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L G Wayne

Publications and source records attributed to L G Wayne.

At least 37 records · Page 2Linked to original sources
PubMed1986

A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels.

Mycobacteria produce two classes of catalase, designated T and M. Only the T-catalase also has a peroxidase-like function. When a 3,3'-diaminobenzidine (DAB) peroxidase stain was applied to polyacrylamide gel electrophoresis gels, followed by a ferricyanide negative stain for catalase, isoenzymes of T-catalase appeared as dark bands within a zone of clearing in the green background; the M-catalase appeared only as a clear zone. Heated and unheated preparations could be used to demonstrate the presence of comigrating bands of M and T. The application of the ferricyanide stain after the DAB stain of T-catalase resulted in marked intensification of the positive bands of T-catalase due to nonenzymatic, peroxide-independent reduction of the ferricyanide by the DAB product.

Catalase↗
PubMed1986

Catalases, peroxidases, and superoxide dismutases in Mycobacterium leprae and other mycobacteria studied by crossed immunoelectrophoresis and polyacrylamide gel electrophoresis.

The five mycobacteria Mycobacterium lepraemurium, M. leprae, M. bovis BCG, M. smegmatis, and M. intracellulare were studied. Catalase and peroxidase activities were demonstrated in polyacrylamide and crossed immunoelectrophoresis gels for M. lepraemurium, M. intracellulare, and BCG, but not for M. leprae. Peroxidase and catalase activities were associated with the same precipitate line in crossed immunoelectrophoresis for M. lepraemurium, M. intracellulare, and BCG, showing that in these mycobacteria the two enzyme activities resided in the same molecule. M. smegmatis peroxidase and catalase activities were closely associated on polyacrylamide gel electrophoresis, but on the crossed immunoelectrophoresis catalase and peroxidase activities were associated with two different precipitate lines. Catalases without peroxidase activity were demonstrated in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in M. intracellulare and M. smegmatis. The catalase without peroxidase activity in M. intracellulare was heat resistant and therefore classified as an m-catalase. In M. smegmatis the catalase without peroxidase activity was only partially heat resistant. All of the catalases with peroxidase activity were heat-sensitive t-catalases. Superoxide dismutase activity in the crossed immunoelectrophoresis was associated with the M. leprae antigen no. 4 and with cross-reacting antigens in the other mycobacteria studied. Several superoxide dismutases were demonstrated in Mycobacterium duvalii. They were antigenically different from the other superoxide dismutases in this study, as shown by lack of reactivity with a monospecific antibody to M. lepraemurium superoxide dismutase. Molecular weights were estimated for all the enzymes in this study by sodium dodecyl sulfate-polyacrylamide gels.

Catalase↗
PubMed1985

Identification of mycobacteria by specific precipitation of catalase with absorbed sera.

Cross-absorbed antisera have been prepared against catalase from reference strains of Mycobacterium asiaticum, Mycobacterium gordonae, Mycobacterium scrofulaceum, Mycobacterium simiae, and Mycobacterium szulgai. A total of 61 strains of mycobacteria were grown in small volumes of liquid medium and disrupted in sealed tubes in a cup horn sonicator, and the extracts were tested by a seroprecipitation technique against each of the reference antibody preparations. All 35 strains that belonged to one of the reference species reacted with the corresponding antibody, and none of the 61 extracts gave a significant cross-reaction with the absorbed antibody to a species to which it did not belong. This method appears to provide a rapid and accurate means of identifying mycobacterial cultures in the diagnostic laboratory.

Animals↗
PubMed1985

The "atypical" mycobacteria: recognition and disease association.

Although techniques based on immunologic or chromatographic analyses have been described for identifying mycobacteria in clinical laboratories, most microbiologists continue to rely on a series of specialized physiological and biochemical tests for this purpose. The recognition of additional significant species over the past decade has required the addition of more tests to the battery used for mycobacterial identification. This paper will review briefly the taxonomic status of species likely to be encountered in clinical specimens and the most useful tests for characterizing them. Strategies will be presented for using these tests in the most efficient way to provide optimal resolution of taxa without use of an unreasonably large battery of tests. A brief survey of techniques that may become more practical in the future will also be included.

