Pneumocystis Carinii Stain Kit (Modified Toluidine Blue O Stain)

Solution I - Toluidine Blue O Stain
Solution 11- 95% Ethyl Alcohol
Solution III - Absolute Alcohol
Solution IV - Xylene

Pneumocystis carinii is an opportunist which causes a diffuse interstitial pneumonia in patients with impaired immune systems. The disease itself can be classified as epidemic or sporadic; the latter occurring in patients who have an underlying immunosuppression. In this procedure, a sulfation reagent of glacial acetic acid and sulfuric acid (not included) is used for the removal of background material. This allows the Pneumocystis carinii cysts to be visualized more easily after staining with Toluidine Blue O. Diagnosis of Pneumocystis carinii pneumonia can frequently be made on bronchoalveolar lavage (BAL) or on touch preparations of pulmonary tissue (open lung biopsies and transbronchial biopsies).

RECOMMENDED PROCEDURES: BAL Specimens:
Using a 50 ml tube, centrifuge for 15 minutes at 2000 x g. Aspirate all but the bottom 5 ml of supernatant. With a Pasteur pipette, aspirate the sediment plus approximately 1 ml of the remaining 5 ml of fluid. Transfer material to a 15 ml centrifuge tube, gently mix and prepare smear by spreading a drop over a I cmm area. If concentrated specimen is very thick or mucoid, spread over entire slide with care being taken not to make the smear too thick. Dry the slides on a heating block at 50 - 55 degrees C. Allow to cool before staining. When dry, process in same manner as slides of BAL. Preparation of Sulfation Reagent: In a dry coplin jar submerged in a plastic tub filled with cool water (not below 10 degrees C), mix 45 ml of glacial acetic acid with 15 ml of concentrated sulfuric acid and stir gently with glass rod. Carefully place smears in reagent for 10 minutes. Mix immediately and again after 5 minutes. Gently rinse with water for 10 minutes. Drain excess water and stain with Sol. I for 3 - 5 minutes. Rinse with Sol. II followed by Sol. III for 10 seconds each. Clear in Sol. IV, two changes each for 10 seconds and mount.

RESULTS: The cyst forms appear as lavender structures (cup-shaped) approximately 5 um in diameter. The cyst outline is distinct, and the internal region stains uniformly.

NOTE: A negative bronchoalveolar lavage does not rule out an infection with Pneumocystis carinii. Other diagnostic procedures may be necessary.

CONTENTS: Toluidine Blue O, HCL, Ethyl Alcohol, Absolute Alcohol, Xylene

These solutions are made from certified dyes (when applicable). For in vitro diagnostic use only.

ENG Scientific, Inc., 82 Industrial East, Clifton, NJ 07012. Tel: 973-472-7200; Fax: 973-472-9460.