Effect of Different Commercial Agar on Inhibitory ... · EFFECT OFAGARSONPHENOLS agar or with deionized distilled water. In each ofthe semisolid media, the final agar concentration

APPLIED MICROBIOLOGY, Mar., 1966Copyright © 1966 American Society for Microbiology

Vol. 14, No. 2Printed in U.S.A.

Effect of Different Commercial Agar Preparationson the Inhibitory Activities of Phenols

J. G. SANDS AND E. 0. BENNETTDepartment ofBiology, University of Houston, Houston, Texas

Received for publication 8 October 1965

ABsTRAcr

SANDS, J. G. (University of Houston, Houston, Tex.), AND E. 0. BENNETr. Effectof different commercial agars on the inhibitory activities of phenols. Appl. Micro-biol. 14:196-202. 1966.-The minimal inhibitory concentrations of 11 phenolicinhibitors were compared in five commercial agars and in nutrient broth. It was

found that the brand of agar affected the end point obtained for a particular inhibi-tor, and that the degree of antagonism varied with each compound studied. The re-sults indicate that there are at least two deleterious factors present in agar, one ofwhich is water-soluble and one which is not. The major portion of the total antago-nism was due to the water-soluble factor, which could be removed by washing theagar in warm distilled water prior to use in the test medium.

During the past several years, this laboratoryhas been interested in the effect of agar on the in-hibitory activities of antimicrobial agents. A re-view of this subject (8) revealed that agar reducedor eliminated the inhibitory activities of manycompounds. Sands, Goers, and Bennett (41, 42)found that one-half of the 30 phenolic inhibitorsstudied were adversely affected by Difco agarwhen Staphylococcus aureus was used as the testorganism. Sands and Bennett (42) and Hanusand Bennett (19) showed that some of the antago-nism can be removed from the product by wash-ing the agar with distilled water. All of the phe-nolic inhibitors affected were antagonized by awater-soluble factor, and 55% of them showedreduced activities in the presence of the water-insoluble factor.

It has also been shown that different agar prepa-rations can produce variable results in inhibitorstudies. Harris and Jacobs (20) compared theeffect of two different agar preparations on thesurvival of Escherichia coli and S. aureus afterexposure to phenol, o-cresol, and p-chloro-m-cresol. They found that more bacteria survivedwhen the cells were plated onto Japanese agarthan on New Zealand agar. Greenberg (18) notedthat a component of Difco agar which interferedwith nitrosoguanidines and 5-diazouracil wasnot present in BBL agar, NBC agar, Ionagar no.2, or in Noble agar. Parag (32) found that Schizo-phyllum commune was sensitive to polymyxin inmedia containing Noble agar, but not in mediacontaining Difco agar.The present study was undertaken to demon-

strate that other commercial agar products inter-fere with the inhibitory activities of phenolic in-hibitors and to show that washing these prepara-tions removes some of the detrimental materialfrom them.

MATERIALS AND METHODSThe culture of S. aureus employed in previous stud-

ies (41) was used throughout this investigation. Theorganisms were maintained on nutrient agar at 4 Cand were transferred periodically. The inoculum wasprepared by growing the organisms for 24 hr at 37 Con nutrient agar and washing them from the agar insterile isotonic saline. A Klett-Summerson photo-electric colorimeter with a blue filter was used to ob-tain a uniform turbidity of 200 Klett units. After a10-fold dilution, this suspension contained approxi-mately 4.7 X 107 cells per milliliter, as determined byplate counts.

Stock solutions of the phenolic inhibitors wereprepared in acetone. Dilutions were made so that 0.1ml contained the desired amount of inhibitor whenadded to 10 ml of medium.The commercial agars used in this study were Difco

agar (Difco Laboratories, Detroit, Mich.), Nobleagar (Difco), BBL agar (Baltimore Biological Labora-tory, Baltimore, Md.), Fisher Flake agar (FisherScientific Co., Fair Lawn, N.J.), and Fisher S.P. agar(Fisher Scientific Co.).The washed agar cubes (3% agar) were prepared in

the same manner as described previously (42). Thecubes were melted under flowing steam in an auto-clave prior to use in the test medium. Aqueous solu-tions (3%) of unwashed Difco agar were melted in asimilar manner.

