Dear Sekar,

Please read the following text:

Table 1. Approximate time bacterial cultures remain viable in different storage conditions.

Condition                   Temp (°C)                                                           Time (approx.)

Agar plates                       4                                                                       4 - 6 weeks

Stab cultures                    4                                                                       3 weeks - 1 year

Standard freezer           -20                                                                      1 - 3 years

Super-cooled freezer   -80                                                                      1 - 10 years

Freeze dried                   ≤4                                                                        15 years+ 

Short-term Storage Working bacterial stocks can be streaked onto agar plates and stored at 4°C for daily or weekly use. Culture dishes should be wrapped with laboratory sealing film (plastic or paraffin) and stored upside down (agar side up) to minimize contamination and to keep both the culture and agar properly hydrated. Some bacterial strains can be stored for up to 1 year at 4°C in agar stab cultures, which are especially useful for transporting samples to other research facilities. Stab cultures are prepared by first sterilizing strain-compatible agar (e.g., lysogeny broth [LB] agar for E. coli) and then transferring the warm liquid agar to screw-cap vials using the appropriate aseptic technique. After the agar has solidified, a single colony is picked from an actively growing culture using a sterile, straight wire. The wire with the bacteria is then plunged deep into the soft agar several times, and the vial is incubated at 37°C for 8-12 hours with the cap slightly loose. The vial is then sealed tightly and stored in the dark at 4°C. - See more at: http://www.thermoscientific.com/content/tfs/en/about-us/general-landing-page/storing-bacterial-samples-for-optimal-viability.html#sthash.FqNKyBns.dpuf

Long-term Storage

As mentioned above, the temperature at which frozen bacteria are stored affects how long they can be stored while remaining viable. Freezing and thawing cells at an appropriate rate and maintaining the frozen stocks at the proper storage temperature help to minimize damage from the freezing process. Also, the greater the cell density, the better the recovery is after thawing the cells. For most bacteria, a density of 107 cells/mL will result in adequate recovery if all conditions are properly maintained.1-2

Cryoprotectants: As water in cells is converted to ice, solutes accumulate in the residual free water. This localized increase in salt concentration can denature biomolecules.3 Furthermore, ice crystal formation can damage cell membranes. Additives that are mixed with the bacterial suspension before freezing lower the freezing point and protect cells during freezing to minimize the detrimental effects of increased solute concentration and ice crystal formation. The most commonly used cryoprotectants are dimethylsulfoxide (DMSO) and glycerol, which are typically used at 5-15% (v/v). Non-permeable additives used as cryopreservants, such as polysaccharides, proteins and dextrans, adsorb to the surface of microorganisms and form a viscous layer that protects membranes, making these agents particularly useful for cryopreservation.4 Other commonly used additives include blood serum, ethylene glycol, methanol, skim milk, yeast extracts and tripticase soy.4

Freezing samples: To prepare glycerol stocks, the glycerol is first autoclaved and allowed to cool. The appropriate volume of glycerol is added to a suspension of log-phase bacteria and vortexed to dissociate the cells and ensure even mixing of the bacteria with the glycerol. After aliquoting the suspension into cryogenic screw-cap vials, the cells are snap-frozen by immersing the tubes in either ethanol-dry ice or liquid nitrogen and then stored in freezers (‑20 to -80°C) or liquid nitrogen (-150°C).5 Repeated thawing and refreezing of the bacterial stocks will reduce cell viability and should be avoided. When recovering strains with antibiotic selection markers, culturing them on selective media will ensure that the bacterial stocks were not contaminated.

Freeze drying: Bacteria can be freeze-dried by suspending log-phase cells in a lyophilization medium and then freeze drying the suspension. Not all bacteria can be successfully freeze-dried.6-8 Certain strains might not survive the process or die rapidly once freeze dried. The best way to determine if a strain is amenable to freeze drying is to empirically evaluate its stability post-freeze drying while maintaining a live culture as a backup. Once freeze dried, it is best to store the bacteria at or below 4°C.

Conclusion

Storing bacterial samples requires careful consideration about how they will be used, availability of the storage unit and space within the unit. The time it takes to prepare a sample for storage, how precious the sample is and whether the strain is compatible with the desired storage condition must also be considered. Furthermore, the storage unit must be carefully monitored, as inconsistent temperature fluxuations can often occur in institutional freezers commonly used in research facilities.9 Aseptic technique must be strictly observed during sample handling and stock preparation to avoid cross-contamination between strains and contamination from air movement and handling. Finally, good record keeping is critical for maintaining bacterial samples. Downloadable forms available online can help keep track of all events surrounding the handling of a specific sample. With careful planning and handling, you can ensure that your bacterial sample remains viable for the entire study period and for future studies.

Cited References

1. Simione, F.P. and Brown, E.M. (1991). ATCC Preservation Methods: Freezing and Freeze Drying. American Type Culture Collection, Rockville, Maryland.

