Vol. 44, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1982, p. 184-1920099-2240/82/070184-09$02.00/0

Bubble Contact Angle Method for Evaluating SubstratumInterfacial Characteristics and Its Relevance to Bacterial

AttachmentMADILYN FLETCHER* AND K. C. MARSHALLt

Department of Environmental Sciences, University of Warwick, Coventry CV4 7AL, England

Received 1 October 1981/Accepted 5 March 1982

A bubble contact angle method was used to determine interfacial free-energycharacteristics of polystyrene substrata in the presence and absence of potentialsurface-conditioning proteins (bovine glycoprotein, bovine serum albumin, fattyacid-free bovine serum albumin), a bacterial culture supernatant, and a bacterialexopolymer. Clean petri dish substrata gave a contact angle of 900, but tissueculture dish substrata were more hydrophilic, giving an angle of 290 or less.Bubble contact angles at the surfaces exposed to the macromolecular solutionsvaried with the composition and concentration of the solution. Modification bypronase enzymes of the conditioning effect of proteins depended on the nature ofboth the substratum and the protein, as well as the time of addition of the enzymerelative to the conditioning of the substratum. The effects of dissolved andsubstratum-adsorbed proteins on the attachment of Pseudomonas sp. strainNCMB 2021 to petri dishes and tissue culture dishes were consistent with changesin bubble contact angles (except when proteins were adsorbed to tissue culturedishes before attachment) as were alterations in protein-induced inhibition ofbacterial attachment to petri dishes by treatment with pronase. Differencesbetween the attachment of pseudomonads to petri dishes and tissue culture dishessuggested that different mechanisms of adhesion are involved at the surfaces ofthese two substrata.

The attachment of bacteria to solid surfaces inaqueous solutions involves three components:the bacterial surface, the solid substratum, andthe surrounding liquid phase. The bacterial sur-face may bear attachment structures, such aspili, or polymers which act as adhesives (18).The properties of these cell surface componentsare determined by the physiology of the orga-nism and vary with growth (3) and environmen-tal conditions (6). The properties of the substra-tum and medium components are probablyeasier to define. Solid surfaces, such as plastics,minerals, and metals, have been described interms of surface charge or surface free energy(21), and these characteristics have then beenrelated to the frequency with which bacteriaattach to the various surfaces (7, 13). Marinebacteria (13; G. Loeb, personal communication)and freshwater bacteria (H. Pringle, personalcommunication) frequently attach more readilyto low-energy (hydrophobic) surfaces than tohigh-energy (hydrophilic) surfaces. However,although clean substrata can be physically and

t Present address: School of Microbiology, The Universityof New South Wales, Kensington, New South Wales 2033,Australia.

chemically characterized, the apparent charac-teristics of the surfaces change rapidly afterimmersion in an aqueous solution containingbacteria and dissolved substances. This is due tosurface adsorption of dissolved materials, par-ticularly macromolecular substances (14). It isnot understood to what extent adsorbed sub-stances condition substratum properties or howsubstratum properties may be expressedthrough an adsorbed macromolecular film.The adsorption of macromolecular substances

to a solid surface should alter both its surfacecharge and its surface free energy. Neihof andLoeb (19, 20) have shown a convergence of theelectrophoretic mobilities (apparent surfacecharge) of heterogeneous particulates in naturalseawater compared with artificial seawater(ASW) and presented evidence that the conver-gence resulted from the presence of organicmolecules (presumably macromolecules) in thenatural seawater. Similarly, the adsorption oforganic materials and colloidal clays in soilsystems alters the apparent surface charge prop-erties of bacteria (15, 17; T. Santoro and G.Stotzky, Bacteriol. Proc., p. 3, 1967).

