Vol. 10, No. 4
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1979, p. 488-491 0095-1 137/79/10-0488/04$02.00/0
Effects of Blood on Blood Culture Medium BEAMAN,`*
BERNARD L. KASTEN,' CYNTHIA L. CORLETT,2 AND THOMAS L. GAVAN2 George H. Scott Research Laboratory, Department of Pathology, Fairvieu General Hospital, Cleveland, Ohio 4411I,' and Department of Microbiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44111
KENNETH D.
Received for publication 23 July 1979
The morphological and biochemical changes that occur after inoculation of sterile blood into a blood culture medium (tryptic soy broth) with sodium polyanetholesulfonate and CO2 were investigated. Cellular changes, pH, PCO2, and PO, were monitored and evaluated. Erythrocytes became crenated and developed precipitated hemoglobin inclusions within 4 h. The lymphocytes appeared morphologically intact at 24 h, and by 48 h a few cells had undergone transformation. Many neutrophils were vacuolated at 24 h. Neutrophils capable of phagocytizing Staphylococcus aureus were observed after 18 h of incubation. Identifiable eosinophiles were present on day 6 of the study. A decrease in P02 in the unvented bottles from 44.4 to 8 mm of Hg occurred by 24 h. PO) remained low for 6 days, after which a slight increase occurred. An increase in P02 in the vented bottle from 51 to 58 mm of Hg occurred by 24 h of incubation. In both the vented and unvented bottles the PCO, increased. This increase was markedly more rapid in the unvented bottle. From a pH of 7.06 a decrease occurred for the first 24 h after inoculation, with the pH stabilizing at 6.8 in the vented bottles and at 6.6 in the unvented bottles. The biochemical changes that occurred in the vented culture bottles stabilized more rapidly than those of the unvented bottles. Changes caused by the addition of sterile blood to a blood culture medium resulted in conditions which departed considerably from accepted optima for the isolation of clinically important microorganisms. The phagocytosis of organisms that occurred may also have reduced the yield.
Recent interest in blood culture systems and, in particular, blood culture media has resulted in many comparative studies on the effectiveness of various experimental media and medium additives (5, 78-10). However, little work has been done on the effect of blood on these culture media. Some attention has been directed toward the removal of blood from the medium, and various other manipulations to decrease the bactericidal activity of blood have been investigated (2, 3, 6, 12). These investigations have been taken undertaken without regard to the effects of the principal variable, the blood itself. The purpose of our investigation was to analyze the events that take place after blood is added to a standard blood culture medium. To approach this problem, two major parameters were studied: (i) soluble gases and hydrogen-ion changes in the system, and (ii) leukocyte viability. The study was undertaken to determine whether the effects of blood alone caused a departure from optimal conditions for microbial growth (4) and to evaluate the morphological and functional integrity of the leukocytes present in the inoculum by observing morphological 488
changes that took place during incubation of the culture medium. MATERIALS AND METHODS The medium used in this study was tryptic soy broth with 0.025'7 sodium polyanethanolesulfonate under 10% CO. and vacuum (Difco Laboratories, Detroit, Mich.) in 50-ml bottles. In the first part of the study, 38 bottles from lots 6221316 and 632944 were used. Initially, each bottle was inoculated with 5 ml of freshly drawn donor blood which was not anticoagulated. Nineteen of the 38 blood culture bottles were vented for 30 s with a cotton-plugged 21-gauge needle. The bottles were then incubated at 35°C. Samples were taken immediately after inoculation, at 2, 4, 6, 8 and 24 h, and once daily for the remaining 6 days. A 5-ml syringe with 20-gauge needle was used to take a 2-ml sample of the culture medium. The rubber bottle stopper was swabbed with isopropyl alcohol (Clinipad, Stamford, Conn.) before each manipulation. Blood smears were made from each sample. Later each slide was Wright stained on a Hematec Slide Stainer (Ames, Elkhart, Ind.) and examined for morphological changes and cellular integrity. The needles were corked, and the syringes were placed in a container of ice. The samples were then immediately tested for soluble gases (PCO2 and PO2) and pH on an
VOL. OF BLOOD ON BLOOD CULTURE MEDIUM 1979EFFECT VOL. 1979 10,10,
489
IL-310 Blood Gas Analyzer (Instrument Laboratories, Lexington, Mass.). The ability of neutrophils to phagocytize live Staphylococcus aureus was studied. In the second part of the study, 10 bottles of tryptic soy broth with 0.025 sodium polyanetholesulfonate under 10% CO2 and vacuum (Difco lot 640840) were each inoculated with 5 ml of freshly drawn blood which was not anticoagulated. The bottles were vented as described above and then incubated at 350C for 30 min and 1, 2, 6 and 18 h. After incubation, 1.0 mil of a stationary-phase culture of S. aureus containing i0' colony-forming units per ml was inoculated into each bottle, and the bottles were then reincubated at 350C. After 30 min, the bottles were opened, the contents were centrifuged, and smears were made of the "buffy-coat" layer of the sediment. Gram and Wright staining was performed on the smeared sediment, which was then examined microscopically for the presence of phagocytized or-
ganisms.
