Absorption and Transport of Fluorescent Brighteners byMicroorganisms

MARJORIE A. DARKEN

Biochemical Research Section, Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York

Received for publication February 13, 1962

DARKEN, MIARJORIE A. (American Cyanamid Co.,Pearl River, N.Y.). Absorption and transport of fluores-cent brighteners by microorganisms. Appl. Microbiol.10:387-393. 1962.-The absorption of brighteners byliving cells and their transport to subsequent growth isdescribed. Brighteners are highly fluorescent, ultraviolet-absorbing compounds which appear to be essentially non-toxic, stable biological markers. They have been effec-tively absorbed by growing cultures of bacteria, yeasts,actinomycetes, and higher fungi, with active growthcenters evidencing the greatest flourescence.

The effects of fluorescent compounds on living micro-organisms have been investigated for over half a centuryby many workers. Some of the early findings, however,have been neglected for decades. The photodynamic actionof the fluorescent acridine dyes, observed with living para-mecia by Raab (1900) and critically evaluated by Blum(1941), has only recently received renewed attention(Freifelder and Uretz, 1960; Hill and King, 1959; Hill,Bensch, and King, 1959, 1960; Wolf and Aronson, 1959,1961). The use of fluors for the expressed purpose of visual-izing cells was initiated by Provazek (1914), but thistechnique remained essentially dormant for a quarter of acentury. A review of research pertaining to inducedfluorescence in microorganisms is available (Darken,1961b). Although the toxicity and mutagenic action offluorescent dyes have been studied, induced fluorescencein actively growing cultures of microorganisms was notinvestigated specifically for cytochemical or cytologicalpurposes until Freifelder and Uretz (1960) reported thatyeasts and bacteria could multiply indefinitely in concen-trations of acridine orange sufficient to permit micro-fluorescent observations of dividing cells. The uptake offluorescent brighteners by active cultures of bacteria,yeasts, actinomycetes, and higher fungi was later reported(Darken, 1961a), as well as the actual transfer of theobserved fluorescence to subsequent growth. The possibleapplication to developmental and genetic studies was

suggested. It is the purpose of this communication toreport details of these preliminary observations, as wellas additional findings, and to present photographic datato illustrate the technique.

MATERIALS AND METHODS

Cultures. The species and collection numbers of theorganisms used in this study follow: Bacillus subtilisATCC 6633, Escherichia coli QM B1457, Mucor murorumQM 776, Neurospora crassa ATCC 9683, Penicilliumchrysogenum Wisc. Q176, Saccharomyces cerevisiae ATCC7753, Streptomyces aureofaciens ATCC 10762, S. griseusATCC 10137, and S. lavendulae ATCC 8664.

Media. The media employed have been described pre-viously. For the higher fungi, a corn steep liquor-glucosemedium (Moyer and Coghill, 1946) and a syntheticmedium (Foster et al., 1945) gave good definition. Withthe actinomycetes, a sucrose-corn steep liquor medium(Niedercorn, 1952) and a synthetic medium (Darkenet al., 1960) were used. To obtain a relatively fine type ofsubmerged.mycelial growth for microscopic study, 1 g ofpartial calcium salt per liter of a modified vinyl acetate-maleic acid copolymer (Monsanto Chemical Co.) wasadded. Trypticase Soy Peptone medium was satisfactoryfor observations on yeast and bacterial cells. For surfacegrowth, yeast-malt agar was used with all but the higherfungi, for which potato dextrose agar was preferable.

Brighteners. The brightener most satisfactory for thesestudies was the disodium salt of 4,4'-bis[4-ani1ino-6-bis-(2-hydroxyethyl)amino-s-triazin-2-ylamino]-2-2'-stilbene-disulfonic acid (I), the structural formula of which isgiven in Fig. 1. Three other brighteners were investigated.One is also a member of the DAS-triazine class (II),another a member of the coumarin class (III), and thelast a member of the benzidine sulfone class (IV). Allexhibit substantivity for cellulosic fibers.

