X. METHODS IN C A M P Y L O B A C T ~ R
447
B. H antigens
l., Preparation of H antigen
The C. fetus strains are cultured as described for production of 0 antigens. The pooled saline suspensions are centrifuged for 20 min at
3000 rev/min and then for 30 min at 15,000 rev/min. The supernatant is
discarded, the sediment is carefully resuspended and washed twice in
phosphate buffered 0.8% NaCl (pH 7.2). Finally the suspension is placed
into an ultramixer (Buhler, Tubingen) for 2 min to shear off the flagella
from cell bodies. For sedimentation of intact cells and broken cell debris,
the suspension must be centrifuged at 21,000 rev/min for 1 h. The flagella
are found in the supernatant fluid (Keeler et al., 1966; Ullmann, 1973).
The purity of the material is investigated by electron microscopy. The
flagella can be enriched by deep freezing 100 ml of the suspension at
- 40°C for 24 h. After thawing out the suspension, the flagella are clumped
in the upper third of the liquid column. After centrifugation at 3000 revlmin,
for 10 min, they can easily be separated (Ullmann, 1973, 1976). For agglutination tests, the flagella are resuspended homogeneously using a Vortex
mixer.
Another possibility for purification of flagella is described by McCoy
et al. (1975). Flagella are separated from cells by five cycles of low and high
speed centrifugation, essentially as performed by Miwatani et al. (1970).
The final product is devoid of cells but contains globular bodies. It is
stored at 5°C with sodium azide (0.02%) as preservative. Flagella are further purified by isopycnic gradient centrifugation in CsCl by the method of
Shapiro and Maize1 (1973) at 25,000 rev/min, for two days at 22°C. Fractions are obtained by collecting drops either through a puncture in the
bottom of the tube, or by upward displacement using an automatic system
(ISCO Gradient Fractionator). CsCl is removed by dialysis.
2. Ultrastructure of jagella
Ultrastructural examination of the flagellum of C. fetus (McCoy et al.,
1975) shows a filament diameter exceeding 17-6 nm during the exponential
growth phase. Filament diameters increase during the course of growth,
reaching a mean width of 21.2 nm about the middle to late stationary phase.
No evidence of a flagellar sheath is observed after different treatments
(0.01 N HCl, 6 M urea, Tris buffer, warm water).
3. The amino-acid composition
The amino-acid composition of flagella from C. fetus subsp. fetus is different from that of C. fetus subsp. intestinalis (Table 111). Cysteine is absent
and proline present in traces only (Ullmann, 1976).
447
B. H antigens
l., Preparation of H antigen
The C. fetus strains are cultured as described for production of 0 antigens. The pooled saline suspensions are centrifuged for 20 min at
3000 rev/min and then for 30 min at 15,000 rev/min. The supernatant is
discarded, the sediment is carefully resuspended and washed twice in
phosphate buffered 0.8% NaCl (pH 7.2). Finally the suspension is placed
into an ultramixer (Buhler, Tubingen) for 2 min to shear off the flagella
from cell bodies. For sedimentation of intact cells and broken cell debris,
the suspension must be centrifuged at 21,000 rev/min for 1 h. The flagella
are found in the supernatant fluid (Keeler et al., 1966; Ullmann, 1973).
The purity of the material is investigated by electron microscopy. The
flagella can be enriched by deep freezing 100 ml of the suspension at
- 40°C for 24 h. After thawing out the suspension, the flagella are clumped
in the upper third of the liquid column. After centrifugation at 3000 revlmin,
for 10 min, they can easily be separated (Ullmann, 1973, 1976). For agglutination tests, the flagella are resuspended homogeneously using a Vortex
mixer.
Another possibility for purification of flagella is described by McCoy
et al. (1975). Flagella are separated from cells by five cycles of low and high
speed centrifugation, essentially as performed by Miwatani et al. (1970).
The final product is devoid of cells but contains globular bodies. It is
stored at 5°C with sodium azide (0.02%) as preservative. Flagella are further purified by isopycnic gradient centrifugation in CsCl by the method of
Shapiro and Maize1 (1973) at 25,000 rev/min, for two days at 22°C. Fractions are obtained by collecting drops either through a puncture in the
bottom of the tube, or by upward displacement using an automatic system
(ISCO Gradient Fractionator). CsCl is removed by dialysis.
2. Ultrastructure of jagella
Ultrastructural examination of the flagellum of C. fetus (McCoy et al.,
1975) shows a filament diameter exceeding 17-6 nm during the exponential
growth phase. Filament diameters increase during the course of growth,
reaching a mean width of 21.2 nm about the middle to late stationary phase.
No evidence of a flagellar sheath is observed after different treatments
(0.01 N HCl, 6 M urea, Tris buffer, warm water).
3. The amino-acid composition
The amino-acid composition of flagella from C. fetus subsp. fetus is different from that of C. fetus subsp. intestinalis (Table 111). Cysteine is absent
and proline present in traces only (Ullmann, 1976).