Unlike measurements of surface charge (aselectrophoretic mobility) that are carried out on

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VOL. 44, 1982

particulates still suspended in the aqueousphase, most evaluations of surface free energy(e.g., critical surface tension) (21) are carried outin air or in a vapor phase. Usually, surface freeenergies are indirectly evaluated by measuringthe contact angles made by sessile drops ofvarious liquids of known surface tensions (i.e.,surface free energies) on the surface being evalu-ated. Theoretically, in a series of liquids ofdecreasing surface tensions, the liquid whichjust wets the solid, thus giving a contact angle of00, should have a surface tension the same as thesurface free energy of the solid. However, sur-face energy measurements of surfaces with ad-sorbed macromolecular films are more difficult,as the adsorbed film must be air dried beforecontact angles can be measured (1). This pro-duces irreversible changes in the configurationof adsorbed macromolecules. Thus, an evalua-tion of the effect of adsorption of macromol-ecules on the surface energy of a solid should becarried out in aqueous solution, as was done inthis investigation by using an air bubble contactangle method.The adsorption of different proteins to a sur-

face can affect subsequent bacterial attachment(8). This investigation attempted to determinewhether the surface-conditioning effect of ad-sorbed macromolecules can be evaluated bymeasuring the contact angles of air bubbles onthe surfaces and whether these contact angledata reflect the surface properties which affectbacterial attachment to protein-coated surfaces.

MATERIALS AND METHODSBubble contact angle measurements. The contact

angles of air bubbles on the test substrata were mea-sured by injecting an air bubble from a syringe (0.25-mm internal diameter, with point removed and filed toa 900 bevel) into a watertight Perspex (Smith BrothersAsbestos Co. Ltd., Leicester, England) chamber con-taining the liquid, so that the bubble floated 6 to 7 mmfrom the point of release at the bottom to rest against atest surface placed horizontally on a stage at the top ofthe liquid. The average bubble size was 2.2 (±0.13)mm in diameter. Contact angles were measured direct-ly with a Vernier microscope with a goniometer eye-piece (Precision Tool and Instruments, ThorntonHeath, England); recorded values represent the meanof 18 observations (both angles of nine bubbles).Errors were within 20, unless otherwise stated, forbubbles which made contact with the surface. Forbubbles that did not make contact, a value of

186 FLETCHER AND MARSHALL

violet. All substrata were tested in replicate. Resultswere expressed both as the absorbance at 590 nm(A590) of each surface and as the index of attachment(Ia), which is the ratio of the A590 of the test substra-tum to that of the control substratum. Thus, valuesnear 1.0 indicate no effect, whereas those significantlybelow 1.0 indicate an inhibition of attachment.

Attachment in the presence of dissolved and adsorbedmacromolecules. The effect of dissolved proteins onattachment was tested by adding 0.5 ml of a BGP,BSA, or BSA-FAF solution in ASW to 4.5 ml ofbacterial suspension to give a final protein concentra-tion of 100 Fg ml-1. Attachment to TCD and PD wasthen measured as above.The effects of proteins and S1, both untreated and

pronase treated, when adsorbed to the attachmentsubstratum were tested by exposing TCD or PD to 5ml of BGP, BSA, or BSA-FAF solution (1 mg ml ofASW-1) for 20, 40, or 60 min at 22 to 24°C. The Si wasadsorbed in the same way for 1 h. For pronase-treatedadsorbed proteins, the proteins and pronase solutionswere mixed immediately before adsorption for 1 h tothe substrata (final concentrations: protein, 100 ,ugml-'; pronase, 2 ,ug ml-'). The dishes were thenrinsed three times with sterile ASW, 5 ml of bacterialsuspension was added to each dish immediately, andattachment was allowed to occur as described above.

Protease enzyme digestions. Pronase P and trypsinwere either added to the bacterial suspension at thebeginning of the 2-h attachment period to see if attach-ment could be prevented (pronase, 67 or 333 ,ug ml ofASW-1; trypsin, 33 or 167 ,ug ml ofASW-1) or used totreat attached bacteria after the 2-h attachment periodand washing with ASW to see if attached cells could beremoved (pronase, 200 ±g ml of ASW-1; trypsin, 100Fg ml of ASW-1). Control suspensions containedenzymes denatured by heating at 100°C for 40 min.

