Production of multivalent extracellular filtrates of <i>Staphylococcus aureus</i>

Baman, Sudhaker I.; Haque, Riaz-Ul

doi:10.1139/m70-210

Cited by 5 publications

(5 citation statements)

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“…Of the 13 cultures of staphylococci used, the Wood 46-white, Wood 46-clear, Foggie, 502A, 146P, and 681C strains of S. aureus have been previously described (Haque, 1967;Murphy and Haque, 1967;Baman and Haque, 1970). S. aureus strains 233, T19, E-delta, and N-delta were supplied by Dr S. K. Maheswaran, Department of Veterinary Bacteriology Public Health, University of Minnesota, St Paul, Minnesota; strain Wood 46M was supplied by Dr A. W. Bernheimer, Department of Microbiology, New York University School of Medicine, New York, NY; and strain PG114 was supplied by Dr F. Kapral, The Ohio State University, Columbus, Ohio.…”

Section: Methodsmentioning

confidence: 99%

“…The haemolytic activities of the filtrates were measured by the tube-titration and the substrate-agar-plate methods (Baman and Haque, 1970). Other biological activities were measured by the substrate-agar-plate method.…”

Section: Methodsmentioning

confidence: 99%

“…For haemolysins, 3% (v/v) of washed erythrocytes of rabbit, sheep, human, and horse; for lipase and esterase, respectively 3% (v/v) of Bacto Lipase Reagent (Difco) and 1 % (w/v) of a-naphthyl acetate and in both cases 0.015 % (w/v) of aniline blue solution (Allied Chemicals) as indicator; for egg-yolk factor, 5 % (v/v) of sterile egg-yolk emulsion (Oxoid); for coagulase, 20% (v/v) of sterile human plasma (BioQuest) (Bentley et al, 1967); and for phosphatase, 1 % (w/v) of phenolphthalein diphosphate (Baman and Haque, 1970).…”

Section: I Ali and Riaz-ul Haquementioning

confidence: 99%

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Recognition by Electrophoretic Localisation of the Extracellular Products of Selected Strains of Staphylococci

Ali

Haque

1974

Journal of Medical Microbiology

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PLATE XVIIT H E aim of this study was to detect a number of haemolytic and non-haemolytic extracellular products formed by selected strains of Staphylococcus aureus. For this purpose we used the electrophoretic localisation technique (Haque, 1967) by means of which the various components of the crude culture filtrates are first separated by electrophoresis in agar gel and then recognised by their biological activities. This technique has the advantages that interactions among the various products are minimised, and that the products can be identsed directly by their electrophoretic mobility and biological activities. We anticipated that the use of these criteria of identification would not only help to define the number and type of extracellular products but would aid us in selecting suitable cultures for future studies of the purification of these substances. MATERIALS AND METHODSOf the 13 cultures of staphylococci used, the Wood 46-white, Wood 46-clear, Foggie, 502A, 146P, and 681C strains of S. aureus have been previously described (Haque, 1967;Murphy and Haque, 1967;Baman and Haque, 1970). S. aureus strains 233, T19, E-delta, and N-delta were supplied by Dr S. K. Maheswaran, Department of Veterinary Bacteriology Public Health, University of Minnesota, St Paul, Minnesota; strain Wood 46M was supplied by Dr A. W. Bernheimer, Department of Microbiology, New York University School of Medicine, New York, NY; and strain PG114 was supplied by Dr F. Kapral, The Ohio State University, Columbus, Ohio. The M3 strain of S. epidermidis, a glucose-fermenting, coagulase-negative staphylococcus, was isolated in our laboratory from a marmoset.All cultures were maintained on Trypticase Soy Agar (Difco) slants. Before use they were checked for haemolytic activity by streaking for isolation on rabbit, sheep, human, and horse blood agar plates.Production of cell-free filtrates. Each culture was grown on dialysis membranes placed over Brain Liver Heart Agar (Difco) plates at 37°C for 20 hours in an atmosphere containing 10-15 % C02 in air. Growth from 25 such membranes was harvested in 12.5 ml of phosphatebuffered saline (PBS), pH 7.3, centrifuged at 3000 g for 15 min. and the supernate filtered through a sterile 0.22-pm membrane-filter.Measurement of biological activities. The haemolytic activities of the filtrates were measured by the tube-titration and the substrate-agar-plate methods (Baman and Haque, 1970). Other biological activities were measured by the substrate-agar-plate method.Preparation of substrate agar. The following substrates were separately added to a sterile 1-5 % (w/v) solution of Bacto Agar (Difco) in PBS, pH 7.3, that had been cooled to

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: I Ali and Riaz-ul Haquementioning

confidence: 99%

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Recognition by Electrophoretic Localisation of the Extracellular Products of Selected Strains of Staphylococci

Ali

Haque

1974

Journal of Medical Microbiology

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“…DNAase activity was determined by the method of Baman and Haque (1970) in which DNA is incorporated in agar; 4-mm wells were bored into the agar, and these were filled with 10, 50, 100 and 200 mg/ml solutions of exoprotein. The plates were incubated at 37°C for 5 days and examined for clearing of the agar.…”

