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. 1986 Jan;23(1):17–21. doi: 10.1128/jcm.23.1.17-21.1986

Techniques for examining Pneumocystis carinii in fresh specimens.

J J Ruffolo, M T Cushion, P D Walzer
PMCID: PMC268565  PMID: 2422197

Abstract

Pneumocystis carinii was examined in fresh preparations of infected rat lung homogenates and tissue culture supernatants by a variety of light microscope techniques, vital dyes, and histologic stains. Phase-contrast microscopy, Nomarski interference-contrast microscopy, and bright-field microscopy with oblique illumination provided excellent views of P. carinii. Erythrosin B, and to a lesser extent trypan blue, were helpful in assessing organism viability. The use of Triton X-100-Giemsa stain permitted differentiation of the developmental stages in the P. carinii life cycle. The techniques developed here are easily adaptable to the microbiology laboratory and thus should have important clinical and research applications.

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Selected References

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  1. Bartlett M. S., Verbanac P. A., Smith J. W. Cultivation of Pneumocystis carinii with WI-38 cells. J Clin Microbiol. 1979 Dec;10(6):796–799. doi: 10.1128/jcm.10.6.796-799.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barton E. G., Jr, Campbell W. G., Jr Pneumocystis carinii in lungs of rats treated with cortisone acetate. Ultrastructural observations relating to the life cycle. Am J Pathol. 1969 Feb;54(2):209–236. [PMC free article] [PubMed] [Google Scholar]
  3. Bowling M. C., Smith I. M., Wescott S. L. A rapid staining procedure for Pneumocystis carinii. Am J Med Technol. 1973 Jul;39(7):267–268. [PubMed] [Google Scholar]
  4. CHALVARDJIAN A. M., GRAWE L. A. A NEW PROCEDURE FOR THE IDENTIFICATION OF PNEUMOCYSTIS CARINII CYSTS IN TISSUE SECTIONS AND SMEARS. J Clin Pathol. 1963 Jul;16:383–384. doi: 10.1136/jcp.16.4.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cushion M. T., Ruffolo J. J., Linke M. J., Walzer P. D. Pneumocystis carinii: growth variables and estimates in the A549 and WI-38 VA13 human cell lines. Exp Parasitol. 1985 Aug;60(1):43–54. doi: 10.1016/s0014-4894(85)80021-7. [DOI] [PubMed] [Google Scholar]
  6. Cushion M. T., Walzer P. D. Cultivation of Pneumocystis carinii in lung-derived cell lines. J Infect Dis. 1984 Apr;149(4):644–644. doi: 10.1093/infdis/149.4.644. [DOI] [PubMed] [Google Scholar]
  7. Cushion M. T., Walzer P. D. Growth and serial passage of Pneumocystis carinii in the A549 cell line. Infect Immun. 1984 May;44(2):245–251. doi: 10.1128/iai.44.2.245-251.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Domingo J., Waksal H. W. Wright's stain in rapid diagnosis of Pneumocystis carinii. Am J Clin Pathol. 1984 Apr;81(4):511–514. doi: 10.1093/ajcp/81.4.511. [DOI] [PubMed] [Google Scholar]
  9. Engelberg L. A., Lerner C. W., Tapper M. L. Clinical features of Pneumocystis pneumonia in the acquired immune deficiency syndrome. Am Rev Respir Dis. 1984 Oct;130(4):689–694. doi: 10.1164/arrd.1984.130.4.689. [DOI] [PubMed] [Google Scholar]
