Hemacytometer Cover Glass

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GerryR
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Hemacytometer Cover Glass

#1 Post by GerryR » Sat Jan 07, 2023 11:59 pm

From what I have read, the cover glass for the standard Hemacytometer is 0.4mm. Most objectives are corrected for 0.17mm. Sooo, is it OK to use 0.17mm cover glass on the Hemacytometer. If not, why not?
Thanks for any input.
GerryR

Alexander
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Re: Hemacytometer Cover Glass

#2 Post by Alexander » Sun Jan 08, 2023 1:11 am

To work properly your hemacytometer needs its special cover glass. It fits the chamber, it is grounded flat for precision and it is stable enough not to be bent by the capillary force in the filled chamber. All those properties are needed to have a defined volume in the chamber. A standard cover glass will not work. Yes, image quality is somewhat reduced but a hemacytometer is used for counting, not for imaging. Objectives with cover glass correction ring will help with image quality issues, if there are any.

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Re: Hemacytometer Cover Glass

#3 Post by GerryR » Sun Jan 08, 2023 1:35 am

Alexander wrote:
Sun Jan 08, 2023 1:11 am
To work properly your hemacytometer needs its special cover glass. It fits the chamber, it is grounded flat for precision and it is stable enough not to be bent by the capillary force in the filled chamber. All those properties are needed to have a defined volume in the chamber. A standard cover glass will not work. Yes, image quality is somewhat reduced but a hemacytometer is used for counting, not for imaging. Objectives with cover glass correction ring will help with image quality issues, if there are any.
I didn't think about the capillary force bending the cover slip, thereby reducing the volume and messing up the count. Thanks for the input!

Tom Jones
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Re: Hemacytometer Cover Glass

#4 Post by Tom Jones » Sun Jan 08, 2023 3:44 am

The CV's of manual chamber counts are pretty high. There is a lot of variability in any manual process. Consistent mixing of the particles/cells in the liquid, consistent sampling technique, consistent loading volume, accurate dilution, are you counting a single type of particle or cell or are you trying to discern between multiple types, are the particles evenly dispersed or are some clumped? Lots of error potential.

I'm skeptical that capillary force will bend the cover glass and cause any change in the volume of the counting chamber, particularly given the counting area is open to air on four sides. Having the specimen lift the the cover glass and increase the chamber volume when the chamber is overloaded, yes, but now you've already screwed up your accuracy.

If anyone has a reference or two to the contrary I appreciate looking at them.

I'm pretty sure the main reason the cover glasses are thicker is that they are reusable and need to be cleanable, and they're heavy enough to not lift up when the chamber is overloaded a little.

dtsh
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Re: Hemacytometer Cover Glass

#5 Post by dtsh » Sun Jan 08, 2023 5:46 am

I'm with Tom Jones on this.
The proper coverglass is more resilient to damage and is easier to handle and clean due to it's size, but I don't think a 0.17mm coverglass would introduce any significant error in volume. I might have a pipette small enough to measure such small volumes; surely someone has and can verify?

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Re: Hemacytometer Cover Glass

#6 Post by GerryR » Sun Jan 08, 2023 1:57 pm

The "standard" Hemacytometer (Haemacytometer ?) cover glasses are 20x26x0.4 mm, and I noticed they are relatively expensive (4 per ~$20.00, 12 per ~$60.00, etc.) Well, I came across some No.1 thickness 20x26 mm cover glasses for an extremely low price, a 10 oz. unopened box for $12.00, and figured they would be good for standard mounting use, if not good for use with the Hemacytometer. I guess I can do a comparison as the Hemacytometer set I have came with 18 of the .4 mm cover glasses. Drawing a blood sample and doing a comparison count should give a relative degree of accuracy between the two cover glasses, though not statistically accurate without a large number of samples. Now all I need are some volunteers. :roll:
Thanks for all the inputs!

