I want to preserve bacterial ATCC strains at -40C. I have E. coli, Pseudomonas aueroginosae, K. pneumoniae, Staph. aureas, Hemophilas influenzae, Strp. pyogenase, Strep. pneumonae. Can anyone suggest a procedure how to do it?
The first thing to do here is to grow your bacteria in he proper medium. Take samples of the rapidly growing bacteria in cryovials. Add enough glycerol to make 15% glycerol final concentration. Keep this right away in -20 and you can store it there. I have never stored at -40 so I don't know whether you need to snap freeze for that first with liquid nitrogen or dry ice before storage which is done when storing at -70. I suggest you try snap freezing and also transfer those that are at -20 into the -40 and see which one works best.
From my experience I grow bacteria cultures in appropriate broth add same quantity of glycerol (50:50 or 1:1), short term or for working stock store in -20 and long term storage in -80.
An alternative method: I grow bacteria in a solid medium (plate culture), where I check it is not contaminated and then I suspend the bacteria in sterile skim milk. Store at -70 ° C.
Hi Ola,
Did you get the answer you are looking for? Did you try my suggestions? If you did, can you share the results with us?
Thank you all for the useful information. I have tried to store my bacterial strains in glycerol after culturing them into nutrient broth from a pure culture on a suitable agar. All my bacteria lives until now except the fastidious one Strep. pneumoniae and Hemphilus influenzae. so if any one have any suggestions for that.
Dear Zoraida Aguilar if you have any farther explanation for the snap freeze I can use it!! I also can use liquid nitrogen for any process if anyone have suggestions.
You can prepare 30% Glycerol and suspense low density culture and stored -80oC. hope it help you.
Use the pre-prepared cryobeads (any company) follow their instructions (harvesting from plates is far better then broths). For the fastidious organisms store at -80, others can be stored at -40 (please note Hemophilas influenzae, Strp. pyogenase, will not preserve for long below -80 for the methods that have been mentioned so far). If you are planning on publishing your work you would be better using NCTC strains as the ATCC dont have as good a traceability. For further information contact me directly.
The best viability results are generally observed with liquid nitrogen storage. If you do not have access to such facility, try to find -140°C freezer or -80°C freezer.
Although some cultures can be stored at -20°C, long-term conservation at temperatures higher than -80°C (and some people say higher than -140°C) might give you unpredictable results. For instance, improper storage may results in interstrain variation of physiological properties and give you several colony types. This is very annoying if you want to have your back up for several experiments to be compared in future. If you are not sure that the culture has retained its properties, you would not be able to compare results of several studies.
Alternatively, you can try getting your cultures freeze-dried.
For the snap freeze, add enough glycerol to make 15% glycerol final concentration. Put this in liquid nitrogen to snap freeze (for at least 30 minutes) with liquid nitrogen or dry ice before storage at -70. If you do not have a -70 freezer, you can keep them in the liquid nitrogen tank but make sure never to run out of liquid nitrogen.
I agreee with Zoraida Aguilar. but you know well many labs have no facility of liquid nitrogen
Then the best solution if there are no -70 or -40 is probably to prepare a stab of the strain for long term storage. For the stab, you must make sure there is no contamination.
All what has being said are acceptable methods of preservation; however, I find the use of cryo-preservation as suggested by Zak Prior very rewarding. I think as others talk about glycerol, equally the cryo-preserve consists of beads and some glycerol. The beads are charged so much so that your organism gets attached to the beads and whenever there is need a single bead is taken from the tube and propagated.
Dear Ola Karmi,
I'm curious... Why at - 40? I find it interesting. Do you have the need for frequent withdrawals of those strains through time. I must tell you that you may be facing a small deggree of risk in viability lost if you're storing for a long time ( how long is long?). However, all the suggestions above are fairly sound and you may not even have to worry about viability. Nevertheless I can think of a long standing technique that proves to be very solid and that ATCC practices to date. Lyophilization of your culture may be the ultimate way, provided you don't need to pull cells frequently. To give you an example, I have found strains from the 1940's maybe even 30's stored at room temperature that have been used succesfully after such long period of time. Quite amazing indeed. The caveat here is you need a specific piece of equipment but once you locate lyophilizer the rest is simple. Wish you the best of luck.
