Sanitizer Comparison

General Homebrew Discussion
1

I recently bought some pre-poured agar plates partly for plating some mixed-culture beers to isolate some samples, and partly just to play around. I had a few plates left over that weren’t going to be used, so I ran a little experiment.

I streaked 4 nutrient agar plates with dregs from Gueuze Fond Tradition, which was one of the beers I was culturing. I let them sit for about 10 minutes to dry. Then I sprayed each with enough of each test solution to visibly wet the entire surface of the plate. I let each plate sit for 2 minutes, then poured off any residual sanitizer. I then stored the plates upside-down to dry. The plates were stored at ambient room temperature, and the results are from 8 days after plating.

The 4 plates were sprayed with:
-Filtered tap water (control solution, and what I use to mix my Iodophor and Star San)
-Iodophor (12.5ppm)
-Star San
-Bacardi 151 rum (75.5% ethanol)

Water:

Water.jpg

Iodophor:

Iodophor.jpg

Star San:

Star%2BSan.jpg

Ethanol:

Ethanol.jpg

The results are pretty clear. The control (tap water) is the big loser and has a significant amount of growth. The ethanol has a surprising amount of growth. Iodophor has a handful of scattered colonies. And Star San is the big winner in this test with no growth after 8 days.

I have to admit, the results were surprising to me. Coming from a medical background, I am very comfortable with iodophors and ethanol for surface disinfection. Povidone-iodine, aka Betadine, is a very common surgical prep and is an iodophor (iodophors are a class of compounds, by the way). But Star San just is not as broad spectrum (regardless of what my results show), and would never be used in the medical field. It is commonly used in dairy and food-processing, however, and certainly has its benefits.

So what do I think is happening here? First of all, what makes Star-San useful in dairy and food processing is that it doesn’t lose its potency in the presence of organic matter the way other sanitizers do. It is also much less volatile than ethanol. That is an important piece, since you need to ensure that your surface stays wet for the entire contact time for the sanitizer in question. And that is what brings me to the last piece - Star San has an added foaming agent. This really helps out in the contact time department.

So what are my personal takeaways from this experiment? The big one is that you can’t make up for poor cleaning practices with sanitization. You must have a surface that is clean of all organic materials for your sanitizers to be effective. My second one is that I will continue to use Star San in my brewery, and that I will go back to using it as my preferred spray sanitizer. Iodophor has a 2-minute contact time and no additives to help it cling to surfaces. I think I’ll take my chances with Star San. I will still use ethanol when I want broad-spectrum coverage (such as stepping up bottle dregs, where I’m starting from a small cell count), but I will be sure to soak the hell out of any surfaces in question and keep it wet by re-applying if needed.

Edit - finally able to get the images in line

2

Great info, Eric. Thanks for posting.

3

Neat experiment.  And really nice that you shared the findings… motivational and informative!  (although, the results are somewhat surprising for the iodophor)  I wonder if the spore characterisitcs of the samples you took allowed the bacteria to wait out the sanitizers?

I know what Star San does to my hands after using it without gloves for long periods of time… so I suspect it is somewhat effective on spores… iodophor and Ethanol… not so sure about them.

Regardless…

Your plating idea has kind of cemented an idea I had to evaluate the cleaning practices in the brewery.  … Take some beer / wort samples and streak plates to evalute the resultant colony formation.

… so that brings me up to a couple questions that you may be able to help me with…

1.) Where did you source your plates?  I just started looking into making plates and I think having pre-poured plates would be a better starting point as I work through the mechanics.  (kind of like doing extract before all grain to get the basics down)  I know there are diferent agar solutions depending on what you are after, but finding a set of pre-poured plates would be a good way to get started into this practice.

2.) Is there any recomendations on a microscope to obtain?  (I have scoured the forums and not seen information on exactly what magnification is adequate for evaluating bacteria vs yeast… seems like the info I find is more geared toward cell counts.  Any places to read or articles to scoop up would be great)

Thanks again for the info!

4

Great info - thank you for posting.

5

That was really cool! Thanks!

