Apr 12, 1976 - pase, esterase, egg yolk factor, lysozyme, deoxyribonuclease, hyaluronidase, penicillinase, and alpha-, beta-, and delta-hemolysins in cell-free ...
APPUED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1976, p.
575-578
Copyright C 1976 American Society for Microbiology
Vol. 32, No. 4 Printed in U.S.A.
Agar Plate Tests of Enhanced Sensitivity for Detecting Biologically Active Products of Staphylococcal Filtrates JEROME J. MADLER, SHENG-HAO LEE, AND RIAZ-UL HAQUE* Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60680 Received for publication 12 April 1976
Optimal conditions for detecting staphylokinase, phosphatase, protease, liesterase, egg yolk factor, lysozyme, deoxyribonuclease, hyaluronidase, penicillinase, and alpha-, beta-, and delta-hemolysins in cell-free filtrates of selected strains of staphylococci by agar plate methods were established by studying the effect of factors such as buffer composition, pH, ionic strength, type of agar, nature and concentration of substrate, and certain metal ions. The final tests that evolved from this study are simple to perform, require only 6 ,u1 of the sample per test, and are capable of detecting microgram and, in some cases, nanogram quantities of the product. The zones of reaction can also be quantitatively related to the amount of material present. The test may also be useful for the detection of extracellular products of other microorganisms. pase,
Biological tests of enhanced sensitivity to detect the presence of biologically active products in the cell-free filtrates of staphylococci are needed for two reasons. First, such tests will help define the components of culture filtrates more fully, and, second, they will aid in establishing the purity of a given product during various steps of its purification. A systematic study to develop and/or adapt agar plate tests of enhanced sensitivity was thus undertaken, and our findings form the subject of this report.
and 8.6; carbonate buffer, pH 9.4, 9.7, and 10.1; and universal buffer (3), pH 5.1, 5.9, 6.7, 7.3, 8.5, 9.6, and 10.6. The pH value was determined by using a Zeromatic pH meter, model SS-3 (Beckman Instruments, Inc., Fullerton, Calif.). The optimal combination of the buffer and pH was selected and used to determine the influence of the ionic strength on the reactions. The following ionic strengths, obtained by adding solid NaCl to the selected buffer system, were used: 0.2, 1.0, 1.5, 3.0, 6.0, 9.0, 15.0, and 30.0 mmho. Ionic strength was measured with a conductivity meter (Radiometer, Copenhagen). Subsequently, the selected buffer system was adjusted to the ionic strength found to be optimal for a particuMATERIALS AND METHODS lar reaction, and this combination of physiological The Wood 46M, Wood 46W, Foggie, PG 114, 681C, conditions was used to test the effects of the types of Smith 5R, 146P, 5662, and Rl strains of Staphylococ- agar and certain metal ions on the reactions. For the cus aureus and the M3 strain of Staphylococcus epi- former, Difco agar at concentrations of 1.5 and 3.0% dermidis were used in this investigation. All of and agarose (Colab Laboratories, Inc., Glenwood, these strains, with the exception of the Smith 5R Ill.) at concentrations of 1.0 and 3.0% were tested. and the Rl strains, have been previously described For the latter, the effects of MgCl2, CaCl2, and (1, 2, 6, 7). The Smith 5R and Rl strains were FeSO4 at concentrations of 0.1, 0.5, 1.0, and 10 mM, obtained from T. Wadstrom, Bacteriology Depart- respectively, were tested. Finally, the effect of substrate concentration was tested by using the followment, Karolinska Institutet, Stockholm. Cell-free filtrates from these cultures were ob- ing concentrations: for staphylokinase, a combinatained by growing the cultures on dialysis mem- tion of fibrinogen (Nutritional Biochemicals Corp. branes placed over brain liver heart (Difco Labora- [NBC], Cleveland, Ohio) and thrombin (NBC), 0.15 tories, Detroit, Mich.) agar plates at 370C for 18 h in and 0.015%, 0.25 and 0.015%, 0.25 and 0.025%, 0.15 an atmosphere containing 10 to 15% C02 in air. The and 0.025%, 0.50 and 0.025%, 0.50 and 0.050%, regrowth from the membranes was harvested as previ- spectively; for phosphatase, phenolphthalein diously described (7) and cell-free filtrates were ob- phosphate (Eastman Organic Chemicals, Rochester, tained by centrifuging and filtering the supernatant N.Y.), 0.15, 0.30, and 0.60%, andp-nitrophenylphosthrough 0.45-Am membrane filters. All filtrates phate (Fisher Scientific Co., Pittsburgh, Pa.), 0.50 were kept frozen and were thawed only when and 1.0%; for protease, gelatin (Difco), 0.15, 0.30, and 0.60%, and casein (Hammersten, Schwarz/ needed. Initially, each of the tests was carried out in agar Mann, Orangeburg, N.Y.), 0.15, 0.30, and 0.60%; for plates separately prepared with: phosphate-buffered lipase, tributyrin (Eastman Organic Chemicals, saline (PBS), pH 7.2; phosphate buffer, pH 6.2, 6.6 Rochester, N.Y.), 0.5, 1.0, and 3.0%, lipase reagent 7.0, 7.2, and 7.4; tris(hydroxymethyl)aminomethane (Difco), 1.0%, and tripalmitin (J. T. Baker Chemical (Tris) buffer, pH 8.4 and 8.8; barbital buffer, pH 8.2 Co., Phillipsburg, N.J.), 3.0%; for esterase, Tween 575
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MADLER, LEE, AND HAQUE
APPL. ENVIRON. MICROBIOL.
