Staining sourdough culture with iodine

Here you can discuss sample and specimen preparation issues.
Post Reply
Message
Author
kwesi
Posts: 28
Joined: Tue Aug 10, 2021 7:08 pm

Staining sourdough culture with iodine

#1 Post by kwesi » Sun Dec 05, 2021 4:37 am

Hello, I'm hoping someone can help me understand what I'm seeing. I took some sourdough culture consisting of flour, water, and wild yeast and bacteria. To the culture I applied dilute Lugol's iodine solution, added this in a thin film to the slide, added a drop of water, and mixed.
As you can see, most of the yeast cells are translucent, but some became quite opaque. The second photo is an image of the same culture with only water added, no iodine. All the yeast cells in that sample were translucent as expected. (Both images 400x magnification)
IMG_8269 copy.jpg
IMG_8269 copy.jpg (127.89 KiB) Viewed 3491 times
IMG_8261 copy.jpg
IMG_8261 copy.jpg (59.33 KiB) Viewed 3491 times
What I was expecting to get from the sample with the iodine, was that the yeast cells would remain translucent but some of the surrounding matrix would turn blue from the iodine reacting with the starch that's dissolved in the water. Instead some of the yeast cells have turned dark, which leads me to believe that they contain glycogen stores. I'm wondering what the difference is between the cells that appear to contain no glycogen and those that do? Does glycogen storage suggest that a cell is more metabolically active, or less?

Any help would be greatly appreciated! These are some preliminary experiments for my neighbour who is a baker, who will be commissioning me to take some photos of his sourdough starter to put up in his shop. I'd love to be able to explain the images I produce for him.

ldflan
Posts: 134
Joined: Wed May 22, 2019 11:36 pm
Location: Morna Moruna

Re: Staining sourdough culture with iodine

#2 Post by ldflan » Mon Dec 06, 2021 1:22 am

Never worked with yeast or looked into this, so just a couple of guesses -

First, are you sure everything imaged is yeast? Could any of it be flour or starch granules from the flour?

Second, my guess would be that if only some yeast are staining in the Lugol's, they are dead. It may be that the Lugol's in this case is acting as a vital stain. Living yeast have intact walls that keep it out, so you don't see any staining; dead ones have deteriorated cell walls, so the Lugol's can enter.

Interested to know what you figure out.

Leonard

Microscopy_is_fun
Posts: 130
Joined: Sun Nov 21, 2021 6:11 pm

Re: Staining sourdough culture with iodine

#3 Post by Microscopy_is_fun » Mon Dec 06, 2021 10:56 pm

Hi kwesi,

Very likely many of the "bubbles" you have in your images are not yeast. Yeast cells are very uniform in size. To better identify what you are seeing, I would do the following:
(1) Put a scale bar in your pictures to give viewers a better idea about the size of the objects.
(2) make a solution of sugar and yeast, one sample stained and one sample unstained. The sugar dissolves in water completely, and you will only see the stained/unstained yeast cells.
(3) make a formulation just with flour and make stained/unstained samples. Then you know how the flower/starch particles look like in your images.

apochronaut
Posts: 6272
Joined: Fri May 15, 2015 12:15 am

Re: Staining sourdough culture with iodine

#4 Post by apochronaut » Tue Dec 07, 2021 3:39 am

I agree with the above posts. The yeasts are the smaller ovoid clustered bodies. Some you can see budding. The larger bodies are starches.