Agglutination Tests↗
PubMed1984

Diagnostic probability matrix for identification of slowly growing mycobacteria in clinical laboratories.

A probability matrix is presented for identification of slowly growing mycobacteria that are likely to be encountered in clinical laboratories. The matrix includes 23 features that are useful for identifying members of 14 species or species complexes. The computer program identifies strains as a function of the ID (identification) score, which measures the discrimination among possible alternative identifications, and the R (ratio) score, which measures the degree of fit to the most likely taxa. It is not necessary to employ all 23 tests when initiating an identification; the program will suggest additional tests to perform when a partial data set fails to yield a definitive identification. Two independent sets of cultures comprising a total of 1,212 strains were used to test the matrix. Correct diagnoses were based on clustering behavior in numerical taxonomic analysis with larger numbers of features. The probable efficiencies with the two sets were 94.2 and 83.4%, respectively, and the accuracy of the definitive identifications for both sets exceeded 95%. A discussion is presented of situations when it may be appropriate to override an R score that has caused the rejection of an identification and to thereby enhance the efficiency.

Catalase↗
PubMed1982

Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions.

Tuberculosis is characterized by periods in which the disease may be quiescent or even clinically inapparent, but in which tubercle bacilli persist and retain the potential to reactivate the disease. The present study was carried out in pursuit of an in vitro model which might contribute to the understanding of the physiology of nonreplicating persisters, with oxygen limitation used as the means of inducing this state. When actively growing aerated cultures of Mycobacterium tuberculosis were suddenly placed under anaerobic conditions the bacilli died rapidly, with a half-life of 10 h. When the bacilli were grown in liquid medium without agitation, they adapted to the microaerophilic conditions encountered in the sediment; the adapted bacilli in the sediment did not replicate there but were tolerant of anaerobiosis, exhibiting a half-life of 116 h. Among the early events associated with the adaptation were the synthesis of an antigen designated URB, the function of which is not known, and a fourfold increase in isocitrate lyase activity. The bacilli later exhibited a 10-fold increase in synthesis of a glycine dehydrogenase that catalyzes the reductive amination of glyoxylate, concomitantly oxidizing NADH to NAD. Specific activities of other enzymes studied were either not affected or moderately diminished in the sedimented bacilli. It is proposed that the glyoxylate synthesis in this model serves mainly to provide a substrate for the regeneration of NAD that may be required for the orderly completion of the final cycle of bacillary replication before oxygen limitation stops growth completely. This orderly shutdown is essential to continued survival of M. tuberculosis in a quiescent form.

Adaptation, Physiological↗
PubMed1982

Microbiology of tubercle bacilli.

Based on conventional taxonomic analyses, as well as molecular level studies on DNA and catalases, the tuberculosis complex (M. tuberculosis, M. microti, M. bovis, and M. africanum) appears to consist of a single species. This species does not occur normally free in nature, but depends on host-to-host transmission for its continued existence. Members of the complex are sufficiently distinct genetically from known free-living mycobacteria so that it is unlikely that they would evolve from the free-living species at a significant frequency in nature, although that is presumably how they arose in the first place. Therefore, eradication of tuberculosis seems to be a realistic hope. The technical means now exist for control of the disease; some improvements in diagnostic technology can be expected to accelerate the control efforts. At a basic science level, more information is needed about the ability of M. tuberculosis to survive in a latent state in a host, only to revive years later, if the control of tuberculosis is to progress to eradication.

Bacteriological Techniques↗
PubMed1979

Antigenic differences between extracts of actively replicating and synchronized resting cells of Mycobacterium tuberculosis.

Protein extracts were prepared from aerobically replicating cells of Mycobacterium tuberculosis and from synchronized non-replicating cells derived from the sediments of non-agitated cultures. Although both preparations share a number of antigens, the extracts of the non-replicating cells also contain antigenic components that are not shared by the replicating cells, and which can be isolated and visualized by immunoaffinity chromatography and immunoelectrophoretic techniques.

