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Horse_Meister
Edible Farmer



Registered: 01/14/07
Posts: 408
Last seen: 5 years, 4 months
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Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice
#7774605 - 12/18/07 05:07 PM (16 years, 2 months ago) |
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After a few weeks of trying to aquire all the necessary equipment and materials, I am finally beginning my works with agar. I have a few books and individuals to get information from, but I thought that keeping an open log of my experience with it from a beginners perspective may help the more experienced individuals who have never delved into the world of agar find an easy place to start. I am looking particularly for experienced agar advice to improve technique. Today is simply about pouring, with no intention of knocking them up with any spores in this run. I may do a finger print or two. Overall I am looking to see how easily they will contaminate without any physical disturbance. I will be leaving them on my desk in an open environment.
Below is the beginning of this log:

Above are the ingredients I have used to make MEA (Malt Extract Agar) I mixed: 1 L H20 1 (25g) bag of agar agar 4 tsp light malt 1 tsp dextrose
to make mixture below:

Loaded into the PC for 45-60 mins at 15 PSI:


I poured the agar into each of an uncounted number of petri dishes. I shook the mixture before pouring. After shaking, the mixture had a large amount of head to it which can be seen here:

The first few plates showed bubbles and a few chunks of agar:

I decided to pour the first few bits from the top of the agar into my mixing cup as using this would be a waste.
Thereafter I had pours that looked like this:

The agar in the mixing cup was solid after maybe 7 minutes. I left an amount in the original PC'ing jar to see how easy it will be to melt it should I decide to prep in the evening and use in the morning in the future.
I did not go through with wiping anything down or attempting to be sterile in my technique. Today was simply about creating a mixture that works and being able to make a nice pour.
Advice and criticism is welcome. What would you do that I have not done today? What would you have done differently? Throw in some sterile technique advice as well. Just explain yourself well in your advice and criticism.
-------------------- KTHXBai2YoU Horse_Meister Life is what happens while you're busy making other plans.
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thedefone
deus ex machina

Registered: 10/06/07
Posts: 1,883
Loc: Gondwana
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Horse_Meister]
#7775059 - 12/18/07 07:08 PM (16 years, 2 months ago) |
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I will be starting this soon. Looking forward to seeing a good learning experience in this. What will the next steps in this process be? Inoculating, and isolating?
Good Luck.
--------------------
I am become death, the destroyer of worlds.
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Jeremy_Davis
Mycelial NetworkAdministrator



Registered: 04/22/05
Posts: 652
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Re: Horse_Meister - Agar Attempts - Log with PHOTOS - Open to Experienced Advice [Re: thedefone]
#7775249 - 12/18/07 07:48 PM (16 years, 2 months ago) |
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First of all, respect for doing this. I love it. I've been using agar for quite some time, so I will try to chime in from time to time if I can give you any advice. When I make agar I like to use a Nalgene Erlenmeyer flask with a screw-top lid. Since it is (totally autoclavable) plastic it has a lot of advantages. Also the shape of the flask really makes pouring agar a breeze. In third world countries, I know people use a lot of glass bottles from local hospitals. They also have a tapered small opening, making pouring really easy. You can probably use any old glass bottle that is made to pour (i.e. for drinks and such, any bottle from alcohol bottles that the local bar emptied last night to ketchup bottles). Trying to pour from a mason jar can be a chore and a bit of a mess for me...
Also when pouring plates, it's good to let the agar cool for a while before pouring it. A few times I poured too hot, and when I stacked the freshly poured plates, something unexpected happened. The heat caused the lid of the dish to push up just a little, making all the dishes solidify at a slight slant. At first I thought the table the flow hood was on was off level, eventually the light bulb went on over my head...
One thing I want to know how to do is reduce condensation. Even when I let it cool to the point that some parts at the bottom begin to gel in the flask, and yet I still get condensation. That's something I'd love to learn in this thread.
Light and Love, JD
-------------------- Jeremy Davis Educational Concerns for Hunger Organization, Inc. Check out the ECHO mushroom blog page to see our lab, growing facility, and more-www.echotech.org/greta
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Vyrk
Stranger


Registered: 12/12/07
Posts: 70
Last seen: 15 years, 6 months
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Re: Horse_Meister - Agar Attempts - Log with PHOTOS - Open to Experienced Advice [Re: Jeremy_Davis]
#7775275 - 12/18/07 07:56 PM (16 years, 2 months ago) |
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and does this mix works?...
Cause I have big problem with all the recipes for doing agar cultures. Someones can`t make spores germinate, other simply doesn't work for me.
Also, you have to consider that you are using only sugars, and a long time of sterilization could burn those sugars. Beware of that.
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Jeremy_Davis
Mycelial NetworkAdministrator



