PREPARATION, STERILIZATION, AND PRESERVATION
`OF OPHTHALMIC SOLUTIONS
`Experimental Studies and a Practical Method
`
`JOHN T. MURPHY, R.Ph.
`HENRY F. ALLEN, M.D.
`AND
`ANITA B. MANGIARACINE, A.B.
`BOSTON
`
`MEDICATIONS available to the ophthalmologist and to his patients usually
`local pharmacies, pharmaceutical
`are obtained from one of
`three sources :
`companies, and hospital pharmacies. Of these, the local pharmacies dispense by far
`the greatest number of preparations. On the other hand, eye medications comprise
`only a small part of
`the total volume of drugs dispensed by local and hospital
`pharmacists. The standards for preparing collyria in an accurate and sterile manner
`are generally considered to be too exacting for the average pharmacist. Unless the
`impetus for improved practices is provided by ophthalmologists, progress will con¬
`tinue to be slow. We shall demonstrate that simple, relatively inexpensive measures
`to achieve accuracy of formulation and sterility of collyria, without
`are sufficient
`sacrifice of drug stability or power of corneal penetration.
`The irony of transmitting disease by measures intended for its prevention is
`to all physicians. A growing awareness among ophthalmologists of the
`abhorrent
`disease-producing role of contaminated eye medications is reflected in recent pub¬
`lications by Theodore,1 King,2 and Vaughan.3 In view of the catastrophic results of
`drug-borne infections, ophthalmic medications must be prepared with aseptic pre¬
`cautions, sterilized after preparation, provided with an antimicrobial preservative,
`handled in such a way as to minimize the possibility of bacterial or viral contami¬
`nation, and cultured at intervals to determine the effectiveness of these measures.
`
`PREPARATION
`The prescribing ophthalmologist has the right to expect a degree of accuracy
`in keeping with U. S. P. standards, namely, within ±5%.i This demand is reason¬
`able in view of the potency of the drugs involved, whereby systemic absorption can
`have serious consequences. Balances and weights should be accurate within these
`limits to weigh the small quantities involved. Measuring devices should be appro-
`Read before the Section on Ophthalmology, at
`the 103rd Annual Meeting of the American
`Medical Association, San Francisco, June 23, 1954.
`Infirmary and Massachusetts General
`From the Pharmacy, Massachusetts Eye and Ear
`Hospital, and the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and
`Harvard Medical School.
`Infirmary and Massachusetts General
`Pharmacist-in-Chief, Massachusetts Eye and Ear
`Hospital (Mr. Murphy).
`Instructor in Ophthalmology, Harvard Medical School; Assistant
`in
`Infirmary (Dr. Allen). Bacteriologist, Mass¬
`Ophthalmology, Massachusetts Eye and Ear
`achusetts Eye and Ear Infirmary (Miss Mangiaracine).
`
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`
` . . . ARCHIVES OF OPHTHALMOLOGY
`
`priate in size for the volumes to be measured. Disregard of these considerations,
`we think, is part of the answer to the ophthalmologist's query why all
`too often
`simple collyria, such as zinc sulfate with boric acid, are too irritating for the patient
`to use.
`Only those ingredients in a proper state of preservation should be used. Par¬
`ticular attention should be paid to salts which carry several moles of water of
`is almost one-half water by weight. The use of
`hydration. Zinc sulfate U. S. P.
`this salt in the fully effloresced state practically doubles the intended concentration
`of active ingredient. This could mean the difference between an acceptable eye
`solution and a distinctly irritating one. Scopolamine hydrobromide is about one-
`eighth water by weight. Use of an effloresced salt of this powerful alkaloid would
`increase the actual concentration of scopolamine by about 12%. Under certain
`circumstances such an increase might have harmful results.
`The principles governing penetration of drugs through the cornea into the
`aqueous humor have been elucidated by Cogan and his co-workers * and by Swan
`and White.7 Passage of dissolved substances is presumed to be a function of phase
`solubility. The epithelium and endothelium are permeable to lipid-soluble and non-
`polar compounds, but not to electrolyes or substances that are exclusively soluble
`in water. The reverse is true of the corneal stroma. For a substance to pass com¬
`it must possess both water and lipid solubility
`pletely through the intact cornea,
` or exist in phases meeting these requirements.
