BDH

Certification No: 181320295
Order Ref: 25565
Date of Issue: 15 February 1995
Serial No: 181320295

CERTIFICATION OF CALIBRATION

The Weight(s) listed below have been compared with known standards of mass. The estimated uncertainties listed are expressed at 95% confidence level.
The value(s) stated is the hypothetical weight of density 8000 kg/m3 which balances the weight when compared in air density of 1.2kg/m3.
The measured values are derived via United Kingdom National Standards. Ref: NAMAS Laboratory 0134 Certified Standards, Certificate Numbers T08243 and T11002.
The weight(s) tested satisfy the accuracy requirements of OIML class F1 standards at the time of testing.
Calibration carried out in accordance with a quality system registered to BS EN ISO 9002:1994 Registration No.FS13125.

Signed

Approved Signatory

BDH Laboratory Supplies
Poole · BH15 1TD · England
Telephone: (01202) 660444
International: +44 1202 660444
Fax: (01202) 666856
Telex: 41186 and 418123 TETRA G

 COULTER

COULTER CALIBRATION STANDARD

BATCH

TYPICAL USAGE RATE:

30µm aperture,
50µm aperture,

CALIBRATION TRACEABILITY - QUALITY ASSURANCE:

ASSAY VALUES have been determined using COULTER COUNTER models MULTISIZER, TA II/PCA, ZM and CHANNELYZER 256 which are verified for performance and calibrated and checked with primary reference standard particles from the National Institute of Standards and Technology, N.I.S.T., (formerly National Bureau of Standards, N.B.S.), and the Community Bureau of Reference, B.C.R. (1).

Property documented records of the calibrations performed with this Coulter Calibration Standard, when used as recommended in the relevant Instrument Instruction Manual, will allow traceability of those calibrations to the N.I.S.T. (N.B.S.) and B.C.R. particle size Reference Materials, as required by some National authorities, such as BS 5750 and NAMAS in the United Kingdom.

CALIBRATION STANDARD SUSPENSION:
The suggested concerntration forboth accuracy and speed of calibration is at a concentration giving around 5% coincidence loss. Particles are suspended at a concentration in aqueous dispersant plus preservative such that the typical usage rate gives approximately 5% coincidence for calibration of an aperture ten times the latex's nominal diameter. Poly (styrenedivinyl benzene) (P.D.V.B) lattices are durable and will not change size upon immerion in most electrolyte solutions used with COULTER COUNTER instruments. Latices may swell or dissolve in some organic liquids, e.g. ketones, chlorinated hydrocarbons and some higher alcohols. With constant current COULTER COUNTER models, calibration may be performed in an electrolyte solution different from that used for sample analysis. There is no evidence of instability with time of Coulter Calibration Standards, as packed, but for maximum security it is recommended that the product is discarded at the expiry date given above.

SIZE CALIBRATION:
COULTER COUNTER instruments can be self calibrated by measuring a suspension of a narrow size range of the material under investigation at a known concentration. From the immersed relative density (g ml) of the material, and from the total volume measured, the calibration factor can be calculated for the COULTER COUNTER instrument and sensing aperture in use (2). This procedure will remove any doubt concerning accurate calibration with very irregularly shaped or conducting particles.  In practice, it is more convenient to calibrate using narrow range particles which have already been sized by other techniques. The most common method is to calibrate with spherical polymer latex particles; Secondary Calibration procedure of BS 3406: Part: 1983 (3).

Using a series of sensing apertures and latex particles. Harfield, Wharton and Lines (4) verified that the response of a COULTER COUNTER Model ZM was linear with particle volume to some 80% of the aperture diameter. By using a series of apertures and verified COULTER COUNTER models, each calibrated to the mode of the size distribution of the relevant N.I.S.T. (N.B.S.) Standard or B.C.R. Certified latex reference materials (1), this Coulter Calibration Standard has been assigned the values given above.

Instruments of high definition size distribution (e.g. MULTISIZER, CHANNELYZER models) are most conveniently calibrated using the number mode size (diameter or volume), see figure A above.

Single or double threshold instruments (e.g. Models A, B, D Industrial, ZB, and ZM without CHANNELYZER accessory) are more conveniently calibrated using the singlet number median diameter, see Figure B above.  The 'weight peak split' method (Figure C) is intended for calibration of TA and TAII Models without a population Count Accessory, whereby the calibration point is the junction of two consecutive channels containing equal weight (volume).  Earlier calibration methods for the TA or TAII used the 1:2 weight peak split method (Figure D) whereby the calibration point is the junction of two consecutive channels containing twice the weight (volume) in the right hand channel as in the left.  The 'number peak split' method (Figure E) is intended for the Model TAII with PCA or PCA 1 population count accessory.
  

CHOICE OF CALIBRATION STANDARD:
COULTER COUNTER instruments need only to be calibrated at one size level for each aperture, preferably between 5 and 20% of the aperture diameter, optimally 10 %, unless a specific calibration point is required by the user.

REDISPERSION OF CALIBRATION STANDARDS:
Coulter Calibration Standards contain both a preservative and a surface active agent specifically chosen to reduce any tendency of the particles to agglomerate or cake upon storage. Before use, shake, and then roll vial between the palm of the hands until any sediment is resuspended. Prepare calibration suspension by adding the appropriate number of drops (see Typical Usage Rate) to the electrolyte solution. Further dispersion by ultrasonic agitation may help, but should not be necessary. The calibration procedures recommended by Coulter Electronics Ltd. are in independent of minor changes in dispersion quality, as doublets, triplets etc. are excluded from the measurements. Stir the calibration suspension during the calibration procedure.

Dry calibration standards must be dispersed by first spatulating with a surfactant, such as Coulter Dispersant. Further dispersion with ultrasonic energy is recommended.

FREQUENCY OF CALIBRATION:
It is expected that the COULTER COUNTER instrument and aperture combination in use will not require calibration at intervals of less than one week so long as the aperture is kept clean. It is recommended that the calibration constant recorded to monitor any long term drift in the electronic components, and that recalibration is performed immediately after any servicing.

