Cant get a clean culture

[chrisbossjake]

Hello folks, I have been playing with agar for a few months now. I cant seem to get a clean culture going though. My plates consist of:

250 ml H2O
5g Agar
1g Dextrose
1g Bee Pollen
.5 g Nutritional Yeast
2.5 g Potato (instant)
1 ml H2O2 (added below 130 deg. F)
The mix is in a pint jar with foil on top and is PCed @ 15psi for 15-20 minutes. When PC pressure comes back to 0psi I take the PC and open it in the flowhood and let the agar jar cool there. I use a alcohol wiped candy thermo. to check the temp of the agar before adding the H2O2. I then use a sterilized 35cc syringe to "pour" my plates. This is divided over about 6-8 plates. I let them cool under the flowhood and then seal the rim with saran wrap or electircal tape.

The myc loves this mix but so does trich. I have been doing my culture transfers in my flowhood and have been torch sterilizing my cutting utensil.

Incubated at 85 deg. F Agar plates are inverted and have electircal tape or saran wrap around the rim to seal them.

Any ideas on what I should/shouldn't be doing with what I am doing?

[calicyco]

You have way too many vectors for contamination for agar work. You should be pouring the agar from the jar into the plate in a quick manner, and thats it. Not sticking in a thermometer, or a syringe. Firstly eliminate all sources of contam, including the peroxide. Check your sterilization process and the flowhood sterility by pouring a plate and sealing it then incubating. If all is well, perhaps add the peroxide step back in, and recheck by incubating. I would eliminate the peroxide until you have established good clean culture procedures.

Does your PC have a stopcock? Does it form a vacuum when it cools? If so, when you open the valve to equalize pressure place an alcohol soaked clean cloth over the stopcock. Thoroughly clean all outside surfaces of the PC as well before opening, with a disinfecting spray, bleach or alcohol, and a sterile cloth. You should PC anything that comes into contact with the agar, plus flame sterilize immediately prior to use.

Forget the thermo and the syringe, unnecessary and a good vector for contams.

Proper testing of each step and procedure with clean uninnoculated agar should help determine your issue.

Take a shower before handling agar. Wear freshly laundered clothing. Wear a mask and gloves. Be as careful and anal as you can be until you have it down pat and are working with pure clean cultures.

Also, how sure are you of the cleanliness of your innoculation source? (Spores, what have you..) Agar is great to isolate clean cultures from contaminated sources, however you have to be able to ensure the sterility of the agar medium itself and your procedures before this will work properly.

Good luck!

[sandman]

Hes right...way too much going on

“Insanity: doing the same thing over and over again and expecting different results.”

-Albert Einstein

Leave out ur pollen, h202, and syringe pouring. Just pc it in a qt jar, open in the hood, wait 30 minutes or so to cool down so you can hold the jar and just pour your stack.

[chrisbossjake]

Originally Posted by calicyco

Also, how sure are you of the cleanliness of your innoculation source? (Spores, what have you..) Agar is great to isolate clean cultures from contaminated sources, however you have to be able to ensure the sterility of the agar medium itself and your procedures before this will work properly.

Good luck!

I started this all from a print which I beleive was contaminated with some sort of bacteria and or trich. My jars will colonize fully but end up with a foul odor similiar to wet spot but they arent wet (I guess it still could be the same bacteria). They also go green sometimes also. Im almost certain it is from the print because I can leave my WBS or grain sitting in incubation temps for a month and they are contam free.

I will cut out all unnecessary steps and just do the bare essencials and check the sterility of my flowhood. Thanks for the tips. Ill giver a go this weekend.

P.S. once I get my technique down, how should I "clean" my cultures?

[SharkieJones]

You could always buy agar with antibiotics in it.

[Mr. Clean]

Originally Posted by SharkieJones

You could always buy agar with antibiotics in it.

sporeworks has some top notch potato dextrose agar, fully sterilized.

can't go wrong.

[Awestruck]

The antibiotic agar will work well against bacterial contams but will be useless against the mean green.
A trick for getting a thermometer reading is to put your agar container in a water bath and take the temps of the water bath to monitor the temo of the agar, since the two will equilibrate. I also use a 10 mL pipette to pour my plates, but the pipettes come pre-sterilized in plastic sheaths. Since you have a flowhood, it is not that bad to have extra steps with pouring your plates or adding H2O2.

Sounds like the print is the most likely suspect here. It is easy to find out.
Pour some plates like you have been doing and keep them for a week without innoculating them. Are they still sterile? Then it's the print! Are they contammed? Then it's your sterile technique... which could be the hood, dishes, syringes, themometer...
I'd also go for 30 mins in the PC for 250 mL. It won't hurt the agar at all.

When you get some viable agar, you can streak it with some spores from your print. You want a fairly dilute solution to do this with since you only want a few colonies on your plate. If you use straight spores, it will be very hard to cut down on contams. An easy way to dilute is to take some sterile water and put some spores in it, mix it up (like a spore syringe) and use that solution to streak your agar. I can explain more if you like.
Hope you get it figured out soon. Keep us posted

[calicyco]

Awestruck is correct, check you technique and then if all is good, and you have a contaminated print, get going on cleaning the culture.

The way to do this is well documented, TMC, MMGG, teks here, etc.

Basically, you want a dilute spore solution, to spread out the contams and good spores. Get them germinating on agar and as soon as you can identify mycelium, transfer it away to a clean plate. Take a very very tiny bit of good mycelium from a contaminated plate and transfer it to a clean one. Then repeat, until all you have growing is a clean plate with good healthy mycelium on it. You want to run away from the contamination as far as possible with multiple transfers. Which is why agar is so useful, you can clean up extremely dirty prints this way. If your print is dirty, you WILL get contams. All is not lost though, as you have a method for getting away from it.

I see no need to measure temperature of agar though, it responds to certain temps by melting/gelling. They do sell infrared laser thermometers to measure temps so you'd never touch anything at all. They are around $60-100 from a quick search. However you should get used to doing it by feel, its not hard and you'll get the hang of it in no time.