Antibiotics.

[pcube]

Hi topiates,I want to try and clean some spores from a contamed jar i made[freaky's b+ contamed with paper from the print] The contam shows up in brf/rye jars as grey/black.The tetracycline i have is for my poultry.I have no docs on it [the farmer i bought the birds from gave it to me in a baby jar]He told me to dose a five gal. bucket with a finger tip full half tsp.So if i mix this and take what i need[half liter] this should be strong enough?Also are there any limits on heat with tetracycline?And lastly any specific recipes that work espeacially with tetracycline? thanks in advance namaste pcube

[spacecowboy]

I have a friend who has had great success using tetracycline in solution form to wash spores with the aid of a syringe filter. It only works to kill bacteria, gram + and -

It is light and heat synsitive, especially in solution, and should be stored in the dark at below freezzing temps. I get my tetracycline from the pet store. It is used as an antibiotic in fish tanks, and comes as a powder contained in a capsule.

I will see if I can find more info.

[spacecowboy]

Tetracycline
Make up in 50%v/v ethanol and water mixture to 12.5mg/ml. Sterile filter
and store in the dark at -20C
this is 1000x use at 12.5micrograms/ml

Obtained from:
http://neelix.molbiol.ox.ac.uk:8080/...reagents.shtml

Normally, gentamicin sulfate is used as an antibiotic due to its ability to withstand sterilization temperatures. Anything you add to agar after it has cooled down greatly adds to your contamination risk. Remember, antibiotics will only help to protect against bacteria. They will do nothing to stop fungi contaminants such as cobweb and trichoderma.
RR

[pcube]

ty very much gentleman.Very much appreciate the prompt reply.

[the jesus]

FOAF has acces to large amounts of laboratory grade pre-sterilized agar in petri dishes and slants. They are all in packs of ten and sealed. They use them in a microbiology lab right out of the package to germinate various bacteria. I am wondering if anyone has a source or link to find out the ph levels of various agars. I believe the ph levels should be between 6 and 7 for cubie myc, is this correct?

Here is a list of the agar available:

SIN (?)
BAP (blood)
chocolate (homogenized blood)
EMB (?)
SAB (sabouraud dextrose)
HE (hectone)
MAC (mackone)
SMAC (sorbitol mackone)
CBA (?)
IMA (inhibitory mold agar)

I belive the IMA and SAB will be the best for germinating psilocybe cubensis spores, but am not sure. If anyone has any experience with any of these your input would be much appreciated.

[siam_jim]

7 is what i use since its neutral....the thing is you don't want your agar to have too much nutes..



siam

[SharkieJones]

If I had to pick one I would pick the Dextrose one but have not had any experience with the above mentioned agar mixes.

[the jesus]

Thats cool. I am going to get a bunch of them all and do some experimenting. I'll post some results. I have read of cases of fungus grown out on SAB, but my microbiologist FOAF seems to think the IMA would be best, but she has no experience cultivating. (the name sounds promising).

[Hippie3]

looks like most of those are intended for culturing specific pathogens,
i'd go with MEA or PDA for cubes.

[fastfred]

Sabouraud's is your best choice there. It's a general medium for fungi and will work alright. MEA or PDA will work a fair bit better though. You should stick with those.

If you're looking to use some lab chems, grab some antibiotics to add to the agar, that will be usefull. Gentamicin can be added before sterilization, but most others have to be added after. Ampicillin works well also and the lab is sure to have it.

IMA should work well also. It has two antibiotics in it. It's an inorganic based media and won't produce as good of results as PDA or MEA.

As for the pH... Standard PDA is 5.6, MEA is 5.5, Sabouraud's is 5.6. Those are FDA specs based on the BAM...
Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.

Of course you can pH them to whatever you want. If you're looking for longer term growth I would pH them to 7 or even add a buffer to keep it up around 7-8.


-FF

Ha! My first post here. I thought I had posted here before... Shroomery seems to be down, so I thought I would check this site out. I haven't been here in a year or two, but I used to read the forums here.

[Lazlo]

Hey Fred and welcome. Do you happen to have that compilation of agar mixtures handy since we're on the subject?

[fastfred]

You must be referring to the infamous "FastFred's Media Cookbook"... Since the shroomery seems to be down it will be good to post another copy here.