INDICATION OF PRODUCT INSTABILITY OR DETERIORATION:
Inability to obtain expected values may indicate product instability or deterioration. Particle suspensions should not be allowed to dry out; calibration powders must be kept dry until use. Discard diluted calibration standard after use.  If three or more consecutive calibrations fall outside your expected range, it may mean that the instrument is not operating properly or that the calibration standard is changing in one or more ways. The calibration of the COULTER COUNTER instrument SHOULD NOT BE CHANGED unless one or more of the following steps make it necessary:

(a) Check that the aperture is clear, and that the wafer and the aperture tube are clean and free from bubbles.
(b) Check the background count of electrolyte solution.
(c) Check the performance of you COULTER COUNTER instrument by using another batch of calibration standard suitable for that aperture tube.
(d) Consult your Coulter Service Representative.

HEALTH AND SAFETY:
COULTER CALIBRATION STANDARDS in suspension contain 0.1% sodium azide (Na N3) as preservative. Contact with heavy hetals may produce explosive deposits.
Avoid inhaling dry calibration standards.

REFERENCES:
1.     (a) SRM 1960, nominal 10µm latex; SRM 1961, Nominal 30µm latex;
        (National Institute of Standards and Technology; formally National Bureau of Standards), Gaithersburg, Maryland 20889, U.S.A.).
        (b) CRM 167, nominal 9.6µm latex; CRM 166, nominal 4.8µm latex; CRM 165, nominal 2µm latex (Community Bureau of Reference- BCR, 200 Rue de la Loi, BrusselsB-1049, Belgium).
2.     Theory of the COULTER COUNTER - Bulletin T-1; Coulter Electronics. Inc. 1957. (reprinted in most COULTER COUNTER instruction Manuals from Coulter Electronics    Ltd.)
3.     BS 3406: Part 5:1983. British Standard Methods for Determination of particle size distribution, Part 5, recommendations for electrical sensing zone method (the Coulter principle), 34pp
4.     Harfield J. G., Wharton R. T., and lines R. W., Part. Charact., 1, 32-34, 1984.

WARRANTY:
This Coulter Calibration Standard is intended for Laboratory use only, as described above, and by trained scientific or technical personnel. It is not intendedfor use in food, drugs or cosmetics, or for haematologu. The sizes quoted are the best obtainable according to the state of the art known to Coulter Electronics Ltd. at the time of assaying.

COULTER CALIBRATION STANDARD

Polymer latex particles intended for the calibration of COULTER COUNTER instruments. Other than those to be used for the red and white blood cell sizing or counting.

SINGLET NUMBER MEDIAN DIAMTER
(Non-multichannel instruments; Figure B).

5.24 µm DIAMETER  

WEIGHT PEAK SPLIT
(Models TA/TA II; Figure C).

5.22 µm DIAMETER

1:2 WEIGHT PEAK SPLIT
(Models TA/TA II; Figure D).

5.12 µm DIAMETER 

NUMBER PEAK SPLIT
(Models TA II/PCA (PCA 1); Figure E).

5.19 µm DIAMETER

TYPICAL USAGE RATE:

70µm aperture,

100µm aperture,

CALIBRATION TRACEABILITY - QUALITY ASSURANCE:
ASSAY VALUES have been determined using COULTER COUNTER models MULTISIZER, TA II/PCA, ZM and CHANNELYZER 256 which are verified for performance and calibrated and checked with primary reference standard particles from the National Institute of Standards and Technology, N.I.S.T., (formerly National Bureau of Standards, N.B.S.), and the Community Bureau of Reference, B.C.R. (1).

Property documented records of the calibrations performed with this Coulter Calibration Standard, when used as recommended in the relevant Instrument Instruction Manual, will allow traceability of those calibrations to the N.I.S.T. (N.B.S.) and B.C.R. particle size Reference Materials, as required by some National authorities, such as BS 5750 and NAMAS in the United Kingdom.

CALIBRATION STANDARD SUSPENSION:
The suggested concerntration forboth accuracy and speed of calibration is at a concentration giving around 5% coincidence loss. Particles are suspended at a concentration in aqueous dispersant plus preservative such that the typical usage rate gives approximately 5% coincidence for calibration of an aperture ten times the latex's nominal diameter. Poly (styrenedivinyl benzene) (P.D.V.B) lattices are durable and will not change size upon immerion in most electrolyte solutions used with COULTER COUNTER instruments. Latices may swell or dissolve in some organic liquids, e.g. ketones, chlorinated hydrocarbons and some higher alcohols. With constant current COULTER COUNTER models, calibration may be performed in an electrolyte solution different from that used for sample analysis. There is no evidence of instability with time of Coulter Calibration Standards, as packed, but for maximum security it is recommended that the product is discarded at the expiry date given above.

SIZE CALIBRATION:
COULTER COUNTER instruments can be self calibrated by measuring a suspension of a narrow size range of the material under investigation at a known concentration. From the immersed relative density (g ml) of the material, and from the total volume measured, the calibration factor can be calculated for the COULTER COUNTER instrument and sensing aperture in use (2). This procedure will remove any doubt concerning accurate calibration with very irregularly shaped or conducting particles.  In practice, it is more convenient to calibrate using narrow range particles which have already been sized by other techniques. The most common method is to calibrate with spherical polymer latex particles; Secondary Calibration procedure of BS 3406: Part: 1983 (3).

Using a series of sensing apertures and latex particles. Harfield, Wharton and Lines (4) verified that the response of a COULTER COUNTER Model ZM was linear with particle volume to some 80% of the aperture diameter. By using a series of apertures and verified COULTER COUNTER models, each calibrated to the mode of the size distribution of the relevant N.I.S.T. (N.B.S.) Standard or B.C.R. Certified latex reference materials (1), this Coulter Calibration Standard has been assigned the values given above.

