Semiautomatic measurement of contact angles on cell layers by a modified axisymmetric drop shape analysis

Colloi& and Sutiacea, 42 (1423) 391-403 Elsevier Science Publishers B.V., Amsterdam -

391 Printed in The Netherlands

Semiautomatic Measurement of Contact Angles on Cell Layers by a Modified Axisymmetric Drop Shape Analysis W.C. DUNCAN-HEWITT’, A.W. NEUMANN’

z. POLICOVA’.

P. CI-IENG’, E-1. VARGHA-BUTLERP

and

‘Department of Mechanical Engineering, Uniuersity of Toronto, 5 King’s College Rod, Toronto, Ontario M6S IA4 (Canada) pCoUege of Pharmacy, Daihousie Uniuarsity, Halifax, Nova Scotia B3H 355 (Canada)

(Received 11 October 1988,accepted 20 February 1989)

ABSTRACT The contact angle between a sessile drop of liquid and a bed of biological par&l- such as bacterial cells provides a measure of the hydrophobicity or, more precisely, the surface tension of the cells. While this surface characteristic plays an important role in diverse proceas~ such as cell adhesion and phagocvtos is, it is difficult to obtain contact angles which are meaningful in a tiermodynamic s4338.eusing traditional methods of measurement (e.g., gcmiometric techniquea) because: (1) the contact angles. which are inherently small on biological substrates in their native hydrated state, are difficult to measure with precision; (2) the contact angle of the sedsik drop invariably decreases as the liquid is absorbed into tbe layer of C&I, and; (3) the hetarogeneous and often rough surfaces produced by any method of cell deposition give rise to sessile dmpa which do not ~OSSMS a perfectly cimularperimeter. A modified a&mmetic drop ehape analyks (ADSACD) permits the contact angle of a sessile drop to be calculated fkom the average diameter of tbe drop aa viewed from above, and provideu the means k circumvent these problems. The ADSA-CD procedure was used to measure the contact angle of water on layem of three different speciesof bacteria. The average contact angles for T. thiooxiduns and Stiph epidermidis were 16.9 and 20.6”, respectively. The contact anglea for two different atraina of T. ferrooxidans were 11.7 and 10.6o and were not stati&icalIy different. The 95% confidence inteervalsfor these means, obtained from fewer than 17 independent measurementswere less than or equal to k 1.0”. We show that this novel strategy providex (1) increased objectivity with respect to the goniometric method; (2) a precise eetimate of the contact angle in spite of the fact that the contact surface quality and the circularity of the angle is time dependent; ( 3 ) a simple means of ming drop periphery on biological surfkes.

INTRGDUCTION

The degree of hydrophobic&y exhibited by cells is an expression of their surface tension. It is characterizedby contact angle measurements and plays an important role in many biological and biotechnologicalprocesses. The phe016%6622/89/$03.50

8 1989E&evier Science Publishera B.V.

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nomenological contact angle, along with the surface tension of the wetting liquid, provides the driving force for the kinetics of cell wetting [1,2 3. The extent of cell adhesion has been shown to be jointly determined by the surface tension of the cells, the surface tension of the second solid phase and the surface tension of the suspending liquid phase 13-51. The intcrfacial tensions in these free energy balances may be obtained from liquid surface tensions and contact angles by means of an equation of state [6]. In principle, the formation provided by the contact angle of a sessile drop of a liquid on a layer of cells ought to be identical with that obtained from contact angles on any surface (e.g. polymers, siliconized glass, etc.). However, the physical nature of cell layers may render it difficult to measure contact angles which are meaningful from a thermodynamic point of view. Previously [7], contact angles were measured on layers of bacteria which initially were wet and which were allowed to dry over time. The contact angle was initially approximately equal to zero, then rose to a plateau value which remained constant for 20-30 min. The plateau value corresponds to the contact angle of the cells in their native, hydrated, state. As the cells dried further, the contact angle changed unpredictably. Briefly, it was concluded that: (1) it is necessary to measure contact angles of sessile drops on layers of cells which are fully hydrated since this is their natural state and, (2) the liquids employed to form the sessile drops must be chosen to be physically and biologically compatible with the cells to avoid altering the nature of the cell membranes in the process of measuring contact angles. Some practical problems persist even when the above requirements are met. Cell layers rnqy be rough and inhomogeneous, are often quite hydrophilic, and tend to absorb liquid from the sessile drop by capillary action. We will discuss the problems arising from these characteristics and the methods we have employed both to identify and circumvent them. Regardless cf the type and quality of surface for which contact angles are being measured, approaches of varying degz ees of sophistication and accuracy may be employed to evaluate the contact angles. In the srmplest approach to contact angle measurement, a tangent is aligned with fhe proEle of a sessile drop where it contacts an underlying surface [S], using either a telescope equipped with a goniometer eyepiece or a protractor on a photograph of the sesaile drop. The accuracy of th;,: direct techniques is nominally +, 2’ on ideal surfaces. If higher accuracy 1s required, alternate methods must be considered [9]. Several strategies based on drop shape analysis have been developed which circumvent the subjective problem of accurately placing a tangent orntb a drop profile at the point where it contacts the solid surface. The axisymmetric drop shape analysis (ADSA) is unique in that it fits an arbitrary set of experimentally determined drop profile coordinates to a curve predicted by the LaPlace equation of capillarity [9]. The earliest version (ADSA-profile, or ADSA-P) required a set of drop profile coordinates and the densities of the two fluid phases as input and calculated the liquid surface tension, drop volumes, and