  10. Felegie T. P., Pasculle A. W., Dekker A. Recognition of Pneumocystis carinii by gram stain in impression smears of lung tissue. J Clin Microbiol. 1984 Dec;20(6):1190–1191. doi: 10.1128/jcm.20.6.1190-1191.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gay J. D., Smith T. F., Ilstrup D. M. Comparison of processing techniques for detection of Pneumocystis carinii cysts in open-lung biopsy specimens. J Clin Microbiol. 1985 Feb;21(2):150–151. doi: 10.1128/jcm.21.2.150-151.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haverkos H. W. Assessment of therapy for pneumocystis carinii pneumonia. PCP Therapy Project Group. Am J Med. 1984 Mar;76(3):501–508. doi: 10.1016/0002-9343(84)90670-3. [DOI] [PubMed] [Google Scholar]
  13. Hughes W. T. Current status of laboratory diagnosis of Pneumocystis carinii pneumonitis. CRC Crit Rev Clin Lab Sci. 1975 Sep;6(2):145–170. doi: 10.3109/10408367509151568. [DOI] [PubMed] [Google Scholar]
  14. Jackson P. R., Pappas M. G., Hansen B. D. Fluorogenic substrate detection of viable intracellular and extracellular pathogenic protozoa. Science. 1985 Jan 25;227(4685):435–438. doi: 10.1126/science.2578226. [DOI] [PubMed] [Google Scholar]
  15. Kovacs J. A., Hiemenz J. W., Macher A. M., Stover D., Murray H. W., Shelhamer J., Lane H. C., Urmacher C., Honig C., Longo D. L. Pneumocystis carinii pneumonia: a comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies. Ann Intern Med. 1984 May;100(5):663–671. doi: 10.7326/0003-4819-100-5-663. [DOI] [PubMed] [Google Scholar]
  16. Levin M., McLeod R., Young Q., Abrahams C., Chambliss M., Walzer P., Kabins S. A. Pneumocystis pneumonia: importance of gallium scan for early diagnosis and description of a new immunoperoxidase technique to demonstrate Pneumocystis carinii. Am Rev Respir Dis. 1983 Jul;128(1):182–185. doi: 10.1164/arrd.1983.128.1.182. [DOI] [PubMed] [Google Scholar]
  17. Libertin C. R., Woloschak G. E., Wilson W. R., Smith T. F. Analysis of Pneumocystis carinii cysts with a fluorescence-activated cell sorter. J Clin Microbiol. 1984 Nov;20(5):877–880. doi: 10.1128/jcm.20.5.877-880.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lim S. K., Eveland W. C., Porter R. J. Development and evaluation of a direct fluorescent antibody method for the diagnosis of Pneumocystis carinii infections in experimental animals. Appl Microbiol. 1973 Nov;26(5):666–671. doi: 10.1128/am.26.5.666-671.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Macher A. M., Shelhamer J., MacLowry J., Parker M., Masur H. Pneumocystis carinii identified by gram stain of lung imprints. Ann Intern Med. 1983 Oct;99(4):484–485. doi: 10.7326/0003-4819-99-4-484. [DOI] [PubMed] [Google Scholar]
  20. Mahan C. T., Sale G. E. Rapid methenamine silver stain for Pneumocystis and fungi. Arch Pathol Lab Med. 1978 Jul;102(7):351–352. [PubMed] [Google Scholar]
  21. Masur H., Jones T. C. The interaction in vitro of Pneumocystis carinii with macrophages and L-cells. J Exp Med. 1978 Jan 1;147(1):157–170. doi: 10.1084/jem.147.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Milder J. E., Walzer P. D., Coonrod J. D., Rutledge M. E. Comparison of histological and immunological techniques for detection of Pneumocystis carinii in rat bronchial lavage fluid. J Clin Microbiol. 1980 Apr;11(4):409–417. doi: 10.1128/jcm.11.4.409-417.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murphy M. J., Pifer L. L., Hughes W. T. Pneumocystis carinii in vitro: A study by scanning electron microscopy. Am J Pathol. 1977 Feb;86(2):387–401. [PMC free article] [PubMed] [Google Scholar]
  24. Murray J. F., Felton C. P., Garay S. M., Gottlieb M. S., Hopewell P. C., Stover D. E., Teirstein A. S. Pulmonary complications of the acquired immunodeficiency syndrome. Report of a National Heart, Lung, and Blood Institute workshop. N Engl J Med. 1984 Jun 21;310(25):1682–1688. doi: 10.1056/NEJM198406213102529. [DOI] [PubMed] [Google Scholar]
