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Methods of Sterilizing Air*
OPHTHALMOLOGY IN THE SOVIET UNION
thalmia by connecting one of the salivary ducts with the conjunctival sac. I have seen one of these cases. Its disadvantage is that the eye tears during mastication. X-ray treat ment to the salivary gland ameliorates the condition. Dr. Shevaloff treats retinal detachment by the ordinary methods. For scierai resection, he uses (1) ordinary resection with under mining of the scierai edges. This allows the
959
stitches to be applied a bit away from the cut scierai border and, when they are tightened, a certain amount of buckling will occur. (2) Scierai resection with insertion of thick cat gut instead of polyethylene tubing. Catgut absorbs fluid and swells, so the stitches should not be much tightened. (3) Scierai buckling using a strip of sclera to indent the choroid. 44 Opera Square.
METHODS O F STERILIZING AIR* F O R U S E I N O P H T H A L M I C SURGERY P H I L I P A.
SHELTON,
M.D.
San Francisco, California AND FREDERICK H .
THEODORE,
M.D.
New York
Recent investigations by one of us1"3 have indicated that so-called saprophytic, con taminant fungi, commonly found in soil and air, are responsible for serious intraocular infections following cataract extractions. The importance of mycotic endophthalmitis as a complication of cataract extraction has also been shown by histopathologic studies,4 as well as in experimental studies.5'6 Since, at best, treatment of mycotic endophthal mitis is unsatisfactory as regards preserving vision, even if the eye is saved, prevention should be stressed. In our original communications, the ob viously many sources from which fungi might enter the eye during ocular surgery were evaluated in some detail. These in cluded: (1) the patient himself; (2) the operating room environment; and (3) his postoperative care. It was indicated in these papers that an often overlooked source of such infection by contaminant fungi is air. * From the Department of Ophthalmology, The Mount Sinai Hospital, New York. This study was aided by a grant from the Kaufman Schlivek Fund.
In one instance, the specific fungus causing the infection was grown from the operating room air, as well as from the eye. Our stressing of this particular factor was not intended in any way to downgrade other possibilities, such as drug contamination, in the prevention of which we were active over a decade ago.7 As a matter of fact, one case that came to our attention showed a strong likelihood of being the result of an infection due to unsterilized cocaine. How ever, the over-all conclusion was that, at the present time, air contamination presents a problem of major importance. It is not generally realized to what extent air contamination by fungi complicates the manufacture of bacterial media and biologic products, and how much effort must be ex pended to insure sterility in this regard. The extremely high fungus content of our outof-door atmosphere is illustrated in Figure 1. While operating room air, fortunately, is far cleaner, routine cultures have shown that, at times, significant numbers of fungi, as well as bacteria, are often present.8 An integral part of modern operating room
960
PHILIP A. SHELTON AND FREDERICK H. THEODORE
methods of obtaining sterile air for this pur pose, a question asked of us by many con-dentious surgeons, once the problem be came apparent to them. For this study, we used air contaminated with a known quantity of Neurospora sitophila, the common red bread mold, obtained from a clinically proved case of postopera tive mycotic endophthalmitis,2 the same strain used in other experiments done by us in this field. EXPERIMENTAL METHOD
Our experiment followed this outline: Fig. 1 (Shelton and Theodore). Mycotic content of outside air. A sterile Sabouraud culture was placed outside a window (sixth floor) for 30 minutes. Hundreds of fungus colonies were grown.
