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Penicillin—The duration of its activity in blood clots
PENICILLIN--DURATION OF ITS ACTIVITY IN BLOOD CLOTS
223
to be investigated, its contents withdrawn and placed in a small sterile polythene specimen jar manufactured for the trial. W h e n both samples were taken, a fitting lid was applied to the iar immediately to prevent evaporation. Subsequent samples were taken from sockets which were undisturbed. All samples were stored at minus 3o°C until the assays were done. At the time o f taking the initial sample from each patient, a series of solutions o f known penicillin concentration were prepared from a standard 30o mg vial o f benzylpenicillin by serial dilutions using sterile distilled water as the solvent. Solutions of penicillin with concentrations ranging from o-oI to o.5//zg/ml were then put into sterile biiou bottles and placed with the serum, saliva and clot samples at minus 3o°C. Subsequent specimens from the same patient were treated similarly and placed with the original samples and standard solutions. After an interval of not more than five days, the samples and standard solutions were removed from the refrigerator. T h e blood clots were cut into fine slices o f approximately 4 ° / z i n thickness using a freezing microtome. These were weighed and an equal weight of distilled water added. T h e samples o f serum were diluted with sterile distilled water to bring the penicillin concentration to between o.oI and 0. 5 /zg per ml, which was considered the most suitable range for accurate reading in the assay m e t h o d used. T w o more different dilutions o f each sample o f serum were used in each case to ensure that at least one fell within the desired range. T h e cup-plate assay m e t h o d was employed using Sarcina Lutea ( A T C C 934I) , as the test organism. Each unknown solution was prepared and tested in duplicate and the average of the two readings recorded.
RESULTS T h e results of the investigations are set out in the accompanying tables (Tables I and II). F r o m the results in both groups it can be seen that penicillin did reach therapeutic concentrations in blood clots formed in tooth sockets, but it would appear from G r o u p I that, without reinforcing the initial dose, the concentration o f TABLE I G r o u p I.
T h e following concentrations o f penicillin are expressed as /xg per ml
Serum Serum Serum Serum Saliva Saliva Saliva Clot Clot Clot
Hours o 6 I2 24 6 I2 24 6 I2 24
r 2.o o'o5 o o 0"07 o o o.x2 o o
2 2. 3 o-15 o o o o o I.io o o
3 4 4.i 3.r o'35 o.2o o o o o 0"03 o-o2 o o o o 0.08 0"08 o o o o
5 3"25 0"25 o o o o o o.Io o o
6 3 .2 o'oI o o 0.02 o o 0.06 o o
7 9"5 1-2o o o 0"6o* 0.02 o o'40 0.06 o
8 2"8 o'I5 o o o o o 0-04 o o
9 6"o o'I5 o o o o o o'I4 o o
xo 5"3 o'55 o o 0"37* o o o'Io o o
* Denotes that the specimen of saliva was heavily contaminated with blood. One megaunit of benzylpenicillin was given intramuscularly one to one and a half hours prior to the extractions. 'Serum o' denotes the time that the extractions were completed.
224
BRITISH
JOURNAL
OF ORAL S U R G E R Y
TABLE I I G r o u p II. Serum Serum Serum Serum Saliva Saliva Saliva Clot Clot Clot
T h e following concentrations o f penicillin are expressed as/zg per ml Hours 2 6 z2 24 6 I2 24 6 I2 24
z 7"0 2"o o.2 o o"o5 o o 0"2 0"22 0"06
2 2"5 o'4 o'9 o'04 o 0"05 o o'18 0"28 o'02
3 7"x5 o.I8 0"24 o"r9 o o'oi o o'08 0"06 0"02
4 5"8 o'95 I-ZO 0"5 o.r6 0"04 0"04 0.42 0"38 0"04
5 4"5 o.6 0"55 o'z 5 o o'o2 o o'16 0"28 0"06
6 4.I5 0-2o o.8 o'6 o 0"02 o 0"20 0"20 0"02
7 2-8 o'55 o'3 o'o8 o o o o.2o 0"20 0"02
8 5"3 o'35 o'3 o'o6 o o-oz o o'i8 0"20 0"04
9 6"25 o'95 o'94 o'45 o'08 o o 0"36 0"40 0"08
ro 3"8 o'3 0"42 o'o6 o'oI o o o'12 O'ZO 0"04
One megaunit of benzylpenicillin was given intramuscularly 15 hours after the extractions, and this was followed by two subsequent intramuscular injections of 800,000 units of Procaine Penicillin Fortified, B.P. The time, as represented above, commenced with the first injection. penicillin in the clot had fallen to zero within 12 hours. T h e r e was one exception (number 7) and in this case, for an unexplained reason, the initial serum concentration was considerably higher than the others in the series, and this was reflected in the results of all the samples taken from this patient. F r o m the results in Group II, it is apparent that penicillin diffused readily into the formed clots, but relatively high doses o f Procaine Penicillin Injection, Fortified B.P. were required at frequent intervals to maintain it at a therapeutic concentration (Figs. I and 2). DISCUSSION T h e cup-plate method was chosen for this investigation as it is probably the most accurate and simple method available for penicillin assays (Bunn et al., 196o). Sarcina Lutea was used as the test organism, as it is extremely sensitive to penicillin (Bunn et al., 196o). T h e method has other advantages in the present context. It is highly adaptable, the samples need not be sterile and the suspended material does not interfere with the diffusion o f solution (Gavin, 1957). T h e sensitivity is dependent upon the thickness of agar used and, provided that meticulous care