Vol. 52, No. 10, October 1963
`
`967
`
`of antihistamines is also of interest. Maietta (14,
`15) has exploited such interactions in using the
`combined antigen-antihistaminic technique in short-
`ening the treatment of hay fever.
`These studies demonstrate that a wide variety of
`hydrocolloids and antihistamines form highly in-
`soluble complexes which probably can serve as
`sustained-release forms of
`these drugs. Con-
`ceivably, soluble complexes which are probably
`formed in most of the systems studied also may be
`of pharmacological importance.
`
`REFERENCES
`( 1 ) Kennon, I... and Higuchi, T., THIS JOURNAL, 45,157
`(1956).
`(2) Miller, 0. H., Prcsniplionisl, 1, 52(1954).
`
`(3) Higuchi, T., and Lachman, L.. THIS JOURNAL, 44,521
`(1955).
`(4) Hirscher, D. A , and Miller, 0. H., J. AM. PHARM.
`Assoc., BSZ 105(1962).
`(5) GrahLm, H. D., Baker, Y. M., and Obi, A. N. N.,
`Tars JOURNAL, 52, 192(1963).
`(6) Smith, P., and Montgomery, R.. “Chemistry of Plant
`Gums and Mucilages and some Related Polysaccharides,”
`Reinhold Publishing Corporation, New York, N. Y., 1959, pp
`15-35.
`(7) Deluca, P. P., and Kostenbauder, H. B., THIS JOUR-
`NAL, 49, 430(1960).
`(8) Hughes, T. R., and Klotz, I. M., “Methods of Bio-
`chemical Analysis,” Vol. 3, Interscience Publishers, Inc..
`New York, N. Y., 1956, p. 265.
`(9) Osol, A., and Sideri, C. N., THIS JOURNAL, 44, 761
`ii axm
`10) Kleckner. L. J., and Osol, A,, ibid., 44, 763(1955).
`11) Harwood P. C., J . Chcm. Soc., 1923,2254.
`(12) Judah, J.’D., Biochcm. Biophys. A d o , 53. 375(1961).
`(13) Kobayashi, Y., Arch. Biochcm. Biophyr.. 96, 20
`nrn,
`,“ur,.
`14) Maietta, A. L., Ann. Allergy, 8,645(1950).
`
`,.
`! 15) Maietta, A. L., Conn. Slafc Mcd. J . , 14, 497(1950).
`
`Effectiveness of Antibacterial Agents Presently
`Employed in Ophthalmic Preparations as Preservatives
`Against Pseudomonas aeruginosa
`
`By S. ROBERT KOHNf, LOUIS GERSHENTELD, and MARTIN BARR
`
`Seven chemical substances or combinations of these substances presently em-
`ployed as preservatives in ophthalmic solutions were studied to note their effective-
`ness as antibacterial agents against 13 different strains of Pseudomonas aeraginosa.
`New i n vitro methods were devised. Among the latter were techniques to (a) dif-
`ferentiate between bacteriostatic and bactericidal activities and (6) determine the
`sterilizing time for each antibacterial agent. The methods presented here have
`several advantages over those previously employed and those now in use. An in
`vivo rocedure was also employed in evaluating these chemical agents to note
`whe&
`the findings were in a reement with the final results obtained in the in
`vit*o studies. The following ctemicals were examined chlorobutanol, benzal-
`konium chloride, thimerosol, combinations of methyl and propylparaben, phenyl-
`mercuric nitrate, phenylethyl alcohol, and polymyxin B sulfate.
`
`B Pseuhmonas aeruginosa (Ps. aeruginosa)
`
`ECAUSE OF THE incidence and seriousness of
`
`infections resulting from the use of contaminated
`ophthalmic solutions (1-1 l), various workers
`have critically investigated
`the antibacterial
`agents employed as preservatives in such prep-
`arations. The findings of these workers have
`been contradictory (12-18). The in vitro pro-
`cedures employed in evaluating the effectiveness
`of the antibacterial agents have been challenged.
`The following observations are noted. There
`is need for methods which will determine the
`effectiveness of (a) antibacterial agents used as
`preservatives in ophthalmic solutions against
`Ps. aeruginosa; and (b) substances which are
`
`Received January 22 1963 from the College of Pharmacy.
`Wa e State Univcrsit; D e t h t Mich.. and the Philadelphia
`Cod& of Pharmacy an: Science Philadelphia, Pa.
`Accepted for publication March’29.1963.
`This study was carried out by S. Robert Kohn, Philadel-
`phia College of Pharmacy and Science, Philadelphia, Pa.,
`in partial fulfillment of Doctor of Philosophy degree require-
`ments.
