The Role of Bacterial Cell Wall Hydrophobicity in Adhesion

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1894 VAN LOOSDRECHT ET AL APPL ENVIRON MICROBIOL, Sartorius Gottingen Federal Republic of Germany to. VAPOUR remove large cell agglomerates, Measurement of bacterial hydrophobicity i Contact angle. measurement Bacterial surfaces for measuring contact an. gles were prepared by collecting bacterial cells on 0 45 km. TSV xSL SOLID pore size micropore filters Sartorius Filters with a con. FIG 1 Relationship between the contact, tinuous bacterial layer were mounted on glass slides and. angle and the different, dried in a desiccator for 0 5 to 3 h Then the contact angle of. interfacial tensions, a 0 1 M NaCl solution with the bacterial surface was.
measured No change in contact angle occurred between 0 5. the difference between the surface free energies of cell PEG and 3 h This is in accordance with findings of Absolom et al. FBPEG and cell DEX FBDEX is smaller than the product of 1 and Busscher et al 3 Incidentally a method developed. the area occupied by the bacterium in the PEG DEX in by Absolom et al 1 was used in which a bacterial film was. terface Ai and the surface free energy of PEG DEX prepared on agar instead of on a micropore filter Contact. FPEGDEX APEG FBPEG FBDEX AiFPEGDEX Using this angles were measured directly with the aid of a microscope. equation and an FPEG DEX of 0 06 mJ m 2 16 it can be with a goniometric eyepiece Kruss GmbH Hamburg Fed. Downloaded from http aem asm org on August 2 2020 by guest. calculated that bacteria move to the interface if the differ eral Republic of Germany Each reported contact angle is. ence between the surface free energies of the bacteria in the the mean of at least six independent measurements. two phases is smaller than 0 036 mJ m2 This condition is ii Partitioning of cells in two phase systems Relative. satisfied when the bacterial surface free energy is about 58 to bacterial hydrophobicity measurements developed by Ro. 62 mJ m 2 FDEX 60 mJ m and FPEG 59 mJ m 2 senberg 13 and Gerson 8 were compared with contact. which would correspond to a contact angle of 34 to 410 11 angle measurements The first method is based on adhesion. The finding that a specific bacterial population concentrates of cells to hexadecane droplets The second method is based. at the interface can be used to check the quantitative validity on the partitioning of cells in a two phase system of an 8. of contact angle measurements DEX Pharmacia T500 6 PEG Merck 6000 solution in. In this paper data on the hydrophobicity of 23 different water The surface tensions of the PEG DEX solutions were. bacterial strains are presented and this hydrophobicity is measured with a Wilhelmy plate tensiometer. related to the adhesion of the cells to negatively charged Preparation of polystyrene disks Negatively charged poly. polystyrene In addition the mentioned methods of measur styrene latex containing OS03 groups was prepared as. ing hydrophobicity are compared and their applicability is described by Goodwin et al 10 and the initiator 3 mM. critically evaluated K2S208 was used The latex obtained was dialyzed freeze. dried and subsequently dissolved in toluene 7 wt wt A. MATERIALS AND METHODS 30 ml portion of this solution was poured into a glass petri. dish diameter 12 cm with a flat bottom which was mounted. Preparation of bacterial suspensions All strains investi horizontally The toluene was allowed to evaporate slowly. gated in this study were obtained from the culture collection for 3 days The polystyrene film obtained was cut into disks. of the Department of Microbiology Agricultural University diameter 1 cm which were stored dust free For adhesion. Wageningen The Netherlands The following strains were experiments the air dried side of the disks was used 20. used Acinetobacter sp 210A Agrobacterium radiobacter This side has a contact angle for water of 70 The number of. Alcaligenes sp A157 Arthhrobacter globiformis Ac8 Art lbro charged groups per unit of surface area could not be estab. bac ter simplex A20 Arthrobacter sp A177 Arthrobacter lished From the electrophoretic mobility of the original. sp A127 Azotiobacter vinelandii A59 Corvnebacteriiumn sp latex particles 7 8 x 10 8 m Vs in 0 01 M phosphate. C125 Escherichia coli NCTC 9002 and K 12 Microscoccus buffered saline it could be inferred that the polystyrene. Iliteus M59 Myc obacterium phlei M9 Pseuidomonas disks had a considerable negative