Instruments of high definition size distribution (e.g. MULTISIZER, CHANNELYZER models) are most conveniently calibrated using the number mode size (diameter or volume), see figure A above.  Single or double threshold instruments (e.g. Models A, B, D Industrial, ZB, and ZM without CHANNELYZER accessory) are more conveniently calibrated using the singlet number median diameter, see Figure B above.  The 'weight peak split' method (Figure C) is intended for calibration of TA and TAII Models without a population Count Accessory, whereby the calibration point is the junction of two consecutive channels containing equal weight (volume).

Earlier calibration methods for the TA or TAII used the 1:2 weight peak split method (Figure D) whereby the calibration point is the junction of two consecutive channels containing twice the weight (volume) in the right hand channel as in the left.  The 'number peak split' method (Figure E) is intended for the Model TAII with PCA or PCA 1 population count accessory.

CHOICE OF CALIBRATION STANDARD:
COULTER COUNTER instruments need only to be calibrated at one size level for each aperture, preferably between 5 and 20% of the aperture diameter, optimally 10 %, unless a specific calibration point is required by the user.

REDISPERSION OF CALIBRATION STANDARDS:
Coulter Calibration Standards contain both a preservative and a surface active agent specifically chosen to reduce any tendency of the particles to agglomerate or cake upon storage. Before use, shake, and then roll vial between the palm of the hands until any sediment is resuspended. Prepare calibration suspension by adding the appropriate number of drops (see Typical Usage Rate) to the electrolyte solution. Further dispersion by ultrasonic agitation may help, but should not be necessary. The calibration procedures recommended by Coulter Electronics Ltd. are in independent of minor changes in dispersion quality, as doublets, triplets etc. are excluded from the measurements. Stir the calibration suspension during the calibration procedure.

Dry calibration standards must be dispersed by first spatulating with a surfactant, such as Coulter Dispersant. Further dispersion with ultrasonic energy is recommended.

FREQUENCY OF CALIBRATION:
It is expected that the COULTER COUNTER instrument and aperture combination in use will not require calibration at intervals of less than one week so long as the aperture is kept clean. It is recommended that the calibration constant recorded to monitor any long term drift in the electronic components, and that recalibration is performed immediately after any servicing.

INDICATION OF PRODUCT INSTABILITY OR DETERIORATION:
Inability to obtain expected values may indicate product instability or deterioration. Particle suspensions should not be allowed to dry out; calibration powders must be kept dry until use. Discard diluted calibration standard after use.  If three or more consecutive calibrations fall outside your expected range, it may mean that the instrument is not operating properly or that the calibration standard is changing in one or more ways. The calibration of the COULTER COUNTER instrument SHOULD NOT BE CHANGED unless one or more of the following steps make it necessary:

(a) Check that the aperture is clear, and that the wafer and the aperture tube are clean and free from bubbles.
(b) Check the background count of electrolyte solution.
(c) Check the performance of you COULTER COUNTER instrument by using another batch of calibration standard suitable for that aperture tube.
(d) Consult your Coulter Service Representative.

HEALTH AND SAFETY:
COULTER CALIBRATION STANDARDS in suspension contain 0.1% sodium azide (Na N3) as preservative. Contact with heavy metals may produce explosive deposits.
Avoid inhaling dry calibration standards.

REFERENCES:
1.     (a) SRM 1960, nominal 10µm latex; SRM 1961, Nominal 30µm latex;
        (National Institute of Standards and Technology; formally National Bureau of Standards), Gaithersburg, Maryland 20889, U.S.A.).
        (b) CRM 167, nominal 9.6µm latex; CRM 166, nominal 4.8µm latex; CRM 165, nominal 2µm latex (Community Bureau of Reference- BCR, 200 Rue de la Loi, BrusselsB-1049, Belgium).
2.     Theory of the COULTER COUNTER - Bulletin T-1; Coulter Electronics. Inc. 1957. (reprinted in most COULTER COUNTER instruction Manuals from Coulter Electronics    Ltd.)
3.     BS 3406: Part 5:1983. British Standard Methods for Determination of particle size distribution, Part 5, recommendations for electrical sensing zone method (the Coulter principle), 34pp
4.     Harfield J. G., Wharton R. T., and lines R. W., Part. Charact., 1, 32-34, 1984.

WARRANTY:
This Coulter Calibration Standard is intended for Laboratory use only, as described above, and by trained scientific or technical personnel. It is not intended for use in food, drugs or cosmetics, or for haematologu. The sizes quoted are the best obtainable according to the state of the art known to Coulter Electronics Ltd. at the time of assaying.

COULTER CALIBRATION STANDARD

Polymer latex particles intended for the calibration of COULTER COUNTER instruments. Other than those to be used for the red and white blood cell sizing or counting.

SINGLET NUMBER MEDIAN DIAMTER
(Non-multichannel instruments; Figure B).

10.0 µm DIAMETER

WEIGHT PEAK SPLIT
(Models TA/TA II; Figure C).

10.1 µm DIAMETER

1:2 WEIGHT PEAK SPLIT
(Models TA/TA II; Figure D).

9.8 µm DIAMETER

NUMBER PEAK SPLIT
(Models TA II/PCA (PCA 1); Figure E).

10.0 µm DIAMETER

TYPICAL USAGE RATE:

100µm aperture,

140µm aperture,

CALIBRATION TRACEABILITY - QUALITY ASSURANCE:
ASSAY VALUES have been determined using COULTER COUNTER models MULTISIZER, TA II/PCA, ZM and CHANNELYZER 256 which are verified for performance and calibrated and checked with primary reference standard particles from the National Institute of Standards and Technology, N.I.S.T., (formerly National Bureau of Standards, N.B.S.), and the Community Bureau of Reference, B.C.R. (1).

Property documented records of the calibrations performed with this Coulter Calibration Standard, when used as recommended in the relevant Instrument Instruction Manual, will allow traceability of those calibrations to the N.I.S.T. (N.B.S.) and B.C.R. particle size Reference Materials, as required by some National authorities, such as BS 5750 and NAMAS in the United Kingdom.