apochronaut
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Re: Hemacytometer Cover Glass

#7 Post by apochronaut » Sun Jan 08, 2023 5:03 pm

Since you are probably using an AO infinity system Gerry, the recommended AO objective for haemocytometer use was the cat.# 1116 45X .66 objective, which has a longer working distance than the cat. #1078 45X .66. When the infinity corrected system was introduced, it began with a numerically ordered set of objectives 1076 10X, 1077 20X, 1078 45X and 1079 100X. The 1078 had a .5mm w.d. and was originally recommended for the haemocytometer initially but very shortly after, the next infinity objective , cat.#1116 came out, also 45X .66 but wuth a .7mm w.d. Obviously the 1078 had some shortcomings during haemocytometer use and AO fast tracked a replacement. Since the 1078 was still produced for microscopy that required a .17mm corrected objective, I have always wondered if the #1116 might be corrected for a thicker cover?

A possible option would be to acquire a #1218 45X .66 w./ .4 -.8 correction collar , such as was made for the inverted scopes.

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Re: Hemacytometer Cover Glass

#8 Post by GerryR » Sun Jan 08, 2023 6:34 pm

I had read (somewhere?) that the 1116 objective was recommended for Hemacytometer use. I have a few of the 1116 objectives on different AO 'scopes. I will be experimenting in the near future to see the differences between the 0.4mm and 0.17mm cover glasses. I also have a 1238 45X objective coming for a phase 'scope I just bought, but can't find any info on the W.D. for it. I'm interested in seeing the differences from BF. I understand that Phase is used for platelet counting. Just experimenting and trying to teach this old dog something new. Again, thanks for all the info!!

Found this:
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GerryR
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Re: Hemacytometer Cover Glass

#9 Post by GerryR » Sun Jan 08, 2023 7:54 pm

If anyone is interested, I copied the AO hematological catalog out of the 1955 Red Book. Scanned the .jpg files and converted to .pdf. It is too large to post here, so if you are interested, PM me with your email, and I will send you a copy.

apochronaut
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Re: Hemacytometer Cover Glass

#10 Post by apochronaut » Sun Jan 08, 2023 10:47 pm

Somewhat like passing a joke around a campfire.
Actually, what you copied to the site is a copy I scanned from an original version of one of the first AO 10 reference manuals, which I still have. It has no date on it but the phone #'s for the regional branch offices in the U.S. are alpha-numeric but the Toronto phone # is numeric only. So on that basis I put a date on it of 1963. I emailed it to Tom Woods upon request, who in turn sent it on to Steve Neeley, which then was credited to Tom Woods on Steve's site. That scan has nothing to do with the 1955 Red Book. There was no series 10 in 1955. The 1955 Red Book probably doesn't even have the series 4 in it yet.
The reference to the # 1078 objective being suitable for haemocytometer work is because that is all they had at the time and it was barely acceptable. There would have been problems with floating and some degree of variability in certain objectives due to shimming. In the next catalogue that is extant, they have added the cat.# 1116 objective, with the same magnification, planarity and N.A. as the #1078 , just with a longer working distance. Why would they need two 45X .66 achromat objectives?
The 1078 objective was clearly not a great objective for the haemocytometer. They couldn't rely on guaranteed performance, so they quickly evolved the #1116, which boasted a .7mm working distance.

GerryR
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Re: Hemacytometer Cover Glass

#11 Post by GerryR » Mon Jan 09, 2023 1:04 am

apochronaut wrote:
Sun Jan 08, 2023 10:47 pm
......... That scan has nothing to do with the 1955 Red Book. There was no series 10 in 1955. The 1955 Red Book probably doesn't even have the series 4 in it yet.
.......
I got the scan about the 1078 off of Neeley's site. Slight misunderstanding, the scan I was referring to out of the 1955 Reb Book, in the above post, is the AO Hematological Catalog, which has a fair amount of information in it about the use of their Hemacytometer, but it was good to learn that the 1078 is not optimal for that purpose. I printed the 6 pages of the catalog and then scanned them to a .pdf file, if anyone is interested in a copy.

