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The Self-Sterilizing Properties of the Vitreous*
T H E SELF-STERILIZING P R O P E R T I E S O F T H E V I T R E O U S * HOWARD REED, M.B.,
ANI ) SHIRLEY PLATTS,
B.Sc.
Winnipeg, Manitoba Since Shafer 1-3 introduced the implanta tion of vitreous in the treatment of retinal detachment, its use has steadily increased. It has, however, brought with it a serious risk. Vitreous contains protein and in the living eye it is maintained at body temperature so that it should act as an ideal culture me dium.4"5 It has no blood supply to promote the rapid mobilization of antibacterial forces and has, therefore, always been considered to have a low resistance to infection. In an attempt to assess the danger of in traocular infection from the use of stored vitreous a series of studies on the self-steril izing properties of vitreous has been under taken in the Winnipeg General Hospital. The first study concerned the storage of contaminated vitreous at 4°C. Shafer 3 was the first to point out that if human vitreous is infected with Staphylococci it becomes ster ile when stored at 4°C. This observation was confirmed and extended by experiments in our laboratory.8 Separate specimens of pooled human vitreous were inoculated with Staphylococcus pyogenes, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, and stored at 4°C. A sample was taken from the infected vitreous every two or three days and cul tured for organisms. The specimens con taining the first four test organisms became sterile in about four weeks but the Bacillus subtilis persisted much longer. We assumed that the persistence of the Bacillus subtilis was due to its ability to form spores under unfavorable conditions. We next investigated the possibility that * From the Departments of Ophthalmology, Uni versity of Manitoba and the Winnipeg Clinic (Dr. Reed) and Winnipeg General Hospital (Miss Shirley Platts). This study was made possible through the generosity of the Canadian National Institute for the Blind.
this self-sterilizing property might be due to a specific antibacterial substance in the vitreous. Bussey, Shafer and Hughes 7 had found that if human vitreous was placed in agar wells, zones of inhibition sometimes ap peared around the wells when the agar was inoculated with bacterial cultures and incu bated at 37°C. This work was confirmed in our laboratory.8 Pooled human vitreous was put in a ditch in a nutrient agar plate. When streak cul tures of Staph. aureus (Wood 46 strain) and M. lysodeikticus were made across the agar plate at right angles to the ditch, a zone of inhibition occurred adjacent to the vitre ous. Cultures of E. coli and A. aerogenes showed confluent growth across the ditch. Control experiments with normal saline, cerebrospinal fluid, dog vitreous, rabbit vit reous and human serum suggested that these substances lacked this antibacterial factor. Since the M. lysodeikticus appeared to be particularly susceptible we suggest that lysozyme might be responsible for this anti bacterial property. Hill 9 considered that the antibacterial property of human vitreous might simply be due to antibiotics which had penetrated the blood-vitreous barrier before death and per sisted in the pooled, stored vitreous. He studied this by putting human vitreous into holes in blood agar plates seeded with Staph. aureus. He found that when the patients had received antibiotics before death a zone of inhibition appeared around the vitreous. No zone of inhibition appeared if the donor had received no antibiotics before death. These experiments appeared to show conclusively that antibiotics administered before death were the sole reason for the disappearance of bacteria infecting stored vitreous. The following experiments were conducted to elucidate this problem further.
502
HOWARD REED AND SHIRLEY PLATTS EXPERIMENT 1
Human vitreous was placed in a depres sion in a blood-agar plate. The plate was then inoculated with a culture of Staph. aureus and incubated at 37°C. This was re peated 25 times using vitreous from 25 pa tients who had received antibiotics before death. As a control the experiment was re peated with 20 specimens of human vitreous obtained from patients who had received no antibiotics.