`Presented to the Pharmacy Subsection, American Associ-
`ation for the Advancement of Science, Philadelphia meeting,
`December 1962.
`+Present address: Johnson and Johnson, New Bruns-
`wick, N. J.
`
`capable of inactivating or inhibiting the anti-
`bacterial action of the preservatives used.
`The purpose of this paper is to report on
`studies which were performed in an effort to
`develop in Yitro methods more effective than
`those employed at present and which will estab-
`lish the efficiency of these antibacterial agents as
`preservatives in ophthalmic solutions against
`Ps. aeruginosa.
`
`GENERAL CONSIDERATIONS
`
`In devising methods for the evaluation of the
`effectiveness of antibacterial agents as preservatives
`in ophthalmic solutions, it is important to develop a
`technique which will determine the time required
`for such agents to produce sterility. Most methods
`which have been used to date did not always take
`this
`into consideration. Indeed,
`they usually
`measured only the bacteriostatic activity of pre-
`servatives.
`Until recently, most workers employed a dilution
`technique to differentiate between the bactericidal
`and bacteriostatic activities of antibacterial agents.
`The basis for this procedure is the dilution of the
`
`

`

`968
`
`preservative far below the concentration necessary
`to have a significant antibacterial effect.
`If growth
`of the bacteria occurred, after inoculation of this
`diluted solution into a subculture medium, the
`antibacterial agent was considered
`to possess
`bacteriostatic activity; if growth did not occur, then
`the antibacterial agent was reported as possessing
`bactericidal properties.
`The above concept is not considered entirely valid
`at present. As Klarmann (19) has reported, in some
`of the dilution techniques, a quantity of the anti-
`bacterial agent may be transferred to the sub-
`culture medium in a concentration which will
`produce stasis, and some may become affixed to the
`cell walls or cells, initially producing stasis. There-
`fore, a bacteriostatic effect may be produced and
`misinterpreted for a bactericidal effect. Another
`disadvantage of the dilution technique is that living
`bacteria present may be diluted to an infinite con-
`centration and may not grow in the subculture
`medium.
`It is therefore apparent that an important factor
`in determining the antibacterial activity of preserva-
`tives in ophthalmic solutions is the complete inhibi-
`tion or inactivation of the antibacterial agent in the
`evaluation procedure, a principle employed by
`Lawrence (16) and Riegelman. et al. (17).
`Until recently, investigators had not attempted to
`correlate the results obtained using the in rifro
`procedures with an in vivo test. As pointed out by
`Riegelman, et al. (17). an in vivo test has several
`advantages and becomes an important consideration
`in procedures used for the evaluation of antibacterial
`agents employed as preservatives in ophthalmic
`solutions.
`
`EXPERIMENTAL
`
`The media employed in the experiments are listed
`in Table I.
`General In Vitro Methods
`To determine
`the effectiveness of presently
`employed antibacterial agents as preservatives for
`ophthalmic solutions against Ps. aeruginosa, four
`experimental in uitro procedures, each with a specific
`objective, were developed. These are described
`below.
`Experiment I
`Purpose.-As
`previously noted, it is important
`to distinguish between bacteriostatic and bacteri-
`cidal activity in evaluating the effectiveness of
`chemical agents for use as preservatives in ophthal-
`mic solutions.
`l'arious
`substances have been
`recommended as inactivating or neutralizing agents
`for the antibacterial activity of the chemicals em-
`ployed. However, data concerning the effective-
`ness of inactivating agents are meager. Therefore.
`Experiment I was devised to determine and compare
`the effectiveness of various media for their ability to
`inhibit the activity of antibacterial agents against
`Ps . aeruginosa .
`Preparation of the Inoculum.-Thirteen
`strains
`of Ps. aeruginosa, obtained from various sources,
`were identified by the usual methods including also
`the cytochrome oxidase test (20, 21), then main-
`tained on agar slants. Two strains of Ps. aerugi-
`nosa. which were found more resistant to many of
`
`Journal of Pharmaceutical Sciences
`
`the antibacterial agents in preliminary studies were
`selected for use in this study. Prior to the day of
`the test, these strains were transferred for at least
`3 consecutive days into 10 ml. of nutrient broth and
`incubated a t 3';' for 24 hours. On the day of the
`
`TABLE I. -MEDIA EMPLOYED
`
`Medium I (pH 6.8)
`Nutrient broth, dehydrated"'
`q.s. ad.
`Purified water
`Medium I1 (pH 6 8)
`q.s. ad.
`Medium 111 (pH 6.8)
`
`Glycerinh
`Medium I
`
`Lecithinc
`Glycerin
`Medium I
`
`q.s. ad.