----------------------------------------
FastFred's Media Cookbook (v0.97)(8-30-2004)
by FastFred
Index
---------------------------
A. Classic Favorite Recipes
---------------------------
Potato Dextrose Agar (PDA)(FDA M127)
Potato Dextrose Yeast (PDY)
Malt Extract Agar (3%)(MEA)(General Microbiology)(FDA M93)
Malt Extract Agar with Yeast (2%)(MEAY)
Sabouraud's Dextrose Broth and Agar (SabDex or SDA)(FDA M133)
-----------------
B. Special Blends
-----------------
"Karo Water" (Corn Syrup Broth)
"Dextrose Tek" Liquid Media (Corn Sugar Broth)
Honey Water (Mycotopia Honey Tek)
Malt-Yeast-Peptone Agar (McKenna's MYP)
Malt Yeast Peptone Agar (Stamets MYP)
Grey Cardboard (instead of agar)
Oatmeal Flake Agar
Moonflower's Rice Malt-Alfalfa-Brewer's Yeast Agar
MycoPsycho's Liquid Mycelial Culture Broth [Malt base]
Ragadinks Liquid Medium
Amaranth Soy Agar
EntheoGenesis No.442
-------------------------------------
C. Food Based Recipies and Variations
-------------------------------------
Corn Meal Agar (CMA)
Cornmeal Dextrose Agar
Potato Flake Agar
Potato Starch Agar
Potato Flake Agar with Yeast
Potato-Carrot Agar
Barley Flour Malt Extract Agar
Barley Flour Modified Sabouraud's
Oatmeal Agar (OA)
Oatmeal Agar A
Oatmeal Agar [B]
V-8 Oatmeal Agar
V8 Medium
Bean Agar
Faba Bean Dextrose Agar (FDA)
Pea Agar
Cabbage Agar
Dr. Pollock's Modified [Dog Food] Agar
-----------------------------------------
D. Other Media and Alternate Formulations
-----------------------------------------
Potato-Glucose Agar 1
Potato-Peptone Medium
Potato-Peptone-Yeast Agar (PPYA)
------
Malt Agar (MA)(FDA M185)[aka 2% MEA]
Difco Malt Extract Broth (FDA M94)
Malt Extract Agar for Yeasts and Molds (MEAYM)(FDA M182)
Malt Extract Peptone Agar
Raper & Thom MEA (RTMEA)
ISP 2 Medium (Malt, Yeast, Glucose)
------
Yeast Extract Agar (YEA)(FDA M181)
Yeast Glucose Agar
Glucose and Yeast Extract Agar
Glycerin Yeast Agar
------
Manure Tincture
Manure Agar
------
Water Agar (aka Starved Agar)
Gelatin Agar (GA)(FDA M54)
Plate Count Agar (SMA)(aka Standard Methods Agar)(FDA M124)
Nutrient Agar (FDA M112)
Starch Agar (FDA M143)(Nutrient agar with starch)
Starch-Yeast Agar
1/5 Starch-Yeast Agar
Long-term Preservation Medium (FDA M85)
Peptone Meat Agar (Meat Water)
-------------------
E. Common Solutions
-------------------
Gentamicin Sulfate Solution (FDA M57)
----------
F. Sources
----------

------------------------
Section A: Basic recipes
------------------------
Potato Dextrose Agar (PDA)(FDA M127)
------------------------------------
200 g Potato infusion [dilute to 1L total, ~4g solids]
20 g Dextrose
20 g Agar
1 L Distilled water (dH2O)
To prepare potato infusion, boil 200 g scrubbed, sliced(unpeeled) potatoes in 1 liter distilled water for 30 min. Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated form). Mix in other ingredients and boil to dissolve. [Dilute to obtain 1 L final volume] Autoclave 15 min at 121°C. Dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes. Final pH, 5.6 ± 0.2.
Medium should not be re-melted more than once. Medium powder is available commercially but may require supplementing with extra agar to a final concentration of 20 g/liter. To BBL or Difco dehydrated medium, add 5 g of agar.
The broth is clear to slightly opalescent and yellowish in color. [1][2]

Potato Dextrose Yeast (PDY)
---------------------------
200 g Potato, infusion from [dilute to 1L total]
20 g Dextrose
2 g Yeast extract
20 g Agar
1 L Distilled water (dH2O)

Gently boil for ten minutes or until the solution is clear.
Autoclave 15 min at 121°C. [13]

Malt Extract Agar (3%)(MEA)(General Microbiology)(FDA M93)
----------------------------------------------------------
30 g Malt extract
20 g Agar
1 L Distilled water
Boil to dissolve ingredients. Avoid overheating, which causes softening of agar and darkening of medium color. Autoclave 15 min at 121°C. Dispense 20-25 ml into sterile 15 x 100 mm petri dishes. Final pH, 5.5 ± 0.2.
This medium is recommended as a general maintenance medium. [1]

Malt Extract Agar with Yeast (2%)(MEAY)
-----------------------------------
20 g extra light malt extract
2 g yeast
15-20 g agar
1 L water [10]

Sabouraud's Dextrose Broth and Agar (SabDex or SDA)(FDA M133)
------------------------------------------------------
40 g Dextrose
10 g Polypeptone or neopeptone
1 L Distilled water