HUMAN VITREOUS INFECTED WITH ONE IOOPFULOF 100,000,000 STAPHYLOCOCCI /ml STORED AT 4»C
O
12
RESULTS
None of the 20 controls showed a zone of inhibition. A zone of inhibition occurred in only four of the 25 specimens of vitreous from persons who had received antibiotics before death. This is not surprising because antibiotics do not readily penetrate the bloodvitreous barrier. That the zone of inhibition appeared in so few cases seemed to cast doubt upon the assumption that antibiotics given before death are responsible for con taminated vitreous becoming sterile when stored at 4°C. We therefore decided to compare the rates at which bacteria disappeared from the fol lowing media: (1) Vitreous from persons who had received antibiotics before death; (2) human vitreous without antibiotics; (3) other liquid media. The other media selected for comparison were: (a) Normal saline, (b) Hank's physi ologic salt solution, (c) rabbit blood serum, (d) human blood serum, (e) homogenized cow's milk previously boiled for 20 minutes. Human vitreous was assumed to contain antibiotics when the patient had received them before death. When there was no rec ord of the administration of antibiotics it was assumed that the vitreous contained none. Of course the nature and dosage of the antibiotic varied from patient to patient. In some specimens of vitreous the antibiotic was probably of low concentration or even absent. Moreover, the great variety of drugs in use made it impossible to correlate the
i WITH ANTISIOTICS WITHOUT ANTIBIOTICS
0
20
40
40
80
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
Fig. 1A (Reed and Platts). Experiment 2. . CONTROLS INFECTED WITH ONE LOOPFUL OF 100,000,000 STAPHLOCOCCI /ml STORED AT 4*C
Z
14'
O
12
0
20
40 ·
40
BO
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
Fig. IB (Reed and Platts). Experiment 2. antibiotic and the rate of disappearance of bacteria. EXPERIMENT 2 (figs. 1A and
IB)
The amount of 0.5 ml. of the human vitre ous and of the various controls was put into
503
SELF-STERILIZING PROPERTIES OF VITREOUS small screw-capped bottles (Bijou) and each was infected with one loopful of a suspen sion of 100,000,000 Staphylococci per ml. and stored at 4°C. The Staphylococci had previously been tested and found to be sen sitive to all antibiotics in common use. Twice each week the medium was subcultured on nutrient agar plates to determine whether it contained viable Staphylococci. At first we attempted to access the number of colonies as we did in our previous experi ments, but we decided it was more reliable to record the time taken for the cultures to become negative.
HUMAN VITREOUS INFECTED WITH ONE LOOPFUL OF 100,000,000 STAPHYLOCOCCI /ml STORED AT 22'C
WITHOUT ANTIBIOTICS WITH ANTIBIOTICS
RESULTS
On the graphs the numbers of specimens are charted vertically and the days taken for the cultures to become negative are charted horizontally. The Staphylococci became nonviable at varying rates. All specimens of normal saline were sterile in about two days but 11 speci mens of cow's milk still contained viable or ganisms at the end of the test period of 100 days. It will be noticed that 60 to 80 days were required for the last of the specimens of vitreous to become sterile. The presence or absence of antibiotics in the vitreous ap peared to make little difference. Presumably the heavy infecting dose of bacteria swamped the small concentration of antibiotic.
0
20
10
60
80
100
110
TIME OF SURVIVAL OF «ACTERIA (DAYS)
Fig. 2A (Reed and Platts). Experiment 3. HUMAN SERUM
CONTROLS INFEaED WITH ONE LOOPFUL OF 100,000,000 STAPHYLOCOCCI A«l STORED AT 22*C
\
MILK
EXPERIMENT 3 (figs. 2A and 2B)
In this experiment the specimens were infected with one loopful of a suspension of 100,000,000 Staphylococci per ml. and stored at room temperature (22°C.) RESULTS
Again there is a wide variation in rates at which the media became sterile. The presence of antibiotics appeared to hasten the death of bacteria in a few vitreous specimens. EXPERIMENT 4 (figs. 3A and 3B)
Vitreous with and without antibiotics was infected with a dose of one loopful of a
TIME OF SURVIVAL OF lACTERIA (OAYSJ
Fig. 2B (Reed and Platts). Experiment 3. suspension of 1,000,000 Staphylococci per ml. One series was stored at 4°C. and the other at 22°C. RESULTS
In this experiment the presence of anti biotics hastened the death of the Staphylo cocci. The other point of interest is that the last of the specimens of vitreous stored at 4°C. were sterile in 40 to 60 days, consider ably less than the time required when the
HOWARD REED AND SHIRLEY PLATTS
504
H U M A N V I I K O U S INFECTED WITH
H U M A N VITREOUS INFECTED WITH
O N E L O O P F U L O F 1 , 0 0 0 , 0 0 0 STAPHYLOCOCCI / m l
O N E L O O P F U l O F 1 0 0 , 0 0 0 , 0 0 0 STAPHYLOCOCCI
STOKED AT 4 ' C
/ml
STORED AT 3 7 " C
Γ
Z
16 WITH ANTIBIOTICS
S ».
