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UNITED STATES EPARTMENT OF iOMMERCE

•UBLICATION Ir*^

NBS TECHNICAL NOTE 579

Testing of Cover Glasses for

Hemacytometer Chambers

U.S.

EPARTMENT

OF

COMMERCE

National

Bureau

of

. ^andards
NATIONAL BUREAU OF STANDARDS

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TiORAL BUREAU OF STANBUi

OCT 2 1971 \

UNITED STATES DEPARTMENT OF COMMERCE

Maurice H. Stans, Secretary

NATIONAL BUREAU OF STANDARDS • Lewis M. Branscomb, Director

ll

NBS TECHNICAL NOTE 579

ISSUED SEPTEMBER 1971

Nat. Bur. Stand. (U.S.), Tech. Note 579, 7 pages (Sept. 1971)

CODEN: NBTNA

Testing of Cover Glasses for

Hemacytometer Chambers

John S. Beers

Optical Physics Division

Institute for Basic Standards

National Bureau of Standards

Washington, D.C. 20234

NBS Technical Notes are designed to supplement the Bureau's regular publications program. They provide a means for making available scientific data that are of transient or limited interest. Technical Notes may be

listed or referred to in the open literature.

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, DC. 20402

(Order by SD Catalog No. C 13.46:579) Price 20 cents Stock Number 0303-0890
CONTENTS

Page

Introduction 1 Specification for Planarity 1 Test for Planarity 2 Equipment 2 Procedure in Testing 3 Interpretation of the Fringe System 3

General Rule for Counting Fringes 4

Prevention of Distortion 4

Determination of Limits 4

Availability of Additional Information 4

Appendix — An Alternate Interferometer 5
Testing of Cover Glasses For Hemacytometer Chambers

John S. Beers

If cover glasses used with hemacytometer chambers are not sufficiently flat, a volume error, causing erroneous blood cell counts, may be introduced in the chamber. This document describes a method of testing cover glasses for conformity to a planarity specification.

Keywords: Cover glass, test method for; hemacytometer; interferometry; planarity.

Introduction

A typical hemacytometer, figure 1, is a single piece

of glass approximately 7 centimeters long, 3.5 centimeters wide and 0.5 centimeter thick. Four transverse grooves are cut in the glass as shown in the side view

leaving three raised lands. A longitudinal groove

bisects the center land and the two center lands thus

created are ground down, lapped optically flat, and a 1 millimeter square ruled on each. The two lands adjacent to the ruled areas form the cover glass supporting surfaces. They are ground down so their upper sur- faces he in a plane precisely 0.1 millimeter above the

ruled areas. Blood, accurately diluted in a pipette, is in- troduced into the chamber between the cover glass and

the ruled surface, and the number of corpuscles

present in the 0.1 cubic millimeter volume bounded by

rr #

TOP VIEW

^COVER GLASS

FIGURE

SIDE VIEW

HEMACYTOMETER ft COVER GLASS

the cover glass and the 1 millimeter square ruling are counted with the aid of a microscope.

An important dimension in a hemacytometer

chamber is the depth from the plane of the surfaces

supporting the cover glass to the plane of the ruled sur- face, measured at the center of the ruling. If the cover

glass used with the chamber has a curved surface, the

portion of the cover glass directly over the center of the

rulings will not, in general, coincide with the plane of the supporting surfaces. Hence, a volume error is in- troduced in the chamber. The planarity specification

for cover glasses limits one aspect of the volume error of chambers when in use. The purpose of this document is to describe a method for testing cover glass

planarity. This method used at the National Bureau of Standards has proven to be practical, relatively simple,

and accurate enough for its purpose.

Specification for Planarity

The specification for hemacytometer cover glasses,

reproduced here, was established at the National Bureau of Standards many years ago and was incorporated into Federal Specification DD-G-426.

Technological advances have substantially reduced the use of hemacytometer chambers. However, those that are used should meet the specifications. While the

specification refers to the National Bureau of Stan- dards precision seal to be marked on items which

comply, this practice is only in effect for each chamber

and its two accompanying cover glasses tested at NBS.