Dissolve completely and dispense 40 ml portions into screw-cap bottles. Final pH, 5.8. Autoclave 15 min at 118-121°C. Do not exceed 121°C.
For Sabouraud's dextrose agar, prepare broth as above and add 15-20 g agar, depending on gel strength desired. Final pH, 5.6 ± 0.2. Dispense into tubes for slants and bottles or flasks for pouring plates. Autoclave 15 min at 118-121 °C. [1]
Sabouraud Dextrose (SabDex) Agar is used for the isolation, cultivation, and maintenance of saprophytic and pathogenic yeasts and fungi.
SabDex Agar is an excellent substitute for Malt or Potato Dextrose Agar, when used by mushroom cultivators to propagate mushroom mycelium.
Sabouraud Dextrose Agar was described by Sabouraud in 1892 and was used for the identification of fungi based on their morphological characteristics. Sabouraud Dextrose Agar is a standard medium used to support the growth of yeasts and molds. It supplies peptone as the protein source and dextrose as the carbohydrate source for nourishment. Bacterial suppression occurs due to the low pH. This media is especially suited for the primary isolation of fungi from normally sterile sites such as cerebrospinal fluid (CSF).
Later, Emmons modified the medium by decreasing the dextrose content and adjusting the pH closer to the neutral range. This modification enhances sporulation and is particularly useful for the subculture of fungi that so not develop fruiting structures on other media, and so is useful in their identification. It also serves as a good holding medium for stock cultures. [3]

------------------
B. Special Blends
------------------
"Karo Water" (Corn Syrup Broth)
-----------------------------
1 Teaspoon Light Corn Syrup (Karo Syrup or store brand Light Corn Syrup)
100 ml Purified Water
Mix well until dissolved, sterilize for 20-30 minutes at 15 psi. [17]

"Dextrose Tek" Liquid Media (Corn Sugar Broth)
----------------------------------------------
1 Teaspoon Powdered Dextrose (corn sugar)
75 ml water [17]

Honey Water (Mycotopia Honey Tek)
---------------------------------
40 g Honey (or roughly 1 tablespoon per pint of water)
1 L Water
The correct mixture for optimum results is 4% sugars (honey) by weight and 96% water.
Water weighs 1 gram per cc/ml so if you use 100 ml as total weight, then 96 grams/ml/cc of water is mixed with 4 grams of honey, etc.
Sterilize for 30 min at 121°C (15 psi). [14]

Malt-Yeast-Peptone Agar (McKenna's MYP)
---------------------------------------
7 g malt extract (powdered or syrup)
1 g peptone or soytone
0.5 g yeast extract
15 g agar [11]

Malt Yeast Peptone Agar (Stamets MYP)
-------------------------------------
20 g extra light malt extract
1 g yeast extract
1 g peptone
15-20 g agar
1 L water [10]

Grey Cardboard (instead of agar)
--------------------------------
Here are the detailed steps for making cardboard plates. Note that you can also use small jars in place of Petri dishes.
1) Measure about 100 mls. of tap water into a small jar.
2) For nutrients, , measure another 100 mls. of tap water into a second jar and add one drop of ordinary soy sauce to the water, and a quarter teaspoon (1.25 mls.) of molasses or light malt powder.
3) Find some gray cardboard, the thicker the better, preferably gray on both sides. Trace a Petri plate onto the cardboard with a pencil and cut out several disks to fit into your plates.
4) Weigh one of your disks and record the weight. Multiply this weight by a factor of 1.3 as a rough guide (you may need to experiment with the amounts here), and add the resulting weight of tap water or nutrient solution to each disk in its Petri plate. (Remember, 1 ounce of water equals 28.35 grams; one gram equals one milliliter.) Example: Suppose my disks weighed 0.17 ounces each. Multiplying 0.17 by 1.3, I get 0.22 ounces. There are 28.35 grams in an ounce, so 0.22 ounces x 28.35 equals 6.3 grams. That means I'll add 6.3 milliliters of solution to each disk.
5) Close up the disks in the plates, and let the water or nutrient solution soak in.
6) Pressure-sterilize the jar of plain water, and the Petri plates with moistened newsprint disks inside, for 10 minutes at 15 psi (allowing the cooker to equilibrate steam for 10 minutes before putting on the pressure regulator).
7) Cool the cooker, and remove the plates and jar of plain water.
8) When the water has cooled, add 3.3 mls. 3% peroxide to the jar, using a pasteurized pipette, to give you a final concentration of about 0.1% peroxide in sterile water.
9) Add about one third of the initial weight of the cardboard as 0.1% peroxide to each disk. Let the solution soak completely into the disks. They are now ready to use. [23]

Oatmeal Flake Agar
------------------
75 g Oatmeal flakes
20-25 g Agar
1 L water
Stir for 5-10 minutes then filter out the larger particles by pouring it through some mesh, save the broth. [Then add the agar]
This is the best medium for Panaeolus cyanescens I've ever encountered. Beware, this medium will be less firm than the other recipes so extra agar has to be added to compensate. [22]

Moonflower's Rice Malt-Alfalfa-Brewer's Yeast Agar
--------------------------------------------------
1 cup alfalfa, infusion from
2 cups rice, infusion from
1 standard dolomite (oyster shell-crushed) tablet
1 pkg of regular baker's yeast
Prepare infusion using approx. 1 1/2 quarts of clean water. Mix in the alfalfa and rice, then allow to soak for 2 hours at room temp with occasional stirring. Filter or strain before adding the infusion.
Prepare yeast by activating in water for around 30 minutes, then strain out solid yeast grains.
Sterilize in pressure cooker for 20 minutes at 15lbs.
This media is reported to produce more "banding" than other media. Will support luxuriant mycelial growth, it is also more than sufficient for starting spores. [19]

