APPLIED MICROBIOLOGYVol. 12, No. 3, p. 204-209 May, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

Effect of Water Extracts of Carob Pods, Tannic Acid, and TheirDerivatives on the Morphology and Growth of Microorganisms'

Y. HENIS, H. TAGARI, AND R. VOLCANI

Faculty of Agriculture, The Hebrew University, Rehovot, Israel

Received for publication 18 December 1963

ABSTRACT

HENIS, Y. (The Hebrew University, Faculty of Agriculture,Rehovot, Israel), H. TAGARI, AND R. VOLCANI. Effect of waterextracts of carob pods, tannic acid, and their derivatives on themorphology and growth of microorganisms. Appl. Microbiol.12:204-209. 1964.-The effect of aqueous extracts of carob (Cera-tonia siliqua) pods, gallotannic acid, gallic acid, and catechol onseveral microorganisms was studied. Carob pod extract and tan-nic acid showed a strong antimicrobial activity toward somecellulolytic bacteria. On the basis of tannin content, to whichantimicrobial effect was related, carob pod extracts inhibitedCellvibrio fulvus and Clostridium cellulosolvens at 15,ug/ml, Sporo-cytophaga myxococcoides at 45,g/ml, and Bacillus subtilis at 75,ug/ml. The inhibiting concentrations for tannic acid were foundto be 12, 10, 45, and 30 ,ug/ml, respectively. Gallic acid and cate-chol were much less effective. Tannic acid and the tannin fractionof carob extract exerted both bacteriostatic and bactericidal ef-fects on C. fulvus. Respiration of C. fulvus in the presence ofbactericidal concentrations of tannic acid or tannin fraction ofcarob extract was inhibited less than 30%. A partial formationof "protoplasts" by C. fulvus was obtained after 2 hr of incuba-tion in a growth medium to which 20% sucrose, 0.15% MgSO4-7H20, and 10 to 50 ,g/ml of tannic acid or 500,ug/ml of peni-cillin, or both, had been added. Tannic acid and the tanninfraction of carob extract protected C. fulvus from metaboliclysis in sucrose solution. Although the growth of other micro-organisms tested was only slightly affected, the morphology ofsome of them was drastically changed in the presence of sub-inhibitory concentrations of carob pod extracts of tannic acid.It is suggested that the site of action of tannins on sensitivemicroorganisms is primarily the cell envelope.

The substances commercially defined as tannins (Nieren-stein, 1934) comprise an important group of natural phe-nolics synthesized by plants (Pridham, 1960). Tanninsand tanninlike substances are well known for their abilityto form complexes with other organic compounds. Theyhave been shown to inhibit decomposition processes bycombining with substrates (Siu, 1951; Basaraba, 1960)or enzymes (Porter et al., 1961; Smart et al., 1961), or byinhibiting the growth of microorganisms involved in theseprocesses (Siu, 1951). The resistance of plants to fungaldiseases was often related to the presence of phenolic sub-stances (Cook and Taubenhaus, 1911; Byrde, Fielding,and Williams, 1959). Tannins have also been reported to

I Contribution from The National and University Institute ofAgriculture, Rehovot, Israel; 1963 Series, no. 577-E.

prevent the infection of plants by tobacco mosaic virusby adsorbing on the virus surface (Cadman, 1960) and toaffect the permeability of erythrocytes to nonelectrolytes(Hunter, 1960). The inhibitory effect of tannins on cellu-lolytic fungi was reported as early as 1912 (Wehmer, 1912).

This report describes the effect of gallotannic acid, aque-ous extracts of carob (Ceratonia siliqua) pods, known tobe rich in tannins of the catechol-condensed type (Nach-tomi and Alumot, 1963), and their derivatives (gallic acidand catechol, respectively) on some cellulolytic as well asnoncellulolytic bacteria.

MATERIALS AND METHODS

The organisms used were: Escherichia coli, Aerobacter aer-ogenes, Vibrio cholerae, Salmonella typhosa, Shigella dysen-teriae, Bacillus subtilis, Streptococcus cremoris, Cellvibriofulvus, Clostridium cellulosolvens, Staphylococcus aureus, andSporocytophaga myxococcoides. The test organism used fordetecting antimicrobial potency was Cellvibrio fulvus.

