Embedding cheese for slicing

Here you can discuss sample and specimen preparation issues.
Post Reply
Message
Author
SuiGenerisBrewing
Posts: 78
Joined: Tue Mar 29, 2022 10:39 pm
Location: Canada
Contact:

Embedding cheese for slicing

#1 Post by SuiGenerisBrewing » Wed Nov 09, 2022 7:01 pm

No, the title is not incorrect.

Aside from amature microscopy, another one of my hobbies is home cheese making. I've been capturing micrographs of some of the organisms found in cheese with some luck, and have found a stain that can be used to give good colour to bacteria and fungi without staining the cheese itself (aqueous methylene blue). I've also has some luck with Gram staining, although the proteins in the cheese stain quite darkly with the safarin.

Where I am running into problems is getting thin sections of the cheese, in the right orientation, without tearing/smearing the section. My goal is to get a "longitudinal" section that allows me to view the distribution of microbes through the "depth" of the cheese - e.g. a slice that allows me to see the spatial distribution of microbes in the cheese from the surface inwards. I don't have a proper microtome, and instead have one of the homemade versions that are essentially a large diameter bolt and nut that have been flattened on one face.

To achieve this, I need to align a small piece of cheese such that my microtome so that I get a cut along the right axis. I've been using a cheese trier to cut a "core" from the cheese. I then use a razer to cut the core such that it will fit sideways in the microtome, so that when I slice, the slice I get a section in the desired orientation. The issue I am having is holding that core still - it's impossible to cut a core that fits tightly enough into my "microtome" to be self-supporting, so I need some sort of material to embed in. I tried paraffin, but that melted and distorted the cheese. I tried "jamming" an over-sized piece of cheese into the microtome, which tore and distorted the cheese. Using pieces of carrots/etc as wedges damages the cheese.

So what I'm wondering is if anyone can think of an embedding material that may work for this? I need quite thin slices to make this work.

Image of a Gram stained cheese below, 1000x magnification, to give you an idea of my progress so far.
20221012_153933.jpg
20221012_153933.jpg (143.62 KiB) Viewed 1860 times

Leitzcycler
Posts: 254
Joined: Mon Sep 09, 2019 11:55 am

Re: Embedding cheese for slicing

#2 Post by Leitzcycler » Wed Nov 09, 2022 7:30 pm

Very interesting project indeed. Your staining looks absolutely great!

Just came to my mind if agar would work.

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

Re: Embedding cheese for slicing

#3 Post by ldflan » Wed Nov 09, 2022 10:07 pm

Maybe try loading the sample in your microtome as usual, then put the whole package in the deep freeze for a day or two, along with the razor. Then cut a few slices at a time, return it to the freezer to cool off, and repeat. If that proves too crumbly, then maybe some sort of embedding. For paraffin I think you'd need to remove the fat with xylene or something before infusing it...

Leitzcycler
Posts: 254
Joined: Mon Sep 09, 2019 11:55 am

Re: Embedding cheese for slicing

#4 Post by Leitzcycler » Thu Nov 10, 2022 10:31 am

If you are able to freeze your microtome, making cryo sections sounds an excellent method to try. Some thoughts:

You may freeze your microtome in the freezer (a few hours may be ok) and then try to keep it cold during your work by wrapping most part of it into some plastic foam. It would perhaps be beneficial if you could somehow increase the cold mass of the microtome e.g. inserting the screw into a massive steel block.

The razor blade reaches the room temperature in seconds so there is no use to put in into the freezer as it will get warm very soon anyway. Instead, you could try to use a cooling spray originally designed to be used to search bad components in electronic circuits. I think you will find it in the local hardware store. You can get down to -50C. You can then spray and keep your razor blade and sample cool during your work. I use this method regularly in many applications.

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

Re: Embedding cheese for slicing

#5 Post by ldflan » Thu Nov 10, 2022 2:23 pm

Leitzcycler wrote:
Thu Nov 10, 2022 10:31 am
The razor blade reaches the room temperature in seconds so there is no use to put in into the freezer as it will get warm very soon anyway.
The blade becoming too warm is a real issue. You could maybe also try putting a bunch in the freezer, and quickly use them one at a time immediately after taking each out. Or perhaps better would be a straight razor with replaceable blades -- you could try setting it on a block of dry ice between cuts, or place it in a bowl of dry ice and alcohol. I've not used the refrigerant spray, but have used liquid CO2 which would be a similar approach. Both are a bit expensive, especially the CO2 option as you would need a tank with a dip tube or way to invert the tank. They say the freeze spray chemical (R-134a or tetrafluoroethane) is better from a climate perspective than CO2, and they put it inhalers, so it's probably reasonably safe.

Leitzcycler
Posts: 254
Joined: Mon Sep 09, 2019 11:55 am

Re: Embedding cheese for slicing

#6 Post by Leitzcycler » Thu Nov 10, 2022 5:41 pm

Or you may construct a poor mans cryo microtome: buy a surplus ice cream cabinet and put your microtome and tools in there :)

SuiGenerisBrewing
Posts: 78
Joined: Tue Mar 29, 2022 10:39 pm
Location: Canada
Contact:

Re: Embedding cheese for slicing

#7 Post by SuiGenerisBrewing » Thu Nov 10, 2022 5:45 pm

Thanks for the ideas everyone. I've tried freezing the samples before, but still had issues with the section "shifting" in the microtome. Cheese becomes crumbly when frozen, so those shifts would cause large pieces to fall off. But I wonder if agar as an embedding medium (plus or minus freezing) may do the trick. I'm pretty sure if I can hold the cheese steady in the right orientation that I can get a good slice. It's just getting it to stay put that has been a PITA.

If I get anything good I'll be certain to post some photos on the forum.

thanks again

Bryan

Post Reply