`including certain
`the more important agents used in ophthalmology,
`Some of
`alkaloids, are weak electrolytes. Salts of these bases, rather than the bases them¬
`selves, are used because of their greater solubility in water, as well as their greater
`stability. Within a range peculiar to each base, these salts progressively dissociate,
`with decrease in hydrogen ion concentration. Eventually a point is reached at which
`the free base is precipitated. In the form of free base most alkaloids are fat-soluble
`these and other organic electro¬
`and variously water-soluble. On the other hand,
`lytes as salts are all water-soluble, and most are lipid-insoluble. Only those sub¬
`stances which possess both water and lipid solubility will penetrate the cornea.
`The interrelationship of hydrogen ion concentration, equilibrium concentration
`of free base, and chemical stability has been well brought out by Hind and Goyan.8
`At the slightly alkaline pH of the tears, increased availability of free base is more
`than offset by lack of chemical stability and by irritating properties of the free base.
`A shift of the reaction toward the acid side, while reducing the concentration of
`increases stability and reduces irritation. These authors use a series of
`free base,
`buffer systems to regulate the pH of various groups of collyria.
`The passage of alkaloids across the corneal barrier was quantitatively studied
`hy Cogan and Hirsch.5 They found increased transfer of atropine, pilocarpine, and
`ephedrine with increase in pH. When a given electrolyte is dissolved in distilled
`water, the pH of the resulting solution is determined by the properties and concen¬
`tration of that electrolyte. The pH of simple solutions of most alkaloidal salts lies
`between 4.0 and 5.0, that is, in a range where the compounds are stable. For reasons
`of increasing stability, therefore, it is seldom necessary to adjust the pH by added
`buffer. When a drop of an unbuffered solution is instilled into the conjunctival sac,
`the buffering action of the tears almost instantly adjusts the pH to neutrality or
`slight alkalinity, thereby making available an increased concentration of free base
`* References 5 and 6.
`
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`
`OPHTHALMIC COLLYRIA—STERILIZATION
`
`for penetration into the corneal epithelium. Any buffer system used to alter the
`inevitably interfere with the buffering action of
`pH of the alkaloidal solution will
`free base available for absorption. For
`the tears, and thus reduce the amount of
`this reason, as well as for reasons of simplicity and economy, we recommend that
`the use of buffers be avoided whenever possible and that only substances of
`low
`buffering capacity be used when it is necessary to use a buffer at all. It is assumed
`that only chemically clean glass containers,
`free of residual detergent substances,
`will be used.
`Although we have not studied experimentally the effects of osmotic properties
`of collyria,
`the empirical use of solutions adjusted approximately to the tonicity
`of 1.3% sodium chloride has proved to be entirely satisfactory in our experience.
`
`STERILIZATION
`Sterilization of collyria has been a problem hitherto unsolved by most
`local
`including auto-
`pharmacies. Morrison and Truhlsen 9 reviewed various methods,
`claving, and found experimentally a variable loss of physiologic activity after one
`or more exposures of collyria for 15 minutes to steam under pressure at 121 C. It
`is to be noted that their experiments were done with solutions buffered on the
`alkaline side of neutrality. On the other hand, Murphy and Stoklosa,10 using
`unbuffered alkaloidal salts at an acid pH, found negligible chemical degradation
`after autoclaving at 121 C. for time intervals appropriate to the size of
`the con¬
`tainers used. Theodore and Feinstein xl advocate the use of filtration under pressure
`for commercially manufactured preparations. This method, although effective,
`is
`not adaptable for use by smaller pharmacies. We shall show that autoclaving under
`the proper conditions of time and pH does not result in significant degradation of
`the principal drugs used in ophthalmology.
`
`PRESERVATION
`Since no known preservative harmless to ocular tissues and compatible with
`the importance
`ophthalmic drugs has virucidal or rapidly bactericidal properties,
`of careful technique in the use of medications cannot be overemphasized. In addi¬
`tion, an antimicrobial substance must be incorporated in ophthalmic formulas. The
`ideal preservative agent for this purpose would be one possessing to the highest
`degree the following properties :
`1. Bacteriostatic and fungistatic power. This must be demonstrated specifically
`against Pseudomonas aeruginosa (Bacillus pyocyaneus), must be maintained in
`the presence of other active ingredients, and must not be inactivated by autoclaving.
`2. Tissue tolerance. The preservative must not be irritating to the cornea or
`conjunctiva on repeated application. No epithelial damage should be demonstrable
`in the concentration used. Moreover, if there is any likelihood that the preparation
`may be used in the operating room, the preservative must be harmless to the intra¬
`ocular tissues as well.