O
I
|2
WITHOUT ANTIBIOTICS
WITHOUT ANTIBIOTICS
WITH ANTIBIOTICS
\
0
20
40
40
80
100 TIME OF SURVIVAL OF BACTERIA (DAYS)
TIME O f SURVIVAL O F «ACTFJIIA (DAYS)
Fig. 4A (Reed and Platts). Experiment 5.
Fig. 3A (Reed and Platts). Experiment 4.
H U M A N SERUM
H U M A N VITREOUS INFECTED WITH O N E LOOPFUL O F 1 , 0 0 0 , 0 0 0 STAPHYLOCOCCI . ,
/ml
STORED AT 2 2 ' C
WITHOUT ANTIBIOTICS
O
*
,2,
II WITH ANTIBIOTICS
0 0
20
40
60
80
100
110
20
40
60
B0
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
TIME OF SURVIVAL O F BACTERIA (DAYS)
Fig. 3B (Reed and Platts). Experiment 4. contaminating dose was 100 times greater. On the other hand when the specimens with the smaller infecting dose of Staphylococci were stored at 22°C, the time required for the Staphylococci to become nonviable was somewhat longer.
Fig. 4B (Reed and Platts). Experiment 5. EXPERIMENT 5 (figs. 4A, 4B and 4C)
Separate specimens of human vitreous and of the controls were infected with one loopful of a suspension of 100,000,000 organ isms per ml. and stored at 37°C. Other speci mens of human vitreous were infected with
SOS
SELF-STERILIZING PROPERTIES OF VITREOUS EFFECT Of INTRAVENOUS CHLOROMYCETIN
HUMAN VITREOUS INFECTES WITH
I LOOPFUL OF 50,030 STAPHYLOCOCCI M.
ONE IOOPFULOF 1,000,000 STAPHYLOCOCCI /ml
STORAGE AT 4»C
STORED AT 37"C
WITHOUT ANTIBIOTICS
2 ,6
WITHOUT ANTIIIOTICS
2 "WITH ANTIBIOTICS
0
S
10
15
30
25
30
35
TIME (DAYS)
Fig. 5 (Reed and Platts). Experiment 6. from each eye and infected with one loopful of 50,000 Staphylococci per ml. and stored at 4°C. 0
20
40
60
80
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
Fig. 4C (Reed and Platts). Experiment 5. one loopful of 1,000,000 Staphylococci per ml. and stored at 37°C. RESULTS
RESULTS
Figure 5 shows the rates of death of the Staphylococci. We cannot explain why the Staphylococci became nonviable jnore rapidly from the vitreous of the rabbits re ceiving the lower dosage of Chloromycetin.