is taken, very small samples may be assayed with a high degree of accuracy. I n the present trial, sterile distilled water was used as the diluent for both the standard solutions and the material under test. Benzylpenicillin (penicillin G) shows less protein-binding than any of the other penicillins in common use (Rowlinson, I967) and errors introduced by the use of water as the diluent are of little significance. T h e results confirm that penicillin does penetrate blood clots in tooth sockets where i t is given before or after the extractions. However, it is clear that a single pre-operative injection o f benzylpenicillin produces a therapeutic level in the clot for a short time only, and would be of little assistance in reducing the incidence of bacterial invasion of tooth sockets after extraction. Group I I produced some interesting findings. It is apparent that, within 6 hours, penicillin had diffused rapidly into the formed clot and had produced a remarkably high concentration with only a single injection of benzylpenicillin. It has been known for years that penicillin diffuses as rapidly into fibrin as it does
PENICILLIN--DURATION
OF ITS A C T I V I T Y IN BLOOD CLOTS
225
into agar (Nathanson & Leibehold, i946); and Weinstein et al. (I95I), by implanting fibrin clots into the backs of rabbits, showed that clot and serum levels were approximately equal after 2 hours. Further, these workers went on to show that penicillin levels were detectable in the clot 4 to 12 hours after they were no longer detectable in the serum. This phenomenon has been noted in other experiments (Ungar, 195o). Thus it would appear that fibrin clots formed in tooth sockets behave in a similar manner to those inserted into experimental animals. Penicillin enters a wound by diffusion and the rate at which it does so is not greatly influenced by the age of the wound (Baker & Hunt, I968). As it has been established that while a concentration gradient exists the movement of antibiotics from serum to tissues will continue (Rowlinson, I967), it would seem to be always prudent to continue to prescribe penicillin after the initial dose if the incidence of tooth socket infection is to be minimised. The figures presented in Table II confirm that penicillin continues to reach the blood clots with successive doses of fortified procaine penicillin, but that the level markedly declines despite a total of 8oo,ooo units of penicillin being given at 7-hourly intervals. It has been noted that with higher systemic doses of penicillin the time that the drug is retained in a fibrin clot is increased, without a significant difference being made in the time that it is detectable in the serum (Weinstcin et al., I95I). It is established that the rate of diffusion of a drug is proportional to its concentration gradient (Rowlinson, I967). T h u s it is interesting to speculate that, on the basis of the figures from the Group II experiment, it might be advantageous to prescribe high doses of benzylpenicillin, at greater inter- 7 vals than is at present customary in order to encourage a high rate of-diffusion into the formed clots, rather than maintain a relatively lower serum level with a depot penicillin with its necessarily low concentration gradient. In the present experiments, conditions of the fibrin clots are rather different from those described in the animal investigations. Although surrounded by highly vascular tissue for the most part, blood clot formed in tooth sockets is being bathed continuously by saliva. The saliva itself might contain enough penicillin to encourage higher readings than might otherwise be found. On the other hand, if the concentration of penicillin were low, the continual passage of saliva over the clot, combined with the constant massage by the tongue and food particles, might cause penicillin to pass out of the clot more rapidly. It was for this reason that it was decided to assay the concentration of penicillin in the saliva on each occasion that a clot was investigated. As can be seen, in each group, saliva levels of penicillin were very low indeed, in many cases not even detectable. These findings correspond fairly well with those of Bender et al. (I953), who, after giving 4oo,0oo units of fortified procaine penicillin, found a peak saliva level of o-o29 units/ml (o'oI7/zg) and this occurred in the first hour. After 6 hours, the saliva level was only o'oo5 t~g/ml, completely undetectable by the cup-plate method of assay. It is clear that any penicillin present in the saliva could not have enhanced that present in the clot. The levels of penicillin in the clots in both groups are low, but it must be remembered that organisms may be inhibited or killed by very small concentrations of this drug. Eagle (I948) has shown that, in vitro, even with doses as low as o'oo4 tzg/ml, streptococcus pyogenes has a significantly decreased division rate, and at a concentration of o.oI6 tzg/ml penicillin is very actively bactericidal to this organism. An increase of Io,ooo-fold from this concentration has little increased q
3.56
~
9 8
~.7
~.6
s erum
GROUP I
clot
~.4
l
8
seliva
.~ .2 I1
6
/2 Time
24
in hours
4"93 serum
GROUP 2
clot
m
.0
so/ivo
§'"
l
!
