- Email: [email protected]
Laboratory evaluation of the Coulter On-line particle Monitor
Powder Technology. 14 (1976) 125 - 130 0 Elsevier Sequoia S-A., Lausanne - Printed in the Netherlands
125
Laboratory Evaluation of the Coulter On-Line Particle Monitor
M. I. BARNETT Welsh School
and E. SIMS
of Pharmacy,
University
of Wales Institute
of Science and Technology.
Cardiff (Gt. Britain)
R. W. LINES Coulter Electronics
Ltd.. Harpenden.
(Received
16, 1975;
October
Hertz
(Gt. Britain)
in revised form November
SUMMARY
The On-line iMonitor system, consisting of a sampling device, control unit and digital printer, is used in conjunction with Model ZB or TA Coulter Counters and is designed primarily for the process control or constant monitoring of particles in fluid systzms. At selected time intervals, samples are taken and particle counts determined at preset size levels. A description of the Monitor and its modes of operation are given and some of its applications and limitations are discussed_
INTRODUCTION
The Coulter Counter@, through its various models, has become an established and well evaluated method of counting and sizing microscopic particles and cells [ 11. Increased interest in process control has led to the development of a fully automatic model, the On-line Monitor. This has been designed specifically to continuously monitor the numbers of particles present in an electrolyte solution at or between preset size levels. The types of application might include examination of sea water, parenteral fluids, plating solutions, cell growth media, crystal growth studies or levels of particulate pollution in river water. The instrument is shown diagrammatically in Fig_ l_ The sample is taken continuously from a suitable outlet from a production plant, or filtration system at pressures ranging from 0 to 345 kN mm2 (0.50 p.s.i.). Three aperture tubes are inset in a perspex block; ell have the same nominal aperture size and each one is
3, 1975)
provided with its own internal electrode, the external elec-crode being common. The sample is fed directly, or via a peristaltic pump, through a coil at the bottom of the unit and then into the sample cell. The action of passing the sample through the coil effectively degasses it so that air bubbles are excluded and not counted. Surplus sample is continuously exhausted through the top of the aperture block, to waste, via the regulator manometer. This system allows a representa-
tive sample of fluid to be present in the sample block. At pre-selected times a sample is allowed through any one aperture tube by opening the two solenoid valves above it_ The rate of sample flow through the cell can be adjusted if necessary by the peristaltic pump, and the dual manometer system on the right-hand side of the unit regulates this flow_ Problems arising from the continuous flow of sample through the apparatus are prevented by an airbreak system. Sampling of the suspension is done by the first aperture in the block, which can be of any diameter, e.g. typically 70 pm for particle monitoring between 2 pm and 30 pm. The sample volume being measured is controlled by a timer built into the control unit and is variable. If an oversize particle reaches the aperture and a blockage results, this is detected electronically and the solenoid valves will close, causing a back pressure at the aperture; also no count will be recorded. Subsequent to a blockage occurring, a second pair of valves will open and the analysis will m-commence with the second aperture_ In the event of the first and second apertures being simultaneously blocked, the third tube will automatically be selected. The
126
,I i
I
PRESSIRE RESERVQIR
TER
SCC:fiE IN -.-____ AFQsuREuN--_~
-
-
_
,SZXPLAG
VALVES
;:
A CLOSED
B ‘&ED
k0
i
6 OFcN
S.‘3.lPLlNG
OPEN
Fig_ 1. Diagrammatic
SAMPLIMj
representation
of the On-line
Monitor.
action of closing the solenoid valves on the fiit aperture has the effect of creating a pressure pulse which in most instances will cIear the blockage. so that this aperture is ready for use i;s and when the other apertures, in their turn, become blocked_ Electrodes El and E2 across the sensing aperture set up the current path and feed back to the controller and Coulter Counter. The model recommended with the On-line Monitor is the ZB. This will count all particles above or betiveen two preset size levels in a given volume of electrolyte_ The two size limits may be no more than 311 by diameter apart, and the general size range above about O-6 pm may be measured_ The upper size limit will be given by the ability to maintain suspension of the particles to and within the sample block, and could be about 100 150 pm for particles or cell; of low density, reducing to perhaps 40 - 50 pm for denser materials such as clays or metal powders. The controller performs the automatic control of the equipment, but can be manually over-ridden if required. The controller takes a particle count every 2,4,8,16,32 or 64 minutes, the duration of count being selectable up to some 30 seconds (typicaliy O-5 - 1.0 ml of sample depending on aperture size). A number can be preset on the controller which, if exceeded by the partic!e count, will activate an audible alarm, for example 999 particles per 0.5 ml above 2 pm for parenteral fluid monitoring_ The audible warning of the count alarm can be replaced by other control equip-
ment which would close down a production plant, if excessive contamination occurred_ The particle count recorded by the Coulter Counter Model ZB is recorded on a printer or other suitable recorder, or can be fed via suitable telemetry to a more distant location.