asepsis should be the elimination of such contamination by proper air filtration, proper air conditioning and the maintenance of posi tive pressure in the operating room suite. However, even under optimal conditions it is probably impossible to achieve complete ste rility of the air in the operating room. As a by-product of our "air-mindedness," an often overlooked defect in sterility tech nique now became obvious: the common practice of most ophthalmic surgeons of in jecting unsterilized operating room air, sim ply obtained by syringe aspiration, during ocular surgery. While we have always felt, on theoretical grounds, that this was wrong, when we established the fact that airborne "saprophytic" fungi could at times consti tute a surgical hazard, it became clear that injection of room air into the anterior chamber during cataract surgery, anterior chamber restoration, freeing of vitreous-corneal adhesions or keratoplasty, or into the vitreous during retinal detachment surgery, without regard to its sterility, was definitely a break in sterility technique. Thus, the dual objectives of the present paper were (1) to highlight the fact that such air used in ophthalmic surgery should be sterile, and (2) to ascertain satisfactory
A. The preparation of Neurospora sitophilacontaminated air in a closed system. B. Quantitative assay of the degree of con tamination of each sample. C. Efficacy of various methods of sterilization of the contaminated air sample. 1. Aspiration through a flamed needle during withdrawal from its container. 2. Aspiration through a coarse filter during withdrawal from its container. 3. Aspiration through a fine filter during with drawal from its container. 4. Autoclaving the air in a syringe after with drawal from its container. 5. Autoclaving the air in its original container. A. P R E P A R A T I O N OF CONTAMINATED AIR
Preparation of the contaminated sample was accomplished by aspiration of two cc. of air overlying a sporulating colony of Neurospora sitophila on an agar slant. This air was then injected into a sterile 25-cc. culture bottle with a rubber diaphragm. Fif teen such samples were prepared and di vided into five groups of three each, to test the -five proposed methods of sterilization already enumerated. B. ASSAY OF THE DEGREE OF CONTAMINA TION
Quantitative assay of the air in the bottle was done by the standard tube dilution tech nique utilizing four bottles. One cc. of con taminated air was injected into the first sterile bottle containing 10 cc. of Sabouraud's liquid medium. The remaining three bottles, each of which contained nine cc. of media,
METHODS OF STERILIZING AIR
received serial infusions of one cc. from the preceding bottle. Thus, a positive culture in bottle No. 1 signified minimum contamina tion of one spore per cc. of the air sample; growth in bottles No. 1 and No. 2 meant minimum concentration of 10 spores per cc. ; growth in bottles No. 1, 2 and 3 indi cated at least 100 spores per cc. ; and growth in all four bottles meant that at least 1,000 spores had originally been present in one cc. of contaminated air. These control assays were done in dupli cate on each air sample. C. METHODS OF STERILIZATION
Experimental sterilizations by the follow ing methods were then performed and cul tures taken in triplicate on each of the three samples allocated to the respective method. Thus for each method nine tests were made. Sterile 25 cc. culture bottles, with rubber diaphragms, containing 10 cc. of Sabouraud's liquid medium, were used throughout. 1. Heating of needle. Aspiration of the contaminated sample was performed through a No. 20 gauge needle heated redhot by a Bunsen burner for 20 seconds. Two cc. of air were aspirated. Then one cc. was expressed from the syringe to rule out possible mixing with room air, and the last one cc. was injected into the culture bottle. 2. Filtration through a coarse filter. A relatively coarse grade filter, (pore diameter 45 microns), was placed in a Swinney hy podermic adapter and mounted between the needle and barrel of a two cc. glass syringe. The entire unit was then autoclaved. Two cc. of contaminated air were withdrawn from its container through the filter. The filter and needle were removed and a fresh needle attached. One cc. of air was expelled through the new needle and the last one cc. was used for the experimental inoculation. 3. Filtration through a fine millipore fil ter. A similar procedure was followed uti lizing a fine grade of Millipore filter (grade HA, having a pore diameter of 0.45 ± 0.02 microns).
961
4. Autoclaving in a syringe. Two cc. of contaminated air were aspirated into a syringe and autoclaved at 15 lb. pressure for six minutes. One cc. was expelled through the needle and the last one cc. used for the cultures. 5. Autoclaving in a vial. The original bot tle with its contaminated air was autoclaved after placing a No. 18 gauge needle through the diaphragm. The autoclave constants were the same as above. The No. 18 needle was removed, then one cc. of air was aspi rated and cultured immediately, and on two additional occasions in the ensuing week after autoclaving. All cultures were incubated at room tem perature and observed daily for signs of growth. A positive culture was indicated by clouding of the media when compared with a sterile control sample. All cultures were observed for three weeks before being clas sified as negative, however, it should be noted that all positive cultures became evi dent within 96 hours. RESULTS
Table 1 indicates the results of the ex perimental cultures for each experimental method. Method 1, flaming a needle, was partially unsuccessful in each series. The greater the degree of initial contamination, the less ef fective was the sterilization. Method 2, aspiration through a coarse fil ter, failed to render the air completely sterile in each of the three series. Methods No. 3, 4, and 5 were uniformly and completely successful in sterilizing the contaminated air. This was true regardless of the initial degree of contamination and in Method No. 5 regardless of the time of inoculation. CONCLUSIONS
It is our feeling that since common air borne contaminant fungi have been shown to cause serious intraocular infections, pre cautions should be taken wherever possible
962
P H I L I P A. S H E L T O N A N D F R E D E R I C K H. T H E O D O R E TABLE 1 SUMMARY OF STUDY
M E T H O D OF STERILIZATION 1
Aspiration through a heated needle Series
Degree of Contamination (spores per cc.)
Culture Results
1st
Over 1,000
Pos. (3)
2nd
10-100
Pos. (1) Neg. (2)
3rd
10-100
Pos. (1) Neg. (2)
Total rendered sterile: 4 of 9. M E T H O D OF STERILIZATION 2
Aspiration through a filter (coarse) c
■
ae
es
Degree of Contamination (spores per cc.)
