how to make frozen stock from streak plate

by Dorris Konopelski I Published 3 years ago Updated 2 years ago

Flash freeze the tube in liquid nitrogen and store at -80. 4. To use, chip out a few pieces of the frozen stock using a sterile pipette tip or sterile toothpick and streak onto a plate containing the appropriate solid media.

Full Answer

How do you streak a plate with a cooled loop?

o With the cooled loop, streak the plate from area ‘A’ across the surface of the agar in three or four parallel lines (area ‘B’). Make sure that a small amount of the culture is carried over.

How do you defrost a frozen stock?

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. Repeat the procedure if you think you need more material. Spread it as normally. The non-thawed stock can be put back to the freezer.

Can you freeze stock for cooking?

Freezing Stock. Some recipes require just a little stock, and the leftovers from a whole can won't keep forever in the refrigerator. Freezing unused stock or a homemade batch in 1-cup muffin tins makes it easy to store in small amounts.

How do you streak agar plates?

n Turn the dish through 90° anticlockwise. o With the cooled loop, streak the plate from area ‘A’ across the surface of the agar in three or four parallel lines (area ‘B’). Make sure that a small amount of the culture is carried over.

How do you streak a plate 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...•

How do you grow bacteria from frozen stock?

Scrape the frozen surface of the culture with a sterile inoculating needle or tip, and immediately immerse it in 2ml growth media within a 2059 snap-cap tube. Grow the bacteria overnight in a 37oc shaker.

How do you thaw glycerol stock?

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).

How do you start a culture from glycerol stock?

Start by adding a ~20 microliters of your Glycerol stock to 3 ml of a nutrient rich broth such as SOC and shake it at 37 celsius for ~8 hours. Then add the starter culture to 250 ml of LB with a selective antibiotic that your plasmid has resistance to.

How do you make a bacteria stock solution?

ProcedureFollow the steps for Inoculating an Overnight Liquid Culture.After you have bacterial growth, add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix.Freeze the glycerol stock tube at -80°C.More items...

Why glycerol is used for the stock?

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. A glycerol stock of bacteria can be stored stably at -80°C for many years.

Why is glycerol used as Cryoprotectant?

Glycerol has good kosmotropic properties; it forms hydrogen bonds with water molecules. This condition makes difficult to form ice crystals by mixture (70% glycerol and 30% water), unless and until the temperature is very low such as −37.8°C. Compare to other cryoprotectant glycerol is less toxic in high concentration.

Can bacteria grow in glycerin?

Putting a happy aqueous bacterium into 100% glycerol will cause immense osmotic shock, probably resulting in death for most bacteria, due to too much of their intracellular water trying to escape to the extracellular, water-free (but glycerol rich) environment.

How do you make glycerol broth?

Dilute pure glycerol in distilled water to create a 50% glycerol solution. Use a sterile pipette to measure out 10 mL of both liquids and combine them in a single flask. Stir or shake the flask thoroughly until the liquids are evenly mixed.

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.

Do you need to autoclave glycerol?

Sterilize glycerol by autoclaving for 20 minutes at 15 pounds per square inch (psi) (1.05 kg/cm2) on liquid cycle.

Can you filter sterilize glycerol?

Sterilize the glycerol in hot air oven at 160C for 60 mins. Alternatively aqueous solutions of glycerol can be either autoclaved(usually the case) or filter sterilized.

Introduction

Culture should be approximately 90% confluent prior to preparation of frozen stock in order to insure the highest number of viable cells from reviving a frozen culture.

Protocol

Trypsinize cells as mentioned in the Subculturing Protocol (Steps 1-4).

Principle of Streaking

The sample/inoculum is diluted by streaking it across the surface of the agar plate. While streaking in successive areas of the plate, the inoculum is diluted to the point where there is only one bacterial cell deposited every few millimeters on the surface of the agar plate.

Procedure

Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is red hot. Allow it to cool.

Background Information

Bacterial glycerol stocks are important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many labs also create bacterial glycerol stocks of their plasmids.

Protocol Video

Watch the protocol video below to learn how to create bacterial glycerol stocks.

Tips and FAQ

The optimal concentration of long-term glycerol storage is unknown. Most labs store bacteria in 15-25% glycerol.

All Answers (79)

I don't know which is better but I have never had any problems with just streaking on agar plate. Why not test both?

Recommendations

The training manual includes information about total nucleic acids (DNA and RNA) from pure soil water samples, genomic RNA and plasmid DNA separation from pure bacterial cultures, quantitative DNA testing, quantitative phylogenetic testing. Methods of cloning, sequencing, bioinformatics analysis of the obtained sequences are presented.

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!

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