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Re: Hemacytometer Cover Glass

#12 Post by SuiGenerisBrewing » Mon Jan 09, 2023 1:03 pm

dtsh wrote:
Sun Jan 08, 2023 5:46 am
I'm with Tom Jones on this.
The proper coverglass is more resilient to damage and is easier to handle and clean due to it's size, but I don't think a 0.17mm coverglass would introduce any significant error in volume. I might have a pipette small enough to measure such small volumes; surely someone has and can verify?
Just did this now, using my labs "clinical grade" (read: overpriced) hemocytometer with its proper coverglass vs a #1.5 thickness 25x50mm. I did three separate counts of the same 1:100 dilution of THP1 (human monocytic cell) culture with each. Counted the four corners and calculated cell density from there:

Standard cover glass
  • Count 1: 1.11 x 10^6/mL
  • Count 2: 1.05 x 10^6/mL
  • Count 3: 1.07 x 10^6/mL
Average: 1.07 x 10^6/mL

#1.5 cover glass
  • Count 1: 9.53 x 10^5/mL
  • Count 2: 1.04 x 10^6/mL
  • Count 3: 9.98 x 10^5/mL
Average: 9.97 x 10^5/mL

So there is a bit of a difference, with the conventional coverslip under-counting by ~7%. On the scale of the 10 ul loading volume, that would be a 0.7 ul difference in internal volume.

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Re: Hemacytometer Cover Glass

#13 Post by Chas » Mon Jan 09, 2023 1:49 pm

What was the case might not be the case now; for example, I have an old Thomas Hawksley with 3 side by side platforms and that has a 2.5mm thick slip.

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Re: Hemacytometer Cover Glass

#14 Post by patta » Mon Jan 09, 2023 2:24 pm

Just a side warning, I've bought "0.4mm" coverslips a while ago from China, "Sail Brand" I think, they were very cheap but also quite crap: thickness random between 0,30 and 0,50mm, and every-single-one well dirty, I had to wash them. Maybe I was unlucky with a bad batch, I can't imagine a technician in an hospital using those things.
It was for use with the Nikon 20x 0.75 objective, no hematocytometer.
One plus of those thick coverslips is that they're quite robust, can be washed without breaking...
You could glue two 0.17 coverslips otherwise.

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Re: Hemacytometer Cover Glass

#15 Post by GerryR » Mon Jan 09, 2023 2:52 pm

SuiGenerisBrewing wrote:
Mon Jan 09, 2023 1:03 pm

.....So there is a bit of a difference, with the conventional coverslip under-counting by ~7%. On the scale of the 10 ul loading volume, that would be a 0.7 ul difference in internal volume.
Thanks for doing all that work!! That would seem to indicate that the thinner glass does have some flex in this application.

I'm not a doctor/lab tech, nor do I play one on TV, but from what I have read, even a 10% variance is not unusual when doing manual counts, even with the proper cover glass. If it is fairly consistent, i.e. always low, or always high, then it can be accounted for during evaluation. An article I read on a medical site (?) stated there are large variances between techs performing the same counts using the same samples and Hemacytometer, I'm guessing due to technique. So, that would tell me that certain results would warrant further testing with, perhaps, different methods.

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Re: Hemacytometer Cover Glass

#16 Post by GerryR » Mon Jan 09, 2023 2:55 pm

Chas wrote:
Mon Jan 09, 2023 1:49 pm
What was the case might not be the case now; for example, I have an old Thomas Hawksley with 3 side by side platforms and that has a 2.5mm thick slip.
Did you mean 0.25mm? 2.5mm seems rather thick.

