Veterinarylmmunology andlmmunopathoiogy, 28 ( 1991 ) 45-56 Elsevier Science Publishers B.V., Amsterdam
45
A colorimetric assay for quantitating bovine neutrophil bactericidal activity Mark G. Stevens a, Marcus E. Kehrli Jr. b and Peter C. Canning~'m National Animal Disease Center. U.S.D.A., A.R.S., "Brucellosis Research Unit and bMinerai Metabolism and Mastitis Research Unit, PO Box 70, Ames, IA 50010. USA
(Accepted 23 April 1990)
ABSTRACT Stevens, M.G., Kehrli Jr., M.E. and Canning, P.C., 199 I. A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet. Immunol. ImmunopathoL, 28: 45-56. A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphyloceccus aureus. The procedure used the tetrazolium compound, 3-[ 4,5-dimethylthiazol-2-yl ]2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophiis, l h e percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate ! 3-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-y) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.
INTRODUCTION
Peripheral blood neutrophils are important in providing defense against bacterial infections in cattle. Bovine diseases such as the Chediak-Higashi (Padgett et al., 1964; Renshaw et al., 1974) and granulocytopathy syndrome Ipresent address: Pfizer Incorporated, Central Research Division, Animal Health Discovery, PO Box 88, Terre Haute, IN 47808, USA.
0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
46
M.G. STEVENS ET AL.
(Nagahata et al., 1987; Takahashi et al., 1987 ) are fatal and are characterized by defective neutrophil bactericidal activity. Neutrophils also represent the predominant population of phagocytic cells in bovine peripheral blood. Therefore, evaluation ofneutrophil bactericidal activity may be an important tool for determining immune status in cattle during disease and drug therapy. Current assays either directly or indirectly measure bactericidal capacity of bovine neutrophils. Techniques which measure iodination, chemiluminescence, and reduction of cytochrome C and nitroblue tetrazolium by bovine neutrophils have become popular methods of indirectly assessing neutrophil bactericidal activity (Roth and Kaeberle, 1981; Lukacs et al., 1985; Steinbeck et al., 1986). These assays indirectly assess bactericidal capacity by measuring neutrophil respiratory burst activity and production of various reactive oxygen intermediates (e.g., hydrogen peroxide, superoxide anion, singlet oxygen, and hydroxyl radicals) which can potentially kill bacteria (Badwey and Karnovsky, 1980; Root and Cohen, 1981 ). Ingestion of bacteria is also commonly used to assess neutrophil immune function (Lukacs et al., 1985; Steinbeck et al., 1986). However, neutrophils can have normal ingestion while possessing severely deficient bactericidal activity as is seen in the ChediakHigashi syndrome in cattle and humans (Root et al., 1972; Renshaw et al., 1974). Hence, measurement of ingestion of bacteria may reveal little about the actual bactericidal capacity of neutrophils. Bactericidal function in neutrophils is a complex process. It involves not only ingestion and respiratory burst activity, but also requires intracellular movement and fusion of phagosomes with lysosomes which results in lysosomal release of at least 60 potential bactericidal compounds (Root and Cohen, 1981; Smolen, 1989). Only a small part of this process is assessed when ingestion of bacteria or respiratory burst function is measured in neutrophiis. Therefore, results from these assays can be difficult to interpret in the context of host protection because bactericidal activity of the neutrophil is not being definitively measured. Traditional assays that measure direct bactericidal activity of neutrophils measure the number of killed bacteria by performing colony counts, which are derived by plating dilutions of bacteria onto nutrient agar plates (Renshaw et al., 1976; Woodard et al., 1983). Measurement of bactericidal activity by colony counting is time consuming and generally limited to analysis of relatively low numbers of test samples. In addition, results can be quite variable because of dilution and plating errors or clumping of bacteria. To overcome problems associated with traditional bactericidal assays, we developed a simple and sensitive colorimetric assay that rapidly measures bactericidal activity of large numbers of neutrophi! samples. The technique uses the tetrazolium compound, 3- [4,5-dimethylthiazol-2-yl ]-2,5-diphenyltetrazolium bromide (MTT), which is reduced to purple formazan by live bacteria in direct proportion to viable cell number. Bacterial killing by neutrophils was measured by colorimetric determination of formazan production.