CALIBRATION
The On-line Monitor must be calibrated for both particle size and liquid volume. The latter calibration is conveniently achieved by measuring the weight or volume of sample passed through to the waste line from the aperture tube during the ccurse of a count. A linear graph of volume sampled against preset size may be constructed_ Calibration for particle size is achieved in the normal manner of the Coulter Counter, except that the calibration suspension is pumped from a stirred beaker into the sample block, and counts are taken marmaNy with the automatic control over-ridden_ Ideally, all three apertures should be calibrated separately, a procedure taking some 20 minutes, but in practice the matching of the apertures is so close that this will not be needed, except in very critical applications. Once calibrated the Monitor will stay in calibration almost indefinitely, but occasional recheck wilI be desirable_ Calibration is commonly made with one narrow range material of known mean size 121, as response is known ta be -I.-sentially
127 TABLE
1
On-line Monitor typical calibration data. Coulter Counter Model ZB, matching switch 10 kilobms, apertures 100 fim diam. Calibration Calibration
material
Instrument
settings
Calibration constant, Kd
Atten. setting, A
Current setting, I
Threshold setting, t
4
I. ?
28.7
2.47
4.35 pm Fixed cells
1 4
1 H
10.5
2_54
2.03 p-n P-V-T_ later
81
1 B
32.0
2.56
9.54 .um S.D.V.B.
latex
AV.
2.53
Diameter settings Particle diameter
A
I
t
2.00 pm
k
1 h
15.8
3.50 pm
r1
1 z
16.6
5.00 ym
1
1 H
15.4
proportional to particle volume [3,4] _ Typical calibration data are shown in Table 1, together with the instrument settings required to obtain given particle sizes.
EVALUATION
1. Monitoring of filtered electrolyte Isotone is a filtered saline produced by Coulter Electronics Ltd., as a diluent primarily for blood, to enable cell counts to be made by a suitable model of Coulter Counter. Reproducibility of the On-line Monitor was checked by sampling directly from a Cubitairier@ of Isoton@, using the peristaltic pump, which was connected by silicone rubber tubing known not to shed particles. The counts having a volume greater than 20 X lW’* cm3, Le. the red blood cell counting volume for a 3.4 pm diam. cell, were recorded at 2-min intervals. Rubber tubing was found to be unsuitable for plumbing as the walls constantly shed particles, and P.V.C. tubing was too rigid for the peristaltic pumpThe output data from the printer consists of two columns, one column showing a sample
identification number which is a consecutive numbering system, and the second column showing the actual particle count- in the preset time interval. A blockage of the monitor would be indicated by a gap in the identification numbers. The counts taken on the Isoton@ from the Cub&airier@ gave a mean of 32.3 with a standard deviation of +9-O. It was noticeable that the mean of the first ten counts was 42.8 f 9.7 and the mean of the last ten counts was 25.8 i 5.9, which indicated that the silicone rubber tubing and the monitor were progressively cleaned up from residual particles. The On-line Monitor was subsequently fitted to the Isoton@ production line, just downstream of the final filter. The line, under low pressure, led again to the peristaltic pump, which boosted the pressure to a value in excess of the 20 kN mm2 required for the monitor The line was subsequently fed to a builGn coil of narrow-bore tubing designed to take out small air bubbles which might interfere with the particle count. The size level was monitored at 3.5 ym diam. using 70-pm apertures, the counts per 0.5 ml being recorded. The mean count from the line was approx-
12s
imately 25 per ml. Initial counts were again high due to the Isoton’% flushing the particles out of the new tubing and the sample block. but after 10 counts at 2-min intervals, i-e. approsimateiy 50 ml of fluid passing through the Monitor, the counts had fallen to a constant level. Any significant increase in the value of the counts from the production line can easily be recosised and the appropriate remedial action can be taken. 2. Monitoring
of intravenous
fluids
