John W. Williams

Scalp Products and Hair as a Culture Medium for Certain Pathogenic Fungi

In the present work observations were made of the growth of certain pathogenic fungi on hair. Bonar and Dreyer 1 used bundles of hair inoculated with trichophyton interdigitale to test antiseptic properties of solutions. Tona 2 observed the growth of trichophyton on hair in the test tube. The growth of the various pathogenic fungi (dermatophytes) on a medium of scalp products and hair seems to have received little attention. The medium used consisted of barber shop hair cleansed with water, placed in petri dishes and test tubes and autoclaved. It was found necessary to keep the medium moist with distilled water by adding an amount sufficient to cover the mat of hair. Growth on this medium was compared with that on conservation Sabourauds. Sabourauds conservation medium was chosen as a standard because of its simplicity. Bacto products were used in making the media. Transplants were made from 30-day-old growths of the organisms on Sabourauds proof agar incubated at room temperature. Approximate time of growth of the following pathogenic fungi and the 2 saprophytes, Lichtheimia sp. and Scopulariopsis brevicaulis were noted: Achorion schoenleinii, Acladium castellcmi, Candida Candida, Endodermophyton tropicale, Endomyces capsultus, Endomyces dermatitidus, Epidermophyton inguinale, Glenospora gammeli, Geotrichum baehmann, Indieila americana, Monilia albicans, Microsporon apiospermum, Microsporon audouini, Oöspora humi, Trichophyton crateriforme, Trichophyton granulosus, Trichophyton japonicum, Trichophyton interdigitate, Willia anomala. Observations were limited to 30 days. Nine organisms which grew well on Sabourauds medium showed no growth on water hair; Achorion schoenleini, Acladium castellani, Candida Candida, Endomyces dermatitidus, Epidermophyton inguinale, Glenospora gammeli, Microsporon audouini, Oöspora humi, and Willia anomala. Since the pathogenicity of the Microsporon audouini manifests itself before, puberty its growth was observed on a medium of childs hair.

Scalp Products and Hair of Men and Women as Culture Media for Certain Pathogenic Fungi.

A study was made of the growth of pathogenic fungi on hair and scalp products before puberty, as collected and after extraction with ether in a Soxhlet for 24 hours. 1 A similar study of hair and scalp products of men and women is here reported. Care was taken that this hair was clean, untreated and undyed. The hair of numerous individuals of different ages was mixed. About 6 cc. of hair was placed in test tubes, covered with distilled water, the tubes plugged with cotton and autoclaved. The following pathogenic fungi and 2 non-pathogenic saprophytes Lichthemia sp. and Scopulariopsis brevicaulis were studied: Achorion schoenleinii, Acladium castellani, Candida candida, Endodermophyton tropicale, Endomyces capsulatus, Endomyces dermatitidus, Epidermophyton cruris, Epidermophyton inguinale, Glenospora gammeli, Geotrichum bachmann, Indiella americana, Microsporon audouini, Microsporon felincum, Microsporon gypseum, Monosporum apiospermum, Monilia albicans, Oöspora humi, Sporotrichum schenkii, Trichophyton crateriforme, Trichophyton granulosum, Trichophyton gypseum asteroides, Trichophyton gypseum lacticolor, Trichophyton interdigitale, Trichophyton japonicum, Trichophyton niveum, Trichophyton sulfureum, Willia anomaia. In reporting results W is used to designate womens hair, M mens, EW extracted womens hair, EM extracted mens. All organisms studied showed growth on our stock 4% peptone, 1% dextrose, 11/2% agar medium in 3 days. Growth was at room temperature in diffuse light. Acladium castellani, Candida candida, Geotrichum bachmann, Lichtheimia sp., Microsporon felineum, Microsporon gypseum, Monilia albicans, Oöspora humi, Sporotrichum schenkii, Trichophyton granulosuni, Trichophyton gypseum asteroides, Trichophyton gypseum lacticolor, Trichophyton interdigitale, Trichophyton japonicum, Trichophyton niveum, Willia anomala showed growth on all hair media in 3 days.