Culture Results
1st
100-1,000
Pos. (3)
2nd
100
Pos. (3)
3rd
100
Pos. (2) Neg. (1)
Total rendered sterile: 1 of 9. M E T H O D OF STERILIZATION 3
Aspiration through a fine (Millipore) filter Series
Degree of Contamination (spores per cc.)
Culture Results
1st
100-1,000
Neg. (3)
2nd
100
Neg. (3)
3rd 10-100 Total rendered sterile: All 9.
Neg. (3)
M E T H O D OF STERILIZATION 4
Autoclaving in a syringe o~.j-
Degree of Contamination (spores per cc.)
Culture Results
1st
100-1,000
Neg. (3)
2nd
100-1,000
Neg. (3)
3rd 100-1,000 Total rendered sterile: All 9.
Fig. 2 (Shelton and Theodore). Results of Method 2 (coarse filtration) third series. The growth in bottles 1-1, 1-2, and 1-3 indicate a spore concentration of between 100 and 1,000 spores/cc. of contaminated air. The experimental series at the right revealed two positive (1 and 2) cultures and one negative (3) culture.
to eliminate these agents and certainly not introduce them into the eye. One possible source of such infection is the utilization of unsterilized operating room air, purpose fully injected into the anterior chamber or into the vitreous, where it remains up to one week or longer. Our studies indicate that autoclaving, either in a bottle or in a syringe, or microfiltration by means of a fine Millipore filter constitute effective methods of sterilizing air as regards fungi. Although no specific studies were done with bacterial spores, since autoclaving is a long-accepted method for sterilization in general, it is felt that air prepared by autoclaving may also be consid ered bacteria-free as well as virus-free. Mi crofiltration by means of the Millipore filter is another accepted method of sterilization but in actual use appears to be somewhat more complicated for clinical ophthalmic surgery, even if a completely disposable unit is used, and is also more expensive.
Neg. (3)
M E T H O D OF STERILIZATION S
Autoclaved bottle containing contaminated air Series
Sterile Spores/cc. Immedi ate
Sterile Two Days
Sterile One Week
1st
100-1,000
All
All
All
2nd
100
All
All
All
All
All
3rd 10-100 All Total rendered sterile: All 9.
Fig. 3 (Shelton and Theodore). Results of Method 4 (autoclaving in a syringe) first series. The four bottles at the left are the quantitative assay series. The growth in bottles 1-1, 1-2, and 1-3 indicate a spore concentration of between 100 and 1,000 spores/cc. contaminated air. T h e experi mental series at the right revealed that all three cultures were negative.
METHODS OF STERILIZING AIR It is our suggestion that room air be autoclaved in a syringe with an anterior cham ber cannula already attached. The plunger of the syringe should be withdrawn to the two cc. mark prior to autoclaving. This pro vides sufficient sterilized air so that prior to injection into the anterior chamber one cc. may be gradually expelled preventing ad mixture of the remainder with unsterile room air. The syringe is autoclaved in a sealed paper bag of the type used for wrap ping sterile supplies. This setup is left un disturbed in its original wrapping to be opened only immediately before use. If not used, it is reautoclaved for the next case. In practice, this presents no technical diffi culties.
963
barrel of the syringe be required at times to obtain a proper flow of air from the needle, it might prove difficult to inject air safely into the anterior chamber by the approved method of gently rotating the plunger rather than pushing it down. Regardless of what method is chosen by the surgeon, when he requires air in oph thalmic surgery, the purposes of this paper will be well achieved if he becomes more air conscious in general and begins to use ster ile air routinely as a step in this direction. Already, in our areas autoclaved air is be ing widely used. SUMMARY
The experiments just detailed confirm the clinical value of autoclaved air in eye surgery as used by us, as well as others, for years. In this connection, we would like to point out the observations of Stallard9 of which we were not aware until the studies were completed. This author, for many years has stressed the importance of using only sterile air in eye surgery. According to him, while bactériologie tests had shown that three methods : ( 1 ) filtration through cotton, (2) flaming of a needle and (3) autoclaving, were all effective ; he preferred the latter. Moreover, he had used autoclaved air for over 10 years in his eye surgery without any untoward sequelae.
Ordinary operating room air cannot be considered sterile and should not be used for intraocular injections. Fungus-free, sterile air for such a purpose may be ob tained by means of autoclaving a partially opened syringe enclosed in a paper wrap ping or by means of microfiltration using a fine Millipore filter. Experiments with air heavily contami nated with Neurospora sitophila, a common airborne fungus, indicate that such auto claving is far superior to coarse filtration or needle flaming as methods of preparing and maintaining a supply of sterile air for use in intraocular surgery. 1855 California Street (9). 625 Park Avenue (21).
