Additives for Growth Medium


There are 6 solutions that are combined and added to growth medium: All should be made up on the same day, except for glucose which is stable and can be mixed before.

  1. D-glucose
  2. ITS: Insulin, transferrin, and selenium
  3. Putrescine
  4. Transferrin (in addition to the transferrin in ITS)
  5. Penicillin/streptomycin
  6. L-glutamine


Preparing tubes:


Do this in advance because you do not want to leave the solutions sitting around as you are doing this. There will be 350 ml of additive solution. Prepare 34 tubes (7 ml plastic tubes) of each of the following 3 types:


D-glucose

Sigma G 7021. 
STERILE: after filtering

Mixing container: 250 ml flask, then stored in a new, sterile 250 ml flask in the refrigerator for now until it is combined with other additives.

INGREDIENT    Amount for 100 ml final solution
     
Autoclaved water       About 75 ml so that the glucose will not clump, as would occur if no water were added first
d-glucose (dextrose)   30.0 g; shake until all is dissolved
Autoclaved water    Fill to 100 ml after all glucose is dissolved

FILTER with pre-washed 0.2 m nylon filter.


ITS

Sigma I-1884. The bottle has bovine insulin 25 mg human transferrin 25 mg, and sodium selenite 25 g. This is used completely so it must be re-ordered each time.

STERILE so this does not need to be filtered.

Mixing container: The bottle that the ITS comes in, then a 50 ml blue top flask.

INGREDIENT        Amount to get 50 ml final solution
     
Start with the bottle of insulin/transferrin/selenium    1 bottle
Autoclaved water    5 ml, mix to dissolve, then transfer to new 50 ml flask.
Autoclaved water    45 ml, then refrigerate until it is mixed with other additives later in the day

This is sterile so it does not need to be filtered.

 

Putrescine

Putrescine is from Sigma # P-7505. This is on the room-temperature chemical shelf.

The 10 X PBS (phosphate buffered saline, Dulbecco's (modified)) is from ICN/Flow # 19-604-54. This is kept in the refrigerator.

STERILE: after filtering

Mixing container: 50 ml blue top flask, then stored in a new, sterile 50 ml blue top flask in the refrigerator for now until it is combined with other additives later in the day.

INGREDIENT        Amount to get 50 ml final solution
     
Putrescine    80 mg
10 X PBS   5 ml
Autoclaved water    45 ml

FILTER with pre-washed 0.2 m nylon filter. Then refrigerate in a new 50 ml blue top flask for now.

 

Transferrin

Transferrin is from Sigma # T-1147. (This is instead of the # T-2252 used before March 93, which was not HIV tested). This is kept in the -20 freezer.

The 10 X PBS (phosphate buffered saline, Dulbecco's (modified)) is from ICN/Flow # 19-604-54. This is kept in the refrigerator. 

STERILE: after filtering

Mixing container: 50 ml blue top flask, then stored in a new, sterile 50 ml blue top flask in the refrigerator for now until it is combined with other additives later in the day.

INGREDIENT    Amount to get 50 ml final solution
     
Transferrin    250 mg
10 X PBS   5 ml
Autoclaved water    45 ml

FILTER with pre-washed 0.2 m nylon filter. Then refrigerate in a new 50 ml blue top flask for now.

 

Penicillin/streptomycin

The penicillin/streptomycin/sodium chloride mixture comes in a sterile 100 ml bottle from Sigma # P-0781. (The bottle contains 100 ml and has penicillin 10,000 U/ml, streptomycin 10 mg/ml, in 0.9% sodium chloride). It is kept in the -80 freezer if any is left. 

STERILE so this does not need to be filtered.

Volume desired: 50 ml.

Leave this in the refrigerator to mix 50 ml in with the other additives later in the day. Caution: if this is a new 100 ml bottle, you will only use half of it. Make a note with tape on the bottle so you don't forget.


L-glutamine

The L-glutamine solution (200 mM) comes in a sterile 100 ml bottle from ICN/Flow # 16-801-49. It is kept in the -80 freezer if any is left. 

STERILE so this does not need to be filtered.

Volume desired: 50 ml.

Prepare this last because the L-glutamine can break down to glutamate. If this is a new 100 ml bottle, you will only use half of it. Remove 50 ml and pipette it into the mixing container for the whole bunch of additives. This will be a sterile disposable container that can hold at least 350 ml of fluid. The container must be discarded afterwards because it contained proteins. Do not use one of the re-usable containers used for other purposes.

 

Combining the six ingredients:

Combine the ingredients in a sterile disposable container that can hold at least 350 ml of fluid. The container must be discarded afterwards because it contained proteins. Do not use one of the re-usable containers used for other purposes.

SOLUTION  Volume  Special notes
     
D-glucose 100 ml   
ITS: Insulin, transferrin, and selenium  50 ml   
Putrescine  50 ml   
Transferrin (in addition to the transferrin in ITS)  50 ml   
Penicillin/streptomycin  50 ml  Caution: this is only half the bottle
L-glutamine  50 ml  Caution: this is only half the bottle
     
TOTAL  350 ml   

 

Filling tubes:


    There will be 350 ml of additive solution, and 34 tubes each labeled "Add 4.9", "Add 3.5", and "Add 1.75". Pipette those numbers of milliliters into each tube. If any solution remains, fill new tubes to those amounts and discard any remaining solution less than 1.75 ml.
    The tubes are then frozen at -20 while being held upright in paper containers. Once the solution has frozen, the tubes can be placed horizontally in the appropriate plastic container, with one box in the in the -20 freezer and the rest in the -80 freezer. 

Copyright 2002 Dr. Michael M. Segal, Department of Neurosurgery, Brigham & Women's Hospital, Boston MA 02115, USA.  Many of the solutions are based on recipes from the Furshpan and Potter labs; the recipes given here are the versions we use now in the Segal lab.  Individual copies may be made of this page for use in educational institutions.