S. cremoris was grown and tested on a medium composedof 5 g of yeast extract (Difco), 5 g of peptone (Difco),and 20 g of glucose in 1 liter of distilled water. Othermicroorganisms were grown and tested on a basal mediumwhich contained 1 g of (NH4)2SO4, 1 g of K2HPO4, 1 g ofKCl, 0.5 g of MgCl2 6H20, and 0.1 g of yeast extract(Difco) in 1 liter of distilled water (pH 7.2). S. myx-ococcoides and C. cellulosolvens were grown and tested onthe basal medium, with strips of Whatman no.1 filterpaper as the sole carbon source. Other microorganismswere grown and tested on a basal medium to which glu-cose at a final concentration of 1 % had been added. Theconcentrated sugar solution was sterilized separately andadded aseptically to the sterilized basal medium.

Estimation of the antimicrobial potency was made withC. fulvus in a basal medium containing 1 % lactose as asole carbon source, and was expressed as minimal inhibi-tory concentration (MIC). Double dilutions of the testedpreparations were run in duplicate in test tubes containing5 ml of assay medium each, inoculated (2 X 104 cells perml, final concentration), and incubated at 30 C for 48 hr.One unit of antimicrobial potency was defined as the

highest dilution of a given preparation that still inhibitsthe growth of C. fulvus under the above-described con-ditions.

Preparations tested for antimicrobial activity were steri-lized by Seitz filtration.

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EFFECT OF TANNINS ON IMICROORGANISAIS

Quantitative tests for tannins were carried out withFolin-Denis reagent, by use of the modification of Rosen-blatt and Peluso (1941). Standard curves were preparedwith tannic acid obtained from the British Drug HousesLtd., Poole, England.Oxygen uptake of bacterial suspensions was determined

manometrically.Optical density of bacterial suspensions was determined

with a Coleman Junior spectrophotometer at 550 mj,.

RESULTSCorrelation between tannins, sugar content, and antimi-

crobial potency of carob pod extracts. In a series of extrac-tions, 100-g samples of carob pod meal were mixed with250 ml of distilled water and incubated at various temper-atures for various periods of timie. The extracts were steri-lized and analyzed for tannic content and antimicrobialactivity as described above (Table 1). Some of the activecomponents were extracted at room temperature, but mostof them could be extracted only by prolonged boiling. Aspart of the activity might have been correlated with the

TABLE 1. Effect of temperature on extraction of antimicrobialactivity and tanninlike substances from carob pod meal

Substance

Carob pod meal

Sucrose, 20%Gallotannic

acid, 1%

Treatment and duration*

25 C, 4 hr40 C, 4 hrBoiling water, 10min

Boiling water, 20min

Boiling water, 40min

Boiling water,100 min

Activity(units/ml)t

3040100

120

165

200

20640

Tannins Tannins(,sg/ml) (jAg/unit)

1,4251,9502,450

2,600

3,500

5,050

10,000

* Samples were mixed with 2.5 ml of water per 1 g of carob podmeal, and treated as indicated.

t For definition, see Materials and Methods.

sugars known to be present in carob pods at concentra-tions of 30 to 40 % (Nachtomi and Alumot, 1963), a 20 %sucrose solution was also tested for antimicrobial activity.This was found to be negligible as compared with the totalactivity of the carob- pods extracted at 100 C for 10 min.On the other hand, a close correlation was observed be-tween the tannin content of the extracts and their anti-microbial potency. To verify this correlation, fractionationby lead acetate was employed. To 100 ml of carob podextract, containing 6 mg of tannins per ml, 65 ml of a

saturated solution of lead acetate were added. The pre-

cipitated tannins were washed with distilled water, andfree tannins were obtained by acidifying the precipitate.A solution of 5 N H2S04 was added dropwise to the con-

stantly stirred precipitate until a pH of 2.0 was reached.The water-insoluble precipitate of PbSO4 was centrifugedoff. The first supernatant which contained the sugars afterprecipitation of the tannins was passed through a mixedbed resin (Amberlite IR-120 and IRA 410) and tested forantimicrobial activity. Simultaneously, all the other frac-tions obtained during this procedure were tested.