In the third part of the study, 24 tryptic soy bottles (Difco lot 640840) were modified to a final pH of 7.5 ± 0.1 by the addition of 2.6 ml of 0.1 N NaOH. Blood was added to the bottles, and the pH values were determined as in the first part of the study.
FIG. 1. Phagocytosis of S. aureus in medium.
Control blood smears and differential cell counts performed on the fresh blood before inoculation to document normal cell morphology. Control samples for soluble-gas analysis were obtained from uninoculated vented and unvented blood culture bottles without blood at the same intervals as the experimental samples were obtained.
100
were
RESULTS The samples taken immediately after inoculation revealed that erythrocyte structural integrity was retained. However, there was evidence of precipitated hemoglobin and slight crenation. After 4 h, the swelling was maximnal and precipitated hemoglobin again became evident. The erythrocytes demonstrated similar morphological features for the next 4 to 5 days, at which timne total lysis was observed. Examination of samples taken immediately after inoculation revealed slight morphological changes in the neutrophils and lymphocytes. By h 6 of incubation, a definite decrease in the number of identifiable leukocytes was apparent. Intact lymphocytes and neutrophils containing phagocytized amorphus material were present at 24 h. Phagocytosis of S. aureus was demonstrated after 18 h of incubation (Fig. 1). After 48 h of incubation, identifiable neutrophils and lymphocytes were rarely observed. Eosinophils remained identifiable until day 6 of incubation; all other leukocytes lost their structural integrity after day 4. The oxygen concentrations i -.i both vented and unvented bottles without blood remained relatively stable during the test period (Fig. 2 and 3). A marked decrease in P02 from 50 mm of Hg
90 80
70
F-
60 CM
o
0L
r
50
7
40 30
20
10 0
6
24
II
48
6 DAYS
HOURS FIG. 2. Mean P02 changes in aerobiG,blood culture medium. Symbols: ci, without blood; M with blood.
Hg occurred in the unvented bottles with blood. A lesser decrease from 50 to 40 mm of Hg occurred in the vented bottles. In the unvented bottles containing blood, a PCO2 concentration equal to 18 ± 2 mm of Hg was observed initially (Fig. 4). Thin value rose abruptly within 24 h and rose slightly during the remaining 6 days. The vented bottles containing blood had initial values approximately equal to those of the unvented bottles but rose continuously throughout the incubation period (Fig. 5). The initial pH in all cases was 7.06 ± 0.05. The pH values of all the control bottles containing only the medium remained stable for 4 to 5 to less than 10 mm of
J. CLIN. MICROBIOL.
BEAMAN ET AL.
490
medium remained nearer accepted optima (4) throughout the 7-day incubation with blood (4). The observed pH change in the modified bottles was from an initial pH of 7.4 ± .05 to a final pH of 7.11 ± .08. The PCO2 and P02 changes were similar to those observed for unmodified bottles.
100
90
80 70 60 N
0
a.