Testing large groups of cover glasses was discontinued

at NBS several years ago because manufacturers or in- spection facilities can easily perform this sorting operation. Those who test cover glasses should state that procedures described in this document were followed.

1
U. S. DEPARTMENT OF COMMERCE

National Bureau of Standards

washington

SPECIFICATIONS FOR HAEMACYTOMETER

CHAMBERS AND COVER GLASSES

CHAMBERS

1. The glass and the ruling of the chamber shall be free from visible defects interfering with the accurate use of the chamber.

2. The chamber shall be of the one-piece or solid type

in which both the cover-glass supports and the ruled

surfaces are integral with the glass slide.

3. The chamber shall be fully, correctly, and permanently marked on the glass slide with the nominal depth

and either the name of the type or ruling or the area of the

smallest squares, also the name of the manufacturer or responsible distributor.

4. The error in the depth of a chamber from the plane of the supporting surfaces for the cover glass to the ruled area shall not exceed plus or minus 2 percent (±0.002 mm

for a chamber having a nominal depth of 0.1 mm, and

±0.004 mm for one having a nominal depth of 0.2 mm). In

the case of a chamber having a nominal depth of 0.020 mm, the error in depth shall not exceed plus or minus 5 percent (±0.001 mm).

5. The error in the length of any side of a central 1-mm

ruling shall not exceed plus or minus 1 percent (±0.01

mm). In the case of a ruling not having a central 1-mm

ruling, the error in the length of any side of each of the NBS Form 80, Aug. 1, 1941

1-mm squares of which it is composed shall not exceed

plus or minus 1 percent (±0.01 mm). In the case of any

ruling not having a central 1-mm ruling and not subdi- vided into 1-mm squares, the error in the length of any

side shall not exceed plus or minus 1 percent. PLAIN COVER GLASSES

Cover glasses shall be free from visible defects and

shall be plane on both sides within 0.002 mm. REINFORCED COVER GLASSES

Reinforced cover glasses shall be free from visible defects and shall be plane within 0.002 mm over the

entire side not having the reinforcement. PRECISION SEAL

Each haemacytometer chamber and each cover glass which on test is found to pass the specifications is marked

with a National Bureau of Standards precision seal. A

polished surface on each slide suitable for the precision

seal about 5 mm in diameter is desirable. SERIAL NUMBER

It is recommended that each slide bear a permanent

serial number.

16—23908-1 OPO

Test for Planarity

Planarity testing of cover glasses uses the inter- ference of light waves. The interference fringes

produced when the cover glass is in contact with an optical flat and illuminated by quasi-monochromatic

light are evaluated to determine acceptability. The

cover glass surface being tested is the one in contact with the optical flat. A spectral light source is used having an intense radiation at one wavelength that predominates over the other radiations. Each of the

two sides of the cover glass is tested in turn. The observed interference fringes represent contour lines on

the cover glass surface in a manner similar to the contour lines on a topographic map.

Equipment

The following equipment is needed:

(1) several optical flats

(2) a monochromatic light source with power

supply

(3) a suitable housing for the above.

It is necessary to have at least three optical flats. Flats become scratched by burrs or sharp edges on the cover glasses and one or more reserves are needed.

Although glass optical flats can be used, those made of

fused quartz do not scratch as easily. Flats 7 to 10 centimeters in diameter are a convenient size. Flatness

within one fringe for the full diameter is sufficient for

this work. With this degree of planarity over the whole

surface the error over an area equal to that of a cover

glass is negligible. A further advantage of having at least three optical flats is that their planarity can be

verified. Two flats placed face to face and illuminated

by quasi-monochromatic light should produce a set of

parallel fringes. The curvature of these fringes is the measure of planarity. The third flat is then compared

with each of the first two to eliminate the possibility that the first comparison involved two surfaces of equal

but opposite curvature, a condition which would

produce straight, equally spaced fringes because points

along any single fringe are points of equal separation

between the two faces being tested. A helium discharge

tube is often used, however, a mercury discharge tube

is equally useful. In either case the lamp can be

operated from the usual 115V a.c. line by means of a

transformer. The interference fringes should be viewed

approximately perpendicular to the optical flat, that is, very nearly in a vertical direction, and a fairly uniform

illumination is desirable over the cover glass surface.