EXPERIMENT 7 (fig. 6) The results are shown in the graphs. It is interesting that in all tests of the controls Experiment 6 was repeated using one the bacteria perished more rapidly in the loopful of 1,000 Staphylococci per ml. to in rabbit serum than in the human serum. At fect the vitreous. 37°C. the presence or absence of the anti biotics in the vitreous appeared to have little RESULTS effect upon the rates at which the Staphylo Again the orginism died more rapidly as a cocci became nonviable on storage. result of the presence of the antibiotic. This time the higher dosage of Chloromycetin was EXPERIMENT 6 (fig. 5) more effective than the lower one. Our next step was to attempt to repro duce the effect of the presence of antibiotics in the vitreous of the experimental animal. We did this as follows : a. Six rabbits were given 0.25 gm. of Chloromycetin intravenously each day for three days. b. Six more were given 0.1 gm. of Chloro mycetin intravenously each day for three days. c. Six control rabbbits received no antibi otics. The rabbits were killed two hours after the last injection, the vitreous was removed Fig. 6 (Reed and Platts). Experiment 7. EFFECT OF INTRAVENOUS CHLOROMYCETIN
^ I LOOPFUL OF 1,000STAPHYLOCOCCI /ml. STORAGE AT 4'C
WITHOUT ANTIBIOTICS
506
HOWARD REED AND SHIRLEY PLATTS CONCLUSIONS
1. If human vitreous which is contami nated with bacteria is stored at 4°C. or at 22°C, it tends to become sterile in a few weeks. 2. The heavier the contaminating dose of bacteria, the longer the time required for the vitreous to become sterile. 3. Contaminated vitreous which contains no antibiotics will become sterile almost as quickly as vitreous from patients who re ceived antibiotics before death. 4. The contaminated specimens of the var ious control media (saline, Hank's salt so lution, rabbit serum, blood serum and milk) also became sterile at varying rates when stored at 4 ° C , some more rapidly than vit
reous and others less rapidly. This suggests that factors other than a specific antibacterial substance are operative. 5. The contaminating doses of bacteria used in our experiments were massive in comparison with the numbers of bacteria likely to be involved when vitreous is used surgically. The danger of infection in vitre ous implantation is therefore small provided that the vitreous is stored at 4°C. for a few weeks and a test culture for sterility is nega tive. Winnipeg Clinic. ACKNOWLEDGMENT
We wish to thank Dr. W. Parker, director of the Virus Laboratory, Winnipeg General Hospital, for much advice, and Mrs. L. Nagel-Leindl for valuable assistance.
REFERENCES
1. Shafer, D. M.: Symposium on retinal detachment. AMA Arch. Ophth., 54:143 (July) 19SS. 2. : Vitreous implant in retinal detachment. New York J. Med., 56:3300 (Nov.) 1956. 3. : The treatment of retinal detachment by vitreous implant. Tr. Am. Acad. Ophth., 61:194 (Mar.-Apr.) 1957. 4. Pischel, D. K.: In discussion of Shafer.3 5. Suie, T., and Sroufe, S.: Letters. Tr. Am. Acad. Ophth., 61:530 (July-Aug.) 1957. 6. Reed, H., Wilt, J. C, and Tushingham, G.: A preliminary report on the self-sterilizing property of vitreous. AMA Arch. Ophth., 60:361 (Sept.) 1958. 7. Bussey, J. L., Shafer, D. M., and Hughes, I. A.: Studies on the antibacterial properties of human vitreous. AMA Arch. Ophth., 61:233 (Feb.) 1959. 8. Reed, H , and Tushingham, G.: The antibacterial property of vitreous. AMA Arch. Ophth., 62:780 (Aug.) 1959. 9. Hill, K.: The nature of the antibacterial effect of human vitreous. Tr. Am. Acad. Ophth., 64:298-307 (May-June) 1960.
A STUDY COMPARING METHODS O F PREVENTING OCULOCARDIAC REFLEX* F R A N K I. M E N D E L B L A T T ^ M.D.,
R A L P H E.
K I R S C H , M.D.,
THE
AND L O U I S LEMBERG,
M.D.
Chicago, Illinois
Cardiac arrest and other arrhythmias have resulted from activation of the oculocardiac reflex during ophthalmic surgery. The re flex was first described in 1908 by Aschner 1 and Dagnini.2 Stimulation of the ophthal mic nerve, the first division of the trigeminal * From the Department of Ophthalmology and the Department of Medicine (Cardiology) of the University of Miami School of Medicine at Jack son Memorial Hospital, Miami, Florida. t Present address: Illinois Eye and Ear Infirmary.
nerve, initiates the reflex arc. 3 The vagus nerve is activated in the medulla oblongata and the resulting cardiac suppression evolves from its parasympathetic effect on the sinoauricular node, the cardiac pacemaker.4 A review of the related literature reveals a disparity of opinion concerning reflex pro phylaxis. Kirsch, et al.,5 from a study of 50 cases, found a retrobulbar injection contain ing lidocaine or procaine adequate to block electrocardiographic changes induced in IS
ANI ) SHIRLEY PLATTS,
B.Sc.