DISCUSSION The data demonstrate that the addition of
50 40 30 20
JLf
blood to a blood culture medium aids in achieving anaerobic conditions by decreasing the amount of soluble oxygen in the medium. This
g
is probably the result of binding of
I0
0
24
6
48
6 DAYS
HOURS FIG. 31. Mean P02 changes in an anaerobic blood culture J ,nedium. Symbols: o., without blood; a% with blood.
oxygen
by
the erythrocytes. However, in general, changes that occur as a result of the addition of blood to a culture medium cause a departure from accepted optima for the growth of most clinically important isolates. The observed decrease in pH from 7.1 to 6.6 probably caused by the formation 50
50 45 40
45 40 -
35 30
35-
O
a-
cli
30
° 25 020
25-
20
1530
15 6-4
4
6DY
I0-
10
5
5
E~~~~~~~~~~~~~~~~~~~
0
6
24
48
6 DAYS
0
6
24
48
6 DAYS
HOURS
HOURS
FIG. 4. Mean PCO2 changes in an anaerobic culture medium. Symbols: c, without blood; rm, with blood.
FIG. 5. Mean PCO2 changes in an aerobic culture medium. Symbols: o, without blood; ma, with blood. 7.4
days, at which time a slight drop was observed 7.2 (Fig. 6). This corresponds to a slight increase in the to blood of addition the PCO2 (Fig. 5). The 7.0 culture medium in all cases caused a marked 6.8 decrease in pH after 24 h. This decrease was I accompanied by an increase in the PCO2 concen- 0f. 6.6 tration in the unvented bottles (Fig. 4). Twenty6.4 eight of 38 bottles demonstrated twofold or 6.2 greater increases in PCO2, with pH changes of discrepancies observed The 0.3 to 0.4 pH unit. 6.0 I can possibly be accounted for by the leaking of 6 DAYS 24 48 6 0 outside air into, and the inside gas mixture out HOURS of, the bottle during sampling. No differences between the lots tested were observed. FIG. 6. Mean pH changes. Symbols: m, without In the series of 24 bottles modified by the blood; c, with blood, aerobic; _, with blood, anaeraddition of 2.6 ml of 0.1 NaOH, the pH of the obic.
VOL. 10, 1979
EFFECT OF BLOOD ON BLOOD CULTURE MEDIUM
of carbonic acid from carbon dioxide may inhibit the growth of gram-positive organisms (1, 11); the decreasing P02 concentration observed during incubation in vented bottles may also hinder the growth of aerobic organisms. The existence of viable phagocytic cells for periods of at least 18 h may decrease the ability of the blood culture medium to support clinically important microorganisms. The antiphagocytic properties of sodium polyanetholesulfonate appears insufficient to prevent phagocytosis under the condition of this study. The major buffer system in the medium used, as with most culture media, was K2HPO4. The buffer has a pKa of 7.2 and is ideal for control of pH values between 7.1 and 7.4, but has little efficacy as a buffer below a pH of 7.1. The data presented indicate that the present buffer system is ineffective in this medium at this pH. Although the removal of blood from a culture system may be desirable for the recovery of most organisms, no practical methods suitable for use in a large clinical laboratory have been proposed (3, 6, 12). The pH decrease which must inhibit the growth of many gram-positive organisms and the overall suitability of the culture medium can be improved by increasing the initial pH to 7.4 (4). This can be done without the increase of buffer by adding small quantities of alkali to the medium. This slight increase allows full utilization of the available buffer system. By increasing the pH to 7.4 and using the same amount of buffer, a change that formerly resulted in a pH decrease from 7.1 to 6.6 results in only a slight change from 7.4 to 7.2. Avoidance of extreme departures from physiological conditions throughout the entire incubation period improves the theoretical suitability of the medium (4) and should enhance the overall ability of the medium to support microbial growth.
491
ACKNOWLEDGMENTS We express our appreciation to Richard Savage, Department of Hematology and Blood Bank, Cleveland Clinic Foundation, who reviewed the blood smears; to Maryanne Povinelli, who provided technical assistance for the phagocytosis studies; and to Hugh F. McCorkle, Department of Pathology, Fairview General Hospital, who photographed the phagocytized S. aureus.
LITERATURE CITED 1.
2. 3.
4. 5.
Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber-
gey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. Dorn, G. L., G. G. Burson, and R. J. Haynes. 1976. Blood culture technique based on centrifugation: developmental phase. J. Clin. Microbiol. 3:251-257. Dorn, G. L, G. G. Burson, and J. R. Haynes. 1976. Blood culture technique based on centrifugation: clinical evaluation. J. Clin. Microbiol. 3:258-263. Frobisher, M., and R. Fuerst. 1973. Microbiology in health and disease, p. 128-129. W. B. Saunders Co., Philadelphia. Hall, M., E. Warren, and J. A. Washington II. 1974. Detection of bacteremia with liquid media containing sodium polyanethanolsulfonate. Appl. Microbiol. 27:
187-191. 6. Kagen, R. L., W. H. Schuette, C. H. Zierdt, and J. D.
MacLowry. 1977. Rapid automated diagnosis of bacteremia by impedance detection. J. Clin. Microbiol. 5: 51-57. 7. Roberts, G. D., C. Horstmeir, M. Hall, and J. A. Washington II. 1975. Recovery of yeast from blood culture bottles. J. Clin. Microbiol. 2:18-20. 8. Roberts, G. D., and J. A. Washington II. 1975. Detection of fungi in blood cultures. J. Clin. Microbiol. 1:309310. 9. Rosner, R. F. 1974. Evaluation of four blood culture methods. Appl. Microbiol. 28:245-247. 10. Rosner, R. F. 1968. Effect of various anticoagulants and no anticoagulants on the ability to isolate bacteria t9 rectly from parallel clinical blood cultures. Am. J. Clin. Pathol. 46:216-219. 11. Smolka, L. R., F. E. Nelson, and L. M. Kelley. 1974. Interaction of pH and NaCl on enumeration of heatstressed Staphylococcus aureus. Appl. Microbiol. 27: 443-447. 12. Sullivan, N. M., V. L. Sutter, and S. M. Finegold. 1975. Practical aerobic membrane filtration blood cultures technique: clinical blood culture trial. J. Clin. Microbiol. 1:37-43.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1979, p. 488-491 0095-1 137/79/10-0488/04$02.00/0
Effects of Blood on Blood Culture Medium BEAMAN,`*
BERNARD L. KASTEN,' CYNTHIA L. CORLETT,2 AND THOMAS L. GAVAN2 George H. Scott Research Laboratory, Department of Pathology, Fairvieu General Hospital, Cleveland, Ohio 4411I,' and Department of Microbiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44111
KENNETH D.
Received for publication 23 July 1979
The morphological and biochemical changes that occur after inoculation of sterile blood into a blood culture medium (tryptic soy broth) with sodium polyanetholesulfonate and CO2 were investigated. Cellular changes, pH, PCO2, and PO, were monitored and evaluated. Erythrocytes became crenated and developed precipitated hemoglobin inclusions within 4 h. The lymphocytes appeared morphologically intact at 24 h, and by 48 h a few cells had undergone transformation. Many neutrophils were vacuolated at 24 h. Neutrophils capable of phagocytizing Staphylococcus aureus were observed after 18 h of incubation. Identifiable eosinophiles were present on day 6 of the study. A decrease in P02 in the unvented bottles from 44.4 to 8 mm of Hg occurred by 24 h. PO) remained low for 6 days, after which a slight increase occurred. An increase in P02 in the vented bottle from 51 to 58 mm of Hg occurred by 24 h of incubation. In both the vented and unvented bottles the PCO, increased. This increase was markedly more rapid in the unvented bottle. From a pH of 7.06 a decrease occurred for the first 24 h after inoculation, with the pH stabilizing at 6.8 in the vented bottles and at 6.6 in the unvented bottles. The biochemical changes that occurred in the vented culture bottles stabilized more rapidly than those of the unvented bottles. Changes caused by the addition of sterile blood to a blood culture medium resulted in conditions which departed considerably from accepted optima for the isolation of clinically important microorganisms. The phagocytosis of organisms that occurred may also have reduced the yield.
