THE USE OF INK BLUE IN THE IDENTIFICATION OF * M. BAXTER, M.Sc. It is known that several species of dermato-over an illuminated source, of the mean colony phytes cause an increase of alkalinity in the sur-radius and the extent of discolouration in cm. be- yond the colony margin. From these values the rounding medium, in contrast to many sapro-relative discoloring powers (discoloring activity) phytes which tend to make the medium moreof different species was expressed as the ratio ex- acid (1—4). Although Goldfarb and Herrmanntent of discoloration:colony radius. (4) suggested a technic involving the flooding Several strains of dermatophytes were also grown of culture plates with a universal indicator toon nutrient agar (Oxoid) to which 4% gelatin had been incorporated. After 5 days growth the plates demonstrate the increased alkalinity occurringwere flooded with a mixture of HgCI2 15 gm, con- around colonies of dermatophytes, the useful-centrated HCI 20 ml and distilled water 100 ml, to ness of a pH indicator as an aid to the identifi-precipitate the unhydrolyzed gelatin. Measure- cation of these fungi would be enhanced if itments of colony radius and extent of gelatin hy- drolysis allowed an expression of proteolytic ac- gave satisfactory results when actually incor-tivity as the ratio extent of hydrolysis:colony porated into the agar. A distinct color changeradius. would have to be produced early in the culture The pathogenic fungi used in this study were period and at a pH value sufficiently above theeither isolated from patients residing in the Bir- mingham area or kindly supplied by the Mycologi- initial pH of the medium to avoid any slightcal Reference Laboratory, London School of Hy- alkalinity which might occur around coloniesgiene and Tropical Medicine. Non-keratinophilic of some saprophytes during the same culturespecies were isolated from soil or as contaminants interval. The dye used would also need to befrom skin cultures. completely non-toxic to both fungal growth and spore production. RESULTS Ink blue has been shown to be of value for Clearing of the ink blue agar around the estimating the pH of culture filtrates of certainfungal colony occurred only in cultures of fungi (5), changing from blue to colorless be- tween pH 6.5 and 7.2. This report records theTrichophyton, , , observations made on the suitability of an agarKeratinomyces and Utenomyces species. Dis- coloration was distinctly visible as a halo around medium incorporating ink blue, illustrates thethe colony and could be seen almost as soon as value of this medium for the early identificationthe began to grow onto the agar from of keratinophilic fungi as a class and investi-the point of inoculation. All cultures except T. gates the possibility of a relationship betweenschoenleini and T. concentricum could be re- the alkalinity produced by dermatophytes andcorded as positive within 3 days. No discolora- their proteolytic activity. tion was recorded with the following fungi: MATERIALS AND METHODS Allescheria boydii fumigatus, A. niger, A. terreus Ink Bluet was incorporated, at a concentration , C. krusei, C. tropicalis of 0.05%, into a medium containing glucose 4%, Cladosporium herbarum Mycological Peptone (Oxoid) 1% and Agar 2% in distilled water. A variety of keratinophilic and sp. non-keratinophilic fungi were grown on this ink Mucor fragilis blue agar in petri-dish culture, using 15 ml of me- Phoma glomerata dium to each petri dish. The presence or absence Pullularia pullulans of discoloration in the agar was recorded after 7 Rhizoctonia solani days' incubation at 25° C, and measurements made, Rhodotorula sp. Received for publication January 28, 1964. *Fromthe Medical Research Council, Unit for Scopulariopsis brevicaulis Research on the Experimental Pathology of the Sepedonium sp. Skin, The Medical School, University of Birming- Trichoderma viride ham, Birmingham 15, England. t As Ink Blue AS Crystals; Imperial Chemical Penicillium frequentens Industries Ltd. Verticillium sp. 23 24 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE I coloration produced. Microsporum as a genus Discolorationof ink blue agar by l4 species ofexhibited low activity while Epidermophyton keratinophilic fungi after 7 days growth and Keratinomyces had high activity. The ink blue was completely non-toxic to the fungi. No Extent ofDiscolor- Discolora-ing Activ- inhibition of growth occurred and the spore tion (cmity (extent from of disco!- forms normally produced on glucose-peptone (cm) oration: colony colony agar were present. margin) radius) The color change of ink blue in liquid medium (blue—colorless) occurs between pH 6.5—7.2 (5). Measurements of the pH of the agar with T.concentricum. 0.25 0.25 1.0 a direct reading pH meter (Electronic Instru- T.equinum 1.5 0.5 0.33 T. gallinae 1.38 0.88 0.64 ments Ltd., Model 23A) using flat-headed glass T. megnini 1.25 1.0 0.8 electrode GFH33 in conjunction with reference T. mentagrophytes electrode RJ23 indicated that discoloration oc- 1.25 1.0 0.8 curred only at pH 6.9 and above. Only the 2 1.5 1.5 1.0 keratinophilic cultures had attained this value. 3 1.88 0.88 0.47 Trichoderma viride, having overgrown the T. persicolor 1.38 0.63 0.46 petri-dish, had raised the pH to 6.8 but no T. quinckeanum 1.44 0.38 0.26 discoloration was produced so that the clearing T. rubrum of ink blue must occur between pH 6.8 and 6.9. 0.94 0.63 0.67 Further culture of T. viride resulted in some 2 0.88 0.63 0.71 discoloration being recorded after 14 days but T. sabouraudi 0.5 0.05 0.1 T. schoenleini 0.14 0.13 0.93 by this time the fungal mat was so well devel- T. soudanense 0.5 0.5 1.0 oped over the entire agar surface that clearing T. suiphureum 0.88 0.63 0.71 was obscure. A culture of T. mentagrophytes T. terrestre 0.5 1.0 2.0 showed discoloration to a distance of 2.5 cm T. tonsurans 0.5 0.25 0.5 from the colony margin. The pH at 1 cm dis- T. verrucosum 0.56 0. 69 1.23 tance was 7.9, at 2 cm 7.0 and at 3 cm 5.9. At T. violaceum 0.385 0.38 1.0 3 cm the agar remained blue. The discolored Micros porum agar could be reconverted to blue by the addi- M. audouini 1.25 0.25 0.2 tion of acid. M. canis 2.0 0.25 0.13 When growing on the ink-blue agar, many M. cookei 2.25 1.0 0.45 fungi became stained on the undersurface of the M. distortum 2.25 0.5 0.22 M. gypseum 1.19 0.25 0.21 , but this in no way interfered with the Epidermophyton floe- cosum 1.25 1.0 0.8 TABLEII 2.25 1.0 0.45 Gtenomyces serratus Proteolyticactivity compared to discoloring Keratinoinyces ajelloi.. 1.0 1.0 1.0 activity of some dermatophytes

