Stressbaby's Sanitizer Experiments

[Stressbaby]

There has been debate in other threads about sanitizers, contact time, and rinsing. I went to Amazon and bought 20 tryptic soy agar plates to test some of these ideas myself. This is the thread in which I'll post my experiments and results. Feel free to suggest experiments. I'll run the tests any way I can until such time as I run out of plates.

An important assumption in all of these experiments is that the pathogens grown on the tryptic soy agar do not differ substantially in their response to the sanitizer interventions than the wine pathogens with which we are concerned. If someone has thoughts on how to verify this assumption, please post below.

 

[Stressbaby]

Quickie experiment #1 - StarSan on the hands

One plate, four quadrants. I made two fingerprints in each quadrant of the agar plate. Each quadrant got fingerprints from different fingers of each hand. So quadrant 1 was index left and pinkie right; quadrant 2 was middle left and ring finger right, etc.
Quadrant 1 - no sanitizer
Quadrant 2 - StarSan, immediate (minimal contact time)
Quadrant 3 - StarSan, 1 min contact time
Quadrant 4 - StarSan, 1 min contact time, rinsed

Will post back in a few days.

 

[Stressbaby]

Experiment #2

In this experiment I'll try to more formally mimic what happens when sanitizing winemaking equipment. The design I have in mind looks like this: I have some leftover country wine, pH 3.3 and SO2 <10 by titrette method. I'll take soda straws and uniformly expose them to pathogens by rolling them in my hands and on the work surface. Straw 1 goes into test tube #1 with 10ml of wine, that's the control. Then each straw will be treated according to a sanitization protocol and go into it's own test tube. All the straws will be swirled thoroughly and 0.1ml of wine drawn from the test tube and placed on the agar plate.

1. Control
2. StarSan, minimal contact time
3. StarSan, 1 min contact time
4. StarSan, 3 min contact time
5. StarSan, left to dry
6. StarSan, 1 min contact time, brief tap water rinse
7. StarSan, 1 min contact time, extended tap water rinse

I would then repeat this with KMS, altering the contact times.
I would also repeat this using a wine with adequate SO2.

I've set up a proof of concept plate to 1) assure my wine is not contaminated; 2) assure that this method yields a reasonable number of control colonies; and 3) to see how much variability there is in the number of colonies produced. I don't want to go through the entire experiment only to find that the control doesn't yield enough colonies to tell whether the interventions had any effect or that there is so much variability that the results aren't reliable. So I put 0.1ml of wine without any other contamination or intervention into quadrant 1 of a fresh agar plate. Then I contaminated a straw and placed it in 10ml of wine and swirled. From that I took 0.3ml and placed 0.1ml in each of the other 4 quadrants. What I'm hoping for here is minimal to no growth in quadrant #1 and some decent number of colonies in each of the other 4 quadrants. Failing to get that, we'll have to revise the methods.

Everyone is welcome to post suggestions.

 

[Stressbaby]

Update on experiment #1: All four quadrants grew lots of colonies. In this experiment, there doesn't appear to be any lower counts using the StarSan on the hands. The quadrant with the rinse looks worse, but the colony count isn't really that much different; some other bug showed up here and these larger colonies just overgrew the smaller ones.

 

[vacuumpumpman]

Thanks for doing this !

There are so many ideas about the proper way to santize, I do believe this is a great wato test those theories

 

[Stressbaby]

Thanks. The proof of concept test plate for experiment #2 didn't work out. Grew one colony on each section, so that is not sufficient numbers. Will need to redesign that. Considering actually inoculating the straws. Stay tuned.

 

[Stressbaby]

Experiment #3
I created a nutrient broth with 1g Opti Malo in 15ml distilled water. I inoculated this with each of the various pathogens which grew from my hands and from the counter top in the first two experiments. This was a mix of bacterial and fungal bugs which seems as reasonable as anything else of the bugs we're trying to get.

Into this nutrient broth I dipped 9 soda straws. I feel the soda straws serve as reasonable proxy for winemaking equipment (tubing, canes, etc). The straws were then treated as follows:

• No treatment
• StarSan, contact time 0'
• StarSan, contact time 1'
• StarSan, contact time 5'
• StarSan, contact time 1', rinsed with tap water
• KMS, contact time 0'
• KMS, contact time 1'
• KMS, contact time 5'
• KMS, contact time 5', rinsed with tap water

After treatment, each straw was vigorously swirled in 30ml of 2016 Syrah. Then 3 drops of each wine placed on the agar plate. I also put 1 drop of the nutrient broth on one plate (and since I messed up one plate, I put 10 drops of nutrient broth on that plate just for fun).

