Hello everyone, I am facing this problem where a few of my bacterial culture are not giving abundant growth as seen previously by the strain (still not identified). I tried growing in the nutrient broth it is growing there but when subcultured on nutrient agar slants the bacteria has very tiny nonvisible colonies. On subsequent subculture, it is nonexistent. I don't want to loose the strains. Please help me revive it.
Hello dear
Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335116/
Stored the liquid culture with 20% glycerol at -80C first.
Can you growth it in liquid again? Prepare genomic DNA, at least you can sequence it.
Test different agar medium, sometimes rich nutrient agar is not good. Test temperature, prolonged incubation, add vitamins or sprout extract, buffer, trace elements.
Try to use Brain Heart Infusion broth supplemented with some growth factors
Hi Ambika Bashisht ,
I agree with Dr. Kien T Nguyen 's answer. As your isolate is growing well in your broth culture you can first store it at -80C. For that, you can add 500 microliters of your culture broth and 500 microliters of 10-15% glycerol into cryovials and store. Then you can try changing the growth parameters of your solid medium. Try to use proper incubation temperature, specific growth requirements. As Dr Kien said it is likely that your culture has specific growth requirements to grow in solid media. Moreover, when you grow isolates in broths you shake it for aeration which usually enhances the growth. That might be another reason, its aeration needs. By the way, it is much better to identify your isolate after growing in broth. When it is identified, you can provide the specific growth requirements on a solid medium.
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