Vol. 129, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 1121-1128 Copyright X) 1977 American Society for Microbiology
Purification of Flagellar Cores of Vibrio cholerae GENE C. H. YANG,1 GORDON D. SCHRANK, AND BOB A. FREEMAN*
Department of Microbiology, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 Received for publication 5 October 1976
A procedure is described for the purification of the cores of flagella sheared from Vibrio cholerae. V. cholerae is a monotrichous organism whose flagellar core (FC) is enclosed within a sheath. The purification procedure consists of several cycles of differential centrifugation and cesium chloride density-gradient ultracentrifugation in the presence of a neutral detergent, Triton X-100. Purity of the FC preparations is assessed by electron microscopy, polyacrylamide gel electrophoresis, and chemical analysis. The purified FC preparations are devoid of flagellar sheaths and free from detectable cell wall and cytoplasmic contamination. Antibody prepared in rabbits against purified FC reacts with the flagellum of intact V. cholerae or purified FC as seen by ferritin-labeled antibody studies. Purified FC is composed of a single protein subunit with an estimated molecular weight of 45,000 g/mol and a density of about 1.3 g/cm3.
Bacterial flagella consist of three morphologically distinct components: a basal body closely associated with the cytoplasmic membrane, a hook with one end attached to the basal body and the other end protruding from the cell surface, and a distal filament portion (7). Although the exact mechanism of bacterial motility is not clearly understood, it is believed that filament and hook are responsible for the rotary movement (3, 22) while the basal body provides the necessary driving action (2, 24). The composition of filaments of many peritrichous and lophotrichous organisms has been extensively analyzed (12). The unsheathed filament uniformly consists of a single protein, called flagellin, with a molecular weight near 40,000 g/mol. Flagellins characteristically lack the amino acids cysteine and tryptophan. Relatively little is known concerning chemical composition of the filament of monotrichous organisms. Pseudomonas rhodos (18) and Vibrio parahaemolyticus (21) possess filaments consisting of a single protein. Flagellar hooks of Salmonella (13, 14) and Pseudomonas (16) are reported to consist of a homogeneous protein. The amino acid composition and antigenicity of hooks and filaments are dissimilar. The chemical composition of the basal body has not been reported. Electron microscopic studies reveal that Vibrio cholerae, as other Vibrio species, possesses a sheathed flagellum (8, 9, 10). The sheaths are readily degraded by autolysis or by brief treat-
ment with acid or urea (8). The core, on the other hand, is resistant to such treatment. The composition of neither sheath nor core of V. cholerae is known. In the present communication, procedures are described for separation of sheathed flagella of V. cholerae in relatively large quantity and further purification of flagellar cores (FC) free of sheath. MATERIALS AND METHODS The bacterial strain used was smooth V. cholerae (eltor) Inaba strain V86 which is highly motile and enterotoxigenic. Maintenance of stock cultures, selection of smooth colonies, and preparation of inocula were described previously (19). Medium. The medium employed was 6% peptone (Difco Laboratories), 0.5% NaCl, and 1.5% agar (pH 6.8 to 7.0). One liter of medium was dispensed into covered, rectangular-shaped vessels (30 by 25 by 13 cm) and autoclaved for 50 min. The solidified medium was inoculated with 10 ml of 12-h broth cultures, containing about 1010 cells per ml, and incubated at 37°C for 24 h. Ten batches of cells were harvested from an equal number of vessels yielding better than 100 g (wet weight) of cells per batch. Cells had better than 90% flagellation, as shown by electron microscopy. Isolation of flagella. A modification of the procedure of DePamphilis and Adler (6) was employed for the separation of sheared flagella. The flagella were initially separated by differential centrifugation followed by isopycnic gradient centrifugation. All procedures were conducted at 4°C; buffers contained 0.001% merthiolate (wt/vol). Bacterial cells were harvested in 0.15 M NaCl by careful scraping from the agar surface. Cells were washed once with 0.15 M NaCl and suspended at a concentration of about 1010 cells per ml in buffer
' Present address: Department of Biology, Saginaw Valley State College, University Center, MI 48710. 1121