`3. Low sensitizing power. Since patients may use certain medications over a
`the preservative used must have a low incidence of
`period of months or years,
`sensitization. This can only be determined by long clinical experience.
`4. Compatibility with active ingredients.
`5. Chemical stability.
`
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` . . . ARCHIVES OF OPHTHALMOLOGY
`
`All the above properties should be maintained under the conditions of use in actual
`these is hydrogen ion concentration.
`important of
`practice. The most
`Although various compounds have been strongly advocated for use as preserva¬
`tives, we know of no previous comparative study of the chemical and antibacterial
`to numerous species and strains of
`these compounds with respect
`behavior of
`organisms under various physical and chemical conditions. The selection of pre¬
`servatives to be tested was based on a consideration of the properties listed above.
`Chlorobutanol and phenylmercuric nitrate are widely used and fulfill many of the
`requirements for the ideal preservative. Phenylethyl alcohol has recently been sug¬
`gested as a possible preservative by Brewer, Goldstein, and McLaughlin 12 on the
`its activity against Gram-negative bacteria.13 Benzalkonium (Zephiran)
`basis of
`chloride, because it has been widely used and is still recommended by some, was
`included in this study, notwithstanding its tendency to be inactivated by other
`compounds present,14 irritation of ocular tissues in bactericidal concentrations,1 r>
`diminished effectiveness at an acid pH range,16 and incompatibility with certain
`anions, notably nitrate, salicylate, and fluorescein ions. The concentrations tested
`in collyria. Chlorobutanol and
`were those generally used for preservative effect
`phenylethyl alcohol, each in 0.5% concentration, were compared with phenylmer¬
`curic nitrate 1 :25,000.
`
`EXPERIMENTAL STUDY
`the laboratory studies reported here was threefold. First,
`it
`The purpose of
`seemed desirable to determine whether and under what conditions solutions of the
`it was desired
`principal ophthalmic drugs would withstand autoclaving. Second,
`to assess and compare the antibacterial properties of
`the substances commonly
`recommended as preservatives for collyria. Third, a comprehensive survey was
`undertaken to determine the effectiveness of the system in use at our hospital.
`
`Materials
`All drugs used in these experiments conformed to U. S. P. or N. F. standards. The boiling
`point of phenylethyl alcohol was 219 C.
`Soft-glass collyrium bottles,f capacity 7.5 cc, were used as containers for the solutions
`during the tests.
`The bottles were sealed with phenolic resin screw caps containing natural
`Freshly distilled water, pH approximately 6.0, was used for all solutions.
`the various organisms were obtained by isolation in our laboratory from clinic
`Strains of
`and private patients. One of
`the Pseudomonas strains was recovered from a corneal ulcer ;
`the strains, however, were
`several others were isolated from conjunctival exudates. Most of
`obtained from ear, nose, or throat sources. The behavior of the eye strains with respect
`to the
`compounds studied did not differ from that of the others.
`infusion broth,
`laboratory.
`freshly prepared in our
`The stock culture medium was meat
`Plates were poured from this with the addition of 2% agar and 5% horse blood. Sugars were
`added by weight to make the appropriate concentrations. Mannitol was used in the concentra¬
`tion usually added to scopolamine solutions. Other media were Brewer's liquid thioglycollate
`medium, Sabouraud's broth, and Mueller's starch agar.
`
`rubber liners.t
`
`Methods
`All experiments in this study were repeated several
`be reproducible.
`All pH determinations were done with a Beckman Model G pH meter.
`
`times, and the results are considered to
`
`t Pennsylvania Glass Company, Pittsburgh.
`} Armstrong Cork Company, Lancaster, Pa.
`
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`OPHTHALMIC COLLYRIA—STERILIZATION
`
`Spectrophotometric determinations were done in the ultraviolet range with a Beckman D U
`quartz spectrophotometer in 1 cm. cells.
`Proximate assays were performed according to U. S. P. methods, using a Watkins con¬
`tinuous alkaloidal extractor.17 All assays were run in duplicate, so that each Table represents
`the average of two or more determinations for each lot tested. More than one lot of each type
`of collyrium was tested.
`Collyria were sterilized by autoclaving in their own sealed glass bottles at 121 C.
`for five
`minutes. This temperature was recorded by a thermometer placed in the autoclave with the
`bottles of collyria.
