By Admin (Admin) on Friday, June 08, 2001 - 05:12 pm: Edit

Materials:
Tapered wide mouth half pint jars
Plastic wide mouth jar lids
Camera lens paper
Aluminum foil
Sterile syringe with needle
Procedure:
1. Trim a piece of lens cleaning paper to a disk roughly the size of the bottom of a jar and place it in the bottom of jar.
2. _Loosely_ cap jar, then wrap the whole jar in aluminum foil.
3. Autoclave at 15 PSI for 20 minutes. Allow jar to cool to room temperature before moving on.
4. Prepare the spore collection "chamber" by cleaning thoroughly (soap and water at least) a glass baking pan and the top half of a 2 liter soda bottle that's been cut in half. The cap should be on the half bottle. Get all the supplies near the terrarium and make sure you're as washed up and sterile as possible.
5. Select a mushroom with a good cap (flat or even just starting to invert) growing in a suitably sterile environment. Go ahead and remove the jar with the lens paper in the bottom from the aluminum foil.
6. Harvest the mushroom and with a sterilised knife or razor cut the stem off as close to the cap as possible. Then, opening the jar as little as is humanly possible, transfer the cap, gills down, into the jar. _Loosely_ cap again and transfer to the pan and cover with the half bottle (the half bottle is used like a bell jar).
ASCII diagram of setup which I'm sure looks horrible in html :
.......................________
.......................|......|
..................____/.......\____
................./.................\
................/...................\
.............../.....................\
............../.......................\
..............|..___________________..|
..............|..'.|.............|.'..|
..............|....|.............|....|
........|.....|....|.shroomcap..|....|.....|
........|.....|....|./---------\.|....|.....|
........|.....|....\_____________/ |.....|
........\__________________________________/
7. Allow setup to remain somewhere cool and relatively draft free for a day or so until sufficient spores have been dropped
8. Remove jar from setup, quickly remove mushroom cap with sterilised forceps, pour in sterile, distilled water. Tighten the jar lid and shake vigorously. The lens paper allows you to get just about all the spores into solution without spending all that time scraping which opens up opportunities for airborne spores to get into your container.
9. Open jar as little as possible again and use freshly sterilised forceps to remove the lens paper. Recap tightly.
10. Make a spore syringe initially by heating the needle red hot and piercing through the jar lid. Suck up the spore solution and remove and cap the syringe. Seal the hole with good quality plastic based tape and store in a light proof container in the fridge, removing the tape and loading syringes as needed. You can re-use lids by taping the hole after a jar is emptied.
*****
The beauty of the this method is that the existence of the one piece cap turns the jar into the equivalent of a culture flask or dish in the lab: the loose cap allows gas exchange but still prevents *almost* any contaminant because of the nature of the air flow. The idea of the soda bottle is great because it's easily obtainable, still allows gas exchange, helps keep humidity high, and contributes to sterility.
Lens paper is very fine, thin, and porous. When it comes time to make spore water, you only have to open the jar long enough to add sterile water then reseal. Shake well and get instant spore solution, schwing!, no need for risky mucking about to scrape the print. You don't even need to remove the paper if you are going to load all your syringes immediately, you just don't want to let the cellulose of the paper become a substrate for the spores to germinate in solution on. The caps are cheap enought (about 25 cents a pop) that they're disposable if you don't want to reuse them.

originally posted by 'spore monkey'

By Hippie3 (Hippie3) on Friday, July 13, 2001 - 03:05 am: Edit

Multiple Spore Printing Tek


Introduction:

This is an improvised version of a popular spore printing idea that already exists. The concept is to allow a mushroom cap to drop its spores on a sterile medium, producing a viable "spore print". The cap must be covered to protect contamination by airborne mould spores and to keep the humidity high so that the mushroom will drop its spores. There are many sterile mediums available but I would recommend aluminium foil above all of them. Paper does not typically allow spores to adhere properly, making it unsuitable for mailing. Also, it is harder to seal without leaving it prone to contamination. The bonus in using this method is the ability to produce multiple sterile/viable spore prints.


Equipment:

Baking Tray (no/slightly raised edges if possible)
Pyrex Baking Dish (upside down it must fit entirely within the tray)
Hand Towel/Paper Towel
Aluminium Foil
An oven
Duct/Masking Tape
Dust Mask
Latex Gloves
Alcohol Aerosol Spray (glen20 is quite a common brand)
Sharp Blade (cheap retractable pen knives are available at most supermarkets)
Little Baggies


Method:

1. The spore printing "setup" needs to be constructed. This is really quite simple. Take the tray, lay down enough towelling to cover the whole tray, turn the pyrex baking dish upside down and place it in the centre.