MycoPsycho's Liquid Mycelial Culture Broth
------------------------------------------
1/2 tsp malt (2.5 ml)
3/4 cup water
1. procure a half-pint jar w/ lid & ring and also a spore syringe.
2. drill/punch a hole in the center of the lid large enough for the syringe.
3. mix 3/4 cup of water with 1/2 tsp.(2.5 ml.) malt.
4. put lid & ring on and put aluminum foil over the top of that and then Pressure Cook (PC) for 20 min. @ 15 psi. , when finished let cool to below 90 degrees F.
5. once cool, setup your sterile environment (flow hood, glove box or even your oven will work) with an alcohol lamp, extra alcohol in a dish, paper towels/napkins, small round bandaid and lastly the spore syringe.
6. remove the foil from the jar, shake the needle and then sterilize the point with the alcohol lamp (get it red hot), cool the needle with a wipe & some alcohol and then inject 1-2 cc/mil spore solution into the jar.
7. once injected wipe the needle w/alcohol and put the guard back on it.
8. cover injection site w/ round bandaid (thanks for that tip magash!).
9. put the jars in your incubator set at 82-84 degrees F. for 4-14 days, you should notice growth by then.
10. sterilize empty syringes in your PC for 15-20 @ 15 psi.
11. remove the bandaid from the jar, sterilize your needle again and then suck myc solution into empty syringe(s) for use on PF style jars or substrate bags. [15]

Ragadinks Liquid Medium
-----------------------
16.5 g dextrose
1.5 g yeast
500 ml tap water [16]

Amaranth Soy Agar
-----------------
20 g amaranth flour
20 g soy flour
9 g agar
500 ml potable or distilled water
Sterilize for 20-30 minutes at 15 psi. [20]

EntheoGenesis No.442
--------------------
10 g amaranth flour
10 g brown rice flour
10 g potato flour
10 g soy flour
2 g malted barley
9 g agar
500 ml potable or distilled water
Sterilize for 20-30 minutes at 15 psi. [20]

---------------------------------------------
Section C: Food Based Recipes and Variations
---------------------------------------------
Corn Meal Agar (CMA)
--------------------
2 g Corn Meal, Infusion from [filter]
15 g Agar
1 L dH2O [4]

Cornmeal Dextrose Agar
----------------------
25 g yellow cornmeal
3 g dextrose
9 g agar
500 ml potable or distilled water [20]

Potato Flake Agar
-----------------
20.0 g Potato Flakes
10.0 g Dextrose
15.0 g Agar
1.0 L Demineralized Water

Potato Dextrose Agar and Potato Flake Agar are formulations developed to promote sporulation of fungi. The potatoes contained in these media provide nutritious bases for luxuriant growth of fungi. Both media contain dextrose as a growth stimulant.
pH 5.6 +/- 0.2 @ 25°C [7]

Potato Starch Agar
------------------
30 g potato starch, soluble
20 g dextrose/glucose
15 g agar, pure (omit for liquid media)
1000 mL (d)H2O
The pH is adjusted following autoclaving to prevent agar hydrolysis by acid. [20]

Potato Flake Agar with Yeast
----------------------------
20 g potato flakes
10 g glucose
1-2 g dried yeast
20 g agar
1 L water [18]

Potato-Carrot Agar
------------------
grated potato 20.0 g
grated carrot 20.0 g
Agar 20.0g
Tap water 1000.0 ml
Boil potato and carrot in 1000.0 ml of water for 1 h, filter, add water to the
initial volume, adjust pH to 7.0 - 7.1 and add agar.
Sterilize at 121°C for 30 min. [6]

Barley Flour Malt Extract Agar
------------------------------
40 g barley flour
2 g malt extract
1 g yeast extract (optional)
9 g agar
500 ml potable or distilled water
Sterilize for 20-30 minutes at 15 psi. [21]

Barley Flour Modified Sabouraud's
---------------------------------
25 g barley flour
5 g dextrose
2 g Polypeptone or neopeptone (optional)
1 g yeast extract
9 g agar
500 ml potable or distilled water
Sterilize for 20-30 minutes at 15 psi. [21]

Oatmeal Agar (OA)
-----------------
60.0 g Oatmeal [filter]
12.5 g Agar [may require more]
1.0 L dH2O
Cook oatmeal 5-10 minutes then filter the liquid into another container using cheesecloth or a metal strainer with a tight mesh. Dilute liquid to 1L, add agar, and heat with swirling until solids dissolve.
This is reported to be a good media for cultivating Panaeolus cyanescens. [5]

Oatmeal Agar A
--------------
Oatmeal 20.0g
Agar 20.0g
Tap water 1000.0 ml
pH 7.2. [6]