`Cultures were incubated aerobically at 37 C. They were read at 48 hours or longer. Delayed
`growth appearing after 48 hours was not observed when cultures were incubated for longer
`periods. The identity of cultures in broth was checked by observation of characteristic growth
`and by periodic plating on blood agar and starch agar.
`In the earlier experiments, surface
`Two methods of contaminating collyria were employed.
`colonies from agar plates were suspended in a small amount of sterile saline. The crude sus¬
`pension was then homogenized by mechanical grinding in a Pyrex tube with a rotating glass
`pestle. One drop of the homogenized suspension was instilled from a Pasteur pipette into each
`bottle of test solution. The second, and simpler, method consisted of using one drop of undiluted
`broth culture to contaminate the solution bottles. No attempt was made to standardize the size
`the drops or the density of the inocula. Neither these factors nor the age of
`of
`the cultures
`appeared to influence the results.
`In testing for residual contamination of collyria, parallel cultures on blood agar and in
`broth were done in the earlier experiments. These revealed that
`inoculation of 0.1 cc. of
`solution into 5 cc. of broth was a more sensitive test for surviving bacteria than inoculation of
`a similar quantity onto the surface of an agar plate. For this reason, only broth tubes were
`tests. Transfers from solutions containing phenylmercuric nitrate were made
`used in subsequent
`into liquid thioglycollate medium in order to neutralize adsorbed traces of
`the mercurial com¬
`pound. All strains grew well on the media used. All strains of Ps. aeruginosa, also, grew well
`in a 10% enzymatic hydrolysate of casein at pH 5.5.
`Tests for bacteriostatic effect were performed by inoculation of one loopful of broth culture
`into tubes of nutrient broth containing the preservative in the concentration to be tested.
`Presence or absence of growth was noted at 48 hours. Subcultures of negative tubes were done
`into 5.0 cc. of liquid medium containing no preservative. However, sub¬
`by transferring 0.5 cc.
`cultures were not done routinely when a bacteriostatic effect was observed in the primary series
`of tubes.
`All comparative experiments were run simultaneously, with parallel controls of each strain
`and each solution. Controls of collyria without preservatives were not
`it had
`included after
`been shown that organisms survived in these indefinitely. However, a saline control of
`the
`inoculum was always included as a check on viability.
`A survey was undertaken of all open bottles of collyria in use wherever they could be found in
`our hospital. A total of 576 individual bottles, representing more than 30 different drugs and
`including 27 bottles of
`fluorescein, were cultured in thioglycollate broth by the method already
`outlined.
`
`Results
`When distilled water with an initial pH of 6.0 was autoclaved at 121 C. for five
`minutes in soft-glass bottles, the pH after autoclaving was found to have risen to
`9.10. Titration of a 50-cc. pooled sample of
`this water with 0.02 N suifuric acid
`in the presence of methyl orange required 0.63 cc. of acid to reach an end-point.
`This amount of acid corresponds to 0.013 mEq. of sodium carbonate in 50 cc, or
`in 7.5 cc. of water.
`0.0019 mEq.
`The same containers were again filled with freshly distilled water at a pH of
`approximately 6.0, sealed as before, and reautoclaved at 121 C. for five minutes.
`The pH of the water after sterilization was found to be 8.4. A pooled sample of
`50 cc. was titrated as before and found to contain 0.003 mEq. of sodium carbonate,
`or 0.00045 mEq. per 7.5 cc.
`
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`
` . . . ARCHIVES OF OPHTHALMOLOGY
`
`To determine whether the relatively small amount of alkali
`transferred from
`the glass to the water would have a significant degrading effect on the alkaloidal
`solutions to be sterilized in these containers, various collyria were prepared accord¬
`ing to the formulae in Table 1. These collyria were checked for pH and assayed
`before autoclaving, immediately after autoclaving, and after 1, 3, 6, and 16 months
`of storage at an average temperature of about 25 C. The results recorded in Tables
`2 and 3 indicate that when solutions of atropine, eucatropine, homatropine, pilo¬
`carpine, and scopolamine were subjected to this treatment, no significant change
`Table 1.—Formulas of Collyria Tested in Experiments
`
`Tetracaine hydrochloride.
`0.5 gm.
`Chlorobutanol. 0.5 gm.
`Sodium chloride. 1.2 gm.
`Dist. water q. s. ad. 100.0 cc.
`
`Atropine sulfate. 1.0 gm.
`Chlorobutanol. 0.5 gm.
`Sodium chloride. 1.1 gm.
`Dist. water q. s. ad. 100.0 cc.
