Do daily transfer in nutrient broth but do it aseptically or you might be culturig the contaminants.

If it hasn't grown by its normal growth rate, then I would really be suspect that the culture contains the desired organism. You can't bring them back from the dead, although cutting-edge research groups (JCVI) have been able to insert synthetic genomes into cells and "boot up" a life form. This won't help you, but I thought you might find it interesting.

Ah, its very difficult to revive the culture from old slants or plates, or say no way to make them alive again.

Well pour some nutrient broth on slant and incubate, may be culturing after that can help you.

In future revive your cultures every 4 weeks if you store them in refrigerator..

Wishes..

As it said before you will not be able to revive you culture if it is dead.

what I will do as Arvind Behal and Hiral Hiral said pour 5-10 ml of nutrient broth on your slant then incubate in shaker for 24h next day inoculate from the broth on Nutrient agar or LB agar media supplemented with antibiotic (if your strain resistant to any).

then incubate (you can tell from the morphology of your strain on agar and its growth rate as well ) if you doubt it, I recommend to subculture again on any selective media specific for it perhaps MacConky or other

Good Luck

All you can do is wash the agar with fresh media and replate to see if colonies grow. I would confirm that it is B. subtilis if you get any growth to rule out contamination.

Pipette a few millilitres of sterilized NB and scrap the slant surface with a sterilized loop. Pipette out the cloudy NB and transfer it into new sterilized NB (can be either in universal bottle or in a small flask). Shake the mixture at 37 degree celc, 150 rpm. If the broth did not turn cloudy the next day, keep on shaking (max 3 days). Just give more time to the bacteria to re-grow. If your broth changed to cloudy, subculture one loopful of the broth onto fresh NA. Keep on repeating subculture the bacteria until you get the single colony. Please perform gram-staining procedure to confirm the bacteria is what you want, not a cross-contaminant

Try MWG medium or add 1% not fat dry milk to a common rich medium. Probably your cultures are in viable but not culturable state and need more nutrients than usual to "resurrect". Could you indicate the species?

First you should perform gram stain to obtain bacteria of your interest and then sub culture it after conforming. You need at least single bacteria for sub culture.

Hi, pity, but you have very low chance to recover the original bacteria. Next time you can add 10% of glycerol to your NA, it helps with most of bacteria. Another old method - to use 10E4 cfu/ml suspension in sterile water at cryovials and keep it at room temperature. Most of bacteria will stay alive up to 15 years. It does not work for strains with latent phages.

my glycerol stocks are from year 2011 of senior and now when am trying to reviving them in new LB they are not growing ..plz can anyone help...

Did you keep them at -80°C? If they were at -20°C, it may be a problem for survival.

You may use TSB as alternative medium.

Please, write the procedure you use for utilisation of glycerol stocks.

If you get growth after 48 hrs, perform Gram stain and compare with your earlier results and confirm.

or

You can prepare nutrient broth, sterilize and cool it. Later add this sterile broth to your old slants aseptically and incubate for 24 hrs at 37oC.  If at least single bacterium is surviving in old slant or any endospore  present , you may get culture again and the culture may be tested for Gram staining.

If you have a sonar bath, you can try in this way..,

make a desired broth solution and cut a small portion of slant containing the organims.

let it grow for 24-18 hrs and there after sonicate it for 30mins only in a sonar bath not in  the probes, and incubate it at 37 C for 24-48 hrs.

Then follow streaking method on a nutrient plate and repeat it for several times.

If you had done a biochemical test earlier of that organisms you can do it again for a confirmation.

You can use mineral oil to store bacteria at room temperature for a period of 12 -15 months.