I am facing a problem. I have isolated few bacteria from soil and preserved them in glycerol stock (50 % glycerol v/v). Now when I am inoculating these cultures in nutrient broth and incubating overnight at 30 degree I am getting the growth but when I am streaking the NA plates either using the glycerol stock or the overnight grown bacterial culture I am not getting any growth. Kindly suggest the solution.
Hi Nidhi, can you give more detail on your methods - particularly for the plate prep/procedure? Best wishes, Ciara
More details would be helpful, but most importantly you need to try freshly made NA plates becase something is probably wrong with them. They may simply have dried too much, and if the organism needs high water percent it may not be able to grow.
Robert John Wolff Ciara Keating I am using nutrient agar from Himedia (Ingredients mentioned on the pack (gms/litre): Peptone-5.00, HM Peptone B-1.50, Yeast extract-1.50, Sodium chloride-5.00 and Agar-15 g, pH-7). I keep the plates just from one day after pouring in the laminar itself then streak. Whenever I inoculate the nutrient broth with bacteria from the glycerol stock and incubate at 30 with shaking (120-150 rpm). I get the growth in nutrient broth. But when I streak the bacteria from this broth to NA plates I don't get any growth.
I doubt of nutrient agar quality, so I suggest you to manufacture the medium yourself with the use of the half of the required amount of agar(8 g agar)
Yes it is possible the NA agar media was stored improperly or is old. Use the nutrient broth powder (that grew the bacteria) and then add to the dissolved agar (use full strength 15 g/l or the soft agar recommended by Ali, though that might be too soft to streak easily). This way using the nutrient media that works, it should also work in agar form.
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