2 Materials
2.1 Mass
Spectrometer
and Software
All procedures described use the Microflex mass spectrometer
(Bruker Daltonics) system and attached software. Leptospira isolates (see Chapter 1) are used as the starting biological material.
1. MALDI-ToF Microflex (Bruker Daltonics).
2. Maldi Biotyper 3.0 (Bruker Daltonics).
2.2 Leptospira
Culture
For MALDI-ToF MS analysis, fresh cultures at the late exponential
phase are used (see Note 1).
1. Leptospira spp.-type strains: These strains are used for the
creation of a MS database.
2. Leptospira isolates: Leptospira are isolated as described and
routinely subcultured in EMJH (Chapter 1).
3. Ellinghausen-McCullough-Johnson-Harris (EMJH) culture
medium [2, 12]: Dissolve 2.3 g of Difco™ Leptospira Medium
Base EMJH (Becton Dickinson) in 900 mL of purified water.
Add 100 mL of Difco™ Leptospira Enrichment EMJH (Becton Dickinson).
2.3 Media
and Reagents
1. Phosphate buffered saline (PBS): 10 mM Na 2 PO 4 , 1.8 mM
KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4.
2. Sterile ultrapure water.
3. Absolute ethanol.
4. Formic acid: 70% solution in water.
5. Acetonitrile.
6. HCCA matrix solution: 10 mg/mL of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/2.5% trifluoroacetic acid
(see Note 2).
2.4 Target Plate,
Other Material,
Plasticware,
and Consumables
1. Ground steel 96-spot target plate.
2. Benchtop centrifuge for microtubes.
3. Micropipettes (1–10; 10–100; 100–1000).
4. Pipette tips.
5. Biosafety cabinet.
6. Microtubes (1.5 mL).
7. Nitrile gloves.
8. Safety glasses.
24
Dominique Girault et al.
2.1 Mass
Spectrometer
and Software
All procedures described use the Microflex mass spectrometer
(Bruker Daltonics) system and attached software. Leptospira isolates (see Chapter 1) are used as the starting biological material.
1. MALDI-ToF Microflex (Bruker Daltonics).
2. Maldi Biotyper 3.0 (Bruker Daltonics).
2.2 Leptospira
Culture
For MALDI-ToF MS analysis, fresh cultures at the late exponential
phase are used (see Note 1).
1. Leptospira spp.-type strains: These strains are used for the
creation of a MS database.
2. Leptospira isolates: Leptospira are isolated as described and
routinely subcultured in EMJH (Chapter 1).
3. Ellinghausen-McCullough-Johnson-Harris (EMJH) culture
medium [2, 12]: Dissolve 2.3 g of Difco™ Leptospira Medium
Base EMJH (Becton Dickinson) in 900 mL of purified water.
Add 100 mL of Difco™ Leptospira Enrichment EMJH (Becton Dickinson).
2.3 Media
and Reagents
1. Phosphate buffered saline (PBS): 10 mM Na 2 PO 4 , 1.8 mM
KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4.
2. Sterile ultrapure water.
3. Absolute ethanol.
4. Formic acid: 70% solution in water.
5. Acetonitrile.
6. HCCA matrix solution: 10 mg/mL of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/2.5% trifluoroacetic acid
(see Note 2).
2.4 Target Plate,
Other Material,
Plasticware,
and Consumables
1. Ground steel 96-spot target plate.
2. Benchtop centrifuge for microtubes.
3. Micropipettes (1–10; 10–100; 100–1000).
4. Pipette tips.
5. Biosafety cabinet.
6. Microtubes (1.5 mL).
7. Nitrile gloves.
8. Safety glasses.
24
Dominique Girault et al.