NOTES 337

INHIBITION OF STAPHYLOCOCCAL BACTERIOPHAGES BY TRYPAN BLUE AND TRYPAN BLUE - CALCIUM COMBINATIONS'

At the present time, there are few known substances which can be used to inhibit bacterial cell lysis by hoiliologous bacteriophages without exerting some toxic effects on host cells or phages, or both. Streptolnycin has been reported to inhibit lysis by a staph~~lococcal (3) and several streptococcal phages (2, 4) without toxic effects, while surainin (7) has been reported to do likewise for a Streptococcz~s lactis phage. T o be added to this list of nontoxic phage inhibitors is the acidic dye trppan blue, whose inhibitory activity is increased in the presence of certain divalent cations.

For the purpose of characterizing the antiphage activity of trypan blue, seven Staphylococcz~s aureus phages of the International Typing Set (1) were used with Trypticase soy agar as plating medium. When the phages were used a t concentrations of around 2 X lop4 to 10-"articles Der milliliter, or routine test dilution, all produced confluent lysis on Trypticase soy agar when concen- trations of 5 X 10-5 M trypan blue and 1.9 X lo-"[ CaClz were added to media alone (Table I , expts. 4 and 5). Overnight incubation of phages in a dye-calcium mixture of the above concentrations did not result in phage inactivation, and thesc levels were completely noninhibitory to host cell growth when measured 1157 growth curves and coinpared to controls without either added inetal ions or dye. However, when these coii~pounds were com- bined a t the same levels, inhibition of all but two group I phages occurred (expt. 7). I t can further be seen that higher concentrations of trypail blue without added CaC12 were inhibitory (expts. 2 and 3) , while lower concentra- tions even in the presence of added CaClz were less inhibitory (expts. 8-10). Group I1 phages tested wcre most sensitive to the trypan blue - CaClz com- bination, while group I phages were most resistant. These results were the saine when phages were used a t 1000 times routine test dilution.

Sodiuin citrate a t a level of 5 X Mcompletely inhibited lysis by phages 29 and 52 in Trypticase soy agar but lysis occurred upon the addition of 1.9 X lo-' hf CaC12, thus ruling out calcium depletion by trypan blue as the cause of its inhibition. Inhibition bv this dve - metal ion coinbination was not affected by the order in which the various coinponents were added to the total system. I t occurred in broth as well as on several other solid media. Other divalent metals (AIg++, Ba++, Sr++, Mni-+, and Fe++) were effective but appeared to be less effective than calciuin. Calcium nitrate was just as effective as the chloride in equal concentrations, while Na2W04, NaJ~Io4, and NaCl showed essentially no activity. The dye-calcium combination in- hibited within the pH ranee of 5.8 to 8.0. -

Trypan blue is ltnown to coilsist of several components detectable by paper chromatography (5). When the dye was fractionated on Whatlnan No. 1 paper with 80% ethanol (v/v), antiphage activity was shown to be associated

'Supported in part by I.C.R.G. Nos. 24 and 32 administered by the Wayne State Univer- sity Medical School. Contribution No. 165 from the Department of Biology, College of Liberal Arts. I am grateful to Jayne C. Bates for technical assistance.

Canadian Journal of Microbiology. Volume 13 (1967)

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CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 13, 1967

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NOTES 339

with the slower moving blue con~ponent. The dye - metal ion combination appears to act by preventing phage adsorption.

I t is of ~ossible interest to note tha t t r v ~ a n blue was once studied as an 2 A

antitumor agent and was shown to display tumor-retarding activity in animals (6). While this author apparently did not use the dye in the presence of added divalent cations, i t is conceivable that the dye - metal ion combination might be more effective in retarding tumor growth.

1. BLAIR, J. E. and WILLIAMS, R. E. 0. 1961. Phage typing of staphylococci. Bull. World Health Organ. 24, 771-784.

2. BROCIC, T. D.. MOSSER, J., and PEACHER, B. 1963. The inhibition bv streptomycin of certain S t ~ e p t o c o c c u ~ bacteriophages, using host bacteria resistant to the andbiotic. - - . J. Gen. ~ilicrobiol. 33, 9-22.

- 3. EDLINGER, E. 1949. Antibiotiques et lyse bacteriophagique. I. Protection des colonies

develoooees a la oerioherie de la zone d'action de la streotomvcine contre I'action du ~adtk~iophage'(~t~phYl~coccus albus wort e t ~ t a p l t ~ l ~ c o c c u ; aureus s ~ K ) . Ann. Inst. Pasteur, 76, 396-400.

4. GRAHAM, D. M. and NELSON, F. E. 1954. Inhibition of lactic streptococcus bacteriophage by crystal violet and other agents. J. Gen. Physiol. 37, 121-138.

5. KELLY, J. W. 1958. Paper chromatography of anionic disazo dyes, especially trypan blue and its red impurity. Stain Technol. 33, 79-88.

6. LUDFORD, R. J. 1929. The vital staining of normal and malignant cells. 11. The staining of malignant tumors with trvoan blue. Rov. Soc. Proc. B. 104. 493-511.

7. REITER, B. a i d ORAM, J. D. 1962.*khibition of a streptococcal bacteriophage by suramin. Nature, 193, 651-652.

RECEIVED JULY 29, 1966. DEPARTMENT OF BIOLOGY, WAYNE STATE UNIVERSITY, DETROIT, MICHIGAN, U.S.A.

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