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Re: Hemacytometer Cover Glass

#17 Post by Chas » Mon Jan 09, 2023 4:58 pm

Yes, 2.5mm :-)
However I recollect making casts of agar, for mounting and later observation/counting, using a counting chamber and a thick slab of glass like this, so perhaps this slip is for that purpose too (?)

It is a 'Hawksley' made Thoma style counting chamber ;-) !
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Last edited by Chas on Mon Jan 09, 2023 10:29 pm, edited 2 times in total.

Tom Jones
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Re: Hemacytometer Cover Glass

#18 Post by Tom Jones » Mon Jan 09, 2023 7:27 pm

GerryR wrote
An article I read on a medical site (?) stated there are large variances between techs performing the same counts using the same samples and Hemacytometer, I'm guessing due to technique. So, that would tell me that certain results would warrant further testing with, perhaps, different methods.
The usual reason (clinical) labs do, or did, manual cell counts, even knowing how variable the results are, and how damn long they take to do, is that generally there are no other methods available. That's either due to cost constraints, or at least a few years ago more likely lack of effective instrumentation. I can't tell you how elated hematology techs were at the advent of automated cell counts. Do a few manually diluted (Sahli mouth pipetted - nice and safe :shock: ) RBC, WBC and Platelet chamber counts and you'll understand what a horrible method it is. It's only relatively recently that cerebrospinal fluid and body fluid cell counts could be done on the available automation.

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Re: Hemacytometer Cover Glass

#19 Post by GerryR » Mon Jan 09, 2023 8:13 pm

Tom Jones wrote:
Mon Jan 09, 2023 7:27 pm
GerryR wrote
An article I read on a medical site (?) stated there are large variances between techs performing the same counts using the same samples and Hemacytometer, I'm guessing due to technique. So, that would tell me that certain results would warrant further testing with, perhaps, different methods.
The usual reason (clinical) labs do, or did, manual cell counts, even knowing how variable the results are, and how damn long they take to do, is that generally there are no other methods available. That's either due to cost constraints, or at least a few years ago more likely lack of effective instrumentation. I can't tell you how elated hematology techs were at the advent of automated cell counts. Do a few manually diluted (Sahli mouth pipetted - nice and safe :shock: ) RBC, WBC and Platelet chamber counts and you'll understand what a horrible method it is. It's only relatively recently that cerebrospinal fluid and body fluid cell counts could be done on the available automation.
I always thought the mouth pipetted method was, shall I say, not the most brilliant method ever devised. A simple hand vacuum pump, with release or stop valve, which has been available for decades, would have made the job of dilution much simpler and safer. In this case, even a syringe could be used to draw in the fluids to eliminate the safety issue. I expect many used that approach but never let it be known.

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Re: Hemacytometer Cover Glass

#20 Post by Tom Jones » Tue Jan 10, 2023 2:56 am

The hand-held pipette bulbs are good for larger volumes, but nothing this small and requiring this much control. The blood dilution pipettes use really small volumes of blood and diluent. I've never seen anyone use a bulb or syringe with one of these pipettes. It would be really difficult to do when using the pipettes to collect a drop of blood from a patient finger stick! :? I've done hundreds and hundreds of these counts. Fortunately this was pre-HIV, and not as much (identified...) hepatitis. No vaccines, no blood-borne pathogen regulations. No gloves either. Hepatitis was an occupation hazard for clinical lab folks.

Here's a primer on old-style hematology chamber cell counts: https://www.klimud.org/public/atlas/idr ... s/fund.htm

Even the pipette kits came with the little rubber hoses and mouth pieces.

Here's an image of an old, unused, Sahli pipette and Hemacytometer set from Clay-Adams I have in my display case. They were in common use until HIV reared it's ugly head. We carried the rubber hoses around in our lab coat pockets and reused them time and time again. :shock: The pipettes are wrapped in the tissue paper. Red is the RBC pipette and white is the WBC pipette. The instructions are for a hemoglobin method, not the chamber. Fun times, and most of us survived!
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