QUANTITATIONOF BOVINENEUTROPHILBACTERICIDALACTIVITY
47
MATERIALS AND METHODS
Reagents RPMI 1640 culture medium was purchased from GIBCO Laboratories (Grand Island, NY). Fetal bovine serum (FBS) containing less than 0.05 ng/ml of endotoxin was obtained from Hyclone Laboratories Inc. (Logan, UT). Colchicine, cytochalasin B, phorbol 12-myristate 13-acetate (PMA), saponin, and MTT were purchased from Sigma Chemical Co. (St. Louis, MO). Saponin was stored at 4°C as a 2% filter-sterilized solution in phosphate buffered saline (PBS) and was diluted with PBS immediately before use. The MTT was prepared as a 2 mg/ml stock solution in PBS and was filter-sterilized and stored in the dark at 4°C. Recombinant bovine interferon-gamma (rBoIFN-7) was a gift from Ciba-Geigy (Basel, Switzerland). The S. aureus Newbould strain 305 (ATCC#29740) was obtained from the American Type Culture Collection (Rockville, MD). Bacteria were grown at 37°C in tryptic soy broth (Difco Laboratories, Detroit, MI) with vigorous aeration until mid-log-phase growth was achieved. Cells were harvested and washed twice in PBS by centrifugation, and then aliquoted and stored in tryptic soy broth at - 7 0 ° C. Thawed bacteria were sonicated to disrupt cell clusters and viable cell number was obtained by plating dilutions of bacteria onto blood agar plates and performing colony counts at 24 h after incubation at 37 oC. Frozen bacteria contained 2 X 109 viable cells/ml and the viability did not change during the experiments. The S. aureus antiserum was obtained from eight Holstein-Friesian cows which had been experimentally infected with S. aureus Newbould strain 305. Pooled antiserum from the eight animals was heat-inactivated by incubating at 56 °C for 30 min. The antiserum was then aliquoted and stored at - 7 0 ° C . Isolation of neutrophils Neutrophils were isolated from peripheral blood of 6-year-old cross-bred steers as previously described (Roth and Kaeberle, 1981 ). Briefly, blood obtained by jugular venipuncture was collected into an acid-citrate-dextrose solution and centrifuged at 1000 × g for 20 min. The plasma and buffy coat layer were removed and i:he pelleted erythrocytes were lysed by short exposure to hypotonic conditions. The remaining cells were placed in RPMI containing 5% FBS. Greater than 90% of the cells in the final preparation were neutrophils based on morphologic examination. The remaining cells were eosinophils. In vitro treatments of neutrophils Fifty/zl of I X 107 neutrophils/ml in RPMI containing 5% FBS were added to flat-bottom wells of a 96-well microtiter plate (Coming Glass Works, Coming NY). Fifty/zl of colchicine, cytochalasin B, PMA, or rBoIFN-y in
48
M.G. STEVENS ET AL.
