Re: alcohol in autoclave

[Lizette Tuason <mmstuason@YAHOO.CA>]

I've observed this to be true as well.  This is why when I'm doing 
flame sterilization, I leave my forcep over the flame for a longer time 
(although not as long as I would when flaming an inoculating loop to 
"red hot").  It gets a tad too warm for my fingers' comfort but it's 
preferable to introducing contaminants.  I keep a fresh plate of sterile 
agar to cool the tip of my forcep or my hockey stick in.

Ethanol, even absolute ethanol, is only a disinfectant because it 
cannot kill the most resistant microorganisms - bacteria that produce 
endospores.  The same is true with other commonly used disinfectants 
in the lab - e.g., bleach.  That's why we autoclave materials at 121C for 
15 minutes (based on the killing time for bacterial endospores;  there 
are always exceptions though) or incinerate them.

Lizette

"agritech@agritechpublications.com" <Agritech@ATT.NET> wrote:
Date: Sat, 15 Oct 2005 15:03:45 +0000
From: "agritech@agritechpublications.com" <Agritech@ATT.NET>
Subject: Re:  alcohol in autoclave?
To: PLANT-TC@LISTS.UMN.EDU

70 % alcohol is not necessarily sterile. See the following excerpt from 
my book Recent Advances in Plant Tissue Culture VIII: Microbial 
"Contaminants" in Plant Tissue Cultures: Solutions and Opportunities 
1996 - 2003

CHAPTER 1
PLANT TISSUE CULTURE CONTAMINANTS

A. ETHANOL

A study by L.R. Schreiber and colleagues at the United States National 
Arboretum suggests that contamination of in vitro plant culture can 
originate in what some tissue culturists might consider to be the most 
unlikely source of contamination (Journal of Environmental Horticulture 
14(2):50-52, 1996).
When a contaminant identified as Bacillus macerans appeared in 
American elm (Ulmus americana) callus cultures after several transfers 
that appeared to be free of contamination, Schreiber?s group tested for 
the presence of bacteria on the forceps used for the transfers by touching 
the forceps tips to culture media in Petri dishes. Schreiber et al. found 
that the contaminating bacteria remained viable on the forceps after they 
were stored in 95% ethanol for several weeks. Furthermore, the 
contaminants remained viable even when the excess alcohol was burned 
off with the flame from an alcohol lamp.
In another experiment, Schreiber et al. artificially contaminated forceps 
with Bacillus macerans by dipping the tips into a bacterial cell suspension 
and air-drying them. The forceps were then stored in 95%, 85%, 80% or 
70% ethanol for up to four hours. Schreiber?s group found that none of the 
alcohol dilutions eliminated the bacteria, with or without alcohol lamp 
flaming. Elimination of the bacterial contaminant could only be achieved 
by autoclaving the forceps at 121°C for 20 minutes or by exposing them 
to a bunsen burner flame for 6-8 seconds.
What is perhaps the most interesting finding of the study, however, is 
that viable bacteria could even be recovered from the alcohol itself. After 
the various alcohol concentrations were inoculated with the bacterial 
suspensions and incubated for 9 and 21 days, sterile filter paper discs 
dipped in each of the solutions showed bacterial contamination when 
incubated in potato dextrose agar. As a matter of fact, a higher percentage 
of discs showed contamination after 21 days than after 9 days, 
suggesting that, not only can bacterial spores survive, but that bacteria 
can actually grow in alcohol.
Since ethanol is itself considered to be a sterilant for in vitro cultures, 
little thought may be given to sterilizing or maintaining the sterility of the 
alcohol used for disinfesting explants of for flaming instruments. It 
appears, however, that for some contaminants at least, ethanol rather 
than being a sterilant, can be a source of contamination during routine 
disinfestation procedures.
--
Agritech Consultants Inc.
Agricell Report
P.O. Box 255
Shrub Oak, NY 10588,
U.S.A.
Phone/Fax: 914 528 3469
E-mail: agritech@agritechpublications.com
Website: http://www.agritechpublications.com

-------------- Original message from "Dr. Joseph Arditti" : --------------
> At 12:22 PM 13-10-05, you wrote:
> >Hi,
> >
> >Why do you need to sterilize 70% alcohol?
>
> Good question. It is already sterile.