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contact angles. Initialiy, the drop profile coordinates were selected manually from an image of the drop. Recent modifications, in which the entire edge of a digitized video image of the drop profile is located objectively through the application of an edge detection algorithm, have permitted substantial automation of the procedure [ 101. The present AIXA-P apprczch, when applied to the measurement of contact angles on smooth, homogeneous surfaces, improves the accuracy of measurement to -+ 0.2 o under ideal circumstances. The nature of the solid surface plays a crucial role in the majority of cases: ideally the surface must be homogeneous, non-reactive, and. smooth [ 2 ]. Obviously, layers of cells are far from ideal: (a) they are necessarily rough, even if the cells are evenly distributed by the method of deposition; (b) they absorb water and other liquids: the drop tends to sink into the layer of cells so that the contact angle is time-dependent. Furthermore, since the layers of cells are typically hydrophilic, the contact angles are generally small [ll].For small contact angles, and especiaily on rough surfaces, the image of the drop appears fuzzy near the surface and it becomes exceedingly difficult to locate the point where the profile of the drop meets the surface (Fig. 1) and operator skill assumes a role of major importance. For such surfaces the alternate ADSA-contact diameter (ADSA-CD) strategy was devised. In this procedure, the drop is viewed from above and the average diameter of the drop is measured. The details ofthe theory and numerical procedure employed in the analysis of the experimental data are described elsewhere [12]. Essentially, ADSA-CD requires the following input: the drop volume, liquid surface tension, the densities of the two fluid phases, and the average diameter of the drop as viewed from above. The diameter may be obtained in several ways depending upon the particular application of ADSA-CD; in the present study an arbitrary set of coordinates characterizing the perimeter of the sessile drop is selected manually and the average contat;ttdiameter is calculated from them. The contact angle of the drop is calculated by numerically minimizing the difference between the volume of a drop predicted by the Laplace equation of capillarity and that prescribed experimentally. At the expense of requiring the liquid surface tension as input, the procedure provides an accurate method to determine small contact angles ( 4 0.2 o; [ 12 ] ), since the position of the drop perimeter, the three-phase line, is much easier to locate when the drop is viewed from above. ADSA-CD possesses the adcled advantage that the advancing contact angle of the drop is measured even if some of the liquid has been removed from the drop by evaporation or by absorption by the underlying substrate: contact angle hysteresis causes the three-phase line, or drop perimeter, to remain at its initial position for a considerable period of time. So far, we have described a new technique with which small cuntact angles mav be measured accurately provided that quantities, such as the surface ten-

Fig. I_ Side and top view of a ~ea~ile drop posnessing accurately

a txnall

contactangle. It is difficult to locate

the point where the profile meets the substrate.

sion of the liquid in the sessile drop, remain constank In this study ADSA-CD was employed to measure the contact angle of sessile drops of water on layers of three types of bacteria, Thi&aciUu.s thiaoxidans, ThiobacZus ferromidans, and Staphylococcus epidermidik. In this illwtion of the AIXA technique in

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a biological sy&em, surface-active substance8 such as proteins could conceivably be excreted by the cells. Adsorption of these substances onto the liquid surface in the aessile drop could cause our input surface tension to be in error. To obviate this possibility, the surface tension of the measuring liquid was checked after it had been in contact with the cella, as described below. We will show that the ADSA-CD strategy provides: (1) increased objectivity; (2) a precise estimate of the contact angle in spite of drop shrinkage; (3) a simple means of assessing surface and drop quality on biological surfaces which are inherently imperfect. MATERIALS

