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By cohy
Posts:  4
Joined:  Mon Oct 13, 2014 2:43 pm
#214470
Hello,
I want o ask you what is your contamination rate during subcultures?
I lose almost one third of my vessels after every subculture. I don't have laminar flow hood. I use only big fish tank. Inside of my aquarium, I have bottle of isopropyl alcohol for my foreceps and scalpel and I always spray everything inside box with isopropyl alcohol. I cut plants on sterilized petri dish (steam sterilized in preassure cooker). Is it normal to suffer such dramatic losses? Contamination rates in good laboratories are under 4 % according to literature. What percentage did you achieve witout LFH?
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By Matt
Location: 
Posts:  20652
Joined:  Mon Apr 21, 2008 11:28 pm
#214478
I don't think it's normal to get such high contamination rates. Before I got a flow hood, less than 10% of my cultures got contamination. Are you sterilizing your forceps between cultures by flaming them off? Also, are you wearing clean clothes and moving slowly and as little as possible when doing the subcultures? And are you keeping the clean cultures as far back in the aquarium as possible?
By cohy
Posts:  4
Joined:  Mon Oct 13, 2014 2:43 pm
#214481
Thank you for response. I don't use flame. I have inside my box only tube full of isopropyl alcohol. I immerse my foreceps and scalpel inside of the tube every two minutes or so. Do you think that's the problem?
I do everything else as you describe.
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By Matt
Location: 
Posts:  20652
Joined:  Mon Apr 21, 2008 11:28 pm
#214500
cohy wrote:Thank you for response. I don't use flame. I have inside my box only tube full of isopropyl alcohol. I immerse my foreceps and scalpel inside of the tube every two minutes or so. Do you think that's the problem?
Yes. Flaming is important to sterilize forceps. Alcohol alone will not do much.
By cvlover
Posts:  447
Joined:  Thu Apr 23, 2009 4:59 pm
#214590
You could reduce the contamination rate in simple ways:
- Modify your fish tank to be a glove box. Cut a plastic board to cover your open tank. Drill/cut 2 holds and connect 2 long gloves (buy them at Asian market for few bucks). Seal the board with plastic tape so you can open and seal your box again and again.
- Add 75 - 91 % alcohol into a long and narrow glass bottle. Heat your forceps and scapels for 2 - 3 minutes on the stove , let they cool a little bit then submerge them into bottle; move them to glove box.
- You clean your box by alcohol then vessels before put them in glove box. Seal the box. Spray alcohol inside. Wait for 15 mintue to kill bacteria and mold then work with you TC.
These ways will reduce a lot your contamination rate when transfer or divide your seedlings. Sometime I open my jars inside box for hours without any problem.
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By cvlover
Posts:  447
Joined:  Thu Apr 23, 2009 4:59 pm
#214652
Some pictures how to reduce contamination rate:

Modify fish tank to glove box
box-front.jpg
box-front.jpg (457.13 KiB) Viewed 1878 times
box-rear.jpg
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Heat equipments on oven
oven.jpg
oven.jpg (390.01 KiB) Viewed 1878 times
Dip into alcohol bottle
bottle.jpg
bottle.jpg (525.49 KiB) Viewed 1878 times
Some pictures of CPs:
Cephalotus
cephalotus.jpg
cephalotus.jpg (423 KiB) Viewed 1878 times
Darlingtonia
darlingtonia.jpg
darlingtonia.jpg (445.33 KiB) Viewed 1878 times
Sundews
cp.jpg
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My tiny garden
garden.jpg
garden.jpg (817.68 KiB) Viewed 1878 times
Smile :)
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By xr280xr
Posts:  2806
Joined:  Wed Jun 22, 2011 3:29 pm
#215142
I'm pretty new to TC but have had good success with sterilization so far (excluding some cases where I made obvious mistakes). When you spray your tank down, make sure to use a generous amount and to let it sit for a minute or two. Spraying it on and then immediately wiping it off will not kill everything. It is a good idea to routinely clean the entire area too and you might try a 10% bleach solution in your tank (but watch out for the fumes). Make sure you're washing your hands & forearms before starting and avoid moving them directly over your cultures where bacteria can fall off of you onto them.

I also use a fish tank and for flaming tools, I use this as a poor-man's touch-o-matic bunsen burner (it is sold under a few different brand names). It's nice because the ignition button is on the top so you can start and stop it with one hand. I chose this over an always-on burner because that would create air circulation in the tank or you would have to move your tools outside the tank to flame them. It has worked well so far. Just be careful of having a flame in the enclosed tank with alcohol & alcohol fumes. I also taped off the air vent in the room I use to make sure there's no air movement in the room.

By the way, are you using any antibiotics/antifungals/PPM in your media?
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By cvlover
Posts:  447
Joined:  Thu Apr 23, 2009 4:59 pm
#215179
I don't use bleach because it damages the glove. Only use alcohol because it' easy on glove. I wipe box, gloves, jars/vessels and equipments with alcohol (not spray, hate inhale too much isopropanol). Before sealing the box I spray with Lysol disinfectant spray and seal it right away. Wait for 15 minutes or more for Lysol, alcohol kill fungi and bacteria before operations.

I never flame tools inside box because it could explode or burn your house. One guy in this forum blows his box by flaming (look at old threat).

I try some anti-fungi/bacteria chemicals in my media but don't like the result so I stop using them.
By cohy
Posts:  4
Joined:  Mon Oct 13, 2014 2:43 pm
#219609
Hi,
I made some changes and now, I have 40 food jars with 0 % contamination - it is 2 months so far.
I found out, the biggest problem is contaminated tissue material. In book Plants From Test Tubes you can also find guide how to transfer plants in sterile conditions using bleach - very easy in kitchen but kind of messy though. Hope this will help guys.
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