Double-strength nutrient broth was prepared anddiluted to single strength with a 3% solution of melted

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agar or with deionized distilled water. In each of thesemisolid media, the final agar concentration was

1.5%.Portions (10 ml) of each medium and 0.1 ml of

inhibitor were pipetted into a series of screw-cap

tubes and were autoclaved for 15 min at 121 C. Thetubes were then placed in a water bath and brought toa temperature of 45 C. Each tube was inoculated with0.1 ml of the standardized suspension of S. aureus

and was vigorously shaken on a Vortex Jr. mixer.The agar was allowed to solidify, and the tubes wereincubated at 37 C for 48 hr. At the end of this period,the tubes were examined under X 10 magnification forgrowth.The minimal inhibitory concentrations of the phe-

nols were first determined by a screening run in in-crements of 100 ppm. Four subsequent experimentswere then performed in which increments of 5 ppmwere employed. All data presented are averages ofthese four determinations with the minimal inhibitoryconcentration given in parts per million (ppm) of in-hibitor (w/v).

RESULTS

The effects of various brands of agar on 11different phenolic inhibitors were studied in thepresent investigation. It may be observed that thedifferent agar preparations can have a significantadverse effect upon the inhibitors (Table 1).Difco agar produced an average 35% reduction;BBL agar, 26% reduction; Fisher S.P. and FisherFlake agars, 22% reduction; and Noble agar, 21%reduction in the inhibitory activities of the com-pounds tested.Table 2 presents the data obtained when washed

and unwashed Difco agar was compared with nu-trient broth. For these compounds, there was an

average 15% reduction with washed Difco agarand an average 41% reduction in their inhibitory

activities when Difco agar was employed. It mayalso be noted that, in some instances, treatment ofthe agar removed all the deleterious material,whereas in other instances this was not the case.

Tables 3 to 6 present the data obtained whenthe various agar products were washed with de-ionized distilled water, as described previously. Itmay be noted that, in certain instances, washingthe agar reduced the deleterious effect of the ma-

terial on phenolic inhibitors.

DISCUSSIONMicrobiologists have employed agar for many

years as a solidifying agent, and it is preferred toall other gel-forming substances in making bac-teriological culture media. Except for an occa-

sional organism which can hydrolyze the ma-terial, agar is considered, in most instances, to bean inactive ingredient of these media. This ideahas persisted for many years, even though a care-ful examination of the literature shows an increas-ing number of reports indicating that this ideamay not always be true.Agar has been shown to contain stimulatory

substances for biological systems. This materialhas been reported in the literature to have thecapacity of stimulating the growth of excised comroots (39), Melanospora destruens (21), Polyporusabietinus and P. adustus (13), Rhodotorula sannei(14), several other fungi (26), Phycomyces blakes-leeanus (11, 27, 34, 35, 48, 49, 50), Fusarium avena-ceum (36, 37), Nitzschia linearis (33), Aspergillusniger (51), Streptomyces (31), and Rhizobium (2,3, 4). In addition, agar has been reported to stim-ulate nitrogen fixation by Azotobacter indicum(25).Agar has been shown to contain substances

TABLE 1. Phenolic inhibitors adversely affected by different agar products*

Smallest concn (ppm) necessary to completely inhibit growth

Compound Nu-trient Difco agar Noble agar BBL agar Fiaher Flake gFiher S-Pbrothagr gr

p-Butoxyphenol ............. ........... 275 335 (18) t 295 (7) 295 (7) 305 (10) 315 (13)2,4,6-Tribromophenol .................. 40 53 (25) 43 (7) 50 (7) 60 (33) 63 (37)2-Methyl-6-t-butyl phenol............... 60 100 (40) 130 (54) 120 (50) 100 (40) 100 (40)o-Dimethyl-aminomethyl-p-butyl phenol. 350 600 (42) 680 (49) 700 (50) 680 (49) 595 (41)p,p'-Biphenol .......................... 30 80 (62) 50 (40) 70 (57) 80 (62) 80 (62)p-Anilinophenol ........................ 75 195 (62) 125 (40) 175 (57) 145 (48) 145 (48)p-Octylphenol ........................... 10 20 (50) 15 (33) 15 (33) 10 (0) 10 (0)p-Benzylphenol ......................... 80 90 (11) 80 (0) 90 (11) 80 (0) 80 (0)2-Bromo-4-phenylphenol ................ 20 30 (33) 20 (0) 20 (0) 20 (0) 20 (0)2-Chloro-4-phenylphenol................ 30 40 (25) 30 (0) 30 (0) 30 (0) 30 (0)4-Nitro-3-trifluoromethyl-phenol ........ 20 25 (20) 20 (0) 20 (0) 20 (0) 20 (0)