Growth. Cultures were grown in the dark at 28 C withthe exception of bacteria, which were incubated at 37 C.For submerged growth, 250-ml Erlenmeyer flasks con-taining 50 ml of medium, or 50-ml Erlenmeyer flasks with10 ml of medium, or test tubes (32 by 175 mm) with10 ml of medium were placed (test tubes slanted) on areciprocating shaker with a 2-in. stroke at 100 cycles permin. For experiments utilizing liquid inoculum, 0.1 % byvolume was transferred for yeasts and bacteria, and 1 %was used for mycelial transfers. Spore suspensions ofP. chrysogenum were prepared by rubbing a mature slantwith a transfer loop in the presence of 10 ml of sterile

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distilled water and filtering by gravity through wet sterilefilter-press cloth for the removal of mycelial fragments.

In addition to test-tube and petri-dish surface cultures,tilted agar plates (Szybalski, 1952, modification of Porter,1924) were prepared, to vary the concentration of bright-ener available for range-finding experiments on the uptakeof brightener by the organisms; pH-gradient agar plates(Sacks, 1956) were also used to study the effect of pH uponbrightener accumulation. Hanging-drop and agar cover-slip preparations were made for growth and reproductionstudies with a heated microscope stage.

Absorption. Brighteners were added to submergedshaken fermentations before or after autoclaving in bothnatural and synthetic media, either before inoculation orafter a period of incubation. Their incorporation in theagar of petri dishes and test tubes was made at the time ofbatching, and they were added to distilled water for ac-cumulation by spores. Brighteners were used at concentra-tions recommended for their absorption by fibers as basedon dry weight. The amount generally indicated is 0.1 %(Villaume, 1958); this percentage of the dry cell weightapproximates 2.5 mg of brightener per 50 ml of liquidculture. To ascertain the uptake of these compounds bycells under refrigerated conditions, cultures were chilledto 4 C, brighteners added, and cultures either left sta-tionary or placed in the shaking head of a refrigeratedcentrifuge operating at 250 rev/min.

Cellular wash. Fluorescent cell suspensions were cen-trifuged at 2,000 rev/min for 2 min, decanted, broughtto original volume with sterile 0.85% NaCl, and resus-pended with a vibrating-type mixer. Three washes inmost instances were sufficient to remove excess brightener,added in accordance with recommendations, as evidencedby lack of fluorescence of the final rinse solutions and non-fluorescence of the organisms transferred to these solutionsas control checks.

Observation. Observations were made in a dark roomwith a magnesium fluoride-coated microscope, with an85-w mercury arc lamp and a no. 5840 Corning exciterfilter of 0.40 mm thickness; a set of Wratten barrier filterswas fitted in the oculars, and an aluminized mirror wasplaced over the substage mirror of the microscope. Car-

gille's immersion oil (Type A) of very low fluorescencewas used. Achromatic objectives with a bright-field con-denser were employed throughout the greater part of thesestudies. The dark-field condenser permits less backgroundluminosity and somewhat sharper contrast, but it is notrecommended for investigations on transport of fluores-cent substances owing to visualization of cells which arenot visible otherwise because of lack of fluorescence.

Photography. Photomicrgraphs were made with anExacta reflex camera and enlarged 2 X. Daylight Super-anscochrome color film was used, with exposures rangingfrom 2 to 20 sec. The binocular head of the microscopewas replaced with a monocular tube, and a 10 X hyper-plane eye-piece was inserted. Shaken cells were mountedin the medium in which they were grown; agar cultureswere suspended in water.

RESULTSThree types of experiments were performed. The first

was designed to study the growth of microorganisms onagar containing fluorescent brighteners. In the second, asubmerged shaken growth was investigated with thesefluors. In the third type of experiment, washed submergedbrightener-labeled cells were transferred to agar and liquidmedia without brightener.

Growth on agar. These experiments were conductedwith a 12% solution of brightener I in 42 % aqueousCellosolve at pH 12. P. chrysogenum and S. aureofacienswere grown on agar slants and petri dishes as describedabove. In a study of the effect of pH upon the absorptionof brightener, no difference was apparent in observedfluorescence between pH 5.6 and 7.8. Growth of P. chryso-genum proceeded on agar without any detectable differencefrom controls when concentrations of brightener I as highas 0.05 ml per 10 ml were used. However, when 0.10 mlwas added, mycelial development was slightly retarded;growth was severely inhibited at a concentration of0.50 ml per 10 ml of medium. S. aureofaciens exhibitedgreater sensitivity towards this brightener. Growth wasnoticeably retarded at a concentration of 0.025 ml andseverely inhibited at 0.10 ml per 10 ml; when 0.010 mlwas added, mycelial development equalled that of the

HN S03Na S03Na

N\ -NH CH = CH N

( HOCH2CH?.)2N

NH

H7N

NHN(CH2CH20H 2

FIG. 1. Structural formula of brightener I.