AND METHODS

The nutritional requirementa and conditions for optimal growth were different for the bacteria studied. Two strains of 77zi&ac&s ferr0o~~idan.s(23270 and 19869) were cultured in a medium which was prepared by mixing a 400 ml aqueous solution containiig 0.4 g ammonium tlulphate, 0.2 g potassium hypophosphate, and 0.08 g magnesium sulphate heptahydrati, and a 100 ml aqueous solution containing 10 g ferric sulphate heptahydrate, and 1 ml of a 1 N ammonium aulphate. The pH of the resulting solution was 2.8 [ 131.The bacteria were grown at 26” C in a water bath for 4 days. They were subsequently harvested by filtration through Whatman #4 filter paper to remove precipitates of ferric salts and then centrifuged at a low apeed to remove any remaining precipitate of ferric salta. The supernatant was then transferred to 60 ml centrifuge tubes and spun down at 3780 g for 26 min. Finally, the bacteria were suspended in 0.001 IV sulfuric acid (PH”3). One strain of T. thiooridam (19377) wa8 cultured in a 1 i aqueous solution containing 0.2 g ammonium eulphate, 0.5 g magnesium aulphate heptahydrate, 0.25 g calcium chloride, 3.0 gpotaaaium hypophosphate, and 0.006 g ferric sulphate. Precipitated gulfu.r (1% w/v) was placed on the Burface of the above medium, which was then boiled for 1 h on 3 consecutive daya 1131. T. thicmwidan.sW~EIgrown in thia medium at 26°C in a water bath for 10 days. The bacteria were harvested by filtration through Whatman #l filter paper, then centrifuged at 60 Hz for 25 min. Finally, they were resuspended in 0.001 N sulfhric acid Staphylococcus epidermidis wan grown in 3% trypticase soy broth at 37°C for approximately 20 h, and then harvested by centrifirgation at 304 g for 16 min. The bacteria were then resuspended in normal saline.

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Filters may contain wetting agents which are empIoyed to facatate the filtration process. Shotid the surfactant contaminate the liquid in the sessile drop the contact angle measurement would be ti error. To explore this possibility a thin pool of liquid was placed on the surEace of an unwashed cellulosic membrane filter (black MS1 Micronsep*, pore size, 0.45 ,can) and was allowed to remain undisturbed f:lr 1 h. The liquid was then aspirated and its surface tension was compared to that of water: theee were found to be indistinguishable. Furthermore, the filter was washed copiously with distilled water prior to use. was placed in a glass filter A 6-6 ml suspension of bacteria ( - 10xocells/ml) holder (MilEpore) equipped with a cellulosic membrane fiiter. Using suction filtration, the bacteria were deposited on the membrane filter. The layer of cells was washed three times with distilled water during the filtration procedure. Suction was terminated when most of the water had been removed, however, care WOES taken to ensure that the cells remained slightly moist. The membrane filter and the layer of cells deposited on it was then rapidly transferred to the surface of a ?reehly prepared 2% solidified agar plate. The purpose of the agar was to act as a water reservoir which decreased the rate at which the cells dried This ensured that the contact angles were measured whiIe the cells were still iu their fully hydrated state,

The surface tension of the water used for contact angle measurements was determined using the WilheImy technique 1141 at 20°C. To ensure that surface-active substances, which might be excreted by the bacteria, had not altered the surface tension of the water during the contact angle measurement procedure, the following tests were performed: (I ) using two different polymer surf&es with defined surface characteristics the contact angle of the liquid aspirated from sessile drops on cell layers was compared to that of pure water; (2) the surface tension of the water in which bacteria were suspended for an hour or more was evaluated using the Wiielmy technique. Contact angles were measured using both the simple goniometric technique and the ADSA-CD approach. In the former approach, a 6-20 fl drop of doubledistilled water was grown from a teflon needle and gently placed upon a region of the bed of cells which had not previously been tested The contact angle of the drop was quickly measure d at specified times after the drop wss deposited, ranging from approximately 6 s to 1 min. While it was possible tn measure both aides of the same drop, an average value could not be calculated since the contact angle changed rapidly. All goniometric measurements were made at 23°C and 30-4096 relative humidity.