Results were recorded by measuring the diameter 20 (Eastman Organic), 0.5, 1.0, and 3.0%, Tween 80 (Eastman Organic), 1.0%, and 1-naphthylacetate of the zones in millimeters. When the sample was (Eastman Organic), 3.0%; for egg yolk factor, con- quantitated on the agar plate, a plot of the diameter centrated egg yolk emulsion (Colab), 0.5, 1.0, and of the zone of activity versus the logarithm of dilu3.0%; for lysozyme, Micrococcus lysodeikticus tion yielded a straight line. When this line was (Difco), 0.15, 0.30, and 0.60%; for deoxyribonuclease extrapolated back to the diameter of the well, it (DNase), Difco DNase test agar, 2.0 and 4.0%; for gave the dilution that should be barely, if at all, dehemolysins, washed human, horse, rabbit, or sheep tectable. This was regarded as the end point, and erythrocytes, 1.0, 2.0, and 4.0%; for hyaluronidase, twice the amount of the crude product contained in potassium hyaluronidate (NBC), 0.1, 0.2, 0.3, 0.4, this well, expressed in terms of its protein content, and 0.5%; for penicillinase, penicillin G (E. R. was taken as the minimum sensitivity of the test. Squibb and Sons, Inc., New York, N.Y.; 0.05% Difco This concentration of the crude product yielded a destarch as indicator), 101, 102, 103, 104, and 105 U/ml. tectable zone of the respective activity. The protein concentration was estimated by the This sequence of experiments led to the selection of optimal conditions, as determined by the size of absorption at 555 nm, using the biuret method (4). the zone of activity or by the minimum amount of RESULTS the active product that could be detected, and these were used in performing the particular test. The best combination of physiological condiAll substrate plates were prepared by adding agar tions as determined experimentally for the deor agarose to the desired buffer containing the metal tection of various biological activities and the ions, if any, and 0.02% NaN3. The agar or the agarose was then dissolved by boiling and cooled to 50°C sensitivity of the test under these conditions before adding the substrate. The mixture was dis- are shown in Table 1. Additional information pensed in 10-ml quantities in petri plates. The agar regarding the characteristics of each of the was allowed to solidify, and the plates were refriger- products is as follows. ated overnight. Staphylokinase. Staphylokinase activity Samples were placed in circular wells, 3 mm in could not be detected by using carbonate buffer diameter, cut in the agar. A sterile metal punch was at any pH tested; likewise, universal buffer at used to cut the wells, and the cut agar cylinder was removed by suction with a Pasteur pipette. The pH 5.1 failed to detect the kinase activity. wells so created were completely filled with the sam- Staphylokinase could be detected using any one ples by using a Pasteur pipette whose tip was previ- of the remaining buffer systems, but the highously drawn to a fine capillary. The volume of this est activity was obtained with 0.001 M phossample was approximately 6 pi. If the sample was to phate buffer at pH 7.2 with added NaCl, to be quantitated, exactly 6 ,lI of the sample was adjust the ionic strenth to 15 mmho. Increasplaced in the well using a Hamilton 25-,ul syringe. ing the phosphate concentration suppressed the After placing the samples, the plates were incubated staphylokinase activity. Phosphate ion in higher at 37°C for 24 h, and, when applicable, the reactions concentration therefore should not be used. were developed with