`Medium IV (pH 6.7)
`q.s. ad.
`Medium V (pH 6.7)
`
`q.s. ad.
`Medium VI (pH 6.7)
`q.s. ad.
`Medium VII (pH 6.7)
`
`q.s. ad.
`Medium VIII ( p H 7.1)
`Fluid thioglycollate medium,
`dehydrated0
`4.s. ad.
`Purified water
`Medium I X (pH 7.0)
`q.s. ad.
`Medium X (pH 7.0)
`
`0.8 Gm.
`100 ml.
`
`4 Gm.
`100 ml.
`0.5 Gm.
`4 Gm.
`100 ml.
`
`3 Gm.
`100 ml.
`0.5 Gm.
`3 Gm.
`100 ml.
`
`3 Gm.
`100 ml.
`
`0.5 Gm.
`3 Gm.
`100 ml.
`
`2.93 Gm.
`100 ml.
`
`3 Gm.
`100 ml.
`0.5 Gm.
`3 Gm.
`100 ml.
`3 Gm.
`100 ml.
`0.5 Gm.
`3 Gm.
`100 ml.
`4 Gm.
`100 ml.
`0 . 5 Gm.
`4 Gm.
`100 ml.
`5 Gm.
`100 ml.
`10 Gm
`100 ml.
`
`5 Gm.
`100
`nil.
`
`Tween SOd
`Medium I
`
`Lecithin
`Tween 80
`Medium I
`Tween 20e
`Medium I
`
`Lecithin
`Tween 20
`Medium I
`
`Tween 80
`Medium VIII
`
`Lecithin
`Tween 80
`Medium \'I11
`Tween 20
`Medium VIII
`
`Lecithin
`Tween 20
`Medium VIII
`
`Glycerin
`Medium VIII
`
`Lecithin
`Glycerin
`Medium VIII
`
`Tween 80
`Medium I
`
`Tween 80
`Medium I
`
`q.s. ad.
`Medium XI (pH 7.0)
`q.s. ad.
`hledium XI1 (pH 7.0)
`
`4,s. ad.
`Medium XI11 (pH 7.1)
`q.s. ad.
`Medium XIV (pH 7.1)
`
`q.s. ad.
`Medium XV (PH 6.7)
`q.s. ad.
`Medium XVI (PH 6.6)
`q.s. ad.
`Medium XVII (pH 6.7)
`Tween 20
`q.s. ad.
`Medium I
`Medium XVIII ( p H 6.6)
`Tween 20
`10 Gm.
`q.s. ad.
`100 ml.
`Medium I
`6 Colgate
`Q Difco Laboratories, Inc., Detroit, Mich.
`Palmolive Co.. New York, N. Y. c Lecithin, (Ex Ovo Sol-
`uble) Pfansteihl Laboratories. Inc.. Waukegan, 111. d Poly-
`oxyethylene (20) sorbitan mono-oleate, Atlas Powder Co..
`Wilmington. Del.
`Polyoxyethylene (20) sorbitan mono-
`laurate. Atlas Powder C o . . Wilmington. Del. I All media
`containing nutrient broth, dehydrated, were sterilized by
`autoclaving at 121' for 15 minutes. a All media containing
`fluid thioglycollate medium dehydrated were autoclaved at
`12l0 for 20 minutes.
`
`Page 2
`
`

`

`Vol. 52, No. 10, October 1963
`
`test, the cultures were shaken by hand approxi-
`mately 1 minute to break up clumps and allowed to
`stand for at least 15 minutes. Dilutions (1: 10 and
`1: 1000) of the 24-hour cultures of the two strains
`were made using nutrient broth, and the approxi-
`mate number of viable bacteria were determined by
`the pour-plate technique. The 1 : 10 and 1 :lo00
`dilutions of the 24hour cultures contained ap-
`proximately 108 and los bacteria per ml., respec-
`tively, as noted after incubating the plates at 37"
`for 48 hours.
`Preparation of the Antibacterial Agent-Medium
`Mixtures.-Four milliliters of each of the various
`test media was placed in culture tubes (approxi-
`mately 15 cm. in length and 13 mm. inside diameter)
`and sterilized. Stock solutions of several concen-
`trations of the antibacterial agents were prepared
`as aqueous solutions:. One milliliter of a sterile
`stock solution of the antibacterial agent was added
`to a tube containing 4 ml. of the sterile medium.
`This was done for each concentration of the anti-
`bacterial agent tested and also for each medium
`being used. The tubes were then shaken by hand
`to insure a uniform mixture.