Oatmeal Agar [B]
----------------
Oats 30.0g
Agar 15.0g
Tap water 1000.0 ml
Keep oats on a water bath at 58°C for 1 h, filter through 2 layers of gauze, dilute
to 1000.0 ml and add 15.0 g agar. [6]

V-8 Oatmeal Agar
----------------
50 ml V-8 juice
25 g Cream of oats
20 g Agar
1 L Water
Be careful to use a container much larger then the volume of medium, i.e., prepare a 500 ml medium in 2 liter flasks or it will tend to boil over no matter how slowly it is cooled down. [20]

V8 Medium
---------
50 ml V8 Juice
.2 g CaCO3
20 g of agar
1 L Water
The commercial V8 juice is occasionally used for tissue cultures of edible mushrooms. It should be noted that most mushrooms prefer neutral to slightly acid range of medium, that is, a pH of about 5.5 to 6.5. However the straw mushroom, Volvariella volvacea, prefers a high pH medium, 6.8 to 7.8. Therefore, it is important to make sure the acidity or pH of the medium is correct for a particular mushroom. Here be careful to use a container much larger then the volume of medium, i.e., prepare a 500 ml medium in 2 liter flasks or it will tend to boil over no matter how slowly it is cooled down. [20]

Bean Agar
---------
Beans (peas or pulse) 100.0 g [infusion from (filter)]
K2HPO2 0.5g
Sucrose 10.0g
Agar 20.0g
Water 1000.0ml
Prepare infusion from beans. Sterilize at 121°C for 30 min. [6]

Faba Bean Dextrose Agar (FDA)
-----------------------------
200 g faba bean seeds or 400 g of faba bean leaves [infusion from]
[autoclave and filter to obtain faba infusion]
20 g of dextrose
18 g agar
1 L water
Method:
a. Weigh out 200 g of faba bean seeds or 400 g of faba bean leaves in a 1.5 1 flask. Add 1L of water, and autoclave at 15 psi. for 30 minutes.
b. Pass the autoclaved beans through a sieve, add 18 g of agar, heat, and stir till dissolved.
c. Add 20 g of dextrose, stir till dissolved, and make up the volume to 11 with tap water. d. Autoclave at 15 psi. for 20 minutes, cool to about 40°C, and pour into petri dishes (normally 40 petri dishes/1).
This medium is used for propagation of B. fabae, A. fabae, and A. tenuis. [12]

Pea Agar
--------
100 g Yellow peas
0.5 g K2HPO2
10.0 g Sucrose
20.0 g Agar
1.0 L Tap water

Boil peas in 1000.0 ml of water, filter through gauze, add water to the
initial volume; add phosphate, sucrose, and agar. Sterilize at 121°C for 30 min. [6]

Cabbage Agar
------------
Cabbage 50.0g
glucose 20.0g
Peptone 10.0g
Agar 20.0g
Tap water 1000.0 ml
Boil 50.0 g of cabbage in 1000.0 ml of water, filter cabbage, adjust the
volume of broth to the initial value. [6]

Dr. Pollock's Modified [Dog Food] Agar
--------------------------------------
10 g dried dog food (ground to flour)
10 g amaranth flour
2 g dextrose or malt extract
9 g agar
500 ml potable or distilled water
Sterilize for 20-30 minutes at 15 psi. [20]

-------------------------------------------------
Section D: Other Media and Alternate Formulations
-------------------------------------------------
Potato-Glucose Agar 1
---------------------
grated potato 200.0 g
glucose 20.0g
Agar 20.0g
Tap water 1000.0 ml
Boil potatoes for 1 h in 1 L of water, filter through gauze. add water to
the initial volume, add glucose and agar.
Sterilize at 105°C for 30 min. [6]

Potato-Peptone Medium
---------------------
Potato decoction 200 ml [infusion from 200g potato]
Yeast extract 1.0 g
Peptone 5.0g
Agar 30.0g
Distilled water 800.0 ml [6]

Potato-Peptone-Yeast Agar (PPYA)
--------------------------------
Potato decoction 200 ml [infusion from 200g potato]
200.0 ml
Peptone 5.0g
Yeast extract 1.0 g
Agar 25.0g
Distilled water to 1000.0 ml
pH 8.0. [6]

Malt Agar (MA)(FDA M185)[aka 2% MEA]
------------------------------------
20 g Malt extract, powdered
20 g Agar
1 L Distilled water
Mix ingredients, steam to dissolve agar and sterilize for 15 min at 121°C. Temper medium to 45°C and pour plates under aseptic conditions.
This medium is recommended as a general maintenance medium. [1]

Difco Malt Extract Broth (FDA M94)
----------------------------------
6.0 g Malt extract base
1.8 g Maltose, technical
6.0 g Dextrose
1.2 g Yeast extract
1.0 L Water
Final pH, 4.7 ± 0.2. [1]

Malt Extract Agar for Yeasts and Molds (MEAYM)(FDA M182)
--------------------------------------------------------
20.0 g Malt extract, powdered
20.0 g Glucose
1.0 g Peptone
20.0 g Agar
1.0 L Distilled water