A housing similar to that shown in figure 2 will provide
LIGHT SOURCE

GROUND GLASS

COVER GLASS

OPTICAL FLAT

4

14'

in iiiiii k——

SIDE VIEW FRONT VIEW

FIGURE 2

these conditions. Some optical supply houses and

precision tool manufacturers carry suitable equipment

for this test, and it may be unnecessary to construct a housing. For example, equipment for flatness testing of gage blocks can be used.

Procedure in Testing

(1) Cleanliness is of utmost importance. Clean the cover glass and the optical flat with alcohol, and dry

with soft, lint free paper toweling or cloth.

(2) Dust the flat and cover glass with a camel's hair

brush.

(3) Place the cover glass on the optical flat. A thin

film of air is formed between them and a system of in- terference fringes will be seen through the cover glass.

(4) Move the cover glass around under light vertical pressure until the fringe system is approximately symmetrical. Count and evaluate the fringes as described

below under sections headed "General Rule for Counting Fringes" and "Determination of Limits."

(5) Turn the cover glass over and repeat the process.

Interpretation of the Fringe System

(1) A series of fringes, parallel and very nearly

equally spaced, indicates a wedge-shaped film of air between the flat and the cover glass, the apex of the

wedge being parallel to the fringes. In figure 3, the apex

could be at the upper left or lower right corner. If only

a few of these parallel fringes are present, eight or less,

the cover glass side in contact with the optical flat is sufficiently plane. Otherwise, move the cover glass

around to see if the number of fringes can be reduced.

Brush again, if necessary, to remove dust particles.

(2) A series of concentric circles indicates that the

surface being tested is a portion of a sphere, the curved

surface being either convex or concave (figs. 4 and 5). When such a fringe system is seen, the cover glass

should, in preparation for counting, be moved around

until the fringe system is approximately symmetrical

about the center of the cover glass.

(3) A series of curved fringes, resembling families of hyperbolas, represents some sort of saddle-shaped sur- face (fig. 6). Again the cover glass should be moved

around until the best symmetry about the cover glass center is attained.

(4) A series of parallel fringes unequally spaced but

symmetrical with respect to one of the fringes in- dicates a portion of a cylindrical surface, but this is sel- dom seen in the testing of cover glasses (fig. 7).

(5) A complex fringe system with many closely

spaced fringes along one or more edges or corners but

not extending into the central portion of the cover glass

indicates either a raised or a rounded edge (fig. 8). Such

Fig. 3 Fig. 4

Fig. 5 Fig. 6

Fig. 7 Fig. 8

Fig. 9
a condition should be investigated carefully because

the optical flat can be very quickly scratched if the

edge is raised. Often the presence of the raised edge

can be detected by the fingernail. A cover glass with a raised edge should be rejected. Also any cover glass

with nicks along the edge should be discarded because

particles of glass may come out of the nicked edge and

scratch the optical flat or the hemacytometer chamber.

A simple rule for checking in such cases is that fringes

will move toward a "high" spot on a cover glass surface

when pressure is applied at that point. If the edge is rounded rather than raised and is limited to a distance

of about 1 millimeter, this effect is negligible as far as accuracy in use is concerned.

(6) A complex fringe system of parallel lines, circles,

and hyperbolas means that the surface is not a simple

geometrical shape (fig. 9). Its departure from planarity can, however, be estimated according to the general

rule for counting.

General Rule for Counting Fringes

The general rule is to count fringes along a line ap- proximately tangent to the center fringe in a direction

to cut the largest number of fringes. This will usually be

from the center toward one corner (fig. 4). If symmetry

cannot be attained by trial and further dusting then the

fringes must be counted in both directions along col- linear lines from the pattern center and then averaged

(fig. 6, with center at X). If the fringe system center is outside the cover glass area count the fringes along a

line cutting the greatest number of fringes (fig. 8).