Winnipeg, Manitoba Since Shafer 1-3 introduced the implanta tion of vitreous in the treatment of retinal detachment, its use has steadily increased. It has, however, brought with it a serious risk. Vitreous contains protein and in the living eye it is maintained at body temperature so that it should act as an ideal culture me dium.4"5 It has no blood supply to promote the rapid mobilization of antibacterial forces and has, therefore, always been considered to have a low resistance to infection. In an attempt to assess the danger of in traocular infection from the use of stored vitreous a series of studies on the self-steril izing properties of vitreous has been under taken in the Winnipeg General Hospital. The first study concerned the storage of contaminated vitreous at 4°C. Shafer 3 was the first to point out that if human vitreous is infected with Staphylococci it becomes ster ile when stored at 4°C. This observation was confirmed and extended by experiments in our laboratory.8 Separate specimens of pooled human vitreous were inoculated with Staphylococcus pyogenes, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, and stored at 4°C. A sample was taken from the infected vitreous every two or three days and cul tured for organisms. The specimens con taining the first four test organisms became sterile in about four weeks but the Bacillus subtilis persisted much longer. We assumed that the persistence of the Bacillus subtilis was due to its ability to form spores under unfavorable conditions. We next investigated the possibility that * From the Departments of Ophthalmology, Uni versity of Manitoba and the Winnipeg Clinic (Dr. Reed) and Winnipeg General Hospital (Miss Shirley Platts). This study was made possible through the generosity of the Canadian National Institute for the Blind.
this self-sterilizing property might be due to a specific antibacterial substance in the vitreous. Bussey, Shafer and Hughes 7 had found that if human vitreous was placed in agar wells, zones of inhibition sometimes ap peared around the wells when the agar was inoculated with bacterial cultures and incu bated at 37°C. This work was confirmed in our laboratory.8 Pooled human vitreous was put in a ditch in a nutrient agar plate. When streak cul tures of Staph. aureus (Wood 46 strain) and M. lysodeikticus were made across the agar plate at right angles to the ditch, a zone of inhibition occurred adjacent to the vitre ous. Cultures of E. coli and A. aerogenes showed confluent growth across the ditch. Control experiments with normal saline, cerebrospinal fluid, dog vitreous, rabbit vit reous and human serum suggested that these substances lacked this antibacterial factor. Since the M. lysodeikticus appeared to be particularly susceptible we suggest that lysozyme might be responsible for this anti bacterial property. Hill 9 considered that the antibacterial property of human vitreous might simply be due to antibiotics which had penetrated the blood-vitreous barrier before death and per sisted in the pooled, stored vitreous. He studied this by putting human vitreous into holes in blood agar plates seeded with Staph. aureus. He found that when the patients had received antibiotics before death a zone of inhibition appeared around the vitreous. No zone of inhibition appeared if the donor had received no antibiotics before death. These experiments appeared to show conclusively that antibiotics administered before death were the sole reason for the disappearance of bacteria infecting stored vitreous. The following experiments were conducted to elucidate this problem further.
502
HOWARD REED AND SHIRLEY PLATTS EXPERIMENT 1
Human vitreous was placed in a depres sion in a blood-agar plate. The plate was then inoculated with a culture of Staph. aureus and incubated at 37°C. This was re peated 25 times using vitreous from 25 pa tients who had received antibiotics before death. As a control the experiment was re peated with 20 specimens of human vitreous obtained from patients who had received no antibiotics.