The Habitat of Trichophyton Interdigitale Outside the Body

Adequate scientific proof of the existence of Trichophyton interdigitale (organism of “Athletes Foot”) outside the body seems lacking. Statements in the literature, however, would lead one to judge otherwise. The U. S. Public Health Service states 1 : “Bath mats are justly blamed and it is probable that ringworm can be acquired from them just as warts on the soles of the feet can be . . . . . it can also originate in hotels and from the use of infected towels or soap.” The Public Health Engineering Section of the American Public Health Association 2 states: “Most if not all of these foot diseases are caused by a fungus which is spread by infection of the floors of dressing rooms, etc., at pools, bath houses, gymnasiums, and other places where persons go barefoot.” While these inferences may be true they are unaccompanied by cultural proof. Bonar and Dryer 3 made 30–40 plants of scrapings of floors, hair, etc., in cracks in the floor and traps on the drains from showers. They were troubled by excessive overgrowth with other organisms. They failed to recover the Trichophyton interdigitale. Because of scarcity of cultural work we felt such a study of showers, boat houses and gymnasia would be valuable. Cotton applicators were moistened with normal saline, placed in test tubes which were plugged with cotton and autoclaved. The applicators were used to mop the area to be studied. They were then cut near the cotton end with a flamed scissors and this portion allowed to drop into a sterile petri dish. Melted Sabourauds proof medium was poured over the swab and the medium agitated by sliding the dish back and forth on the table. In this manner an even distribution of colonies was obtained. The petri dishes were observed over a period of a month and transplants made on Sabourauds proof slants of colonies which appeared the least suspicious.

Effect of Age of a Specific Medium on Morphology of Colonies of Certain Pathogenic Fungi.

In this work batches of media were allowed to age at laboratory temperature, planted with organisms and morphological differences observed. In aging, the medium loses water and the nutrient concentrates so that while the ratio of ingredients is probably approximately the same the moisture content, as will be noted below, becomes much less. Our routine W medium, consisting of 4% peptone, 1% dextrose, 1 1/2% agar and adjusted to pH 5.6, was used. The age of media which was placed on laboratory bench tops varied by approximately 10-day intervals from fresh medium to 90-day-old medium. The following representative fungi and 2 non-pathogenic saprophytes, Lichtheimia sp. and Scopulariopsis brevicaulis, were studied: Achorion schoenleinii, Acladium castellani, Candida candida, Endodermophyton indicum (Castellani), Endodermophyton tropicale, Endomyces capsulatus, Endomyces dermatitidus, Epidermophyton cruris, Epidermophyton inguinale, Epidermophyton rubrum (Castellani), Glenospora gammeli, Geotrichum bachmann, Indiella americana, Microsporon audouini, Microsporon felineum, Microsporon gypseum, Monosporum apiospermum, Monilia albicans, Oöspora humi, Sporotrichum schenkii, Trichophyton balcaneum (Castellani), Trichophyton crateriforme, Trichophyton decalvans (Castellani), Trichophyton granulosum, Trichophyton gypseum, Trichophyton gypseum asteroides, Trichophyton gypseum lacticolor, Trichophyton interdigitale, Trichophyton japonicum, Trichophyton louisiana (Castellani), Trichophyton niveum, Trichophyton purpureum, Trichophyton sulfureum, Willia anomala. With age there was retraction and contraction of the medium. This was accompanied by less space for the growth to spread over, with some resultant lessening of growth. In 13-weeks-old medium there was enough shrinkage so that after a month of growth only a dry scale remained. Yet growth occurred in all instances with the exception of Trichophyton louisiana. Thirteen-week growths were smaller, much dried yet retained in most instances some of the typical morphology seen on fresh medium. In many instances the growths on 11- and 12-week media were much dried but showed good differentiation.

I. Relation of Bile Salts, Cholesterin, Sodium Citrate and Sodium Bicarbonate to Toxicity of Pneumococcus

Experiments were performed to determine certain facts relative to the action of sodium citrate, sodium bicarbonate, and some of the constituents of the bile, namely cholesterin and sodium taurocholate, on the toxicity of the pneumococcus. These substances were used separately and in combinations and were injected intraperitoneally into the white mouse. In all, approximately 75 such mice were employed. It is thought by some that bile salts increase the toxicity of the pneumococcus and that cholesterin acts as a buffer reducing this toxicity. The results obtained by various workers 1 , 2 , 3 , 4 have, however, not agreed in all features and for that reason it seems justifiable to place on record all experiments pertinent to this subject. Previous workers 4 have reported that when sodium taurocholate was dissolved in distilled water and injected into the peritoneal cavity of the mouse, the largest dose tolerated was 0.009 gm. In those experiments in which normal saline was used in place of distilled water the sodium taurocholate product∗ showed approximately this toxicity. We, therefore, employed one-half the lethal dose or 0.005 gm. of the product. In comparison with sodium taurocholate dissolved in normal saline, a like solution with 1 % sodium citrate added, was made up and it was found that the toxicity was practically doubled while the sodium citrate injected alone had no toxic effect. Twenty-four hour growths of the pneumococcus, Type I, on blood agar were washed down with sterile saline. Suspensions and dilutions thereof were injected into a number of mice in varying dosage in order to standardize the virulence. To determine the relationship of the number of microorganisms to their virulence, a dilution of 1:8 was made. While 0.05 cc. of the undiluted suspension killed in 18 to 24 hours, equal amounts of the diluted suspension produced death in one-half of the animals in 48 hours.

