So, a little while ago, I posted about an experiment I was developing as a STEM education program aimed at high school students…
And balloons. 🎈
And I called them the Yeasty Bois.
If you didn’t catch that post, I recommend reading it first before reading this one.
I mentioned in the previous post that you can add a twist to the typical ‘yeast balloon experiment’ to explore common household items that slow or inhibit yeast growth.
And that’s basically what I’ve been trialling, these last few weeks.
But first, I couldn’t quite remember how to best set up the standard experiment. The notes I had on hand also had different figures for some of the measurements, so I went back to basics and started off by trialling different amounts of water, yeast, and sugar.
And because the only difference between science and mucking around is writing stuff down, I made notes about each attempt. 😂
Attempt 1: Standard control as per current protocol (that I had on hand).
- 250 ml 40 degree (Celsius) water, 1 tsp sugar, 2 tsp yeast
- No gas
- Yeast in sachets may be old (should be stored in freezer in future, once opened)
- Balloon inflated at 40 min (but no where near enough)
New can of yeast opened and used subsequently
Attempt 2: Standard control with 1 tsp yeast (half yeast) (I’d already started this before Attempt 1 was done 😅)
- Less clumping of yeast
- Balloon did not inflate at all (beyond 30 min)
Attempt 3: Standard control as per current protocol (with fresher yeast)
- Balloon inflated at 32 min but was very small in size
Attempt 4: Standard control with 300 ml 40 degree water (more water)
- Balloon was inflated more but was still not enough at 30 min
Attempt 5: Standard control with 2tsp sugar (double the food)
- Not enough inflation at 30 min
Noticed at this point that unopened cans of yeast expired on 13/05/2022 (three days prior) 😅😂
Attempt 6: Standard control with 300 ml 40 degrees water (more water), 2tsp sugar (double)
- Not enough inflation at 30 min
Attempt 7: Standard with 1 tbsp sugar (triple)
- Not enough inflation at 30 min
Attempt 8: Standard with 300 ml 40 degrees water (more water), 1 tbsp sugar (triple)
- Not enough inflation at 30 min
Now, at this point, some of you may be able to see that some of those balloons look inflated enough. It’s large enough that the circumference could be measured.
But the problem is, those are the balloons at 45 min+ after addition of yeast, with no growth inhibitors what so ever. What I wanted were large balloons at under 30 min. I had to make it better.
At this point, I actually went back to my blog post to check the measurements, and realised the major difference.
The yeast. I had it written down on the blog as one whole tbsp. Not 2 tsp.
So with that in mind, I continued.
Attempt 9: Standard with 1tbsp yeast (1.5 times more)
- Not bad, but could be bigger (~25cm at 30 min)
Attempt 10: Standard with 1tbsp yeast (1.5 times more), 2tsp sugar (double)
- Yeast sludge hits balloon before 30 min
- 25 cm at 20 min
- 30 cm at 30 min
- 37 cm at 35 min
Attempt 11: Standard with 1tbsp yeast (1.5 times more), 1 tbsp sugar (triple)
- Not as inflated at 30 min (22cm)
Overall summary notes:
- 300 ml water is too much (250 ml is the right amount in a 500ml bottle)
- 3 tsp sugar is too much (2tsp is best amount in the above conditions)
- More yeast = faster expansion of balloon
And so it turned out that attempt number 10 was the way to go!
Now, the reason why I wrote out my notes here isn’t to overwhelm you.
It’s just to show you the process of optimising something by tweaking different conditions and seeing how it impacts the system.
And that’s pretty much science in a nutshell.
First you start with standard, then you change certain parameters.
Change the yeast so it’s more fresh, because the old stuff might not be as active, and therefore won’t produce as much gas to inflate the balloon.
Add more sugar to see if it’ll encourage more growth (and therefore gas production). Add more water to change the water to air ratio in the bottle. Add more yeast to produce more gas…
All of those are different variables that I’m changing to see if I get a better result, and I hope you can see why I changed them (i.e. what underlying theories made me tweak them in the way that I had).
Now that I had an idea of what the standard protocol should be, I decided to trial the different growth inhibitors I wanted to use.
For each growth inhibitor I tested, there was a bottle each for:
- Standard conditions + 5 ml growth inhibitor
- Standard conditions + 10 ml growth inhibitor
- Standard conditions + 15 ml growth inhibitor
- Standard conditions + 20 ml growth inhibitor
And obviously there was a bottle with no addition of growth inhibitor (control)
What I wanted to see was the balloon going from big (30 cm circumference) for the control bottle, then with increasing amounts of inhibitor, that circumference getting smaller- because the inhibitor was slowing the growth of the yeast inside the bottle.