RESULTS

Effects of macromolecules on contact angles.The effects of dissolved proteins on contact

TABLE 1. Bubble contact angles obtained whenTCD and PD were exposed to various dissolved

proteinsContact angle (degrees) at following

Protein and protein concn (±g ml-'):substratum

0 0.1 1.0 10 100

BGPTCD 29 29 23 18

BACTERIAL ATTACHMENT 187

corresponding bacterial attachment experiment(see Table 5). With TCD, contact angles werenot affected by adsorbed proteins, whereas withPD, contact angles were reduced to between 54and 70°. The length of the adsorption time didnot influence the extent of reduction. With Si,contact angles on TCD were reduced, but therewas no effect on PD.

Effects of enzymes on conditioning macromol-ecules. Pronase and trypsin dissolved in ASWhad no effect on the contact angles made bybubbles on either TCD or PD surfaces. WithTCD, the addition of pronase after exposure ofthe surfaces to Si, BGP, BSA, or BSA-FAFfailed to produce any change in contact angles.However, if pronase was added to the macro-molecules before exposure of the surfaces, thenenzymatic digestion appeared to reduce the ef-fect of Si, BGP, and BSA-FAF on contactangles. This result was somewhat inconsistent,as most bubbles did not make contact with thesurface (contact angle,

188 FLETCHER AND MARSHALL

TABLE 4. Attachment to TCD and PD at 23 to 24°C and at 15°C with proteins added to the Pseludomnonassuspension

A,91) (X 103) of bacteriaTemp ('C) Protein attached to:

TCD PD TCD PD

23-24 Not added 123 (+5)" 170 (±8)BGP 72 (±4) 180 (±8) 0.59 1.06BSA 75 (±4) 144 (±7) 0.61 0.35BSA-FAF 73 (±5) 108 (±6) 0.59 0.64

15 Not added 75 (±2) 121 (±9)BGP 56 (±5) 138 (±6) 0.75 1.14BSA 62 (±4) 94 (±6) 0.83 0.78BSA-FAF 60 (±3) 69 (±2) 0.80 0.57

a Parenthetical values are standard errors of the means.

of time allowed for adsorption (TS1 from the Pseiudomonas cultureto both surfaces, attachment to TCed, whereas attachment to PD wcantly affected.

Effects of pronase digestion oftioners on their ability to inhibi

TABLE 5. Effects of adsorbed protsubsequent attachment at 23

Adsorbed Time ofsubstance adsorption (min)

BGP 20

40

60

BSA 20

40

60

BSA-FAF 20

40

60

60S1

able 5). When Although pronase adsorbed to TCD or PD hadwas adsorbed no effect on subsequent attachment, its addition'D was inhibit- to protein conditioners could affect their ability/as not signifi- to inhibit attachment (Table 6). With PD, the

addition of pronase to BGP, BSA, and BSA-protein condi- FAF before adsorption reduced their abilities toit attachment. inhibit subsequent attachment, as indicated in

Table 6 as an increase in I,. With TCD, howev-er, pronase digestion sometimes reduced the

emns and S1 on abilities of adsorbed BGP and BSA to inhibitto 24'C attachment, but the effect was not consistent

I( and varied with the attachment temperature.TCD PD Pronase had no effect on inhibition by BSA---

0.11 FAF. With SI, pronase had no significant effect0 0061 on inhibition of attachment to TCD.00.06 Effects of pronase and trypsin on bacterial0.01 011 attachment. When pronase was added to the0 0.03 bacterial suspension at the beginning of the

attachment period, attachment to both TCD and0 0.13 PD was consistently inhibited (Table 7). By0.006 0.01 contrast, denatured pronase had considerably

less effect, and at the higher pronase concentra-tion, attachment to TCD even increased. The

0.02 0.21 reason for this increase is not known. Trypsin0.02 0.12 also appeared to inhibit attachment to PD and0.005 0.16 TCD, but not to the same extent as pronase.0 0.10 Moreover, denatured trypsin also inhibited at-

tachment (Table 7). When bacteria were first0.006 0.20 allowed to attach and then were treated with0 0.11 enzymes (Table 8), pronase digestion removed

appreciable numbers of bacteria which had at-tached to TCD and PD at 20 to 25'C and those

0 0.11 which had attached to PD at 15'C. Pronase was0) 0.06 not consistently effective in removing bacteria0.02 0.10 attached to TCD at 15'C. Trypsin had little or no0 0.04 effect on the removal of attached bacteria.