Dear Angel very interesting i suggest you share it with us: I have found strains from the 1940's maybe even 30's stored at room temperature that have been used successfully after such long period of time.
Dear Ola,
Preservation can be done satisfactorily using Microbank Bead System at -20C or -70C. Follow instructions of using Microbank Bead System provided by manufacturer.
Its easy method of preserving strains.
One of the best ways to long time preservation is the lyophilization, also called freeze-drying. When bacteria are lyophilized there is no requirement of low temperatures, which facilitates storage and transportation. This process is commonly used to ATCC. To perform the lyophilization process you will need a freeze-dryer. If there is none in your lab, look to the neighbors.
Job is right, the requirements of low temperature in developing countries is an issue.
Hi Ola,
I am attaching an ATCC official bacterial culture guide recently published by ATCC team. I think this would help you at large in understanding and maintaining different ATCC bacterial cultures. Best of luck.
Having used the glycerol - broth method myself for many of your bacteria it turned to be very reliable. There was no problem in "wakening" the bacteria after 5 years.
By freezing at -70 or -80 degrees in Celsius; first, prepare
a suspension of your bacterial or yeast strain in skimed milk with 10% glycerol and
put small volumes in cryovials; than just put them in the freezer.
-40 is curious température, for our knewlegment we use -80. Thé culture is stores ni booth containing glycérol (3/1 v/v).
According to ATCC, use 5-10% glycerol and recommended temperature is -70C...some bacteria is glycerol sensitive...
Most pathogens can be stored at-20 in a 50:50 glycerol:broth mixture rather than in 10 or 20% glycerol. 50% glycerol does not solidify and hence there is no cycle of freeze thaw. Freeze thaw cycle decreases cell survival by a log or two. As others have pointed out, some species are not amenable to glycerol preservation eg some spirochetes but most pathogens can be stored in 50% glycerlol. Freeze Drying (FD) can be tedious and there is no guarantee that bacteria can be preserved for long periods especially if one is a novice at FD procedures.
Dr B. Patel, can you give some general recommendations what is the suitable preservative method for fastidious and non fastidious bacteria since there are so many school of taught?I found difficulties in maintenance of fastidious bacteria...
Dear Mhd Razali, the following may be of some assistance. Cryotubes for aerobe preservation eg from Nunc - comes in sterlie packs. For anaerobes, use 2ml glass vials which come with butyl septum - usually used in HPLC / GC. These vials come non-sterile and can be sterilized using normal sterliization procedures. Grow cultures in liquid to early to mid log phase for aerobes using the best conditions for growth. You can also use solid medium. For anaerobes grow directly in sterile vials. Harvest cells using low centrifugation and remove supernatant. For anaerobes use a stream of sterile OFN for all manipulations but is not required for aerobes. Resuspend in a 50:50 pre-sterlized mixture of glycerol-Trypticase Soy Broth (TSB). The final concentration of TSB should be 2%. you can also use serum, sucrose, lactose to the mixture in which case reduce the concentration of TSB. Store at -20, at -80 and liq Nitrogen (all or whatever is available). Check CFU counts before and after preservation. If the count has decreased substantially than use another method by cecking the literature. There are a lot of misconceptions / miths about preservation techniques in the literature but the most important is to question what each component does and why they are important. For example, Loss of viability can be due to cells being accessed from stationary phase or decline phase in which case the viability is already poor and the cultures will not preserve well. Lab work isby trial and error and with reasoning you should get the required level of confidence. Preservation of cultures is very important as you may need a collection in future to determine eg epidemiology, evolution / changes in the strains over time, new techniques applied to understand strain differences, identify gene markers for developing rapid diagnostic tests etc. All the best.
Dear Dr. B. Patel...Thank you very much for a very tremendous explanation...
Bacterial cultures can be preserved for long by suspending in glycerol broth and storing at -20 to -70 degree Celsius.
The protocol as below:
To 0.85 ml of bacterial culture, add 0.15 ml of sterile glycerol (sterilized by autoclave, final glycerol concentration : 15%, v/v).
Vortex the culture to ensure that the glycerol is evenly dispersed.
Transfer the culture to a labeled storage tube equipped with a screw cap.