* Data based on the average of four determinations.t Per cent difference from nutrient broth.

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TABLE 2. Comparison of the effects of washed andunwashed Difco agar on the inhibitory

activities of phenolic inhibitors

Smallest concn (ppm) necessaryto completely inhibit growth

CompoundNu-

Washed Unwashed

broth Difco agar Difco agar

p-Octyl phenol 10 10 (0)* 20 (50)*

4-Nitro-3-trifluoro-methylphenol. 20 20 (0) 25 (20)

p-Benzylphenol. 80 80 (0) 90 (11)p,p'-Biphenol 30 50 (40) 80 (62)

p-Anilinophenol 75 115 (35) 195 (62)

* Per cent difference from nutrient broth.

TABLE 3. Comparison of the effects of washed andunwashed Noble agar on the inhibitory

activities of phenolic inhibitors

Smallest concn (ppm) necessaryto completely inhibit growth

CompoundNu- Washed Unwashedtrient Noble Noblebroth agar agar

2,4,6-Tribromophenol.. 35 35 (0)* 35 (0)*2-Bromo-4-phenylphe-

nol.20 20 (0) 20 (0)2-Methyl-6-t-butyl phe-

nol.60 115 (48) 120 (50)p,p'-Biphenol.30 63 (52) 68 (56)

* Per cent difference from nutrient broth.

TABLE 4. Comparison of the effects of washed andunwashed BBL agar on the inhibitory

activities of phenolic inhibitors*

Smallest concn (ppm) necessary tocompletely inhibit growth

CompoundNutri- Washed BBL Unwashed

broth agar BBL agar

2,4,6-Tribromophe-nol.35 35 (0) t 45 (22) t

2-Bromo-4-phenyl-phenol.20 20 (0) 20 (0)

2-Methyl-6-t-butylphenol.60 110 (45) 110 (45)

p,p'-Biphenol 30 50 (40) 80 (62)

* All data based on the average of four deter-minations.

t Per cent difference from nutrient broth.

TABLE 5. Comparison of the effects of washed andunwashed Fisher Flake agar on the inhibitory



CompoundNutri- Washed Unwashedbroth Fisher agar Fisher agar

2,4, 6-Tribromophe-nol.35 33 (6) t 45 (22)t

2-Bromo-4-phenyl-phenol.20 20 (0) 20 (0)


p,p'-Biphenol. 30 60 (50) 80 (62)



TABLE 6. Comparison of the effects of washed andunwashed Fisher S.P. agar on the inhibitory



CompoundNutri- Washed Unwashedent Fisher Fisher

broth S.P. agar S.P. agar

2,4,6-Tribromophe-nol.35 35 (0) t 50 (30) t

2-Bromo4-phenyl-phenol.20 20 (0) 20 (0)


p,p'-Biphenol. 30 73 (59) 80 (62)



which are inhibitory for microorganisms. Gould,Kane, and Mueller (16), Ley and Mueller (28),and Casman (9) found that Difco and Merckagar products contained inhibitors for Neisseriagonorrhoeae which could be neutralized by theaddition of starch to the medium. Robbins andMcVeigh (38) demonstrated that a component ofhydrolyzed agar inhibited the growth of Tricho-phyton mentagrophytes, S. aureus, E. coli, Pseu-domonas aeruginosa and several other microor-ganisms, including fungi. Lynn and Morton (30)showed that certain agar products inhibited thegrowth of pleuropneumonia-like organisms.Yaphe (52) reported that acid hydrolysis prod-ucts of agar inhibited the growth of E. coli. Dukesand Gardner (12) found that agar contained awater-soluble factor which inhibited the growth