CALIBRATION STANDARD SUSPENSION:
The suggested concerntration forboth accuracy and speed of calibration is at a concentration giving around 5% coincidence loss. Particles are suspended at a concentration in aqueous dispersant plus preservative such that the typical usage rate gives approximately 5% coincidence for calibration of an aperture ten times the latex's nominal diameter. Poly (styrenedivinyl benzene) (P.D.V.B) lattices are durable and will not change size upon immerion in most electrolyte solutions used with COULTER COUNTER instruments. Latices may swell or dissolve in some organic liquids, e.g. ketones, chlorinated hydrocarbons and some higher alcohols. With constant current COULTER COUNTER models, calibration may be performed in an electrolyte solution different from that used for sample analysis. There is no evidence of instability with time of Coulter Calibration Standards, as packed, but for maximum security it is recommended that the product is discarded at the expiry date given above.

SIZE CALIBRATION:
COULTER COUNTER instruments can be self calibrated by measuring a suspension of a narrow size range of the material under investigation at a known concentration. From the immersed relative density (g ml) of the material, and from the total volume measured, the calibration factor can be calculated for the COULTER COUNTER instrument and sensing aperture in use (2). This procedure will remove any doubt concerning accurate calibration with very irregularly shaped or conducting particles.  In practice, it is more convenient to calibrate using narrow range particles which have already been sized by other techniques. The most common method is to calibrate with spherical polymer latex particles; Secondary Calibration procedure of BS 3406: Part: 1983 (3).

Using a series of sensing apertures and latex particles. Harfield, Wharton and Lines (4) verified that the response of a COULTER COUNTER Model ZM was linear with particle volume to some 80% of the aperture diameter. By using a series of apertures and verified COULTER COUNTER models, each calibrated to the mode of the size distribution of the relevant N.I.S.T. (N.B.S.) Standard or B.C.R. Certified latex reference materials (1), this Coulter Calibration Standard has been assigned the values given above.  Instruments of high definition size distribution (e.g. MULTISIZER, CHANNELYZER models) are most conveniently calibrated using the number mode size (diameter or volume), see figure A above.  Single or double threshold instruments (e.g. Models A, B, D Industrial, ZB, and ZM without CHANNELYZER accessory) are more conveniently calibrated using the singlet number median diameter, see Figure B above.  

The 'weight peak split' method (Figure C) is intended for calibration of TA and TAII Models without a population Count Accessory, whereby the calibration point is the junction of two consecutive channels containing equal weight (volume).  Earlier calibration methods for the TA or TAII used the 1:2 weight peak split method (Figure D) whereby the calibration point is the junction of two consecutive channels containing twice the weight (volume) in the right hand channel as in the left.  The 'number peak split' method (Figure E) is intended for the Model TAII with PCA or PCA 1 population count accessory.

 
CHOICE OF CALIBRATION STANDARD:
COULTER COUNTER instruments need only to be calibrated at one size level for each aperture, preferably between 5 and 20% of the aperture diameter, optimally 10 %, unless a specific calibration point is required by the user.

REDISPERSION OF CALIBRATION STANDARDS:
Coulter Calibration Standards contain both a preservative and a surface active agent specifically chosen to reduce any tendency of the particles to agglomerate or cake upon storage. Before use, shake, and then roll vial between the palm of the hands until any sediment is resuspended. Prepare calibration suspension by adding the appropriate number of drops (see Typical Usage Rate) to the electrolyte solution. Further dispersion by ultrasonic agitation may help, but should not be necessary. The calibration procedures recommended by Coulter Electronics Ltd. are in independent of minor changes in dispersion quality, as doublets, triplets etc. are excluded from the measurements. Stir the calibration suspension during the calibration procedure.  Dry calibration standards must be dispersed by first spatulating with a surfactant, such as Coulter Dispersant. Further dispersion with ultrasonic energy is recommended.

FREQUENCY OF CALIBRATION:
It is expected that the COULTER COUNTER instrument and aperture combination in use will not require calibration at intervals of less than one week so long as the aperture is kept clean. It is recommended that the calibration constant recorded to monitor any long term drift in the electronic components, and that recalibration is performed immediately after any servicing.

INDICATION OF PRODUCT INSTABILITY OR DETERIORATION:
Inability to obtain expected values may indicate product instability or deterioration. Particle suspensions should not be allowed to dry out; calibration powders must be kept dry until use. Discard diluted calibration standard after use.  If three or more consecutive calibrations fall outside your expected range, it may mean that the instrument is not operating properly or that the calibration standard is changing in one or more ways. The calibration of the COULTER COUNTER instrument SHOULD NOT BE CHANGED unless one or more of the following steps make it necessary:

(a) Check that the aperture is clear, and that the wafer and the aperture tube are clean and free from bubbles.
(b) Check the background count of electrolyte solution.
(c) Check the performance of you COULTER COUNTER instrument by using another batch of calibration standard suitable for that aperture tube.
(d) Consult your Coulter Service Representative.

HEALTH AND SAFETY:
COULTER CALIBRATION STANDARDS in suspension contain 0.1% sodium azide (Na N3) as preservative. Contact with heavy metals may produce explosive deposits.
Avoid inhaling dry calibration standards.

REFERENCES:

1.     (a) SRM 1960, nominal 10µm latex; SRM 1961, Nominal 30µm latex;
        (National Institute of Standards and Technology; formally National Bureau of Standards), Gaithersburg, Maryland 20889, U.S.A.).
        (b) CRM 167, nominal 9.6µm latex; CRM 166, nominal 4.8µm latex; CRM 165, nominal 2µm latex (Community Bureau of Reference- BCR, 200 Rue de la Loi, BrusselsB-1049, Belgium).
2.     Theory of the COULTER COUNTER - Bulletin T-1; Coulter Electronics. Inc. 1957. (reprinted in most COULTER COUNTER instruction Manuals from Coulter Electronics    Ltd.)
3.     BS 3406: Part 5:1983. British Standard Methods for Determination of particle size distribution, Part 5, recommendations for electrical sensing zone method (the Coulter principle), 34pp
4.     Harfield J. G., Wharton R. T., and lines R. W., Part. Charact., 1, 32-34, 1984.