appropriate reagents and the A positive reaction is indicated by a sharp results were recorded. clearing zone and/or the punctuate Muller pheThe reagents for developing the various reactions were as follows. For detecting hyaluronidase, the nomenon. Hazy zones may not be due to staphyplates were flooded with 1% cetylpyridinium chlo- lokinase and should be checked by noting their ride (Fisher) or 1% cetyltrimethylammonium bro- inhibition by e-amino caproic acid (5%) and mide (Fisher), and the reagent was allowed to react trypsin soybean inhibitor, both of which inhibit for 1 to 12 h before reading; for detecting penicillin- the staphylokinase reaction. ase, the plates were flooded with a 0.5 dilution of Phosphatase. Phosphatase activity was inGram's iodine (1 g of I2, 2 g of Kl to 600 ml of water); by carbonate buffer at all pH values for detecting DNase and protease, the plates were hibited tested and by universal buffer at pH 5.1 and flooded with 1 N HCl and acidified mercuric chloride (100 ml of 0.2 M HgCl2 and 20 ml of concentrated 10.6. The best substrate was phenolphthalein HCl), respectively. Positive reactions on the above diphosphate since it yielded a clearer reaction plates consisted of clear zones against an opaque than p-nitrophenylphosphate. CaCl2 and FeSO4 background. For detecting the phosphatase activity, did not enhance phosphatase activity and were the plates were exposed to NH4OH vapors for 1 min. even inhibitory in certain cases. MgCl2 in a 10 A positive reaction was indicated by a pink color mM concentration potentiated phosphatase acwhen phenolphthalein diphosphate was the sub- tivity. strate and by a yellowish color when p-nitrophenylProtease. The best conditions for detecting phosphate was the substrate. All other plates could protease activity differed with the culture used. be read directly. Staphylokinase, lipase, lysozyme, and the blood agar plates had clear zones around the The best buffer system, nevertheless, was PBS wells when the samples contained these activities. (pH 7.2) with ionic strength at 15 mmho. CalEsterase and egg yolk plates yielded opaque zones cium chloride was not a universal requirement around wells when the samples contained these ac- for detecting the protease activity. Since it did tivities. potentiate the proteolytic activity of filtrates of
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AGAR PLATE TESTS OF ENHANCED SENSITIVITY
VOL. 32, 1976
TABLE 1. Parameters of agar plate tests used to detect selected extracellular products of staphylococci Product
Buffer
pH
NaCl (g/ Ionic liter) strength added (mmho)
Staphylokinase 0.001 M phosphate
7.2
8.4
Phosphate
0.1 M phosphate
6.6
0
Protease Lipase Esterase Egg yolk factor
PBS 0.05 M Tris 0.05 M Tris 0.05 M Tris
7.2 8.8 8.8 8.8
0 7.5 7.5 7.5
15 15 15 15
Lysozyme
PBS
7.2
0
15
Penicillinase
PBS
7.2
0
15
Hyaluronidase
0.05 M Tris
8.8
0
1
15 9
MiniMetal ions
Substrate
mum
itya (,g) 10 mM MgCl2
Fibrinogen, 0.15%, + thrombin, 0.025% 10 mM MgC12 Phenolphthalein diphosphate, 0.15% 0.5 mM CaCl2 Gelatin, 0.3% Tributyrin, 1% (vol/vol) Tween 20, 1% (vol/vol) Concentrated egg yolk emulsion, 1% (vol/vol) 1.0 mM CaCl2 M. lysodeikticus, 0.15% (Difco) Penicillin G (103 U/ml) with 0.05% starch as indicator Potassium hyaluronate, 0.2% 10 mM MgC12 Erythrocytes, 2% (vol/vol) DNase agar (Difco), 4%
10 50 10 5 5 10 25
2
Hemolysins PBS 7.2 0 15 1 DNase 0.05 M Tris 8.8 0 1 0.003 a Micrograms of crude product expressed in terms of protein concentrations that yielded a detectable reaction.