`Homatropine hydrobromide . 2.0 gm.
`Chlorobutanol. 0.5 gm.
`Sodium chloride. 0.9 gm.
`Dist. water q. s. ad. 100.0 cc.
`Eucatropine hydrochloride
`4.0 gm.
`Chlorobutanol. 0.5 gm.
`Sodium chloride. 0.6 gm.
`Dist. water q. s. ad. 100.0 cc.
`
`.
`
`Physostigmine salicylate . 0.5 gm.
`Chlorobutanol. 0.5 gm.
`Sodium bisulfite. 0.1 gm.
`Boric acid. 2.2 gm.
`Dist. water q. s. ad. 100.0 cc.
`Atropine sulfate. 4.0 gm.
`Chlorobutanol. 0.5 gm.
`Sodium chloride. 0.7 gm.
`Dist. water q. s. ad. 100.0 cc.
`Scopolamine hydrobromide . 0.2 gm.
`Chlorobutanol. 0.5 gm.
`Mannitol. 8.0 gm.
`Dist. water q. s. ad. 100.0 cc.
`Phenylephrine hydrochloride . 10.0 gm.
`Chlorobutanol. 0.5 gm.
`Sodium bisulfite. 0.3 gm.
`Dist. water q. s. ad. 100.0 ce.
`
`Table 2.—Determinations by Method of Alkaloidal Extraction*
`
`Atropine
`Sulfate
`
`Atropine
`Sulfate
`
`Assay
`3.969
`
`pH
`4.3
`
`2.9
`3.6
`4.3
`5.Y
`
`Assay
`1.010
`
`0.999
`0.986
`1.010
`0.997
`
`1.020
`
`pH
`4.9
`
`3.7
`3.5
`4.3
`
`5.2
`
`Eucatropine
`Hydrochloride
`pH
`Assay
`4.058
`
`Homatropine
`Hydrobromide
`PH
`Assay
`4.7
`2.008
`
`2.9
`4.3
`4.2
`4.4
`
`4.079
`3.957
`4.088
`
`1.999
`1.948
`2.023
`
`2.6
`3.9
`4.1
`
`4.0
`
`Solution
`
`Before autoclaving
`Months after
`autoclaving
`
`0 1
`
`11
`12
`IS
`
`least
`
`* Assays in grams per 100 cc.
`in potency occurred. Stability was maintained during periods of storage of at
`14 months.
`Table 3 indicates that solutions of phenylephrine were stable for a period of at
`least three months after heat sterilization. The only evidence of degradation at the
`end of this time was a slight change to a pink color. Tests for the presence of sulfite
`ion in the formula after three months were negative, showing complete oxidation
`of sulfite to sulfate. The possibility of prolonging the stability of these solutions by
`increasing the sulfite content of the formula is being explored.
`Table 4 indicates that 0.5% solutions of physostigmine salicylate are stable for
`the only change at the end of seven months was a very
`at least several months ;
`slight discoloration. Analysis of this solution also revealed absence of sulfite ion.
`Here, again, studies are under way to determine whether increasing the sulfite
`content will prolong stability.
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`OPHTHALMIC COLLYRIA—STERILIZATION
`
`tetracaine showed no significant degradation at
`Solutions of
`the end of six
`the end of 14 months, at which time
`months. No further assay was done until
`significant degradation was found. Studies are in progress to ascertain at about
`time interval degradation becomes significant. The results are recorded in
`what
`Table 4.
`Since the pH of chlorobutanol solution falls after autoclaving, and because an
`autoclaved solution gives a positive test for chloride ion, it is concluded that hydro¬
`lysis of chlorobutanol occurs during sterilization. To determine the degree of
`hydrolysis occurring at various hydrogen ion concentrations, 0.5% solutions of
`chlorobutanol were prepared in citric acid-sodium phosphate buffers in 100 cc.
`
`Table 3.—Determinations by Method of Alkaloidal Extraction*
`
`Solution
`
`Before autoclaving
`Months after
`autoclaving
`
`Pilocarpine
`Hydrochloride
`PH
`Assay
`4.0
`
`Scopolamine
`Hydrobromide
`PH
`Assay
`4.5
`0.301
`
`Phenylephrine
`Hydrochloride
`PH
`Assay
`4.7
`9.717
`
`3.0
`2.9
`3.9
`
`3.795
`3.790
`3.767
`
`3.679
`
`2.8
`4.6
`5.0
`
`5.2
`
`0.301
`0.310
`0.304
`
`0.296
`
`11
`14
`
`2.7
`3.6
`4.4
`
`9.717
`9.717
`9.757 (slight change
`in color)
`
`1 Assays in grams per 100 cc.