Registered: 04/22/05
Posts: 652
Loc: Florida
Last seen: 11 years, 10 months
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Re: Horse_Meister - Agar Attempts - Log with PHOTOS - Open to Experienced Advice [Re: Vyrk]
#7775432 - 12/18/07 08:32 PM (16 years, 2 months ago) |
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30-45 minutes should definitely do the trick for sterilizing agar.
-------------------- Jeremy Davis Educational Concerns for Hunger Organization, Inc. Check out the ECHO mushroom blog page to see our lab, growing facility, and more-www.echotech.org/greta
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caricapapaya
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Registered: 04/10/06
Posts: 258
Last seen: 1 year, 8 months
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Re: Horse_Meister - Agar Attempts - Log with PHOTOS - Open to Experienced Advice [Re: Jeremy_Davis]
#7776954 - 12/19/07 09:47 AM (16 years, 2 months ago) |
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the time needed sterilzation depends on the volume to be sterilized.
15 min at 15 psi (121 C/about 252 F) will do the trick for very small volumes.
I usually go about 30-45, like jeremy said, for a liter. 20 mins for 500 ml.
also, you can use 15g agar per liter, and save a little there.
good luck.
carica
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Workman
1999 Spore War Veteran



Registered: 03/01/01
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Loc: Oregon, USA
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Re: Horse_Meister - Agar Attempts - Log with PHOTOS - Open to Experienced Advice [Re: caricapapaya]
#7777407 - 12/19/07 11:59 AM (16 years, 2 months ago) |
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I used to PC 500ml of agar at 10 psi for 20 minutes.
But recently I have been using the microwave with suprisingly good results. You just have to watch it very closely as it will foam up and boil over early in the heating process. When it foams up, stop the microwave, swirl it around if there are any unmixed additives and start again. You may have to do this several times. Once you get past the foaming stage, nuke it hard for about 5 minutes, let cool and you are good to go. I do 500 ml at a time in a 1 Liter erlenmeyer flask with a polyfil plug.
This is much faster than using the PC. If I hadn't tried it myself I wouldn't have believed that this would work. Now, its all I do. It is worth trying at least once to see if it will work for you. Watch it real close though, boil over is a real mess.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification 
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caricapapaya
Stranger



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Re: Horse_Meister - Agar Attempts - Log with PHOTOS - Open to Experienced Advice [Re: Workman]
#7778008 - 12/19/07 02:55 PM (16 years, 2 months ago) |
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I can second workman's comments about microwaving. I have done it, and it works.
It is commonly used in hobbyist plant tissue culture too...
try it out.
carica
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Horse_Meister
Edible Farmer



Registered: 01/14/07
Posts: 408
Last seen: 5 years, 4 months
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Horse_Meister]
#7798219 - 12/25/07 12:55 PM (16 years, 1 month ago) |
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It has been some seven days since pouring the agar into the plates. I will break down what I can understand. For everything else, please add your suggestions and experience to this thread.
When the dishes were poured, condensation immediately formed on the covers. Obviously this came out of the difference in air temperature. The real trouble I have noticed as a result of these droplets forming is that if they are allowed to sit on the agar, the following holes develop:

Notice the mold near the sides. I am not sure how to elimated droplets of H20 from forming, other than having the agar poured at a cooler temp than I had allowed it to.
There is a trend in the contamination in each of the photos. They are never in the immediate center of the dish, purely the sides. The petris were sitting on my desk for the last seven days with no parafilm of ziploc bag to protect the incoming air. In the same room is a ceiling fan. I am led to believe that these contams came as a result of not protecting where air could be exchanged. I cannot reason it to be from when I poured them. Below are some more splendid photos:




^^ This one. Look closely at the Yellow contamination. What is this one? Then you see a white droplet as well, what is this one? Please identify if you can what each of these molds are.
I'll be doing my second attempt shortly, quite possibly today. More photos to follow. This time I will be using ziploc bags to protect the dishes.
Thanks for your responses and advice so far. Workman, I will be sticking to PCing it because I can. My AA is simple and not messy. Had I had some no name brand PC which offered a mess every use, I would more than likely go with your route. I'll keep it in mind should I ever need to.
-------------------- KTHXBai2YoU Horse_Meister Life is what happens while you're busy making other plans.
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Workman
1999 Spore War Veteran