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control slants. A concentration of 0.01 ml per 10 ml wasselected for observations on both these organisms.

Actively growing mycelium, especially the hyphal tips,evidenced the greatest fluorescence of brightener (Fig. 2).A brilliant blue fluorescent differentiation was observedwithin the cells, as well as in the side walls and cross wallsof the mycelium (Fig. 3). Highly fluorescent spores werealso seen in mature agar cultures (Fig. 3). The accen-tuated uptake of brightener by the hyphal tips and itsdifferentiation within the cell was somewhat less pro-nounced when cultures were not grown in complete dark-ness.

Submerged growth. Preliminary studies were conductedwith the four brighteners described above. A 12 % solutionof brightener I in 42% aqueous Cellosolve at pH12 was usedfor further experimentation. Bacteria (B. subtilis andE. coli), yeasts (S. cerevisiae), actinomycetes (S. aureo-faciens, S. griseus, and S. lavendulae), and higher fungi(including M. murorum, N. crassa, and P. chrysogenum)were all found to fluoresce both when grown in the pres-ence of this brightener and when the brightener was addedas a direct stain. Although absorption of the fluor by thecell wall was essentially instantaneous, distribution withinthe cell required a longer period of time. In the filamentousfungi, side walls and cross walls, when present, glowedinstantly and there was marked emphasis of fluorescenceat the hyphal tips (Fig. 4). When synthetic media wereused, less background fluorescence was noted. In yeasts,the cell walls fluoresced instantly, with greater fluorescenceof brightener in the prebudding region (Fig. 5). Approxi-mately 2 hr were required for the differentiation by thefluor within the cell. Actively growing vegetative yeastcells (Fig. 5) appeared to fluoresce more differentiallythan older (Fig. 6) or dead (Fig. 7) cells, which exhibitedan opaque glow. In bacteria, fluorescence could be seeninstantaneously. Cultures of all organisms exposed tobrightener in the light evidenced a somewhat less efficientuptake, and differential fluorescence at the active growthcenters was slightly less pronounced.

Toxicity tests for brightener I were conducted in sub-merged growth. The results (Table 1) show a specifictolerance by individual organisms. The tolerance shownby P. chrysogenum and S. aureofaciens was slightly in-creased when 48-hr liquid mycelial inoculum was used, incontrast to spores. With both of these organisms, a con-centration of 0.10 ml brightener I per 50 ml was essen-tially nontoxic with mycelial transfers; a marked delayin growth occurred with 0.50 ml per 50 ml. When sporeswere employed, the delay in mycelial growth was apparentat a concentration of 0.10 ml per 50 ml. If the brightenerwas not incorporated into the medium initially but wasadded 24 or 48 hr after inoculation, a greater tolerancewas found. For general observations, a concentration of0.025 ml brightener I per 50 ml was selected for initialaddition to the higher fungi and actinomycetes; this

amount was effectively absorbed and did not leave avisible residue of brightener in the medium.Although B. subtilis and E. coli showed an initial toler-

ance of 0.20 ml brightener I per 10 ml, a concentration of0.0025 ml was found to be effectively absorbed withouta visible excess. Yeast cells tolerated markedly less bright-ener. A concentration of 0.0025 ml per 10 ml was toxic, andupon transfer of these cells at 24 hr to fresh medium with-out brightener no growth occurred. At a level of 0.00025 mlper 10 ml, growth was delayed and at 48 hr was equivalentto the control at 24 hr; however, at this low concentrationof brightener some of the cells did not fluoresce. With an18-hr addition of the fluor, greater tolerance was found(due probably to the increased cellular weight). Whenbrightener was added at 18 hr and transfer was made at24 hr to fresh medium without brightener, a concentrationof 0.025 ml per 10 ml permitted subsequent growth. Atthis level, however, considerable excess fluor was presentin the medium. A concentration of 0.0025 ml brightener Iadded at 18 hr or 0.00025 ml initially was adopted formicroscopy of growing yeast cells.The effect of pH upon the uptake of brightener was

