Detection of fungi using fluorescent brighteners

Introduction

Certain fluorescent dyes such as Blankophor have a high affinity for the  b -glycosidically linked polysaccharides such as glucan and chitin, which are main the constituents of the fungal cell wall.  Therefore, these fluorescent dyes can be used for screening clinical samples for the presence of fungal elements.  This procedure can be performed using the following specimens: Nail, Skin, Bronchial alveolar lavage fluid, Sputum and Biopsies.

Materials

  • Blankophor P (Bayer, Cat. No. C52349) OR Fluorescent brightener (Sigma, Cat. No. F-3543)
  • 20% Potassium hydroxide (Sigma, Cat. No. P-1767)

Equipment

  • Fluorescent microscope (excitation below 400nm and a barrier filter at 420nm)
  • Slides
  • Coverslips

 Procedure

1) Prepare a 0.25mg/ml working solution of the fluorescent brightener in 20% potassium hydroxide.  Store in a polyethylene tube in the fridge, in the dark.

2) Place a couple of drops of the fluorescent brightener working solution onto a clean, labelled slide and add the specimen.  Lay a coverslip over the specimen, avoiding air bubbles.

3) Leave for 1-15 minutes to allow for maceration of the specimen.

4) Scan the slide using a fluorescent microscope on low magnification (x10).
Stained mounts can be kept in a moist chamber in the dark room at room temperature for several days.

Tips and general comments

1) Fungal elements in extremely alkaline milieu will produce a bright yellowish fluorescence, while at a moderate alkaline pH a bright blue fluorescence will appear. Elements exhibiting yellow or blue fluorescence should be examined at a higher magnification to confirm the presence of fungal elements.

2) Experience is required in scanning slides as other elements, such as clothing fibres, will also fluoresce.

3) High background may result from: a preparation that is too thick, insufficient incubation time or  incorrect strength of KOH.  It is recommended that you gently tap the preparation with the end of a spatula, increase the incubation time for the specimen or repeat test with a fresh preparation of KOH.

4) We use a strain of Candida albicans as a positive control with each batch of isolates to be tested.

5) If the positive control does not fluoresce this may be due to incorrect strength or deterioration of fluorescent brightener.  Repeat the test using a fresh batch of fluorescent brightener.

6) Standard microbiological technique is adequate for safety whilst performing this procedure.  Gloves should be worn whilst handling any patient sample containing body fluids.  All sputum and bronchial alveolar lavage fluid samples should be tested at containment level 3 (U.K standards), using the appropriate precautions.

References

Monod M, Baudraz-Rosselet F, Ramelet AA and Frenk E (1989) Direct Mycological Examination in Dermatology: a Comparison of Different Methods.  Dermatologica, 179, 183-186

Image of Fluorescence microscopy with A.fumigatus

Image of A.fumigatus stained with Blankophor®

Year prepared: 

2003