Filtration through cotton was not tested by us. While this method certainly is sim ple enough, should excess pressure on the
We wish to thank Dr. M. L. Littman for his aid and advice in this study.
ACKNOWLEDGMENT
REFERENCES
1. Theodore, F. H., Littman, M. L., and Almeda, E.: The diagnosis and management of fungus endophthalmitis following cataract extraction. AMA Arch. Ophth., 66:163-175 (Aug.) 1961. 2. : Endophthalmitis following cataract extraction due to Neurospora sitophila: A so-called nonpathogenic fungus. Am. J. Ophth., 53:35-39 (Jan.) 1962. 3. Theodore, F. H.: The role of so-called saprophytic fungi in eye infections. In Fungi and Fungous Diseases. (Edited by Gilbert Dalldorf.) Springfield, 111., Thomas, 1962, chap. 3. 4. Fine, B. S., and Zimmerman, L. E.: Exogenous intraocular fungus infections: With particular ref erence to complications of intraocular surgery. Am. J. Ophth., 48:151-165 (Aug.) 1959. 5. Foster, J. B. T., Almeda, E., Littman, M. L., and Wilson, M. E.: Some intraocular and conjunctival effects of amphotericin B. in man and in the rabbit. AMA Arch. Ophth., 60:555-564 (Oct. Pt. 1) 1958. 6. Prenner, E„ Laval, J., and Theodore, F. H.: Mycotic endophthalmitis. Am. J. Ophth., 54:817-821 (Nov.) 1962. 7. Theodore, F. H.: (a) Contamination of eye solutions. Am. J. Ophth., 34:1764 (Dec.) 1951; (b)
964
PHILIP A. SHELTON AND FREDERICK H. THEODORE
Theodore, F. H., and Feinstein, R. R.: Preparation and maintenance of sterile ophthalmic solutions. J.A.M.A., 152:1631-1633 (Aug. 22) 19S3. 8. Theodore, F. H.: Personal observations. 9. Stallard, H. B.: Eye Surgery. Baltimore, Williams & Wilkins, 1958, ed. 3, p. 43.
VARIATION IN T H E D E P T H OF T H E ANTERIOR CHAMBER AND INTRAOCULAR PRESSURE* G. M. BLEEKER,
M.D.
Amsterdam, Holland
In previous articles,1'2 a method was de scribed to measure the depth of the anterior chamber of the eye from photographs made with the Zeiss slitlamp. Continuous record ing at short intervals revealed variations in this depth, ranging from about 0.05 to 0.2 mm. Accommodation and pilocarpine were seen to reduce the distance between the an terior surface of lens and cornea, presum ably by increasing the axial diameter of the lens. When precautions were taken to abol ish accommodation and drugs were avoided, records of normal eyes still demonstrated variability of the depth of the anterior cham ber. Since under these circumstances there was no reason to believe that a change in lens diameter was responsible, these varia tions were assumed to be an indication of slight forward or backward displacement of the ocular diaphragm. This suggested proportional alteration in contents of the an terior or posterior segment of the eye. These preliminary findings were of suffi cient importance to encourage further in vestigations. The present study provides more extensive material as to enable an at tempt to answer the following questions: 1. Are these measurements of sufficient accuracy to substantiate the assumption of variability of the anterior chamber depth? 2. Is there any relation between the depth ♦From the University Eye Hospital. Director: Prof. Dr. A. Hagedoorn. This is a Blaauwfund publication. Financial support was received from the Netherlands Organisation of Pure Scientific Research and from the Amsterdamsche Universiteitsvereniging.
of the anterior chamber and the intraocular pressure? 3. What may be the origin of these fluctu ations in the depth of the anterior chamber? METHODS AND MATERIAL
Serial recording of the anterior chamber depth was performed by taking four pictures at a time, at two-hour intervals, of the an terior chamber by means of a Zeiss slitlamp equipped with a photographic outfit. The depth of the anterior chamber was measured from the enlarged prints (fig. 1). This method proves of value only if every pic ture is taken under absolutely identical cir cumstances. To meet this necessity the pa tient has to focus a fixation mark that has been inserted into the center of the illumi nating unit of the slitlamp in such a way that its virtual image seems to be in the far distance. This insures perfect alignment of the optical axis of the eye with that of the illuminating set. In this way, accommoda tion is excluded in emmetropie and myopic eyes or is leveled to a constant factor in cases of hypermetropia. The observer's binocular together with the camera is invariably put at an angle of 64 degrees with the common optical axis of eye and illuminating set (fig. 2). Correct focus ing of the narrow beam in the center of the lens completes this almost mathematically accurate setup and insures uniform pictures. The life-size negatives are enlarged 10 times in a fixed focus magnifier. From these prints, the distance between the anterior sur-
thalmia by connecting one of the salivary ducts with the conjunctival sac. I have seen one of these cases. Its disadvantage is that the eye tears during mastication. X-ray treat ment to the salivary gland ameliorates the condition. Dr. Shevaloff treats retinal detachment by the ordinary methods. For scierai resection, he uses (1) ordinary resection with under mining of the scierai edges. This allows the
959
stitches to be applied a bit away from the cut scierai border and, when they are tightened, a certain amount of buckling will occur. (2) Scierai resection with insertion of thick cat gut instead of polyethylene tubing. Catgut absorbs fluid and swells, so the stitches should not be much tightened. (3) Scierai buckling using a strip of sclera to indent the choroid. 44 Opera Square.