`Preparation of the Controls.-Controls were em-
`ployed to determine bacterial contamination (nega-
`tive controls) and to determine whether the media
`were capable of supporting the growth of Ps. aerugi-
`nosa (positive controls). Media prepared with the
`antibacterial agent but without the presence of
`Ps. aeruginosa were employed as negative controls.
`Media prepared without the antibacterial agents
`but with Ps. aeruginosa were employed as positive
`controls.
`the 1: 10
`Procedure.-One-tenth milliliter of
`dilution of each strain of Ps. aeruginosa was added
`separately to a tube containing a single concentra-
`tion of the sterile antibacterial agent in the individual
`medium being tested. This was repeated for each
`concentration of
`the antibacterial agent in each
`medium being tested. Each tube was shaken by
`hand to insure an even distribution of the bacteria.
`After 1 hour of contact a t 24", 0.5 ml. was trans-
`ferred into tubes containing 4.5 ml. of the subculture
`medium, and the mixture was shaken by hand.
`After 24 hours of contact at 24". another transfer of
`0.5 ml. was made into tubes containing 4.5 ml. of the
`subculture medium. The same procedure was
`repeated with the 1 :lo00 dilution of the strain used
`and with the two dilutions of the other strain. All
`tubes were incubated at 37" for a period of 7 days
`and observed for the presence or absence of growth.
`The procedure was repeated to verify the h d h g s .
`
`Experiment II
`previously indicated in the evalua-
`Purpose.-As
`tion of the antibacterial activity of chemical agents
`intended for use as preservatives in ophthalmic
`solutions, an important consideration is the time
`required for such agents to kill the bacteria after
`contact (sterilizing time). Experiment IZ was there-
`fore devised to determine whether the antibacterial
`agent is fast-acting or slow-acting. This experiment
`was also designed to show the necessity of developing
`subculture media containing suitable inactivating
`agents.
`Preparation of the Inoculum.-A 1 : 10 dilution of
`each of the 24-hour-old cultures of the 13 strains of
`
`969
`Ps. aeruginosa was used in this study. Each was
`prepared as described previously.
`Preparation of
`containing
`the Media.-Tubes
`4.5 ml. of each of the various test media described
`previously were prepared and sterilized.
`Preparation of
`the Antibacterial Solutions.-
`Tubes containing 5 ml. of the sterile aqueous anti-
`bacterial solutions to be tested were prepared.
`Preparation of Controls.-Controls
`were em-
`ployed to determine bacterial contamination (nega-
`tive controls) and to determine whether the media
`were capable of supporting the growth of Ps. aerugz-
`nosa (positive controls). Tubes containing 5 ml.
`of the antibacterial solutions, without the presence
`of Ps. aeruginosa, were employed as negative
`controls. Solutions prepared without the anti-
`bacterial agent but containing Ps. aeruginosa were
`employed as positive controls.
`a 1: 10
`Procedures.-One-tenth
`milliliter of
`dilution of each strain of Ps. aeruginosa was sepa-
`rately added to a tube containing 5 ml. of the sterile
`antibacterial solution. This was repeated for all
`13 strains. Each tube of the antibacterial agent-
`bacteria mixture was then shaken by hand. After
`1-hour of contact at 24', 0.5 ml. was transferred into
`the subculture
`the tube containing 4.5 ml. of
`medium. This was then shaken by hand. After
`24 hours of contact at 24O, another transfer of 0.5
`ml. was made into 4.5 ml. of the subculture medium.
`All subculture tubes were incubated at 37" for a
`period of 7 days and observed for the presence or
`absence of growth. The procedure was repeated to
`verify the hdmgs.
`Experiment III
`purpose of this experiment was
`Purpose.-The
`similar to that of Experiment 11, except that the
`exact sterilizing time (as noted under Procedure)
`was determined.
`Preparation of
`dilutions
`the Inoculum.-Two
`(1 : 10 and 1 : 1000) of the 24-hour-old cultures of 13
`dzerent strains of Ps. aeruginosa were used in this
`experiment. They were prepared as previously
`described.
`Preparation of the Medium-Four
`and one-half
`milliliters of
`the medium, which had shown the
`maximum inactivation of
`the antibacterial agent
`being tested, was employed.
`Preparation of
`the Antibacterial Solutions-
`Tubes containing 5 ml. of the sterile antibacterial
`solution to be tested were prepared.
`Preparation of Controls.-Negative
`controls were prepared.
`procedure was similar to that
`Procedure.-The
`employed in Experiment 11, except for changes in
`If the results of Experiment I1
`the contact times.