2. Simione, F.P. (1992). Key issues relating to the genetic stability and preservation of cells and cell banks. J Parenter Sci Technol 46:226-32.

3. De Paoli, P. (2005). Biobanking in microbiology: From sample collection to epidemiology, diagnosis and research. FEMS Microbiology Reviews 29:897-910

4. Huba'lek, Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology 46: 205-29.

5. Moore, L.W. and Rene, V. (1975). Liquid nitrogen storage of phytopathogenic bacteria. Phytophathology 65:246-50.

6. Miyamoto-Shinohara, Y., et al. (2008). Survival of freeze-dried bacteria. J Gen Appl Microbiol 54(1):9-24.

7. Miyamoto-Shinohara, Y., et al. (2006). Survival curves for microbial species stored by freeze-drying. Cryobiology. 52(1):27-32.

8. Miyamoto-Shinohara, Y., et al. (2000). Survival rate of microbes after freeze-drying and long-term storage. Cryobiology. 41(3):251-5.

9. Su, S.C., et al. (1996). Temperature variations in upright mechanical freezers. Cancer Epidemiol Biomarkers 5(2):139-40.

- See more at: http://www.thermoscientific.com/content/tfs/en/about-us/general-landing-page/storing-bacterial-samples-for-optimal-viability.html#sthash.FqNKyBns.dpuf

Hoping this will be helpful,

Rafik

Dear Rafik,

Thanks very much for your detailed answer. It is very much informative and useful.

i especially want to know whether i can store the cultured liquid phase e-coli in LB medium in normal refrigerator for 2 days or so. because for me to use the liquid culture, currently i am culturing daily from agar. if the liquid culture can be directly stored for say 2-3 days without much cell death ratio, then my daily culture work may reduce.

Thanks

hari

Dear Sekar,

I think if you store the liquid at 4 C for two days, it will still give you good results.

Rafik

Dear Rafik,

Thanks  a lot for clarification !

hari

Yes you should be able to store for a few days at 4 degrees, but still be sure you dilute back so that they are growing enough generations to fully be in the growth phase you need. 

One note I would add to Dr. Harikrishnan's table is that those survivals are reasonable estimates for wild type strains, but RecA mutants strains (and most strains used for molecular biology are RecA minus) have much shorter survival periods and won't last nearly as long on plates or in liquid at 4C as the wild type. 

Last week (Monday 1st Dec),  I grew an overnight culture in 10mL LB+carbenicillin and stored it at 4oC. Yesterday (8th December) I added 2mL of that culture to 20mL LB+C and within 3 hours there was sufficient growth to seed a large scale culture.  So my answer (for XL1-blue, anyway) is at least 1 week is OK, probably much longer.

But if it will save you so much time, why not find out by experiment!!!

It all depends on what you are going to do.  Whenever cells go stationary, they accumulate mutations at about 10e-6 /locus per day.  So bulk cultures are unaffected and if you are choosing individual cells, you would be unlucky to choose a mutant.  Generally in the lab we would not use refrigerated colonies for more than two to three weeks. But this is arbitrary. 

A two-day old broth culture is perfectly OK for most purposes. It may take a little time to come out of lag phase (maybe an hour) when you subculture it, but otherwise it will be fine. 

This question and answers are very valuable for me aswell!

So i need to strike my plate on monday morning, put on refrigirator,  go back to my home town on thursdays and come back to working city on monday. Its a recombinant b strain ecoli in my case. So they would still be alive in mentioned timeline right?

Thank you. I'm guessing the same is applicable to my case as well. I'm supposed to do a maxi prep today (Saturday) but someone has kindly locked the door to the giant centrifuge that I need. So I keep my cultures in 4'C now until Monday. (Lesson lernt: Don't work on weekends. Haha).

Very useful answers here. let me put a question differently. if I store my LB broth cultures in the refrigerator for 3days, If I do not sub-culture it but instead choose to do a plasmid prep directly from the stored culture, would I still get a good yield? Rafik Karaman and Michael J. Benedik

Roosevelt Anyanwu it would probably be ok to do your mini preps after few days, plasmid DNA is quite stable. However what I normally do when I don't have time to complete a mini prep is to pellet the cells and store the cell pellet frozen and then when time to do the plasmid prep just resuspend into the plasmid prep lysis buffer.

You have been very helpful. Thank you very much Michael J. Benedik .

Roosevelt Anyanwu Actually, I was looking for a good answer to this question too. I know you will get a good yield. The question is whether or not some of the cells will be dying, breaking down, and releasing contaminants that will cause the resulting DNA to be less pure, perhaps increasing the endotoxin concentration. As Michael Benedik said, we usually freeze bacterial pellets for future use. Someone in the lab now wants to store the liquid, and I suspect it will be OK for our purposes, but I am not certain it is a good idea.

https://www.thermofisher.com/in/en/home/industrial/microbiology/microbiology-learning-center/storing-bacterial-samples-optimal-viability.html

Rafik Karaman which is the appropriate shipping temperature for agar slants? They told me they can go at room temperature. Do you think it's necessary to be transferred at 4oC?