Influence of time allowed for attachment on0.02 0.13 numbers of bacteria attached. When bacteria0 0.05 were allowed to attach to PD and TCD for 0.25,

1, or 2 h, there was a considerable increase in0.3 0.83 the numbers attached to PD with time, but very0 0.84 little increase in the numbers attached to TCD(Table 9). By 0.25 h, there were considerably

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BACTERIAL ATTACHMENT 189

TABLE 6. Effects of pronase on the ability of adsorbed conditioners to inhibit attachment of Pseidormonassp. at 23 to 26°C and at 15°C

Temp ( C) Adsorbed TCD PDTempt0C) ~ substanceWithout With Without Withpronase pronase pronase pronase

23-26 BGP 0.21 0.13 0.22 0.77BGP 0.03 0.07 0.39 0.54BSA 0.16 0.43 0.52 0.82BSA 0.16 0.19 0.54 0.64BSA-FAF 0.13 0.10 0.28 0.72BSA-FAF 0.02 0.07 0.33 0.59Si 0.61 0.53 0.94 0.81

15 BGP 0.14 0.79 0.05 0.99BSA 0.07 0.36 0.31 0.47BSA-FAF 0.43 0.57 0.04 0.56

0.49 0.44 0.99 1.01

more bacteria attached to TCD then to PD. By 2h, however, the numbers of bacteria attached toPD had markedly increased and exceeded thenumbers attached to TCD.

DISCUSSIONThe advantage of the bubble contact angle

technique, as compared with measurements ofcritical surface tensions (21), is that the interac-tion between an air bubble and a solid immersedin an aqueous phase is analogous to the bacteri-um-solid interaction during attachment and thusmay have some bearing on bacterial attachment.With this technique, moreover, the influence of

TABLE 7. Attachment of Pseudomonas sp. at 20 to24°C in the presence of pronase or trypsin

Treatmenta C'TCD PD

Pronase 0.06b 0.47b0.02 0.070.02 0.07

Denatured pronase 1.45b 0.89b0.75 1.020.68 0.84

Trypsin 0.48b 0.75b0.17 0.690.54 0.78

Denatured trypsin 0.08b 0.53b0.26 0.550.34 0.38

Unless stated otherwise, the concentrations wereas follows: pronase, 67 ,ug ml-'; and trypsin, 33 p.gml-'.

b Pronase concentration, 333 ,ug ml-'; trypsin con-centration, 167 ,ug ml-'.

the dissolved macromolecules on the propertiesof the substratum may be observed withoutpredrying and denaturation of the adsorbed mac-romolecules.With the bubble contact angle method, a wa-

ter-wettable (hydrophilic) surface is indicated byan angle of

190 FLETCHER AND MARSHALL

TABLE 8. Effect of pronase on Pseudomonas sp.attached at 20 to 25°C and at 15°C and then

subsequently removed by washing

Temp (°C) TreatmentTCD PD

20-25 Pronase 0.62 0.520.56 0.280.15 0.170.35 0.23

Denatured 1.19 1.23pronase 0.68 1.22

0.89 1.000.64 1.17

15 Pronase 0.53 0.341.09 0.321.04 0.20

Denatured 0.83 0.95pronase 1.39 0.90

1.44 0.79

tum surface free energy since no protein waspresent in the adjacent liquid phase. Differencesbetween contact angles achieved when the pro-teins were preadsorbed (Table 2) and dissolvedin the liquid phase (Table 1) would have beendue to differences in liquid tension and possiblydifferences in substratum properties due to dif-ferent concentrations or configurations of ad-sorbed proteins. Other examples of the influenceof adsorbed proteins on contact angles are (i) theinfluence of pronase digestion of proteins oncontact angles and the difference between con-tact angles achieved when pronase was addedbefore and after exposure of the substrata toproteins and (ii) the influence of exposure ofsubstrata to two successive proteins or mixturesof the same proteins on contact angles (Table 3).In both cases, differences in contact angles wereachieved, although the protein composition ofthe liquid phase was the same. No attempt wasmade to evaluate the surface tensions of theprotein solutions, as protein adsorption at theliquid-air interface prevents accurate measure-ment with the available techniques.