studied with P. chrysogenum and S. aureofaciens. Noapparent difference was noted when flasks of mediumcontaining 0.025 ml brightener I per 50 ml were adjustedwith H2SO4 or NaOH, before inoculation, to pH 5.0, 5.56.0, 6.5, 7.0, 7.5, and 8.0. The adjustment of 3-day-oldmycelium to these pH levels before the addition of bright-ener was also without effect during observation periods of10 min, 1 hr, 2 hr, and 24 hr. It is of interest that totalantibiotic production by P. chrysogenum, S. aureofaciens,and S. griseus was not delayed or reduced when 0.025 mlof brightener I was added to 50 ml of medium beforeinoculation. Concentrations as high as 0.50 ml delayed,but did not reduce, the total antibiotic yields. When sub-merged cultures were chilled before the addition of bright-ener and then kept under refrigeration, both shaken andunshaken cells evidenced uptake of the fluorescent com-pound. It should be noted that the normally active growthcenters were no longer sharply delineated by their intenseglow, and cellular differentiation was somewhat obscuredunder these conditions.

Transport of brightener. A distinction is needed betweenthe simple diffusion of brighteners through establishedmycelium independently of hyphal growth, as in the caseof typical movement of dyes (Schutte, 1956), and theactive transport by uptake of material at one site and itstransference to another site in the mycelium due to hyphalextension, as is apparently the case in radioactive tracerexperiments with cobalt-60 and caesium-137 (Grossbardand Stranks, 1959) and with phosphorus-32 (Lucas,1960). It appears that the transport of brighteners isactually via the extending mycelium itself, and the pres-ent paper offers evidence for this interpretation.When divided plates were prepared with brightener I-

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FIG. 2. Penicillium chrysogenum, 1-month-old agar culture with 0.01 ml of-brightener I. Note accentuated hyphal tips. 10 X 43 a sec.FIG. 3. Penicillium chrysogenum, 1-month-old agar culture with 0.025 ml of brightener I. Note cellular differentiation with conidiophore

and spore formation. 10 X 97 8 sec.FIG. 4. Penicillium chrysogenum, 8-day-old submerged growth with 0.025 ml of brightener I. Note accentuated hyphal tips. 10 X 43 9 sec.FIG. 5. Saccharomyces cerevisiae, 1-day-old submerged growth with 0.00026 ml of brightener I. Note cellular differentiation. 10 X 43 8 sec.FIG. 6. Saccharomyces cerevisiae, 3-day-old submerged growth with 0.00025 ml of brightener I. Note lack of differentiation and opaque

glow of older cells. 10 X 97 12 sec.FIG. 7. Saccharomyces cerevisiae, 3-day-old submerged growth; 0.0025 ml of brightener I added after autoclaving. Note universal lack of

differentiation and general opaque glow. 10 X 97 20 sec.

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labeled (0.025 ml per 10 ml) and unlabeled agar sectionsand the brightener-containing area alone was inoculatedwith M. murorum, P. chrysogenum, or S. aureofaciens,the mycelial growth which extended over the glass divisiononto the section with no brightener was fluorescent. Whenwashed mycelium of P. chrysogenum or S. aureofacienswhich had been grown for 3 days in shaken culture in thepresence of 0.025 ml of brightener I per 50 ml was trans-ferred to agar cover slips or to hanging-drop slides with no

brightener, fluorescence was evident in the new growth(Fig. 8). When a suspension of Penicillium spores was

shaken for 2 hr in 5 ml of sterile distilled water containing0.01, 0.02, or 0.05 ml of brightener I, washed, and trans-ferred to agar or liquid medium with no brightener,fluorescent germ tubes formed and were observed todevelop into fluorescent young mycelial strands. Thesewere also seen when spores produced on agar containing0.05 ml of brightener per 10 ml were washed and trans-ferred to agar or liquid medium with no brightener(Fig. 9). When washed mycelium of P. chrysogenum,grown in the presence of brighteners as previously de-scribed, was used as inoculum for liquid medium contain-ing no brightener, the new growth showed a blue fluores-cence. Indications are that leaching of the fluorescentcompound into the medium did not occur to an extentsufficient to account for this fluorescence, since bacterialor yeast cells which had previously been observed tofluoresce with the brightener did not do so when introducedinto the liquid medium simultaneously with the ungerm-