The On-line Monitor was fitted with ‘iO-pm aperture tubes that had been previously calibrated and the Model ZB controls set to take particle counts above 5 pm and 2 pm respectively. These levels were chosen to correspond with the limit test for particulate matter in large volume injections stated in the British Pharmacopoeia [5] _ A sample time of 10 seconds was used, which corresponded to a sample volume of O-15 ml; all the determinations were subsequently converted to number of particles per ml. One-litre flesible plastic ampoules containing normal saline intravenous fluid were used for the study. Using iaminar flow conditions_ the projection at the lower end of the container w’s cut and inserted immediately into the silicone rubber tubing, which was connected through a peristaltic pump to the monitor. A series of determinations at the 2-pm and 5-pm levels gave mean counts of 157 = 18 and 15 2 3 respectively in one container, and 159 + 19 and 17 f 3 in a second container, both containers originating from the same production batch- This method for determining particulate contamination in intravenous fluids is to be preferred to the normal methods that have been described in the literature [6], as the fluid is fed directly from its container to the monitoring system in a closed circuit, hence the chances of contamination from the surrounding environment are reduced. 3. Monitoring sets and
of intrauenour
infrauenous
administration
cannulae
The level of particulate contamination in administration sets was determined by taking counts at 5Km and 2-pm particle diameter levels at 2-min intervals. The sample volume was the same as that stated above- The 5-grn count was initiated automatically by the instrument, and the 2-urn count initiated
TABLE
2
Monitoring zpertures Above
3 pm
of sea
water. Counts per ml. lOO-pm Above
3.5 ,xm
Above
0083s 00803 00826 OOS39 OOS16 0OSl-I OOSlS 00638 00615 OOS-t3 00853 OOS3S OOiS6 ooss5
0230s 02566 02459 0232s 02351 02302 Ogvii 05333 02315 02360
052-?3 05613 05188 05506 05520 05652 05608 05592 05628 05192 0567’7 0527-i 05785 06862 066li 05’732 05670 05933
Mean = 815 SD. = ~-11
hlean = 9-156 SD. = 29’7
b-S:.=
OG962 OOSS8 008’78 00908
02-13-I 02339 02293 02393 02522 02162 0233’7 022%
2-O ,um
hlenn = 5'716 +1c)8
manually, imm4iately following the termination of the 5pm count_ By utilising this technique, counts at the two levels represent particulate contamination in a similar volume of fluid essentially at the same time. The alternative to this method is to allow counts to be taken automatically at one level only. The normal saline was monitored as indicated in the previous section, prior to the administration sets being fitted to the ampoule. The saline passing through the administration set was then monitored against time. The particulate count was initially at a high level, appros. 36.5 X lo3 particles/ml at 2 ,um (cfmonitored saline). and gradually decreased to a constant level _ It is possible by this technique to follow the rate of change of particulate contamination with both the volume of fluid passing through the administration set, and the flow rate being used, both of which have been shown by Williams and Bamett [7] to be significant. The application of the monitor was extended to the determination of particle contamination in butterfly cannulae with a thin-walled siliconed needle. The cannula was connected directly to the Polyfusor under laminar flow conditions, and the needle inserted into the previously cleaned silicone rubber tube which
129
TABLE 3 Monitoring apertures
do-
of river water.
Counts
00040 00046 00030 00033 oov19 00027 00022 00026 00031 00027 0004S
00556 006i4 00630 00665 00591 00660 00569 00572
00036
00623-
&lean = 32 SD. = 19
Mean
per ml.
.
-.
100~pm
0057 1 006-&O
5
00531
= 60s S.D. = -50
PARTICLE DIAMETER
Fig.
was connected to the monitor. Counts 5-pm and 2-pm levels were determined before.
at the as
4. Monitoring of sea and river water Sea water was sampled close to the shore, and although it contaked more than average suspended solids, monitoring was achieved using 100~pm aperture tubes and taking counts at 3.5~pm, 5-r_cm and lo-r.rrn levels_ River water was analysed in a similar manner, again using 100-pm aperture tubes. Successful monitoring was achieved, although the low conductivity of the river water gave rise to some noise generation at low instrument settings (below 4 - 5% of the aperture diameter)_ The mean results of these determinations, and sets of data produced, are shown in Tables 2 and 3_ 5. Monitoring of microbial growth (volume distribution) Saccharomyccs cerevisiae was cultivated at 37 “C in a modified Pasteur’s medium comprising glucose 75.0 g/l, potassium dihydrogen phosphate 2.0 g/l, sodium potassium tartrate 5.0 g/I and magnesium sulphate 0.1 g/l_ The monitor was attached to a Model TA Coulter Counter, the starting and resetting of which was performed manually, as automatic
control for this model was not yet developed, while the monitor regulated the time intervals. The Model TA allows a 14 - 16 point size distribution histogram of the particle size
2. Typical
calibration
(,,m)
drita.