1122
J. BACTERIOL.
YANG, SCHRANK, AND FREEMAN
containing 0.1 M tris(hydroxymethyl)aminomethane (Tris), 0.1 mM ethylenediaminetetraacetate (EDTA), sodium salt, and 1% (wt/vol) Triton X-100 (Sigma Chemical Co.; pH 7.8; TET buffer). The flagella were sheared in a Waring blender at 19,500 rpm for 45 s. The suspension was diluted with one volume of TET buffer and flagella were purified by differential centrifugation. Cell debris was removed by centrifugation in a fixed angle rotor at 10,000 x g for 10 min. The flagella were pelleted at 30,000 x g for 2 h. The differential centrifugation cycle was repeated 4x. Crude flagella were then suspended in TET buffer containing half the concentration of Triton X-100 (0.5%). Flagella purified in this manner (crude fraction) were still contaminated with vesicle-like cell debris. Crude fraction flagella were placed in cellulose acetate tubes containing 15 g of optical grade CsCl (Schwarz/Mann). The salts were quickly dissolved, and the volume was brought to 39 ml with TrisEDTA buffer (0.1 M Tris, 0.1 mM EDTA, pH 7.8). These tubes were centrifuged at 64,000 x g for 44 h in an SW 27.1 rotor (Beckman Instruments). Fractions containing 1 ml were collected and the density of representative fractions was determined by refractometer (Bausch & Lomb, Inc.). Fractions were examined by electron microscopy to identify those containing only FC. Fractions containing FC were pooled, dialyzed against Tris-EDTA buffer to remove CsCl, centrifuged at 82,000 x g for 4 h, suspended in a small quantity of Tris-EDTA buffer, and stored at 4°C. Lipopolysaccharide. Lipopolysaccharide (LPS) of V. cholerae strain V86 was prepared by the method of Westphal (26). Immunization. Adult white New Zealand outbred rabbits were immunized with 80 ,g of intact FC according to the following schedule: on days 1 and 10, 10 ,ug was injected subcutaneously; on days 4, 7, 13, and 17, 15 ,ug was injected intravenously. Ten days after the last injection animals were exsanguinated and serum was separated and frozen in 5ml portions. The same immunization schedule was used for LPS with 20 ,tg given subcutaneously and 40 ug given intravenously. Electron microscopy. Copper grids, 300 mesh, were coated with 0.4% Formvar in dichloroethane (wt/vol; Ladd Research Industries, Inc.) and carbon stabilized. Grids were inverted on drops of the following reagents, drained on filter paper, and transferred to the next reagent as follows: (i) suspension of bacteria or purified FC, 45 s; (ii) distilled water, 10 s; and (iii) negative staining with uranyl acetate (1% wt/vol), 45 s. Negative staining for sheaths was by the method of Follett and Gordon (8); grids were floated and drained with the following reagents: (i) suspension of bacteria or purified FC, 45 s; (ii) 1% potassium iodide (wt/vol), 10 s; (iii) two consecutive drops of distilled water, 10 s each; and (iv) 1% ammonium molybdate (wt/vol), 10 s. Ferritin-antibody staining was used to show localization of antibody on whole bacterial cells. Bacteria were grown in heart infusion broth (Difco), washed twice with 5 mM phosphate-buffered saline (PBS), and suspended at an optical density at 525 nm of 0.340.