1
I
|
l
|
) ~lmm
6)
| 0
e
[]
~B 24
Ti/ne in hours FIGS. I a n d 2 X d e n o t e s t h e t i m e t h e extractions were carried out. T h e arrows indicate t h e t i m e s at w h i c h t h e penicillin injections were given.
PENICILLIN--DURATION
OF
ITS A C T I V I T Y
IN B L O O D
CLOTS
227
bactericidal effect. In a later paper, Eagle and others (1953) confirm that larger doses of the antibiotic are no more bactericidal than smaller ones, but do have the advantage that the effective concentration is maintained for a longer time. The minimum inhibitory concentration of penicillin for Staph. aureus is o.o2 /zg/ml (Rowlinson, 1967) and for B. fusiformis, Strep. viridans and N . catarrhalis it is of the order of o'o3 /zg/ml (Garrod, I97I). It is clear that the minimum level achieved in the clots in Group II are of certain value in inhibiting organisms in the blood clots formed within the mouth after tooth extraction. CONCLUSION Benzylpenicillin, when given systemically, pre-operatively, is retained in the blood clots formed in tooth sockets after extraction, but its presence is very shortlived. When injected intramuscularly, post-operatively, after the cIots have formed, it diffuses rapidly into them and reaches a very adequate level within 6 hours and can be maintained there with successive doses of intramuscular penicillin. From the results of these investigations, it would seem to be unnecessary to continue the practice of giving pre-operative systemic penicillin at the time of the premedication, as post-operative doses achieve a therapeutic level of the drug within a very short time. It is possible that it may be more advantageous to use high doses of benzylpeniciUin which encourage rapid diffusion into avascular areas, rather than a depot penicillin preparation which produces a much lower serum concentration, albeit for a longer period, but gives a lower concentration in blood clot. ACKNOWLEDGEMENT S M y thanks are due to Professor Killey, Mr N. L. Rowe and Mr L. W. Kay for permission to perform these investigations on their patients. In particular I would like to thank Dr G. Blake, Head of the Department of Bacteriology at the Eastman Dental Hospital, for his advice and encouragement during the investigations, and Mrs Vidic, his Chief Technician, for her great technical assistance.
REFERENCES BAKER,G. & HUNT, T. (I968). American Journal of Surgery, II5, 53I. BENDER,I. B., PRESSMAN,R. S. & TASCHMAN,S. G. (1953). Journal of the American Dental Association, 46, I64. BUNN, P., KNIGHT,R. & AMBERG,J. (196o). Antimicrobial Agents Annual. EAGLE, H. (I948). Annals of Internal Medicine, z8, 26o. GAVIN, J. J. (I957). Applied Microbiology, 5, 25. GARROD, L. P. & O'GRADY, F. (197I). Antibiotics and Chemotherapy, 3rd ed., p. 56. Edinburgh: E. & S. Livingstone, Ltd. NATHANSON,M. H. & LIEBEHOLD,R. A. (1946). Proceedings of the Society of ExperimentM Biology and Medicine, 6z, 83. ROWLINSON,G. N. (I967). Recent Advances in Medical Microbiology, Chapter 7. Edited by A. P. Waterson. London: J. & A. Churchill. UNGAR,J. & PRAGUE,M. D. (I95o). Lancet, I, 56. W~INSTZIN, L., DAmOS, G. K. & PERRIN, T. S. (I951). Journal Laboratory and Clinical Medicine, 38, 712.