distribution, over a range of some 2 - -10% of the aperture diameter to be accumulated, in the time a Model ZB takes to obtain a single size level count. Thus the change in size distribution of the yeast cells with time could be monitored_ The results of this study were plotted by the X-Y recorder integral with the TA, and typical data shown in Fig. 2 as relative volume (weight) in particle diameter size ranges, replotted from the original output. Alternatively, a total count at 2-min intervals could be taken to follow the growth rate by number.
DISCUSSION A minor problem encountered with the monitor was that interference from estemal sources, and instrument noise, became apparent at a Ieve: above that found with the same aperture t:Lbes operating directly on the ZB. However, this problem only occurred towards the lower limits of the tube, giving a lower limit of some 3% of aperture diameter for super-clean fluid monitoring where electronic no’ ie can be a significant part of the particle count, instead of the normal 2% in the standard laboratory models. The Online Monitor has obvious applications in the particulate contamination field and facilitates the counting of particles in such systems as intravenous fluids and water pollution. Additionally, when coupled with the Model TA,
it has direct application to the studies of growth in microbiological systems, and dissolution/floccuIation/crystallisation processes.
3
REFERENCES
4 5 6
1 Coulter Counter Irxdustrial Bibliography, July 1974. 2 W. >I. Wood and J_ G. Harfield, Proc. Particle Size
7
Anaiysis Conf., Sot. Anal. Chem., 1970, pp_ 293 300. W_ H. Coulter, Proc. Sot. Natl. Electron. Conf., 12 (1957) 1034. B. A_ Batch, J_ Inst. Fuel (Oct. 1965) 455 - 461. British Pharmacopoeia 1973, Appendix XVIC. M. J. Groves. Parenteral Products, Heinemann, London, 1973. A Williams and M. I_ Barn&t. Pharm. J., 190 (1974) 191,204.
125
Laboratory Evaluation of the Coulter On-Line Particle Monitor
M. I. BARNETT Welsh School
and E. SIMS
of Pharmacy,
University
of Wales Institute
of Science and Technology.
Cardiff (Gt. Britain)
R. W. LINES Coulter Electronics
Ltd.. Harpenden.
(Received
16, 1975;
October
Hertz
(Gt. Britain)
in revised form November
SUMMARY
The On-line iMonitor system, consisting of a sampling device, control unit and digital printer, is used in conjunction with Model ZB or TA Coulter Counters and is designed primarily for the process control or constant monitoring of particles in fluid systzms. At selected time intervals, samples are taken and particle counts determined at preset size levels. A description of the Monitor and its modes of operation are given and some of its applications and limitations are discussed_
INTRODUCTION
The Coulter Counter@, through its various models, has become an established and well evaluated method of counting and sizing microscopic particles and cells [ 11. Increased interest in process control has led to the development of a fully automatic model, the On-line Monitor. This has been designed specifically to continuously monitor the numbers of particles present in an electrolyte solution at or between preset size levels. The types of application might include examination of sea water, parenteral fluids, plating solutions, cell growth media, crystal growth studies or levels of particulate pollution in river water. The instrument is shown diagrammatically in Fig_ l_ The sample is taken continuously from a suitable outlet from a production plant, or filtration system at pressures ranging from 0 to 345 kN mm2 (0.50 p.s.i.). Three aperture tubes are inset in a perspex block; ell have the same nominal aperture size and each one is
3, 1975)
provided with its own internal electrode, the external elec-crode being common. The sample is fed directly, or via a peristaltic pump, through a coil at the bottom of the unit and then into the sample cell. The action of passing the sample through the coil effectively degasses it so that air bubbles are excluded and not counted. Surplus sample is continuously exhausted through the top of the aperture block, to waste, via the regulator manometer. This system allows a representa-
tive sample of fluid to be present in the sample block. At pre-selected times a sample is allowed through any one aperture tube by opening the two solenoid valves above it_ The rate of sample flow through the cell can be adjusted if necessary by the peristaltic pump, and the dual manometer system on the right-hand side of the unit regulates this flow_ Problems arising from the continuous flow of sample through the apparatus are prevented by an airbreak system. Sampling of the suspension is done by the first aperture in the block, which can be of any diameter, e.g. typically 70 pm for particle monitoring between 2 pm and 30 pm. The sample volume being measured is controlled by a timer built into the control unit and is variable. If an oversize particle reaches the aperture and a blockage results, this is detected electronically and the solenoid valves will close, causing a back pressure at the aperture; also no count will be recorded. Subsequent to a blockage occurring, a second pair of valves will open and the analysis will m-commence with the second aperture_ In the event of the first and second apertures being simultaneously blocked, the third tube will automatically be selected. The
126
,I i
I
PRESSIRE RESERVQIR
TER
SCC:fiE IN -.-____ AFQsuREuN--_~
-
-
_
,SZXPLAG
VALVES
;:
A CLOSED
B ‘&ED
k0
i
6 OFcN
S.‘3.lPLlNG
OPEN
Fig_ 1. Diagrammatic
SAMPLIMj
representation
of the On-line
Monitor.