`showed the antibacterial agent to be effective
`against Ps. aeruginosa within 1 hour, i.e., no growth
`in the subculture tubes containing the 1-hour trans-
`fer. then the agent was tested at contact times of
`If the antibacterial
`15, 30, 45, and 60 minutes.
`agent was shown to be effective between 1 and 24
`hours, i.e., no growth in the subculture tubes con-
`taining the 24-hour transfers, tests were carried out
`at 3,6, 9, and 12 hours of contact. If necessary 15,
`18, and 21 hours of contact also were employed.
`If the antibacterial agent was shown to be ineffective
`after 24 hours of contact, further studies were not
`considered warranted.
`
`and positive
`
`Page 3
`
`

`

`970
`Experiment IV
`Experiment ZZZ, the sterilizing time
`-se.-In
`of each chemical agent employed as a preservative
`in ophthalmic solutions was determined against Ps.
`aeruginosa. Obviously, the sterilizing time must be
`In
`based on the total destruction of all bacteria.
`Experiment III, only 0.5 ml. of the antibacterial
`agent-bacteria mixtures was transferred
`to the
`subculture medium. The purpose of this experi-
`ment was to determine whether this volume of
`inoculum was as effective as would be transplants of
`larger volumes.
`Preparation of the Inoculum.-The most resistant
`strains of Ps. aeruginosa, as noted in Experiment IIZ,
`were selected and used in this experiment. A 1: 10
`and a 1: lo00 dilution of the 24-hour-old cultures of
`the selected strains were used. They were prepared
`as described previously.
`Preparation of the Medium.-The medium used
`in Experiment IIZ was employed here. Four and
`one-half milliliters, 9 ml., 18 xnl., and 45 ml. of the
`medium, respectively, were placed separately in
`appropriate containers and sterilized.
`Preparation of
`the Antibacterial Solutions.-
`The concentration of the antibacterial solution used
`in this study depended upon the strains of Ps.
`oeiuginosa which were selected. A series of four
`tubes containing 5 ml. of the sterile antibacterial
`solution was prepared for each dilution of each strain
`of Ps. aeruginosa employed in this experiment.
`Preparation of the Controls.-Controls were pre-
`pared as described in Experiment ZI.
`contact time used in this ex-
`Procedure.-The
`periment was the same as that which revealed no
`growth immediately following a contact time period
`which had shown growth in the subculture medium
`for the strains selected. On the day of the experi-
`ment, 0.1 ml. of each of the diluted strains was added
`separately to each tube in the series containing the
`antibacterial solution. This was repeated for each
`strain used in this experiment. Transfers were
`made from the series of tubes at the selected contact
`time, using a 0.5 ml., 1 ml., 2 ml., and 5 ml. inoculum
`into 4.5 ml., 9 ml., 18 ml., and 45 ml. of subculture
`medium, respectively. The containers of subculture
`mediumantibacterial agent-bacteria mixtures were
`shaken by hand. All containers of this subculture
`medium of the antibacterial agent-bacteria mixture
`were incubated a t 37' for a period of 7 days and
`observed for the presence or absence of growth.
`The entire procedure was repeated to verify the
`findings.
`Antibacterial Agents Studied
`The antibacterial agents studied for their effective-
`ness as preservatives for ophthalmic solutions
`against Ps. aeruginosa were as follows: chloro-
`butano1.l benzalkonium chloride,* thimerosol,s com-
`binations of methylparaben and propylparaben,'
`phenylmercuric nitrate,' phenylethyl alcohol! and
`polymyxin B s ~ l f a t e . ~ Aqueous solutions of these
`
`~. 1-Chlorobutanol. Hydrous, Merck and Co.. Inc.. Rahway.
`N . J.
`2 Zephiran Chloride. Winthrop Laboratories. Inc., New
`York. N . Y.
`8 Mann Fine Chemicals, Iac., New York. N . Y.
`4 Heyden Cheuucal Corp., New York, N . Y.
`6 Metalsalts Corp., Hawthorne, N . J.
`6 S. B. Penick and Co., Inc., New York, N . Y.
`7 Chps. Pfizu and Co.. New York, N. Y.
`
`Journal of Pharmaceutual Sciences
`
`Fig. 1.-Appearance of the rabbit eye infected with
`Ps. aeruginosa.
`
`Fig. 2.-Appearance of the normal rabbit eye.
`
`agents were prepared, sterilized by appropriate
`procedures, and assayed for content of active
`ingredient (22).
`In Vivo Method
`purpose of the in vivo study is to
`Purpose.-The
`prove that ( a ) the results obtained by employing
`Experiment ZII (in d r o method) for noting the
`efficiency of the antibacterial agents are valid, i.e.,
`the bacteria are incapable of growing in the sub-
`culture medium and accordingly are also incapable
`of producing an ocular infection in vivo; and (b)
`the inactivating media utilized in this study are ade-
`quate.