Mix ingredients, heat to dissolve agar and sterilize at 121°C for 15 min. Temper media to 45°C and pour plates under aseptic conditions. Dehydrated MA is commercially available, but since several MA formulas exist, check for the correct composition.
Final pH 5.4. [1]

Malt Extract Peptone Agar
-------------------------
30 g Malt extract
3 g Soya peptone
15 g Agar
1 L Distilled water
Adjust pH to 5.6. Sterilize at 121°C for 10 min. [8]

Raper & Thom MEA (RTMEA)
------------------------
To 2% MEA add:
Glucose 10g
Soy Peptone 5g [9]

ISP 2 Medium (Malt, Yeast, Glucose)
-----------------------------------
Malt extract 10.0 g
Yeast extract 4.0 g
Glucose 4.0g
Agar 15.0g
Distilled water 1000.0 ml
pH 7.2. [6]

Yeast Extract Agar (YEA)(FDA M181)
----------------------------------
10.0 g Proteose peptone
3.0 g Yeast extract
5.0 g NaCl
15.0 g Agar
1.0 L Distilled water
Adjust pH to 7.2-7.4. Autoclave at 121°C for 15 min. [1]

Yeast Glucose Agar
------------------
Yeast extract 5.0 g
Glucose 10.0g
Peptone 5.0g
Agar 20.0g
Distilled water 1000.0 ml
pH 7.2. Sterilize at 121°C for 15 min. [6]

Glucose and Yeast Extract Agar
------------------------------
Glucose 20.0g
Yeast extract 10.0 g
CaCO2 20.0g
Agar 17.0g
Distilled water 1000.0 ml [6]

Glycerin Yeast Agar
--------------------
Yeast extract 5.0 g
Glycerin 50.0g (also called glycerine or glycerol)
CaCO2 1.0g
Agar 20.0g
Distilled water 1000.0 ml [6]

Manure Tincture
---------------
Cow manure (fresh) 1.0 kg
Distilled water 3000.0 ml
Boil, squeeze through gauze into a bottle and dilute 3 to l. [6]

Manure Agar
-----------
Horse manure 100-125 g
Agar 25.0g
Distilled water 1000.0 ml
Boil manure in 1000.0 ml of water for 10 min, then keep for 16-20 h, filter
through 1-2 layers of filter paper, adjust to the initial volume, add agar.
Sterilize at 121°C for 15 min. [6]

Water Agar (aka Starved Agar)
-----------------------------
Agar 20.0g
Distilled water 1000.0 ml
Sterilize at 121°C for 15 min. [6]

Gelatin Agar (GA)(FDA M54)
--------------------------
4 g Peptone
1 g Yeast extract
15 g Gelatin
15 g Agar
1 L Distilled water
Suspend ingredients with constant stirring to prevent scorching gelatin, and boil to dissolve gelatin and agar. Adjust to pH 7.2 ± 0.2. Autoclave 15 min at 121°C. Cool to 45-50°C. Pour plates. [1]

Plate Count Agar (SMA)(aka Standard Methods Agar)(FDA M124)
---------------------------------------------
5.0 g Tryptone
2.5 g Yeast extract
1.0 g Dextrose
15.0 g Agar
1.0 L Distilled water
Heat to dissolve ingredients. Dispense into suitable tubes or flasks. Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2.
For viable yeasts and molds, dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes. [1]

Nutrient Agar (FDA M112)
------------------------
3 g Beef extract
5 g Peptone
15 g Agar
1 L Distilled water
Heat to boiling to dissolve ingredients. Dispense into tubes or flasks. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2. [1]

Starch Agar (FDA M143)(Nutrient agar with starch)
-------------------------------------------------
23 g Nutrient agar (FDA M112)
10 g Potato starch
1 L Distilled water

Heat to dissolve agar in 500 ml water. Dissolve starch in 250 ml water. Combine and dilute to 1 liter. Autoclave 15 min at 121°C.
Note: add 3 g agar to Difco's dehydrated starch agar. [1]

Starch-Yeast Agar
-----------------
Yeast extract 2.0 g
Starch (soluble) 10.0 g
Agar 20.0g
Tap water 1000.0 ml
pH 7.3. [6]

1/5 Starch-Yeast Agar
---------------------
Yeast extract 0.4 g
Soluble starch 2.0 g
Agar 20.0 g
Distilled water 1000.0 ml
pH 7.3. [6]

Long-term Preservation Medium (FDA M85)
---------------------------------------
3 g Yeast extract, 0.3%
10 g Peptone
30 g NaCl
3 g Agar
1 L Distilled water
Heat to dissolve ingredients. Dispense 4 ml portions to 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121°C. Cool and tighten caps for storage. No pH adjustment is necessary. [1]