2. Now the actual printing squares have to be cut out. You want each individual square of foil to be slightly wider than the cap you want to print from, and twice as long as it is wide. Cut out as many rectangles as will fit in the setup, or as will be printed. Lift the pyrex baking dish and neatly place the foil squares underneath. (I prefold them in half, then unfold them before I put them down. This will make more sense later on.)

3. Place the entire setup in the oven at 150C for 30-60 minutes. All materials used (with the exception of the towelling) are good conductors of heat so 150C is plenty hot enough to sterilise the setup. 30 minutes is fairly short, yet I have had no problems with contamination using this time period. Be sure not too sterilise with too high a temperature for too long or the towelling may ignite

4. Remove the setup from the oven and while (relatively) hot create a "handle" from the tape. I find this necessary because unlike small, single print covers a curved edge pyrex baking dish is quite awkward to lift with minimal contact. Cut a measure of tape, fold it in half, but not completely! Only fold it to about halfway down the length so that you end up with a sort of "T" shaped piece of tape. The top of this "T" should be adhesive whilst the sides are not. Stick one sticky side on the top of the pyrex baking dish, and the other on the side of the pyrex baking dish. If you can not understand this handle concept (I know it is not very clear!) then feel free to improvise your own. The reason this is done while hot is to ensure a more permanent contact between the tape and the dish. If it is done later the tape often becomes unstuck after being sprayed with aerosol alcohol.

5. So now you have a nice sterile setup! Well, except for the tape of course! So leave it somewhere relatively clean to cool. Room temperature is what you are aiming for here. You will find that the pyrex baking dish will take quite a while to emit all heat and return to room temperature (approximately 2 hours).

6. Now take your setup and place it somewhere partially enclosed to minimise chances of contamination by airborne particles. Bring your terrarium closeby if possible, this is where a nice portable terrarium comes in handy. (If one is not available you might consider making a mini-terrarium for transporting casing/cake to allow printing, or alternatively, place your casing/cake in a clean plastic container. Thoroughly spray down the surrounding area with aerosol alcohol. Don your dust mask and gloves. Spray the gloves for extra sterility. Wipe down the sharp blade with an alcohol wipe (make your own by spraying a tissue) and hold it in the enclosed area while the alcohol evaporates. Give the immediate area another liberal spraying (can't hurt to be overcautious

7. Now you have a relatively sterile area. Aseptic is quite a difficult goal for the avid home cultivar, so relatively sterile will have to do (in my experience it works fine most of the time anyway). Remove the lid from the terrarium, select the choicest specimens (any that developed larger, faster, or generally more roust than the others are desirable). The veil should have released itself, leaving it drooping around the stem. The cap should be hemispherical, approaching a slight flatness. While this will result in a somewhat smaller print than an upturned cap it means that only the rim of the cap actually touches the printing medium, thereby lowering chances of contamination.

8. Now the cap must be cut from the mushrooms. Using the ethanol sterilised blade make a cut at rought the same stem height as the edge of the cap. It is best to cut as close to the gills as possible, but it is also best not to touch the gills with anything at all if possible. I figure edge of cap height is a good compromise. Some people sterilise the cap by wiping the top down with a hydrogen peroxide soaked cotton wool ball, then the same with ethanol. Some people even *LIGHTLY* wipe the gills. I have never found any of these necessary and I advise that they are not attempted unless contamination has become an issue.

9. Slowly lift the dish using the tape "handle". Slow is the key here because a quick lift will result in a large influx of air, raising chances of contamination. Once lifted to a minimum height (enough to allow a gloved hand to fit in) place the cap in the centre of one side of the foil rectangle. Once the process is completed for all caps the setup must sit for 12-48 hours undisturbed. The longer you leave it the heavier the print. This is, of course, just a rule of thumb and mileage will vary between individuals. Each individual microscopic spore takes between 30-60 seconds to drop, so do the calculations and you can work out the time needed for a nice visibly thick print

10. After waiting 12-48 hours, spray down the entire area with aerosol alcohol again, put on the dust mask and gloves, and spray the gloves again. Have your baggies nearby and open the first one. Slowly lift the dish, remove the cap carefully and quickly fold the foil over. Now fold each side over slightly, and finally, fold the end. Place the print in the ziplock baggy and you have just created a spore print. Repeat the process with each cap and you will have a nice batch of prints. Some people suggest removing the caps with a sterilised pin, but again I have never found this necessary. Plus, the outside of the print has to be touched to be folded anyway. By lifting the cap carefully your hands should not come into contact with the printing surface.

11. If you regularly make batches of prints like this for the purpose of distribution (be it sale, trade, competition etc.) a randomly selected print should be made into a syringe as a sort of "quality testing procedure". Label your prints with small adhesive labels that include species, race, printing time, print date, and source. This will be appreciated by all who receive your prints. Good luck and happy printing!


originally posted by "auto59009"