then that could occur almost immediately. I would like to believe that lost of purity would imply that after some sub-culturing, the pure culture that was stocked becomes a mixed culture and the candidate that was stocked requires to be re-isolated in pure culture. Apart from lyophilization, my culture collection stores in frozen-glycerol state and sub-culturing is done once in one and half to two years, depending on the organism. Yes, there is a difference in length of vitality period. Fungi traditionally grow slower than bacteria and so retain their vitality longer. This is more or less like the first question; extension of vitality. As long as organisms are conducting metabolism, there will always exist a chance or chances of variants arising from sub-culturing. Yes, it is very possible to re-isolate the same strain from soil after inoculation into the soil. Yes, the vitality will remain the same, after all, a good number of microbial strains are maintained in sterile soil.
The activity and performance of a newly prepared culture depend on many factors. A very important one is, how culture acts. For example, if we prepare bacterial culture for remediation of organic pollutants, the cultivation of bacterial biomass for inoculation must be cultivated under selective pressure. The reason: all enzymes participating in pollutant degradation are plasmid coded and when the selective pressure is not present during growth and multiplication, no enzymes are produced and biodegradation activity is lost.
It is only one example, how required activity can be lost.
Regards
Vit
Suggest you prepare large volume culture expressing the character you want for distribution is spearate vials for feeze drying . Rehydrate and culture as needed. The drug industry specifies no more than 5 transfers from lyophil for its standard QC isolates to maintain their resistance. http://www.pharmacopeia.cn/v29240/usp29nf24s0_c51.html
Maintenance of purity of microbial culture through sub-culturing cycles will primarily depend upon the nature of microbe and the upkeep. The transfer is always subject to aseptic conditions to avoid contamination. But there is always risk of genetic changes also. Refrigeration, paraffin method, cryopreservation, and lyophilization (freeze-drying) could be adopted for maintaining master culture and using it as and when contamination is noticed.
But bacteria may loose the P solubilizing ability while sub culturing and fungi do not lose. Among bacteria, aerobic spore forming bacteria are more effective P solubilizers. Mr. Phil above has mentioned 5 transfers. yes but it is stanardized depending upon nature of microbe.
Suresh
I'm not aware and do not think the 5 transfer rule in USP was based on data. Re the microorganisms involved, I'm aware of some testing and recall the bacteria did not show change within the parameters addressed but think the Aspergillus brasiliensis did show some change.
This question always ponders that vitality of regained microbial inoculant will be the same to that of earlier inoculant ?
More elaboration needed whether the vitality of regained microbial inoculant will be the same to that of earlier inoculant.
Microbial cultures as biocontrol agents for post harvest perishable fruits and vegetables possibilities could be worked out
Agreed , no doubt about it . Role of additives is mandatory to be worked out to add an additional element of bioefficacy...
The cultures can remain viable and with unchanged characteristics for 10 to 30 years or more. Many species which can't be preserved by lyophilization are successfully preserved with liquid nitrogen method. The nutrients consumed by the cells are continuously replenished by an inflow of fresh medium. A constant inflow of fresh medium is balanced by a constant efflux of spent medium plus cells. Working bacterial stocks can be streaked onto agar plates and stored at 4°C for daily or weekly use. Culture dishes should be wrapped with laboratory sealing film and stored upside down to minimize contamination and to keep both the culture and agar properly hydrated. Freeze-drying is the preferred method keeping reference strains and other isolates for long periods and is standard practice for large commercial and national culture collections. Bacterial and yeast cultures have been maintained at -140°C for 15 years without significant loss of viability. Storing cells in cryogenic freezers is the most effective and, as compared to freeze drying, the easiest method for long-term storage. Most cultures can be stored in cryogenic vials at -20°C for at least a year, with some bacterial stocks remaining viable for several decades when stored at -80°C and in liquid nitrogen
Rk Naresh
For general purposes, you are correct. However it is specified in appropriate Pharmacopoeia that cultures be transferred no more than five times from lyphil. This involves specific isolates* for which studies have found extensive subculture producing detectable changes only in one - Aspergillus brasiliensis.
*see http://www.uspbpep.com/usp29/v29240/usp29nf24s0_c51.html
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