chrisskee
Posts: 7
Joined: Fri Mar 08, 2019 4:42 pm

Re: Staining sourdough culture with iodine

#5 Post by chrisskee » Tue Dec 07, 2021 7:52 pm

Hi.Just I repeated your experiment with staining sourdough culture with iodine -only starch grains were stained dark,yeast and bacteria very lightly yellowish.It is very difficult to see unstained yeast between small starch grains and other particle under 400x.I had to use 1000x and oil imerrsion to differentiate yeast from other particles unless you make very fine smear,dry,fix and stain eg:gram stain or giemsa stain.I used both in the past with good result and examin the smear under oil imersion.
Greetings Chris

kwesi
Posts: 28
Joined: Tue Aug 10, 2021 7:08 pm

Re: Staining sourdough culture with iodine

#6 Post by kwesi » Fri Dec 10, 2021 3:18 am

Thanks for the input everyone, I think I mistakenly thought yeasts were more pleiomorphic than they are. I'm going to try to get a pair of linear polarizers which might help me differentiate the starch grains from the yeast cells better.
I'm still not totally sure why most of the starch didn't stain with the Lugol's, but I could keep tinkering and see what I can figure out.

ldflan
Posts: 134
Joined: Wed May 22, 2019 11:36 pm
Location: Morna Moruna

Re: Staining sourdough culture with iodine

#7 Post by ldflan » Sat Dec 11, 2021 1:23 am

If the large structures in your photo are just starch granules, then yes a pair of polarizers will show them when crossed as birefringent disks with a maltese cross interference pattern. If you can lay hands on a first order compensator (even a plastic one), you'll get some pretty interference colors as well. But if the large chunks in the picture are bits of flour, then the polarizers won't be much use. Hard to know what's going on without knowing the scale on your picture... If you could grow the yeast on a pure liquid diet or centrifuge it out and put it in water, or what have you, you might be able to tell how it reacts to the Lugol's.

Also, bear in mind that starch does not all react the same to Lugol's. The black/blue reaction is indicative of amylose (long chain starch). Amylopectin (branched starch) stains reddish brown.

kwesi
Posts: 28
Joined: Tue Aug 10, 2021 7:08 pm

Re: Staining sourdough culture with iodine

#8 Post by kwesi » Mon Dec 13, 2021 3:13 am

I'm kind of new to this - what's a compensator and how would I set my microscope up with one?

ldflan
Posts: 134
Joined: Wed May 22, 2019 11:36 pm
Location: Morna Moruna

Re: Staining sourdough culture with iodine

#9 Post by ldflan » Wed Dec 15, 2021 1:14 am

It's a piece of birefringent material that shifts the polarization state of light and "retards" the extraordinary ray a specified fraction or multiple of a wavelength (at a specified wavelength - for microscopy, usually green). The phase shift results in cancellation of some wavelengths when the two rays are interact, in effect imparting color by virtue of interference where there was none before. When the light is further modified by other birefingent materials in the sample, you get more distinctive interference colors. You would want a 1 lambda, full, or first order waveplate, most likely.

It's not easy stuff to get your head around, but the effects are easy to see in use. A good place to start is a decent book on optical mineralogy.

https://en.wikipedia.org/wiki/Waveplate

http://www.microscopy-uk.org.uk/mag/ind ... jcomp.html

Precision waveplates are cut from crystal minerals and fairly expensive. However, you can use a sheet of polycarbonate plastic of the right thickness if you don't need a lot of precision. There's a guy on ebay selling them laser cut from sheet polycarbonate in various diameters - $30 or so, I think, which is a lot considering it's just a piece polycarbonate, but on the other hand he has gone to the effort of cutting them to size and selecting the right plastic, so... Anyway I got one for a friend as a gift some months ago, and they do work pretty well. Better if you sandwich it between glass to keep it from getting too scratched up.

I don't know how your microscope is laid out. Assuming it's a simple compound microscope, you could get a plastic one big enough to cover the condenser.

If your scope has a slot for two filters between the head and the objectives, you can perhaps find a purpose-made one, but again they are often expensive. The slot for the waveplate is set at 45 degrees to the polarizer slot. It's mostly either pretty high end research stands that will have these two slots for a polarizer and retarder plate between the objectives and the head. All petrographic scopes will be set up to accept both. However, as I said for general use the retarder plate can be on the condenser side, too, so you don't actually have to have an extra filter slot for it if you can find a plate that is large enough to cover your lightsource at the condenser.

Hope that all makes some sense...

Leonard

Post Reply