Anaerobiosis↗
PubMed1978

A co-operative numerical analysis of Mycobacterium gastri, Mycobacterium kansasii and Mycobacterium marinum.

A co-operative taxonomic study has been performed on slowly growing photochromogenic mycobacteria (Runyon Group I) and closely related organisms. Phenetic data on 54 strains, studied in seven laboratories, were collected and analysed by numerical taxonomic methods. Immunological properties and phage susceptibility patterns were analysed independently to establish correlation with numerical classification. Mycobacterium gastri, M. kansasii and M. marinum appeared as distinct well-defined clusters and the serological and phage data supported the resolution of these three species. A table of definitive properties is presented. Two strains each of M. simiae and M. asiaticum formed a loose cluster which was clearly separated from the previously mentioned three species; the small number of strains examined precluded the establishment of a list of definitive properties of these two species. It is concluded that the Runyon Groups, which provided a practical though arbitrary basis for establishment of a series of co-operative studies, have served their purpose and should now be supplanted by classification and nomenclature based on species.

Bacteriophage Typing↗
PubMed1978

Functional heterogeneity of rabbit peritoneal and alveolar macrophages activated with Mycobacterium bovis BCG.

The biological activity of rabbit peritoneal and alveolar macrophages activated with M. bovis BCG was investigated by use of tests measuring their phagocytic and staphylocidal properties. Macrophages were separated in a discontinuous albumin gradient which yielded 4 to 5 bands depending on the source of the exudate. The peritoneal exudate had about 40% of cells recovered in band E and the alveolar exudate had about 35% in band C. There were not significant differences between the uptake by three subpopulations of macrophages. The alveolar macrophages from normal rabbits were less active than peritoneal macrophages. They were also less active those from vaccinated animals. Subpopulations of band B macrophages killed S. aureus more efficient than those from band C. Such differences reflect the number of mature or immature cells in each subpopulations. Mature cells (bond B and C) possess phagocytic and bactericidal activities whereas immature cells (bond D) are capable of ingesting bacteria but no killing them.

Animals↗
PubMed1977

Altered surface properties of Escherichia coli associated with a specific amino acid change in the S12 ribosomal protein of streptomycin-resistant mutants.

Escherichia coli mutants resistant to streptomycin exhibited differences in countercurrent distribution from the parental strains. The degree of difference from the parental strain correlated with the degree of restriction of translation and thus the particular strA allele. The changes in countercurrent distribution in the phase systems used probably resulted predominantly from surface charge alterations. The differences in countercurrent distribution in these and other mutants may be a useful selective technique to obtain different types of mutants for which specific selective techniques may not be available. In addition, it appears that the surface properties of cells, which determine their position in countercurrent distribution, are a function of the translational efficiency and fidelity, and that the surface of cells consists of a mosaic that is an expression of this translational fidelity.

Countercurrent Distribution↗
PubMed1977

Synchronized replication of Mycobacterium tuberculosis.

When Mycobacterium tuberculosis was grown in Tween-albumin broth without any agitation, the bacilli replicated in the upper, oxygen-rich portion of the medium at a rate that was just balanced by the rate at which the bacilli settled toward the bottom of the tube. When the organisms that accumulated in the sediment were suspended and diluted into fresh medium, they exhibited synchronous replication. The bacilli initiated ribonucleic acid synthesis immediately upon suspension, but marked deoxyribonucleic acid synthesis was not apparent until after the first cellular division was completed, about 14 h after suspension.

DNA↗
PubMed1976

Dynamics of submerged growth of Mycobacterium tuberculosis under aerobic and microaerophilic conditions.