METHODS O F STERILIZING AIR* F O R U S E I N O P H T H A L M I C SURGERY P H I L I P A.
SHELTON,
M.D.
San Francisco, California AND FREDERICK H .
THEODORE,
M.D.
New York
Recent investigations by one of us1"3 have indicated that so-called saprophytic, con taminant fungi, commonly found in soil and air, are responsible for serious intraocular infections following cataract extractions. The importance of mycotic endophthalmitis as a complication of cataract extraction has also been shown by histopathologic studies,4 as well as in experimental studies.5'6 Since, at best, treatment of mycotic endophthal mitis is unsatisfactory as regards preserving vision, even if the eye is saved, prevention should be stressed. In our original communications, the ob viously many sources from which fungi might enter the eye during ocular surgery were evaluated in some detail. These in cluded: (1) the patient himself; (2) the operating room environment; and (3) his postoperative care. It was indicated in these papers that an often overlooked source of such infection by contaminant fungi is air. * From the Department of Ophthalmology, The Mount Sinai Hospital, New York. This study was aided by a grant from the Kaufman Schlivek Fund.
In one instance, the specific fungus causing the infection was grown from the operating room air, as well as from the eye. Our stressing of this particular factor was not intended in any way to downgrade other possibilities, such as drug contamination, in the prevention of which we were active over a decade ago.7 As a matter of fact, one case that came to our attention showed a strong likelihood of being the result of an infection due to unsterilized cocaine. How ever, the over-all conclusion was that, at the present time, air contamination presents a problem of major importance. It is not generally realized to what extent air contamination by fungi complicates the manufacture of bacterial media and biologic products, and how much effort must be ex pended to insure sterility in this regard. The extremely high fungus content of our outof-door atmosphere is illustrated in Figure 1. While operating room air, fortunately, is far cleaner, routine cultures have shown that, at times, significant numbers of fungi, as well as bacteria, are often present.8 An integral part of modern operating room
960
PHILIP A. SHELTON AND FREDERICK H. THEODORE
methods of obtaining sterile air for this pur pose, a question asked of us by many con-dentious surgeons, once the problem be came apparent to them. For this study, we used air contaminated with a known quantity of Neurospora sitophila, the common red bread mold, obtained from a clinically proved case of postopera tive mycotic endophthalmitis,2 the same strain used in other experiments done by us in this field. EXPERIMENTAL METHOD
Our experiment followed this outline: Fig. 1 (Shelton and Theodore). Mycotic content of outside air. A sterile Sabouraud culture was placed outside a window (sixth floor) for 30 minutes. Hundreds of fungus colonies were grown.
