Laminar flow cabinet

[John]

I have been doing more with flasks and would like to make a laminar flow cabinet to work in. Does anybody have ideas as to what size and flow rate to shoot for? I have seen homemade ones on the web so it can be done. Hopefully under $200.

[adriponic]

First of all find your hepa filter, then build a box around. Open a hole on top to accomodate a air blower which can
be operated through a variable power device to set the
blower speed at will.

If you can get hold of a book from Paul Stamets "Growing
gourmet mushrooms" (i don't remember the exact title)
you'll find there all you need to build a home made laminar flow cabinet.

Good Luck

[John]

Thank you for your help. I will look for the book.
John

[adriponic]

and yet John do not trust the laminar flow cabinet too
much. I have 4 of those 2 home built and 2 from the best
industry standards ( the home built are as good as the
famous brands) and yet they can't beat my 20 years
old sterile box.

Would you believe?

Good Luck

[John]

I have been using a sterile box but it seems that one or two out of ten flasks have contamination. I think that maybe there are spores under the foil that is over the lids. I was assuming that it would be sterile under there since it was in the pressure cooker. I have of course sprayed the jars with bleach solution but not around the threads. I was thinking that the flow cabinet would speed things up since I wouldn't have to load it and then wait 30-40 min for the bleach to sterilize everything. Do you have any pictures of your setup?

John

[adriponic]

Ok John

As soon as i can lay my hands on my digi camera in my
never ending mess, I will post pics of my setup.

Meanwhile we have to detremine the source of contamination. Its a good practice to keep a few
witness flasks from each batch (do not sow and keep
these for 45 to 60 days) If the do not get contaminated
then you can assume that contamination comes at
sowing or transferring tissue/replating.

If on the other side the witnesses become contaminated
then you have to rework your cooking/sterilizing
procedures.

Once everything is inside my sterile box, I spray with
25-30% bleach and start working after 5 to 10 mins.

Hope this helps

[newflasker]

Originally Posted by adriponic

Once everything is inside my sterile box, I spray with 25-30% bleach and start working after 5 to 10 mins.
Hope this helps

Hi Adriponic,
I spray with Lysol and wait for 30 minutes (I like Lysol odor). 1 out of 10 I got contamination with my sterile box. How good is your process? If yours is better I will try it.
Nguyen

[John]

Hi Jean Pierre... thanks for hanging in there with me. I have pepared flasks and let them sit for 30-40 days and there hasn't been any contamination so I believe that the problem is when I replate. This last time I had trouble with the sixth and eighth mother flask that I did. So I sit here wondering what I could have done diff between flasks. These flasks came from two diff sources, one in Malaysia and the other from Ecuador. The ones from Malaysia were doubled bagged in sterile conditions and the ones from Ecuador were in the original flasks. The Ecuador ones gave me trouble. I must not be sterilizing the outside of the replate flasks good enough and maybe I touch the edge when I am inserting the plants. I probably will not get any more flasks until spring so I have a lot of time to ponder this and maybe change my procedure. Do you feel the need to vent the replate flasks? It would make things simpler if I didn't.
John

[adriponic]

hi John - Hi Nguyen

Ng 10% is, I would say, a high contamination rate. Going
to my records for the past 5 years the average contamination is as follows:-

Mother flasks (inclusive of tissue culture) 1,457
Mother flasks contaminated 36 = 2.5%

Replate flasks = 8,613
Replates contaminated = 69 = 4.75%

I work in a sterile room cleaned with a laminar flow
30 mins ahead of operations which are carried in a
sterile box.

John - I do not vent - All my flaskings are done in 800 ml
micro oven plastic containers filled with 100 - 150 ml
media. There is lot of air space inside so i do not have to
vent. The containers & media are sterilised in a 20 years
old home made autoclave which is gas fired. One run
can accomodate 20 flasks + utensils. Heat goes up to
123 celcius in 14 mins with just about 1.2 litres of water
inside. One run from firing to harbouring the flasks and
utensils inside the sterile room takes less than 40 mins.
However the flasks & covers have to be sealed with
sterile cling film in front of the laminar flow IMMEDIATELY
upon removal to the sterile room. Quite a hard job to
handle very hot plastic but I am now at ease with it
after some 7 years practice.

My son will visit me in a couple of hours. I told him to
bring his digital camera and i will have him take pics of
whole set up for posting.

Hope this helps - Kind Regards

[newflasker]

Originally Posted by adriponic

Mother flasks (inclusive of tissue culture) 1,457
Mother flasks contaminated 36 = 2.5%
Replate flasks = 8,613
Replates contaminated = 69 = 4.75%

I work in a sterile room cleaned with a laminar flow 30 mins ahead of operations which are carried in a sterile box.

Hi adriponic,
WOW, 1,457 mother flasks + 8,613 relate flasks + Tissue Culture = flask Master . What kind of Tissue Culture have you been working with? I have no problem with orchid seed mother flask but with replate flask. The problem happens when I transfer seedlings from one jar to another jar. I think I need to use Clorox/NaDCC but I hate chlorine odor.
Nguyen