Freeze the culture in dry ice or in liquid nitrogen, and then transfer the tube to –70 degree Celsius for long-term storage.
You can refer Molecular Biology by Sambrook Russel for more details
Lyophilisation is the best method for prserving cultures on long term basisThis is my observation after adopting all methods
I agree with the asnwers provided; lyophilization and glycerol stock at very low temperature. The best way is to plate frequently if use contineously and keep at low temperature.
The main problem in maintaining cultures is the need for repeated subcultures. So if available nitrogen cylinder the best.
Lyophilization is by far the best method I have used or come across until now. My own bacterial culture (related to a US patent) is preserved lyophilized. I prefer this over other methods since it is easy to use and revive culture for regular/intermittent use. You can otherwise consider Dr. Patel's (and also some others) suggestion on using glycerol stock-cryo preservation after making sure that your culture comes from the log phase (grow you liquid cultures and plate them to find out the exact timing of this log phase that you want to use your culture from). However, for regular use it is preferable to use frequent sub-culturing method. You can once in a while check for the purity of your bacterium by using your cryo-preserved stock as a reference. Good luck..:-)
I think the previous researchers were right in suggesting lyophilized cultures for preservation as the conventional methods require repeated plating and that may prove to be a hinderence to originality of the bacterium
I also would like to know more about lyophilized bacteria. At what stage in their growth cycle do you do this? Any stabilizer needed? What solvent should they be in? What is the survival rate? How long can you keep them in the lyophilized state? What temperature do you keep them after lyophilization? Is there a reference material you can post or send privately in a message? All answers will be appreciated.
Preservation
Most bacterial strains can be stored for many years as freeze-dried cultures (lyophilization) or at temperatures
below -130˚C (cryopreservation)⁴. There are many advantages of preservation that far outweigh the required
investment in the equipment and reagents needed to maintain living cultures. These advantages include:
• Overall safety of bacterial stocks against loss due to equipment failure or contamination by other
microbial organisms.
• Elimination of time, energy, and material costs associated with the maintenance of bacterial strains not
currently in use.
• Insurance against phenotypic drift associated with prolonged passage, due to genetic instability and/
or selective pressures.
• Creating a standard working stock that can be used for a series of experiments.
Cryopreservation
Overview
Generally, freezing a suspension of living cells may result in a decrease in viability16, 17. As the bacterial cell
suspension is cooled below the freezing point, ice crystals begin to form and the concentration of solutes
in the suspension increases. The formation of intracellular ice crystals can result in the damage of cellular
structures. This effect can be minimized if water within the cells is allowed to escape by osmosis during
the cooling process. A slow cooling rate, generally -1˚C to -10˚C per minute, facilitates this progression.
However, as the cells lose water, they shrink in size and will quickly lose viability if they surpass a minimum
threshold volume. The addition of cryoprotectant agents, such as glycerol or dimethylsulfoxide (DMSO),
will mitigate these effects18, 19. For the preservation of bacterial cultures, ATCC recommends using glycerol
as a cryoprotectant.
The standard procedure for cryopreservation is to freeze cells slowly until they reach a temperature below
-70˚C in medium that contains a cryoprotectant. Vials are then transferred to a liquid-nitrogen freezer to
maintain cultures at temperatures below -130˚C.
The recovery of cryopreserved cells requires the rapid thawing of the bacterial suspension in a 37˚C water
bath. The entire contents of the vial are then transferred to an appropriate growth medium.
There are numerous factors that can affect the viability of recovered bacterial strains. These critical
parameters can include the composition of the freeze material, the growth phase of the bacterial strain,
or the concentration of bacterial cells within the solution. To obtain optimal cell viability upon recovery,
modify the cryopreservation procedure for each bacterial strain, being sure to harvest cultures during the
late logarithmic phase of growth.
Contact ATCC for more information on the cryopreservation of bacterial strains. ATCC bacterial strains that
are frozen are commonly cryopreserved with a medium consisting of a final concentration of 10% sterile
glycerol.