First up was household disinfectant (Pine-o-cleen).
Now, this appears to be doing what I want it to, but I still had a problem with it.
Even the balloon with the least amount of disinfectant (orange) looked too small. It meant that all the subsequent balloons had hardly inflated.
If I was a student that had set this up, and I came back to see this… I’d be a little disappointed. I’d want the balloons to be inflated a bit more- across the board.
So, what would you change, if you were in my situation?
Well, given I can’t pause for dramatic effect, just pretend that I did. 😅😂
But I just diluted the disinfectant by half with water. Because the problem was that it was so concentrated that even a small amount was inhibiting yeast growth too much.
Now that’s a whole lot better, visually.
Having one bottle out of five not quite as inflated is better than four out of five being shrivelled.
Next up was salt 🧂
Now, I wanted to try salt because it’s always been in my head that salt is inhibitory to yeast growth. Anyone who’s done some baking would have heard or read a lot of notes on how you have to add the yeast and keep it separate, or at least, away from the salt as much as possible. So I figured, it would just be super bad for yeast growth.
There was a solution of salt in water, so I added the same volumes as before and waited 30 minutes to see what would happen.
Not quite what I had in mind.
In fact, the addition of 5 ml stock salt solution was actually producing a larger balloon than without. 😅😂
I didn’t know what concentration of salt there was in the stock, so I made a 5% (w/v) solution and repeated the experiment.
Which was even worse! 😂😂😂
In hindsight, it made sense.
Salt has ions, and ions are great for metabolism.
But obviously only in small amounts- which this was.
And while that’s a great concept to explain to students, it’s not a simple thing to explain in the fifteen minutes we have to debrief.
So I’m going to ditch the salt for now and replace it with something less… complicated.
Now, I chose bleach because we use bleach solutions all the time in the lab to disinfect things.
Alcohol is useful to a certain extent, but when you’re dealing with genetic material (e.g. like when you’re extracting genetic material from a nose swab), alcohol just smears the genetic material across the surface and doesn’t actually get rid of it.
Bleach, on the other hand, degrades genetic material, and makes it fall to pieces. In essence, it nukes everything.
So I thought I’d give a 1/4 strength solution a go (diluted in water), and it looked pretty good…
But then I was absentmindedly looking at my jumper later on, and…
Yeah, I can’t have students going home with bleach stains on their expensive school uniforms!
So I’m going to ditch the bleach solution for now and try something else.
Don’t worry about the jumper- I wore it because it was okay to be damaged. 😅
I actually used normal dishwashing detergent to begin with, but it barely had an effect, so I ditched it (data not shown 😂).
It makes sense, in all honesty, because detergents are primarily used to lubricate a surface, so that you can physically remove germs using water and manual action. They’re not designed to ‘kill’ germs by contact.
But what about all those ‘kills 99.99% of germs’ handwashes??
So I did the same, and…
It’s not as drastic as the disinfectant- but it’ll do!
At this point I was just desperate for something that wasn’t going to bleach/damage clothing, so while it’s not the most dramatic of differences… it’s fine for an experiment like this.
It’ll be a nice discussion point, either way.
Vinegar is another common household item that’s long been used to inhibit microbial growth.
Think of all the preserves that are in vinegar (e.g. pickles). The very low pH, or acidic nature of vinegar (otherwise known as acetic acid), should inhibit yeast growth to some extent, because yeast (and many other organisms) prefer a neutral pH- something that isn’t too acidic or alkaline/basic.
So, I repeated the experiment using white vinegar, and…
Like it was planned!
Note the paper towel covering up all the liquid that spilt when I accidentally knocked over the 20 ml vinegar condition.
Always keep your workspace clear.
Or at least, make sure you can wipe it up quickly. 😂
Methanol (Methylated spirits)
I chose methanol as my last substance as a stand in for alcohol sanitisers. I would love to be able to use ethanol, but the idea of having somewhat drinkable alcohol available to underage students just seemed too… dicey. 😅
Isopropanol/isopropyl alcohol/rubbing alcohol will also suffice, here, but for some reason I find the smell of isopropanol more offensive than methanol, so… I just went with personal preference. 😂
Either way, when there was a massive hand sanitiser shortage here, and people were flocking to hardware stores to make their own batches, even the metho was completely gone, so.
Clearly it’ll do (in a pinch)!
So, I added the same volumes of straight up methanol from the bottle, and…
Again, couldn’t have asked for a more photogenic line up.
Now, given this program is on a very tight schedule, there’s not a lot of room to cover underlying theories in any great depth (as mentioned).
So with this in mind, there are some obvious gaps/limitations to the above methodology, that I won’t be able to address during the actual experiment itself.
I wonder if you can think of them, now?