`Preparation of the Inoculum.-The
`inoculum of
`Ps. aeruginosa was prepared as previously de-
`scribed. For the most part, the strains of Ps.
`aeruginosa which proved to be the most resistant to
`the antibacterial agents in the in uitro experiments
`were employed in this study. Occasionally an
`intermediate strain, with regard to resistance toward
`the antibacterial agent, and also a mixture of
`resistant strains were employed. The mixture of
`resistant strains was prepared on the day of the
`test by mixing 1 ml. of each of the respective 24-
`hour-old nutrient broth cultures in a sterile tube and
`
`Page 4
`
`

`

`Vol. 52, No. 10, October 1963
`
`
`TABLE II.-HIGHEST CONCENTRATION OF PRESERVATIVES INACTIVATED BY VARIOUS MEDIA
`
`971
`
`Preserva-
`tive
`Medium 1
`II
`I11
`IV
`V
`VI
`VII
`VIII
`IX
`X
`XI
`XI1
`_ _ _ _
`XI11
`XIV
`XV
`XVI
`XVII
`XVIII
`
`Chloro-
`butanol,
`'%
`
`0 . 4
`...
`...
`...
`...
`...
`...
`...
`...
`...
`...
`. . .
`...
`. . .
`...
`0.5
`0.6"
`0.6"
`0.6"
`
`b
`
`...
`...
`b
`b
`
`Methylparaben, '%
`Propylparaben, '%
`0.14
`0.02
`. . .
`. . .
`...
`...
`...
`. , .
`...
`. , .
`...
`. . .
`...
`. , .
`...
`. , .
`. . .
`. . .
`...
`...
`...
`. , .
`...
`. . .
`...
`. , .
`...
`_ . .
`0.2"
`0. 04a
`0.2"
`0.04"
`0.2a
`0.04"
`0.04"
`0.2a
`
`Phenyl-
`mercuric
`Nitrate
`
`b
`b
`...
`1:50,000
`. . .
`1:6250
`1:6250
`1:3125"
`1:6250
`1:3125*
`1:6250
`1:3125a
`...
`...
`...
`...
`
`Phenylethyl Polymyxin B
`Alcohol, %
`Sulfate. Units
`0.2
`31.25
`. . .
`15.6
`2000"
`. . .
`- - . --
`. . .
`21 25
`. . .
`31.25
`31.25
`. . .
`. . .
`31.25
`. . .
`...
`. . .
`. . .
`. . .
`. . .
`. . .
`. . .
`. . .
`. . .
`. . .
`. . .
`. . .
`...
`...
`0.5
`1.05
`. . .
`0.5
`. . .
`. . .
`1.0"
`
`Benz-
`alkonium
`Chloride Thimerosol
`I:~O.OOO
`i:3o;ooo
`1:2500
`-. ----
`1 . innna
`1 : 1OOoa
`...
`1:loOo"
`1:1OOoa
`. . .
`1:20,000
`...
`1:20,000
`...
`1:2500a
`...
`1:2o,OOo
`. . .
`1:250@
`...
`1:20,000
`...
`1:2500a
`...
`...
`...
`...
`...
`...
`...
`...
`0 Highest concentration tested. b Growth was not evident in this medium when lthe preservative was in a concentration of
`1 : 50,000 (the smallest concentration studied).
`proceeding as when a single strain of Ps. aeruginosa
`was used.
`Preparation of the Antibacterial Agent-Bacteria
`Mixtures.-One-tenth milliliter of the dilution of
`the strain or mixture of strains was added to 5 ml.
`of the sterile preservative solution being investi-
`gated. The tube was shaken by hand for approxi-
`mately 15 seconds and allowed to remain at 24'
`for a specific contact time, each time period depend-
`ing on the results obtained in the in d r o experiments
`for the antibacterial agent being studied.
`Preparation of the Controls.-A
`sterile solution of
`the preservative was employed as a positive control,
`not only for accidental contamination, but also to
`detect any action and untoward reaction the pre-
`servative might have on the rabbit's eye. A dilu-
`tion of Ps. aeruginosa, containing approximately
`10,OOO bacteria per ml., was employed as a negative
`control to determine pathogenicity of Ps. aeruginosa
`in the rabbit's eye.
`time periods were
`contact
`Procedure.-Two
`employed for each antibacterial agent-bacteria
`mixture studied. The contact time periods were
`selected, as mentioned previously, from the findings
`of the in Gtro experiments for the various anti-
`bacterial agents tested. The f i s t contact time
`period selected was the longest period of time which
`still showed growth of the bacteria in the subculture
`medium; also the contact time period immediately
`following the first contact time period was chosen.
