Culture Medium

2.2.1. Amoeba: Proteose-Yeast Extract-Glucose Broth (PYG)

1. 20 g Proteose peptone (Beckon Dickinson, Franklin Lakes, NJ).

2. Yeast extract (Difco, Detroit, MI).

3. 900 mL Deionized H2O.

4. Adjust pH to 6.5 with 1 M HCl and autoclave for 15 min at 121.5°C.

5. After autoclaving, add 50 mL of filter-sterilized 2 M glucose.

6. Add autoclaved and filter-sterilized stock salt solutions of the following: 10 mL 0.4 M MgSO4-7H2O, 8.0 mL 0.05 M CaCl2, 10 mL sodium citrate (Na3C6H5O7-2H2O), 10 mL 0.25 M Na2HPO4-7H2O, 10 mL 0.25 M KH2PO4.

1. Potato dextrose agar (PDA; Difco).

2. Sabouraud's dextrose agar (SAB).

1. Bacto yeast nitrogen base (Difco) supplemented with 0.5% glucose (DYNB). Adjust pH to 5.5 with 1 M HCl.

2. Bacto Sabouraud's dextrose agar (SAB; Difco).

Fig. 1. The principle of the FACSCalibur™ Optical System (16). (1) A sample consisting of particles in suspension is injected into a nozzle, and the fluid containing the particles is hydro-dynamically compressed to a thin stream, forcing the particles to flow in a single file. (2) The stream of cells intercepts the beam of the laser and absorbs the laser light. (3) As particles flow through the beam, light is scattered; scattered light is either deflected unmodified laser light or fluorescent light emitted from either a fluorochrome indigenous to the particle or one that has been attached to it. (4) Unmodified scattered laser light is measured at a low angle (FSC, forward angle light scatter) given a measurement of particle size. Scattered light can also be measured as SSC (side angle light scatter), which gives a measurement of granularity or cytoplasmic complexity. (5) The emitted light is captured by lenses and filters to measure selectively emitted light of particular wavelengths in a given detector. (6) Pulse signals sent by the detectors are converted into voltages and stored by computer software. Software integrates information from each detector for each particle.

Fig. 1. The principle of the FACSCalibur™ Optical System (16). (1) A sample consisting of particles in suspension is injected into a nozzle, and the fluid containing the particles is hydro-dynamically compressed to a thin stream, forcing the particles to flow in a single file. (2) The stream of cells intercepts the beam of the laser and absorbs the laser light. (3) As particles flow through the beam, light is scattered; scattered light is either deflected unmodified laser light or fluorescent light emitted from either a fluorochrome indigenous to the particle or one that has been attached to it. (4) Unmodified scattered laser light is measured at a low angle (FSC, forward angle light scatter) given a measurement of particle size. Scattered light can also be measured as SSC (side angle light scatter), which gives a measurement of granularity or cytoplasmic complexity. (5) The emitted light is captured by lenses and filters to measure selectively emitted light of particular wavelengths in a given detector. (6) Pulse signals sent by the detectors are converted into voltages and stored by computer software. Software integrates information from each detector for each particle.

2.3. Solutions l.

Was this article helpful?

0 0

Post a comment