Mouthpiece sterilizing test results

[Philip Jensen]

I earn a living conducting research on plants, but as I use molecular biology tools to do this work, I actually work with bacteria a lot more than I do actual plants.

I was working late last night, and happend to have some extra E. coli (a disarmed strain - DH5alpha) so I set up an experiment.

(1) I took a Bach 18 mouthpiece and placed it in 3 ml of a solution containing 5X10[8] bacterial (500 million) for 10 sec. I gently blotted the mouthpiece on some sterile filter paper and then set the mouthpiece on a pertri plate containing Lennox agar.

(2) I repeated the dip in the bacteria solution and the blot and then dipped it for 1 second in a 10 ml solution of 70% Isopropyl alchohol (this is what you find in the stores), blotted it on sterile filter paper and then onto the same petri plate. Unfortunately the petri plates are smaller than 2 tuba mouthpiece diameters, so there is some overlap.

I repeated (1) and (2) above on a second plate, this time dipping for 1 minute in the 70% isopropanol solution.

The plates were incubated overnight at 37 degrees C.

Results:

I got a nice ring of bacterial growth on both of the (1) treatments.

For the 1 second dip, the were NO colonies. Zero, zip, zilch, nada.

For the 1 minute dip I did see a short arc of colonies from the Isopropyl treated test. However, this arc comes out from where the two treatments overlapped on the petri plate and is most likely the result of wicking of moisture from the (1) treatment along the rim when the (2) treatment was placed on the plate.

I'll continue the incubations in case any slower growing bacteria are present.

I took some pictures and will post them later.

Philip "double whammy - science nerd & band nerd" Jensen

[Rick Denney]

I got a nice ring of bacterial growth on both of the (1) treatments.

For the 1 second dip, the were NO colonies. Zero, zip, zilch, nada.

So, rubbing alcohol kills bacteria on mouthpiece effectively.

If you could, try the experiment with hydrogen peroxide intead of alcohol, heh, heh.

I love this stuff.

Rick "who figures a similar test with virii will be asking too much" Denney

[Philip Jensen]

Rick, I meant to do the H2O2 experiment, but forgot mine at home and I don't have it in the lab. Maybe next week. I'm not set up to do viruses.

Here's a picture of the plates. The areas labeled control is the dip in bacteria only. You can easily see the mouthpice was covered with bacteria.

The 1 min dip was a 1 min soak, not an in and out, repeat, treatment.

The wicking is probably from liquid that was on the sides of the rim. This was a quick and dirty experiment and I didn't have a supply of sterilized paper towels handy, but I did have some sterile filter paper that I just set the mouthpiece on after dipping, - no wiping. The mouthpiece sinks a bit into the agar so liquid on the sides could be left on the plate. That plus the plates themselves are moist and the weight of the mouthpiece could force some liquid out of the agar.



I'm going to take one of these plates and flood it with 5 ml of 70% isopropanol. I'll let the alchohol evaporate and return the plate to the incubator for the weekend.

I talked to one of my colleagues who just happend to be preparing a talk for mushroom producers on sanitizing their facilities. Mushroom producers can have problems with contaminating fungi so my colleague tested various treatments, including ethyl and isopropyl alcohols - including several well know brands of booze.

He was working with fungi, not bacteria. He took liquid suspensions of fungi and added the treatment dujour to the culture, then spun down the contents, resuspended the culture and plated it. Any alcohol treatment over 26% killed ALL of the fungi. He has a nice slide showing 5 different booze bottles and underneath the results from using that as a disinfectant. As straight booze, they all gave 100% kill.

The problem with alcohol, is that while very effective, the cost would be prohibitive on an industrial scale.

The problem with references, is that the effectiveness is common knowledge so nobody writes about it. I'm sure if you went back 75 year in Biological Abstracts you could find plenty of work. The web resources don't got back that far - yet.

I did a quick search and found 2 interesting papers:

Clin Microbiol Rev. 2004 Oct;17(4):863-93,
Epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs.

Kampf G, Kramer A.

Bode Chemie GmbH & Co., Scientific Affairs, Melanchthonstrasse 27, 22525 Hamburg, Germany. guenter.kampf@bode-chemie.de.

The etiology of nosocomial infections, the frequency of contaminated hands with the different nosocomial pathogens, and the role of health care workers' hands during outbreaks suggest that a hand hygiene preparation should at least have activity against bacteria, yeasts, and coated viruses. The importance of efficacy in choosing the right hand hygiene product is reflected in the new Centers for Disease Control and Prevention guideline on hand hygiene (J. M. Boyce and D. Pittet, Morb. Mortal. Wkly. Rep. 51:1-45, 2002). The best antimicrobial efficacy can be achieved with ethanol (60 to 85%), isopropanol (60 to 80%), and n-propanol (60 to 80%). The activity is broad and immediate. Ethanol at high concentrations (e.g., 95%) is the most effective treatment against naked viruses, whereas n-propanol seems to be more effective against the resident bacterial flora. The combination of alcohols may have a synergistic effect............

http://www.ncbi.nlm.nih.gov/entrez/quer ... s=15489352

And:

Appl Environ Microbiol. 2002 May;68(5):2576-9.
Efficacy of common laboratory disinfectants on the infectivity of Cryptosporidium parvum oocysts in cell culture.

Weir SC, Pokorny NJ, Carreno RA, Trevors JT, Lee H.

Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1.

Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts.

http://www.pubmedcentral.nih.gov/articl ... d=11976138

Second author on this one is Pokorny!

Cryptosporidium (not a bacteria) is really a tough bugger. If you read the paper, it is interesting that it takes an average of 132 oocytes to cause an infection in 50% of the people