how do you not unfreeze bacteria stock

by Prof. Muriel Koepp Jr. Published 3 years ago Updated 2 years ago

Subsequent freeze and thaw cycles reduce shelf life. To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw!

Don't thaw your glycerol stock, merely dip a (sterile) pipette tip in it and put it in 2 mL LB medium (or equivalent) pre-culture. Then after a few hours either transfer it to a larger volume for midi/maxy prep. I used to start the pre-culture in the morning, and then transfer at the end of the day to 150 mL LB medium.

Full Answer

What happens when you add glycerol to frozen bacteria?

The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years. Watch the protocol video below to learn how to create bacterial glycerol stocks.

How do you remove bacteria from glycerol stock?

Subsequent freeze and thaw cycles reduce shelf life. To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria onto an LB agar plate.

How to store frozen bacteria for a long time?

However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive.

How do you make stock of a bacterial strain?

To make stocks of a bacterial strain I suspend fresh bacteria, grown on plate or in culture, in a 1:1 solution of LB and glycerol. To make chemically competent cells I use the following protocol:

Can you not let the glycerol stock Unthaw?

Try not to freeze/thaw your glycerol stock too many times. Placing the glycerol stock on dry ice while streaking onto LB agar will prevent it from thawing completely and will improve the shelf life. It is very important that you shake the glycerol before freezing (5-6 times).

How do you grow bacteria from frozen stock?

Bacterial Glycerol StocksPut 0.5ml bacterial culture in a sterile eppendorf tube.Add 0.5ml of sterile 80% (v/v) glycerol soution.Freeze on dry ice or directly into –70oc .Store at –70oC. Cells are best for about 4-6 months, but will probably work ok for a whole year.

What happens if your glycerol stock thaws?

To my knowledge, glycerol is not toxic to cells (bacterial or eukaryotic). However, it does inhibit growth. The thing that is indeed toxic to bacteria is the repeated freeze/thaw cycles. A glycerol stock from -80C should never be completely thawed.

How do you preserve bacteria for a long time?

Freezing is a good way to store bacteria. Generally, the colder the storage temperature, the longer the culture will retain viable cells. Freezers can be split into three categories: laboratory, ultralow, and cryogenic. The problem faced by bacteria (and other cells) stored in freezers is ice crystals.

How do you Plate bacteria from glycerol stock?

Streaking bacteria on an agar plate from a glycerol stockTransfer the glycerol stock onto dry ice next to a flame.Sterilise the loop in the flame.Rub the loop in the glycerol stock.Streak the bacteri in zigzag over half the agar plate.Sterilise the loop.More items...•

Does glycerol need to be refrigerated?

While glycerin is shelf stable and does not require preservation or refrigeration in concentrations above 50 percent, it is important to know that using any concentration less than 50 percent poses several challenges.

How do you reactivate bacteria?

1:283:12MicroKwik Culture® Reactivation Instructions - YouTubeYouTubeStart of suggested clipEnd of suggested clipCulture from the culture vial and transfer it to the tube containing. The rehydration medium cap theMoreCulture from the culture vial and transfer it to the tube containing. The rehydration medium cap the tube. Allow the culture to reactivate for the maximum.

How do you revive a dead bacterial culture?

make a desired broth solution and cut a small portion of slant containing the organims. let it grow for 24-18 hrs and there after sonicate it for 30mins only in a sonar bath not in the probes, and incubate it at 37 C for 24-48 hrs. Then follow streaking method on a nutrient plate and repeat it for several times.

How do you thaw frozen bacteria?

These are temperatures where bacteria multiply rapidly. Thaw food in the refrigerator at 40°F or less, in cold running water less than 70°F, or in the microwave if you'll be cooking or serving it immediately. Thawing in the refrigerator takes the longest time and advance planning.

How do you thaw glycerol stocks?

Thaw bacterial glycerol stock(s), mix by pipetting, and transfer 5 µL per well into the deep well growth plate. 2. After inoculation, seal the growth plate with a gas-permeable seal and shake (300 rpm) at 37 °C for at least 16 hours (hairpin-pLKO, sgRNA-pXPR, or ORF-pLEX clones), or 18 hours (ORF-EntryClone clones).

Why is glycerol used to freeze bacteria?

Glycerol is used as a cryoprotectant as it disrupts the hydrogen bonding between water molecules, hence preventing the formation of ice crystals during freezing. This protects yeast and bacterial cells from ice crystal damage, and maintains their viability for future reculture.

How do you store bacterial culture in broth?

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.

How do you maintain stock culture?

Frozen storage on cryoprotectant beads Perhaps the most practical method of long term microbial stock culture storage for smaller laboratories and those engaged in routine testing is to freeze cultures onto porous beads designed to allow cells and spores to attach to their surface – providing some degree of protection ...

How do we store bacteria?

Bacteria are best preserved by freezing at ultralow to cryogenic temperatures, or by freeze drying. However, some bacterial cultures may be used daily, thus having a readily available active culture can be practical. Cultures stored at 4°C are sensitive to more rapid die off than frozen or freeze dried bacteria.

How much glycerol does it take to freeze bacteria?

15% glycerolBacteria can be frozen using a solution of 15% glycerol. The process is simple and requires screw cap microfuge tubes and sterile glycerol. The glycerol is diluted to 30% so that it is easy to pipette. Equal amounts of 30% glycerol and culture broth are mixed, dispensed into tubes and then frozen.

How long do glycerol stocks last?

Obviously different strains of bacteria fare differently in cryogenic conditions, but for the most part, bacteria in glycerol at -80C should be good for multiple years.

How do you make a bacteria stock solution?

