Hi Srichandrasekar Indhuprakash

You can streak your bacteria in agar slants and store them in a refrigerator at 0 - 4C. It is a short term preservation technique and bacterial cells are not completely metabolically inactive in a fridge. Some of my cultures lost their viability. I would recommend storing your cultures at -80C with a cryoprotectant (cryopreservation). It will secure the metabolic capacity of the bacterial cells. No mutations taking place either. Cells stay in a dormant state until revived again. What you have to do is growing your bacteria in its most favourable broth culture medium and mix 500 microliters of the grown broth culture with 500 microliters of sterilized 10-20% glycerol in a cryovial and store it in a -80C freezer. Do it in replicates for each isolate. You can recover the cells again by adding the vial contents aseptically into a sterilized broth medium.

Hi

You should be preculturing before any test of bacteria. The age of bacteria culture must at 18-24 hours.

My doctor stored bacteria from 2017, within nutrient slants with added Glycerol 20 ml and kept within freezer.then she wanted to throw them away by scientific way.

BUT WE NEED IT FOR PRACTICAL MICROBIOLOGY LECTURE,

I subcultured them 70 Percents of them growths very well with all their characters.

This picture is just after first its subculture Pseudomonas aeruginosa.

use Glycerol or DMSO preparation and store at - 80.

You can preserve the bacteria using 20 to 25% Glycerol in -70 oC temperature for several months.

Prepare a cell suspension in TSB and centrifuge 1.5 mL of that suspension in centrifuge tubes, remove the supernatant and repeat the procedure to get more biomass. At the end add 500 mL of TSB with 15% glycerol and you can freeze it at -80ºC

Hi Srichandrasekar, the best way to preserve the bacteria for long term use to make the glycerol stocks and freeze it in -80 (20-25% glycerol in LB). This way the bacteria would be perfectly fine for several months given that you won't thaw and freeze them again and again. However, if you want to use them in subsequent days and you are in resource limited settings, you can keep the culture in 4 degree fridge and subculture next day to grow into desired growth phase and number and use them, they would work fine. I won't recommend to subculture the liquid culture more than 4-5days old. Agar slants and plates can be preserved little longer than liquid culture up to few weeks without altering their properties. Though if the experiment is related with plasmid or protein expression, you might require fresh transformation and fresh culture rather than the stored cells depending upon your plasmid and protein. I hope this helps.

Making glycerol stocks and freezing them in -80C is the safest way to keep bacterial cells for a long time (20-25 % glycerol in TLB). Since you won't be thawing and freezing the bacteria again and again because it is too much costly to freeze again and again , they'll be fine for long time (several months )

If you are doing physiological studies then you need to have bacteria in an identical state each time to compare. It is ok to store a culture in the refrigerator to use as a starter culture but not as the experimental culture. I would often make a culture grown to mid log phase and then put in the fridge. Then you can dilute 1:10 into fresh media and grow for a few hours depending upon the growth phase you want your cells. This starter is good for a week or two but not much longer. Storing in glycerol at -80 is of course the way to go for making a permanent stock of your strain but I am presuming you have that already.

Dear Sir

You can add glycerol to the culture and store it at freezing conditions. If I don't make a mistake, you can make a 50% v/v suspension of bacterial cells and glycerol.

You can store your bacterial strains at -20°C by preparing a liquid culture and adding glycerol until a concentration of 20% glycerol in the final solution is reached. In this way you can store your bacterial strains for about four weeks.