2 Materials
2.1 Bacterial
Virulence Attenuation
by Passaging [8]
1. Potato slices.
2. Beef bile.
3. Glycerol.
4. Mycobacterium bovis.
2.2 Bacterial
Virulence Attenuation
by Mutagenesis [9]
2.2.1 Isolation of galE
Mutants
1. S. typhi, strain Ty 2 (obtained from the World Health
Organization).
2. Brain heart infusion (BHI) broth.
3. Endo agar: Bacto peptone 10 g, galactose 10 g, K2HPO4
3.5 g, sodium sulfite 2.5 g, basic fuchsin 0.4 g, and agar 5 g
dissolved in 1 L of distilled water.
4. N-methyl-N
0 -nitro-N-nitrosoguanidine (NG).
5. JCR-170 (obtained from Dr. H.J. Creech—Chemotherapy
Laboratory, Institute for Cancer Research, Philadelphia, PA)
rIO], or ultraviolet light.
2.2.2 Stability Test
1. Swiss white mice.
2. 0.85% NaCl.
3. Potter-Elvehjem homogenizer (Arthur H. Thomas, Philadelphia, PA).
4. Endo Agar (see Subheading 2.2.1).
2.2.3 Galactose-Induced
Bacteriolysis
1. Infusion broth (Baltimore Biological Laboratories, Baltimore,
MD).
2. Shaking incubator.
3. Spectrophotometer.
4. Galactose (0.1%).
2.2.4 Determination
of Galactose Within
Bacteria
1. [14C]-galactose (18000 counts per min/mg).
2. S. typhi galE mutants.
3. BHI broth, centrifuge.
4. 0.85% NaCl.
5. Glass beads (0.17–0.18 mm).
6. Sorvall Omni-Mixer (DuPont, Norwalk, CT).
7. 1 N HClO 4 .
8. Ion exchange chromatography with Dowex 1.
Live Vaccine Attenuation
333
2.1 Bacterial
Virulence Attenuation
by Passaging [8]
1. Potato slices.
2. Beef bile.
3. Glycerol.
4. Mycobacterium bovis.
2.2 Bacterial
Virulence Attenuation
by Mutagenesis [9]
2.2.1 Isolation of galE
Mutants
1. S. typhi, strain Ty 2 (obtained from the World Health
Organization).
2. Brain heart infusion (BHI) broth.
3. Endo agar: Bacto peptone 10 g, galactose 10 g, K2HPO4
3.5 g, sodium sulfite 2.5 g, basic fuchsin 0.4 g, and agar 5 g
dissolved in 1 L of distilled water.
4. N-methyl-N
0 -nitro-N-nitrosoguanidine (NG).
5. JCR-170 (obtained from Dr. H.J. Creech—Chemotherapy
Laboratory, Institute for Cancer Research, Philadelphia, PA)
rIO], or ultraviolet light.
2.2.2 Stability Test
1. Swiss white mice.
2. 0.85% NaCl.
3. Potter-Elvehjem homogenizer (Arthur H. Thomas, Philadelphia, PA).
4. Endo Agar (see Subheading 2.2.1).
2.2.3 Galactose-Induced
Bacteriolysis
1. Infusion broth (Baltimore Biological Laboratories, Baltimore,
MD).
2. Shaking incubator.
3. Spectrophotometer.
4. Galactose (0.1%).
2.2.4 Determination
of Galactose Within
Bacteria
1. [14C]-galactose (18000 counts per min/mg).
2. S. typhi galE mutants.
3. BHI broth, centrifuge.
4. 0.85% NaCl.
5. Glass beads (0.17–0.18 mm).
6. Sorvall Omni-Mixer (DuPont, Norwalk, CT).
7. 1 N HClO 4 .
8. Ion exchange chromatography with Dowex 1.
Live Vaccine Attenuation
333