Peptone Meat Agar (Meat Water)
------------------------------
Peptone 10.0g
NaCl 5.0g [optional]
Agar 20.0g
Meat water 1000.0 ml
Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons,
add 1000.0 ml of tap water and leave for 12 h at room temperature or in a
thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or
cloth and boil the filtrate for 5 min. The proteins are denatured. Filter the cooled down
mass through a cotton-wool filter and add water to the initial volume. pH 7.2 - 7.4.
Sterilize at 121°C for 30 min. [6]

-------------------
E. Common Solutions
-------------------
Gentamicin Sulfate Solution (FDA M57)
-------------------------------------
5.00 g Gentamicin sulfate
1.00 L Distilled water
Sterilize by filtration through 0.20 µm membrane. Store at -20°C. [1]

----------
F. Sources
----------
[1] Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.
[2] EMD Chemicals [aka Merck]
[3] PML Microbiologicals
[4] Sigma Fine Chemicals
[5] Sigma Fine Chemicals, instructions by FastFred
[6] VKM Media Catalog 2003
[7] remel
[8] DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany
[9] Xenova Limited, UK
[10] Paul Stamets (GGMM and/or TMC)
[11] Terence McKenna
[12] Screening Techniques for Disease Resistance in Faba Beans
[13] FastFred
[14] Mycotopia Honey Tek
[15] MycoPsycho (Shroomery)
[16] Ragadinks (Shroomery)
[17] Nan (Nanook)
[18] Pinback (Shroomery)
[19] Moonflower (???)
[20] Unknown (Shroomery)
[21] Unknown (Shroomery) edited by FastFred
[22] Unknown (Shroomery), comments by Una
[23] Rush Wayne's Peroxi Manual Volume II (Via Hippie3)(Mycotopia)
[24] USDA Complete Guide to Home Canning

[fastfred]

Any comments, suggestions, or submissions about the cookbook are welcome.


-FF

[Lazlo]

That's the one. Thanks!

[fastfred]

Maybe Hippie can archive that for everyone... I think it's a pretty handy source and should keep your shroomies eating well for a long time to come.


-FF

[GoVols]

Has anyone ever tried adding antibiotics to the mix to reduce contamination?

I have been thinking about this, sure Im not the first to though, and I think given the right antibiotic it could work.

Of course one would have to find the correct antimicrobial agent and antibiotics work in many ways.

I know using the kind that work on the rhibosimes couldnt work because they would inhibit growth by effecting the mitchondria of the fungal cell.

I was thinking something allong the lines of penecillin which effects the formation of new cell walls. Effectivly killing bacteria it effects by their own reproduction.

Anyway I could go on, but has anyone ever tried this and has it worked?

[cyberd0c]

Originally Posted by GoVols

I know using the kind that work on the ribosomes couldnt work because they would inhibit growth by effecting the mitchondria of the fungal cell.

?

bacteria and fungal cells are so different that they are in two different kingdoms. one is prokaryote and the other is a eukaryote, so antibiotics that work on bacterial ribosomes are harmless to the fungal mitochondria. as a matter of fact many antibiotics are produced by fungi including penicillin, but you can use gentamicin with a broader spectrum. it is being used routinely and is available from commercial mycosuppliers mixed with agar known as antibiotic agar.

[Hippie3]

most antibiotics can't survive the pressure cooker,
and are of no use against molds.
not to mention rather expensive, difficult to get
and each is only effective against certain bacteria,
most of which are bacteria that attack humans or domestic animals.
the kinds of contams common is our hobby
would not be controllable just by dumping some
amoxicillin in with the substrate mix.
aside from limited use in agar culture as was mentioned,
antibiotics are not the answer to contams.

[GoVols]

Originally Posted by cyberd0c

bacteria and fungal cells are so different that they are in two different kingdoms. one is prokaryote and the other is a eukaryote, so antibiotics that work on bacterial ribosomes are harmless to the fungal mitochondria. as a matter of fact many antibiotics are produced by fungi including penicillin, but you can use gentamicin with a broader spectrum. it is being used routinely and is available from commercial mycosuppliers mixed with agar known as antibiotic agar.

The eukaryotic and prokaryotic cells have 80S and 70S rhibosomes, with the eukaryotic having 60S and 40S subunits and the prokaryotic having 50S and 30S sub units.

The problem arises when you look at the mhitocondria of the eukaryotic cell. It aslo has 70S with 40S and 30S sub units, same as prokaryotic.

This is why many believe that the mhitochodria is an ancient prokaryotic organism that formed a symobytic relationship with eukaryotic cells.

The reason Im asking is that my girlfriend is a pharmacist so I have access to any antibiotic I want.

[cyberd0c]

Originally Posted by GoVols

The eukaryotic and prokaryotic cells have 80S and 70S rhibosomes, with the eukaryotic having 60S and 40S subunits and the prokaryotic having 50S and 30S sub units.

The problem arises when you look at the mhitocondria of the eukaryotic cell. It aslo has 70S with 40S and 30S sub units, same as prokaryotic.