2:135:25Creating Bacterial Glycerol Stocks - YouTubeYouTubeStart of suggested clipEnd of suggested clipAnd the labels can easily unstick from tubes at low temperatures snap top tubes are not recommended.MoreAnd the labels can easily unstick from tubes at low temperatures snap top tubes are not recommended. Because they can open and expectedly at very cold temperatures. Once you have your bacterial.

What is the difference between glycerin and glycerol?

glycerols are the triol compound used for many purposes in pure or mixed form , but glycerine is the commercial name of glycerol, which is not pure ,which contain mostly 95% of glycerol , it can't be used when pure glycerol is required .

What temperature does glycerin freeze at?

Mixing water with Glycerin lowers the freezing point of the mixture below that of either pure water (0°C) or pure glycerin (17°C). Please note that although pure glycerin freezes at 17°C, it will typically supercool at temperatures below this, and become very thick instead of freezing.

Does glycerin go rancid?

If you have an old bottle of vegetable glycerin on your shelf, you're likely wondering, does vegetable glycerin go bad? The short answer is yes, vegetable glycerin does degrade over time. Most manufacturers suggest using VG within 1–2 years of the manufacture date.

Most recent answer

Thandokazi Sam very good question and most of the experts have already answered the right way and when we used to work in the laboratories we used to do like whenever we had to recover the bacteria from the glycerol stock generally because of its wsoftnesse have to open the tube in thebegining and slowly and then use a sterile loop which is a important step Raphael Hans Lwesya shared the video is quite clear itself as well and then with the help of any glass rod or pipette if using glass rod then consider very thin rods and scrape some of the frozen bacteria from the top most portion and do not pierce into it.

All Answers (13)

I don't know much about filamentous bacterial isolates but for bacterial in glycerol stocks, we streak the bacteria on a plate with the necessary antibiotics and grow them overnight at 37. Once it has grown, you grow a colony in the broth (still containing the necessary antibiotics) overnight and use cultures for the DNA isolation. Hope this helps!

All Answers (5)

For making bacterial glycerol stocks and the suggested percentage of glycerol please check the attachment.

What is the best medium for freezing fungal spores?

Suspensions of bacterial cells or fungal spores are prepared in a cryoprotectant medium, generally containing 10-15% glycerol to minimise damage during freezing. The suspension is then dispensed into suitable containers, such as small screw-capped vials, which are then immersed in, or suspended above, liquid nitrogen.

What temperature does nitrogen freeze dry?

The temperature of liquid nitrogen is -196 o C , well below the temperature at which all metabolic activity is thought to cease. As with freeze-drying, not all microbial cultures will survive the process, but those that do may survive for many years.

How long does it take for a freeze dried vial to dry?

Once frozen, the plugged or loosely capped vials are placed in the drying chamber of a freeze dryer and dried under vacuum for 2 – 24 hours to remove water in the frozen state. A secondary drying step may also be applied by attaching the vials to the manifold of the freeze-dryer for a further 2 – 12 hours.

How are subcultures stored?

After incubation at a suitable temperature, the subcultures are stored in a refrigerator until required, or until the next subculturing is due.

What is a thick suspension of bacterial cells?

A thick suspension of bacterial cells or fungal spores is first prepared in a suitable suspending medium, such as 10% skim milk, or a specific lyophilisation buffer. This suspension is then dispensed into small glass vials and frozen.

How many passages of culture should be made from the original strain?

In fact, the American Type Culture Collection (ATCC) recommends that no more than five passages (subcultures) should be made from the original type strain, with the first passage being defined as the culture prepared from the vial supplied by ATCC.

Can you thaw a vial of culture?

The vial should be resealed and returned to the freezer immediately so that the remainder of the culture is not allowed to thaw. This method allows stable stock cultures to be kept for extended periods, providing that the temperature is kept at -70 o C or less. The cultures are also less vulnerable to contamination.

How to revive glycerol?

How I used to revive a glycerol stock: Take the glycerol stock out of the freezer but do not let it thaw. Take a sterile (autoclaved) pipet tip or something similar to scratch some bacteria-containing ice from the stock and place it on the agar plate, where it will melt immediately.

Is it better to do streak on agar plate or agar plate?

Saroj Kumar. All India Institute of Medical Sciences. One can do the both but it better to do first the streak on agar plate as it also help to avoid the contamination. As everyone agree with the streak one but both are fine as long as contamination is not an issues.

Can glycerol be made from a single colony?

Glycerol stocks can then be made from a single isolated colony. Some workers will take this single colony and streak again then continue with growth and glycerol stocks. Upon re-growth it is best to still take the approach of streaking the stock onto agar plates to isolate single colonies.

Should antibiotics be added to plasmids?

Antibiotics should be added if the cells you are trying to revive contains plasmid with the antibiotic resistance. I have also seen people directly adding the glycerol stock to the culture broth, sometimes the cells grows but most of the time it does not. So I prefer streaking is better. Cite. 6th May, 2014.

Can you inoculate glycerol from streaking?

You have to make sure the strain you are inoculate is not contaminated. Frequently used glycerol stocks may have some possibility of contamination.

How to thaw frozen cells in liquid nitrogen?

Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial. Transfer the vial it into a laminar flow hood.

How to thaw cells before incubating?

Dilute the thawed cells slowly before you incubate them, using pre-warmed growth medium. Plate thawed cells at high density to optimize recovery. Always use proper aseptic technique and work in a laminar flow hood. Always wear personal protective equipment, including a face mask or goggles.

What temperature should cryovirus be?

Cryovial containing frozen cells. Complete growth medium, pre-warmed to a temperature of 37°C. Disposable, sterile centrifuge tubes. Water bath at a temperature of 37°C. 70% ethanol.

Is thawing a good technique?

The thawing procedure is stressful to a frozen culture. Using good technique and working quickly ensures that a high proportion of the cells survive the procedure. As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cells and other reagents for best results when freezing and thawing.