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of Haemophilus vaginalis. This inhibitory effectcould be overcome by using Noble agar or byadding 0.1% cornstarch to the regular agar me-dium.Agar has been shown to interfere with the

growth of viruses. Liebhaber and Takemoto (29)and Takemoto and Liebhaber (46) noted that asulfated polysaccharide which inhibited thegrowth of EMC virus was released from agarduring the process of autoclaving the material.These workers found that dextran neutralized theeffect of the inhibitor on the virus. Wallis,Melnick, and Bianchi (47) reported that plaqueformation by echoviruses 2, 3, 5, and 6 and reo-virus 1 was inhibited in Noble agar but not inDifco agar. These workers found that the addi-tion of MgCl2 neutralized the viral inhibitors inthe agar. Schulze and Schlesinger (43) and Schulze(44) noted that the infectivity and hemagglutinat-ing activities of dengue-2 virus were inhibited bya sulfated polysaccharide present in agar.

It has even been reported that agar can inter-fere with immunological reactions. Arima andKurokawa (5) studied the effect of 11 differentsamples of agar on the precipitation of diphtheriatoxin with antitoxin. More than half of the agarsamples gave unsatisfactory reactions. The au-thors concluded that agar made from certain kindsof marine plants was not satisfactory for use inthis procedure.Agar can also influence the effects of physical

agents upon microorganisms. Geissler (15) notedthat a nutritive substance in agar protected Sac-charomyces carlsbergensis from the lethal effect ofradiation.

In some instances, agar may potentiate theeffects of inhibitors on microorganisms. Cook(10) noted that chlorocresol inhibited Hae-mophilus bronchiseptious 10 times more effectivelyin the presence of agar than in its absence. Iyerand Iyer (23) demonstrated that B.D.H. agar(British Drug House, Ltd., London, England)contained a factor which increased the inhibitoryactivity of chlortetracycline. These workers foundthat washing the agar after it was autoclaved re-moved the potentiating material, and that S.aureus was as sensitive to the antibiotic in thewashed agar medium as it was in the presence ofthe antibiotic alone. Harris and Jacobs (20) andJacobs and Harris (24) demonstrated that agarcontained a component which increased the detri-mental effect of phenol, o-cresol, and p-chloro-m-cresol against E. coli and S. aureus. The activityof this substance was reduced when the agar wastreated with Norit and ferric chloride. Sands et al.(41) and Sands and Bennett (42) noted that p-sec-butylphenol and o-bromophenol were slightly

more effective in the presence of agar than in itsabsence.Agar can interfere with the inhibitory activities

of antimicrobial agents. The literature containsreports demonstrating that this material reducedthe inhibitory activities of acridine (6, 7), octylalcohol (1), crystal violet (40), 5-diazouracil (18),fatty amines (19), 1-nitroso-2-nitramino-2-imida-zoline (18), laurylamine saccharinate (17), lecithin(1), nitrosoguanidines (18), oleates (1), phenols(17, 41), proflavine (7), quaternary ammoniumcompounds (22, 45), stearates (1), and sulfosuc-cinates (1).The results of this investigation provide addi-

tional evidence that agar has a deleterious effecton phenolic inhibitors. The effect of agar on theantimicrobial properties of 39 phenolic inhibitorshas been studied by Sands et al. (41), Sands andBennett (42), and in this investigation. It wasfound that agar has an adverse effect on 19 ofthese compounds, or approximately one-half ofall the compounds studied.

It has also been shown that different agar prepa-rations can produce variable results in inhibitorstudies. Harris and Jacobs (20) and Jacobs andHarris (24) compared the effect of two differentagar preparations on the survival of E. coli andS. aureus after exposure to phenol, o-cresol, andp-chloro-m-cresol. They found that more bacteriasurvived when the cells were plated onto nutrientbroth solidified with New Zealand agar.Greenberg (18) noted that a component of Difcoagar which interfered with the inhibitory activitiesof nitrosoguanidines, 1-nitro-2-nitramino-2-imid-azoline, and 5-diazouracil was not present inBBL agar, NBC agar, Ionagar no. 2, or in Nobleagar. Parag (32) studied the inhibitory activityof polymyxin against S. commune in media con-taining Difco or Noble agar. He found that theorganism was sensitive to the antibiotic in Nobleagar, but not in Difco agar.The results of the present investigation also