Ioanna Eleftheriadou stabs can certainly be stored for a long time at room temperature so shipping at room temperature should be fine. I have had stabs that were stored at room temp for many years and were still fine (so long as they were well sealed). The problem with stabs is mostly that of their drying out.

Michael J. Benedik thank you very much for your help. The only problem is that the courier says that due to summer the temperature can be higher. I ask because there is a big difference in shipping cost between Environmental and room temperature.

Summer shipping temperatures is an entirely different matter. E. coli for example will do fine at 43C but will die off pretty rapidly at 45-46C (depending upon the strain). Other bacterial strains may be higher or lower. So it would very much depend upon the actual temperatures the samples would be held at.

If this was something very important I would probably ship at environmental controlled temperature. Sitting in a hot truck during a sunny afternoon could easily exceed 45C.

If not critical then you could send normally and see if they make it or not. If they arrived dead, then resend at controlled temperatures (assuming time is not critical).

I'm grateful for your answer. The strains will come from Spain to Greece and are Pseudomonas aeruginosa. If they arrive dead I will have to pay the shipping cost twice. So I will ask controlled shipping temperature. Thank you again!!

As Michael indicated P. aeruginosa grows well at 37°C, and can also survive at a wide range of temperatures from 4°C to 42°C.

or more details, please see:

Article Growth and Laboratory Maintenance of Pseudomonas aeruginosa

Article Growth and Laboratory Maintenance of Pseudomonas aeruginosa

Rafik

Michael J. Benedik Rafik Karaman could you help me with this ? Bacterial strains of Pseudomonas aeruginosa on agar slants are considered as category B for shipping ? Thank you in advance

It depends upon the strain and the origin. If it is a clinical isolated and infectious then it likely is Category B. If it is a soil or environmental isolate or a lab strain then it is probably exempt. Essentially if it is a BSL-2 strain then it should be Category B, if it is a BSL 1 strain then it should be exempt. But EU rules might be stricter than US rules.

Michael J. Benedik thank you very much!! particularly are efflux pump mutants that are originated from PAO1.

Based on the type of strain you have, short-term preservation can be different. If your strain is mutant it is better to freeze-dry or cryopreserve it . Otherwise, by using a slant culture you can maintain E.coli for c.a. two weeks in the refrigerator.

Sharareh Harirchi Do you know if Pseudomonas aeruginosa efflux pump mutants on agar slants can be stored at room temperature for several weeks ?

Ioanna Eleftheriadou

Hi,

On the basis of my experiment in Iranian Biological Resource Center (IBRC), room temperature is not suitable for bacterial culture and also, P. aeruginosa can not be store for a long time in the refrigerator. It may be survive 2 weeks. I recommend to preserve it in a deep freezer.

I can only say from my experience, but a culture of E.Coli cells can be easily stored in a fridge for a day. At this temperature, they are unlikely to divide, but their metabolism also slows down. Before dying they will be starving, so you might end up having cells with certain genes being up- or downregulated. When cells starve or being stressed, they alter not only metabolism but also DNA protein interactions and other biophysical stuff is changing.

It is more from a publication point of view, that we should not alter too much things, and everything what we do must be justified. Like why would you keep cultures for few days in a fridge? But it makes perfect sense, if you would like to study, for example starvation of cells.

Ioanna Eleftheriadou - I'm sure this is too late to be relevant to you, but JIC anyone in the future has a similar question...I feel like there is a cheap & easy answer that I didn't see brought up.

Since your case is mostly about survivability of the bacteria over a few days (a week at most). Why not just mail/ship it (expedited 1-3 day) in a nice thick Styrofoam cooler loaded up with ice packs (or dry ice if you're planning to send it frozen)?

I frequently use pseudo aeruginosa (ATCC 15442) in our lab. Compared to the many other organisms we use, pseudo has been pretty hardy, and multiplies quickly, which to us makes it a great challenge organism for the antimicrobial testing we do. I can't help but think that if pseudo didn't like a little heat (we incubate at 35-37C/body temp) and if it didn't survive in cooler temps as well (18-21C, ex. hospitals), it wouldn't cause so many infections.

In a worst case scenario, I would say it's still more likely than not, that if even just a few of your bacteria survive the trip, once given a few days of TLC, there will be more than enough to grow frozen and working stocks. I would just suggest that upon its arrival, to resuspend it in enrichment broth & incubate it near body temp @ >/= RH, stationary/or not (both work, but stationary will be slower), in a vented container (if not rocked).

Hi everyone, I also have a question regarding storage of E.coli culture in ampicillin medium. I was wondering whether I could store the 3ml cultures on 4 degrees over the weekend to conduct a miniprep plasmid isolation on Monday morning.

Thanks for the help!

@Michael, I wish to prepare glycerol stock of DH5 alpha strain, and I have overnight grown culture. Can I store this culture at 4 deg for a day and use it for banking the next day?

yes