`Obviously, the latter contact time period would
`have shown no growth of the bacteria in the sub-
`culture medium. The fist exposure period was
`employed to prove that when growth of Ps.
`aerugitzosa occurs in the subculture medium, an
`infection could also be produced in the rabbit's eye.
`The second contact time period was utilized to fulfill
`the purpose of the in vivo experiment.
`Usually six rabbits8 were employed for each anti-
`bacterial agent studied. The rabbits were placed by
`hand in individual cages which were thoroughly
`cleaned with a disinfectant and live steam after each
`
`experiment. The antibacterial agent-bacteria mix-
`ture was injected intracorneally into the rabbit's
`eye. Approximately 0.01 ml. was injected from a
`one-quarter-ml. tuberculin syringee graduated in
`0.01 ml. One-quarter-inch 27-gauge necdles'
`were employed. Approximately 5 minutes befor?
`each injection, two drops of a 2% butacaine sulfatelo
`solution were placed in the conjunctival sacs of each
`rabbit.
`At the f i s t contact time period selected an intra-
`corneal injection of the antibacterial agent-bacteria
`mixture was given into one eye of each of two rabbits.
`At the next contact time, another intracorneal
`injection was given into the other eye of each of the
`same two rabbits. A third rabbit received an injec-
`tion of the sterile antibacterial solution (positive
`control) in one eye and an injection of the diluted
`bacteria (negative control) in the other eye. The
`rabbits were observed for a period of 7 days for
`evidence of ocular infections (see Fig. 1). Figure 1
`shows the appearance of an eye infected with Ps.
`aeruginosa 5 days after the injection. Figure 2
`shows the appearance of a normal eye. Most eyes
`infected with Ps. aeruginosa were completely closed
`within 7 days after the injection.
`As a further control, swabs were periodically taken
`of any infected eyes to recover and identify the
`presence of Ps. aeruginosa.
`The above procedure was repeated, employing
`another strain or a mixture of strains of Ps. aerugi-
`nosa.
`
`RESULTS
`From the data obtained from Experiments I and
`I1 employing the different antibacterial agents as
`preservatives (Tables I1 and 111), the following
`media were selected to be employed as the inactivat-
`ing media in Experiments 111 and IV: Medium
`XVIII for chlorobutanol, Medium V for benzakon-
`ium chloride, Medium X for thimerosol, Medium
`XVIII for combinations OF methylparaben and
`propylparaben, Medium X for phenylmercuric
`9 Becton. Dickinson and Co., Rutherford, N. J.
`10 Butyn Sulfate, Abbott Laboratories, Chicago, Ill.
`
`a M d e white New
`Philadelphia, Pa.
`
`Zealand rabbits, Huntinpdon
`
`Page 5
`
`

`

`Journal of Pharmaceutical Sciences
`solutions against Ps. aeruginosa, the time required
`for such an agent to kill the bacteria must be known.
`The experiments employed in these studies deter-
`mine the sterilizing time for each antibacterial agent
`against Ps. aeruginosa. In addition, other important
`factors are evident in the experiments previously
`described which are not contained in those proce-
`dures in current use or those employed in the past.
`One advantage is that the procedures do not rely
`upon a dilution technique to differentiate between
`bacteriostatic and bactericidal effects of the anti-
`bacterial agents being tested.
`Inactivating agents
`were employed for this purpose. The latter, in-
`corporated in the subculture media, function by
`inactivating or neutralizing the antibacterial pro-
`perties of the chernical agents employed for their
`preservative activity.
`Another advantage of the test procedures em-
`ployed in this study is that they establish the
`inactivating or neutralizing ability of the subculture
`media toward the antibacterial activity of
`the
`chemical agents studied. Such data have not been
`reported previously by the various workers in the
`field.
`In the evaluation of
`the effectiveness of anti-
`bacterial preservatives, it is not only important to
`determine the time required for sterilization, but it
`is also necessary to determine whether the bacteri-
`DISCUSSION
`cidal effect was complete. A method which evalu-
`ates the destruction of bacteria but neglects to
`In the evaluation of the effectiveness of anti-
`bacterial agents used as preservatives in ophthalmic
`determine whether a total kill has occurred may
`OF STRAINS OF Ps. aeruginosa WHICH DISPLAYED GROWTH IN BEST TEST MEDIA
`TABLE III.-NuE~ER
`AFTER CONTACT WITH PRESERVATIVESO
`-
`Contact
`Medium
`XVI
`XVII
`XVIlI
`Time, hr.
