Use of a Detergent–Acid Mixture to rupture Tissue Culture Cells

Fildes, Celia; Hart, P. D’Arcy

doi:10.1038/214624a0

Cited by 5 publications

(3 citation statements)

References 4 publications

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“…Assessment of Intracdlular Bacterial Multiplication.--At appropriate intervals the frequency distribution of acid-fast bacilli in the cells on the cover slips was determined, together with a parallel enumeration of the proportion of cells shed into the medium that were heavily infected (10). Since for the most part the cell layers were substantially intact at the times of assessment, these counts agreed with others obtained by total recovery of bacteria from the whole cell culture (13). The mean number of acid-fast bacilli per macrophage on the cover slips was recorded graphically.…”

Section: Methodssupporting

confidence: 82%

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RESPONSE OF CULTURED MACROPHAGES TO MYCOBACTERIUM TUBERCULOSIS, WITH OBSERVATIONS ON FUSION OF LYSOSOMES WITH PHAGOSOMES

Armstrong

Hart

1971

The Journal of Experimental Medicine

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917

536

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The cytological response to the ingestion of tubercle bacilli by cultured mouse peritoneal macrophages has been studied by electron microscopy. Methods included a quantitative assessment based on systematic surveying of cell profiles, and of phagosomes and their contained bacteria, encountered in thin sections; classification of the sectioned bacteria into visibly damaged and apparently intact categories; prelabeling of dense granules (secondary lysosomes) with ferritin as an aid to identifying the occurrence and frequency of phagosome-lysosome fusion; and monitoring of bacterial growth and viability by light microscopy and cultural counts. The situations studied were as follows: progressive infection with the multiplying virulent strain H37Rv; ingestion of the same strain previously inactivated by gamma radiation; infection with an attenuated strain (BCG); and a stabilized virulent infection induced by the surfactant Macrocyclon. In the bacterial suspensions used routinely for inoculation, about half the bacilli were viable, matching closely the proportions of intact and damaged organisms identified with the electron microscope. In the inoculated macrophages, some phagosomes containing intact bacilli and others containing damaged bacilli were always to be found; but the proportion of organisms scored as damaged increased, and that of intact organisms decreased, in situations where the population as a whole had been rendered nonviable before inoculation, or where they became so intracellularly as in the late stages of a BCG infection. Evidence of fusion of ferritin-marked lysosomes with some bacterium-containing phagosomes was obtained in all experiments, but a significant difference was regularly observed according to whether the bacilli were damaged or intact. Virtually all phagosomes containing damaged bacilli showed signs of fusion; but when many phagosomes were present containing apparently intact organisms (as with actively multiplying strain H37Rv or with this strain held at a steady level of viability by Macrocyclon, and also with strain BCG at an early stage of that infection), signs of fusion of lysosomes with these phagosomes were infrequent. From these findings it is inferred that intracellular survival of M. tuberculosis in cultured macrophages is associated with a tendency to nonfusion of dense granules with the phagosome, thus avoiding direct exposure of the bacilli to the contents of these organelles. It is suggested, further, that fusion of dense granules with the phagosome, leading to digestion, is determined by recognition of the bacillus as nonviable. The possibility is discussed that the cytological response to different mycobacterial infections may reflect differences of a basic nature between facultative and obligate intracellular parasitism.

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Section: Methodssupporting

confidence: 82%

“…Thus at least up to halfway through the monolayer survival period after a heavy" tuberculous infection there was a marked tendency for the phagosomes containing intact bacilli not to have fused with ferritin-marked lysosomes (Figs. [11][12][13], at a time when those containing FIGS. 11-13.…”

Section: Quantitative Assessment Of Fusionmentioning

confidence: 99%

RESPONSE OF CULTURED MACROPHAGES TO MYCOBACTERIUM TUBERCULOSIS, WITH OBSERVATIONS ON FUSION OF LYSOSOMES WITH PHAGOSOMES

Armstrong

Hart

1971

The Journal of Experimental Medicine

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917

536

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“…Then the macrophages, with intracellular M. leprae, were incubated in situ in the Leighton tubes for 9 days with 1 ~tCi [Me-3H]thymidine or 1 #Ci [Me-3H]thymine (both at 0.14 /LM) in 1 ml macrophage medium. Medium was changed at 3 days and 6 days, then at 9 days radioactivity was determined in acid insoluble material of M. leprae after lysing macrophages by incubating for 1 h at 37°C in 0.1% cetrimide in 0.1 M HC1 [7] and washing M. leprae organisms on GFC filters at least four times with buffered Tween 80 [5]. Aseptic technique and materials were used throughout; incubations and media were at 37 ° C and Leighton tubes allowed to equilibrate with an atmosphere of 5% CO2/95% humidity for 20 rain before sealing.…”

Section: Incubations Of Intracellular M Leprae With Radioisotopicallmentioning

confidence: 99%

Pyrimidine scavenging by Mycobacterium leprae

Wheeler

1989

FEMS Microbiology Letters

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Mycobacterium leprae incorporated exogenously supplied pyrimidenes as bases or nucleosides, but not as a nucleotide, into its nucleic acids. Notably, thymine was incorporated ∼5 times more rapidly than thymidine by bith suspensions of, or intracellular M. leprae. Thymine incorporation was significantly inhibited by clofazamine and dapsone at near‐pharmacological levels. Therefore, incorporation of thymine is preferable as an activity for assessing viability of M. leprae. Nucleosides were converted to nucleotides through kinases, bases through phosphoribosyltransferases. Alternatively, thymine and uracil could first be converted to nucleosides. Cytosine and uracil bases were interconvertable, and uracil alone could supply all the pyrimidine requirements of M. leprae, though conversion to the thymine base was extremely slow. Overall, pyrimidine scavenging occurs at a slower rate than, and appears not to be so important as purine scavenging in M. leprae.

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Mycobacterium tuberculosis in Macrophages: Effect of Certain Surfactants and Other Membrane-Active Compounds

Hart

1968

Science

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Some compounds, not directly inhibitory or enhancing, nevertheless influence growth of tubercle bacilli in macrophages in cell culture. They include certain surfactants whose effects can be varied by their structural design. The compounds are probably stored in cell lysosomes. They can interact with various membranes to affect permeability. The anti- and protuberculous surfactants differ in such interaction and also in effect on lysosomal enzyme activity in infected macrophages. A link between the effect on lysosomal membranes and on tuberculous infection is suggested.

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Use of a Detergent–Acid Mixture to rupture Tissue Culture Cells

Cited by 5 publications

References 4 publications

RESPONSE OF CULTURED MACROPHAGES TO MYCOBACTERIUM TUBERCULOSIS, WITH OBSERVATIONS ON FUSION OF LYSOSOMES WITH PHAGOSOMES

RESPONSE OF CULTURED MACROPHAGES TO MYCOBACTERIUM TUBERCULOSIS, WITH OBSERVATIONS ON FUSION OF LYSOSOMES WITH PHAGOSOMES

Pyrimidine scavenging by Mycobacterium leprae

Mycobacterium tuberculosis in Macrophages: Effect of Certain Surfactants and Other Membrane-Active Compounds

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