For the ADSA-CD measurements, a &x1 af a knowa volume of double-distilled water was placed on a bed of bacterial cells using a Gilmont micrometer syringe (accuracy 0.022 fl; [ 121) eguipped with a stainless steel needle. The volume of the drop was varied as a diction of the drop diameter observed in the previous measurement and ranged from 0.60 fl to 2.70 ,uL This permitted optimal utilization of the entire bed of cells. A video image of the seaaile drop, viewed from the top, was then obtained using a Wild-Heerbrugg M2S Zoom ~tereomicroacope at a magnification of 6 to 8 times. The images were usually obtained within 6 s after drop formation. However, two runs were performed in which images of the same drop were saved at defined times ranging up to 1 min, in order to compare the reeults with those obtained using goniometric measurements. This latter procedure was performed on a layer of T. thimxidam which had been allowed to drs_ The initial contact angle for a S~BB& drop of water on the dried cells was substantially higher than that for hydrated cells which permitted the decrease in contact angle to be followed for up to a minute. The contact angle of sessile drops on hydrated cells decreased to less than 5” wit&in 10 a. The experiment was timed from the moment that suction of the bacterial layer was discontinued The fiwt contact angle measurement was performed 5-10 min after the filtration was discontinued. The first contact angle measurement was performed 5-10 min after the filtration was discontinued At regular time intervals a drop was placed on a previously untested portion of the surface. This procedure was repeated for approximately 2 h, or until the entire surface had been sampled. As discussed previously, this strategy is adopted to determine the contact angle of cells which are still in their native, hydrated, etata [7]. The scale of the drop image was calibrated using the image of a precisely manufactured grid which concurrently provided a reference to correct any distortion of the video image. The images were later recalled onto a Vax station II/GPX video screen and manually digitized by selecting 20 to 30 points on the perimeter of the drop using a mouse. These points were selected to estimate the average drop diameter. For instance, on a gently undulating contact line, care was taken to select points which characterised both minimum and maximum contact diameters, while a sharp protrusion or intrusion was excluded. The ADSA-CD program uses fhe arbitrary points on the drop perimeter to calculate an average drop diameter and from this value, provides the average contact angle [16]. RESULTS

AND DISCUSSION

Contact angles forlayersof bacteria The surface tension of thy water which wan employed in the contact angle measurements was 72.68 mJ mm2 at 20” C. This value did not change after the water had been in contact with the bacterial cells.

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The quality of the layers of bacterial cells varied considerably, depending upon the uniformiLy of the filtration procese. Some layers were visibly rough and undulating (Fig. Za), and it was often impossible to form axieymmetric dropa on such m&aces (Fig. Zb). Lack of axiqmmetry provides a very eimple

Fig. 2. Imae of seede drops on a layer of T. thiooxidarz8 cds. and a non-axieymmetric drop (b)am shown.

Examples

of a rough 8wrfhce (a)

criterion to disregard certain drops; clearly, the drop depicted in Fig. 2b was discarded The sum of the squared radial distances of the experimentally measured coordinates on the drop perimeter from the circle, the location of which is defined by the mean drop diameter, would provide a simple statistical measure of the circularity of the drop. Surface roughness, such as that depicted in Fig. 2a, presents a difficult problem. Although the drop may appear quite circular, the measured contact angle tends to increase as the roughness increases, as ahown by Vargha-Butler et al. [ 161. While this measured angle is the correct phenomenological contact angle, it cannot he used in the Young equation and therefore cannot be used to characterize the interfacial energetics of the surface being tested. Quantitative and completely objective means for discarding certain drops because of the roughness visible (seen when viewed from above) are not yet available. More frequently, draps such as are depicted in Figs 3a and 3b are observed. For these drops, points at the drop perimeter are selected that estimate the average drop diameter best. Small invaginations, possibly a result of “sticking” of the three-phase line at some inhomogenity, or “fingers”, which could form in particularly rough regions due to wicking, were avoided (Fig. 4). The results of these measurements are shown in Fig. 6. From the figures, it is apparent that the measured contact angle remained within a fairly narrow range as the layers of cells dried slowly over approximately 2 h. These observations must be contrasted with earlier observations which were obtained using a different method of cell deposition [ 171. In the earlier approach, the initial contact angle was approximately zero, increased to a plateau value as the cells dried, then deviated unpredictably thereafter. In the present approach, excess water is removed somewhat more before filtration is discontinued and the agar plate decreases the rate of drying considerably. The contact angle reaches its plateau value immediately and, due to the water reservoir of the agar plate, this value is observed for a long time. Nevertheless, the common feature of both methods is the occurrence of contact angles which are independent of time, and which we believe indicate the contact angle of the cells in their native, hydrated, state. The average contact angle for the &z..pk t?@ermidis in this study was found to be 20.6 -+ 0.9 o (n= l&,95% confidence interval, C.I. ) , and is comparable to that determined earlier, i.e. 24.5 o [ 111.The contact aIlg18 of the T. thiouxidans WCS 16.9 2 0.9 O (n = 16; 95% C-I. ) . This is slightly larger than 15.6 + 0.6O (n= 30; 96% C-I.), the average value determined on the same strain in the present study by goniometric measure merits. An important advantage of the ADSA-CD approach is readily observed from these figures: a relatively small number of measurements provides a mean contact angle with relatively high precision. When the contact angles characteristic of two strains of 7’. fermoxidaraa employed in this study were compared (strain 23270: Il.72 LO”, strain 19869: 10.6+0.9”; n=l6 and 18, respectively; 95% C.I.), no statktically significant difference was found.

