Re: alcohol in autoclave?

["agritech@agritechpublications.com" <Agritech@ATT.NET>]

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.
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-------------- Original message from "Dr. Joseph Arditti" <jarditti@UCI.EDU>: --------------
> At 12:22 PM 13-10-05, you wrote:
> >Hi,
> >
> >Why do you need to sterilize 70% alcohol?
>
> Good question. It is already sterile.
>
> >
> >But going back to the question, I don't think the autoclave will get
> >harmed since it's only a small amount. Your alcohol, however, will not be
> >70% anymore after autoclaving since a lot of the alcohol in it will volatilize.
>
> We used to add heat labile compounds in 70% ethanol to media after
> sterilization. I am sure that some of the alcohol evaporated on contact
> with the hot solution.
>
> >
> >Joseph Arditti
>
> Dr. Joseph Arditti, Professor Emeritus
> University of California, Irvine, CA 92604
> E-mail: jarditti@uci.edu