Prevention of Distortion

When moving the cover glass around on the optical

flat, be careful not to exert pressure that will distort by

"wringing" the cover glass to the flat. This condition can be detected by a tendency of the cover glass to stick to the flat. Wringing distortion can be avoided by

insuring that the cover glass slides easily. Keeping the cover glasses and flats completely dry will also minimize this effect.

Determination of Limits

The effective wavelength of helium light is the yellow

0.588 micrometer spectral line, and for mercury light it is the green 0.546 micrometer line. Therefore, in passing from one fringe to the next, there is a dif- ference in separation of the surfaces of 1/2 of .0588 =

0.294 micrometer with helium light, and 1/2 of 0.546 =j

0.273 micrometer with mercury light. A tolerance of 2 micrometers is equivalent to 2/0.294 or 2/0.273 = 7

fringes. Hence, using the procedure outlined above,

whenever more than 7 fringes are intersected along a tangent to a centrally located symmetrical fringe

system, counting only between the center and the edge

or corner, the cover glass should be rejected. If the

fringe system is not symmetrical, count in both

directions along a single straight line through the

system center, and divide the total by two. The limit- ing number of fringes for the quotient is again 7.

Availability of Additional Information

The National Bureau of Standards will, on request,

furnish information about sources of supply for the

necessary equipment, or any other information that may be required.
Appendix

An Alternate Interferometer

A modification of the apparatus for cover glass test- ing is shown in figure 10. Scratching of optical flats is eliminated in this interferometer by supporting the cover glass on three matched steel spheres cemented

to the optical flat in a triangular configuration. The two

lenses cause light coming through the entrance aperture to fill a 5-centimeter circle on the optical flat. The

plate glass and plate glass mirror allow viewing the

VIEWING APERTUREPLATE GLASS MIRROR

PLATE GLASS

LENS No- I LIGHT SOURCE

ENTRANCE

APERTURE

LENS No- 2

3 SPHERES

fringes in a perpendicular direction to the optical flat. Two fringe patterns are often seen when inspecting a cover glass in this interferometer. One of these pat- terns results from interference between the two faces

of the cover glass and can be identified as the pattern

that remains when a slip of paper is inserted between

the cover glass and the optical flat. The pattern formed

between the bottom cover glass face and the optical flat

is the only one read.

BASEPLATE

SIDE VIEW

COVER GLASS

OPTICAL FLAT

3 POINT ADJUSTABLE

SUPPORT FOR BASEPLATE

Z-Z

FRONT VIEW

FIGURE 10

U.S. GOVERNMENT PRINTING OFFICE : 1971 OL—439-632
FORM NBS-114A (1-71) U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

1. PUBLICATION OR REPORT NO. NBS-TN-579

2. Gov't Accession No.3. Recipient's Accession No.

4. TITLE AND SUBTITLE

Testing of Cover Glasses for Hemacytometer Chambers

5. Publication Date September 1971

6. Performing Organization Code

7. AUTHOR(S) 8. Performing Organization

John S. Beers

10. Project/Task/Work Unit No. 2320190

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

11. Contract/Grant No.

12. Sponsoring Organization Name and Address 13. Type of Report & Period Covered Final

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.)

If cover glasses used with hemacytometer chambers are not

sufficiently flat, a volume error, causing erroneous blood

cell counts, may be introduced in the chamber. This document describes a method of testing cover glasses for

conformity to a planarity specification.

17. KEY WORDS (Alphabetical order, separated by semicolons)

Cover glass, test method for; hemacytometer; inter ferome try, planarity.

18. AVAILABILITY STATEMENT

FxH UNLIMITED.

I I FOR OFFICIAL DISTRIBUTION. DO NOT RELEASE

TO NTIS.

19. SECURITY CLASS

(THIS REPORT)

UNCLASSIFIED

20. SECURITY CLASS

(THIS PAGE)

UNCLASSIFIED

21. NO. OF PAGES

7

22. Price

20 cents

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