In my lab, we lost the identification sign of Agrobacterium stock. So i tried to prepare stock by the following two procedures:
1. First i cultured Agrobacterium on solid YEP media and then transfer it to liquid media.Then after incubate it in shaker almost 2days, i take 8 mL of liquid culture + 2 mL 100% glycerol in a falcon tube. Then take 1 mL to each 10 eppendrof tube and store it at -20.
2. In this procedure i used 20% glycerol instead of 100% by diluting glycerol in distilled water.
Both times liquid stock in eppendrof freeze out. I read a article that if stock become freeze it means the solution is not appropriate.
I've two inquiries.
Is my procedure correct? If not than what'll be standard one.
Thanks for your co-operation.
I guess Amanda is right. I usually add 1 ml overnight culture of my agro strains to 0.5 ml undiluted glycerol, vortex and freeze. So it's about 1/3 glycerol - might make a difference. Since I never had any trouble reviving the stocks (even after years) I never tried any other dilution. Works with E.coli as well.
standard inoculums were prepared based on the method of Tingay et al., (1997). A single colony of agro was selected to inoculate two 10 ml vials of MGL media. This was incubated at 28°C, shaking at 180 rpm for approximately 48 hours. Standard inoculums were prepared by adding 200 µl of culture to 200 µl of 15% glycerol in an eppendorf tube and stored frozen at -80°C.
an otherwise, you can confirm whether the bacterial overgrowth was actually Agrobacterium using Benedict test. The LYM medium (1% lactose, 0.1% yeast extract, and 2% agar) containing plates were streaked with the overgrown bacteria and incubated overnight at 37°C. Benedict reagent was added to these plates and yellow ring of cuprous oxide formed around the growth confirming positive results (Bernaerts and De, 1963).
Benedict’s solution
Solution I
Mix the following: 17.3g Na Citrate
10.0g Na2CO3
60-70ml hot deionized water (~55 deg.)
Solution II
1.73g /10 ml H2O CuSO4 . 5H2O
Cool Solution I, add 10 ml Solution II, bring up to 100ml with
deionized water. Stable at room temperature.
For these practicalities I find the information resources from Addgene quite invaluable. After their guidelines (https://www.addgene.org/recipient-instructions/create-glycerol-stock/) 25% glycerol in the final stock solution has been working well for me.
However, I do think that it is not of utmost importance of having this process optimised, since usually you only need to recover one colony out of a streak.
I usually add 500ul of bacterial liquid culture to 500ul of 80% glycerol. I plunge this stock into liquid nitrogen and store it frozen at -80 . When I need to use the stock I keep it frozen and use a wire loop (flamed) or sterile toothpick to take a small amount of frozen cells which I then streak out on a plate.
What do you mean by freeze out? If you are storing the stocks at -80 (which you definitely should be) then the stocks will freeze whatever your glycerol concentration.
(N.B. I always make 2 glycerol stocks, a regular use one and a backup, if my regular use one ever becomes non-viable I use the backup to make 2 more - a new regular use one and a new backup)
Hi Niloy,
I follow this protocol for Agro stock.
1. I streak the Agro strain in YEP solid media (you can also use LB agar), keep it in 28'c, generally It takes 24-48hrs for growth. I prepare fresh YEP Broth(LB broth is also enough), I take a single colony in 10ml of Broth with required antibiotic,keep it at 28'c with 160-180rpm continous shaking.
2. I usually add 1ml from the culture to 1ml of 50% glycerol(autoclaved-hood open) in a cryovial (autoclaved-hood open), then I store it in -80'C.
Note- Repeated freeze thaw can degrade the culture, so i suggest you to make aliquots.
All the best...
Hi Niloy,
I follow this protocol for Agro stock.
1. I streak the Ag strain in YEP solid media, keep it in 28'c, generally It takes 3 days to get single colony. I prepare fresh YEP Broth(LB broth is also enough), I take a single colony in 3ml of Broth with required antibiotic,keep it at 28'c with 160-180rpm continuous shaking.
2. I usually add 1ml from the culture to 0.8ml of 98% glycerol(autoclaved-hood open) in a cryovial (autoclaved-hood open), then I store it in -80'
Hey Niloy Kumar Das ,
I use 25% (Final concentration of glycerol in the stock). I will suggest you not to freeze the vial immediately after preparing it.
Mix glycerol and bacterial broth, vortex them, keep them aside for some time (~30 minutes) Such that glycerol can enter the cells, and then freeze them using liquid nitrogen.
Good Luck
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