ProteolyticDiscoloring Activity Activity In subsequent tests a large number of unidenti- (Extent of (extent of gelatin ink blue fied non-keratinophilic fungi also proved nega- hydrolysis: discolora- colony tion: colony tive. radius) radius) The measurements shown in Table I of the discoloration produced after 7 days' culture Trichophyton schoenleini.. 3.0 0.93 indicated no direct relationship between the T. rubrum 2.3 0.67 colony size and the extent of clearing. Tricho- T. terrestre 2.0 2.0 phyton as a genus showed varying activity at T. mentagrophytes...... 0.85 1.0 species level. Similar colony types, e.g. T. mentagrophytes and T. quinckeanum showed 1.5 0.13 1.5 no similarity of activity and strains within one M. gypseum 0.21 Epidermophyton floccosum. 1.25 0.8 species were as variable as different species. T. Keratinomycesajelloi...... 0.13 1.0 terrestre was outstanding in the amount of dis- IDENTIFICATION OF DERMATOPHYTES 25 clearing of the agar. Some species, e.g. Sco-phenomenon complicates the use of ink blue in pulariopsis brevicaulis and Candida albicansinitial isolation media but if pigment produc- absorhed the dye throughout the colony andtion is not important, any fungus growing from assumed a blue color but no discoloration waskeratin samples can immediately be identified as produced in the agar. This staining phenomenonkeratinophilic and so potentially pathogenic. obscured pigment production in all but the mostThis fact is especially useful in cases where powerful pigment producers e.g. M. coolcei. heavy contamination is expected, such as the Table II shows there to be no correlation be-isolation of dermatophytes from shower stalls. tween the proteolytic activities of several der-The keratinophilic species can be picked out matophytes and the extent of clearing producedimmediately and a pure culture obtained. The in ink blue agar. T. terrestre and M. gypseum,isolation of a fungus strain which is temporarily both with high proteolytic activity had high andor permanently unclassifiable as a low discoloring activities respectively, while K.on a morphological basis is a not uncommon ajelloi with a low proteolytic activity had a highoccurrence and in these cases ink blue agar rep- discoloring activity. resents a useful preliminary isolation medium before subculturing onto more selective media. DISCUSSION The experiments with ink blue agar con- SUMMARY firmed for a wide range of species the property 1. The inclusion of ink blue into an agar me- of dermatophytes to produce alkaline condi- tions in the medium. This property was adium allowed the release of alkali by cultures characteristic not only of dermatophytes butof 24 species of keratinophilic fungi to be meas- also of other species classified amongst theured quantitatively after 7 days growth. 2. Cultures of non-keratinophilic fungi failed Gyrnnoascaceae and usually regarded as non- to discolor this pH indicator. pathogenic keratinophils. The early release of 3. Alkali production was not correlated with alkali has been suggested to be of importance in the attack of keratin by these fungi (6). Thethe proteolytic activity of the fungus. discoloration produced in ink blue agar is a 4. It is suggested that use may be made of measure not only of pH changes but also of thethe discoloration produced by keratinophilic fungi for the rapid identification of morphologi- quantity of alkali produced, assuming that thecally uncertain strains as keratinophilic and so thickness of the agar is nearly constant. The comparison of discoloration produced and thepotentially pathogenic. proteolytic activity of the fungus was made to REFERENCES examine the possibility of a relation between 1. MALLINCKRODT-HAUPT, A. voN: Der wert der this release of alkali and the enzymic activity pH messung bel piltzkulturen. Abt. Bkt., but the results indicated no correlation between 125:368, 1932. the two. 2.STOCKDALE, P.M.: Nutritional requirements ofthe dermatophytes. Biol. Rev., 28:84, The halo of discolored agar present around 1953. the keratinophil cultures was a reflection of a3.NICKERSON,W. J.:Biology of pathogenic fungi. Waltham, Mass. Chronica Botanica rise in pH to 6.9 and above. None of the non- Co. 1947. keratinophilic species produced this discolora-4. GOLDFARB, N. J. AND HERRMANN, F.: A study tion after 7 days but both types of fungi ex- of pH changes by in culture media. J. Invest. Derm., 27: 193, 1956. hibited staining of the undersurface of the5. BAXTER, M. AND COOPER, J. R.: Ink blue as a mycelium. This indicates that the hydrogen ion pH indicator for certain biological systems. concentration within the individual hyphae of To be published. 6. CHESTERS, C. G. C. AND MATTITSON, G. E.: The keratinophils is not sufficiently high to main- decomposition of wool keratin by Keratino- tain discoloration of the dye. The staining myces ajelloi. Sabouraudia, 2: 225, 1963.