Agar culture info

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By Admin (Admin) on Thursday, August 23, 2001 - 11:37 am:

Agar culture info
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Cooking & Usage Instructions
The flask or bottle in which the medium is sterilized should never be more than 2/3 full in order to avoid boilover.
Plug the flask or bottle with non-absorbent cotton and cover with aluminum foil to help prevent entry of external contaminants.
Sterilize all formulas for 15 minutes at 15 psi.
Remove the medium as soon as the pressure reaches "0." Over sterilization or prolonged heating will change the composition of the medium. Sugars such as dextrose or malt can "caramelize," resulting in a medium that can inhibit mycelial growth and encourage sectors (mutations). Excessive heating of medium can also cause a drop in pH, resulting in a more acid medium. It is possible to destroy completely the gelling properties of agar by prolonged heating, and this destruction is hastened as the acidity increases.
For long-term health and maintenance of sacred strains, alternate any two of the above media. This will help prevent senescence, which can occur when cultures are grown on one medium only. Store strains at 35° - 40°F; transfer every six months. In this manner, sacred strains can be maintained for years.
Never compromise the integrity of your strain library. Strains showing even a hint of senescence should be removed from the collection.

Potato Dextrose Yeast Agar
250 grams of washed, unpeeled potatoes
10 grams dextrose
1.5 grams yeast
15 grams agar
Slice potatoes 1/8 inch thick
Rinse slices clean in cool tap water
Final rinse with distilled water
Cook in distilled water until tender
Strain and set aside cooking water
Rinse potatoes with distilled water
Retain rinse water, discard potatoes
Add distilled water to make 1 litre
Bring liquid to slow boil
Add dry ingredients

PDA (Potato Dextrose Yeast Agar): Wash 250 grams of unpeeled potatoes and slice them 1/8 inch thick. Wash these several times in cool tap water until the water is clear. Drain the slices in a collander and rinse once with distilled water. Cook the potato slices in distilled water until tender. Strain the cooking liquids through a flannel cloth or several layers of cheesecloth and collect the liquid in a flask. Rinse the boiled potatoes several times with distilled water, add these rinse waters to the liquid in the flask, and discard the potatoes. Add enough distilled water to the flask to make one liter. Bring the liquid to a boil and add 15 grams of agar, 10 grams of dextrose, and 1.5 grams of yeast extract. The agar must be added slowly and carefully to prevent boiling over. While the liquid is hot, pour it into petri dishes or other culture containers. Each should be filled half way.
PDY (Potato Dextrose Yeast broth): PDY broth is made in exactly the same manner except the agar is omitted. Mason jars are filled half way with the hot or cool liquid.

Malt Extract Agar
20 grams malt extract
100 mg potassium phosphate
dibasic (K2HPO4)
100 mg calcium carbonate
9.5 grams agar
1 L distilled water

MEA (Malt Extract Agar): To one liter of gently boiling water (distilled) add 20 grams of malt extract, 20 grams of agar (slowly, carefully to prevent boiling over), 100 mg of potassium phosphate dibasic (K2HPO4), and 100 mg of calcium carbonate. While still hot pour the liquid into the culture dishes.

Cornmeal Dextrose Agar
25grams yellow cornmeal
2.5 grams dextrose
9.5 grams agar
500 mL distilled water

Dung extract 100.00 ml
Malt extract 5.00 g
MgSO4 x 7 H2O 0.50 g
Ca(NO3)2 x 4 H2O .72 g
K2HPO4 .25 g
Peptone 0.10 g
Agar 15.00 g
Distilled water 900.00 ml

Dung extract: Boil an average sized piece of horse dung (fresh or frozen) for two hours in 150 ml
water using a water bath; filter and use immediately


more formulas....


Agar Formulas and Info
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Agar Formulas and Info


The following are proven agar recipes specifically formulated for the in vitro culturing and long-term maintenance of Psilocybe cubensis strains:

Amaranth* Soy Agar
20 grams amaranth flour
20 grams soy flour
9.5 grams agar
500 mL distilled water
Sterilize fifteen minutes at 15 psi pressure; final pH is 6.0 and requires no adjustment.

*Amaranth, from South America, is the only grain that contains all essential amino acids; it is extremely high in L-lysine, containing up to five percent.


Oatmeal Neopeptone Agar
40 grams oatmeal or oat flour
2 grams neopeptone (optional)
9.5 grams agar
500 mL distilled water
Sterilize fifteen minutes at 15 psi pressure; final pH is 6.0 and requires no adjustment.


Modified Sabouraud's Medium
25 grams barley flour
5 grams dextrose
2 grams neopeptone (optional)
1 gram yeast extract
9.5 grams agar
500 mL distilled water
Sterilize fifteen minutes at 15 psi pressure; final pH is 5.8 and requires no adjustment.


Cornmeal Dextrose Agar
25 grams yellow cornmeal
2.5 grams dextrose
9.5 grams agar
500 mL distilled water
Sterilize fifteen minutes at 15 psi pressure; final pH is 6.0 and requires no adjustment.


Barley Malt Extract Agar
40 grams barley flour
2 grams malt extract
1 - 2 grams yeast extract (optional)
9.5 grams agar
500 mL distilled water
Sterilize fifteen minutes at 15 psi pressure; final pH is 6.0 and requires no adjustment.