WARRANTY:
This Coulter Calibration Standard is intended for Laboratory use only, as described above, and by trained scientific or technical personnel. It is not intended for use in food, drugs or cosmetics, or for haematologu. The sizes quoted are the best obtainable according to the state of the art known to Coulter Electronics Ltd. at the time of assaying.

COULTER CALIBRATION STANDARD

MATERIAL

TYPICAL USAGE RATE:

CALIBRATION TRACEABILITY - QUALITY ASSURANCE:
ASSAY VALUES have been determined using COULTER COUNTER models MULTISIZER, TA II/PCA, ZM and CHANNELYZER 256 which are verified for performance and calibrated and checked with primary reference standard particles from the National Institute of Standards and Technology, N.I.S.T., (formerly National Bureau of Standards, N.B.S.), and the Community Bureau of Reference, B.C.R. (1).

Property documented records of the calibrations performed with this Coulter Calibration Standard, when used as recommended in the relevant Instrument Instruction Manual, will allow traceability of those calibrations to the N.I.S.T. (N.B.S.) and B.C.R. particle size Reference Materials, as required by some National authorities, such as BS 5750 and NAMAS in the United Kingdom.

CALIBRATION STANDARD SUSPENSION:
The suggested concentration for both accuracy and speed of calibration is at a concentration giving around 5% coincidence loss. Particles are suspended at a concentration in aqueous dispersant plus preservative such that the typical usage rate gives approximately 5% coincidence for calibration of an aperture ten times the latex's nominal diameter. Poly (styrenedivinyl benzene) (P.D.V.B) lattices are durable and will not change size upon immerion in most electrolyte solutions used with COULTER COUNTER instruments. Latices may swell or dissolve in some organic liquids, e.g. ketones, chlorinated hydrocarbons and some higher alcohols. With constant current COULTER COUNTER models, calibration may be performed in an electrolyte solution different from that used for sample analysis. There is no evidence of instability with time of Coulter Calibration Standards, as packed, but for maximum security it is recommended that the product is discarded at the expiry date given above.

SIZE CALIBRATION:
COULTER COUNTER instruments can be self calibrated by measuring a suspension of a narrow size range of the material under investigation at a known concentration. From the immersed relative density (g ml) of the material, and from the total volume measured, the calibration factor can be calculated for the COULTER COUNTER instrument and sensing aperture in use (2). This procedure will remove any doubt concerning accurate calibration with very irregularly shaped or conducting particles.

In practice, it is more convenient to calibrate using narrow range particles which have already been sized by other techniques. The most common method is to calibrate with spherical polymer latex particles; Secondary Calibration procedure of BS 3406: Part: 1983 (3).  Using a series of sensing apertures and latex particles. Harfield, Wharton and Lines (4) verified that the response of a COULTER COUNTER Model ZM was linear with particle volume to some 80% of the aperture diameter. By using a series of apertures and verified COULTER COUNTER models, each calibrated to the mode of the size distribution of the relevant N.I.S.T. (N.B.S.) Standard or B.C.R. Certified latex reference materials (1), this Coulter Calibration Standard has been assigned the values given above.

Instruments of high definition size distribution (e.g. MULTISIZER, CHANNELYZER models) are most conveniently calibrated using the number mode size (diameter or volume), see figure A above.  Single or double threshold instruments (e.g. Models A, B, D Industrial, ZB, and ZM without CHANNELYZER accessory) are more conveniently calibrated using the singlet number median diameter, see Figure B above.  The 'weight peak split' method (Figure C) is intended for calibration of TA and TAII Models without a population Count Accessory, whereby the calibration point is the junction of two consecutive channels containing equal weight (volume).

Earlier calibration methods for the TA or TAII used the 1:2 weight peak split method (Figure D) whereby the calibration point is the junction of two consecutive channels containing twice the weight (volume) in the right hand channel as in the left.  The 'number peak split' method (Figure E) is intended for the Model TAII with PCA or PCA 1 population count accessory.

CHOICE OF CALIBRATION STANDARD:
COULTER COUNTER instruments need only to be calibrated at one size level for each aperture, preferably between 5 and 20% of the aperture diameter, optimally 10 %, unless a specific calibration point is required by the user.

REDISPERSION OF CALIBRATION STANDARDS:
Coulter Calibration Standards contain both a preservative and a surface active agent specifically chosen to reduce any tendency of the particles to agglomerate or cake upon storage. Before use, shake, and then roll vial between the palm of the hands until any sediment is resuspended. Prepare calibration suspension by adding the appropriate number of drops (see Typical Usage Rate) to the electrolyte solution. Further dispersion by ultrasonic agitation may help, but should not be necessary. The calibration procedures recommended by Coulter Electronics Ltd. are in independent of minor changes in dispersion quality, as doublets, triplets etc. are excluded from the measurements. Stir the calibration suspension during the calibration procedure.  Dry calibration standards must be dispersed by first spatulating with a surfactant, such as Coulter Dispersant. Further dispersion with ultrasonic energy is recommended.

FREQUENCY OF CALIBRATION:
It is expected that the COULTER COUNTER instrument and aperture combination in use will not require calibration at intervals of less than one week so long as the aperture is kept clean. It is recommended that the calibration constant recorded to monitor any long term drift in the electronic components, and that recalibration is performed immediately after any servicing.

INDICATION OF PRODUCT INSTABILITY OR DETERIORATION:
Inability to obtain expected values may indicate product instability or deterioration. Particle suspensions should not be allowed to dry out; calibration powders must be kept dry until use. Discard diluted calibration standard after use.  If three or more consecutive calibrations fall outside your expected range, it may mean that the instrument is not operating properly or that the calibration standard is changing in one or more ways. The calibration of the COULTER COUNTER instrument SHOULD NOT BE CHANGED unless one or more of the following steps make it necessary:

(a) Check that the aperture is clear, and that the wafer and the aperture tube are clean and free from bubbles.
(b) Check the background count of electrolyte solution.
(c) Check the performance of you COULTER COUNTER instrumentby using another batch of calibration standard suitable for that aperture tube.
(d) Consult your Coulter Service Representative.