There is some correlation between the effectof proteins on bubble contact angle and bacterialattachment. Solutions of BGP, BSA, and BSA-FAF all reduced contact angles on TCD and PD,but the effect of BGP on PD was comparativelysmall (Table 1). When bacteria attached in thepresence of each of these proteins, attachmentto TCD was reduced by all of the proteins,whereas attachment to PD was reduced only byBSA and BSA-FAF (Table 4). Moreover, prot-amine sulfate had no effect on the bubble contactangle (Table 1), and in a previous study withPseudomonas sp. strain NCMB 2021 (8), prot-amine sulfate did not inhibit attachment, where-as attachment was reduced by BSA, gelatin,fibrinogen, and pepsin. These results suggestthat the bubble contact technique can detectalterations in substratum interfacial propertieswhich may influence bacterial adhesion.When BGP, BSA, and BSA-FAF were ad-

sorbed to the substrata before contact anglemeasurement, contact angles were reduced onPD but were not affected on TCD. By contrast,when S1 was preadsorbed, contact angles werereduced on TCD but were not affected on PD(Table 2). When the proteins and S1 were ad-sorbed to the substrata before bacterial attach-ment, all proteins inhibited attachment to bothsurfaces, and S1 inhibited attachment to TCDonly. Thus, the results from the contact anglemeasurements and the attachment experimentsare consistent, except for the effect of proteinson TCD. It is unclear how the adsorbed proteinscan inhibit attachment to TCD but not affectbubble contact angle. Possibly, attachment inhi-bition is due to a steric effect (16) which isdifficult to detect at such low contact angles.The interactions among the different types of

molecules (Table 3) are complex and impossibleto interpret at this stage yet are indicative ofwhat might occur in natural habitats. The inter-actions could be occurring either at the solid-liquid interface or in solution, followed by ad-sorption of the interacting molecules.Attachment inhibition was not modified by ad-sorbing BGP and BSA-FAF in succession or byadsorbing the two together.As determined by bubble contact angle mea-

TABLE 9. Increase in numbers of attached Pseudomonas sp. with time

A590 (X103) of bacteria attached to:Expt TCD PD

0.25 h 1 h 2 h 0.25 h 1 h 2 h

1 42 (+2)- 31 (+3) 41 (+1) 6 (+2) 14 (+2) 78 (+1)2 28 (±8) 40 (±4) 51 (±7) 2 (±1) 127 (+13) 170 (±10)3 34 (±1) 40 (+1) 45 (±9) 7 (±2) 93 (±15) 178 (+3)4 60 (±2) 82 (+5) 97 (±3) 34 (±4) 109 (±16) 157 (±3)

a Parenthetical values are standard errors of the means.

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BACTERIAL ATTACHMENT 191

surements, the extent of pronase modification ofconditioning properties of proteins depends onthe nature of the protein and the substratum, aswell as on the time of addition of the enzymes.The effect of proteins on contact angles wasreduced considerably when pronase was addedto the proteins before release of the bubble.When pronase was added after the bubble hadalready approached the substratum conditionedby proteins, Si or polymer fraction A, thenmacromolecules adsorbed to TCD appeared tobe protected somewhat from enzymatic action,whereas those adsorbed to PD were more ex-posed to attack. This suggests that the config-uration of the adsorbed macromolecules wasdifferent on the two surfaces and possibly thatdifferent side chains may anchor the macromol-ecules on the different substrata. These resultswere supported by studies on the effect of pro-nase on the ability of proteins to inhibit attach-ment. Pronase reduced inhibitions of attachmentto PD by BGP, BSA, and BSA-FAF (Table 6).With TCD, however, the effects of pronase oninhibition by BGP were not consistent (Table 6).This may have been related to the irregularity inthe contact angle measurements that occuredbecause the bubbles did not always make con-tact, suggesting the presence of an adsorbedlayer of water or other material which was noteasily displaced by the bubble. With TCD andthe effects of pronase on BSA and BSA-FAF,there was no correlation between contact anglemeasurements and attachment experiments, andat this point, this disparity cannot be explained.Pronase not only affected the conditioning