asepsis should be the elimination of such contamination by proper air filtration, proper air conditioning and the maintenance of posi tive pressure in the operating room suite. However, even under optimal conditions it is probably impossible to achieve complete ste rility of the air in the operating room. As a by-product of our "air-mindedness," an often overlooked defect in sterility tech nique now became obvious: the common practice of most ophthalmic surgeons of in jecting unsterilized operating room air, sim ply obtained by syringe aspiration, during ocular surgery. While we have always felt, on theoretical grounds, that this was wrong, when we established the fact that airborne "saprophytic" fungi could at times consti tute a surgical hazard, it became clear that injection of room air into the anterior chamber during cataract surgery, anterior chamber restoration, freeing of vitreous-corneal adhesions or keratoplasty, or into the vitreous during retinal detachment surgery, without regard to its sterility, was definitely a break in sterility technique. Thus, the dual objectives of the present paper were (1) to highlight the fact that such air used in ophthalmic surgery should be sterile, and (2) to ascertain satisfactory
A. The preparation of Neurospora sitophilacontaminated air in a closed system. B. Quantitative assay of the degree of con tamination of each sample. C. Efficacy of various methods of sterilization of the contaminated air sample. 1. Aspiration through a flamed needle during withdrawal from its container. 2. Aspiration through a coarse filter during withdrawal from its container. 3. Aspiration through a fine filter during with drawal from its container. 4. Autoclaving the air in a syringe after with drawal from its container. 5. Autoclaving the air in its original container. A. P R E P A R A T I O N OF CONTAMINATED AIR
Preparation of the contaminated sample was accomplished by aspiration of two cc. of air overlying a sporulating colony of Neurospora sitophila on an agar slant. This air was then injected into a sterile 25-cc. culture bottle with a rubber diaphragm. Fif teen such samples were prepared and di vided into five groups of three each, to test the -five proposed methods of sterilization already enumerated. B. ASSAY OF THE DEGREE OF CONTAMINA TION
Quantitative assay of the air in the bottle was done by the standard tube dilution tech nique utilizing four bottles. One cc. of con taminated air was injected into the first sterile bottle containing 10 cc. of Sabouraud's liquid medium. The remaining three bottles, each of which contained nine cc. of media,
METHODS OF STERILIZING AIR
received serial infusions of one cc. from the preceding bottle. Thus, a positive culture in bottle No. 1 signified minimum contamina tion of one spore per cc. of the air sample; growth in bottles No. 1 and No. 2 meant minimum concentration of 10 spores per cc. ; growth in bottles No. 1, 2 and 3 indi cated at least 100 spores per cc. ; and growth in all four bottles meant that at least 1,000 spores had originally been present in one cc. of contaminated air. These control assays were done in dupli cate on each air sample. C. METHODS OF STERILIZATION
Experimental sterilizations by the follow ing methods were then performed and cul tures taken in triplicate on each of the three samples allocated to the respective method. Thus for each method nine tests were made. Sterile 25 cc. culture bottles, with rubber diaphragms, containing 10 cc. of Sabouraud's liquid medium, were used throughout. 1. Heating of needle. Aspiration of the contaminated sample was performed through a No. 20 gauge needle heated redhot by a Bunsen burner for 20 seconds. Two cc. of air were aspirated. Then one cc. was expressed from the syringe to rule out possible mixing with room air, and the last one cc. was injected into the culture bottle. 2. Filtration through a coarse filter. A relatively coarse grade filter, (pore diameter 45 microns), was placed in a Swinney hy podermic adapter and mounted between the needle and barrel of a two cc. glass syringe. The entire unit was then autoclaved. Two cc. of contaminated air were withdrawn from its container through the filter. The filter and needle were removed and a fresh needle attached. One cc. of air was expelled through the new needle and the last one cc. was used for the experimental inoculation. 3. Filtration through a fine millipore fil ter. A similar procedure was followed uti lizing a fine grade of Millipore filter (grade HA, having a pore diameter of 0.45 ± 0.02 microns).
961
4. Autoclaving in a syringe. Two cc. of contaminated air were aspirated into a syringe and autoclaved at 15 lb. pressure for six minutes. One cc. was expelled through the needle and the last one cc. used for the cultures. 5. Autoclaving in a vial. The original bot tle with its contaminated air was autoclaved after placing a No. 18 gauge needle through the diaphragm. The autoclave constants were the same as above. The No. 18 needle was removed, then one cc. of air was aspi rated and cultured immediately, and on two additional occasions in the ensuing week after autoclaving. All cultures were incubated at room tem perature and observed daily for signs of growth. A positive culture was indicated by clouding of the media when compared with a sterile control sample. All cultures were observed for three weeks before being clas sified as negative, however, it should be noted that all positive cultures became evi dent within 96 hours. RESULTS
Table 1 indicates the results of the ex perimental cultures for each experimental method. Method 1, flaming a needle, was partially unsuccessful in each series. The greater the degree of initial contamination, the less ef fective was the sterilization. Method 2, aspiration through a coarse fil ter, failed to render the air completely sterile in each of the three series. Methods No. 3, 4, and 5 were uniformly and completely successful in sterilizing the contaminated air. This was true regardless of the initial degree of contamination and in Method No. 5 regardless of the time of inoculation. CONCLUSIONS
It is our feeling that since common air borne contaminant fungi have been shown to cause serious intraocular infections, pre cautions should be taken wherever possible
962
P H I L I P A. S H E L T O N A N D F R E D E R I C K H. T H E O D O R E TABLE 1 SUMMARY OF STUDY
M E T H O D OF STERILIZATION 1
Aspiration through a heated needle Series
Degree of Contamination (spores per cc.)
Culture Results
1st
Over 1,000
Pos. (3)
2nd
10-100
Pos. (1) Neg. (2)
3rd
10-100
Pos. (1) Neg. (2)
Total rendered sterile: 4 of 9. M E T H O D OF STERILIZATION 2
Aspiration through a filter (coarse) c
■
ae
es
Degree of Contamination (spores per cc.)