surface potential. flluorescens P9 Pseiudomonas aeruiginosa P8 Pseiudoinonas Adhesion experiments Freshly prepared bacterial cell sus. piatida P11 Pseudomonas sp 26 3 Pse idomnonas sp P52 pensions were incubated together with polystyrene disks on. Pseiudomonas sp P80 Rhizobiiim legutminosariuin R6 a rotary shaker at 25 C After incubation for 0 5 h the disks. Rhodopseudomonas palitstris and Thiobac illus v ersutils were taken from the suspension and rinsed gently for 30 s in. ATCC 25364 0 1 M phosphate buffered saline to remove nonattached. Bacteria were grown in mineral salts medium containing cells The rinsing was performed by moving the disks slowly. the following per liter of distilled water 1 93 g of KH2PO4 through the water to prevent detachment of cells due to. 7 93 g of K2HPO4 0 75 g of NH4Cl 0 05 g of MgSO4 and 1 shear forces Possible transfer of the cells from the polysty. ml of trace element solution 24 Ethanol 4 ml liter was rene surface to the air water interface during the washing. used as the sole carbon and energy source because it has procedure could not occur because a drop of liquid always. minimal interactions with surfaces it is uncharged and has a remained on the disk during the washing procedure Rinsed. low octanol water coefficient Strains showing no growth on disks were dried and colored with Erythrocyne red The. ethanol A v inelandii E co li and M liuteius were grown on number of cells adhering to the surface were counted under. nutrient broth The incubation temperature was 30 C a light microscope with a calibrated eyepiece Surface cov. After 40 h of incubation bacteria were harvested by erage was calculated by multiplying the number of cells per. centrifugation and washed twice in 0 1 M phosphate square meter by the cross sectional area of the cell. buffered saline containing 0 29 g of KH2PO4 per liter 1 19 g. of K2HPO4 per liter and 4 93 g of NaCl per liter For. adhesion experiments cells were suspended in phosphate RESULTS. buffered saline to a final concentration of 1 x 109 to 3 x 109 In a first attempt we tried to measure contact angles of. cells per ml Before the cell suspensions were used they bacterial deposits by the method described by Absolom et al. were filtered through an 8 p m pore size micropore filter 1 Although the procedure was followed closely we were. VOL 53 1987 BACTERIAL ADHESION 1895, TABLE 1 Contact angles of water for different bacteria Three of four bacterial strains expected to concentrate at the. Contact interface actually did so The contact angle measurements. angle also have a predictive value for the adherence of bacteria to. Strain no and name,1 Pseudomonas fluorescens 21 2 1 5. negatively charged polystyrene Fig 5 Correlation between. 2 Pseudomonas aeruiginosti 25 7 0 9 coverage of a surface and contact angle measurements on. 3 Pseuidomonis piMtida 38 5 1 0 these surfaces has also been reported elsewhere 1 3 5 14. 4 Pseuidomonas sp strain 26 3 20 1 0 8 23 A good correlation between bacterial adhesion and the. S Pseudomonas sp strain 52 19 0 1 0 hexadecane test has already been reported earlier 13. 6 Pseudomonas sp strain 80 29 5 0 5,7 Escherichia coli NCTC 9002 15 7 1 2 DISCUSSION. 8 Escherichia coli K 12 24 7 0 4 Measurement of bacterial hydrophobicity can be of impor. 9 Arthrobacter globiform nis 23 1 0 7, 10 Arthrobacter simplex 37 0 0 9 tance in many research areas e g biofouling oral microbi. 11 Arthrobacter sp strain 177 60 0 1 5 ology 3 phagocytosis 22 soil microbiology etc There. 12 Arthrobacter sp strain 127 38 0 1 3 fore a good measure for bacterial hydrophobicity is needed. 13 Micrococcus lutelus 44 7 0 9 The use of a broad range of various tests 14 makes it. 14 Acinetobacter sp strain 210A 32 6 0 5 difficult to compare the outcomes of the different studies It. Downloaded from http aem asm org on August 2 2020 by guest. 15 Thiobacillius versutus 26 8 0 8 may be worthwhile to initiate some test series in different. 16 Alcaligenes sp strain 175 24 4 0 5 laboratories with a few reference strains A thorough evalu. 17 Rhodopselidomonas paliustr is 34 3 0 5 ation of the results may lead to a generally accepted standard. 18 Agrobacterilon radiobacter 44 1 0 5 hydrophobicity test In the following part we will evaluate. 19 Bacillius licheniformnis 32 6 0 5 the three methods used to measure surface hydrophobicity. 20 Corynebacter sp strain 125 70 0 3 0, 21 Azotobacter vinelandii 43 8 0 5 and discuss possible practical problems and shortcomings.