strains PG 114 and 681C, CaCl2 at 0.5 mM was added to all media designed to detect protease activity. Lipase, egg yolk factor, and esterase. Lipase could be detected under all the conditions tested. Universal buffer at pH 5.1 and 5.9 and phosphate buffer at pH 6.2 and 6.6, however, inhibited egg yolk factor and esterase activity. The best combination of buffer and pH values that enabled the detection of all three of these activities was Tris buffer, pH 8.8, at an ionic strength of 15 mmho. None of the metal ions were essential for detecting these activities. Lysozyme. Lysozyme activity could be detected only with PBS, pH 7.2, with an ionic strength of 15 mmho. All other buffers were inhibitory with the exception of Tris buffer, pH 8.8, which was best for the detection of lysozyme activity of the Smith 5R strain. Lysozyme activity of this strain, however, could be detected with PBS, pH 7.2, and this buffer was therefore selected for detecting the lysozyme activity in general. Addition of 1 mM CaCl2 was important for the detection of lysozyme activity of M3 and 681C strains of staphylococci. Since CaCl2 did not adversely affect the detection of lysozyme activity of other strains, CaCl2 was routinely included in the preparation of plates for detecting the lysozyme activity. DNase. Media prepared with Tris buffer, pH 8.6 (ionic strength, 1 mmho), without the addition of any metal ions was best for the detection of DNase activity. Reactions were also detectable on media prepared with other buffers between the pH range of 5.9 to 10.1 with the exception of PBS, which yielded faint zones. Hemolysins. Use of Tris, barbital, and car-
bonate buffers and of universal buffer, pH 5.1, 9.6, and 10.6, is not recommended because these buffers lysed horse erythrocytes and, in some cases, also human and rabbit erythrocytes. Sheep erythrocytes, however, were not affected by these buffers. The best combination of buffer, pH, and ionic strength for the detection of hemolytic activity was PBS, pH 7.2, with an ionic strength of 15 mmho to which was added 10 mM MgCl2 to detect the presence of beta-hemolysin. Hyaluronidase. Hyaluronidase activity could be detected only when agarose was utilized as the solidifying agent. The best conditions for detecting the hyaluronidase activity were Tris buffer, pH 8.8, and ionic strength of 1 mmho. None of the added metal ions tested were required. Penicillinase. Tris, barbital, and carbonate buffers inhibited the penicillinase activity. Difco agar was likewise inhibitory. Best conditions for detecting the penicillinase activity were PBS, pH 7.2, ionic strength of 15 mmho, and agarose as the solidifying agent. DISCUSSION The study reported here is an attempt to develop and/or adapt agar plate tests for detecting selected extracellular products of S. aureus in a simple, sensitive, and reproducible manner. As such it is expected to provide means to better characterize strains of S. aureus in terms of their biologically active extracellular products. These methods will also be useful to establish the purity of isolated extracellular products. In the past, preparations of certain staphylococcal products considered to be "pure"
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MADLER, LEE, AND HAQUE
by immunological and physical means of testing purity were later found to contain biologically active impurities (5; S. H. Lee, T. Sarauskas, E. S. Hoy, and R. Haque, J. Med. Microbiol., in press). Now these and other preparations can be characterized more fully. Regarding sensitivity of the methods reported here, it should be pointed out that sensitivity as reported here is a reflection ofthe crude nature of the filtrates used. Precise information about their sensitivity should await the availability of purified preparations. LITERATURE CITED 1. Ali, M., and R. Haque. 1974. Recognition by electrophoretic localization of the extracellular products of se-
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2.
3. 4. 5.
6.
7.
lected strains of staphylococci. J. Med. Microbiol. 7:375-382. Baman, S. I., and R. Haque. 1970. Production of multivalent extracellular filtrates of Staphylococcus aureus. Can. J. Microbiol. 16:1255-1261. Britton, H. T. S., and G. Welford. 1937. The standardization of some buffer solutions at elevated temperatures. J. Chem. Soc. 2:1848-1852. Campbell, D., J. Garvey, N. Cremer, and D. Sussdorf. 1970. Methods in immunology, 2nd ed. W. A. Benjamin, Inc., New York. Gladstone, G. P. 1966. Staphylococcal hemolysins. Postepy Mikrobiol. 5:145-161. Haque, R. 1967. Identification of staphylococcal hemolysins by an electrophoretic localization technique. J. Bacteriol. 93:525-530. Murphy, R. A., and R. Haque. 1967. Purification and properties of staphylococcal delta-hemolysin. I. Production of delta hemolysin. J. Bacteriol. 94:13271333.