Culture Results
1st
100-1,000
Pos. (3)
2nd
100
Pos. (3)
3rd
100
Pos. (2) Neg. (1)
Total rendered sterile: 1 of 9. M E T H O D OF STERILIZATION 3
Aspiration through a fine (Millipore) filter Series
Degree of Contamination (spores per cc.)
Culture Results
1st
100-1,000
Neg. (3)
2nd
100
Neg. (3)
3rd 10-100 Total rendered sterile: All 9.
Neg. (3)
M E T H O D OF STERILIZATION 4
Autoclaving in a syringe o~.j-
Degree of Contamination (spores per cc.)
Culture Results
1st
100-1,000
Neg. (3)
2nd
100-1,000
Neg. (3)
3rd 100-1,000 Total rendered sterile: All 9.
Fig. 2 (Shelton and Theodore). Results of Method 2 (coarse filtration) third series. The growth in bottles 1-1, 1-2, and 1-3 indicate a spore concentration of between 100 and 1,000 spores/cc. of contaminated air. The experimental series at the right revealed two positive (1 and 2) cultures and one negative (3) culture.
to eliminate these agents and certainly not introduce them into the eye. One possible source of such infection is the utilization of unsterilized operating room air, purpose fully injected into the anterior chamber or into the vitreous, where it remains up to one week or longer. Our studies indicate that autoclaving, either in a bottle or in a syringe, or microfiltration by means of a fine Millipore filter constitute effective methods of sterilizing air as regards fungi. Although no specific studies were done with bacterial spores, since autoclaving is a long-accepted method for sterilization in general, it is felt that air prepared by autoclaving may also be consid ered bacteria-free as well as virus-free. Mi crofiltration by means of the Millipore filter is another accepted method of sterilization but in actual use appears to be somewhat more complicated for clinical ophthalmic surgery, even if a completely disposable unit is used, and is also more expensive.
Neg. (3)
M E T H O D OF STERILIZATION S
Autoclaved bottle containing contaminated air Series
Sterile Spores/cc. Immedi ate
Sterile Two Days
Sterile One Week
1st
100-1,000
All
All
All
2nd
100
All
All
All
All
All
3rd 10-100 All Total rendered sterile: All 9.
Fig. 3 (Shelton and Theodore). Results of Method 4 (autoclaving in a syringe) first series. The four bottles at the left are the quantitative assay series. The growth in bottles 1-1, 1-2, and 1-3 indicate a spore concentration of between 100 and 1,000 spores/cc. contaminated air. T h e experi mental series at the right revealed that all three cultures were negative.
METHODS OF STERILIZING AIR It is our suggestion that room air be autoclaved in a syringe with an anterior cham ber cannula already attached. The plunger of the syringe should be withdrawn to the two cc. mark prior to autoclaving. This pro vides sufficient sterilized air so that prior to injection into the anterior chamber one cc. may be gradually expelled preventing ad mixture of the remainder with unsterile room air. The syringe is autoclaved in a sealed paper bag of the type used for wrap ping sterile supplies. This setup is left un disturbed in its original wrapping to be opened only immediately before use. If not used, it is reautoclaved for the next case. In practice, this presents no technical diffi culties.
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barrel of the syringe be required at times to obtain a proper flow of air from the needle, it might prove difficult to inject air safely into the anterior chamber by the approved method of gently rotating the plunger rather than pushing it down. Regardless of what method is chosen by the surgeon, when he requires air in oph thalmic surgery, the purposes of this paper will be well achieved if he becomes more air conscious in general and begins to use ster ile air routinely as a step in this direction. Already, in our areas autoclaved air is be ing widely used. SUMMARY
The experiments just detailed confirm the clinical value of autoclaved air in eye surgery as used by us, as well as others, for years. In this connection, we would like to point out the observations of Stallard9 of which we were not aware until the studies were completed. This author, for many years has stressed the importance of using only sterile air in eye surgery. According to him, while bactériologie tests had shown that three methods : ( 1 ) filtration through cotton, (2) flaming of a needle and (3) autoclaving, were all effective ; he preferred the latter. Moreover, he had used autoclaved air for over 10 years in his eye surgery without any untoward sequelae.
Ordinary operating room air cannot be considered sterile and should not be used for intraocular injections. Fungus-free, sterile air for such a purpose may be ob tained by means of autoclaving a partially opened syringe enclosed in a paper wrap ping or by means of microfiltration using a fine Millipore filter. Experiments with air heavily contami nated with Neurospora sitophila, a common airborne fungus, indicate that such auto claving is far superior to coarse filtration or needle flaming as methods of preparing and maintaining a supply of sterile air for use in intraocular surgery. 1855 California Street (9). 625 Park Avenue (21).