Section: Bioiogical and Biochemical Assaysmentioning

confidence: 99%

Haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii

Barclay

Threlfall²,

Leighton³

1989

Journal of Medical Microbiology

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Summary. Of 120 laboratory-maintained strains of Listeria rnonocytogenes and two of L. ivanovii examined for haemolytic and lipolytic activity, 62 exhibited haemolytic activity alone, 20 of these showed haemolytic and lipolytic activity and 40 had neither activity. The L. ivanovii strains showed both activities. The results indicated a relationship between haemolysin production and lipolytic activity which was not explained by the serotype of the organism. In addition, the following hydrolytic activities were detected in the cell-free growth media of strains L. rnonocytogenes Boldy and L. ivanovii (formerly L. rnonocytogenes) Type 5 (substrates acted upon are given in parentheses) : acid phosphatase (4-nitrophenylphosphate, naphthyl phosphate, glycerophosphate, phosphorylcholine and GTP); neutral phosphatase (4-nitrophenylphosphate, naphthyl phosphate, phosphorycholine, NADP and UDPG); phosphodiesterase (bis-4-nitrophenylphosphate, ATP and NADP) ; NADase (NAD) ; phospholipase C (4-nitrophenylphosphoryl-choline, phosphatidyl choline and ethanolamine, and sphingomyelin) ; and lipase and esterase (triacetin, tributyrin, triolein, naphthyl-laurate, -myristate, -caprylate, -palmitate and -oleate, 4-nitrophenyl-acetate -1aurate and Tween 80). The preparations also showed weak catalase activity. No evidence was found for the presence of RNAase, DNAase, peptidase/amidase, phosphoamidase, a-amylase, glucosidase, galactosidase, pyranosidase or glucose aminidase. IntroductionThe clinical features of the diseases caused by Listeria spp. suggest that toxins and enzymes produced by the bacteria contribute to their pathogenicity. The toxicity is associated partly with the cell envelope (Stanley, 1949 ;Patocka and Mara, 1973) but much of it is associated with the extracellular products. The discovery that L. monocytogenes produces a thiol-activated haemolysin (Harvey and Faber, 1941) helped to explain the nature of the extracellular toxic activities of this organism. However, in early attempts to differentiate the toxic activities of the haemolysin from those of the cell envelope, cell-free culture filtrates rather than purified haemolysin were used. Later attempts to ascribe an enzyme activity to the haemolysin showed that the culture filtrates also contained lipolytic (Girard et al., 1963) and nicotin-

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“…DISCUSSION The results of this study indicate that some of the serologically different proteinases of staphylococci and micrococci show different electrophoretic mobilities, and that in most instances the relative order of enzyme migration was the FIG. 4. Macroelectrophoretic separation of S. aureus proteinases, group A, on cellulose acetate strips in 0.05 M barbital buffer, pH8.6, at 200 Vafter development of their reactions on casein agar.…”

Section: Scherer and Brownmentioning

confidence: 99%

Differentiation of Staphylococcal and Micrococcal Proteinases by Electrophoresis

Scherer

Brown

1974

Applied Microbiology

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The electrophoretic separation of the proteinases produced by staphylococci and micrococci was studied in four buffers. The duration of electrophoresis was based on the migration of a marker dye for a predetermined distance. The migration distances of the enzymes and dye were measured, and enzyme-dye values were calculated. A comparison of enzyme-dye values showed that complete separation of eight serologically different proteinases did not occur in any one buffer; however, in most instances, their relative order of migration was the same in all buffers. Certain strains of Staphylococcus epidermidis produced two proteinases that were different serologically as well as electrophoretically. Staphylococcus aureus strains, on the other hand, produced up to four proteinases that were serologically the same. The proteinases of staphylococci and micrococci can be best characterized by both electrophoretic and serological methods. The serological differentiation of extracellular proteinases has been used for classifying organisms of the family Micrococcaceae (7, 13). Electrophoresis has been used for the separation and identification of various proteinases (9; 0. Sandvik, Ph.D. thesis, Veterinary College of Norway, Oslo, 1962). Because eight serologically different proteinases of staphylococci and micrococci have been identified, we wanted to determine whether the enzymes also differed in their electrophoretic mobilities. The possibility existed that a preliminary identification of a proteinase could be made by electrophoresis and then confirmed with a specific antiserum. This procedure would obviate the need to test each proteinase with all eight antisera before an identification was made and thus conserve the antisera. This report describes the separation of staphylococcal and micrococcal proteinases by electrophoresis with different buffers and support media. MATERIALS AND METHODS Cultures. Fifteen cultures of Staphylococcus aureus, 67 of Staphylococcus epidermidis, and 11 of Micrococcus sp. were used in this study. All cultures were classified by the method of Baird-Parker (3) and by serological differentiation of their proteolytic enzymes (12, 13). Proteinase production by the different organisms was as follows: group A by S. aureus; groups B, C, D, F, and H by S. epidermidis; and groups E and G by Micrococcus sp. Five cultures classified as proteinase groups A, B, C, D, and E were 76E obtained from Olav Sandvik, Veterinary College of Norway, Oslo, and the other cultures, including strains of groups F, G, and H, were isolated at the National Animal Disease Center, Ames, Iowa. All cultures were isolated from milk samples of infected bovine udders except six cultures of S. aureus; five of these six were of porcine origin obtained from Richard Shuman, and one was of avian origin obtained from Kenneth Heddleston of this Center. Enzyme production. Heart infusion agar (Difco

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Production of multivalent extracellular filtrates of Staphylococcus aureus

Cited by 5 publications

References 8 publications

Recognition by Electrophoretic Localisation of the Extracellular Products of Selected Strains of Staphylococci

Recognition by Electrophoretic Localisation of the Extracellular Products of Selected Strains of Staphylococci

Haemolysins and extracellular enzymes of Listeria monocytogenes and L. ivanovii

Differentiation of Staphylococcal and Micrococcal Proteinases by Electrophoresis

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