HUMAN VITREOUS INFECTED WITH ONE IOOPFULOF 100,000,000 STAPHYLOCOCCI /ml STORED AT 4»C
O
12
RESULTS
None of the 20 controls showed a zone of inhibition. A zone of inhibition occurred in only four of the 25 specimens of vitreous from persons who had received antibiotics before death. This is not surprising because antibiotics do not readily penetrate the bloodvitreous barrier. That the zone of inhibition appeared in so few cases seemed to cast doubt upon the assumption that antibiotics given before death are responsible for con taminated vitreous becoming sterile when stored at 4°C. We therefore decided to compare the rates at which bacteria disappeared from the fol lowing media: (1) Vitreous from persons who had received antibiotics before death; (2) human vitreous without antibiotics; (3) other liquid media. The other media selected for comparison were: (a) Normal saline, (b) Hank's physi ologic salt solution, (c) rabbit blood serum, (d) human blood serum, (e) homogenized cow's milk previously boiled for 20 minutes. Human vitreous was assumed to contain antibiotics when the patient had received them before death. When there was no rec ord of the administration of antibiotics it was assumed that the vitreous contained none. Of course the nature and dosage of the antibiotic varied from patient to patient. In some specimens of vitreous the antibiotic was probably of low concentration or even absent. Moreover, the great variety of drugs in use made it impossible to correlate the
i WITH ANTISIOTICS WITHOUT ANTIBIOTICS
0
20
40
40
80
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
Fig. 1A (Reed and Platts). Experiment 2. . CONTROLS INFECTED WITH ONE LOOPFUL OF 100,000,000 STAPHLOCOCCI /ml STORED AT 4*C
Z
14'
O
12
0
20
40 ·
40
BO
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
Fig. IB (Reed and Platts). Experiment 2. antibiotic and the rate of disappearance of bacteria. EXPERIMENT 2 (figs. 1A and
IB)
The amount of 0.5 ml. of the human vitre ous and of the various controls was put into
503
SELF-STERILIZING PROPERTIES OF VITREOUS small screw-capped bottles (Bijou) and each was infected with one loopful of a suspen sion of 100,000,000 Staphylococci per ml. and stored at 4°C. The Staphylococci had previously been tested and found to be sen sitive to all antibiotics in common use. Twice each week the medium was subcultured on nutrient agar plates to determine whether it contained viable Staphylococci. At first we attempted to access the number of colonies as we did in our previous experi ments, but we decided it was more reliable to record the time taken for the cultures to become negative.
HUMAN VITREOUS INFECTED WITH ONE LOOPFUL OF 100,000,000 STAPHYLOCOCCI /ml STORED AT 22'C
WITHOUT ANTIBIOTICS WITH ANTIBIOTICS
RESULTS
On the graphs the numbers of specimens are charted vertically and the days taken for the cultures to become negative are charted horizontally. The Staphylococci became nonviable at varying rates. All specimens of normal saline were sterile in about two days but 11 speci mens of cow's milk still contained viable or ganisms at the end of the test period of 100 days. It will be noticed that 60 to 80 days were required for the last of the specimens of vitreous to become sterile. The presence or absence of antibiotics in the vitreous ap peared to make little difference. Presumably the heavy infecting dose of bacteria swamped the small concentration of antibiotic.
0
20
10
60
80
100
110
TIME OF SURVIVAL OF «ACTERIA (DAYS)
Fig. 2A (Reed and Platts). Experiment 3. HUMAN SERUM
CONTROLS INFEaED WITH ONE LOOPFUL OF 100,000,000 STAPHYLOCOCCI A«l STORED AT 22*C
\
MILK
EXPERIMENT 3 (figs. 2A and 2B)
In this experiment the specimens were infected with one loopful of a suspension of 100,000,000 Staphylococci per ml. and stored at room temperature (22°C.) RESULTS
Again there is a wide variation in rates at which the media became sterile. The presence of antibiotics appeared to hasten the death of bacteria in a few vitreous specimens. EXPERIMENT 4 (figs. 3A and 3B)
Vitreous with and without antibiotics was infected with a dose of one loopful of a
TIME OF SURVIVAL OF lACTERIA (OAYSJ
Fig. 2B (Reed and Platts). Experiment 3. suspension of 1,000,000 Staphylococci per ml. One series was stored at 4°C. and the other at 22°C. RESULTS
In this experiment the presence of anti biotics hastened the death of the Staphylo cocci. The other point of interest is that the last of the specimens of vitreous stored at 4°C. were sterile in 40 to 60 days, consider ably less than the time required when the
HOWARD REED AND SHIRLEY PLATTS
504
H U M A N V I I K O U S INFECTED WITH
H U M A N VITREOUS INFECTED WITH
O N E L O O P F U L O F 1 , 0 0 0 , 0 0 0 STAPHYLOCOCCI / m l
O N E L O O P F U l O F 1 0 0 , 0 0 0 , 0 0 0 STAPHYLOCOCCI
STOKED AT 4 ' C
/ml
STORED AT 3 7 " C
Γ
Z
16 WITH ANTIBIOTICS
S ».