Dr. Pollock's Modified Agar*
10 grams dried dog food
10 grams amaranth flour
2 grams dextrose or malt extract
9.5 grams agar
500 mL distilled water
Sterilize fifteen minutes at 15 psi pressure; final pH is 6.2 and requires no adjustment.

*The above formula is a modification of one first used by the late Dr. Stephen H. Pollock, discoverer of the extremely rare Psilocybe tampanensis, Psilocybe wassoniorum, and ethnomycologist par excellence.

Tips

*The flask or bottle in which the medium is sterilized should never be more than 2/3 full in order to avoid boilover.

*Plug the flask or bottle with non-absorbent cotton and cover with aluminum foil to help prevent entry of external contaminants.

*Remove the medium as soon as the pressure reaches "0."
Over sterilization or prolonged heating will change the composition of the medium.
Sugars such as dextrose or malt can "caramelize," resulting in a medium that can inhibit mycelial growth and encourage sectors (mutations).
Excessive heating of medium can also cause a drop in pH, resulting in a more acid medium.
It is possible to destroy completely the gelling properties of agar by prolonged heating, and this destruction is hastened as the acidity increases.

*For long-term health and maintenance of sacred strains, alternate any two of the above media. This will help prevent senescence, which can occur when cultures are grown on one medium only. Store strains at 35° - 40°F; transfer every six months. In this manner, sacred strains can be maintained for years.

*Never compromise the integrity of your strain library. Strains showing even a hint of senescence should be removed from the collection.

Required Reading
Difco Manual Tenth Edition. (First Edition published in 1927.) Difco Laboratories, Detroit, MI (1984).
 



Saving Strains On Agar : Spore Longevity : Shroom Glossary

 
Posted by: Mycota Dec 29 02, 04:00 AM GMT
Just house cleaning the drive & ran into this forgotten tidbit. I copied it from somewhere (can't remember) & saved it for a reason. Good info.

MALT EXTRACT AGAR (MEA)
1. Boil 20 g malt extract in one (1) liter water until dissolved.
2. Add 20 g agar.
3. Boil until agar is dissolved.
4. Sterilize at 15 psi for 15 minutes.

POTATO DEXTROSE AGAR (PDA)
You will need:
Potato 200g
Dextrose 20 g
Agar 20 g
Tap water 1 l

1. Take 200g potatoes.
2. Scrub the potatoes clean - DO NOT PEEL.
3. Cut into 12 mm cubes.
4 .Weigh out 200g potatoes.
5. Rinse rapidly in running water.
6. Place in one (1) litre water.
7. Boil until soft (1 hour).
8. Mash and squeeze as much of the pulp as possible through a fine sieve.
9. Add 20 g agar and boil till dissolved.
10. 10. Add 20 g dextrose and stir till dissolved.
11. Make up to one (1) liter with water.
12. Sterilize at 15 psi for 20 minutes.

SABOURAUD DEXTROSE AGAR (SDA)

You will need:
D-glucose (Dextrose or Maltose) 200 g
Peptone 20 g
Agar 20 g
Tap water 1 l

1. Dissolve the D-glucose, peptone and agar in the tap water by boiling them together.
2. Autoclave at 15 psi for 15-20 minutes.

READY-MIXED POWDERED AGAR PRODUCTS
Follow the instructions given by the manufacturer.

POURING AGAR PLATES
1. Autoclave the agar (to melt and sterilize). Cool the agar until hand hot.
2. Lay out newly opened plastic Petri dishes OR sterile glass Petri dishes in a clean area.
3. Pour the agar into the dishes to a depth of 0.5 in (approximately 15 ml in a 9 cm Petri plate)
4. Allow to cool uncovered in a sterile air cabinet, keeping the lids within the sterile air flow (do not touch the inside surface of the lids as this will cause contamination).

Note: If you do not have a sterile air flow cabinet, replace the lid of each Petri plate immediately after adding the agar and allow the agar to set. Once cool, any condensation which has collected on the lids of the agar plates can be removed by taking the lid and giving it a short sharp shake. Replace the lids immediately.
5. Cover with lids.
6. If not needed immediately, store in the refrigerator (5C) for several weeks.

PREPARING SLOPES IN BOTTLES
Use 25-30 ml Universal bottles; use SDA for Metarhizium and PDA, SDA or MEA for Beauveria

1. Prepare agar.
2. Put approximately 7 ml of agar in each Universal bottle.
3. Place the bottles in the autoclave with lids loose and autoclave as instructed in the agar recipe.
4. Tilt the bottles so that the agar forms a slope inside the bottle and let them cool.
5. Tighten the lids and store in a refrigerator until use.




Posted by: CharlieBrown Jun 19 03, 06:06 PM GMT
Go to a health food store and pick up agar agar and experiment with different additives.

This is probably one of the best I've found.

Oatmeal agar:
steep 30 g oatmeal for 15 min in 1 L steaming distilled water,
decant supernate into fresh 1 L flask;
add 20 g agar and bring total volume to 1 L with distilled water;
autoclave and pour into Petri dishes

This stuff hosts mycelium very well wink.gif


Posted by: czen Dec 05 02, 10:49 AM GMT
Here's Workman's method using instant potato flakes. I'm going to give it a shot next:


Here is my suggested formula:
18-25 grams of agar/dextrose premix
5 grams of instant potatos
500 ml of water
This should do around 20 standard petri dishes if you pour them fairly thin. Good luck.

Have a good one,
czen