223
to be investigated, its contents withdrawn and placed in a small sterile polythene specimen jar manufactured for the trial. W h e n both samples were taken, a fitting lid was applied to the iar immediately to prevent evaporation. Subsequent samples were taken from sockets which were undisturbed. All samples were stored at minus 3o°C until the assays were done. At the time o f taking the initial sample from each patient, a series of solutions o f known penicillin concentration were prepared from a standard 30o mg vial o f benzylpenicillin by serial dilutions using sterile distilled water as the solvent. Solutions of penicillin with concentrations ranging from o-oI to o.5//zg/ml were then put into sterile biiou bottles and placed with the serum, saliva and clot samples at minus 3o°C. Subsequent specimens from the same patient were treated similarly and placed with the original samples and standard solutions. After an interval of not more than five days, the samples and standard solutions were removed from the refrigerator. T h e blood clots were cut into fine slices o f approximately 4 ° / z i n thickness using a freezing microtome. These were weighed and an equal weight of distilled water added. T h e samples o f serum were diluted with sterile distilled water to bring the penicillin concentration to between o.oI and 0. 5 /zg per ml, which was considered the most suitable range for accurate reading in the assay m e t h o d used. T w o more different dilutions o f each sample o f serum were used in each case to ensure that at least one fell within the desired range. T h e cup-plate assay m e t h o d was employed using Sarcina Lutea ( A T C C 934I) , as the test organism. Each unknown solution was prepared and tested in duplicate and the average of the two readings recorded.
RESULTS T h e results of the investigations are set out in the accompanying tables (Tables I and II). F r o m the results in both groups it can be seen that penicillin did reach therapeutic concentrations in blood clots formed in tooth sockets, but it would appear from G r o u p I that, without reinforcing the initial dose, the concentration o f TABLE I G r o u p I.
T h e following concentrations o f penicillin are expressed as /xg per ml
Serum Serum Serum Serum Saliva Saliva Saliva Clot Clot Clot
Hours o 6 I2 24 6 I2 24 6 I2 24
r 2.o o'o5 o o 0"07 o o o.x2 o o
2 2. 3 o-15 o o o o o I.io o o
3 4 4.i 3.r o'35 o.2o o o o o 0"03 o-o2 o o o o 0.08 0"08 o o o o
5 3"25 0"25 o o o o o o.Io o o
6 3 .2 o'oI o o 0.02 o o 0.06 o o
7 9"5 1-2o o o 0"6o* 0.02 o o'40 0.06 o
8 2"8 o'I5 o o o o o 0-04 o o
9 6"o o'I5 o o o o o o'I4 o o
xo 5"3 o'55 o o 0"37* o o o'Io o o
* Denotes that the specimen of saliva was heavily contaminated with blood. One megaunit of benzylpenicillin was given intramuscularly one to one and a half hours prior to the extractions. 'Serum o' denotes the time that the extractions were completed.
224
BRITISH
JOURNAL
OF ORAL S U R G E R Y
TABLE I I G r o u p II. Serum Serum Serum Serum Saliva Saliva Saliva Clot Clot Clot
T h e following concentrations o f penicillin are expressed as/zg per ml Hours 2 6 z2 24 6 I2 24 6 I2 24
z 7"0 2"o o.2 o o"o5 o o 0"2 0"22 0"06
2 2"5 o'4 o'9 o'04 o 0"05 o o'18 0"28 o'02
3 7"x5 o.I8 0"24 o"r9 o o'oi o o'08 0"06 0"02
4 5"8 o'95 I-ZO 0"5 o.r6 0"04 0"04 0.42 0"38 0"04
5 4"5 o.6 0"55 o'z 5 o o'o2 o o'16 0"28 0"06
6 4.I5 0-2o o.8 o'6 o 0"02 o 0"20 0"20 0"02
7 2-8 o'55 o'3 o'o8 o o o o.2o 0"20 0"02
8 5"3 o'35 o'3 o'o6 o o-oz o o'i8 0"20 0"04
9 6"25 o'95 o'94 o'45 o'08 o o 0"36 0"40 0"08
ro 3"8 o'3 0"42 o'o6 o'oI o o o'12 O'ZO 0"04
One megaunit of benzylpenicillin was given intramuscularly 15 hours after the extractions, and this was followed by two subsequent intramuscular injections of 800,000 units of Procaine Penicillin Fortified, B.P. The time, as represented above, commenced with the first injection. penicillin in the clot had fallen to zero within 12 hours. T h e r e was one exception (number 7) and in this case, for an unexplained reason, the initial serum concentration was considerably higher than the others in the series, and this was reflected in the results of all the samples taken from this patient. F r o m the results in Group II, it is apparent that penicillin diffused readily into the formed clots, but relatively high doses o f Procaine Penicillin Injection, Fortified B.P. were required at frequent intervals to maintain it at a therapeutic concentration (Figs. I and 2). DISCUSSION T h e cup-plate method was chosen for this investigation as it is probably the most accurate and simple method available for penicillin assays (Bunn et al., 196o). Sarcina Lutea was used as the test organism, as it is extremely sensitive to penicillin (Bunn et al., 196o). T h e method has other advantages in the present context. It is highly adaptable, the samples need not be sterile and the suspended material does not interfere with the diffusion o f solution (Gavin, 1957). T h e sensitivity is dependent upon the thickness of agar used and, provided that meticulous care is taken, very small samples may be assayed with a high degree of accuracy. I n