inated spores or mycelium.Fluorescent yeast cells, which had shaken for 18 hr in

the presence of 0.00025 ml of brightener I per 10 ml ofmedium, were washed and transferred to medium withoutbrightener in test tubes and in hanging-drop slides. Bluefluorescence was observed in the buds and in the newlyformed cells (Fig. 10); observations on the development ofascospores were greatly facilitated (Fig. 11). Cells ofE. coli did not become fluorescent when added simul-taneously to this medium at the time of transfer of thewashed yeast cells.

DISCUSSIONIn a search for fluorescent compounds which would act

not only as vital stains but also as markers for genetic anddevelopmental studies, certain requirements must be met.Such compounds should be essentially nontoxic at theconcentration employed, efficiently absorbed by the cell,and sufficiently stable for detection in subsequent growth.Attempts to use such well-known vital fluors as acridineorange, fluorescein, rhodamine, and Thioflavin were un-

successful in preliminary trials (Darken, 1961a). Muchwork has been done in the field of direct vital staining withacridine orange. This compound, however, has been foundto be loosely bound to cytoplasmic nucleic acid in vitallystained monkey-kidney cells (Mayor, 1961), to be some-

what toxic to fibroblast cells at levels sufficiently high forcellular fluorescence (Wolf and Aronson, 1961), to have a

reversible absorption spectrum which became apparentas the dye was eluted from Ehrlich ascites tumor cells(Loeser, West, and Schoenberg, 1960), and to have a

highly reversible degree of binding within fibroblast cellswhich was to a large extent determined by the concentra-tion in the medium (Hill et al., 1960). These latter workersalso noted that it was necessary to make observationsand photographs immediately, since exposure to lightcaused rapid bleaching and loss of crisp clear outlines.

Fluorescent antibiotics and steroids were also unsatis-factory in preliminary trials. Other workers have foundthe fluorescent antibiotic tetracycline, for example, to beloosely bound. Milch, Rall, and Tobie (1957) reportedthat, although bone fluorescence persisted for at least 10weeks when a single small parenteral dose of tetracyclinewas administered to freshly frozen sections of severalspecies of laboratory animals, this induced fluorescencedisappeared from all other tissues within 6 hr after injec-tion. Working with monkey kidney tissue culture cells,DuBuy and Showacre (1961) noted that tetracycline-treated material, when resuspended in tetracycline-freemedium, evidenced a gradual decrease in fluorescenceduring a period of several hours.

In the present study, some of the commercially available

TABLE 1. Effect of brightener I on growth in submerged culture

Relative growth after incubation with brightener* in the darkOrganisms Inoculum Medlum

0.00025 0.0025 0.025 0.10 0.25 0.50

Penicillium chrysogenum ... Liquid 50 +++t +++ +++ +++ ++ +P. chrysogenum ............ Spore 50 +++ ++ ++ + + 0Streptomyces aureofaciens. . Liquid 50 +++ +++ +++ +++ ++ +S. aureofaciens ............. Spore 50 +++ +++ ++ + + 0Bacillus subtilis ............ Liquid 10 +++ +++ +++ +++ +++ +++Escherichia coli ............ Liquid 10 +++ +++ +++ +++ +++ +++Saccharomyces cerevisiae Liquid 10 + 0 0 0 0 0

* Concentrations expressed as ml per volume noted.t Growth equal to control without brightener at 1 day for bacteria and yeast, and at 3 days for other organisms.

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FIG. 8. Penicillium chrysogenum, 1-day-old hanging-drop slidepreparedfrom washed 3-day mycelium grown in the presence of 0.026ml of brightener I. Note transport of fluorescence to new mycelialgrowth. 10 X 10 9 sec.

FIG. 9. Penicillium chrysogenum, 1-day-old hanging-drop slideprepared from washed and filtered ungerminated spore suspensionrubbed from agar culture grown in the presence of 0.05 ml of brightenerL. Note transport offluorescence to germ tubes. 10 X 43 4 sec.