Equal volumes of the bacterial suspension and 1:10 dilution of test antisera were mixed and incubated at 37°C for 30 min. The reacted bacteria were washed three times with PBS, suspended to the original volume, mixed with an equal volume of a 1:4 dilution of ferritin-conjugated antibody (goat anti-rabbit globulin, Cappel Laboratories, Inc.), and incubated at 37°C for 30 min. The organisms were washed three times with PBS and suspended. Copper grids were floated on 1 drop of this suspension for 45 s and drained. All preparations were viewed with a Phillips E. M. 200 electron microscope at 60 kV. Polyacrylamide gel electrophoresis. FC were solubilized by incubation in 3 mM HCl and 1% sodium dodecyl sulfate (SDS) for 30 min at 60°C and subjected to electrophoresis on 7% polyacrylamide gels (5) at constant current of 2 mA per tube. Untreated FC were also analyzed in the alkaline and acidic gels of Davis (5) and Reisfeld (17), respectively. For SDS-polyacrylamide gel electrophoresis, FC were dialyzed against 10 mM sodium phosphate buffer (pH 7.0) and solubilized by 1% SDS and 1% 2mercaptoethanol for 3 h at 37°C. These preparations were analyzed electrophoretically with continuous 10% polyacrylamide-SDS gels as described by Weber and Osborn (25) at a constant current of 8 mA per tube. Gels were stained with Coomassie blue stain (0.05% Coomassie brilliant blue, 25% isopropanol, 10% ethanol) and destained with 7.5% acetic acid. Molecular weights of solubilized FC were determined by comparison to a plot of log molecular weights versus electrophoretic mobilities of marker proteins (25). Chemical analysis. Total carbohydrate was measured as glucose equivalents by the anthrone method of Scott and Melvin (20), with purified dextran 500 (Sigma) as a standard. Total inorganic phosphorus was determined by the procedure of Chen et al. (4) after hydrolysis in 6 N H2SO4 at 100°C for 2 h. Protein concentrations were estimated by the method of Lowry et al. (15).
RESULTS Electron microscopic observation of pellets of crude fraction flagella following five cycles of differential centrifugation in the presence of Triton X-100 revealed about an equal number of vesicles and FC. Although these vesicles did not adhere to the FC in the presence of the neutral detergent, it was not possible to separate the two by this procedure. In the absence of Triton X-100 the vesicles appeared to be bound to the FC. This detergent was essential for the initial separation of vesicle and FC. CsCl gradient centrifugation of the above crude fraction flagella was necessary for purification of FC. The crude fraction formed two translucent bands in the gradient. The upper band, located near a density of 1.3 g/cm3 of CsCl, contained purified FC as shown in Fig. 1. About 400 mg of the purified FC was obtained
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FIG. 1. Electron micrograph of V. cholerae, strain V86, flagellar cores from a representative fraction after CsCl density gradient ultracentrifugation. Bar represents 0.1 ttm. 1123
1124
from 1,000 g (wet weight) of vibrios. The lower band located at a density of 1.33 g/cm3 of CsCl contained vesicles without detectable FC (Fig. 2). The results of isopycnic gradient centrifugation density determinations for FC are shown in Fig. 3. The resolution of this density determination depended upon the sample size used and was very reliable when the crude fraction material was obtained from approximately 70 g (wet weight) of washed cells. As the initial sample preparation size increased, the linearity of the gradient was somewhat disrupted. In contrast to the findings of DePamphilis and Adler (6), Triton X-100 was not restricted to the top of the gradient, but it appeared to be distributed throughout the gradient. The morphology of intact flagella present on bacterial cells and of isolated FC was studied by electron microscopy. The measurement of these structures as seen on electron micrographs (Fig. 1 and 4) revealed that the diameter of the intact flagellum (i.e., sheath and core) was 23.2 nm (±+0.29 nm, n = 30). The cores of the intact flagella measured 10.5 nm (+0.12, n = 30). The diameter of the purified FC (Fig. 1) was 10.0 nm (±0.36 nm, n = 30). Therefore, the diameters of the intact cores and the FC were not significantly different, suggesting the absence of sheath material on the purified FC. This conclusion was supported by the finding that 0.01 N HCl or 6 M urea produced swelling of sheath material of flagella on intact cells but did not affect the morphology of purified FC. Antibody to purified FC was shown to react with flagella on intact bacterial cells as detected by the indirect test with ferritin-conju-
:4
J BACTERIOL
YANG, SCHRANK, AND FREEMAN
J t;
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gated antiglobulin (Fig. 5). Antibody was shown to react with the purified FC by the same technique. This FC antibody immobilized viable cholera organisms and was not reactive with LPS in capillary tube precipitation tests. When viable organisms were treated with antibody to FC and observed with phase contrast microscopy, the bacteria formed loose agglutinates; individual cells occasionally dissociated and demonstrated altered motility with nondirectional rotation. However, antibody to LPS did not precipitate FC in capillary tube precipitation tests; as expected, the LPS antibody agglutinated viable vibrios in a characteristic granular clump which resulted in mechanical immobilization. These findings also support the
0.4
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JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 1121-1128 Copyright X) 1977 American Society for Microbiology
Purification of Flagellar Cores of Vibrio cholerae GENE C. H. YANG,1 GORDON D. SCHRANK, AND BOB A. FREEMAN*
Department of Microbiology, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 Received for publication 5 October 1976
A procedure is described for the purification of the cores of flagella sheared from Vibrio cholerae. V. cholerae is a monotrichous organism whose flagellar core (FC) is enclosed within a sheath. The purification procedure consists of several cycles of differential centrifugation and cesium chloride density-gradient ultracentrifugation in the presence of a neutral detergent, Triton X-100. Purity of the FC preparations is assessed by electron microscopy, polyacrylamide gel electrophoresis, and chemical analysis. The purified FC preparations are devoid of flagellar sheaths and free from detectable cell wall and cytoplasmic contamination. Antibody prepared in rabbits against purified FC reacts with the flagellum of intact V. cholerae or purified FC as seen by ferritin-labeled antibody studies. Purified FC is composed of a single protein subunit with an estimated molecular weight of 45,000 g/mol and a density of about 1.3 g/cm3.