`
`Table 4.—Determinations by Spectrophotometric Method*
`
`Homatropine
`Hydrobromide
`pH
`Assay
`4.7
`2.003
`
`3.8
`3.8
`
`2.003
`2.000
`
`Solution
`
`Before autoclaving
`Months after
`autoclaving
`
`0 1 2 3 6 7 9
`
`Physostigmine
`Salicylate
`pH
`Assay
`0.497
`4.0
`
`Tetracaine
`Scopolamine
`Hydrochloride
`Hydrobromide
`-"-s ,-^—
`pH
`pH
`Assay
`0.479
`5.3
`5.0
`
`0.294
`
`3.2
`
`3.0
`
`3.4
`5.7
`
`0.497
`
`0.497
`
`0.480t
`
`0.477
`0.481
`
`0.484
`0.474
`
`0.406Î
`
`14
`
`* Assays in grams per 100 cc.
`t Solution slightly pink.
`I Significant degradation.
`
`tightly stoppered glass bottles. The chlorobutanol was dissolved with the aid of
`magnetic stirrers, using Teflon-coated magnets. The solutions were autoclaved in
`the same bottles at 121 C. for different periods of time. Titration for chloride ion
`with standard silver nitrate solution was carried out according to the U. S. P.
`modification of Volhard's method. Table 5 shows that hydrolysis of chlorobutanol
`is slight at pH values below 5.0 but increases sharply in less acid solutions. Since
`liberation of chloride ion reduces the pH of unbuffered solutions, hydrolysis of
`chlorobutanol tends to be self-limited. The autoclaved solutions were not irritating
`to the eyes of human volunteers. The results are recorded in Table 5.
`It is apparent from the Tables accompanying the text that all compounds studied
`exerted an antibacterial effect against the Pseudomonas strains used in these experi¬
`ments. Table 6 represents the protocol of a single experiment. It suggests that the
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`
` . . . ARCHIVES OF OPHTHALMOLOGY
`
`effect of these compounds is not only bacteriostatic, but even bactericidal over a
`24-hour period of exposure. Actually, out of a large number of trials, rare instances
`occurred of growth in tubes containing chlorobutanol, phenylmercuric nitrate, and
`phenylethyl alcohol
`in the concentrations listed.
`In the case of chlorobutanol, these
`failures were rare and sporadic. No instance was observed in which a single strain
`was able to grow consistently on serial
`transfer in chlorobutanol broth,
`the first
`subculture being invariably negative. The incidence of growth in the presence of
`phenylethyl alcohol 0.5% was less fortuitous, in that strains 4K and 32S were able
`transfer in phenylethyl alcohol broth. Successful
`to grow consistently on serial
`
`Table 5.—Percentage Hydrolysis of Buffered 0.5% Chlorobutanol Solutions
`Before and After Sterilization at 121 C.
`
`Before
`Sterilization
`
`0 Min.
`pH
`Time at 121 C.
`
`%
`
`3.0
`4.0
`5.0
`
`'6.0
`7.0
`
`8.0
`
`8.0
`
`0
`0
`0
`
`0
`
`0.6
`
`1.8
`
`2.4
`
`5 Min.
`
`After Sterilization
`10 Min.
`15 Min.
`
`pH
`
`2.5
`
`3.0
`3.9
`4.8
`5.7
`6.4
`
`6.3
`
`6.8
`
`%
`
`0.6
`
`1.8
`1.8
`2.4
`
`13.0
`
`52.2
`61.7
`
`62.7
`
`pH
`
`2.5
`
`3.0
`3.9
`4.7
`5.7
`
`6.4
`
`6.3
`
`6.6
`
`%
`
`2.4
`2.4
`2.4
`4.7
`
`16.3
`
`61.7
`69.0
`
`88.9
`
`pH
`
`%
`
`2.4
`
`2.9
`3.7
`4.4
`
`5.4
`
`6.1
`
`6.1
`
`6.6
`
`3.3
`
`2.4
`2.4
`5.3
`26.7
`74.0
`76.0
`
`92.4
`
`20 Min.