HEALTH AND SAFETY:  
COULTER CALIBRATION STANDARDS in suspension contain 0.1% sodium azide (Na N3) as preservative. Contact with heavy hetals may produce explosive deposits.
Avoid inhaling dry calibration standards.

REFERENCES:
1.     (a) SRM 1960, nominal 10µm latex; SRM 1961, Nominal 30µm latex;
        (National Institute of Standards and Technology; formally National Bureau of Standards), Gaithersburg, Maryland 20889, U.S.A.).
        (b) CRM 167, nominal 9.6µm latex; CRM 166, nominal 4.8µm latex; CRM 165, nominal 2µm latex (Community Bureau of Reference- BCR, 200 Rue de la Loi, BrusselsB-1049, Belgium).
2.     Theory of the COULTER COUNTER - Bulletin T-1; Coulter Electronics. Inc. 1957. (reprinted in most COULTER COUNTER instruction Manuals from Coulter Electronics    Ltd.)
3.     BS 3406: Part 5:1983. British Standard Methods for Determination of particle size distribution, Part 5, recommendations for electrical sensing zone method (the Coulter principle), 34pp
4.     Harfield J. G., Wharton R. T., and lines R. W., Part. Charact., 1, 32-34, 1984.

WARRANTY:
This Coulter Calibration Standard is intended for Laboratory use only, as described above, and by trained scientific or technical personnel. It is not intendedfor use in food, drugs or cosmetics, or for haematologu. The sizes quoted are the best obtainable according to the state of the art known to Coulter Electronics Ltd. at the time of assaying.   

COULTER CALIBRATION STANDARD

Polymer latex particles intended for the calibration of COULTER COUNTER instruments. Other than those to be used for the red and white blood cell sizing or counting.

TYPICAL USAGE RATE:

CALIBRATION TRACEABILITY - QUALITY ASSURANCE:
ASSAY VALUES have been determined using COULTER COUNTER models MULTISIZER, TA II/PCA, ZM and CHANNELYZER 256 which are verified for performance and calibrated and checked with primary reference standard particles from the National Institute of Standards and Technology, N.I.S.T., (formerly National Bureau of Standards, N.B.S.), and the Community Bureau of Reference, B.C.R. (1).  Property documented records of the calibrations performed with this Coulter Calibration Standard, when used as recommended in the relevant Instrument Instruction Manual, will allow traceability of those calibrations to the N.I.S.T. (N.B.S.) and B.C.R. particle size Reference Materials, as required by some National authorities, such as BS 5750 and NAMAS in the United Kingdom.

CALIBRATION STANDARD SUSPENSION:
The suggested concentration for both accuracy and speed of calibration is at a concentration giving around 5% coincidence loss. Particles are suspended at a concentration in aqueous dispersant plus preservative such that the typical usage rate gives approximately 5% coincidence for calibration of an aperture ten times the latex's nominal diameter. Poly (styrenedivinyl benzene) (P.D.V.B) lattices are durable and will not change size upon immerion in most electrolyte solutions used with COULTER COUNTER instruments. Latices may swell or dissolve in some organic liquids, e.g. ketones, chlorinated hydrocarbons and some higher alcohols. With constant current COULTER COUNTER models, calibration may be performed in an electrolyte solution different from that used for sample analysis. There is no evidence of instability with time of Coulter Calibration Standards, as packed, but for maximum security it is recommended that the product is discarded at the expiry date given above.

SIZE CALIBRATION:
COULTER COUNTER instruments can be self calibrated by measuring a suspension of a narrow size range of the material under investigation at a known concentration. From the immersed relative density (g ml) of the material, and from the total volume measured, the calibration factor can be calculated for the COULTER COUNTER instrument and sensing aperture in use (2). This procedure will remove any doubt concerning accurate calibration with very irregularly shaped or conducting particles.  In practice, it is more convenient to calibrate using narrow range particles which have already been sized by other techniques. The most common method is to calibrate with spherical polymer latex particles; Secondary Calibration procedure of BS 3406: Part: 1983 (3).

Using a series of sensing apertures and latex particles. Harfield, Wharton and Lines (4) verified that the response of a COULTER COUNTER Model ZM was linear with particle volume to some 80% of the aperture diameter. By using a series of apertures and verified COULTER COUNTER models, each calibrated to the mode of the size distribution of the relevant N.I.S.T. (N.B.S.) Standard or B.C.R. Certified latex reference materials (1), this Coulter Calibration Standard has been assigned the values given above.  Instruments of high definition size distribution (e.g. MULTISIZER, CHANNELYZER models) are most conveniently calibrated using the number mode size (diameter or volume), see figure A above.  Single or double threshold instruments (e.g. Models A, B, D Industrial, ZB, and ZM without CHANNELYZER accessory) are more conveniently calibrated using the singlet number median diameter, see Figure B above.

The 'weight peak split' method (Figure C) is intended for calibration of TA and TAII Models without a population Count Accessory, whereby the calibration point is the junction of two consecutive channels containing equal weight (volume).  Earlier calibration methods for the TA or TAII used the 1:2 weight peak split method (Figure D) whereby the calibration point is the junction of two consecutive channels containing twice the weight (volume) in the right hand channel as in the left.  The 'number peak split' method (Figure E) is intended for the Model TAII with PCA or PCA 1 population count accessory. 
 

 
CHOICE OF CALIBRATION STANDARD:
COULTER COUNTER instruments need only to be calibrated at one size level for each aperture, preferably between 5 and 20% of the aperture diameter, optimally 10 %, unless a specific calibration point is required by the user.