In Table 2, the distribution of the antimicrobial activityof the various fractions is shown. It may be concludedthat the antimicrobial activity of the carob pod extractsis closely correlated with the fraction precipitated by leadacetate and reacting to tannin reagents. The sugar fractionof the carob pod extract inhibited growth only at dilutionslower than 1:20, as compared with 1:300 dilutions ofthe whole extract, which still inhibited the growth of C.fulvus.

Antimicrobial spectrum of carob pod extract, tannic acid,gallic acid, and catechol. Antimicrobial spectra are shownin Table 3. Most of the organisms tested seemed to berelatively resistant to the preparations used. However,growth of Cellvibrio fulvus and Clostridium cellulosolvens,both cellulolytic organisms, was inhibited at very lowconcentrations of gallotannic acid and carob pod extract.Sporocytophaga myxococcoides and B. subtilis were some-

what less sensitive. All other microorganisms tested were

not affected by the preparations used. Gallic acid, thebuilding unit of gallotannic acid, and catechol, from which

TABLE 2. Antimicrobial activity and tannin content of 1 :2.5 carob pod extract fractionated with lead acetate

Potency (units)* TanninsFraction Amt

Units/ml Total Per cent Amt Total Per cent

ml mg/mlUntreated ...................................... 100 390 39,000 100 6.0 600 100Washed precipitate ............................ 30 680 20,400 52.3 11.0 330 55Supernatant ................................... 135 80 10,800 27.7 0.76 102.6 17Washing water of washed precipitate ........... 77 - - - 26.9 4.48Residue of washed precipitate adsorbed onPbSO4 .............................. 83 - 16.6 2.76

Supernatant passed through ion-exchangecolumn ...................................... 20 0.049

Control, lead acetate precipitated with H2SO4 - 20

* For definition, see Materials and Methods.

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HENIS, TAGARI, AND VOLCANI

condensed tannins, including those present in carob ex-

tracts, are supposedly derived (Nachtomi and Alumot,1963), were much less effective. In the case of Streptococ-cus cremoris, precipitation reaction took place betweenthe peptone and tannin preparations, which made growthobservations and interpretation of the results more diffi-cult.

Bacteriostatic and bactericidal effect of tannic acid andtannin fraction of carob pod extract on C. fulvus. C. fulvuscells (2 days old) grown on lactose-basal agar were sus-

pended in lactose-basal medium containing various con-

centrations of tannic acid or tannin fraction of carob ex-

tract and incubated at 30 C. Samples were taken atintervals, and the number of survivors was determined bythe plate count method, with lactose-basal agar (Fig.1).Both tannic acid and the tannin fraction of carob ex-

tract showed a bacteriostatic effect on C. fulvus at tanninconcentrations up to 10 ,ug/ml, and a bactericidal effectat higher concentrations. However, whereas the carobextract fraction caused an immediate decrease in cell via-bility at bactericidal concentrations, tannic acid did notaffect cell viability to a significant degree during the first2 hr of incubation.

Effect of tannic acid and the tannin fraction of carob ex-

tract on the respiration of C. fulvus and E. coli. With boththe tannin-resistant E. coli and the tannin-sensitive C.fulvus, inhibition of respiration did not exceed 30 % even

after 3 hr of incubation and at concentrations highly bac-tericidal for C. fulvus. No significant difference in the effectof tannins on the respiration of the resistant E. coli as

compared with the tannin-sensitive C. fulvus could be ob-served.