22 Rhizobiium legluminosaruin 31 0 1 0 The measurement of contact angles of an aqueous 0 1 M. 23 Mvcobacterphlei 70 0 5 0 NaCl solution with a layer of bacteria gave reproducible. results although the bacterial layer had to be dried slightly. before measurements could be performed Contact angles. correlated relatively well r 2 0 8 with the adhesion of. not able to obtain reasonable contact angles The bacteria bacteria to negatively charged polystyrene Fig 5 From. were washed away from the agar by the drop of water placed these findings and the data reported in the literature 1 3 it. on them The measured contact angles about 170 did not. can be concluded that contact angles are very useful for. differ very much for the bacteria tested and closely resem estimating the hydrophobicity of the cell surface of a given. bled the contact angle of clean agar Other investigators organism and consequently provide an important factor for. H J Busscher personal communication have had the predicting its adhesion to various surfaces Analyses of such. same experience Measurement of contact angles on bacteria. data in terms of individual surface free energies or surface. collected on micropore filters gave more meaningful results tensions as done in the equation of state and geometric. The results of these contact angle measurements with 0 1 mean approaches involves a nonthermodynamic assump. M NaCl solution and a range of different strains are summa tion and should therefore be avoided the more so as the use. rized in Table 1 The variation in contact angle was relatively of surface free energies to calculate adhesion energy 1 3 is. small 10 indicating that the bacterial film surface was restricted to cases in which bacteria and a solid make direct. rather homogeneous The contact angles for different strains contact whereby the original phase boundaries are replaced. can deviate strongly from one to another even within the. by new ones In the experiments reported here cells may. same genus No direct correlation between contact angles. adhere at a certain distance from the solid surface at the. for gram positive and gram negative cell walls was ob so called secondary minimum of the DLVO theory 15 21. served To test the agreement between the geometric mean. 3 and equation of state 1 approaches for estimation of TABLE 2 Comparison of calculated surface free energies by the. surface tension ysv or its dispersive part d the contact equation of state the geometric mean approaches. angles of c bromonaphthalene a completely apolar liquid. and 0 1 M NaCl solution on a bacterial layer were measured Equation Geometric. of state mean, Both approaches gave almost identical results Table 2 This Contact angle. approach approach, is not surprising since the geometric mean and equation of Organism mJi m2 mJ m2. state approaches have essentially the same theoretical basis. model proposed by Fowkes 7 ox Bromo,natphthalene, To examine to what extent the preparation procedure of a. bacterial layer for contact angle measurements influences Pseiudotionacs sp strain 25 20 41 68 40 70. cell surface hydrophobicity a comparison was made be 26 3. Arthlrobaicter globiforinis 20 23 42 67 42 72, tween the contact angle measurement and the behavior of. Arthirobacter sp strain 37 60 36 47 36 48, bacteria in two different two phase systems The experimen 177.
tal setup of both measurements is shown in Fig 2 From the Mi r1 o ocs lrcutells 31 44 38 56 39 60. relationship between the contact angle measurements and. the adhesion to hexadecane droplets Fig 3 we concluded Veilloniella alcales cens 57 20 28 68 27 68. that bacteria with a contact angle below 300 do not adhere to Strepto occu1s sonanulis 41 42 34 57 34 59. the hydrocarbon phase Above this critical contact angle Strepto ocse zS salivalrils 44 26 33 65 33 67. adhesion increased concomitantly with the contact angle Strepto o c us mirtio 31 55 38 49 38 53. Although important deviations occur the general trend in S sanoiis S salivarei is and S mnitior were. I The data for v aIcalesceCns, the partition of bacteria in the PEG DEX system approxi taken from Busscher et al 3 and are used here Is additional data to showr the. mately follows the contact angle measurements Fig 4 agreement between the equation of state and geometric mean approaches. 1896 VAN LOOSDRECHT ET AL APPL ENVIRON MICROBIOL,4 198 16215 19. Downloaded from http aem asm org on August 2 2020 by guest. contact angle 0, FIG 3 Relationship between contact angle and adhesion of. bacteria to hexadecane Numbers refer to the numbering of the. different bacteria in Table 1, number of hexadecane droplets obtained in the aqueous. Cell wall hydrophobicity wasmeasuredas the contact angle ofwaterona bacterial layercollected ona microfilter The contact angles ranged from 15 to 700 This method was compared with procedures based upon adhesion to hexadecane and with the partition of cells in a polyethylene glycol dextran two phase system The results obtained with these three methodsagreed reasonably well Theadhesion of 16

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