You may try with storage of bacteria in simple sterile distilled water, at room temperature.

Hi Dr. Kothari

thank you for your answer, but how long our isolates can be stored in sterile distilled water? and can we secure all of my isolates? 

Best regards

Hi,

you may stock your cultures using ratio 1:1 (culture:glycerol 80%). If your cultures grow slowly after the defrosting is quite normal. To revitalize them, try to use an enriched broth.

Best regards

Hi

Yes, I use ratio 1:1 and 1:10, when I culture last day, it grow slow (approx 3 day), thank you for your suggestion,

Best regards

Halo Pak Pramono, apakah mutlak harus menggunakan gliserol? Di lab saya sekarang, saya menggunakan cryoprotectant skim 10% dan gliserol 20%. Hasilnya cukup bagus untuk penyimpanan di freezer. Bila dirasa tumbuhnya kurang, maka bisa dilakukan reaktivasi sebanyak dua kali dari kultur beku pada media MRS Broth.

hi

kebetulan kemarin saya coba menggunakan 50% gliserol, sebelumnya saya pernah menggunakan 5% gliserol dan 10% skim juga. problem utama kemarin ialah freezer yang naik suhu sampai -10C dan begitu saya coba plate (pada MRS broth juga) dua kali, kurang begitu bagus hasinya. ada solusi?

best regards

Hi,

I would say that the storage temperature you are using is way too high for good survival. You should store them at -18 C (for short periods, max. 1 month) or -80 C if longer periods are intended. A lower temperature works much better in this case.

Concerning cryoprotectants for your culture, you can use three types of materials:

a) glycerol, that acts by modifying the rate of ice crystal growth and cell membrane permeability

b) skim milk or gelatin, which are protective colloids.

c)sugars (like lactose), that reduce the amount of intracellular water (thus reducing the damage caused by its crystallization).

It is possible to combine several of these ingredients - a quick search in a widely available search engine (such as Google Scholar) will provide you with several references on the matter.

I mostly use glycerol (100 microL glycerol; 800 microL culture) and it works well - but I work with much lower temperatures than you do. I strongly suggest that you use a lower temperature - and ensure that freezing is fast, by freezing only very small amounts at each time - you want to keep those ice crystals tiny, not to damage your bacterial cells.

Good luck!

Hi

Thank you very much for your explanation, it really helpful

yes, i do concern about the temperature since this is my first experience to storing microbes in -10C. in my old lab, we usually employ -80 C, :) 

Best regards

Hi there,

Your main problem is that you are not storing it your samples at a low enough temperature. -10 is simply too high, you could potentially attempt to flash freeze your sample (with glycerol) in liquid nitrogen before transferring to the -10 freezer if that is your only option. As this will ensure the glycerol vitrifies to try and protect your bacteria for as long a time as possible.

And it is relatively normal for your growth to be slow, you might want to consider increasing the density which you freeze your samples especially if there is a high chance of loss of sells because of the high-temperature you are storing it at.

I hope this helps somewhat.

Hi

Thank you very much Mahbubani. Its really help me :)

Dear Heru-Pramono,

I stock my LAB cultures using ratio 1:1 (bacterial culture:glycerol 50%) but at a very low -80C. It works very well. I recommned you to stıck your cultures at below -10 C. In order to revitalize the cultures, I use M17 or MRS broth (depends on the culture).

I hope this helps you.

Hi Birce Taban

Thank you very much for your kindly inform me. Yes, we already find some method to overcome high temperature (-10 C) refrigerator by centrifuge it and store it with 1:1 glycerol 50%. they grow fairly good

:)

Hello

Have you ever tried to store lactic acid bacteria at 4 degrees? Is there any recomended media?

Thank you.

Hello Rodrigues

We only store at 4°C for short term like one week evaluation. But, sometimes the activity reduced considerably