Effect of tannic acid and carob pod extract on the morphol-

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TABLE 3. Antimicrobial spectrum (MIC in,ug/ml) of somenatural substances containing phenol groups

Organism Tannic acid Carob pd Gallic Catecholextract* acid

Cellvibrio fulvus ......... 12 15 250 500Sporocytophagamyxococcoides .......... 45 75 _

Clostridiumcellulosolvens .10 60

Bacillus subtilis... 30 75 500 250Shigella dysenteriae 500 600 500 600Salmonella typhosa. ........... > 1, 000 > 600 > 1, 000 1,000Vibrio cholerae ........... > 1, 000 > 600 > 1, 000 > 1, 000Streptococcus cremoris 250t 600t 1,000 500Pseudomonas fluorescens.. > 1,000 >600 > 1,000 > 1,000

Fit F+++ F- F-Escherichia coli .......... > 1, 000 > 600 > 1, 000 > 1, 000

F+++ F+ F- F-Aerobacter aerogenes ..... > 1, 000 > 600 > 1, 000 > 1, 000

F- F- F- F-

* Expressed as Mg/ml of tannic acid.t Medium became opaque as a result of complex formation be-

tween tannic acid and peptone.t F = various degrees of chain or filament formation.

FIG. 1. Effect of tannic acid and tannin fraction of carob extracton the viability of Cellvibrio fulvus. Counts were made after 48 hrof incubation at 30 C.

TABLE 4. Effect of tannins on the respiration* of Cellvibriofulvus and Escherichia coli

E. coli C. fulvus

Tan- Tannic acid Carob extract Tannic acid Carob extract onins

02 Inhibi- 02 Inhibi- 02 Inhibi- 02 Inhibi-tion tion tion 2 tion

pg/ml pliters/hr % %difersl % plifers/hr % jlditers/hr %

0 48 48 31 - 31 -10 47 2.0 47 2.0 28 9.6 28 9.650 45 6.3 47 2.0 26 16.1 28 9.6100 41 14.5 46 4.2 22 29.0 23 25.8

* Each Warburg vessel contained 0.5 ml of cell suspension (opti-cal density = 0.1 X 10; X = 550 m,), 0.5 ml of 0.2 M glucose, 0.5 mlof 0.5 M phosphate buffer (pH 7.0), and 0.5 ml of either tannin solu-tion or distilled water. The center well of each vessel contained0.2 ml of 15% KOH. Temperature, 30 C. Gas phase, air. Incuba-tion period, 3 hr.

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FIG. 2. Pseudomonasfluorescens, 48-hr-old culture, stained with methylene blue. X 1,200. Most of the cells are single, although some appearin pairs.

FIG. 3. Pseudomonas fluorescens, 48-hr-old culture, grown in the presence of carob pod extract (600 ,ug/ml of tannin). The cells appear in

long chains and adsorb more stain. Methylene blue. X 1,200.FIG. 4. Pseudomonas fluorescens, 48-hr-old culture, grown in the presence of tannic acid (1,000 ,ug/ml). A strong adsorption of the stain is

noted, but very little, if any, chain formation (more than two cells held together). Methylene blue. X 1,200.FIG. 5. Escherichia coli, 48-hr-old-culture. Methylene blue. X 1,200.FIG. 6. Escherichia coli, 48-hr-old culture, grown in the presence of tannic acid (100 sAg/ml). Long filaments are clearly seen and the stain

is strongly adsorbed on the cells. Methylene blue. X 1,200.FIG. 7. Escherichia coli, 48-hr-old culture, grown in the presence of carob pod extract (600 iAg/ml of tannin). Chain formation is much less

than in the case of Pseudomonas fluorescens (Fig. 2). Methylene blue. X 1,200.

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HENIS, TAGARI, AND VOLCANI

ogy of E. coli, P. fluorescens, and C. fulvus. When the testedorganisms were grown in the presence of subinhibitoryconcentrations of tannic acid and carob pod extract andexamined under a microscope, a drastic change in themorphology and growth pattern of some of them was ob-served (Fig. 2 to 7). In the presence of carob pod extract,P. fluorescens showed a strong tendency to grow in chains.C. fulvus also showed some degree of chain formation atsubinhibitory concentrations. On the other hand, tannicacid caused E. coli to grow in filaments, whereas P. fluo-rescens was only slightly affected. No change in the mor-phology of A. aerogenes, V. cholerae, S. aureus, and B. sub-tilis could be observed.