properties of macromolecules, but also the at-tachment process (Tables 7 and 8). Pronase and,to a lesser extent, trypsin remove some strainsof attached marine pseudomonads (5), butFletcher (10) reported that pronase, trypsin, andseveral other enzymes did not remove Pseiudo-monas sp. strain NCMB 2021 when it wasattached to glass. In this study, the removal ofattached bacteria from PD and TCD after pro-nase digestion (Table 8) indicated that a proteincomponent is involved in attachment to thesesubstrata. This is supported by the inhibition ofattachment by pronase added to the suspensionof attaching bacteria (Table 7). Although bacteri-al adhesive polymers are usually considered tobe polysaccharides (4) and polysaccharide com-ponents have been demonstrated by electronmicroscopy (4, 12), a nonpolar constituent alsomay be involved. An adhesive comprising anonpolar protein or lipid could account for thetendency of this organism to attach more readilyto hydrophobic surfaces than to hydrophilicones. The inhibition of attachment by trypsin isapparently not an enzymatic effect, as denaturedtrypsin also prevented attachment, and normal

trypsin could not remove attached cells. Anyinhibition was probably due to the effect oftrypsin on interfacial tension, as occurred withthe proteins.An extracellular polymer was isolated in an

attempt to identify the components of the super-natant responsible for decreases in bubble con-tact angle. Fraction A was similar to Si in itseffects on contact angles, and the same compo-nents could be involved. Further work is neces-sary before the similarities between fraction Aand Si and the differences between fractions Aand B can be explained.

Previous experiments have shown that anincrease in temperature can promote attachment(9), but it is not known whether this is due to atemperature influence on bacterial physiology oron the cell surface adhesive or whether thethermodynamics of the adsorption process areaffected. Hydrophobic interactions are favoredby an increase in temperature (2), but on thatbasis, temperature might be expected to affectattachment to TCD and PD somewhat different-ly. In combined results from 11 experiments,attachment to TCD and PD at 15° was lower byfactors of 0.52 ± 0.06 and 0.70 ± 0.10, respec-tively, than that at 20 to 250C (M. Fletcher,unpublished data). This suggests that tempera-ture may have a greater effect on attachment toPD than on attachment to TCD; this is consist-ent with hydrophobic interactions being thedominant type of interaction in attachment tohydrophobic substrata.

In a number of the experiments, results dif-fered markedly between the TCD and PD sub-strata (Table 4, BGP; Table 5, Si; Table 6). Thisindicates that there may be at least two possiblemodes of attachment for this organism, involv-ing either two separate adhesive polymers ortwo adsorption sites on the same polymer.Fletcher (11) also found that inhibition of attach-ment by metabolic inhibitors depended onwhether the substratum was TCD or PD and thatinhibition was more frequent with PD. Therewas little increase in the numbers of bacteriaattached to TCD between 0.25 and 2 h of expo-sure of the bacteria to the substratum, whereaswith PD there was a large increase (Table 9).The time dependence of attachment to PD.combined with inhibitor studies (11), suggeststhat physiological activity is required for attach-ment to this substratum, whereas attachment toTCD may be determined primarily by physio-chemical adsorption.

ACKNOWLEDGMENTS

We thank L. Richardson for excellent technical assistance.G. I. Loeb for very helpful discussions. and ICI Ltd. for theloan of the Vernier microscope.

This work was supported in part by a Science and Engineer-ing Research Council Visiting Fellowship research grant.

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