Registered: 03/01/01
Posts: 3,598
Loc: Oregon, USA
Last seen: 5 hours, 14 minutes
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Horse_Meister]
#7798345 - 12/25/07 01:45 PM (16 years, 1 month ago) |
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The plates look good. Bubbles that form holes don't hurt anything although there is a product you can add to agar to prevent that.
The contaminates are from they air as you suspected. The white and yellow dots are bacteria or yeasts. I rarely try to identify the contaminates since they are all bad. Ziplock bags help but if you place more than one plate in the bags and one goes bad, you lose them all in that bag. I like to cut rolls of glad clingwrap into two inch mini rolls. These can be stretched over the edge after cooling just like parafilm. They breathe enough to keep the cultures going and slow the drying of the plate as well as keep airborne molds and bacteria out.
Looks like you are doing well so far. Keep at it.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification 
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Horse_Meister
Edible Farmer



Registered: 01/14/07
Posts: 408
Last seen: 5 years, 4 months
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Workman]
#7798952 - 12/25/07 06:12 PM (16 years, 1 month ago) |
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I don't believe it was bubbles that formed the holes. I believe it was the condensation build up that dripped and then ate a hole in the agar. I only really messed up one plate with a bubble in it from pouring it.
-------------------- KTHXBai2YoU Horse_Meister Life is what happens while you're busy making other plans.
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Yrat
Hello

Registered: 11/08/07
Posts: 2,312
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Horse_Meister]
#7799025 - 12/25/07 06:51 PM (16 years, 1 month ago) |
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once the agar is solid turn the plates upside down so that the dish cover rests on the bottom and the agar is suspended above. any condensation that forms pools in the lid and not on your agar surface. i remember doing this in my microbiology courses. there will be no effect on growth.
-------------------- "There are a thousand hacking at the branches of evil to one who is striking at the root." -Henry David Thoreau Strike The Root
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Horse_Meister
Edible Farmer



Registered: 01/14/07
Posts: 408
Last seen: 5 years, 4 months
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Yrat]
#7799411 - 12/25/07 09:20 PM (16 years, 1 month ago) |
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What do you do with this pool of water now?
-------------------- KTHXBai2YoU Horse_Meister Life is what happens while you're busy making other plans.
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Horse_Meister
Edible Farmer



Registered: 01/14/07
Posts: 408
Last seen: 5 years, 4 months
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Horse_Meister]
#7870415 - 01/12/08 07:12 PM (16 years, 1 month ago) |
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So here comes a multitude of photos of my failures. I am posting this quickly, I may edit when I have time to think about what I have done. I am looking for inspiring photos of small clean rooms/laminar flow hoods/ what people use to incubate petri dishes. Please inspire me!

I did a finger print. Look what came from it ^. I shouldn't be fingering any girls, they may get a yeast infection...lol


Nice mold




The trich I know came from my oyster bag that I had previously grown out. I was hoping to keep it going.

This oyster looks good, except the red and snotty looking paste on the topside of the red.



^ The above photo is 1 of 2 successful dishes without contam. These were immediately put into freezer ziploc bags after the agar solidified. The chunky centre came from the liquid agar being slightly chunky at the top of the pour.
I firmly believe that I can do agar. It looks like I definately need some sort of flow hood seeing as all my transfers or spreads turned out contam.
Any and all advice
-------------------- KTHXBai2YoU Horse_Meister Life is what happens while you're busy making other plans.
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Blutjager
Inhuman


Registered: 06/11/06
Posts: 9,220
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Re: Horse_Meister - Agar Attempts - Log with PHOTS - Open to Experienced Advice [Re: Horse_Meister]
#7870691 - 01/12/08 08:08 PM (16 years, 1 month ago) |
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Quote:
Horse_Meister said: So here comes a multitude of photos of my failures. I am posting this quickly, I may edit when I have time to think about what I have done. I am looking for inspiring photos of small clean rooms/laminar flow hoods/ what people use to incubate petri dishes. Please inspire me!

I did a finger print. Look what came from it ^. I shouldn't be fingering any girls, they may get a yeast infection...lol


Nice mold




The trich I know came from my oyster bag that I had previously grown out. I was hoping to keep it going.

This oyster looks good, except the red and snotty looking paste on the topside of the red.



^ The above photo is 1 of 2 successful dishes without contam. These were immediately put into freezer ziploc bags after the agar solidified. The chunky centre came from the liquid agar being slightly chunky at the top of the pour.
I firmly believe that I can do agar. It looks like I definately need some sort of flow hood seeing as all my transfers or spreads turned out contam.
Any and all advice
Looks like the 1st time I tried to grow on agar
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