Effect of penicillin and tannic acid on C. fulvus cells sus-pended in media containing high concentrations of sucrose.A glucose basal medium containing 20 % sucrose, 0.15 %MIgSO4*7H20, and penicillin or tannic acid, or both, wasused. Test tubes, each containing 5 ml of medium, wereinoculated with a 48-hr-old culture of C. fulvus and incu-bated at 30 C. Changes in optical density were followedspectrophotometrically (Fig. 8). A decrease in optical den-sity, accompanied by cell lysis, occurred in all the suspen-sions tested. It was more pronounced when penicillin waspresent in the medium, but could be prevented by tannicacid after incubation periods of 2 hr or more. In addition,formation of "protoplasts" (MIcQuillen, 1960) was followedunder a phase microscope.

In the presence of penicillin or tannic acid, or both,formation of spherical bodies, as well as intermediaryforms of rods with a bulb-shaped form at their end, wasclearly observed. However, this phenomenon was limitedto a small fraction (ca. 10 %) of the bacterial population.

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H O U R S

FIG. 8. Turbidity changes of Cellvibrio fulvus suspended insucrose and sucrose-penicillin media as affected by tannic acid.Initial optical density = 0.20; X = 550 m,u. Composition of sucrose

medium: 20% sucrose and 0.15% MgSO4-7H20 in glucose-basalmedium. Incubation temperature: 30 C.

Similar results were obtained with the tannin fractionof carob extract.

DIscussIONGallotannic acid and the tannin fraction of carob pod

extract inhibited the growth of saprophytic cellulolyticbacteria, especially C. fulvus. The growth of noncellulo-lytic saprophytic, as well as pathogenic, bacteria was notinhibited by the preparations tested. This is partially incontrast with the findings of Masquelier (1959), whostated that the tannins present in wines exhibit a strongbactericidal effect on pathogenic but not on saprophyticbacteria. However, it may be expected that tannins ofdifferent origin and composition will affect microorgan-isms differently.

In considering the possible mechanism by which tanninsaffect the growth and morphology of microorganisms, itshould be borne in mind that these are substances of ahigh molecular weight, which form insoluble complexeswith proteins in a nonspecific way. Tannins may affectbacteria either by adsorbing on their surface, or by pene-trating into the bacterial cell and reacting with its com-ponents. Being composed of proteins as well as of poly-saccharides, lipids, and mucopeptide components (Perkins,1963), cell walls of gram-negative bacteria such as C.fulvus may react with tannins. This is also true for the lipo-proteins containing cytoplasmic membrane. However, itis rather difficult to test these possibilities directly, asthere is no adequate technique available at present forthe isolation of pure cell walls or cytoplasmic membranesof gram-negative bacteria (Perkins, 1963). Indirect evi-dence that tannins really affect the cell envelope is demon-strated by the formation of "protoplasts" (McQuillen,1960) by C. fulvus in the presence of tannic acid or tanninfraction of carob extract. It seems that inhibition of cell-wall synthesis is one of the early effects exerted by tanninson sensitive bacteria. The lysis of C. fulvus cells in glucosebasal medium containing 20 %o sucrose is apparently iden-tical with the "metabolic lysis" of S. faecalis and E. colidescribed by Abrams (1959). Tannic acid and tannin frac-tion of carob extract protected C. fulvus from metaboliclysis at bacteriostatic as well as bactericidal concentrations.On the other hand, respiration of C. fulvus was onlyslightly affected even at bactericidal concentations. This,as well as the formation of protoplasts mentioned above,would perhaps indicate that cell-wall synthesis and theintegrity of the osmotic barrier of C. fulvus are impairedby tannins prior to their penetration into the bacterialcell. Once cell permeability is irreversibly affected, viabilityis also lost. Therefore, it is unnecessary to assume a harm-ful effect of tannins on the inner components of the bac-terial cell. An adsorption of tannic acid on the surface oftobacco mosaic virus and a harmful effect of tannins onthe permeability of erythrocytes have been observed byother authors (Cadman, 1960; Hunter, 1960). Anotherindication of the effect of tannins on cell envelope is the

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EFFECT OF TANNINS ON MICROORGANISMS

formation of chains and filaments at subinhibitory con-

centrations, which resembles the effect of penicillin (Prattand Dufrenoy, 1948) and magnesium deficiency (Webb,1951) on microorganisms.No explanation is available for the extreme differences

in sensitivity to tannic acid observed among the micro-organisms tested. Presumably, this reflects unknown differ-ences in cell-envelope function and composition. Furtherstudies are required to evaluate these problems.