Filtration through cotton was not tested by us. While this method certainly is sim ple enough, should excess pressure on the
We wish to thank Dr. M. L. Littman for his aid and advice in this study.
ACKNOWLEDGMENT
REFERENCES
1. Theodore, F. H., Littman, M. L., and Almeda, E.: The diagnosis and management of fungus endophthalmitis following cataract extraction. AMA Arch. Ophth., 66:163-175 (Aug.) 1961. 2. : Endophthalmitis following cataract extraction due to Neurospora sitophila: A so-called nonpathogenic fungus. Am. J. Ophth., 53:35-39 (Jan.) 1962. 3. Theodore, F. H.: The role of so-called saprophytic fungi in eye infections. In Fungi and Fungous Diseases. (Edited by Gilbert Dalldorf.) Springfield, 111., Thomas, 1962, chap. 3. 4. Fine, B. S., and Zimmerman, L. E.: Exogenous intraocular fungus infections: With particular ref erence to complications of intraocular surgery. Am. J. Ophth., 48:151-165 (Aug.) 1959. 5. Foster, J. B. T., Almeda, E., Littman, M. L., and Wilson, M. E.: Some intraocular and conjunctival effects of amphotericin B. in man and in the rabbit. AMA Arch. Ophth., 60:555-564 (Oct. Pt. 1) 1958. 6. Prenner, E„ Laval, J., and Theodore, F. H.: Mycotic endophthalmitis. Am. J. Ophth., 54:817-821 (Nov.) 1962. 7. Theodore, F. H.: (a) Contamination of eye solutions. Am. J. Ophth., 34:1764 (Dec.) 1951; (b)
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PHILIP A. SHELTON AND FREDERICK H. THEODORE
Theodore, F. H., and Feinstein, R. R.: Preparation and maintenance of sterile ophthalmic solutions. J.A.M.A., 152:1631-1633 (Aug. 22) 19S3. 8. Theodore, F. H.: Personal observations. 9. Stallard, H. B.: Eye Surgery. Baltimore, Williams & Wilkins, 1958, ed. 3, p. 43.
VARIATION IN T H E D E P T H OF T H E ANTERIOR CHAMBER AND INTRAOCULAR PRESSURE* G. M. BLEEKER,
M.D.
Amsterdam, Holland
In previous articles,1'2 a method was de scribed to measure the depth of the anterior chamber of the eye from photographs made with the Zeiss slitlamp. Continuous record ing at short intervals revealed variations in this depth, ranging from about 0.05 to 0.2 mm. Accommodation and pilocarpine were seen to reduce the distance between the an terior surface of lens and cornea, presum ably by increasing the axial diameter of the lens. When precautions were taken to abol ish accommodation and drugs were avoided, records of normal eyes still demonstrated variability of the depth of the anterior cham ber. Since under these circumstances there was no reason to believe that a change in lens diameter was responsible, these varia tions were assumed to be an indication of slight forward or backward displacement of the ocular diaphragm. This suggested proportional alteration in contents of the an terior or posterior segment of the eye. These preliminary findings were of suffi cient importance to encourage further in vestigations. The present study provides more extensive material as to enable an at tempt to answer the following questions: 1. Are these measurements of sufficient accuracy to substantiate the assumption of variability of the anterior chamber depth? 2. Is there any relation between the depth ♦From the University Eye Hospital. Director: Prof. Dr. A. Hagedoorn. This is a Blaauwfund publication. Financial support was received from the Netherlands Organisation of Pure Scientific Research and from the Amsterdamsche Universiteitsvereniging.
of the anterior chamber and the intraocular pressure? 3. What may be the origin of these fluctu ations in the depth of the anterior chamber? METHODS AND MATERIAL
Serial recording of the anterior chamber depth was performed by taking four pictures at a time, at two-hour intervals, of the an terior chamber by means of a Zeiss slitlamp equipped with a photographic outfit. The depth of the anterior chamber was measured from the enlarged prints (fig. 1). This method proves of value only if every pic ture is taken under absolutely identical cir cumstances. To meet this necessity the pa tient has to focus a fixation mark that has been inserted into the center of the illumi nating unit of the slitlamp in such a way that its virtual image seems to be in the far distance. This insures perfect alignment of the optical axis of the eye with that of the illuminating set. In this way, accommoda tion is excluded in emmetropie and myopic eyes or is leveled to a constant factor in cases of hypermetropia. The observer's binocular together with the camera is invariably put at an angle of 64 degrees with the common optical axis of eye and illuminating set (fig. 2). Correct focus ing of the narrow beam in the center of the lens completes this almost mathematically accurate setup and insures uniform pictures. The life-size negatives are enlarged 10 times in a fixed focus magnifier. From these prints, the distance between the anterior sur-