Fig. 3. Images of sessile drops of water on a layer of T. ferrooxidans cells. The conta.ct angles calculated using ADSA-CD were 12.7’ (a) and 11.3” (b).

The effect of drop shrinkage on goniometric and AESA-CD

measuremel 2ts

Drops of water on layers of cells are inherently unstable hecause thLewater tends to penetrate between the cells by capillary action. As a result, the contact

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Fig. 4. Example of the manner in which the perimeter of a fictitious manually on a video screen using a cursor controlled by a mouse.

25 9

t? a,

F m 5 m e

sessile drop would be located

Slash. ekdermidis

T. thiaoxidans (19377) 20

0

0

15

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0

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0

0

0

0

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8

T.

ferrcwxicians ( 923270, 019859) 0

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4

. , . , . ,, , , , . .. . . , . , . 10

20

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time (minutes)

Fig. 5. The contact angles of sessiie drops of water on layers of different bacterial cells. The horizontal axis represents that time after formation of the layer at which the drops were deposited. The mean contact angles ( + 95% confidence interval) were: (a) 20.6 f. 0.9” (Staph. epidermidk ); (b) 16.9f0.9” (T. thiooxiduns); (c) 11.7k 1.0” (T.fcrrooxidansstrain 23270),and (d) 10.5AO.9” (T. ferrooxicians strain 19859).

angle decreases rapidily, so that accurate and reproducible contact ar.gles may be assessed with a goniometer only with much experience and skill. Examination of Fig. 6, which shows the change with time of the contact angle of four separate drops, as measured using both the goniometric and ADSA-CD meth-

402 0 ADSA-CD n QOfllOmet9r e

QOniometer

0or

(run 1) 0 ADSA-CD (WI

(estimate

1)

l QOIIiOInet9r

(run 2) (rUtI

2)

from drop QrOwth experiment)

b mesEnvementf3with goniometer measurements as a function of Fig. 6. Comparison of ADSA-CD drop age. The draining away of the measuring liquid from the drop doee not cause the three-phase line to move for a considerable time.

ads, makes this fact readily apparent. Tt is very difficult to assess the contact angle earlier than 5 s after the drop is formed without encountering kinetic effects. Zero-time goniometric contact angles may be estimated only by back extrapolation of the contact anglea for a given drop measured over time, or by measuring the contact angle of the drop as it is being grown on the surface. In the first case, one encounters the uncertainty associated with any extrapolation, while in the latter case, the contact angle is dynamic and may be different from the true static contact angle [ 18 J_ For the layers of cells tested in this study, the drop had virtu&y vanished within 1 min. The perimeter of the drop remained in approximately the same position for at least 40 8, due to the propeneity of the three-phase line to “stick” in its initial equilibrium position. After approximat;ely 40 a, the drop perimeter sometimes appeared to increase rather than to decrease as the water was drawn between the cells by capillary action. CONCLUSIONS

The contact angle of water on layers of bacteria is accurately determined using me ADSA-CD approach. In particuhu, the following benefits were established: (1) increased objectivity; (2) precise estimates of the mean population contact angle are derived from relatively few measurements, (3) the interactive approach permits the assessment of surface and drop quality, an important feature for contact angle measurements on biological surfaces which are inherently imperfect; (4) it is possible to measure contact angles accurately in spite of the fact that the contact angle decreases continually due to wickiug since the important input parameter is the average radial position of the three-

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phase line; the latter has been shown to remain essentially stationary for at leaat 40 8, i.e. a span of time more than adequate for image acquisition. Software for ADSA-CD can be obtained from the authors. ACKNOWLEDGEMENTS

The financial support from the Natural Science and Engineering Research Council of Canada (strategic grant #STRM32434) is gratefully acknowledged.

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