show that the brand of agar employed in a studyof antimicrobial agents can have an effect on theresults obtained (Table 1). It is interesting to notethat the average detrimental effect of agar is aboutthe same for all the products studied, althoughtwo of them were supposedly more purified thanothers.Two workers have attempted to remove from

agar the material which has an adverse effect onantimicrobial agents. Greenberg (18) reportedthat the material could be extracted from Difcoagar with water or methanol, but not with ethylalcohol, acetone, or butanol. The extract lost allactivity within 24 hr at room temperature, butwas stable for at least 4 weeks at -10 C. It was

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ninhydrin-negative, and polarographic analysisrevealed no -SH or S-S compounds. There wasno detectable amount of ascorbic acid, and it didnot absorb ultraviolet light. Sands and Bennett(42) found that there are at least two factorspresent in Difco agar which interfere with the in-hibitory activities of phenolic compounds. One ofthese factors is water-soluble and can be removedfrom the agar by extraction with warm distilledwater. Hanus and Bennett (19) also showed thatthese two factors can interfere with the inhibitoryactivities of fatty amines. This water-soluble fac-tor may be similar to the material reported byGreenberg (18). The second factor is not water-soluble and has not been previously detected inagar. The data presented in this publication giveadditional verification that Difco agar contains atleast two deleterious factors for phenolic inhibi-tors.The results also indicate that the various agar

preparations may contain different water-solubleantagonists. It may be observed that, in Difcoagar, the water-soluble factor alone interfereswith p-octyl phenol, 4-nitro-3-trifluoro-methyl-phenol, and p-benzyl phenol (Table 2). In addi-tion, Sands and Bennett (42) reported that 2-bromo-phenylphenol and 2-chloro-4-phenylphe-nol were antagonized only by the water-solublefactor found in Difco agar. These observationswere confirmed in this investigation. There was,however, no reduction of the effectiveness of theseinhibitors in some of the other unwashed agarpreparations (Table 1). One could then concludefrom these observations that the other agar prepa-rations do not contain the water-soluble factorpresent in Difco agar.These agar preparations, however, do contain

water-soluble factors which antagonize other in-hibitors (Tables 3 to 6). Since washing thesepreparations with deionized distilled water failedto remove all of the antagonism, it appears thatthese products also contain water-insoluble fac-tors which interfere with phenolic inhibitors.Noble agar does not contain any of these water-

soluble factors (Table 3). Its presence in the testmedium does reduce the activity of most phenolicinhibitors (Table 1), indicating the presence of awater-insoluble factor. Similar results were ob-tained when Difco purified (certified) agar wasemployed.The differences observed in the various agar

preparations may be due in part to differentsources of raw agar and in part to differences inthe refining processes. During the course of theseinvestigations and others previously reported, atleast three different batches of Difco agar wereused, and the results have been duplicated. It

appears, therefore, that the antagonists found inDifco agar are consistently present in this prod-uct.The most alarming aspect of this information

and that presented previously is related to studiesdesigned to discover new antimicrobial agents. Itis common practice to screen new and unusualchemicals against microorganisms in a semisolidmedium. The question then arises as to how manybiocides or chemotherapeutic agents have escapeddiscovery because of a faulty practice which isused extensively in many laboratories.

ACKNOWLEDGMENTSThe authors thank the Ethyl Corp., New York,

N.Y., Dow Chemical Co., Midland, Mich., AbbottLaboratories, N. Chicago, Ill., Rohm and Haas Co.,Philadelphia, Pa., Maumee Chemical Co., Toledo,Ohio, Roberts Chemicals, Inc., Nitro, W. Va., E. I.DuPont de Nemours & Co., Wilmington, Del., andDifco Laboratories, Detroit, Mich. for kindly fur-nishing materials used in this investigation.

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Effect of Different Commercial Agar on Inhibitory ... · EFFECT OFAGARSONPHENOLS agar or with deionized distilled water. In each ofthe semisolid media, the final agar concentration