Techniques for examining Pneumocystis carinii in fresh specimens

Ruffolo, John J.; Cushion, M T; Walzer, Peter D.

doi:10.1128/jcm.23.1.17-21.1986

Cited by 26 publications

(9 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There is a distinct possibility that these elements of the cytoskeleton are lacking, or are poorly developed in P. carinii. This is consistent with the observation that living P. carinii cells, when viewed by light microscopy in fresh preparations, do not show any sign of motility [2, 3,6].…”

Section: Resultssupporting

confidence: 92%

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

Ruffolo

Cushion

Walzer

1989

The Journal of Protozoology

View full text Add to dashboard Cite

An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristae that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a "fuzzy coat" that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytes of the lung alveolus.

show abstract

Section: Resultssupporting

confidence: 92%

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

Ruffolo

Cushion

Walzer

1989

The Journal of Protozoology

View full text Add to dashboard Cite

show abstract

“…One of the problems associated with establishing a culture for P. carnii is assessment of the viability of the organisms used as inocula. Many procedures for the purification of organisms from infected rat lungs have been described (4,12,38,42,49), but few have focused on the vitality of the P. carinii postisolation (23,41). As an initial step towards defining culture conditions for P. carinii, we have explored various methods for the evaluation of organism viability.…”

mentioning

confidence: 99%

“…In previous studies, fluorogenic stains, such as calcein acetoxymethyl ester-ethidium homodimer (Live/Dead kit; Molecular Probes, Eugene, Oreg. ), and classical vital stains have been reported to be useful for this purpose (21,22,26,41). The advantage of these staining techniques is that they permit a cell-by-cell evaluation of the viability of the total cell population present.…”

mentioning

confidence: 99%

“…The advantage of these staining techniques is that they permit a cell-by-cell evaluation of the viability of the total cell population present. However, manual counting (21,22,41) or flow cytometry (26) is required to assess the percent dead versus live cells, and thus the process can be quite time consuming. In addition, preliminary in vitro drug screening studies using fluorescent staining methods in…”

mentioning

confidence: 99%

See 1 more Smart Citation

Use of an ATP bioluminescent assay to evaluate viability of Pneumocystis carinii from rats

Chen

Cushion

1994

J Clin Microbiol

Self Cite

View full text Add to dashboard Cite

A bioluminescent assay which employs the luciferin-luciferase ATP-dependent reaction was used to evaluate the viability of populations of Pneumocystis carinii derived from infected rat lungs. Contamination with host cells was reduced by a purification method which involved a combination of lowand high-speed centrifugations resulting in a 1,000-fold reduction of the rat cells while enriching for the trophic form of P. carinii. A linear correlation for the number of P. carinii nuclei versus the amount of ATP was observed. The ATP content of the organism populations could be maintained at inoculum levels for one week, although the number of organisms did not increase. Addition of respiratory chain inhibitors dramatically decreased the ATP content of the P. carinii after 24 h of incubation, with the exception of the antibiotic oligomycin B. Low concentrations of trimethoprim-sulfamethoxazole and pentamidine isethionate reduced the organism ATP content by over 50% after 24 h of exposure, while no effect was observed with 100-fold greater concentrations of ampicillin. The bioluminescent assay was found to be a more sensitive indicator of viability than a dual fluorescent staining technique. This assay does not require replication of P. carinii and should be a useful method for in vitro drug screening and viability assessment of P. carinii populations.

show abstract

“…Aliquots of these specimens were also stained with Diff-Quik and examined by light microscopy. The effects of enzyme treatment on P. carinii morphology were compared with effects on control (untreated) preparations by using procedures and criteria detailed in earlier reports (4,29).…”

mentioning

confidence: 99%

Properties of the major antigens of rat and human Pneumocystis carinii

1989

Self Cite

View full text Add to dashboard Cite

The major rat and human Pneumocystis carinii antigens were analyzed for their susceptibility to treatment with enzymes and other procedures by immunoblotting, immunofluorescence, and light microscopy. Carbohydrate residues were further analyzed by lectin-binding experiments. The 116-kilodalton (kDa) band of rat P. carinii was susceptible to proteolytic (e.g., trypsin) and glycolytic (e.g., Zymolyase) treatments but not to a variety of other procedures (e.g., lipase). This moiety reacted strongly with concanavalin A and wheat germ agglutinin, indicating the presence of mannosyl or glucosyl and N-acetylglucosamine residues. Immunofluorescence staining and surface labeling suggested that the 116-kDa antigen was located on the P. carinii cell wall. The 45-and 50-kDa bands were as sensitive as the 116-kDa band to degradative treatments when studied after immobilization onto nitrocellulose but were more resistant to proteolytic enzymes when studied in situ on whole organisms. These moieties exhibited poor binding to lectins and reactivity by surface-labeling procedures. The 116-kDa band of human P. carinii appeared to be a glycoprotein with characteristics similar to those of its counterpart in rats, whereas the human P. carinii 40-kDa band exhibited protein and carbohydrate properties more closely related to those of the 45-and 50-kDa rat-derived antigens. We conclude that P. carinii antigens are complex glycoproteins and that this information will be helpful in developing strategies for their isolation and purification and study of their function.

show abstract

Techniques for examining Pneumocystis carinii in fresh specimens

Cited by 26 publications

References 31 publications

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

Ultrastructural Observations on Life Cycle Stages of Pneumocystis carinii

Use of an ATP bioluminescent assay to evaluate viability of Pneumocystis carinii from rats

Properties of the major antigens of rat and human Pneumocystis carinii

Contact Info

Product

Resources

About