  25. Ognibene F. P., Shelhamer J., Gill V., Macher A. M., Loew D., Parker M. M., Gelmann E., Fauci A. S., Parrillo J. E., Masur H. The diagnosis of Pneumocystis carinii pneumonia in patients with the acquired immunodeficiency syndrome using subsegmental bronchoalveolar lavage. Am Rev Respir Dis. 1984 Jun;129(6):929–932. doi: 10.1164/arrd.1984.129.6.929. [DOI] [PubMed] [Google Scholar]
  26. Pifer L. L., Hughes W. T., Murphy M. J., Jr Propagation of Pneumocystis carinii in vitro. Pediatr Res. 1977 Apr;11(4):305–316. doi: 10.1203/00006450-197704000-00010. [DOI] [PubMed] [Google Scholar]
  27. Pintozzi R. L. Modified Grocott's methenamine silver nitrate method for quick staining of Pneumocystis carinii. J Clin Pathol. 1978 Aug;31(8):803–805. doi: 10.1136/jcp.31.8.803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rosen P. P., Martini N., Armstrong D. Pneumocystis carinii pneumonia. Diagnosis by lung biopsy. Am J Med. 1975 Jun;58(6):794–802. doi: 10.1016/0002-9343(75)90634-8. [DOI] [PubMed] [Google Scholar]
  29. Shelhamer J. H., Ognibene F. P., Macher A. M., Tuazon C., Steiss R., Longo D., Kovacs J. A., Parker M. M., Natanson C., Lane H. C. Persistence of Pneumocystis carinii in lung tissue of acquired immunodeficiency syndrome patients treated for pneumocystis pneumonia. Am Rev Respir Dis. 1984 Dec;130(6):1161–1165. doi: 10.1164/arrd.1984.130.6.1161. [DOI] [PubMed] [Google Scholar]
  30. Stover D. E., Zaman M. B., Hajdu S. I., Lange M., Gold J., Armstrong D. Bronchoalveolar lavage in the diagnosis of diffuse pulmonary infiltrates in the immunosuppressed host. Ann Intern Med. 1984 Jul;101(1):1–7. doi: 10.7326/0003-4819-101-1-1. [DOI] [PubMed] [Google Scholar]
  31. Thomson R. B., Jr, Smith T. F. Acridine orange staining of Pneumocystis carinii. J Clin Microbiol. 1982 Jul;16(1):191–192. doi: 10.1128/jcm.16.1.191-192.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Von Behren L. A., Pesanti E. L. Uptake and degradation of Pneumocystis carinii by macrophages in vitro. Am Rev Respir Dis. 1978 Dec;118(6):1051–1059. doi: 10.1164/arrd.1978.118.6.1051. [DOI] [PubMed] [Google Scholar]
  33. Walzer P. D., Powell R. D., Jr, Yoneda K. Experimental Pneumocystis carinii pneumonia in different strains of cortisonized mice. Infect Immun. 1979 Jun;24(3):939–947. doi: 10.1128/iai.24.3.939-947.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Walzer P. D., Powell R. D., Jr, Yoneda K., Rutledge M. E., Milder J. E. Growth characteristics and pathogenesis of experimental Pneumocystis carinii pneumonia. Infect Immun. 1980 Mar;27(3):928–937. doi: 10.1128/iai.27.3.928-937.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Walzer P. D., Rutledge M. E. Humoral immunity in experimental Pneumocystis carinii infection. I. Serum and bronchial lavage fluid antibody responses in rats. J Lab Clin Med. 1981 Jun;97(6):820–833. [PubMed] [Google Scholar]
  36. Yoneda K., Walzer P. D. Attachment of Pneumocystis carinii to type I alveolar cells studied by freeze-fracture electron microscopy. Infect Immun. 1983 May;40(2):812–815. doi: 10.1128/iai.40.2.812-815.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yoshida Y., Shiota T., Yamada M., Matsumoto Y. Further light microscopic studies on morphology and development of Pneumocystis carinii. Zentralbl Bakteriol Mikrobiol Hyg A. 1981;250(1-2):213–218. [PubMed] [Google Scholar]

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