Recent interest in blood culture systems and, in particular, blood culture media has resulted in many comparative studies on the effectiveness of various experimental media and medium additives (5, 78-10). However, little work has been done on the effect of blood on these culture media. Some attention has been directed toward the removal of blood from the medium, and various other manipulations to decrease the bactericidal activity of blood have been investigated (2, 3, 6, 12). These investigations have been taken undertaken without regard to the effects of the principal variable, the blood itself. The purpose of our investigation was to analyze the events that take place after blood is added to a standard blood culture medium. To approach this problem, two major parameters were studied: (i) soluble gases and hydrogen-ion changes in the system, and (ii) leukocyte viability. The study was undertaken to determine whether the effects of blood alone caused a departure from optimal conditions for microbial growth (4) and to evaluate the morphological and functional integrity of the leukocytes present in the inoculum by observing morphological 488
changes that took place during incubation of the culture medium. MATERIALS AND METHODS The medium used in this study was tryptic soy broth with 0.025'7 sodium polyanethanolesulfonate under 10% CO. and vacuum (Difco Laboratories, Detroit, Mich.) in 50-ml bottles. In the first part of the study, 38 bottles from lots 6221316 and 632944 were used. Initially, each bottle was inoculated with 5 ml of freshly drawn donor blood which was not anticoagulated. Nineteen of the 38 blood culture bottles were vented for 30 s with a cotton-plugged 21-gauge needle. The bottles were then incubated at 35°C. Samples were taken immediately after inoculation, at 2, 4, 6, 8 and 24 h, and once daily for the remaining 6 days. A 5-ml syringe with 20-gauge needle was used to take a 2-ml sample of the culture medium. The rubber bottle stopper was swabbed with isopropyl alcohol (Clinipad, Stamford, Conn.) before each manipulation. Blood smears were made from each sample. Later each slide was Wright stained on a Hematec Slide Stainer (Ames, Elkhart, Ind.) and examined for morphological changes and cellular integrity. The needles were corked, and the syringes were placed in a container of ice. The samples were then immediately tested for soluble gases (PCO2 and PO2) and pH on an
VOL. OF BLOOD ON BLOOD CULTURE MEDIUM 1979EFFECT VOL. 1979 10,10,
489
IL-310 Blood Gas Analyzer (Instrument Laboratories, Lexington, Mass.). The ability of neutrophils to phagocytize live Staphylococcus aureus was studied. In the second part of the study, 10 bottles of tryptic soy broth with 0.025 sodium polyanetholesulfonate under 10% CO2 and vacuum (Difco lot 640840) were each inoculated with 5 ml of freshly drawn blood which was not anticoagulated. The bottles were vented as described above and then incubated at 350C for 30 min and 1, 2, 6 and 18 h. After incubation, 1.0 mil of a stationary-phase culture of S. aureus containing i0' colony-forming units per ml was inoculated into each bottle, and the bottles were then reincubated at 350C. After 30 min, the bottles were opened, the contents were centrifuged, and smears were made of the "buffy-coat" layer of the sediment. Gram and Wright staining was performed on the smeared sediment, which was then examined microscopically for the presence of phagocytized or-
ganisms.
In the third part of the study, 24 tryptic soy bottles (Difco lot 640840) were modified to a final pH of 7.5 ± 0.1 by the addition of 2.6 ml of 0.1 N NaOH. Blood was added to the bottles, and the pH values were determined as in the first part of the study.
FIG. 1. Phagocytosis of S. aureus in medium.
Control blood smears and differential cell counts performed on the fresh blood before inoculation to document normal cell morphology. Control samples for soluble-gas analysis were obtained from uninoculated vented and unvented blood culture bottles without blood at the same intervals as the experimental samples were obtained.
