Letter to the Editor Cellufluor Staining of Bronchoalveolar Lavage Samples Baselski et al. (1) have reported that Cellufluor (Calcofluor white) can be used for rapid detection of Pneumocystis carinii cysts in bronchoalveolar lavage samples (BALs). We have also found this method to be extremely useful for the rapid diagnosis of Pneumocystis infecton and infections by other opportunistic fungi in BALs from immunocompromised patients. Contrary to their results, however, we have found that Calcofluor white from suppliers other than Polysciences, Inc., (Fungi-Fluor Kit; Polysciences, Inc., Warrington, Pa.) bring about comparable staining of P. cannii cysts. We have found that the Calcofluor white reagent supplied by Remel (Lenexa, Kans.) also beautifully demonstrates the characteristic "double-parenthesis-like" structure in P. cannii cysts in BALs from immunocompromised patients from our hospital. The Remel reagent contains an "A" solution consisting of glycerol and KOH and a "B" solution which contains the Calcofluor white reagent and Evan's blue. We found that staining only occurs if reagents A and B are both placed on the slide (for approximately 1 min) and are then rinsed off for examination. Addition of reagent B alone (without KOH) will result in staining of other fungal elements (hyphae and pseudohyphae) and not of P. cannii. According to Remel, they have recently changed the grade of Calcofluor white and are obtaining it from Polysciences, Inc., which is an alternative explanation for why it stains P. cannii cysts. From a practical standpoint, however, it is important to note that high pH, which is present in the Polysciences kit (the pH of their solution is between 11 and 12), appears to be at least one critical factor in staining of P. carinii.

Author's reply Since our initial publication on the usefulness of Cellufluor (Calcofluor white) for the rapid and sensitive detection of Pneumocystis carinii cysts in bronchoalveolar lavage samples (1), there have been several independent verifications (2, 3, 4). Two controlled clinical studies have also used Fungi-Fluor (Polysciences, Inc.) (3, 4). However, the third report by Harrington and Hageage (2) noted some variability in staining activity of various preparations and included results which differed from ours. This report also reviewed many of the variables which may potentially affect stain performance. We believe the conflicting results (including the experience by Dr. Milligan with the Remel product) and the many performance variables underscore the need for each laboratory to verify the performance of any Calcofluor white product with P. carinii control slides before implementing routine use. REFERENCES

1. Baselski, V. S., M. K. Robison, L. W. Pifer, and D. R. Woods. 1990. Rapid detection of Pneumocystis cannii in bronchoalveolar lavage samples by using Cellufluor staining. J. Clin. Microbiol. 28:393-394.

1. Baselski, V. S., M. K. Robison, L. W. Pifer, and D. R. Woods. 1990. Rapid detection of Pneumocystis carinii in bronchoalveolar lavage samples by using Cellufluor staining. J. Clin. Microbiol. 28:393-394. 2. Harrington, B. J., and G. J. Hageage. 1991. Calcofluor white: tips for improving its use. Clin. Microbiol. Newsl. 13:3-5. 3. Kim, Y. K., S. Parulekar, P. K. W. Yu, R. J. Pisani, T. F. Smith, and J. P. Anhalt. 1990. Evaluation of calcofluor white stain for detection of Pneumocystis carinii. Diagn. Microbiol. Infect. Dis. 13:307-310. 4. Stratton, N., J. Hryniewicki, S. L. Aarnaes, G. Tan, L. M. de la Maza, and E. M. Peterson. 1991. Comparison of monoclonal antibody and calcofluor white stains for the detection of Pneumocystis carinii from respiratory specimens. J. Clin. Microbiol 29:645-647.

Tom Milligan Department of Pathology Saint Louis University Hospital St. Louis, Missouri 63110

Vickie Baselski Department of Pathology University of Tennessee, Memphis Memphis, Tennessee 38163

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