6

Good to know, thanks!  I’ll continue using PBW/Star San with confidence.

7

Things like this are what makes this the best forum on the web.  I learn something every time I click here, thanks for the information.

8

Excellent demo!

9

The results are not useful because the test is horribly flawed. The flaw is not the organic load.  Star San provided a vicious barrier between the media and the microflora during streaking, resulting in a much lower cell count transfer.  That barrier also prevented the cells that managed to get transferred from the loop to the plate from getting to the nutrient source, an the phosphoric acid weakened and already weak culture.  The other sanitizers do not produce a film, alcohol has a high flash rate, and iodine sublimates quickly into gas when there is only a thin layer on the surface of an item (sublimation is why iodophor solution turns clear).  A better test would have been to streak first, allow CFUs to form, carefully overlay santizers on top of the colonies, allow the plates to sit for the santizer used per plate’s stated kill time, and then test for CFU viability. That’s the way that sanitizers are normally used and tested.

I am willing to bet there there are still viable yeast colonies on the Star San plate that did not form visible CFUs.  If you still have the plates, perform an aseptic transfer from the Star San plate to an another blank plate. Streak only the first quadrant from the Star San plate, and then perform overlapping dilution streaks in the other three quadrants.

10

Mark, by that logic, wouldn’t that same viscous barrier that Star San creates on our fermenters afford us the protection that it clearly afforded Eric’s agar plates ?

11

A microscope is nice to have, but it is one of least useful things in a home brewing lab.  Microscope is really only useful for performing cell counts and viability testing (both of which are more important to commercial brewers than home brewers).  Most microbiology work is performed via plating onto various types of media that suppress the growth of one microorganism while allowing another to grow, or using a dye to differentiate different between microorganisms.  For example, WLN uses bromocresol green.  Different microflora take of the dye differently, which is also useful when determining if one is working with a multi-strain yeast culture.  WLD is WLN with cycloheximide added.  WLD is useful when counting bacteria CFUs on a plate, which will give one an idea of the level of bacteria contamination in one’s culture.  Lin’s Wild Yeast Medium and Lin’s Cupric Sulfate Medium are used to identify wild yeast.

12

No, for a couple of reasons.  The surface of the plate was aseptic (absolutely sterile) before the sanitizer was applied.  I have streaked enough plates to know that a wet surface generally makes cell transfer more difficult, which is one of the reasons why I am careful to control condensation when pouring plates.  Add in the fact that Star San is slippery, and the friction coefficient goes down even further.  Now, add in a non-evaporating barrier that contains phosphoric acid, and any cells that managed to get transferred were further weakened.

Now, the big difference between Eric’s test and a fermentation vessel is that the residual film was not diluted by several gallons of nutrient rich medium.  The wort buffers the pH up, allowing any microflora that managed to make it through the sanitization step to grow.  Most yeast strains are not going to grow very quickly at 2.9 or lower pH.  In fact, one of the risks that one takes when acid washing yeast is that the culture is usually weakened afterwards.  The phospohic acid in Star San is the only deterrent to yeast growth, as the killing agent (the surfactant) relies on electrical charge differences to enter a cell.  Star San is negatively charged (the reason why Star San is known as an acid-anionic sanitizer).  Yeast cells are also negatively charged.  To kill yeast cells, the polarity of the surfactant would have to be changed to cationic.  That’s how quaternary ammonium compounds (quats) work.  Quats are cations.

As I mentioned earlier, a more conclusive test would be to inoculate and incubate the plates before overlaying the colony-forming units (CFUs) with sanitizer.  Sanitizers are not applied to sterile surfaces in a brewery.  They are applied to non-sterile surfaces.  A sanitizer cannot rely on pH to control yeast growth because the residual film will be rinsed away and diluted by a much larger volume of liquid with a higher pH.

As an aside, finings also take advantage of cellular charge.  Finings used to clear beer of yeast hold a positive charge.  Finings used to beer and wine of bacteria hold a negative charge.

13

Eric streaked the plate before before he applied the sanitizer.