The reason Im asking is that my girlfriend is a pharmacist so I have access to any antibiotic I want.

true, but in real life the antibiotics in the concentrations we use do not affect mitochodrial ribosomes significantly (people and animals are given antibiotics all the time). antibiotic agar does not solve the mold problem as mentioned but is certainly active against bacteria, and I use it with other agents such as peroxide or bleach and physical barriers such as micron filter and aseptic techniques even UV light and ionizers. despite all that you still get contaminants although to a much much lesser extent. it is not practical for the purposes of growing mushrooms to achieve a zero level of contamination, but you can come close.

[thepope]

Foaf finds that a search for 100% gentamycin sulfate for koi ponds,
shows that 25 grams can be had for like $48 + shipping
On sporeworks I think it says that gentamycin is autoclavable and used at .05 grams per liter of agar, correct me if I'm wrong
His is $8 for .05g, and 25g for $48,
I couldn't find anything inbetween
but the large amount is much greater than ones personal needs

[spacecowboy]

Originally Posted by GoVols

Has anyone ever tried adding antibiotics to the mix to reduce contamination?

Not personally, but it only really works to control bacteria

I have been thinking about this, sure Im not the first to though, and I think given the right antibiotic it could work.

Of course one would have to find the correct antimicrobial agent and antibiotics work in many ways.

Tetracycline - works good against almost all gram + and - bacteria. It was not until recently that there have been discoveries of bacteria resistant to TC. But I think they were found in the deep sea.

I know using the kind that work on the rhibosimes couldnt work because they would inhibit growth by effecting the mitchondria of the fungal cell.

I was thinking something allong the lines of penecillin which effects the formation of new cell walls. Effectivly killing bacteria it effects by their own reproduction.

Anyway I could go on, but has anyone ever tried this and has it worked?

Not tried it yet, but others have with great results. Catch 22 is that it only works to control bacteria not other contams like mold and fungi, therefore you still need to isolate a good clean culture after germinating the contaminated spores.

Originally Posted by Hippie3

...rather expensive, difficult to get...

Not really....

TC capsules available at Wally World or the local pet store. "For use in freshwater aquariums for the treatment of these tropical fish diseases: gill disease, cotten mouth disease, bacterial tail rot."

Cost about $5-$10 a pak. However, as H3 said about antibiotics, TC is a very sensitive antibiotic - light and heat are destructive to it. Plus you still need to isolate a pure strain due to the fact it is only effective against bacteria and not mold or fungi.

[homobrassinolide]

Looking online, SWIM thinks about ordering some media. Remel has Sabouraud Dextrose Agar, Emmons w/Chloramphenicol, Gentamicin and
Sabouraud Dextrose Agar, Emmons w/Penicillin, Streptomycin that appear to be the best suited for SWIM's purposes. What has worked the best for those who have used pre-made media?

FOAF recently visited a co-op for veterinary supplies to be used in agar. Co-op has mostly duramycin, oxytetracycline, and a few others. If FOAF wanted to make their own media what are/is the best and what proportion(s)?

[dial8]

Here is a decent link...
http://archives.mycotopia.net/discus...tml?1072710648

[joystik]

Has anyone tried to use gentamicin or other antibiotic in your LC water to prevent bacteria contamination?

[Lazlo]

You should never need to. They and the spores/culture should be super sterile in order to work successfully. For inoculating sterile mediums that is. I've heard peop's mention using salt in pump sprayers filled with liquid cultures for the spraying down of wood chip beds. Whole different story there.

[Aleph]

There is some problem in germinating spores if I use agar with Gentamicin sulphate?

Awaiting that Workman sends me new prints, the tropicalis spores that I have are contaminated of bacterias.

[hyphaenation]

Welcome to the Mycotopia neighborrhood.

All will be revealed...

[Myc]

Some information I've read recently suggests that there should be no problem germinating spores on agar with gentamicin.

Hope this link helps out with your question:

Tabletop Mushroom Cultivation

[Hippie3]

btw
it's only effective against bacteria,
not molds nor yeasts

[Aleph]

Thanks to all for the answers.

To verify it:
- I made 6 petri dishes with MEA-Getamicin (50ppm).
- Spores Panaeolus Tropicalis (origin holanda).
- Temperature 79ºF.
- Three days later, show micelium three of the six petri dishes.

Result:
The spores germinate well in agar with getamicin sulfate.

[joystik]

Id would like to show you a Pan Goliath culture started from spores. The first image is a plate that has two layers of agar. The first layer was normal MEA, the second is gentamicin MEA poured on top still hot after discovering bacterial infection. No visible growth was perceptible before the second layer was added. Antibiotic agar is indeed very helpful.


The second picture shows a TC isolate taken from spore culture completely grown in gentamicin agar. I dont have the original plate anymore so i could not take the picture to show it here.

Im going antibacterial from now on, since bacteria has been my main problem. Although no antibiotic will prevent mold growth.

I wonder if i should keep the spore plates after i take the isolates from it. I just normally toss them.

Peace,

Joy.

P.S. I used 0.5% Gentamicin by volume. 5ml Gentamicin per Liter of Agar.

[joystik]

One more pic, antibiotic agar Pan Tex isolate.

Joy.