 

[Stressbaby]

Experiment #3 48 hour results in form of colony counts:

No treatment - 21 colonies
StarSan, contact time 0' - 0 colonies
StarSan, contact time 1'- 0 colonies
StarSan, contact time 5'- 0 colonies
StarSan, contact time 1', rinsed with tap water- 0 colonies
KMS, contact time 0'- 0 colonies
KMS, contact time 1'- 0 colonies
KMS, contact time 5'- 0 colonies
KMS, contact time 5', rinsed with tap water- 0 colonies

 

[Stressbaby]

The blob in the upper left is one drop of the inoculated nutrient broth. The colonies in the "no treatment" section are visible upper right.

 

[sour_grapes]

No treatment - 21 colonies
StarSan, contact time 0' - 0 colonies

Help me understand: does "contact time 0' " mean just dipping it in StarSan?

 

[Stressbaby]

Help me understand: does "contact time 0' " mean just dipping it in StarSan?

Poorly worded in retrospect. Perhaps I should say "minimal."
This arm and the KMS "contact time 0'" arm were handled by spraying with StarSan or KMS solution (inside the straw and outside) and immediately placing in the 30ml of wine from which the sample was drawn.

 

[Stressbaby]

72 hours:

No treatment - 78 colonies
StarSan, contact time 0' - 0 colonies
StarSan, contact time 1'- 0 colonies
StarSan, contact time 5'- 0 colonies
StarSan, contact time 1', rinsed with tap water- 0 colonies
KMS, contact time 0'- 1 colony
KMS, contact time 1'- 0 colonies
KMS, contact time 5'- 0 colonies
KMS, contact time 5', rinsed with tap water- 0 colonies

Should have done one with rinse only...

 

[Stressbaby]

Some folks on other threads have gotten feisty.

This started with the contention from @BernardSmith that tap water is "full of bacteria" and that one was "neutralizing" the sanitizer, presumably by reinfecting the equipment, if you rinse after sanitizing. I said I didn't think that this was true. Thankfully, BernardSmith is an open-minded dude who understands the scientific method, is receptive to new information, and appears to be willing to change his mind when presented with valid data.

There are reasons to believe that rinsing sanitized equipment might not be harmful or might even be helpful. Those include 1) bacterial colony counts in tap water in the US are fairly low. Cleveland has shown 0.2-2 CFU/ml, lower than many samples of bottled water; 2) new medical evidence related to wound irrigation demonstrating tap water to be safe and more effective than other irrigation therapies because of the mechanical flushing action (lavage), link, link, making the notion of rinsing to reduce infection biologically plausible; 3) established, respected winemakers who follow this practice.

I'm not definitively saying that rinsing is "proper procedure." I'm not saying this is a BETTER way of doing things. I'm making no comment on the reasons one might want to rinse sanitizer. I'm not suggesting that residual sanitizer is harmful - any data supporting the notion that sanitizer residue is not harmful is gratefully acknowledged but is not relevant.

I'm saying that it is not possible to state with certainty that this rinsing practice is harmful because there are reasons to think otherwise. This thread is an attempt to find out.

Constructive comments are welcome. @1d10t points out that rinsing prior to sanitizing is another approach to consider. S/he is exactly right - rinsing prior to sanitizing could in theory be superior! Tap water lavage followed by sanitizer could be the best approach of any. The next experiment shall have an arm testing this.

 

[Ajmassa]

Constructive comments are welcome. @1d10t points out that rinsing prior to sanitizing is another approach to consider. S/he is exactly right - rinsing prior to sanitizing could in theory be superior! Tap water lavage followed by sanitizer could be the best approach of any. The next experiment shall have an arm testing this.

Isn’t this already assumed though? Typically anything I’m sanitizing is coming fresh off a clean&rinse or at minimum a rinse with piping hot tap water.
I would think a situation of sanitizing equipment that has not been recently rinsed in some way would be extremely uncommon anyway.

 

[bstnh1]

Whenever you gather a water sample for testing, you are advised to disinfect the faucet from which you're drawing the sample because bacteria on the faucet can easily be introduced into the sample when the water flows. If you choose to use tap water for any wine making purpose, you run the risk of contaminating what may otherwise be bacteria-free water unless you wipe off the faucet with bleach or another disinfectant prior to drawing the water. This would also be true of the container into which you are collecting the water. If it's not sanitized or disinfected prior to filling with water, it's very possible that bacteria could be introduced.

 

[CDrew]

It would be interesting if there were a way to set up your experiment, to determine if StarSan or KMBS solutions were superior to one another. I use StarSan for everything but bottles, but willing to be convinced that another way is superior.

Good job on your culture experiments. It looks like all of your protocols work to sanitize your straws!

 

[Bts]

It looks like better is pretty much irrelevant. Momentary contact with either is good enough. Which is really nice to hear since sitting there like an ***** for half a minute every time I forget to presoak something in the sanitizer bucket is pretty annoying.....and I may have occasionally counted 1, 2, skipafew, 30

 

[Stressbaby]

I appreciate the feedback.
Experiments still ongoing.
I put 1ml of tap water on a plate Sunday, will check on it tonight.
Next round of experiments will use some discs or solid flat surfaces and transfer those directly to the plates instead of into wine first.