O
I
|2
WITHOUT ANTIBIOTICS
WITHOUT ANTIBIOTICS
WITH ANTIBIOTICS
\
0
20
40
40
80
100 TIME OF SURVIVAL OF BACTERIA (DAYS)
TIME O f SURVIVAL O F «ACTFJIIA (DAYS)
Fig. 4A (Reed and Platts). Experiment 5.
Fig. 3A (Reed and Platts). Experiment 4.
H U M A N SERUM
H U M A N VITREOUS INFECTED WITH O N E LOOPFUL O F 1 , 0 0 0 , 0 0 0 STAPHYLOCOCCI . ,
/ml
STORED AT 2 2 ' C
WITHOUT ANTIBIOTICS
O
*
,2,
II WITH ANTIBIOTICS
0 0
20
40
60
80
100
110
20
40
60
B0
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
TIME OF SURVIVAL O F BACTERIA (DAYS)
Fig. 3B (Reed and Platts). Experiment 4. contaminating dose was 100 times greater. On the other hand when the specimens with the smaller infecting dose of Staphylococci were stored at 22°C, the time required for the Staphylococci to become nonviable was somewhat longer.
Fig. 4B (Reed and Platts). Experiment 5. EXPERIMENT 5 (figs. 4A, 4B and 4C)
Separate specimens of human vitreous and of the controls were infected with one loopful of a suspension of 100,000,000 organ isms per ml. and stored at 37°C. Other speci mens of human vitreous were infected with
SOS
SELF-STERILIZING PROPERTIES OF VITREOUS EFFECT Of INTRAVENOUS CHLOROMYCETIN
HUMAN VITREOUS INFECTES WITH
I LOOPFUL OF 50,030 STAPHYLOCOCCI M.
ONE IOOPFULOF 1,000,000 STAPHYLOCOCCI /ml
STORAGE AT 4»C
STORED AT 37"C
WITHOUT ANTIBIOTICS
2 ,6
WITHOUT ANTIIIOTICS
2 "WITH ANTIBIOTICS
0
S
10
15
30
25
30
35
TIME (DAYS)
Fig. 5 (Reed and Platts). Experiment 6. from each eye and infected with one loopful of 50,000 Staphylococci per ml. and stored at 4°C. 0
20
40
60
80
100
110
TIME OF SURVIVAL OF BACTERIA (DAYS)
Fig. 4C (Reed and Platts). Experiment 5. one loopful of 1,000,000 Staphylococci per ml. and stored at 37°C. RESULTS
RESULTS
Figure 5 shows the rates of death of the Staphylococci. We cannot explain why the Staphylococci became nonviable jnore rapidly from the vitreous of the rabbits re ceiving the lower dosage of Chloromycetin.