100
were
RESULTS The samples taken immediately after inoculation revealed that erythrocyte structural integrity was retained. However, there was evidence of precipitated hemoglobin and slight crenation. After 4 h, the swelling was maximnal and precipitated hemoglobin again became evident. The erythrocytes demonstrated similar morphological features for the next 4 to 5 days, at which timne total lysis was observed. Examination of samples taken immediately after inoculation revealed slight morphological changes in the neutrophils and lymphocytes. By h 6 of incubation, a definite decrease in the number of identifiable leukocytes was apparent. Intact lymphocytes and neutrophils containing phagocytized amorphus material were present at 24 h. Phagocytosis of S. aureus was demonstrated after 18 h of incubation (Fig. 1). After 48 h of incubation, identifiable neutrophils and lymphocytes were rarely observed. Eosinophils remained identifiable until day 6 of incubation; all other leukocytes lost their structural integrity after day 4. The oxygen concentrations i -.i both vented and unvented bottles without blood remained relatively stable during the test period (Fig. 2 and 3). A marked decrease in P02 from 50 mm of Hg
90 80
70
F-
60 CM
o
0L
r
50
7
40 30
20
10 0
6
24
II
48
6 DAYS
HOURS FIG. 2. Mean P02 changes in aerobiG,blood culture medium. Symbols: ci, without blood; M with blood.
Hg occurred in the unvented bottles with blood. A lesser decrease from 50 to 40 mm of Hg occurred in the vented bottles. In the unvented bottles containing blood, a PCO2 concentration equal to 18 ± 2 mm of Hg was observed initially (Fig. 4). Thin value rose abruptly within 24 h and rose slightly during the remaining 6 days. The vented bottles containing blood had initial values approximately equal to those of the unvented bottles but rose continuously throughout the incubation period (Fig. 5). The initial pH in all cases was 7.06 ± 0.05. The pH values of all the control bottles containing only the medium remained stable for 4 to 5 to less than 10 mm of
J. CLIN. MICROBIOL.
BEAMAN ET AL.
490
medium remained nearer accepted optima (4) throughout the 7-day incubation with blood (4). The observed pH change in the modified bottles was from an initial pH of 7.4 ± .05 to a final pH of 7.11 ± .08. The PCO2 and P02 changes were similar to those observed for unmodified bottles.
100
90
80 70 60 N
0
a.
DISCUSSION The data demonstrate that the addition of
50 40 30 20
JLf
blood to a blood culture medium aids in achieving anaerobic conditions by decreasing the amount of soluble oxygen in the medium. This
g
is probably the result of binding of
I0
0
24
6
48
6 DAYS
HOURS FIG. 31. Mean P02 changes in an anaerobic blood culture J ,nedium. Symbols: o., without blood; a% with blood.
oxygen
by
the erythrocytes. However, in general, changes that occur as a result of the addition of blood to a culture medium cause a departure from accepted optima for the growth of most clinically important isolates. The observed decrease in pH from 7.1 to 6.6 probably caused by the formation 50
50 45 40
45 40 -
35 30
35-
O
a-
cli
30
° 25 020
25-
20
1530
15 6-4
4
6DY
I0-
10
5
5
E~~~~~~~~~~~~~~~~~~~
0
6
24
48
6 DAYS
0
6
24
48
6 DAYS
HOURS
HOURS
FIG. 4. Mean PCO2 changes in an anaerobic culture medium. Symbols: c, without blood; rm, with blood.
FIG. 5. Mean PCO2 changes in an aerobic culture medium. Symbols: o, without blood; ma, with blood. 7.4
days, at which time a slight drop was observed 7.2 (Fig. 6). This corresponds to a slight increase in the to blood of addition the PCO2 (Fig. 5). The 7.0 culture medium in all cases caused a marked 6.8 decrease in pH after 24 h. This decrease was I accompanied by an increase in the PCO2 concen- 0f. 6.6 tration in the unvented bottles (Fig. 4). Twenty6.4 eight of 38 bottles demonstrated twofold or 6.2 greater increases in PCO2, with pH changes of discrepancies observed The 0.3 to 0.4 pH unit. 6.0 I can possibly be accounted for by the leaking of 6 DAYS 24 48 6 0 outside air into, and the inside gas mixture out HOURS of, the bottle during sampling. No differences between the lots tested were observed. FIG. 6. Mean pH changes. Symbols: m, without In the series of 24 bottles modified by the blood; c, with blood, aerobic; _, with blood, anaeraddition of 2.6 ml of 0.1 NaOH, the pH of the obic.