the present trial, sterile distilled water was used as the diluent for both the standard solutions and the material under test. Benzylpenicillin (penicillin G) shows less protein-binding than any of the other penicillins in common use (Rowlinson, I967) and errors introduced by the use of water as the diluent are of little significance. T h e results confirm that penicillin does penetrate blood clots in tooth sockets where i t is given before or after the extractions. However, it is clear that a single pre-operative injection o f benzylpenicillin produces a therapeutic level in the clot for a short time only, and would be of little assistance in reducing the incidence of bacterial invasion of tooth sockets after extraction. Group I I produced some interesting findings. It is apparent that, within 6 hours, penicillin had diffused rapidly into the formed clot and had produced a remarkably high concentration with only a single injection of benzylpenicillin. It has been known for years that penicillin diffuses as rapidly into fibrin as it does
PENICILLIN--DURATION
OF ITS A C T I V I T Y IN BLOOD CLOTS
225
into agar (Nathanson & Leibehold, i946); and Weinstein et al. (I95I), by implanting fibrin clots into the backs of rabbits, showed that clot and serum levels were approximately equal after 2 hours. Further, these workers went on to show that penicillin levels were detectable in the clot 4 to 12 hours after they were no longer detectable in the serum. This phenomenon has been noted in other experiments (Ungar, 195o). Thus it would appear that fibrin clots formed in tooth sockets behave in a similar manner to those inserted into experimental animals. Penicillin enters a wound by diffusion and the rate at which it does so is not greatly influenced by the age of the wound (Baker & Hunt, I968). As it has been established that while a concentration gradient exists the movement of antibiotics from serum to tissues will continue (Rowlinson, I967), it would seem to be always prudent to continue to prescribe penicillin after the initial dose if the incidence of tooth socket infection is to be minimised. The figures presented in Table II confirm that penicillin continues to reach the blood clots with successive doses of fortified procaine penicillin, but that the level markedly declines despite a total of 8oo,ooo units of penicillin being given at 7-hourly intervals. It has been noted that with higher systemic doses of penicillin the time that the drug is retained in a fibrin clot is increased, without a significant difference being made in the time that it is detectable in the serum (Weinstcin et al., I95I). It is established that the rate of diffusion of a drug is proportional to its concentration gradient (Rowlinson, I967). T h u s it is interesting to speculate that, on the basis of the figures from the Group II experiment, it might be advantageous to prescribe high doses of benzylpenicillin, at greater inter- 7 vals than is at present customary in order to encourage a high rate of-diffusion into the formed clots, rather than maintain a relatively lower serum level with a depot penicillin with its necessarily low concentration gradient. In the present experiments, conditions of the fibrin clots are rather different from those described in the animal investigations. Although surrounded by highly vascular tissue for the most part, blood clot formed in tooth sockets is being bathed continuously by saliva. The saliva itself might contain enough penicillin to encourage higher readings than might otherwise be found. On the other hand, if the concentration of penicillin were low, the continual passage of saliva over the clot, combined with the constant massage by the tongue and food particles, might cause penicillin to pass out of the clot more rapidly. It was for this reason that it was decided to assay the concentration of penicillin in the saliva on each occasion that a clot was investigated. As can be seen, in each group, saliva levels of penicillin were very low indeed, in many cases not even detectable. These findings correspond fairly well with those of Bender et al. (I953), who, after giving 4oo,0oo units of fortified procaine penicillin, found a peak saliva level of o-o29 units/ml (o'oI7/zg) and this occurred in the first hour. After 6 hours, the saliva level was only o'oo5 t~g/ml, completely undetectable by the cup-plate method of assay. It is clear that any penicillin present in the saliva could not have enhanced that present in the clot. The levels of penicillin in the clots in both groups are low, but it must be remembered that organisms may be inhibited or killed by very small concentrations of this drug. Eagle (I948) has shown that, in vitro, even with doses as low as o'oo4 tzg/ml, streptococcus pyogenes has a significantly decreased division rate, and at a concentration of o.oI6 tzg/ml penicillin is very actively bactericidal to this organism. An increase of Io,ooo-fold from this concentration has little increased q
3.56
~
9 8
~.7
~.6
s erum
GROUP I
clot
~.4
l
8
seliva
.~ .2 I1
6
/2 Time
24
in hours
4"93 serum
GROUP 2
clot
m
.0
so/ivo
§'"
l
!