Lets cover some of the really big ones.
Limitation number 1: Volume issues
I’ve shown above how I added increasing volumes of growth inhibitor to the corresponding bottle- the underlying theory being that, the more growth inhibitor I add, the smaller the resulting balloon.
But if I were to make the experiment better, what’s an obvious thing I should be doing to… say… the control bottle?
I’m adding more volumes of growth inhibitor/liquid to the other bottles, right?
So… what if the decreasing size of the balloon is also because, I’m adding extra liquid into the bottle? Especially given that 300 ml of water (instead of 250 ml) made the balloon smaller.
How could I address this, and eliminate this possibility from the equation?
Well, an easy solution is to basically get the maximum volume of growth inhibitor added (20 ml)…
And add the same amount to the control bottle…
But using water.
That way, the volume of the control bottle will be the same as the bottle with the maximum volume of growth inhibitor added.
Now, you could also go even further with this, and add 15 ml of extra water to the 5 ml, 10 ml of extra water to the 10 ml, and so on- so that all bottles essentially have 20 ml of extra ‘liquid’ added.
Limitation number 2: Temperature
Temperature is a really important factor when you think about microbial growth.
Most microbes that cause human disease have an optimum growth temperature around 37 degrees (human body temperature).
Optimum meaning that they grow best at that temperature. They can also grow outside of that temperature zone, just a little more slowly.
If it’s too cold, they tend to just go into stasis. Actually, this is a really good public service announcement (PSA) opportunity:
Most microbes (including those that cause foodborne illnesses – e.g. food poisoning, gastro) do not ‘die’ from being frozen.
I feel like a lot of people don’t know this. 😓
They just go into stasis, essentially. They’re alive, they just don’t grow as well.
But as soon as the temperature comes back up, they’ll become active again and start replicating as per usual. So please, don’t ‘sterilise’ foods/clothes/objects by chucking them in the freezer- it does not work.
What does work for sterilisation is heat.
Ever heard of pasteurisation? Well that’s the process of sterilising food by exposing it to heat- usually above 57 degrees Celsius for a period of time.
Why does that work?
Well, it’s because the basic building blocks of life (proteins, genetic material) start to fall apart at high temperatures. That’s why you boil things for 15 minutes or so to sterilise them. You can also do this to smelly dish cloths so you get more usage out of them.
I digress. 😅😂
So with temperature being such an important factor in optimum microbial growth…
What do you think of the experimental procedure/methodology, where I leave the bottles for 30 minutes at room temperature?
Sure, the water was 40 degrees…
But will it still be 40 degrees when we come back to measure the balloons?
Does it have to be at 40 degrees the whole time?
Well, for one thing, 40 degrees was chosen so that, by the time the yeast gets introduced to the water, everything will be around 37 degrees. At least, above 30 degrees, minimum. 40 degrees itself it a little too high for happy yeast growth.
Sure, I could do something to keep the bottles at a warmer temperature…
But it’s not really necessary to do that.
The balloons are big enough for this experiment. The largest circumference (30 cm) is the length of a standard ruler. The other balloons (with growth inhibitors) will be smaller than that.
If I kept the temperature more stable, the balloons would presumably get bigger- which would make it a little more finicky to measure. Not impossible, by any means- just finicky. Not what I want when we’re so short on time.
But there’s something else to keep in mind when we’re adding the growth inhibitors.
They’re all at room temperature (~22 degrees), so they’ll likely cool the water in the bottle, to an extent.
It’s not a large volume we’re adding (less than 10% of the total volume), but it’s still a variable that we could account for, if we were to be more precise.
So… how would you address this?
Well, we could keep the growth inhibitors at around 40 degrees, too? Although I’d recommend looking into the stability of each growth inhibitor at that temperature. Some of them might degrade (i.e. won’t be as effective) at such high temperatures.
We could use an incubator or water bath- some sort of heating device to keep the temperature of the bottle stable, too.
Those are some of the really obvious limitations, but can you think of any others?
Being able to critically analyse something so that you can identify issues and solve them… those are the traits that make a really good scientist.
No experiment is ever going to be ‘perfect’, but you can try to cover all bases by trying to:
- Alter the experimental approach to ‘fix’ the problem areas
- Do additional experiments that uses different methodologies to cover those ‘gaps’, and show the same result
All of those add robustness to your data- adding weight to your claims and making it more likely that what you’re seeing is ‘real’.
And that brings us to the end of this giant post!
If you want to give the experiments a go (especially with kids or teens), I highly recommend it. Again, everything is designed so that you can do the experiments with easy to purchase items from the supermarket.
I hope it sparks some sort of curiosity. ☺️🥼🧪⚗️🧬🔭🧫
A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