RPMI plus 5% FBS were added to neutrophils in triplicate at the following final concentrations: cytochalasin B, 10.0, 1.0, 0.I and 0.01 #g/ml; colchicine, 1.0, 0 l, 0.01 and 0.001 gg/ml; PMA, 10.0, 1.0, 0.1 and 0.01 ng/ml; and rBolFN-7, 100, l0 and I pg/ml. All of these compounds were soluble in RPMI except for cytochalasin B which was first dissolved by preparing a l 0 mg/ml solution in dimethyl sulfoxide (DMSO) before addition to RPMI. The highest tested level of cytochalasin B (10 gg/ml) contained 0.1% DMSO. This level of DMSO had no effect on neutrophil bactericidal activity. Neutrophils were incubated for 2 h at 37°C with the various levels of colchicine or cytochalasin B before measurement of bactericidal activity. Bactericidal activity of neutrophils was assessed at 2, 6, and 24 h after incubation at 37 °C with the indicated concentrations of PMA and rBolFN-7. Neutrophils were used in the bactericidal assay without washing. Colorimetric bactericidal assay
The S. aureus and S. a u r e u s antiserum were rapidly thawed by agitation in a 37°C water bath. Bacteria were opsonized with antibody by incubating l X 10s bacteda/ml in RPMi containing 5% FBS and 1% S. a u r e u s antiserum for 30 rain at room temperature. Previous analysis established this antiserum concentration optimally opsonized S. a u r e u s without agglutinating the bacteria. Fifty 91 of opsonized bacteria were added to the neutrophils resulting in a ratio of 10 bacteria per neutrophil. Microtiter plates were then placed on a Micro Shaker II (Dynatech Laboratories Inc, Alexandria, VA) and mixed at an instrument setting of 3.5 for l h at 37°C to allow killing of bacteria by neutrophils. The opsonized S. aureus suspension, which was added to neutronhils_ wa~ caincuhatad with th~ plates. This suspension was used to . . . . *~-'~ a standard curve of bactericidal activity. The neutrophils were lysed by adding 50 91 of 0.2% saponin at 1 h after incubation with S. aureus. A standard curve of bactericidal activity was then prepared from the opsonized S. aureus suspension. Bacteria were diluted in RPMI containing 5% FBS and 1% S. aureus antiserum to correspond to a 0, 30, 60, and 90% reduction in cell number. Fifty 91 of each dilution was added in quadruplicate to separate microtiter wells containing 100 91 of RPMI plus 5% FBS. Fifty 91 of 0.2% saponin was then added to each well. All wells then received 50 91 of 2 mg/ml of MTT. Plates were incubated at room temperature for 10 rnin to allow reduction of MTT to formazan by viable bacteria. The formazan-laden bacteria were pelleted on the bottom of microtiter wells by centrifuging plates at 1600 × g for 5 min. Fluid from the wells was removed by inverting and gently blotting plates onto absorbent paper. One hundred fifty 91 of isopropanol was added to the bacteria-formazan precipitate and plates were allowed to sit for 10 rain. The formazan was then dissolved by vigorously agitating plates for 5 min with the Micro Shaker II using a setting of 6.0. Any remaining formazan precipitate was completely
QUANTITATION OF BOVINE NEUTROPHIL BACTERICIDAL ACTIVITY
49
dissolved by mixing the well contents. Fifty gl of PBS was then added to the solubilized formazan. Formazan produced by bacteria was quantitated by measuring optical density (OD) at 560 nm. The OD readings were obtained using an automated 96-channel microtiter plate spectrophotometer (Vmax, Molecular Devices Corporation, Palo Alto, CA) interfaced to a computer. The OD corresponding to 0 and 90% killing of bacteria was established by linear regression analysis. The percentage of bacteria killed by neutrophils was determined by the formula: 1
(OD sample) - (OD 90% killing) ×90% (OD 0% killing) - (OD 90% killing)
The controls for each neutrophil sample tested included incubation of neutrophils for 1 h with no bacteria before lysing neutrophils with saponin. Lysed neutrophils were then incubated for 10 rain with MTT alone and with MTT plus the challenge dose of S. aureus. Statistical analysis Data were analyzed by analysis of variance (ANOVA) using Tukey's honesty sum difference test for multiple group comparisons. Neutrophil samples obtained from each animal were used as blocking factors in the ANOVA. Blocking was achieved by determining statistical differences among treated and untreated (control) neutrophils obtained from the same animal. RESULTS
Stqndard curve o f bactericidal activity Co!aversion of MTT to formazan by S. aureus was directly related and highly correated (R=0.997, P_
45
A colorimetric assay for quantitating bovine neutrophil bactericidal activity Mark G. Stevens a, Marcus E. Kehrli Jr. b and Peter C. Canning~'m National Animal Disease Center. U.S.D.A., A.R.S., "Brucellosis Research Unit and bMinerai Metabolism and Mastitis Research Unit, PO Box 70, Ames, IA 50010. USA
(Accepted 23 April 1990)
ABSTRACT Stevens, M.G., Kehrli Jr., M.E. and Canning, P.C., 199 I. A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet. Immunol. ImmunopathoL, 28: 45-56. A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphyloceccus aureus. The procedure used the tetrazolium compound, 3-[ 4,5-dimethylthiazol-2-yl ]2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophiis, l h e percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate ! 3-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-y) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.