FIG. 10. Saccharomyces cerevisiae, 1-day-old hanging-drop slideprepared from washed 1-day cells grown in the presence of 0.00025ml of brightener I. Note cellular differentiation with transport offluorescence to new cell. 10 X 97 8 sec.

FIG. 11. Saccharomyces cerevisiae, 3-day-old culture preparedfromwashed 1-day submerged growth with 0.00025 ml of brightener I. Noteascospore formation. 10 X 97 12 sec.

optical bleaching agents or brighteners were found to acteffectively not only as vital stains but also as stable bio-logical markers that were bound by the cell and weretransferred by it to subsequent growth. These brightenersappear to be highly fluorescent, able to pass through cellwalls, substantive to proteins, fluorescent at pH 5.0 to8.5, and stable as regards fluorescence when bound. Thequestion of possible mutagenic action of these fluors andother ultraviolet-absorbing compounds, both alone andin conjunction with known mutagens, including ultra-violet light, is currently under investigation in thislaborator*y.

Brighteners are a relatively new class of chemical com-pounds; their biological applications were first noted byDarken (1961a). Earlier, Paine, Radley, and Rendell(1937) had described the ultraviolet fluorescence of textilefibers when treated with N,N'-diacyl derivatives of 4,4'-diaminostilbene-2,2'-disulfonic acid. Eggert and Wendt(1939) disclosed the use of N,N'-bis-triazinyl derivativesof diaminostilbene as protective agents against deteriora-tion of paper by the absorption of ultraviolet light. Al-though the affinity of some of these compounds for cellu-losic material, with a subsequent whitening effect, wasthus definitely established, it was not until 1948 thatextensive research programs culminated in their com-mercial availability.

Brighteners do not absorb any significant amount ofvisible light. They convert absorbed ultraviolet light tolonger wavelengths, which are emitted as visible light.In this process, free electrons are excited to a higherenergy level, and, upon return to the original energystate, part of the absorbed light is in the visible blueregion of the spectrum. Thus, blue light is added to thereflectance of brightener-treated substances.The binding of brighteners is a function of two factors,

the concentration-fluorescence relationship and the per-centage of exhaust (Villaume, 1958). When the concentra-tion of a triazinyl stilbene brightener is plotted vs. itsfluorescent intensity, a sigmoid-type curve is obtained,and the fluorescence efficiency decreases as the concentra-tion increases beyond a critical level. It becomes apparentthat at a concentration of 0.002 % a specific brightenerproduces more than twice the fluorescence obtained withone-half of this concentration; however, a concentrationof 0.10% gives only about 8% more intensity than one-half of this amount. This concentration-fluorescence rela-tionship varies with different brighteners. The degree ofexhaustion (the ratio of the affixed brightener to thebrightener present in the solution) also varies with thetype of brightener, and ranges between 40 and 95%. Fac-tors which influence the exhaust include time, tempera-ture, light intensity, concentration, and pH. Many differ-ences exist among brighteners in regard to substantivity,degree of exhaustion, and fastness properties.Of the four brighteners investigated, brightener I has

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proven to be the most satisfactory. This brightener hasexcellent exhaustion, solubility, and binding properties;its resistance to heat and light while in solution is alsoexcellent. Its effective pH range is 5 to 11. That the bind-ing of this fluor to microorganisms is stable is shown bythe observation that washed, refrigerated, brightener-labeled cells suspended in 0.85 % NaCl continued tofluoresce strongly for 18 months without detectableleaching.The applications of brighteners and other ultraviolet-

absorbing compounds would appear to merit furtherstudy, and work is continuing in this laboratory. It ishoped that other workers will find these compounds usefulas tools for investigation of the many problems whichmight be successfully probed with highly fluorescent,stable markers.

ACKNOWLEDGMENTS

The author wishes to thank E. J. Backus, M. K. Nadel,and N. E. Rigler for their interest and encouragementthroughout these investigations, K. Hsu of ColumbiaUniversity for his helpful guidance in photographic detail,and W. Allen and J. Leavitt for supplying the brightenersfrom the Organic Chemicals Division, American CyanamidCo., Bound Brook, N.J.

LITERATURE CITED

BLUM, H. F. 1941. Photodynamic action and diseases caused bylight. Reinhold Publishing Corp., New York.

DARKEN, M. A. 1961a. Applications of fluorescent brighteners inbiological techniques. Science 133:1704-1705.