action of closing the solenoid valves on the fiit aperture has the effect of creating a pressure pulse which in most instances will cIear the blockage. so that this aperture is ready for use i;s and when the other apertures, in their turn, become blocked_ Electrodes El and E2 across the sensing aperture set up the current path and feed back to the controller and Coulter Counter. The model recommended with the On-line Monitor is the ZB. This will count all particles above or betiveen two preset size levels in a given volume of electrolyte_ The two size limits may be no more than 311 by diameter apart, and the general size range above about O-6 pm may be measured_ The upper size limit will be given by the ability to maintain suspension of the particles to and within the sample block, and could be about 100 150 pm for particles or cell; of low density, reducing to perhaps 40 - 50 pm for denser materials such as clays or metal powders. The controller performs the automatic control of the equipment, but can be manually over-ridden if required. The controller takes a particle count every 2,4,8,16,32 or 64 minutes, the duration of count being selectable up to some 30 seconds (typicaliy O-5 - 1.0 ml of sample depending on aperture size). A number can be preset on the controller which, if exceeded by the partic!e count, will activate an audible alarm, for example 999 particles per 0.5 ml above 2 pm for parenteral fluid monitoring_ The audible warning of the count alarm can be replaced by other control equip-
ment which would close down a production plant, if excessive contamination occurred_ The particle count recorded by the Coulter Counter Model ZB is recorded on a printer or other suitable recorder, or can be fed via suitable telemetry to a more distant location.
CALIBRATION
The On-line Monitor must be calibrated for both particle size and liquid volume. The latter calibration is conveniently achieved by measuring the weight or volume of sample passed through to the waste line from the aperture tube during the ccurse of a count. A linear graph of volume sampled against preset size may be constructed_ Calibration for particle size is achieved in the normal manner of the Coulter Counter, except that the calibration suspension is pumped from a stirred beaker into the sample block, and counts are taken marmaNy with the automatic control over-ridden_ Ideally, all three apertures should be calibrated separately, a procedure taking some 20 minutes, but in practice the matching of the apertures is so close that this will not be needed, except in very critical applications. Once calibrated the Monitor will stay in calibration almost indefinitely, but occasional recheck wilI be desirable_ Calibration is commonly made with one narrow range material of known mean size 121, as response is known ta be -I.-sentially
127 TABLE
1
On-line Monitor typical calibration data. Coulter Counter Model ZB, matching switch 10 kilobms, apertures 100 fim diam. Calibration Calibration
material
Instrument
settings
Calibration constant, Kd
Atten. setting, A
Current setting, I
Threshold setting, t
4
I. ?
28.7
2.47
4.35 pm Fixed cells
1 4
1 H
10.5
2_54
2.03 p-n P-V-T_ later
81
1 B
32.0
2.56
9.54 .um S.D.V.B.
latex
AV.
2.53
Diameter settings Particle diameter
A
I
t
2.00 pm
k
1 h
15.8
3.50 pm
r1
1 z
16.6
5.00 ym
1
1 H
15.4
proportional to particle volume [3,4] _ Typical calibration data are shown in Table 1, together with the instrument settings required to obtain given particle sizes.
