I am investigating the effect of different culture media (with different amounts of ingredients) on the urease activity of the S.pasteurii. In order to prevent the effect of the initial ingredients (which are used for dilution after the first use) on urease activity, I am going to dilute the bacterial powder with 0.5mL distilled water, shake it gently by hand (15 min) and then add 0.1 of the water-diluted bacterial solution to the culture solutions with different ingredients. Can I do this? What are your other suggestions?
The bacteria in freeze -dried form are in stress condition so it is better to use broth, e.g., LB broth to dilute and revive it before plating on the respective agar plate.
It would be better to first dilute the bacterial powder in distilled water but then centrifuge to remove any components of the lyophilized mixture (often contains sugars as lyoprotectants). Resuspend the bacterial pellet in physiological saline and add to the different culture solutions in whichever cell concentration you wish. You may also wish to resuspend the bacterial pellet directly with the culture solutions (separate your lyophilized mixture prior to centrifuging). I, personally, would do a couple wash steps with physiological saline (by resuspending-recentrifuging) to ensure that all remnants of lyohpilized media is removed.
Hope that helps!
Whether before freeze drying that culture was added with any stabilizer/cryoprotectent/antibiotic/ if it is present the trace will be thare during culturing. If it is not, should diluted with appropriate diluents like normal saline/ Phoshate buffer saline/selective broth. And also you may do the their IPQC tests like sterility means without any contamination, motility, and other basic microbial tests..
Tnx Dear Arjan; yes, culturing on a plate is possible with this strain but how can I culture the powder bacteria on a plate directly and without dilution?
yes... you can add distilled water in your conserved bacteria. It is actually how it is.
HI
Oxoid manufactures de Maximum Recovery Diluent, this buffer is recommended by the HPA from Uk for the recovery of freeze dried bacterial lenticules . For some strains really improves the recovery rate.
Depending on the microbe, I would rather use a buffered solution to minimize osmotic stress...
Concerning the last comment, I don't believe that osmotic stress is that much of a concern when re-hydrating lyophilized bacteria with distilled water as all of the solutes/salts from the original bacterial suspension are maintained in the dried powder during the lyophilization process (water is lost). Hence, when you add the water at a similar volume as the previous suspension (pre-lyophilization), the initial osmotic conditions are restored.
Prior to dilution it should be centrifuged with Normal saline /PBS and then take very small part of the pellet undergone for plating with selective medium, for colony morphology (Colour,motile, transference, etc) the other part again centrifuged with NS and then start your other process, in the mean time those bacteria was inoculated on the selective media would be compare with known standard. this my suggestion Mr.Hamed..
I would grow them on a solid medium and, then, suspended the colonies on saline (0.85%), centrifuged them and resuspended the cells directly in the solutions with different ingredients.
The bacteria in freeze -dried form are in stress condition so it is better to use broth, e.g., LB broth to dilute and revive it before plating on the respective agar plate.
I am also suggesting you to dilute in normal saline solution or culture media , not in distill water.
Hi Hamed Khodadadi. I wonder if the cells were washed before the lyophilization? In other words I wonder whether the cell powder is really only cells or there is organic material from any broth? If the cells were previously washed, then you definitely want to recover them using the desired broth (LB is the most common one, but it doesn't need to be the best broth for your bacterium, it just need to have nutrients and salts). If you try to dilute them in water, the efficiency of the procedure will be certainly lower. The cells will run out their nutrient reservoir quite rapidly and will end up dying for starvation. In contrast, if you use broth, you can always spin them down and refill the tube with water or solutions with the different ingredients that you want to test. I always use broth to resuspend lyophilized cells. I hope it helps.
I suppose it can be done . Normally thas what we do serial dilution of the original stock solution, and we use buffer instead of just distilled water.
i experienced a lot of difficulty in reviving freeze-dried cultures. we used BHI Broth
I did not mean that you should culture the bacterial powder directly on a plate without any dehydration. I do not think that is a good idea because it would be more difficult to spread the dry powder on the surface of the agar. You should try to add some distillated water (or physiological saline), pour in the surface of the medium and then spread with a Drigalski spatula or with an inoculation loop.
For all practical purposes we should not directly pate the powder as the cells require nutrition for reviving
Agreed with samuel, You need to revive your bacteria first in medium broth and then use it.
Through the experiment, it is better to added freeze dried bacterial to liquid media preserving cell rupture and adaptation to new condition. Since the additive material with bacterial cells gradually dissolved later the bacterial cells were faced .
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