Bacterial flagella consist of three morphologically distinct components: a basal body closely associated with the cytoplasmic membrane, a hook with one end attached to the basal body and the other end protruding from the cell surface, and a distal filament portion (7). Although the exact mechanism of bacterial motility is not clearly understood, it is believed that filament and hook are responsible for the rotary movement (3, 22) while the basal body provides the necessary driving action (2, 24). The composition of filaments of many peritrichous and lophotrichous organisms has been extensively analyzed (12). The unsheathed filament uniformly consists of a single protein, called flagellin, with a molecular weight near 40,000 g/mol. Flagellins characteristically lack the amino acids cysteine and tryptophan. Relatively little is known concerning chemical composition of the filament of monotrichous organisms. Pseudomonas rhodos (18) and Vibrio parahaemolyticus (21) possess filaments consisting of a single protein. Flagellar hooks of Salmonella (13, 14) and Pseudomonas (16) are reported to consist of a homogeneous protein. The amino acid composition and antigenicity of hooks and filaments are dissimilar. The chemical composition of the basal body has not been reported. Electron microscopic studies reveal that Vibrio cholerae, as other Vibrio species, possesses a sheathed flagellum (8, 9, 10). The sheaths are readily degraded by autolysis or by brief treat-
ment with acid or urea (8). The core, on the other hand, is resistant to such treatment. The composition of neither sheath nor core of V. cholerae is known. In the present communication, procedures are described for separation of sheathed flagella of V. cholerae in relatively large quantity and further purification of flagellar cores (FC) free of sheath. MATERIALS AND METHODS The bacterial strain used was smooth V. cholerae (eltor) Inaba strain V86 which is highly motile and enterotoxigenic. Maintenance of stock cultures, selection of smooth colonies, and preparation of inocula were described previously (19). Medium. The medium employed was 6% peptone (Difco Laboratories), 0.5% NaCl, and 1.5% agar (pH 6.8 to 7.0). One liter of medium was dispensed into covered, rectangular-shaped vessels (30 by 25 by 13 cm) and autoclaved for 50 min. The solidified medium was inoculated with 10 ml of 12-h broth cultures, containing about 1010 cells per ml, and incubated at 37°C for 24 h. Ten batches of cells were harvested from an equal number of vessels yielding better than 100 g (wet weight) of cells per batch. Cells had better than 90% flagellation, as shown by electron microscopy. Isolation of flagella. A modification of the procedure of DePamphilis and Adler (6) was employed for the separation of sheared flagella. The flagella were initially separated by differential centrifugation followed by isopycnic gradient centrifugation. All procedures were conducted at 4°C; buffers contained 0.001% merthiolate (wt/vol). Bacterial cells were harvested in 0.15 M NaCl by careful scraping from the agar surface. Cells were washed once with 0.15 M NaCl and suspended at a concentration of about 1010 cells per ml in buffer
' Present address: Department of Biology, Saginaw Valley State College, University Center, MI 48710. 1121
