How do you clean your coverslips?

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Wes
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How do you clean your coverslips?

#1 Post by Wes » Sat Jun 27, 2020 4:56 pm

Whenever I open a new pack of coverslips they are always dirty (mostly specks but sometimes residue as well). Normally I'd give them an alcohol wipe and that's fine for wet mounts.

Sometime ago I made attempts to arrange diatoms on adhesive-coated coverslips. I cleaned them in 2M sodium hydroxide for a few hours, then rinse extensively with DI water and eventually store them in ethanol. The coverslips were really clean, the only issue was that their thickness was hovering around 0,14-0,15 mm (it was not indicated but I suspect these are #1 thickness)

Now I tried the same procedure with high precision #1.5H coverslips from a different manufacturer and it seems that the sodium hydroxide significantly alters the glass surface. There is an irregularly distributed hazy fog-like layer. I have the feeling that the hydroxide is etching the surface, maybe destroying the nice finish (the longer I leave them in the hydroxide solution the more noticeable it gets). I don't know why it wasn't a problem with the previous coverslips, its likely different type of glass used to make them.

What protocol do you follow when you have to clean coverslips for more critical applications like darkfield microscopy or diatom arrangements? Boiling piranha mix is something I'd prefer not to deal with but it probably works best.

Hobbyst46
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Re: How do you clean your coverslips?

#2 Post by Hobbyst46 » Sat Jun 27, 2020 6:30 pm

1. NaOH definitely dissolves glass, to an amount that depends on the concentration of the NaOH and type of glass (and time and temperature).
2. 2M NaOH is certainly much too strong, for cleaning purposes. 0.1M is the strongest I would use - if use NaoH at all (see below).
3. NaOH is useful to decompose biological residues like fat, proteins. It dissolves some inorganic materials as well.
3. A strong acid would be a better choice than NaOH since it does not dissolve glass. Certainly, Pirana water would be among the best.
4. I WOULD NOT RECOMMEND ANY OF THE ABOVE FOR HOME USE. Rubbing them lightly between the fingers in a dish washing solution (Palmolive or similar) works fine for me. Then DW and alcohol - in that order.
5. If the soap does not work, try maybe Barkeeper's Friend. In solution.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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mrsonchus
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Re: How do you clean your coverslips?

#3 Post by mrsonchus » Sat Jun 27, 2020 9:14 pm

Hi, this is an issue I've struggled with to varying extents for the 5 years or so that I've been making slides.
The haze that the dratted old and/or cheapo coverslips almost always seem to have to the effect of making them useless has plagued me also.

The sure(est)-fire and only way I've found to achieve near-perfect coverslips is to keep buying different sets until one box (or indeed many) proves to be (near) perfect. When it comes to cleaning slides, the same is to some extent true but not even close to being so critical. After all manner of different protocols for cleaning them I now have an acceptable and hands-free as it were method for slides.

I find that if a coverslip has haze - it's useless, no matter how thoroughly and carefully it is cleaned. If however a coverslip comes 'out of the box' without haze, regardless of any other dust etc, I find that hand-hot soapy-water (I use 'Fairy-Liquid' - a well known brand here in the U.K) is the best. Gently rubbing in this with a sponge then rinsing in COLD water (I always use tapwater but where I live, there's no residue from drying).

The amount of rinsing has proved to be the key - it's simply not the same to rinse a couple of times for perhaps 30 seconds each time, that is, until there's clearly no trace of soapy-water left. Intuitively they look clean, should be clean - but, they're not. Rinse for perhaps 2-3 minutes in a fast-flowing (fast not forceful) cold water source (tap for me...) with the glass almost parallel to the flow of the water (i.e. the incident-angle of water on glass is shallow).

Cloth, tissue or (non-yellow) flame dries them perfectly - followed with a brief 'puffer' (e.g. the 'rocket shaped' one) of both sides immediately before use. Standingthem up to dry never works for me.

I've had quite a bit of success with the 'look for then buy' branded coverslips regardless of age, as long as they're still in their original unopened packets/boxes-of-packets. They must however be cheao if there's very many of them as a lot in order to buy speculatively....

Here are some images - as you see, snapping-up boxes of old coverslips at great prices does tend to leave a little..er...extra-stock on my lab-shelves!
The following images vary from the size 2 25x25mm Cornings (great size & shape but their thickness (size 2 is nominally 0.19->0.25mm thick) is a great problem with my UIS2 40x 0.65n.a. objective). Below this power it's no real problem-factor - above I have a dry 60x with coverglass thickness collar that in practice eliminates the problem, and a 100x which is oil-immersion so (theoretically at least - I think..) has no problem. They're very good however for any type of temporary gel or wet mount observations.

Coverglass sizes,
Image

My 'coverglass mountain'...
Image

The size 2 25x25,
Image

Then I have some lovely Leica Surgipath size 1.5 24x50s - bought in sets of 2 or 3 boxes of 100 - a little large but useful as some of my (Botanical) slides have serial sections - these are, as are all of them I think, fit for use with automatic coverslipping machines, so slide apart perfectly when picking a single slip out of the box - never and stuck ones beyond the attraction of clean & super-smooth glass to itself.
Image

Then there are the Fisherfinest premium 24x50s of thickness size 1 - manufactured in 2005 but still perfect out of the box!
Image

Some Fisherfinest premium 24x30s of thickness size 1 manufactured 22 years ago - back in 1998!
Image
Just shows, age is not a guarantee of degradation with coverslips....