When Mycobacterium tuberculosis is grown in detergent-containing medium under continous agitation, multiplication is known to follow a logarithmic mode. When the cultures are not continuously shaken, but only agitated a few times a week to resuspend the bacilli and permit turbidity to be measured, the net increase suggests an arithmetic growth mode. It is shown here that a single pulse of aeration of an unshaken submerged culture of M. tuberculosis causes an almost instantaneous acceleration of growth, followed rapidly by a cessation of growth. Whether or not the bacilli will subsequently resume growth depends on the bacillary population density of the cuture at the time of application of the pulse of aeration. If the bacilli are permitted to grow in the depths of Dubos Tween Albumin broth without any agitation, they exhibit net arithmetic growth and attain a maximal population density greater than is seen in cultures exposed to occasional pulses of aeration. By the use of isotopically labeled cells, it has been shown that replication occurs ar a logarithmic rate amoung the small proportion of the bacilli that remain suspended in nonagitated cultures. This replication is balanced by settling of cells, resulting in a net appearance of arithmetic multiplication. The cells that have settled into the sediment replicate at a very slow rate, if at all, but do retain their viability for 4 weeks or longer. This suggests a possible analogy to quiescent tubercle bacilli in vivo.

Aerobiosis↗
PubMed1975

Laboratory services for mycobacterial diseases.

The philosophy of the recently proposed "Levels of Laboratory Service" program, which will be so vital to the conduct of a successful outpatient tuberculosis treatment and control program, is presented. The hallmark of this program is the decentralization of the diagnostic/monitoring services as they involve laboratory participation. In the long run this could mean more efficient operation, more reliable reporting, and probably less work for the participating laboratories. The greater emphasis on smear examination (Level I) as a monitoring tool will mean fewer cultures, thereby lessening the load for those laboratories that once went through countless clinically requested exercises of repetitively proving by culture the existence of M. tuberculosis in a given patient. Doubtless, the bulk of the work will be conducted in Level II laboratories; but here, too, identification of the most easily defined pathogen, M. tuberculosis, will minimize the over-all workload for these investigators while decreasing their concern about mycobacteria other than tubercle bacilli. Expertise gained in frequent repetitions of a limited number of tests (niacin, nitrate reduction, and pH 7/68 degrees C catalase) will ensure reliable speciation of the clinically most important Mycobacterium. The work of Level III laboratories should eventually be reduced primarily to organisms other than M. tuberculosis, thereby ensuring that a number of highly competent reference institutions will not only attain proficiency in taxonomic aspects of mycobacteria, but will also reflect the regional picture of the changing patterns in mycobacterial pathogens of man. Participation of laboratories in proficiency testing programs will encourage top-level performance in all areas. Additionally, such testing programs will serve a teaching role; a laboratory need not feel "locked in" at a given service level, but may increase its proficiency and move up a step in terms of the service it provides. In contrast, no laboratory need feel compelled to increase its activities; if daily workloads limit the extent of their involvement with mycobacteria, these laboratories can be confident that other institutions are providing needed services. The success of the entire "Levels of Laboratory Service" program depends on the recognition by individual laboratories of their own workload limitation, the directed motivation of personnel, and the maintenance of a free and open pipeline of communication to laboratories at the next higher level of service.

Bacteriological Techniques↗
PubMed1974

Separation of erythromycin-resistant and -susceptible subpopulations of Escherichia coli 15 by partition in two-polymer aqueous phases.

Partition of cells in two-polymer aqueous phases depends on subtle differences in the cells' surface properties (primarily surface charge). A culture of Escherichia coli 15 arg(-) was subjected to countercurrent distribution in a dextranpolyethylene glycol aqueous phase system and found to consist of two well-differentiated subpopulations. Clones derived from these two subpopulations (designated clones 5 and 6) exhibited characteristic partitions and were stable on subculture. Clone 5 cells were found to be susceptible to erythromycin and clone 6 cells were resistant. When a culture of clone 5 was exposed to erythromycin, resistant mutants were selected with the same partition as clone 6. Countercurrent distribution in two-polymer aqueous phase systems is thus shown to be a sensitive method for detecting some heterogeneities of bacterial populations and resolving such mixtures. Possible clinical implications of changes in bacterial surface properties associated with acquired drug resistance are discussed.

Chemical Phenomena↗