DARKEN, M. A. 1961b. Natural and induced fluorescence in micro-scopic organisms. Appl. Microbiol. 9:354-360.

DARKEN, M. A., H. BERENSON, R. J. SHIRK, AND N. 0. SJOLANDER.1960. Production of tetracycline by Streptomyces aureofaciensin synthetic media. Appl. Microbiol. 8:46-51.

DuBuy, H. G., AND J. L. SHOWACRE. 1961. Selective localization oftetracycline in mitochondria of living cells. Science 133:196-197.

EGGERT, J., AND B. WENDT. 1939. Wrapping materials. U. S. Patent2,171,427.

FOSTER, J. W., L. E. McDANIEL, H. B. WOODRUFF, AND J. L.STOKES. 1945. Microbiological aspects of penicillin. V. Coni-diophore formation in submerged cultures of Penicilliumnotatum. J. Bacteriol. 50:365-368.

FREIFELDER, D., AND R. B. URETZ. 1960. Inactivation by visiblelight of yeast sensitized by a nucleic acid fluorochrome. Na-ture 186:731-732.

GROSSBARD, E., AND D. R. STRANKS. 1959. Translocation of cobalt-60 and caesium-137 by fungi in agar and soil cultures. Nature184:310-314.

HILL, R. B., JR., K. G. BENSCH, AND D. W. KING. 1959. Unimpairedmitosis in cells with modified deoxyribonucleic acid. Nature184:1429-1430.

HILL, R. B., JR., K. G. BENSCH, AND D. W. KING. 1960. Photo-sensitization of nucleic acids and proteins. The photodynamicaction of acridine orange on living cells in culture. Exptl.Cell Research 21:106-117.

HILL, R. B., JR., AND D. W. KING. 1959. The effect of acridineorange on L strain fibroblasts. Federation Proc. 18:481.

LOESER, C. N., S. S. WEST, AND M. D. SCHOENBERG. 1960. Absorp-tion and fluorescence studies on biological systems: nucleicacid-dye complexes. Anat. Record 138:163-178.

LUCAS, R. L. 1960. Transport of phosphorus by fungal mycelium.Nature 188:763-764.

MAYOR, H. D. 1961. Cytochemical and fluorescent antibody studieson the growth of poliovirus in tissue culture. Texas Repts.Biol. Med. 19:106-122.

MILCH, R. A., D. P. RALL, AND J. E. TOBIE. 1957. Bone localiza-tion of the tetracyclines. J. Natl. Cancer Inst. 19:87-93.

MOYER, A. J., AND R. D. COGHILL. 1946. Penicillin. VIII. Produc-tion of penicillin in surface cultures. J. Bacteriol. 51:57-78.

NIEDERCORN, J. G. 1952. Process for producing Aureomycin. U.S.Patent 2,609,329.

PAINE, C., J. A. RADLEY, AND L. P. RENDELL. 1937. Fabrics fluores-cent to ultraviolet light. U.S. Patent 2,089,413.

PORTER, C. L. 1924. Concerning the characteristics of certainfungi as exhibited by their growth in the presence of otherfungi. Am. J. Botany 11:168-188.

PROVAZEK, S. VON. 1914. Uber die Fluoreszenz der Zellen. Klein-welt 6:30, 37.

RAAB, 0. 1900. Ueber die Wirkung fluorescirender Stoffe aufInfusorien. Z. Biol. 39:524-546.

SACKS, L. E. 1956. A pH gradient agar plate. Nature 178:269-270.SCHUTTE, K. H. 1956. Translocation in the fungi. New Phytolo-

gist 55:164-182.SZYBALSKI, W. 1952. Gradient plate technique for study of bac-

terial resistance. Science 116:46-48.VILLAUME, F. G. 1958. Optical bleaches in soaps and detergents.

J. Am. Oil Chemists' Soc. 35:558-566.WOLF, M. K., AND S. B. ARONSON. 1959. Growth, fluorescence and

metachromasy of cells cultured in the presence of acridineorange. (Abstr.) Anat. Record 133:441.

WOLF, M. K., AND S. B. ARONSON. 1961. Growth, fluorescence andmetachromasy of cells cultured in the presence of acridineorange. J. Histochem. Cytochem. 9:22-29.

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