`%
`pH
`
`Buffer
`
`2.3
`
`2.8
`3.6
`4.3
`
`5.4
`
`6.1
`
`6.0
`
`6.6
`
`4.1
`4.1
`7.7
`30.2
`78.8
`79.4
`
`94.4
`
`f 0.02 M citric acid
`l O.C).04 M sodium phosphate
`Same as above
`Same as above
`Same as above
`0.1 M citric acid
`0.2 M sodium phosphate
`Same as above
`0.05 M citric acid
`0.1 M sodium phosphate
`/ 0.1 M citric acid
`1 0.2 M sodium phosphate
`
`Table 6.—Growth of Pseudonwnas in Nutrient Broth, ph 7.4 ter 7.6
`
`(35 Strains)
`
`Primary
`Control. 35/35
`Chlorobutanol 0.5%. 0/35
`Phenylmercuric nitrate 1:25,000.
`0/35
`Phenylethyl alcohol 0.5%. 2/35
`
`Subculture
`into
`Thioglycollate
`
`_
`
`0/35
`0/35
`2/35
`
`passage was not obtained in phenylmercuric nitrate broth, nor were static organisms
`recovered from it in thioglycollate broth.
`When broth containing chlorobutanol 0.5% was autoclaved in sealed containers
`the hydrogen ion concentration increased, pre¬
`at 15 lb. pressure for 10 minutes,
`sumably because of hydrolysis of chlorobutanol. Although the broth became slightly
`turbid, no growth was obtained in the autoclaved broth, as shown by negative
`subcultures in plain broth. The results are shown in Table 7. Strain 4K again
`demonstrated its ability to grow in phenylethyl alcohol broth.
`As seen in Table 8, addition of fluorescein sodium to chlorobutanol broth inter¬
`fered with the bacteriostatic effect of the latter upon Pseudomonas strains. This
`effect is considered to be due to degradation of chlorobutanol at higher pH values.
`That phenylethyl alcohol
`is not an effective bacteriostatic agent against Gram-
`positive species has already been mentioned and is confirmed by the data presented
`in Table 9.
`70
`
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`OPHTHALMIC COLLYRIA—STERILIZATION
`
`Eight strains of Candida were completely inhibited by chlorobutanol 0.5% in
`Sabouraud's broth. Controls of each strain grew readily in plain Sabouraud's broth.
`Two strains of Aspergillus niger were likewise inhibited.
`Four strains of Proteus vulgaris which grew readily in ordinary media were
`inhibited in the presence of chlorobutanol 0.5% and phenylethyl alcohol 0.5%.
`
`Table 7.—Growth of Pseudomonas in Broth
`
`Broth control .
`pH control .
`Chlorobutanol 0.5% .
`Phenylethyl alcohol 0.5%.
`
`(10 Strains)
`Unautoclaved
`Growth/Strains
`10/10
`
`pH
`7.0
`
`6.6
`
`0/10
`1/10
`
`Autoclaved
`Growth/Strains Subculture
`
`pH
`
`10/10
`0/10
`1/10
`
`0/10
`
`Table 8.—Growth of Pseudomonas in Broth
`
`(10 Strains)
`
`Broth control.
`Chlorobutanol 0.5%.
`Chlorobutanol 0.5% and fluorescein 2.2%.
`
`pH
`7.4
`6.9
`7.4
`
`Growth/Strains
`10/10
`0/10
`3/10
`
`Table 9.—Grozuth of Staphylococcus Aureus in Broth
`
`(10 Strains)
`
`Control .
`Cholorobutanol 0.5% .
`Phenylethyl alcohol 0.6%.
`
`Growth/Strains
`1Q/10
`0/10
`4/10
`
`Table 10.—Growth of Pseudomonas in Different Media
`
`Infusion broth
`1% dextrose broth.
`
`_
`
`Thioglycollate broth
`1% mannitol broth...
`8% mannitol broth...
`
`* Not repeatable.
`
`(22 Strains)
`
`Control
`
`With Chlorobutanol O.i
`
`PH
`7.4
`7.0
`7.0
`7.3
`7.7
`6.9
`
`Growth
`22/22
`22/22
`5/5
`22/22
`5/5
`10/10
`
`PH
`7.5
`7.1
`7.1
`7.2
`7.6
`6.9
`
`Growth
`0/22
`0/22
`1/5*
`1/22*
`0/5
`0/10
`
`As shown in Table 10,
`the bacteriostatic effect of chlorobutanol was demon¬
`strated in the presence of various sugars and reducing substances. Two isolated
`instances of growth could not be repeated in the same media.