REDISPERSION OF CALIBRATION STANDARDS:
Coulter Calibration Standards contain both a preservative and a surface active agent specifically chosen to reduce any tendency of the particles to agglomerate or cake upon storage. Before use, shake, and then roll vial between the palm of the hands until any sediment is resuspended. Prepare calibration suspension by adding the appropriate number of drops (see Typical Usage Rate) to the electrolyte solution. Further dispersion by ultrasonic agitation may help, but should not be necessary. The calibration procedures recommended by Coulter Electronics Ltd. are in independent of minor changes in dispersion quality, as doublets, triplets etc. are excluded from the measurements. Stir the calibration suspension during the calibration procedure.  Dry calibration standards must be dispersed by first spatulating with a surfactant, such as Coulter Dispersant. Further dispersion with ultrasonic energy is recommended.

FREQUENCY OF CALIBRATION:
It is expected that the COULTER COUNTER instrument and aperture combination in use will not require calibration at intervals of less than one week so long as the aperture is kept clean. It is recommended that the calibration constant recorded to monitor any long term drift in the electronic components, and that recalibration is performed immediately after any servicing.

INDICATION OF PRODUCT INSTABILITY OR DETERIORATION:
Inability to obtain expected values may indicate product instability or deterioration. Particle suspensions should not be allowed to dry out; calibration powders must be kept dry until use. Discard diluted calibration standard after use.  If three or more consecutive calibrations fall outside your expected range, it may mean that the instrument is not operating properly or that the calibration standard is changing in one or more ways. The calibration of the COULTER COUNTER instrument SHOULD NOT BE CHANGED unless one or more of the following steps make it necessary:

(a) Check that the aperture is clear, and that the wafer and the aperture tube are clean and free from bubbles.
(b) Check the background count of electrolyte solution.
(c) Check the performance of you COULTER COUNTER instrument by using another batch of calibration standard suitable for that aperture tube.
(d) Consult your Coulter Service Representative.

HEALTH AND SAFETY:
COULTER CALIBRATION STANDARDS in suspension contain 0.1% sodium azide (Na N3) as preservative. Contact with heavy metals may produce explosive deposits.
Avoid inhaling dry calibration standards.

REFERENCES:
1.     (a) SRM 1960, nominal 10µm latex; SRM 1961, Nominal 30µm latex;
        (National Institute of Standards and Technology; formally National Bureau of Standards), Gaithersburg, Maryland 20889, U.S.A.).
        (b) CRM 167, nominal 9.6µm latex; CRM 166, nominal 4.8µm latex; CRM 165, nominal 2µm latex (Community Bureau of Reference- BCR, 200 Rue de la Loi, BrusselsB-1049, Belgium).
2.     Theory of the COULTER COUNTER - Bulletin T-1; Coulter Electronics. Inc. 1957. (reprinted in most COULTER COUNTER instruction Manuals from Coulter Electronics    Ltd.)
3.     BS 3406: Part 5:1983. British Standard Methods for Determination of particle size distribution, Part 5, recommendations for electrical sensing zone method (the Coulter principle), 34pp
4.     Harfield J. G., Wharton R. T., and lines R. W., Part. Charact., 1, 32-34, 1984.

WARRANTY:
This Coulter Calibration Standard is intended for Laboratory use only, as described above, and by trained scientific or technical personnel. It is not intended for use in food, drugs or cosmetics, or for haematologu. The sizes quoted are the best obtainable according to the state of the art known to Coulter Electronics Ltd. at the time of assaying.

NIST

UNITED STATES DEPARTMENT OF COMMERCE
National Institute of Standards and Technology
Gaithersburg, Maryland 20899-

REPORT OF CALIBRATION
NIST Test No. 821/263573-00

For:     
            
            

Item:     
         

This stage micrometer was calibrated with the NIST Line Scale Interferometer (LSI). The LSI consists of a scanning electro-optical line detector, a high precision one-axis motion system, and a high accuracy heterodyne interferometer for determining the displacement of the test artifact beneath the line detector. The wavelength of a stabilized helium-neon laser corrected for temperature, humidity and atmospheric pressure is used as the length standard. The instrument is housed in an environmental chamber in which all environmental properties are carefully monitored. The complete description of the design and operation of the NIST LSI is given in the Journal of Research of the National Institute of Standards and Technology Volume 104, Number 3, May-June 1999, "The NIST Length Scale Interferometer".

Results of the calibration are given on the following pages of this report. Each length value is the mean of 4 measurements and the expanded uncertainty in each value is

U=ku_c

u_c = Ö (u_i² +u_j²)

Where u_i is the standard uncertainty arising from random effects and u_j is the standard uncertainty arising from systematic effects in the measurement process. The coverage factor k=2 was used which gives for each reported value a level of confidence of approximately 95 percent.

Measurements were made from line center to line center using a 0.16 mm segment of each.

NIST Test No. 821/263573-00
10 MM Stage Micrometer
Graticules, Ltd., NIST #5594
  

Graduation line midway between the line tips. During measurement the stage micrometer was placed on a flat surface.

During the measurement the environmental chamber and the artifact temperature were held within +/-0.005°C of 20°Celsius. All lengths are reported at a temperature of 20°Celsius (68°Fahrenheit). A coefficient of linear thermal expansion of 8.5 x 10-6 / °Celsius was used in normalizing the lengths to 20°Celsius.
  