VOL. 10, 1979
EFFECT OF BLOOD ON BLOOD CULTURE MEDIUM
of carbonic acid from carbon dioxide may inhibit the growth of gram-positive organisms (1, 11); the decreasing P02 concentration observed during incubation in vented bottles may also hinder the growth of aerobic organisms. The existence of viable phagocytic cells for periods of at least 18 h may decrease the ability of the blood culture medium to support clinically important microorganisms. The antiphagocytic properties of sodium polyanetholesulfonate appears insufficient to prevent phagocytosis under the condition of this study. The major buffer system in the medium used, as with most culture media, was K2HPO4. The buffer has a pKa of 7.2 and is ideal for control of pH values between 7.1 and 7.4, but has little efficacy as a buffer below a pH of 7.1. The data presented indicate that the present buffer system is ineffective in this medium at this pH. Although the removal of blood from a culture system may be desirable for the recovery of most organisms, no practical methods suitable for use in a large clinical laboratory have been proposed (3, 6, 12). The pH decrease which must inhibit the growth of many gram-positive organisms and the overall suitability of the culture medium can be improved by increasing the initial pH to 7.4 (4). This can be done without the increase of buffer by adding small quantities of alkali to the medium. This slight increase allows full utilization of the available buffer system. By increasing the pH to 7.4 and using the same amount of buffer, a change that formerly resulted in a pH decrease from 7.1 to 6.6 results in only a slight change from 7.4 to 7.2. Avoidance of extreme departures from physiological conditions throughout the entire incubation period improves the theoretical suitability of the medium (4) and should enhance the overall ability of the medium to support microbial growth.
491
ACKNOWLEDGMENTS We express our appreciation to Richard Savage, Department of Hematology and Blood Bank, Cleveland Clinic Foundation, who reviewed the blood smears; to Maryanne Povinelli, who provided technical assistance for the phagocytosis studies; and to Hugh F. McCorkle, Department of Pathology, Fairview General Hospital, who photographed the phagocytized S. aureus.
LITERATURE CITED 1.
2. 3.
4. 5.
Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber-
gey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. Dorn, G. L., G. G. Burson, and R. J. Haynes. 1976. Blood culture technique based on centrifugation: developmental phase. J. Clin. Microbiol. 3:251-257. Dorn, G. L, G. G. Burson, and J. R. Haynes. 1976. Blood culture technique based on centrifugation: clinical evaluation. J. Clin. Microbiol. 3:258-263. Frobisher, M., and R. Fuerst. 1973. Microbiology in health and disease, p. 128-129. W. B. Saunders Co., Philadelphia. Hall, M., E. Warren, and J. A. Washington II. 1974. Detection of bacteremia with liquid media containing sodium polyanethanolsulfonate. Appl. Microbiol. 27:
187-191. 6. Kagen, R. L., W. H. Schuette, C. H. Zierdt, and J. D.
MacLowry. 1977. Rapid automated diagnosis of bacteremia by impedance detection. J. Clin. Microbiol. 5: 51-57. 7. Roberts, G. D., C. Horstmeir, M. Hall, and J. A. Washington II. 1975. Recovery of yeast from blood culture bottles. J. Clin. Microbiol. 2:18-20. 8. Roberts, G. D., and J. A. Washington II. 1975. Detection of fungi in blood cultures. J. Clin. Microbiol. 1:309310. 9. Rosner, R. F. 1974. Evaluation of four blood culture methods. Appl. Microbiol. 28:245-247. 10. Rosner, R. F. 1968. Effect of various anticoagulants and no anticoagulants on the ability to isolate bacteria t9 rectly from parallel clinical blood cultures. Am. J. Clin. Pathol. 46:216-219. 11. Smolka, L. R., F. E. Nelson, and L. M. Kelley. 1974. Interaction of pH and NaCl on enumeration of heatstressed Staphylococcus aureus. Appl. Microbiol. 27: 443-447. 12. Sullivan, N. M., V. L. Sutter, and S. M. Finegold. 1975. Practical aerobic membrane filtration blood cultures technique: clinical blood culture trial. J. Clin. Microbiol. 1:37-43.