1122
J. BACTERIOL.
YANG, SCHRANK, AND FREEMAN
containing 0.1 M tris(hydroxymethyl)aminomethane (Tris), 0.1 mM ethylenediaminetetraacetate (EDTA), sodium salt, and 1% (wt/vol) Triton X-100 (Sigma Chemical Co.; pH 7.8; TET buffer). The flagella were sheared in a Waring blender at 19,500 rpm for 45 s. The suspension was diluted with one volume of TET buffer and flagella were purified by differential centrifugation. Cell debris was removed by centrifugation in a fixed angle rotor at 10,000 x g for 10 min. The flagella were pelleted at 30,000 x g for 2 h. The differential centrifugation cycle was repeated 4x. Crude flagella were then suspended in TET buffer containing half the concentration of Triton X-100 (0.5%). Flagella purified in this manner (crude fraction) were still contaminated with vesicle-like cell debris. Crude fraction flagella were placed in cellulose acetate tubes containing 15 g of optical grade CsCl (Schwarz/Mann). The salts were quickly dissolved, and the volume was brought to 39 ml with TrisEDTA buffer (0.1 M Tris, 0.1 mM EDTA, pH 7.8). These tubes were centrifuged at 64,000 x g for 44 h in an SW 27.1 rotor (Beckman Instruments). Fractions containing 1 ml were collected and the density of representative fractions was determined by refractometer (Bausch & Lomb, Inc.). Fractions were examined by electron microscopy to identify those containing only FC. Fractions containing FC were pooled, dialyzed against Tris-EDTA buffer to remove CsCl, centrifuged at 82,000 x g for 4 h, suspended in a small quantity of Tris-EDTA buffer, and stored at 4°C. Lipopolysaccharide. Lipopolysaccharide (LPS) of V. cholerae strain V86 was prepared by the method of Westphal (26). Immunization. Adult white New Zealand outbred rabbits were immunized with 80 ,g of intact FC according to the following schedule: on days 1 and 10, 10 ,ug was injected subcutaneously; on days 4, 7, 13, and 17, 15 ,ug was injected intravenously. Ten days after the last injection animals were exsanguinated and serum was separated and frozen in 5ml portions. The same immunization schedule was used for LPS with 20 ,tg given subcutaneously and 40 ug given intravenously. Electron microscopy. Copper grids, 300 mesh, were coated with 0.4% Formvar in dichloroethane (wt/vol; Ladd Research Industries, Inc.) and carbon stabilized. Grids were inverted on drops of the following reagents, drained on filter paper, and transferred to the next reagent as follows: (i) suspension of bacteria or purified FC, 45 s; (ii) distilled water, 10 s; and (iii) negative staining with uranyl acetate (1% wt/vol), 45 s. Negative staining for sheaths was by the method of Follett and Gordon (8); grids were floated and drained with the following reagents: (i) suspension of bacteria or purified FC, 45 s; (ii) 1% potassium iodide (wt/vol), 10 s; (iii) two consecutive drops of distilled water, 10 s each; and (iv) 1% ammonium molybdate (wt/vol), 10 s. Ferritin-antibody staining was used to show localization of antibody on whole bacterial cells. Bacteria were grown in heart infusion broth (Difco), washed twice with 5 mM phosphate-buffered saline (PBS), and suspended at an optical density at 525 nm of 0.340.
Equal volumes of the bacterial suspension and 1:10 dilution of test antisera were mixed and incubated at 37°C for 30 min. The reacted bacteria were washed three times with PBS, suspended to the original volume, mixed with an equal volume of a 1:4 dilution of ferritin-conjugated antibody (goat anti-rabbit globulin, Cappel Laboratories, Inc.), and incubated at 37°C for 30 min. The organisms were washed three times with PBS and suspended. Copper grids were floated on 1 drop of this suspension for 45 s and drained. All preparations were viewed with a Phillips E. M. 200 electron microscope at 60 kV. Polyacrylamide gel electrophoresis. FC were solubilized by incubation in 3 mM HCl and 1% sodium dodecyl sulfate (SDS) for 30 min at 60°C and subjected to electrophoresis on 7% polyacrylamide gels (5) at constant current of 2 mA per tube. Untreated FC were also analyzed in the alkaline and acidic gels of Davis (5) and Reisfeld (17), respectively. For SDS-polyacrylamide gel electrophoresis, FC were dialyzed against 10 mM sodium phosphate buffer (pH 7.0) and solubilized by 1% SDS and 1% 2mercaptoethanol for 3 h at 37°C. These preparations were analyzed electrophoretically with continuous 10% polyacrylamide-SDS gels as described by Weber and Osborn (25) at a constant current of 8 mA per tube. Gels were stained with Coomassie blue stain (0.05% Coomassie brilliant blue, 25% isopropanol, 10% ethanol) and destained with 7.5% acetic acid. Molecular weights of solubilized FC were determined by comparison to a plot of log molecular weights versus electrophoretic mobilities of marker proteins (25). Chemical analysis. Total carbohydrate was measured as glucose equivalents by the anthrone method of Scott and Melvin (20), with purified dextran 500 (Sigma) as a standard. Total inorganic phosphorus was determined by the procedure of Chen et al. (4) after hydrolysis in 6 N H2SO4 at 100°C for 2 h. Protein concentrations were estimated by the method of Lowry et al. (15).