Certified correct by

William B Penzes

 For the Director

Dr. Michael T. Postek, Group Leader
Nano-Scale Metrology Group
Precision Engineering Division
Manufacturing Engineering Laboratory

NIST Test No. 821/263573-00
10 MM Stage Micrometer
Graticules, Ltd., NIST #5594

     INTERVAL
  (MILLIMETER)

 LENGTH
    (mm)

CORRECTION
    (mm)

       ui
     (mm)

       uj
    (mm)

       U
    (mm)

0.000 TO 0.100

0.099837

-0.000163

0.000002

0.000000

0.000004

0.000 TO 0.200

0.199853

-0.000147

0.000003

0.000000

0.000006

0.000 TO 0.300

0.299811

-0.000189

0.000003

0.000000

0.000006

0.000 TO 0.400

0.399828

-0.000172

0.000003

0.000000

0.000007

0.000 TO 0.500

0.499904

-0.000096

0.000005

0.000000

0.000009

0.000 TO 0.600

0.600004

0.000004

0.000005

0.000000

0.000011

0.000 TO 0.700

0.700009

0.000009

0.000005

0.000000

0.000010

0.000 TO 0.800

0.800053

0.000053

0.000003

0.000000

0.000006

0.000 TO 0.900

0.900036

0.000036

0.000003

0.000000

0.000006

0.000 TO 1.000

1.000075

0.000075

0.000002

0.000000

0.000005

NIST Test No. 821/263573-00
10 MM Stage Micrometer
Graticules, Ltd., NIST #5594

0.000080

  NPL

NATIONAL PHYSICAL LABORATORY
Teddington Middlesex UK TW11 0LW Switchboard 0181-977 3222

CENTRE FOR MECHANICAL AND OPTICAL TECHNOLOGY

Certificate of Calibration

REFERENCE STAGE GRATICULE

 FOR

DESCRIPTION
The NPL reference stage graticule consists of a 25 mm x 75 mm glass slide bearing a design, in chromium, produced by direct copying of a master drawn using electron-beam lithography. The design consists of a central area, in the middle of which is the symbol "NPL", surrounded by the following four test areas.

1) A square grid comprising boxes varying in size from 25 µm to 400 µm. The numbers in the centre of the squares represent the nominal length of the sides of the square in which they appear.
2) A 20 x 17 array of spots of nominally the same 15 µm diameter.
3) A row of nine spots forming a log-normal distribution with diameters varying from 4.5 µm - 27 µm.

The uncertainties are for a confidence probability of not less than 95%.

DATE OF CALIBRATION 18 June 1997
IDENTIFICATION NPL Reference Stage Graticule 126
Reference: 08A038/970127/106-66

This certificate provides traceability of measurement to recognised national standards, and to the units of measurement realised at the NPL or other recognised national standards laboratories. This certificate may only be published in full, unless permission for publication of an approved extract has been obtained in writing from the Managing Director. It does not itself impute to the subject of calibration any attributes beyond those shown by the data contained herein.

NATIONAL PHYSICAL LABORATORY
Continuation Sheet

MEASUREMENTS
The grid was measured along all four of the 400µm sides with a length measuring microscope, using a 50x objective and a refractive index compensated laser interferometer fitted to the stage. The lengths were measured between the centres of the two parallel lines forming opposite sides of the grid.

The spots were measured using a microscope with a 100x objective and an image analyser. They were compared with spots measured using an image-shearing microscope, calibrated against an interferometrically measured stage micrometer. The diameters are calculated from the area of the spots and thus represent an average spot diameter.

RESULTS:
1) The lengths of the sides of the grid are 399.99 µm.
2) The monosize array has a mean diameter of 14.70 µm. The largest single spot is 14.84 µm and the smallest is 14.58 µm.
3) The diameters of the spots forming the root-2 array are:

NATIONAL PHYSICAL LABORATORY
Continuation Sheet

UNCERTAINTIES:
The uncertainty in the grid measurement is +/- 0.50 µm.
The uncertainty in the diameters of the spots forming the monosize array and the log-normal array is +/- 0.30 µm.
The uncertainty in the diameters of the spots forming the root-2 array is +/- 0.30 µm.
The uncertainty in the mean of the log-normal distribution is +/- 0.30 µm.

NOTES:
A guide to the practical use of the reference stage graticule and the formulae used to calculate the log-normal parameters are given in the enclosed sheets.

NPL

USE OF THE NPL REFERENCE STAGE GRATICULE

 1. THE GRID

The grid is used to calculate the image analyser and to detect any gross image distortion. An appropriate sized square should be imaged on the screen and the analyser calibrated across the square in the normal way. The squareness of the calibration can be checked by using the other two sides of the square (on some analysers calibration in a second direction is not possible and a variable frame can be used). Many image analysers can produce a software generated grid. If this grid is superimposed on the image of the graticule grid, any large-scale image distortions will become evident.

The monosize array is used to check for localised distortions of the image. The distortions are quite common at the edgeof the field of view and a knowledge of where and by how much the scaling breaks down allows determination of the usable measuring area of the image. The slide is positioned so that the 20x17 array of spots fill the screen. The height and width of the spots (usually feret 90 and feret 0 respectively) are then measured and either by printing out these sizes with the spot position or by labelling the image on the screen if this is possible, the deviation in the sizes of

2. THE MONOSIZE ARRAY

 

the spots at the edges can be seen by comparison with spots in the middle of the screen. Measurements must be made rather than direct visual observations because screen distortions may affect judgement.

3. THE ROOT-2 ARRAY

The root-2 array is used to determine the threshold level required to measure the spots correctly. The image analyser works by the user choosing a grey level at which anything darker is spot and anything lighter is 

background. As the edge of a spot is not a clean edge but a blurred one due to the limited resolution of the camera and the physical limits of optical microscopy, the specified threshold level affects the measured size of the spots. This array of spots provides a useful research tool for investigating the effects of background lighting, detect level, focus etc. on measured spot size.

The final test area is the log-normally distributed array. This is an idealised distribution of maximum dynamic range for a full screen and is used as a final check on the analyser when all other variables have been corrected for evaluated. The 100 spots should be arranged to fill the screen and are measured. Using the software that may be provided with the image analyser, the mean and standard deviation of the log-normal distribution can be determined and compared with the certified values. The mean and standard deviations have been calculated using the following equations.

4. THE LOG-NORMAL ARRAY

The equations that define the mean and standard deviation of the log-normal distribution are:
MEAN = esp

  STANDARD DEVIATION = exp

(

100

Where d_i is the diameter of spot number I.

Please contact NPL if you wish the log-normal parameters to be calculated using alternative formulae.

CENTRE FOR MECHANICAL AND OPTICAL TECHNOLOGY
NATIONAL PHYSICAL LABORATORY