Marienfeld thickness size 1s in 22x22mm and 18x18mm (my favourite size I think)
Image

Image

I use a micrometer to measure slips when I'm mounting sections directly onto the coverslip - not just to find a 0.17mm thickness, but to enable the actual thickness of the coverslip to be included in the slide's label or notes, for the higher n.a. observations.
Image

Accurate & readable to about 0.001mm (marked to 0.01mm) - the image below may be read as 0.159mm as seen at this angle.
Image

Over 17000 coverslips all-in - but bought well and very, very nice to use. The resin-based mounting media that I use ('Omnimount' and latterly 'Histomount') run beautifully under these when mounting onto solvent-wet ('Histoclear' limone-based solvent) slides.

Apologies for this long post - I'll move it to it's own post if it's in the way - just let me know.
Last edited by mrsonchus on Sun Jun 28, 2020 7:44 pm, edited 1 time in total.
John B

MicroBob
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Re: How do you clean your coverslips?

#4 Post by MicroBob » Sun Jun 28, 2020 7:19 am

Nice mountain, John!😂
This discussion on glass surfaces would sound like whitch craft to many people. But yes, this really is a topic and a very interesting one too. I had lots of problems with slides, even not so old, newly bought brand slides. The surfaces were etched, probably by moisture dissolving sodium out of the surface. Cover slips are usually made from borosilicate glass and should be more resistant. Their hydrolysis class is better. It also was my impression that the slides suffer when in the package, but not so much as a finished slide in a box.

What I also find fascinating is the magic of glass surfaces. Two look alike but their surface acts different. This might also be the case with John's slide rinsing. The detergent might already be gone but a change in physical properties takes place.

Bob

Wes
Posts: 630
Joined: Sat Mar 09, 2019 12:58 pm

Re: How do you clean your coverslips?

#5 Post by Wes » Sun Jun 28, 2020 8:24 am

Thank you everyone for the information and ideas and special thanks to John for showing his mountain.
Hobbyst46 wrote:
Sat Jun 27, 2020 6:30 pm
1. NaOH definitely dissolves glass
A long time ago, I used to seed cells on coverslips for immunofluorescence and I clearly remember we used to clean the slips in 2M NaOH (or maybe KOH) and that allowed the cells to adhere really nicely to the slides. I wonder why we used this approach if strong hydroxides dissolve glass. Yesterday I ended up trying the home detergent approach and I did get nice and clean slides in the end, but there is probably an extremely thin layer of detergent stuck to the coverslips, I don't know if that would be a problem for diatom mounting but its certainly a problem for things like mass spectrometry (irrelevant in my case but I recall MS technicians complaining about detergent trace contamination on glass surfaces in the past).
mrsonchus wrote:
Sat Jun 27, 2020 9:14 pm
Apologies for this long post - I'll move it to it's own post if it's in the way - just let me know.
I really liked seeing your mountain so please keep it here. I noticed you have plenty of #1 coverslips, was there a special need for these? And out of curiosity did you buy the Marienfeld slips on Ebay? I've been trying to find a small package of round 15 or 18 mm #1.5H coverslips from that company but they sell packs of 1000 for 240 euro only.
MicroBob wrote:
Sun Jun 28, 2020 7:19 am
Nice mountain, John!😂
This discussion on glass surfaces would sound like whitch craft to many people. But yes, this really is a topic and a very interesting one too. I had lots of problems with slides, even not so old, newly bought brand slides. The surfaces were etched, probably by moisture dissolving sodium out of the surface. Cover slips are usually made from borosilicate glass and should be more resistant. Their hydrolysis class is better. It also was my impression that the slides suffer when in the package, but not so much as a finished slide in a box.

What I also find fascinating is the magic of glass surfaces. Two look alike but their surface acts different. This might also be the case with John's slide rinsing. The detergent might already be gone but a change in physical properties takes place.

Bob
Regarding the physical and surface properties of different glass types I found the following brochure from Schott to be quite interesting. Glass science has a come long way since the last century.

https://www.us.schott.com/d/tubing/ffed ... ses_us.pdf

Hobbyst46
Posts: 2927
Joined: Mon Aug 21, 2017 9:02 pm

Re: How do you clean your coverslips?

#6 Post by Hobbyst46 » Sun Jun 28, 2020 9:54 am

Wes wrote:
Sun Jun 28, 2020 8:24 am
Hobbyst46 wrote:
Sat Jun 27, 2020 6:30 pm
1. NaOH definitely dissolves glass
A long time ago, I used to seed cells on coverslips for immunofluorescence and I clearly remember we used to clean the slips in 2M NaOH (or maybe KOH) and that allowed the cells to adhere really nicely to the slides. I wonder why we used this approach if strong hydroxides dissolve glass. Yesterday I ended up trying the home detergent approach and I did get nice and clean slides in the end, but there is probably an extremely thin layer of detergent stuck to the coverslips, I don't know if that would be a problem for diatom mounting but its certainly a problem for things like mass spectrometry (irrelevant in my case but I recall MS technicians complaining about detergent trace contamination on glass surfaces in the past).
As said above, the extent of etching of the glass by NaOH depends on several variables.
I also, years ago, cleaned cover slips, especially slip-bottom petri dishes, with NaOH. NaOH efficiently hydrolyses biochemicals. IMHO, 0.1M (or 0.2M, at the most) for 1-2 h at room temperature is just fine to remove the remains of the cells (especially if they had been removed with trypsin). Thereafter, the slips were examined under brightfield and fluorescence and no objectionable effect was visible. I admit that I did not study the glass surface with any other sensor, such as SEM or AFM.
While many molecules are easily adsorbed on glass - for example stains, and NaOH itself - I think that detergents can be totally removed; immersion in vinegar followed by boiling in DW as a final cleaning step can be practiced. Member MicroBob has suggested flaming; I also found this method to be very effective, but it has to be very rapid, since the coverslips tend to either blow out or, worse, bend due to the heat.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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