EVALUATION
1. Monitoring of filtered electrolyte Isotone is a filtered saline produced by Coulter Electronics Ltd., as a diluent primarily for blood, to enable cell counts to be made by a suitable model of Coulter Counter. Reproducibility of the On-line Monitor was checked by sampling directly from a Cubitairier@ of Isoton@, using the peristaltic pump, which was connected by silicone rubber tubing known not to shed particles. The counts having a volume greater than 20 X lW’* cm3, Le. the red blood cell counting volume for a 3.4 pm diam. cell, were recorded at 2-min intervals. Rubber tubing was found to be unsuitable for plumbing as the walls constantly shed particles, and P.V.C. tubing was too rigid for the peristaltic pumpThe output data from the printer consists of two columns, one column showing a sample
identification number which is a consecutive numbering system, and the second column showing the actual particle count- in the preset time interval. A blockage of the monitor would be indicated by a gap in the identification numbers. The counts taken on the Isoton@ from the Cub&airier@ gave a mean of 32.3 with a standard deviation of +9-O. It was noticeable that the mean of the first ten counts was 42.8 f 9.7 and the mean of the last ten counts was 25.8 i 5.9, which indicated that the silicone rubber tubing and the monitor were progressively cleaned up from residual particles. The On-line Monitor was subsequently fitted to the Isoton@ production line, just downstream of the final filter. The line, under low pressure, led again to the peristaltic pump, which boosted the pressure to a value in excess of the 20 kN mm2 required for the monitor The line was subsequently fed to a builGn coil of narrow-bore tubing designed to take out small air bubbles which might interfere with the particle count. The size level was monitored at 3.5 ym diam. using 70-pm apertures, the counts per 0.5 ml being recorded. The mean count from the line was approx-
12s
imately 25 per ml. Initial counts were again high due to the Isoton’% flushing the particles out of the new tubing and the sample block. but after 10 counts at 2-min intervals, i-e. approsimateiy 50 ml of fluid passing through the Monitor, the counts had fallen to a constant level. Any significant increase in the value of the counts from the production line can easily be recosised and the appropriate remedial action can be taken. 2. Monitoring
of intravenous
fluids
The On-line Monitor was fitted with ‘iO-pm aperture tubes that had been previously calibrated and the Model ZB controls set to take particle counts above 5 pm and 2 pm respectively. These levels were chosen to correspond with the limit test for particulate matter in large volume injections stated in the British Pharmacopoeia [5] _ A sample time of 10 seconds was used, which corresponded to a sample volume of O-15 ml; all the determinations were subsequently converted to number of particles per ml. One-litre flesible plastic ampoules containing normal saline intravenous fluid were used for the study. Using iaminar flow conditions_ the projection at the lower end of the container w’s cut and inserted immediately into the silicone rubber tubing, which was connected through a peristaltic pump to the monitor. A series of determinations at the 2-pm and 5-pm levels gave mean counts of 157 = 18 and 15 2 3 respectively in one container, and 159 + 19 and 17 f 3 in a second container, both containers originating from the same production batch- This method for determining particulate contamination in intravenous fluids is to be preferred to the normal methods that have been described in the literature [6], as the fluid is fed directly from its container to the monitoring system in a closed circuit, hence the chances of contamination from the surrounding environment are reduced. 3. Monitoring sets and
of intrauenour
infrauenous
administration
cannulae
The level of particulate contamination in administration sets was determined by taking counts at 5Km and 2-pm particle diameter levels at 2-min intervals. The sample volume was the same as that stated above- The 5-grn count was initiated automatically by the instrument, and the 2-urn count initiated
TABLE
2
Monitoring zpertures Above
3 pm
of sea
water. Counts per ml. lOO-pm Above
3.5 ,xm
Above
0083s 00803 00826 OOS39 OOS16 0OSl-I OOSlS 00638 00615 OOS-t3 00853 OOS3S OOiS6 ooss5
0230s 02566 02459 0232s 02351 02302 Ogvii 05333 02315 02360
052-?3 05613 05188 05506 05520 05652 05608 05592 05628 05192 0567’7 0527-i 05785 06862 066li 05’732 05670 05933
Mean = 815 SD. = ~-11
hlean = 9-156 SD. = 29’7
b-S:.=
OG962 OOSS8 008’78 00908
02-13-I 02339 02293 02393 02522 02162 0233’7 022%
2-O ,um
hlenn = 5'716 +1c)8
manually, imm4iately following the termination of the 5pm count_ By utilising this technique, counts at the two levels represent particulate contamination in a similar volume of fluid essentially at the same time. The alternative to this method is to allow counts to be taken automatically at one level only. The normal saline was monitored as indicated in the previous section, prior to the administration sets being fitted to the ampoule. The saline passing through the administration set was then monitored against time. The particulate count was initially at a high level, appros. 36.5 X lo3 particles/ml at 2 ,um (cfmonitored saline). and gradually decreased to a constant level _ It is possible by this technique to follow the rate of change of particulate contamination with both the volume of fluid passing through the administration set, and the flow rate being used, both of which have been shown by Williams and Bamett [7] to be significant. The application of the monitor was extended to the determination of particle contamination in butterfly cannulae with a thin-walled siliconed needle. The cannula was connected directly to the Polyfusor under laminar flow conditions, and the needle inserted into the previously cleaned silicone rubber tube which
129
TABLE 3 Monitoring apertures
do-
of river water.