`13
`13
`13
`1
` 0
`0
`0
`24
`Medium
`IV
`3
`0
`
`I
`0
`0
`x
`13
`0
`
`I
`2
`0
`
`I
`13
`13
`
`I
`0
`0
`
`IV
`0
`0
`
`XI
`3
`0
`
`X V
`3
`0
`
`I1
`0
`0
`
`111
`13
` 0
`
`VIII
`0
`0
`
`XI11
`0
`0
`
`V
`4
`0
`
`VI
`3
`0
`
`VII
`4
`0
`
`
`
`
`IX
`3
`0
`
`XIV
`3
`0
`-
`XVIII
`6
`0
`
`IX
`3
`0
`
`XIV
`2
`0
`
`XVIII
`13
`13
`
`V
`0
`0
`
`VI
`0
`0
`
`VII
`0
`0
`
`
`
`
`972
`nitrate, and Medium 111 for polymyxin B sulfate.
`Media XVI and XVIII were found to be the best
`neutralizing media for phenylethyl alcohol
`in
`Experiment Z. Since the sterilizing time for phenyl-
`ethyl alcohol was found to be greater than 24 hours,
`further experiments were not carried out on this
`chemical in Experiment II.
`In Table IV a summary of the results obtained in
`Experiment IIZ is presented. The sterilizing times
`which are listed against Ps. aeruginosa for each of
`the chemical agents are those which have been
`obtained under severe in nitro
`test conditions
`(dilution of bacterial inoculuni was 1 :lo). They
`indicate the time required to kill all of the strains of
`Ps. aeruginosa employed in this study.
`The results of Exfieriment I V are not recorded
`here, but in all cases they indicate that 0.5 ml. was
`adequate as the volume of antibacterial agent-
`bacteria mixture used as the transfer in Experiments
`ZZ and 111.
`The results of the in vivo experiments indicate that
`they are in agreement in all instances with the in
`vitro findings of Experinrent IZZ, i.e., those solutions
`which produced growth following transfer into
`subculture media produced ocular infections; those
`which did not produce growth did not produce ocular
`infections.
`
`Preservative
`Chlorobutanol, 0.5%
`
`I
`13
`0
`
`XV
`13
`0
`
`Benzalkonium chloride, 1 : 5OOO
`
`0 . 5
`24
`
`Thimerosol, 1 : 5000
`
`Thimerosol, 1 : 5000
`
`Methylparaben, 0.27,
`Propylparaben, 0.047,
`
`Phenylmercuric nitrate,
`1 : 10,000
`
`Phenylmercuric
`1:10,000
`
`nitrate,
`
`Phenylethyl alcohol, 0.57,
`
`Polymyxin B sulfate, 1000
`units/ml.
`
`0 Thirteen strains tested.
`
`1
`24
`
`1
`24
`
`1
`24
`
`1
`24
`
`1
`24
`
`1
`24
`
`1
`24
`
`I
`2
`0
`
`I1
`1
`0
`
`111
` 1
` 0
`Medium
`V
`VIII
` 0
`3
` 0
`0
`Medium
`XI1
`XI11
`13
`3
` 0
`0
`Medium
`XVI
`XVII
` 6
`3
` 0
`0
`Medium
`V
`IV
`I
` 0
`0
`0
` 0
`0
`0
`c htedium--
`x
`XI
`XI1
` 6
`i
`2
`0
`0
` 0
`xv XVI
`Medium
`XVII
`13
`13
`13
`13
`13
`13
`Medium
`IV
`0
`0
`
`Page 6
`
`

`

`Vol. 52, No. 10, October 1963
`
`In the experiments employed
`prove to be in error.
`in this investigation, the adequacy of the volume of
`the antibacterial agent-bacteria mixture transferred
`to the subculture media has been evaluated. This
`has not usually been taken into consideration.
`It was noted that the results of the in oioo test
`agreed a t all times with the results of the in rritro
`procedures. This was additional evidence of the
`effectiveness of the inactivating media employed
`for the various antibacterial chemical agents under
`study.
`An antibacterial agent intended for use as a
`preservative in ophthalmic solutions should have
`quick-acting bactericidal activity. There is no
`exact definition for the term quick-acting. ‘ I n this
`discussion, an antibacterial agent will arbitrarily be
`to possess quick-acting bactericidal
`considered
`activity against Ps. aeruginosa if the sterilizing time
`of that agent was 1 hour or less under the extreme
`test conditions employed in this study.
`Chlorobutanol is not a quick-acting antibacterial
`preservative aginst Ps. aeruginosa. A 12-hour
`period of contact was required for a 0.5% solution of