From ahicks@nmt.edu Wed Nov 13 20:20:32 1996
To: ahicks@quartz.nmt.edu


Date: Tue, 12 Nov 1996 11:24:21 -0600
From: r-halgren@nwu.edu (Robert G. Halgren)
Subject: [11162] re: [11138] Flasking and Physan

>Date: Sun, 10 Nov 1996 12:21:30 -0800
>From: Stephen Gaudin <talin69@loop.com>
>
>I tried adding a drop of physan to a couple of flasks at the first sign
>of mold, but it is too soon to tell what will happen.  I haven't seen
>any more mold, but I only just figured out to even try this.  Up to now,
>I've just thrown the flask and contents out.  I also decided to find out
>if the mold was coming from the seed, or my semi-clean processes.  I
>kept 10 flasks without seed, and set them aside.  One flask has develped
>a reddish tint and shows some sort of coagulating growth in the medium
>(mold of some sort I figure).  I therefore can assume that though the
>seed may be a factor, my semi-clean area WAS contaminated.  I can deal
>with 10% loss, but would like to eliminate as many losses as I can.

        I may have discussed this experiment previously.  It isn't a good
experiment, but it is a start.  I took a flask of contaminated Paphiopedilum
(hybrid) seedlings (a good spot of mold in the center, usually certain death
to a replate no matter where you take the protocorms from).  Protocorms were
removed from the flask, leaving anything that was within about .5cm of the
mold (I'm curious, not stupid), and put in a 3%H2O2 solution with a small
amount of detergent added.  Protocorms were soaked for about five minutes
before beginning replate.  Protocorms were replated on three different
media, my normal media with no addition, media with the addition of 2.5mg/ml
Amphotericin B (Fungizone), or media with 1.0mg/ml Amphotericin B.  I
started with the high concentration of antibiotic.  Obviously protocorms
were in H2O2 longer for the "normal" media than for the antibiotic media, it
probably took me 15 minutes to replate all the flasks.  For the normal
flask, the remainder of the protocorms were dumped, with the sterilant, into
the flask, and some of the extra sterilant removed by aspiration - so take
that as you will.
        Conclusions so far.  Amphotericin B does not seem to inhibit the
growth of plants at the concentrations used. More importantly, the H2O2
alone was sufficient to prevent mold carryover into the replate flask.  In
other words, all plants are doing fine. The ones in the antibiotic
containing media seem to be doing better, in fact, but that is almost
certainly an artifact of the replating procedure.  Obviously this needs
repeating, preferrably a couple of dozen times...  I'm not going to go out
of my way to get contaminated flasks to do the experiment, however.
        So, Stephen, to your problem.  While Paphiopedilums at the protocorm
stage do not seem to be affected by Amphotericin B, I do not know about
seed.  Germination may be inhibited (heck, it may be enhanced).  I do not
know if there has been much work done on the subject (my university library
isn't adequate to the task).  Most flasking protocols assume you are working
with good quality, clean seed, in an ideal flasking environment.  Don't need
antibiotics in that case, now do you? Of course not all of us get to work
under ideal conditions, and nobody can be guaranteed clean seed at all
times.  Sounds like a worthwhile line of investigation.  As is Fred
Bergman's work with using "alternative" sterilants.  A combination of both
might be necessary for truely recalcitrant seed.  As for using Physan...
I'm going to guess that that would be bad for plants at the concentrations
you would need to use.  It is phytotoxic at the higher levels (trust me...),
I would argue that even lower levels could be phytotoxic if given in a
continuous exposure.  Just a guess, I was almost certain that Amphotericin
was going to be toxic too, so what do I know? *grin*

Rob
*********************************************************************** ****
Rob's Rules:  http://pubweb.acns.nwu.edu/~rhalgren |Send me orchid seeds!
   1)There is always room for one more orchid.       |
   2)There is no Rule 2.                             |Future Starving PhD.
    2a)When one has insufficient credit to buy       |
       more orchids, obtain more credit.             |Will flask for food.

------------------------------