INTRODUCTION
Peripheral blood neutrophils are important in providing defense against bacterial infections in cattle. Bovine diseases such as the Chediak-Higashi (Padgett et al., 1964; Renshaw et al., 1974) and granulocytopathy syndrome Ipresent address: Pfizer Incorporated, Central Research Division, Animal Health Discovery, PO Box 88, Terre Haute, IN 47808, USA.
0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
46
M.G. STEVENS ET AL.
(Nagahata et al., 1987; Takahashi et al., 1987 ) are fatal and are characterized by defective neutrophil bactericidal activity. Neutrophils also represent the predominant population of phagocytic cells in bovine peripheral blood. Therefore, evaluation ofneutrophil bactericidal activity may be an important tool for determining immune status in cattle during disease and drug therapy. Current assays either directly or indirectly measure bactericidal capacity of bovine neutrophils. Techniques which measure iodination, chemiluminescence, and reduction of cytochrome C and nitroblue tetrazolium by bovine neutrophils have become popular methods of indirectly assessing neutrophil bactericidal activity (Roth and Kaeberle, 1981; Lukacs et al., 1985; Steinbeck et al., 1986). These assays indirectly assess bactericidal capacity by measuring neutrophil respiratory burst activity and production of various reactive oxygen intermediates (e.g., hydrogen peroxide, superoxide anion, singlet oxygen, and hydroxyl radicals) which can potentially kill bacteria (Badwey and Karnovsky, 1980; Root and Cohen, 1981 ). Ingestion of bacteria is also commonly used to assess neutrophil immune function (Lukacs et al., 1985; Steinbeck et al., 1986). However, neutrophils can have normal ingestion while possessing severely deficient bactericidal activity as is seen in the ChediakHigashi syndrome in cattle and humans (Root et al., 1972; Renshaw et al., 1974). Hence, measurement of ingestion of bacteria may reveal little about the actual bactericidal capacity of neutrophils. Bactericidal function in neutrophils is a complex process. It involves not only ingestion and respiratory burst activity, but also requires intracellular movement and fusion of phagosomes with lysosomes which results in lysosomal release of at least 60 potential bactericidal compounds (Root and Cohen, 1981; Smolen, 1989). Only a small part of this process is assessed when ingestion of bacteria or respiratory burst function is measured in neutrophiis. Therefore, results from these assays can be difficult to interpret in the context of host protection because bactericidal activity of the neutrophil is not being definitively measured. Traditional assays that measure direct bactericidal activity of neutrophils measure the number of killed bacteria by performing colony counts, which are derived by plating dilutions of bacteria onto nutrient agar plates (Renshaw et al., 1976; Woodard et al., 1983). Measurement of bactericidal activity by colony counting is time consuming and generally limited to analysis of relatively low numbers of test samples. In addition, results can be quite variable because of dilution and plating errors or clumping of bacteria. To overcome problems associated with traditional bactericidal assays, we developed a simple and sensitive colorimetric assay that rapidly measures bactericidal activity of large numbers of neutrophi! samples. The technique uses the tetrazolium compound, 3- [4,5-dimethylthiazol-2-yl ]-2,5-diphenyltetrazolium bromide (MTT), which is reduced to purple formazan by live bacteria in direct proportion to viable cell number. Bacterial killing by neutrophils was measured by colorimetric determination of formazan production.
QUANTITATIONOF BOVINENEUTROPHILBACTERICIDALACTIVITY
47
MATERIALS AND METHODS
Reagents RPMI 1640 culture medium was purchased from GIBCO Laboratories (Grand Island, NY). Fetal bovine serum (FBS) containing less than 0.05 ng/ml of endotoxin was obtained from Hyclone Laboratories Inc. (Logan, UT). Colchicine, cytochalasin B, phorbol 12-myristate 13-acetate (PMA), saponin, and MTT were purchased from Sigma Chemical Co. (St. Louis, MO). Saponin was stored at 4°C as a 2% filter-sterilized solution in phosphate buffered saline (PBS) and was diluted with PBS immediately before use. The MTT was prepared as a 2 mg/ml stock solution in PBS and was filter-sterilized and stored in the dark at 4°C. Recombinant bovine interferon-gamma (rBoIFN-7) was a gift from Ciba-Geigy (Basel, Switzerland). The S. aureus Newbould strain 305 (ATCC#29740) was obtained from the American Type Culture Collection (Rockville, MD). Bacteria were grown at 37°C in tryptic soy broth (Difco Laboratories, Detroit, MI) with vigorous aeration until mid-log-phase growth was achieved. Cells were harvested and washed twice in PBS by centrifugation, and then aliquoted and stored in tryptic soy broth at - 7 0 ° C. Thawed bacteria were sonicated to disrupt cell clusters and viable cell number was obtained by plating dilutions of bacteria onto blood agar plates and performing colony counts at 24 h after incubation at 37 oC. Frozen bacteria contained 2 X 109 viable cells/ml and the viability did not change during the experiments. The S. aureus antiserum was obtained from eight Holstein-Friesian cows which had been experimentally infected with S. aureus Newbould strain 305. Pooled antiserum from the eight animals was heat-inactivated by incubating at 56 °C for 30 min. The antiserum was then aliquoted and stored at - 7 0 ° C . Isolation of neutrophils Neutrophils were isolated from peripheral blood of 6-year-old cross-bred steers as previously described (Roth and Kaeberle, 1981 ). Briefly, blood obtained by jugular venipuncture was collected into an acid-citrate-dextrose solution and centrifuged at 1000 × g for 20 min. The plasma and buffy coat layer were removed and i:he pelleted erythrocytes were lysed by short exposure to hypotonic conditions. The remaining cells were placed in RPMI containing 5% FBS. Greater than 90% of the cells in the final preparation were neutrophils based on morphologic examination. The remaining cells were eosinophils. In vitro treatments of neutrophils Fifty/zl of I X 107 neutrophils/ml in RPMI containing 5% FBS were added to flat-bottom wells of a 96-well microtiter plate (Coming Glass Works, Coming NY). Fifty/zl of colchicine, cytochalasin B, PMA, or rBoIFN-y in
48
M.G. STEVENS ET AL.
RPMI plus 5% FBS were added to neutrophils in triplicate at the following final concentrations: cytochalasin B, 10.0, 1.0, 0.I and 0.01 #g/ml; colchicine, 1.0, 0 l, 0.01 and 0.001 gg/ml; PMA, 10.0, 1.0, 0.1 and 0.01 ng/ml; and rBolFN-7, 100, l0 and I pg/ml. All of these compounds were soluble in RPMI except for cytochalasin B which was first dissolved by preparing a l 0 mg/ml solution in dimethyl sulfoxide (DMSO) before addition to RPMI. The highest tested level of cytochalasin B (10 gg/ml) contained 0.1% DMSO. This level of DMSO had no effect on neutrophil bactericidal activity. Neutrophils were incubated for 2 h at 37°C with the various levels of colchicine or cytochalasin B before measurement of bactericidal activity. Bactericidal activity of neutrophils was assessed at 2, 6, and 24 h after incubation at 37 °C with the indicated concentrations of PMA and rBolFN-7. Neutrophils were used in the bactericidal assay without washing. Colorimetric bactericidal assay
The S. aureus and S. a u r e u s antiserum were rapidly thawed by agitation in a 37°C water bath. Bacteria were opsonized with antibody by incubating l X 10s bacteda/ml in RPMi containing 5% FBS and 1% S. a u r e u s antiserum for 30 rain at room temperature. Previous analysis established this antiserum concentration optimally opsonized S. a u r e u s without agglutinating the bacteria. Fifty 91 of opsonized bacteria were added to the neutrophils resulting in a ratio of 10 bacteria per neutrophil. Microtiter plates were then placed on a Micro Shaker II (Dynatech Laboratories Inc, Alexandria, VA) and mixed at an instrument setting of 3.5 for l h at 37°C to allow killing of bacteria by neutrophils. The opsonized S. aureus suspension, which was added to neutronhils_ wa~ caincuhatad with th~ plates. This suspension was used to . . . . *~-'~ a standard curve of bactericidal activity. The neutrophils were lysed by adding 50 91 of 0.2% saponin at 1 h after incubation with S. aureus. A standard curve of bactericidal activity was then prepared from the opsonized S. aureus suspension. Bacteria were diluted in RPMI containing 5% FBS and 1% S. aureus antiserum to correspond to a 0, 30, 60, and 90% reduction in cell number. Fifty 91 of each dilution was added in quadruplicate to separate microtiter wells containing 100 91 of RPMI plus 5% FBS. Fifty 91 of 0.2% saponin was then added to each well. All wells then received 50 91 of 2 mg/ml of MTT. Plates were incubated at room temperature for 10 rnin to allow reduction of MTT to formazan by viable bacteria. The formazan-laden bacteria were pelleted on the bottom of microtiter wells by centrifuging plates at 1600 × g for 5 min. Fluid from the wells was removed by inverting and gently blotting plates onto absorbent paper. One hundred fifty 91 of isopropanol was added to the bacteria-formazan precipitate and plates were allowed to sit for 10 rain. The formazan was then dissolved by vigorously agitating plates for 5 min with the Micro Shaker II using a setting of 6.0. Any remaining formazan precipitate was completely
QUANTITATION OF BOVINE NEUTROPHIL BACTERICIDAL ACTIVITY
49
dissolved by mixing the well contents. Fifty gl of PBS was then added to the solubilized formazan. Formazan produced by bacteria was quantitated by measuring optical density (OD) at 560 nm. The OD readings were obtained using an automated 96-channel microtiter plate spectrophotometer (Vmax, Molecular Devices Corporation, Palo Alto, CA) interfaced to a computer. The OD corresponding to 0 and 90% killing of bacteria was established by linear regression analysis. The percentage of bacteria killed by neutrophils was determined by the formula: 1
(OD sample) - (OD 90% killing) ×90% (OD 0% killing) - (OD 90% killing)
The controls for each neutrophil sample tested included incubation of neutrophils for 1 h with no bacteria before lysing neutrophils with saponin. Lysed neutrophils were then incubated for 10 rain with MTT alone and with MTT plus the challenge dose of S. aureus. Statistical analysis Data were analyzed by analysis of variance (ANOVA) using Tukey's honesty sum difference test for multiple group comparisons. Neutrophil samples obtained from each animal were used as blocking factors in the ANOVA. Blocking was achieved by determining statistical differences among treated and untreated (control) neutrophils obtained from the same animal. RESULTS
Stqndard curve o f bactericidal activity Co!aversion of MTT to formazan by S. aureus was directly related and highly correated (R=0.997, P_