`Phosphate-citrate and phosphate buffer systems containing chlorobutanol 0.5%,
`when heavily contaminated with Pseudomonas organisms, tended to become sterile
`within a 24-hour period. This tendency was apparent over a pH range from 3.0 to
`8.0. At the lowest pH values, death of some controls was presumed to be due to
`acidity alone. When neutral or alkaline solutions of sufficiently high buffering
`capacity were autoclaved in the presence of chlorobutanol,
`the antibacterial effect
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`

`
` . . . ARCHIVES OF OPHTHALMOLOGY
`
`•of the latter was definitely impaired. It was presumed that chlorobutanol had been
`hydrolyzed by heat at these higher pH values. In the more acid pH range, up to
`pH 6.0, autoclaving did not
`inactivate the antibacterial effect of chlorobutanol.
`The results of these experiments are summarized in Table 11.
`As shown in Table 12, comparative tests of the antibacterial effect against 15
`strains of Pseudomonas of the three test substances after autoclaving in phosphate
`buffer pH 5.0 demonstrated an apparent superiority of chlorobutanol.
`Tetracaine 0.5% proved rapidly toxic to Pseudomonas organisms, as evidenced
`by the appearance of negative cultures in most
`instances within 20 minutes after
`the introduction of a contaminating drop of broth culture or saline suspension of
`
`Table 11.—Survival of Pseudomonas in Buffer Solutions
`
`(9 Strains)
`pH
`3
`Buffer controls. 2/5
`Chlorobutanol 0.5%, autoclaved.
`0/5
`Chlorobutanol 0.5%, unautoclaved.
`
`...
`
`4
`5/5
`0/5
`
`...
`
`5
`9/9
`0/9
`0/4
`
`6
`9/9
`0/9
`0/4
`
`7
`9/9
`9/9
`0/4
`
`8
`8/9
`9/9
`0/4
`
`Table 12.—Twenty-Four Hour Survival of Pseudomonas in Autoclaved Phosphate Buffer ph 5.0
`
`(15 Strains)
`
`Survival/Strains
`(no preservative).
`15/15
`Control
`Chlorobutanol 0.6%. 0/15
`Phenylmercuric nitrate 1:25,000.
`2/15
`Phenylethyl alcohol 0.5%. 3/15
`
`Table 13.—Twenty-Four Hour Survival of Pseudomonas in Sodium Fluorescein,
`2.2%, Unautoclaved
`
`pH
`Control. 7.2
`Chlorobutanol 0.5%. 7.3
`Phenylmercuric nitrate 1:26,000.
`7.4
`Phenylethyl alcohol 0.5%. 7.3
`
`Survival/Strains
`6/6
`1/6
`2/6
`1/6
`
`bacteria. Addition of benzalkonium chloride did not enhance the bactericidal effect
`of tetracaine.
`is destroyed
`As has been mentioned,
`the antibacterial effect of chlorobutanol
`by autoclaving in the presence of sodium fluorescein. Table 13 shows that when
`unautoclaved aqueous solutions of fluorescein were contaminated with Pseudomonas,
`the test preservatives was able consistently to sterilize the drug solution
`none of
`within 24 hours.
`in 2.2% sodium fluorescein were
`Instances of 72-hour survival
`the preservatives studied. As has been mentioned, benzal¬
`recorded for each of
`konium chloride is incompatible with fluorescein.
`When collyria other than fluorescein, unbuffered and containing chlorobutanol
`0.5%, were autoclaved in sealed soft-glass bottles, a fall
`in pH was noted, but the
`antibacterial effect of chlorobutanol was not inactivated. The theoretical basis for
`this observation has been presented. The results are recorded in Table 14.
`The bactericidal effect of benzalkonium chloride was decidedly variable, in that
`survival often occurred in lower dilutions when higher dilutions were sterile. Even
`72
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`

`
`OPHTHALMIC COLLYRIA—STERILIZATION
`
`in the region of neutrality or slight alkalinity, benzalkonium did not appear to be
`a uniformly effective bactéricide, as seen from Table 15. We are currently explor¬
`ing its anti-Pseudomonas effect at lower pH values.
`
`Comment
`The technique employed in the experimental studies constituted a severe chal¬
`lenge to the antibacterial properties of
`the test substances. Plate counts showed
`the inocula contained several million organisms.
`It is inconceivable that
`in
`that
`actual practice any such degree of contamination would occur. Obviously, no
`pharmaceutical preparation offers any more favorable opportunity for bacterial
`growth than the culture media used in