RESULTS Electron microscopic observation of pellets of crude fraction flagella following five cycles of differential centrifugation in the presence of Triton X-100 revealed about an equal number of vesicles and FC. Although these vesicles did not adhere to the FC in the presence of the neutral detergent, it was not possible to separate the two by this procedure. In the absence of Triton X-100 the vesicles appeared to be bound to the FC. This detergent was essential for the initial separation of vesicle and FC. CsCl gradient centrifugation of the above crude fraction flagella was necessary for purification of FC. The crude fraction formed two translucent bands in the gradient. The upper band, located near a density of 1.3 g/cm3 of CsCl, contained purified FC as shown in Fig. 1. About 400 mg of the purified FC was obtained
*:. fl
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.:
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'.
4
Il
, ms t
7
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1.
.:
.
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t,
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.\
.i
i@&J6X: i.:!
.4*q
FIG. 1. Electron micrograph of V. cholerae, strain V86, flagellar cores from a representative fraction after CsCl density gradient ultracentrifugation. Bar represents 0.1 ttm. 1123
1124
from 1,000 g (wet weight) of vibrios. The lower band located at a density of 1.33 g/cm3 of CsCl contained vesicles without detectable FC (Fig. 2). The results of isopycnic gradient centrifugation density determinations for FC are shown in Fig. 3. The resolution of this density determination depended upon the sample size used and was very reliable when the crude fraction material was obtained from approximately 70 g (wet weight) of washed cells. As the initial sample preparation size increased, the linearity of the gradient was somewhat disrupted. In contrast to the findings of DePamphilis and Adler (6), Triton X-100 was not restricted to the top of the gradient, but it appeared to be distributed throughout the gradient. The morphology of intact flagella present on bacterial cells and of isolated FC was studied by electron microscopy. The measurement of these structures as seen on electron micrographs (Fig. 1 and 4) revealed that the diameter of the intact flagellum (i.e., sheath and core) was 23.2 nm (±+0.29 nm, n = 30). The cores of the intact flagella measured 10.5 nm (+0.12, n = 30). The diameter of the purified FC (Fig. 1) was 10.0 nm (±0.36 nm, n = 30). Therefore, the diameters of the intact cores and the FC were not significantly different, suggesting the absence of sheath material on the purified FC. This conclusion was supported by the finding that 0.01 N HCl or 6 M urea produced swelling of sheath material of flagella on intact cells but did not affect the morphology of purified FC. Antibody to purified FC was shown to react with flagella on intact bacterial cells as detected by the indirect test with ferritin-conju-
:4
J BACTERIOL
YANG, SCHRANK, AND FREEMAN
J t;
,.r
>
gated antiglobulin (Fig. 5). Antibody was shown to react with the purified FC by the same technique. This FC antibody immobilized viable cholera organisms and was not reactive with LPS in capillary tube precipitation tests. When viable organisms were treated with antibody to FC and observed with phase contrast microscopy, the bacteria formed loose agglutinates; individual cells occasionally dissociated and demonstrated altered motility with nondirectional rotation. However, antibody to LPS did not precipitate FC in capillary tube precipitation tests; as expected, the LPS antibody agglutinated viable vibrios in a characteristic granular clump which resulted in mechanical immobilization. These findings also support the
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