Counts
00040 00046 00030 00033 oov19 00027 00022 00026 00031 00027 0004S
00556 006i4 00630 00665 00591 00660 00569 00572
00036
00623-
&lean = 32 SD. = 19
Mean
per ml.
.
-.
100~pm
0057 1 006-&O
5
00531
= 60s S.D. = -50
PARTICLE DIAMETER
Fig.
was connected to the monitor. Counts 5-pm and 2-pm levels were determined before.
at the as
4. Monitoring of sea and river water Sea water was sampled close to the shore, and although it contaked more than average suspended solids, monitoring was achieved using 100~pm aperture tubes and taking counts at 3.5~pm, 5-r_cm and lo-r.rrn levels_ River water was analysed in a similar manner, again using 100-pm aperture tubes. Successful monitoring was achieved, although the low conductivity of the river water gave rise to some noise generation at low instrument settings (below 4 - 5% of the aperture diameter)_ The mean results of these determinations, and sets of data produced, are shown in Tables 2 and 3_ 5. Monitoring of microbial growth (volume distribution) Saccharomyccs cerevisiae was cultivated at 37 “C in a modified Pasteur’s medium comprising glucose 75.0 g/l, potassium dihydrogen phosphate 2.0 g/l, sodium potassium tartrate 5.0 g/I and magnesium sulphate 0.1 g/l_ The monitor was attached to a Model TA Coulter Counter, the starting and resetting of which was performed manually, as automatic
control for this model was not yet developed, while the monitor regulated the time intervals. The Model TA allows a 14 - 16 point size distribution histogram of the particle size
2. Typical
calibration
(,,m)
drita.
distribution, over a range of some 2 - -10% of the aperture diameter to be accumulated, in the time a Model ZB takes to obtain a single size level count. Thus the change in size distribution of the yeast cells with time could be monitored_ The results of this study were plotted by the X-Y recorder integral with the TA, and typical data shown in Fig. 2 as relative volume (weight) in particle diameter size ranges, replotted from the original output. Alternatively, a total count at 2-min intervals could be taken to follow the growth rate by number.
DISCUSSION A minor problem encountered with the monitor was that interference from estemal sources, and instrument noise, became apparent at a Ieve: above that found with the same aperture t:Lbes operating directly on the ZB. However, this problem only occurred towards the lower limits of the tube, giving a lower limit of some 3% of aperture diameter for super-clean fluid monitoring where electronic no’ ie can be a significant part of the particle count, instead of the normal 2% in the standard laboratory models. The Online Monitor has obvious applications in the particulate contamination field and facilitates the counting of particles in such systems as intravenous fluids and water pollution. Additionally, when coupled with the Model TA,
it has direct application to the studies of growth in microbiological systems, and dissolution/floccuIation/crystallisation processes.
3
REFERENCES
4 5 6
1 Coulter Counter Irxdustrial Bibliography, July 1974. 2 W. >I. Wood and J_ G. Harfield, Proc. Particle Size
7
Anaiysis Conf., Sot. Anal. Chem., 1970, pp_ 293 300. W_ H. Coulter, Proc. Sot. Natl. Electron. Conf., 12 (1957) 1034. B. A_ Batch, J_ Inst. Fuel (Oct. 1965) 455 - 461. British Pharmacopoeia 1973, Appendix XVIC. M. J. Groves. Parenteral Products, Heinemann, London, 1973. A Williams and M. I_ Barn&t. Pharm. J., 190 (1974) 191,204.