LITERATURE CITED

ABRAMS, A. 1959. Reversible metabolic swelling of bacterial pro-

toplasts. J. Biol. Chem. 234:383-388.BASARABA, F. 1960. Effects of vegetable tannins on decomposi-

tion of some organic compounds. Ph.D. Thesis, Rutgers,The State University, New Brunswick, N.J.

BYRDE, R. J. W., A. H. FIELDING, AND A. H. WILLIAMS. 1960. Therole of oxidized polyphenols in the varietal resistance ofapples to brown rot, p. 95-99. In J. B. Pridham [ed.],Phenolics in plants in health and disease. Pergamon Press,Inc., New York.

CADMAN, C. H. 1960. Inhibition of plant virus by tannins, p. 101-105. In J. B. Pridham [ed. ], Plant phenolics group. PergamonPress, Inc., New York.

COOK, M. T., AND J. J. TAUBENHAUS. 1911. The relation of para-

sitic fungi to the contents of the cells of the host plants. I.The toxicity of tannin. Delaware Univ. Agr. Expt. Sta. Bull.91, p. 3-77.

HUNTER, F. R. 1960. The effect of tannic acid on the permeabilityof erythrocytes to nonelectrolytes. J. Cellular Comp. Physiol.55:175-188.

McQUILLEN, K. 1960. Bacterial protoplasts, p. 249-359. In I. C.

209

Gunsalus and R. Y. Stanier [ed.], The bacteria, vol. 1, Struc-ture. Academic Press, Inc., New York.

MASQUELIER, J. 1959. The bactericidal action of certain phenolicsof grapes and wine, p. 123-131. In J. W. Fairbairn [ed.], Thepharmacology of plant phenolics. Academic Press, Inc.,New York.

NACHTOMI, E., AND E. ALUMOT. 1963. Tannins and polyphenols incarob pods (Ceratonia siliqua). J. Sci. Food Agr. 14:464-468.

NIERENSTEIN, N. 1934. The natural organic tannins. J. & A.Churchill Ltd., London.

PERKINS, H. R. 1963. Chemical structure and biosynthesis ofbacterial cell walls. Bacteriol. Rev. 27:18-55.

PORTER, W. L., J. H. SCHWARTZ, T. A. BELL, AND J. L. ETCHELLS.1961. Probable identity of the pectinase inhibitor in grapeleaves. J. Food Sci. 26:600-605.

PRATT, R., AND J. DUFRENOY. 1948. Cytochemical interpretationof the mechanism of penicillin action. Bacteriol. Rev. 12:79-103.

PRIDHAM, J. B. 1960. Plant phenolic group. Symposium, Bristol,April 1959. Symposium Publications Division, PergamonPress, Inc., New York.

ROSENBLATT, M., AND J. V. PELUSO. 1941. Determination of tan-nins by photocolorimeter. J. Assoc. Offic. Agr. Chemists 24:170-181.

SIu, R. G. H. 1951. Microbial decomposition of cellulose. ReinholdPublishing Corp., New York.

SMART, W. W. G., T. A. BELL, N. W. STANLEY, AND W. A. COPE.1961. Inhibition of rumen cellulase by an extract from Sericeaforage. J. Dairy Sci. 44:1945-1946.

WEBB, M. 1951. The influence of magnesium on cell division. Theeffect of magnesium on the growth of bacteria in chemicallydefined media of varying complexity. J. Gen. Microbiol. 5:485-495.

WEHMER, C. 1912. Tannic acid as protection for oak wood againstdry rot. Mycol. Centr. 1:138-148.

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