bob-bob Posted February 19, 2007 (edited) I have used the SE and could not find an answer so here is this question, Would this recipe for agar Boil ¼ cup of water. Add one teaspoon of gelatin and one teaspoon of sugar. Stir for one minute until everything is dissolved. 2. Cool the solution for ten minutes. 3. Fill a petri dish to a little below the top edge. 4. Close the petri dish by putting on the cover immediately. Let the covered petri dish stand for at least four hours. be sufficient to use with lets say Shiitake spores? Edited February 19, 2007 by bob-bob Share this post Link to post Share on other sites
DuG Posted February 19, 2007 (edited) try this link http://www.atcc.org/common/documents/mediapdfs/336.pdf for Potato Dextrose Agar or PDA. You'll want to sterilze the agar after you mix it to stop infections, or if your using glass petri dishes bung them in the pressure cooker. edit: wrong link Edited February 19, 2007 by DuG Share this post Link to post Share on other sites
Fenris Posted February 19, 2007 Definately go to the health food store and get some agar-agar its not too expensive and it works fine. 15g/L agar 20g/L glucose Boil the L of water with a couple of chopped up potato's and throw in a tsp of vegemite for good measure. Works fine. And you have some tasty potato bits to eat while you are making things up. The agar needs to come to the boil otherwise it will not polymerise and it won't set properly. If you cant sterilise the agar you can add 5ml of H2O2 per L but the agar must have cooled to 55Deg C before adding the H2O2, this should keep mould spores away but you may not germinate your mushroom spores too easily but it can be done however this method works better for agar transfer. Share this post Link to post Share on other sites
bob-bob Posted February 20, 2007 Thanks y'all... much appreciated.. Share this post Link to post Share on other sites
DocAzz Posted February 20, 2007 I remember something about using Vegemite in agar from Year12 science....??? Can anyone confirm or deny this? Share this post Link to post Share on other sites
ENtiTY Posted February 20, 2007 I remember something about using Vegemite in agar from Year12 science....???Can anyone confirm or deny this? Yep. Its in Fenris's post above . I know the yeast extract/vitamin b complex would be beneficial, not to sure about the salt levels though. Any info on how salt affect Myc? Share this post Link to post Share on other sites
watertrade Posted February 20, 2007 A little bit of salt will be ok and will actually inhibit bacterial growth. Saying that it would be easy to add too much. I have never used vegemite but want to give it a try. Milo too. Share this post Link to post Share on other sites
ENtiTY Posted February 21, 2007 Ok, I tried fenris's recipe last night, half quantities and dextrose instead of glucose. One potato, approx weight 150g, was peeled and chopped finely. 500ml hot tap water was added to sauce pan with potato and 1/3 teaspoon vegemite. Simmered until potato had softened through and then the liquid was strained through a paper towel and hot water was added to make up to 500ml. 10g Dextrose and 7.5g Agar was weighed out and added with stirring to the potato filtrate. The batch is then split into 2 PC'able containers, tops covered with Al foil and PC'd for 15 min at 15 PSI. PC removed from heat and left to bleed pressure for 2 minutes. Remaining pressure is carfully equalised and HOT Agar jars are removed to cool to handling temp. I used plastic perti dishes that really don't like heat so the temp was left to drop to about 60-70 deg C before pouring. The first jar was poured and left to cool. Into the second jar I added 1.5ml 3% H2O2 (I assume that fenris was talking about 10 vol H2O2) at about 50 deg C. The agar was then poured and left to cool. There is a very fine line between the temperature at which you can add the peroxide and the temperature at which the agar starts to solidify. About 5 deg C. When I went to stir in the peroxide I noticed the Agar in the bottom of the jar had started to solidify so you need to be quick or have a container of hot water around to keep the agar liquid for proper mixing through of the peroxide and pouring. Once the agar has dried for 2 days I'll inocculate and see how it performs. Performance report to follow. Share this post Link to post Share on other sites
Ace Posted February 21, 2007 Awesome write up Hazza Should be interesting to see the results of this one! Share this post Link to post Share on other sites
Fenris Posted February 21, 2007 Looks good The point at which the peroxide can be added is close to the point at which the agar wants to start coagulating but if you go much higher in temp, the peroxide will degrade very quickly. I am interested to hear how the peroxide plates go, I got about a 70% germination rate with no contams. it takes a few more days for them to start showing growth as well. Share this post Link to post Share on other sites
ENtiTY Posted February 21, 2007 Hey Fenris, Hmmm... Not to sure about spores on P plates, I mainly poured them for myc cleaning. I'll try and streak a couple tomorrow night maybe. Can always pour some more How do you calculate your germination rates? I'm guessing over multiple plates yeah? I'll keep ya'll posted... Share this post Link to post Share on other sites
mescalito Posted February 21, 2007 Nice write up Fenris and thanks for thr vegemite tip too,I've heard of adding yeast and B vitamins but never thought of the old vege. Could you somehow pre-treat the vegemite and geet the salts to fall out of supension? How much does adding the vegemite alter the opacity of the mix? Does it make it easier or harder to distinguish contams early on? I'm going to try my hand at liquid culture soon then I'll move onto pouring plates and slants. Best of luck Harry Share this post Link to post Share on other sites
occidentalis Posted February 22, 2007 Have a look at my recipe for pea agar here http://www.shaman-australis.com/forum/inde...showtopic=13558 Share this post Link to post Share on other sites
ENtiTY Posted February 23, 2007 Ok, took some pics of the plates after 2 days. Full Size Image This is an image of 2 non-peroxide plates side by side. As you can see the one on the left is almost totally covered in contam. The one on the right has contam starting on the edges, its nearly impossible to see in the pic but its there. The rest of the non-peroxide plates were completely covered with contam. I was quite surprised to see such a colonisation in such a short period of time. At first I thought it was some of the potato starch coming out of solution, but after checking the peroxide plates and finding them all clear I realised it was contam. Not what I expected at all ! Full Size Image Here we have the contaminated plate from the first pic next to a peroxide plate. Big difference . I know its hard to see from the images but the peroxide plates were nice and clear as apposed to the cleanest of the non peroxide plates having a white haze on the surface and contam around the edges. These petri dishes are plastic and don't stand a run in the PC, they even start to deform in the microwave at any thing over a minute. A proper set of glass dishes would make it easier to dodge the contam but this goes to show how much protection the peroxide really does give I'm sold ! Share this post Link to post Share on other sites