14

I agree, Eric streaked prior to sanitizer application and the test seems to represent a condition that may be similar to actual conditions. I’m not sure I see the flaw that Mark points out. However, I still agree with Mark’s other contention that StarSan may not provide all the broad spectrum sanitation that we brewers should have on occasion.

15

I would have thought a better test would have been to streak some sterile ss coupons, apply sanitizer as per directions, then immerse the coupons in some sterile wort.  Then perhaps one could plate the wort after some amount of time?

16

And dried it.

17

Yes, but then Eric immediately applied sanitizer.  That’s not a valid test.  The low pH of the Star San solution prevented the culture from multiplying.  It did not necessary kill the yeast culture.  The real test is to streak the plate, incubate the culture until visible colony-forming units appear, overlay the colony with sanitizer, wait until the santiizer’s stated killing period has elasped, and then test for viability by restreaking another plate or staining yeast from the plate on a hemocytometer after diluting it with sterile water.  The ratio of dead cells to live cells is the only valid test for a sanitizer.

18

OK, Mark’s approach makes sense. Eric, get going!  :slight_smile:

19

While this may be true when you are only considering the definition of “sanitary”, I don’t think it really matters practically. If the star-san did the best job of getting rid of yeast and bacteria, I don’t care if it killed the cells or just washed them away, as long as the undesirable microbes are gone.

20

However, the test does not simulate actual conditions.  The test was flawed the moment that the surface of the plate was allowed to remain at Star San pH during the incubation period.  That’s not what happens when one sanitizes with Star San due to the pH buffering effect of adding a much larger volume of liquid with a higher pH to the Star San sanitized vessel.

The other plates tested the sanitizer’s ability to kill viable cells because the sanitizers have no residual effect.  Alcohol ceases to be effective the moment that it flashes off, and so does iodophor when the iodine sublimates into iodine gas.  Those two plates tested the sanitizers’ ability to truly kill vegetative cells because nothing was left to hinder the growth of cells after the sanitizers left the surface as gases.

One of the terminology problems that we routinely see in home brewing is the intermixing of the terms sanitation and sterilization.  The goal of a sanitizer is not kill every vegetative cell.  The goal of a sanitizer is to kill most of the vegetative cells.  True sterilization kills 100% of the vegetative cells and 100% of the spores that can germinate into vegetative cells. Boiling is in between sanitizing and sterilizing in that it kills 100% of vegetative cells, but does not kill spores.

Let’s look at a commercial sanitizer for one’s home; namely, Lysol.  It’s maker claims that Lysol kills 99.9% of viable cells.  It only takes one viable cell to produce a colony-forming unit (CFU), which is why CFUs are known as pure cultures.  That claim means that we are almost guaranteed to have one CFU form if the sample that we use to streak a plate contains at least 1,000 viable vegetative cells.  A  CFU contains a large number of cells.  A Saccharomyces cell is 5 to 10 µm in diameter, which means yeast cells are at least 1/5th of the smallest size that is visible to the naked eye.

While yeast cells will not replicate very well at Star San solution pH, it does not mean that they are dead.  A yeast culture can contain a bacteria load.  Yet, the bacteria load will not rear its ugly head as long as the pH remains below 4.6, which is the pH at which many bacteria species stop replicating (one of the reasons why we do not want to rinse yeast with and store it under boiled water is because doing so raises the pH of the culture).  Yeast is also susceptible to pH-based growth limiting.  It’s just that domesticated brewer’s yeast can withstand a lower pH than most bacteria species.  Anyone who has made a batch of mead where the pH dropped into the low threes has noticed sluggish to non-existent fermentation. The yeast cells are not dead.  All one needs to is to buffer the pH of the mead back up to over 3.5 or so, and the fermentation will usually restart.

I shot the photo shown below.  It is a batch of Star San that I made with water that contains 130ppm as CaCO3 alkalinity. Yet, the pH is way below the pH at which most brewer’s yeast will replicate or even function, and the pH of Star San that is made with distilled water is even lower (< 2.0).

CloudyStarSan_zpsdd6f365b.jpg