1
I
|
l
|
) ~lmm
6)
| 0
e
[]
~B 24
Ti/ne in hours FIGS. I a n d 2 X d e n o t e s t h e t i m e t h e extractions were carried out. T h e arrows indicate t h e t i m e s at w h i c h t h e penicillin injections were given.
PENICILLIN--DURATION
OF
ITS A C T I V I T Y
IN B L O O D
CLOTS
227
bactericidal effect. In a later paper, Eagle and others (1953) confirm that larger doses of the antibiotic are no more bactericidal than smaller ones, but do have the advantage that the effective concentration is maintained for a longer time. The minimum inhibitory concentration of penicillin for Staph. aureus is o.o2 /zg/ml (Rowlinson, 1967) and for B. fusiformis, Strep. viridans and N . catarrhalis it is of the order of o'o3 /zg/ml (Garrod, I97I). It is clear that the minimum level achieved in the clots in Group II are of certain value in inhibiting organisms in the blood clots formed within the mouth after tooth extraction. CONCLUSION Benzylpenicillin, when given systemically, pre-operatively, is retained in the blood clots formed in tooth sockets after extraction, but its presence is very shortlived. When injected intramuscularly, post-operatively, after the cIots have formed, it diffuses rapidly into them and reaches a very adequate level within 6 hours and can be maintained there with successive doses of intramuscular penicillin. From the results of these investigations, it would seem to be unnecessary to continue the practice of giving pre-operative systemic penicillin at the time of the premedication, as post-operative doses achieve a therapeutic level of the drug within a very short time. It is possible that it may be more advantageous to use high doses of benzylpeniciUin which encourage rapid diffusion into avascular areas, rather than a depot penicillin preparation which produces a much lower serum concentration, albeit for a longer period, but gives a lower concentration in blood clot. ACKNOWLEDGEMENT S M y thanks are due to Professor Killey, Mr N. L. Rowe and Mr L. W. Kay for permission to perform these investigations on their patients. In particular I would like to thank Dr G. Blake, Head of the Department of Bacteriology at the Eastman Dental Hospital, for his advice and encouragement during the investigations, and Mrs Vidic, his Chief Technician, for her great technical assistance.
REFERENCES BAKER,G. & HUNT, T. (I968). American Journal of Surgery, II5, 53I. BENDER,I. B., PRESSMAN,R. S. & TASCHMAN,S. G. (1953). Journal of the American Dental Association, 46, I64. BUNN, P., KNIGHT,R. & AMBERG,J. (196o). Antimicrobial Agents Annual. EAGLE, H. (I948). Annals of Internal Medicine, z8, 26o. GAVIN, J. J. (I957). Applied Microbiology, 5, 25. GARROD, L. P. & O'GRADY, F. (197I). Antibiotics and Chemotherapy, 3rd ed., p. 56. Edinburgh: E. & S. Livingstone, Ltd. NATHANSON,M. H. & LIEBEHOLD,R. A. (1946). Proceedings of the Society of ExperimentM Biology and Medicine, 6z, 83. ROWLINSON,G. N. (I967). Recent Advances in Medical Microbiology, Chapter 7. Edited by A. P. Waterson. London: J. & A. Churchill. UNGAR,J. & PRAGUE,M. D. (I95o). Lancet, I, 56. W~INSTZIN, L., DAmOS, G. K. & PERRIN, T. S. (I951). Journal Laboratory and Clinical Medicine, 38, 712.