Freeze Medium
Glycerol and DMSO are the most common cryoprotectant agents. ATCC predominantly recommends using
a 20% glycerol stock at a final concentration of 10%. However, if the bacterial strain is sensitive to glycerol,
a 50% DMSO stock can be used at a final concentration of 5%. In contrast, glycerol can be sterilized via
autoclavation whereas DMSO must be sterilized by filtration. Care should be used when handling any
DMSO solution as it will rapidly penetrate intact skin and may carry toxic contaminants along with it.
Phone 800.638.6597 www.atcc.org page 18
Preservation
Use only reagent-grade DMSO or glycerol. Store both in aliquots protected from light. ATCC offers DMSO
(ATCC® No. 4-X) that has been thoroughly tested for use, as well as TSB with 10% glycerol (ATCC® No. 20-
2200) for the cryopreservation of non-fastidious bacteria. Overall, the optimum formulations for individual
bacterial strains must be determined empirically.
Equipment
A. Cryopreservation vials
There are two materials to choose from for cryopreservation vials: glass or plastic. Glass vials are
more difficult to work with; they need to be sterilized before use, they need to be sealed with a hot
flame, and they can be difficult to open. However, they are preferred for long-term storage (many
years) of valuable cultures and are considered fail-safe once properly sealed.
If cryopreservation in glass ampoules is not possible, plastic vials can be used. Plastic vials come in
two varieties: those with an internal thread and silicone gasket, and those with an external thread.
Vials with an internal-thread were the first commercially available, but have some disadvantages
over the external-thread version. For example, while the silicone gasket provides an excellent seal,
it needs to be tightened just right; the vial will leak if the seal is too tight or too loose. For the
storage of cryopreserved stocks, ATCC uses plastic vials.
B. Controlled-rate freezing chambers
There are several means to achieve a cooling rate of -1˚C per minute. The best method involves the
use of a computer controlled, programmable electronic freezing unit (such as Thermo Scientific*
CryoMed Freezers), which rigorously maintains this rate of cooling. This is the method used
exclusively at ATCC. Such equipment is relatively expensive and absolutely necessary for only the
most sensitive strains.
A less costly approach is to place the cryopreservation vials into an insulated chamber and cool
for 24 hours in a mechanical freezer at -70˚C or colder. There are several commercially available
freezing chambers which achieve a cooling rate very close to the ideal -1˚C per minute (CoolCell®
LX; ATCC® ACS-6000). Alternatively, the vials can be placed into a polystyrene box, with 15 mm
(3/4 inch) thick walls and 1L capacity that is packed with paper, cotton wool, or foam peanuts for
insulation.
Liquid Nitrogen Freezing Storage
The ultra-low temperatures (below -130˚C) required for long-term storage can be maintained by specialized
electric freezers, or more commonly, by liquid nitrogen freezers. There are two basic types of liquid nitrogen
storage systems: immersing vials in the liquid or holding vials in the vapor phase above the liquid. The
liquid-phase system holds more nitrogen and thus requires less maintenance. However, there is always a
chance that some liquid will enter improperly sealed vials, which may then explode when retrieved. For this
reason, ATCC strongly recommends storage in vapor-phase.
Vapor-phase storage systems create a vertical temperature gradient within the liquid nitrogen container.
The temperature at the bottom of the container will be -196˚C, whereas the temperature at the top will
vary depending upon the amount of liquid nitrogen at the bottom and the length of time the container is
open. To ensure the safe storage of cells, maintain sufficient levels of liquid nitrogen in the container so that
the temperature at the top is -130˚C or colder. All storage systems should be equipped with temperature
alarms.
Refrence:-https://www.atcc.org/~/media/PDFs/Culture%20Guides/ATCC_Bacterial_Culture_Guide.ashx
A hi tech labs can use liquid nitrogen for storing cultures,I am giving methods for ordinary labs The best method for such labs is stab culture and every month subcultures can be done and store carefully
Hello All! Could you please suggest method of preparation of frozen stocks of anaerobic microbe F. nucleatum (ATCC 25586) other than suggested by ATCC.
Aslamo alaikum! Can anyone suggest me the method for the preservation of bacterias at -80 for at least 6 months. the bacterias i want to preserve are E. coli, Pseudomonas stutzeri, Staph. aureas, Bacillus subtilus and Enterobacter sp.. Can anyone suggest a procedure how to do it?
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology