JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1976, p. 327-329 Copyright © 1976 American Society for Microbiology
Vol. 3, No. 3 Printed in U.SA.
Acceleration of Tetrazolium Reduction by Bacteria RAYMOND C. BARTLETT, MARY MAZENS,*
AND
BRIAN GREENFIELD
Department of Pathology, Hartford Hospital, Hartford, Connecticut 06115,* and Trinity College, Hartford, Connecticut 06106 Received for publication 25 July 1975
Conditions were assessed which would permit more rapid recognition of bacterial growth than has been previously reported using tetrazolium salts. Microtitration trays were used. 2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride is rapidly reduced by respiring cells in tissue homogenates but is more toxic than other tetrazoliums when added to growing bacterial cultures. Phenazine methosulfate (PMS), an intermediate electron carrier, potentiates tetrazolium reduction. Growth was readily detected by the addition of these compounds after 3 to 4 h of incubation in Schaedler broth. The final concentration prior to addition to tray wells was 1.0 mg/ml for 2-(p-iodophenyl)-3(pnitrophenyl)-5-phenyltetrazolium chloride and 0.06 mg/ml for PMS. Addition of 0.5 to 0.8 g of agar per liter of broth enhanced subsequent tetrazolium reduction.
Microbiological applications of reduction of tetrazolium salts to visible formazan precipitates as a measure of microbial growth have been reviewed by Pegram (12). These compounds usually have been introduced into actively metabolizing cultures. 2, 3, 5-Triphenyltetrazolium chloride has been most commonly used because it is less toxic than other tetrazolium salts (10, 14, 16). Knowledge of the chemistry of tetrazolium reduction has been derived primarily from studies by using tissue homogenates and purified hexosemonophosphate as a substrate. There is no proof that all of the observations are applicable to bacterial cultures. It has been suggested that reduction is mediated by dehydrogenases linked with coenzyme 1 nicotinamide adenine dinucleotide, coenzyme 2 nicotinamide adenine dinucleotide phosphate (9), and flavoproteins (5). Dickens and McIlwain reported that an intermediate electron carrier, phenazine methosulfate (PMS), could be employed as a substitute for flavoprotein (6). Barnett used this compound to demonstrate tetrazolium reduction of lactate dehydrogenase isoenzymes following electrophoresis (3). Nachlas et al. (11) and Atkinson et al. (2) reported that 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) was more rapidly reduced than other tetrazolium salts. INT has been shown to be more toxic than other tetrazoliums when added to growing bacterial cultures by May et al. who found 64 ,ug/ml lethal for Staphylococcus aureus (10). Tetrazolium reduction by bacteria is enhanced under anaerobic conditions (1, 5, 13, 15) and is retarded by aeration (7). Although Pegram reported a decrease in tetrazolium reduction by Escherichia coli when lactose was
added to the medium, Somerson and Morton found lactate to enhance tetrazolium reduction by Mycoplasma (13). Increased amounts of glucose might enhance growth and the accumulation of reduced compounds in bacterial culture accelerating reduction of tetrazolium salts. Fred and Knight (7) reported no enhancement of tetrazolium reduction by Penicillium after the addition of glucose, but an evaluation of the effect on bacterial cultures has not been reported. Kopper reported that a pH of 7.5 was optimum and that concentrations of NaCl of 1% or less were desired for young metabolizing cells (8). Others have established a quantitative relationship between bacterial growth and tetrazolium reduction (8, 14). There is a need for a rapid and sensitive means for detection of bacterial growth in the presence of various inhibitors such as antibiotics. This is a report of studies that were conducted to obtain maximum sensitivity for detection of bacterial growth after a 3-h period of incubation. The inhibitory effect of the tetrazolium salt was avoided by adding the compound after the period of incubation. An optimal basal medium was selected and controlled amounts of PMS and INT comparable to concentrations used by other investigators were added (3, 6, 10). Subsequently, controlled amounts of glucose and agar were added to determine their effect on the amount of formazan produced. EXPERIMENTAL Evaluation of broths, combinations of INT and PMS and incubation time. A solution was prepared approximating the maximum solubility of INT in saline (2.0 mg/ml) by dissolving 200 mg in 5 ml of warm 100% ethyl alcohol and adding physiological 327
328
J. CLIN. MICROBIOL.
BARTLETT ET AL.
saline to produce a total volume of 100 ml. This was without supplements. This broth was employed stored in the dark at 4 C and was used within 1 without supplements at a pH of 7.2 to 7.4 in further month. Stationary phase cultures for each portion of studies. Effect of different concentrations of INT and this investigation were prepared by inoculating several colonies into broth from blood agar plates PMS. A solution consisting of 0.11 mg of PMS per ml which had been incubated with the test culture for in physiological saline was prepared and stored at 18 to 24 h. The inoculum density was equilibrated 4 C in the dark. This was used within 30 days. with the barium sulfate standard described by Mixtures of INT and PMS were prepared immediBauer et al. (4) by the addition of inoculum or dilu- ately before use. Any remaining was discarded betion with broth. This yielded a concentration of cause the mixture was observed to produce small about 1.5 x 108 colony-forming units/ml. This was amounts of formazan spontaneously after 30 to 60 subsequently diluted with broth to a final concen- min. All possible combinations of four concentratration of 10" colony-forming units/ml. This suspen- tions of each compound were employed. Prior to the sion (50 Au) was added to the wells of plastic microti- addition of the mixture to the tray wells final contration trays (Ames Co., Division of Miles Laborato- centrations of INT were 0.125, 0.25, 0.5, and 1.0 mg/ ries, Elkhart, Ind.) which already contained 50 /.d of ml and 0.005, 0.01, 0.03, and 0.06 mg/ml for PMS. S. broth in each well. The purpose of this was to simu- aureus ATCC 25923 (ATCC, American Type Culture late the use of these plates for biochemical or anti- Collection) and E. coli ATCC 25922 were incubated microbial susceptibility tests in which the trays at 37 C for 4 h. Fifty microliters of each INTwould contain previously prepared inhibitory solu- PMS combination was added to wells containing the tions in broth. culture. The color development was observed after Selection of broth. A comparison was made of 15, 30, and 45 min of further incubation at 37 C. brain heart infusion broth, tryptic digest casein soy Controls were prepared by inoculating wells with 50 broth, Mueller-Hinton broth, Schaedler broth, Col- ,ul of freshly prepared unincubated inoculum of each umbia broth, and Todd-Hewitt broth. All of these test culture. Blank broth controls were also used. were evaluated with and without the addition of RESULTS horse serum (2.5% vol/vol), Isovitalex (Baltimore The most intense color development was obBiological Laboratory, Cockeysville, Md.) and yeast extract (0.25% vol/vol) (Baltimore Biological Labo- served with mixtures containing the highest ratory). Following a 4-h incubation of test cultures, concentrations of INT and PMS (Table 1). No 50 ,l of a 0.5-mg/ml solution of INT in saline was color was observed up to 45 min after addition added to each microtitration tray well. The plates of INT and PMS to wells containing unincuwere reincubated for 30 to 60 min to observe color development. Preliminary studies revealed that dif- bated inocula of control cultures. Spontaneous ferences were more marked when a slowly growing color development was observed occasionally organism such as Staphylococcus epidermidis was afcer 30 and 45 min in control wells containing used. For this reason eight separate isolates of this uninoculated broth. Because of the insolubility species were tested. Schaedler broth demonstrated of INT and risk that more rapid and intense distinctly more color development both with and spontaneous reduction might result from
TABLE 1. Formazan precipitate after addition of INT and PMS S. aureus (ATCC 25923) Final concn (mg/ml) E. coli (ATCC 25922) (min) Broth controls (min) (min) INT
PMS
15
30
45
15
30
45
15
30
45
0.125 0.125 0.125 0.125 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5
0.005 0.01 0.03 0.06 0.005
+ +
+ + + + + + + + + + ++ ++ ++ ++ ++ +++
+ + + + + + ++ ++ ++ ++ ++ ++ ++ ++ +++ +++
+ ++ + ++ + + ++ +++ + ++ +++ +++ + + +++ +++
+ ++ + ++ ++ ++ +++ +++ ++ ++ +++ +++ ++ ++ +++ +++
++ ++
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
0
1.0 1.0 1.0 1.0
0.01
0.03 0.06 0.005 0.01
0.03 0.06 0.005 0.01
0 + + + + + + + + + + + + ++
++ ++ ++ ++ +++ +++
++ +++ +++ +++ +++ +++ +++
0 0 0 0 0 0 0 0 0
0/+ 0/+ 0
0/+ 0.03 0/+ 0/+ 0.06 +++ 0/+ 0/+ a Amount of formazan precipitate 15, 30, and 45 min after the addition of varying concentrations of INT and PMS to cultures which had been incubated for 4 h. Unincubated inoculum controls produced no precipitate after 45 min. Uninoculated broth controls showed variable precipitation after 30 and 45 min. Symbols: (0) no color; (+) pink; (++) rose; (+++) red.
VOL. 3, 1976
TETRAZOLIUM REDUCTION BY BACTERIA
higher concentrations of PMS, solutions containing final concentration of 1.0 mg of INT per ml and 0.06 mg of PMS per ml were used in subsequent studies. The more intense and rapid color development afforded by this mixture of PMS and INT suggested a shortening of incubation of test cultures from 4 to 3 h. Repeat studies employing these concentrations of INT and PMS produced the same amount of color development in a 3-h culture that had been observed previously using only INT after 4 h of incubation. Therefore subsequent studies were performed after 3 h of incubation of the test cultures. Effect of varying concentrations of glucose and agar in Schaedler broth. Addition of glucose to Schaedler broth. Schaedler broth was prepared containing controlled amounts of glucose resulting in final concentrations ranging from 1 to 10 g/liter. An increase in formazan production was observed as the concentration was increased to 5 g/liter with the test cultures of S. aureus and E. coli. No additional formazan production was observed when higher concentrations of glucose were used (Table 2). Freshly prepared unincubated inoculum and TABLE 2. Effects ofvarying concentrations ofglucose in Schaedler brotha Amount of glucose in broth (g/liter) Culture
Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922) Broth control Unincubated inoculum controls
0
1.0
2.5
5
7.5
10
+
+
+
++
++
++
+
+
++
++
++
++
0 0
0 0
0 0
0 0
0 0
0 0
a A mixture consisting of final concentrations of 1 mg of INT per ml and 0.06 mg of PMS per ml was added after a 3-h incubation of cultures. Color development was evaluated 30 min after reincubation at 37 C. Symbols: (0) no color change; (+) pink; (++) rose.
TABLE 3. Effect of addition of agar to Schaedler
brotha Amount of Noble agar in broth
Organism 0
(g/liter) 0.5 0.8
1.0
Staphylococcus +++ +++ aureus + ++ (ATCC 25923) Escherichia coli (ATCC + +++ +++ ++ 25922) Control broth 0 0 0 0 Unincubated inoculum 0 0 0 0 controls a INT and PMS were added after 3 h of incubation. Inspection for color development was 30 min after reincubation at 37 C. Symbols: (0) no color change; (+) pink; (+ +) rose; (+ + +) red.
329
uninoculated broth controls demonstrated no formazan production. The original glucose content of the medium (5 g/liter) appeared to be optimal. Addition of agar to Schaedler broth. Noble agar was added to the broth to provide concentrations of 0.5 to 1.0 g/liter in the well during the period of incubation prior to addition of INT and PMS. Maximal formazan precipitate was produced with concentrations of 0.5 and 0.8 g/ liter (Table 3). Less formazan production was observed when 1 g/liter was used. This may have been caused by the increased viscosity of the broth at this concentration. Blank broth controls and freshly prepared unincubated inoculum demonstrated no formazan production. LITERATURE CITED 1. Altman, F. P. 1970. Oxygen-sensitivity of various tetrazolium salts. Histochemie 22(3):256-261. 2. Atkinson, E., S. Melvin, and S. W. Fox. 1950. Some properties of 2,3,5-triphenyltetrazolium chloride and several Iodo derivatives Science 111:385-387. 3. Barnett, H. 1962. Electrophoretic separation of lactate dehydrogenase isoenzymes on cellulose acetate. Biochem. J. 84:83p-84p. 4. Bauer, A. W., W. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standarized single disk method. Am. J. Clin. Pathol.
45:493-496. 5. Brodie, A. F., and J. S. Gots. 1951. The effects of an isolated dehydrogenase enzyme and flavoprotein on the reduction of triphenyltetrazolium chloride. Science 114:40-41. 6. Dickens, F., and H. Mcllwain. 1938. Phenazine compounds as carriers in the hexosemonophosphate system. Biochem. J. 32:1615-1625. 7. Fred, R. B., and J. G. Knight. 1949. The reduction of 2,3,5-triphenyltetrazolium chloride by Penicillium chrysogenum. Science 109:169-170. 8. Kopper, P. H. 1952. Studies on bacterial reducing activity in relation to age of culture. J. Bacteriol. 63:639645. 9. Mattsen, A. M., C. 0. Jensen, and R. A. Dutcher. 1947. Triphenyltetrazolium chloride as a dye for vital tissues. Science 106:294-295. 10. May, P. S., J. W. Winter, G. H. Fried, and W. Antopol. 1960. Effect of tetrazolium salts on selected bacterial species. Proc. Soc. Exp. Biol. Med. 105:364-366. 11. Nachlas, M. M., S. S. Karmarkar, and A. M. Seligman. 1960. Variations in reduction of tetrazolium salts by dehydrogenase systems. Proc. Soc. Exp. Biol. Med. 104:407-409. 12. Pegram, R. G. 1969. Microbiological uses of 2,3,5-triphenyltetrazolium chloride. J. Med. Lab. Technol. 26:175-198. 13. Somerson, N. L., and H. E. Morton. 1953. Reduction of tetrazolium salts by pleuropneumonialike organisms. J. Bacteriol. 65:245-251. 14. Tengerdy, R. P., J. G. Nagy, and B. Martin. 1967. Quantitative measurement of bacterial growth by the reduction of tetrazolium salts. Appl. Microbiol. 15:954-955. 15. Throneberry, G. O., and F. G. Smith. 1953. The effect of triphenyltetrazolium chloride on oat embryo respiration. Science 117:13-15. 16. Weinberg, E. D. 1953. Selective inhibition of microbial growth by the incorporation of triphenyl tetrazolium chloride in culture media. J. Bacteriol. 66:240-242.
Vol. 3, No. 3 Printed in U.SA.
Acceleration of Tetrazolium Reduction by Bacteria RAYMOND C. BARTLETT, MARY MAZENS,*
AND
BRIAN GREENFIELD
Department of Pathology, Hartford Hospital, Hartford, Connecticut 06115,* and Trinity College, Hartford, Connecticut 06106 Received for publication 25 July 1975
Conditions were assessed which would permit more rapid recognition of bacterial growth than has been previously reported using tetrazolium salts. Microtitration trays were used. 2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride is rapidly reduced by respiring cells in tissue homogenates but is more toxic than other tetrazoliums when added to growing bacterial cultures. Phenazine methosulfate (PMS), an intermediate electron carrier, potentiates tetrazolium reduction. Growth was readily detected by the addition of these compounds after 3 to 4 h of incubation in Schaedler broth. The final concentration prior to addition to tray wells was 1.0 mg/ml for 2-(p-iodophenyl)-3(pnitrophenyl)-5-phenyltetrazolium chloride and 0.06 mg/ml for PMS. Addition of 0.5 to 0.8 g of agar per liter of broth enhanced subsequent tetrazolium reduction.
Microbiological applications of reduction of tetrazolium salts to visible formazan precipitates as a measure of microbial growth have been reviewed by Pegram (12). These compounds usually have been introduced into actively metabolizing cultures. 2, 3, 5-Triphenyltetrazolium chloride has been most commonly used because it is less toxic than other tetrazolium salts (10, 14, 16). Knowledge of the chemistry of tetrazolium reduction has been derived primarily from studies by using tissue homogenates and purified hexosemonophosphate as a substrate. There is no proof that all of the observations are applicable to bacterial cultures. It has been suggested that reduction is mediated by dehydrogenases linked with coenzyme 1 nicotinamide adenine dinucleotide, coenzyme 2 nicotinamide adenine dinucleotide phosphate (9), and flavoproteins (5). Dickens and McIlwain reported that an intermediate electron carrier, phenazine methosulfate (PMS), could be employed as a substitute for flavoprotein (6). Barnett used this compound to demonstrate tetrazolium reduction of lactate dehydrogenase isoenzymes following electrophoresis (3). Nachlas et al. (11) and Atkinson et al. (2) reported that 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) was more rapidly reduced than other tetrazolium salts. INT has been shown to be more toxic than other tetrazoliums when added to growing bacterial cultures by May et al. who found 64 ,ug/ml lethal for Staphylococcus aureus (10). Tetrazolium reduction by bacteria is enhanced under anaerobic conditions (1, 5, 13, 15) and is retarded by aeration (7). Although Pegram reported a decrease in tetrazolium reduction by Escherichia coli when lactose was
added to the medium, Somerson and Morton found lactate to enhance tetrazolium reduction by Mycoplasma (13). Increased amounts of glucose might enhance growth and the accumulation of reduced compounds in bacterial culture accelerating reduction of tetrazolium salts. Fred and Knight (7) reported no enhancement of tetrazolium reduction by Penicillium after the addition of glucose, but an evaluation of the effect on bacterial cultures has not been reported. Kopper reported that a pH of 7.5 was optimum and that concentrations of NaCl of 1% or less were desired for young metabolizing cells (8). Others have established a quantitative relationship between bacterial growth and tetrazolium reduction (8, 14). There is a need for a rapid and sensitive means for detection of bacterial growth in the presence of various inhibitors such as antibiotics. This is a report of studies that were conducted to obtain maximum sensitivity for detection of bacterial growth after a 3-h period of incubation. The inhibitory effect of the tetrazolium salt was avoided by adding the compound after the period of incubation. An optimal basal medium was selected and controlled amounts of PMS and INT comparable to concentrations used by other investigators were added (3, 6, 10). Subsequently, controlled amounts of glucose and agar were added to determine their effect on the amount of formazan produced. EXPERIMENTAL Evaluation of broths, combinations of INT and PMS and incubation time. A solution was prepared approximating the maximum solubility of INT in saline (2.0 mg/ml) by dissolving 200 mg in 5 ml of warm 100% ethyl alcohol and adding physiological 327
328
J. CLIN. MICROBIOL.
BARTLETT ET AL.
saline to produce a total volume of 100 ml. This was without supplements. This broth was employed stored in the dark at 4 C and was used within 1 without supplements at a pH of 7.2 to 7.4 in further month. Stationary phase cultures for each portion of studies. Effect of different concentrations of INT and this investigation were prepared by inoculating several colonies into broth from blood agar plates PMS. A solution consisting of 0.11 mg of PMS per ml which had been incubated with the test culture for in physiological saline was prepared and stored at 18 to 24 h. The inoculum density was equilibrated 4 C in the dark. This was used within 30 days. with the barium sulfate standard described by Mixtures of INT and PMS were prepared immediBauer et al. (4) by the addition of inoculum or dilu- ately before use. Any remaining was discarded betion with broth. This yielded a concentration of cause the mixture was observed to produce small about 1.5 x 108 colony-forming units/ml. This was amounts of formazan spontaneously after 30 to 60 subsequently diluted with broth to a final concen- min. All possible combinations of four concentratration of 10" colony-forming units/ml. This suspen- tions of each compound were employed. Prior to the sion (50 Au) was added to the wells of plastic microti- addition of the mixture to the tray wells final contration trays (Ames Co., Division of Miles Laborato- centrations of INT were 0.125, 0.25, 0.5, and 1.0 mg/ ries, Elkhart, Ind.) which already contained 50 /.d of ml and 0.005, 0.01, 0.03, and 0.06 mg/ml for PMS. S. broth in each well. The purpose of this was to simu- aureus ATCC 25923 (ATCC, American Type Culture late the use of these plates for biochemical or anti- Collection) and E. coli ATCC 25922 were incubated microbial susceptibility tests in which the trays at 37 C for 4 h. Fifty microliters of each INTwould contain previously prepared inhibitory solu- PMS combination was added to wells containing the tions in broth. culture. The color development was observed after Selection of broth. A comparison was made of 15, 30, and 45 min of further incubation at 37 C. brain heart infusion broth, tryptic digest casein soy Controls were prepared by inoculating wells with 50 broth, Mueller-Hinton broth, Schaedler broth, Col- ,ul of freshly prepared unincubated inoculum of each umbia broth, and Todd-Hewitt broth. All of these test culture. Blank broth controls were also used. were evaluated with and without the addition of RESULTS horse serum (2.5% vol/vol), Isovitalex (Baltimore The most intense color development was obBiological Laboratory, Cockeysville, Md.) and yeast extract (0.25% vol/vol) (Baltimore Biological Labo- served with mixtures containing the highest ratory). Following a 4-h incubation of test cultures, concentrations of INT and PMS (Table 1). No 50 ,l of a 0.5-mg/ml solution of INT in saline was color was observed up to 45 min after addition added to each microtitration tray well. The plates of INT and PMS to wells containing unincuwere reincubated for 30 to 60 min to observe color development. Preliminary studies revealed that dif- bated inocula of control cultures. Spontaneous ferences were more marked when a slowly growing color development was observed occasionally organism such as Staphylococcus epidermidis was afcer 30 and 45 min in control wells containing used. For this reason eight separate isolates of this uninoculated broth. Because of the insolubility species were tested. Schaedler broth demonstrated of INT and risk that more rapid and intense distinctly more color development both with and spontaneous reduction might result from
TABLE 1. Formazan precipitate after addition of INT and PMS S. aureus (ATCC 25923) Final concn (mg/ml) E. coli (ATCC 25922) (min) Broth controls (min) (min) INT
PMS
15
30
45
15
30
45
15
30
45
0.125 0.125 0.125 0.125 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5
0.005 0.01 0.03 0.06 0.005
+ +
+ + + + + + + + + + ++ ++ ++ ++ ++ +++
+ + + + + + ++ ++ ++ ++ ++ ++ ++ ++ +++ +++
+ ++ + ++ + + ++ +++ + ++ +++ +++ + + +++ +++
+ ++ + ++ ++ ++ +++ +++ ++ ++ +++ +++ ++ ++ +++ +++
++ ++
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0
0
1.0 1.0 1.0 1.0
0.01
0.03 0.06 0.005 0.01
0.03 0.06 0.005 0.01
0 + + + + + + + + + + + + ++
++ ++ ++ ++ +++ +++
++ +++ +++ +++ +++ +++ +++
0 0 0 0 0 0 0 0 0
0/+ 0/+ 0
0/+ 0.03 0/+ 0/+ 0.06 +++ 0/+ 0/+ a Amount of formazan precipitate 15, 30, and 45 min after the addition of varying concentrations of INT and PMS to cultures which had been incubated for 4 h. Unincubated inoculum controls produced no precipitate after 45 min. Uninoculated broth controls showed variable precipitation after 30 and 45 min. Symbols: (0) no color; (+) pink; (++) rose; (+++) red.
VOL. 3, 1976
TETRAZOLIUM REDUCTION BY BACTERIA
higher concentrations of PMS, solutions containing final concentration of 1.0 mg of INT per ml and 0.06 mg of PMS per ml were used in subsequent studies. The more intense and rapid color development afforded by this mixture of PMS and INT suggested a shortening of incubation of test cultures from 4 to 3 h. Repeat studies employing these concentrations of INT and PMS produced the same amount of color development in a 3-h culture that had been observed previously using only INT after 4 h of incubation. Therefore subsequent studies were performed after 3 h of incubation of the test cultures. Effect of varying concentrations of glucose and agar in Schaedler broth. Addition of glucose to Schaedler broth. Schaedler broth was prepared containing controlled amounts of glucose resulting in final concentrations ranging from 1 to 10 g/liter. An increase in formazan production was observed as the concentration was increased to 5 g/liter with the test cultures of S. aureus and E. coli. No additional formazan production was observed when higher concentrations of glucose were used (Table 2). Freshly prepared unincubated inoculum and TABLE 2. Effects ofvarying concentrations ofglucose in Schaedler brotha Amount of glucose in broth (g/liter) Culture
Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922) Broth control Unincubated inoculum controls
0
1.0
2.5
5
7.5
10
+
+
+
++
++
++
+
+
++
++
++
++
0 0
0 0
0 0
0 0
0 0
0 0
a A mixture consisting of final concentrations of 1 mg of INT per ml and 0.06 mg of PMS per ml was added after a 3-h incubation of cultures. Color development was evaluated 30 min after reincubation at 37 C. Symbols: (0) no color change; (+) pink; (++) rose.
TABLE 3. Effect of addition of agar to Schaedler
brotha Amount of Noble agar in broth
Organism 0
(g/liter) 0.5 0.8
1.0
Staphylococcus +++ +++ aureus + ++ (ATCC 25923) Escherichia coli (ATCC + +++ +++ ++ 25922) Control broth 0 0 0 0 Unincubated inoculum 0 0 0 0 controls a INT and PMS were added after 3 h of incubation. Inspection for color development was 30 min after reincubation at 37 C. Symbols: (0) no color change; (+) pink; (+ +) rose; (+ + +) red.
329
uninoculated broth controls demonstrated no formazan production. The original glucose content of the medium (5 g/liter) appeared to be optimal. Addition of agar to Schaedler broth. Noble agar was added to the broth to provide concentrations of 0.5 to 1.0 g/liter in the well during the period of incubation prior to addition of INT and PMS. Maximal formazan precipitate was produced with concentrations of 0.5 and 0.8 g/ liter (Table 3). Less formazan production was observed when 1 g/liter was used. This may have been caused by the increased viscosity of the broth at this concentration. Blank broth controls and freshly prepared unincubated inoculum demonstrated no formazan production. LITERATURE CITED 1. Altman, F. P. 1970. Oxygen-sensitivity of various tetrazolium salts. Histochemie 22(3):256-261. 2. Atkinson, E., S. Melvin, and S. W. Fox. 1950. Some properties of 2,3,5-triphenyltetrazolium chloride and several Iodo derivatives Science 111:385-387. 3. Barnett, H. 1962. Electrophoretic separation of lactate dehydrogenase isoenzymes on cellulose acetate. Biochem. J. 84:83p-84p. 4. Bauer, A. W., W. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standarized single disk method. Am. J. Clin. Pathol.
45:493-496. 5. Brodie, A. F., and J. S. Gots. 1951. The effects of an isolated dehydrogenase enzyme and flavoprotein on the reduction of triphenyltetrazolium chloride. Science 114:40-41. 6. Dickens, F., and H. Mcllwain. 1938. Phenazine compounds as carriers in the hexosemonophosphate system. Biochem. J. 32:1615-1625. 7. Fred, R. B., and J. G. Knight. 1949. The reduction of 2,3,5-triphenyltetrazolium chloride by Penicillium chrysogenum. Science 109:169-170. 8. Kopper, P. H. 1952. Studies on bacterial reducing activity in relation to age of culture. J. Bacteriol. 63:639645. 9. Mattsen, A. M., C. 0. Jensen, and R. A. Dutcher. 1947. Triphenyltetrazolium chloride as a dye for vital tissues. Science 106:294-295. 10. May, P. S., J. W. Winter, G. H. Fried, and W. Antopol. 1960. Effect of tetrazolium salts on selected bacterial species. Proc. Soc. Exp. Biol. Med. 105:364-366. 11. Nachlas, M. M., S. S. Karmarkar, and A. M. Seligman. 1960. Variations in reduction of tetrazolium salts by dehydrogenase systems. Proc. Soc. Exp. Biol. Med. 104:407-409. 12. Pegram, R. G. 1969. Microbiological uses of 2,3,5-triphenyltetrazolium chloride. J. Med. Lab. Technol. 26:175-198. 13. Somerson, N. L., and H. E. Morton. 1953. Reduction of tetrazolium salts by pleuropneumonialike organisms. J. Bacteriol. 65:245-251. 14. Tengerdy, R. P., J. G. Nagy, and B. Martin. 1967. Quantitative measurement of bacterial growth by the reduction of tetrazolium salts. Appl. Microbiol. 15:954-955. 15. Throneberry, G. O., and F. G. Smith. 1953. The effect of triphenyltetrazolium chloride on oat embryo respiration. Science 117:13-15. 16. Weinberg, E. D. 1953. Selective inhibition of microbial growth by the incorporation of triphenyl tetrazolium chloride in culture media. J. Bacteriol. 66:240-242.