EXPERIMENT 7 (fig. 6) The results are shown in the graphs. It is interesting that in all tests of the controls Experiment 6 was repeated using one the bacteria perished more rapidly in the loopful of 1,000 Staphylococci per ml. to in rabbit serum than in the human serum. At fect the vitreous. 37°C. the presence or absence of the anti biotics in the vitreous appeared to have little RESULTS effect upon the rates at which the Staphylo Again the orginism died more rapidly as a cocci became nonviable on storage. result of the presence of the antibiotic. This time the higher dosage of Chloromycetin was EXPERIMENT 6 (fig. 5) more effective than the lower one. Our next step was to attempt to repro duce the effect of the presence of antibiotics in the vitreous of the experimental animal. We did this as follows : a. Six rabbits were given 0.25 gm. of Chloromycetin intravenously each day for three days. b. Six more were given 0.1 gm. of Chloro mycetin intravenously each day for three days. c. Six control rabbbits received no antibi otics. The rabbits were killed two hours after the last injection, the vitreous was removed Fig. 6 (Reed and Platts). Experiment 7. EFFECT OF INTRAVENOUS CHLOROMYCETIN
^ I LOOPFUL OF 1,000STAPHYLOCOCCI /ml. STORAGE AT 4'C
WITHOUT ANTIBIOTICS
506
HOWARD REED AND SHIRLEY PLATTS CONCLUSIONS
1. If human vitreous which is contami nated with bacteria is stored at 4°C. or at 22°C, it tends to become sterile in a few weeks. 2. The heavier the contaminating dose of bacteria, the longer the time required for the vitreous to become sterile. 3. Contaminated vitreous which contains no antibiotics will become sterile almost as quickly as vitreous from patients who re ceived antibiotics before death. 4. The contaminated specimens of the var ious control media (saline, Hank's salt so lution, rabbit serum, blood serum and milk) also became sterile at varying rates when stored at 4 ° C , some more rapidly than vit
reous and others less rapidly. This suggests that factors other than a specific antibacterial substance are operative. 5. The contaminating doses of bacteria used in our experiments were massive in comparison with the numbers of bacteria likely to be involved when vitreous is used surgically. The danger of infection in vitre ous implantation is therefore small provided that the vitreous is stored at 4°C. for a few weeks and a test culture for sterility is nega tive. Winnipeg Clinic. ACKNOWLEDGMENT
We wish to thank Dr. W. Parker, director of the Virus Laboratory, Winnipeg General Hospital, for much advice, and Mrs. L. Nagel-Leindl for valuable assistance.
REFERENCES
1. Shafer, D. M.: Symposium on retinal detachment. AMA Arch. Ophth., 54:143 (July) 19SS. 2. : Vitreous implant in retinal detachment. New York J. Med., 56:3300 (Nov.) 1956. 3. : The treatment of retinal detachment by vitreous implant. Tr. Am. Acad. Ophth., 61:194 (Mar.-Apr.) 1957. 4. Pischel, D. K.: In discussion of Shafer.3 5. Suie, T., and Sroufe, S.: Letters. Tr. Am. Acad. Ophth., 61:530 (July-Aug.) 1957. 6. Reed, H., Wilt, J. C, and Tushingham, G.: A preliminary report on the self-sterilizing property of vitreous. AMA Arch. Ophth., 60:361 (Sept.) 1958. 7. Bussey, J. L., Shafer, D. M., and Hughes, I. A.: Studies on the antibacterial properties of human vitreous. AMA Arch. Ophth., 61:233 (Feb.) 1959. 8. Reed, H , and Tushingham, G.: The antibacterial property of vitreous. AMA Arch. Ophth., 62:780 (Aug.) 1959. 9. Hill, K.: The nature of the antibacterial effect of human vitreous. Tr. Am. Acad. Ophth., 64:298-307 (May-June) 1960.
A STUDY COMPARING METHODS O F PREVENTING OCULOCARDIAC REFLEX* F R A N K I. M E N D E L B L A T T ^ M.D.,
R A L P H E.
K I R S C H , M.D.,
THE
AND L O U I S LEMBERG,
M.D.
Chicago, Illinois
Cardiac arrest and other arrhythmias have resulted from activation of the oculocardiac reflex during ophthalmic surgery. The re flex was first described in 1908 by Aschner 1 and Dagnini.2 Stimulation of the ophthal mic nerve, the first division of the trigeminal * From the Department of Ophthalmology and the Department of Medicine (Cardiology) of the University of Miami School of Medicine at Jack son Memorial Hospital, Miami, Florida. t Present address: Illinois Eye and Ear Infirmary.
nerve, initiates the reflex arc. 3 The vagus nerve is activated in the medulla oblongata and the resulting cardiac suppression evolves from its parasympathetic effect on the sinoauricular node, the cardiac pacemaker.4 A review of the related literature reveals a disparity of opinion concerning reflex pro phylaxis. Kirsch, et al.,5 from a study of 50 cases, found a retrobulbar injection contain ing lidocaine or procaine adequate to block electrocardiographic changes induced in IS