Growth of Certain Pathogenic Fungi on Asparagin Medium

Since asparagin is used frequently as a Substitute for peptone, it was substituted in the following observations and the differential characteristics of the organisms planted were observed. The media used differed in that a portion contained 1% dextrose while another portion did not. The constituents were: Asparagin 10 gm., dextrose 10 gm., MgSO4.7H2O 0.4 gm., NaCl 2.0 gm., KH2PO4 6.0 gm., K2HPO4 1.0 gm., agar 1.5 gm., distilled water 1000 cc. The salts as used resulted in a pH of 5.5. A control medium of 4% peptone, 1% dextrose and 1 1/2% agar was planted. The following pathogenic fungi and 2 saprophytes, Lichtheimia sp. and Scopulariopsis brevicaulis were observed: Achorion schoenleini, Acladium castellani, Candida candida, Endodermophyton tropicale, Endomyces capsulatus, Endomyces dermatitidus, Epidermophyton inguinale, Glenospora gammeli, Geotrichum bachmann, Indiella americana, Monosporum apiospermum, Microsporon audouini, Monilia albicans, Oöspora humi, Trichophyton crateriforme, Trichophyton granulosum, Trichophyton interdigitale, Trichophyton japonicum, Willia anomala. Observations were made over 30 days. The tubes were kept at room temperature. The table gives the number of days within which growth occurred. One plus indicates a scant growth, 2, 3, 4 plus successively larger growth, and 5 plus the entire slant covered; v plus indicates very scant, a growth which we have been able to duplicate on plain agar. In no instance were the control growths less than 4 plus nor was their appearance delayed more than 4 days.

II. Effect of Dyes on Colonies of Certain Pathogenic Fungi

In a previous paper observations were made on the growth of certain pathogenic fungi on a medium containing alcoholic nigrosine and one containing litmus. 1 In the present paper a like medium (peptone 4%, dextrose 1%, agar 1 1/2%) containing 2% Eosin Y and one containing 2% Eosin B∗ were used. The following pathogenic fungi and 2 saprophytes, Lichtheimia sp. and Scopulariopsis brevicaulis were observed: Achorion schoenleinii, Acladium castellani, Candida candida, Endodermophyton tropicale, Endomyces capsulatus, Endomyces dermatitidus, Epidermophyton inguinale, Glenospora gammeli, Geotrichum bachmann, Indiella americana, Monosporum apiospermum, Microsporon audouini, Monilia albicans, Oöspora humi, Trichophyton crateriforme, Trichophyton granulosum, Trichophyton interdigitale, Trichophyton japonicum, Willia anomala. Observations were made over 30 days. The tubes were kept at room temperature. Growth occurred within 4 days in all tubes of the control media and in the dye tubes with the exception of Achorion schoenleinii, Endodermophyton tropicale—Eosin B, Glenospora gammeli, Microsporon audouini—Eosin Y where it occurred in 6 days, Endodermophyton tropicale—Eosin Y, Microsporon audouini—Eosin B, where it occurred within 11 days and Trichophyton crateriforme, Trichophyton japonicum—Eosin B, where it occurred within 8 days, Growth was as a rule somewhat less marked on the media containing dye but only markedly so in the instance of Trichophyton japonicum.

Effect of Human Blood Serum on the Toxicity of Bile Salts.

Approximately two dozen white mice were used in these experiments. The effect on the toxicity of bile salts when injected intraperitoneally was observed, using normal saline and blood serum as vehicles. The lethal dose of stock bile salts 1 , 2 dissolved in normal saline had previously been found to be 0.009 gm. This product contained proteins, etc., which were removed, since they might influence the results. The procedure consisted of cleaning with chloroform and ether and dissolving the solid matter in alcohol. The alcoholic solution cleared by standing was pipetted off, filtered, and evaporated and the residue dried in a hot air oven. The residue was then immediately weighed so as to obviate as much as possible the adsorption of water which factor would necessarily influence the weight. The solutions when made up contained 0.001 and 0.002 gm. of bile salts to the cc. Their slight variation in acidity was not sufficiently marked in the authors experience to influence the results. The surface tension estimated by the drop method for the serum and saline salt mixtures was virtually the same for like concentrations of the salt. When normal saline was used as a vehicle 0.009 gm. of bile salts were found lethal as in previous experiments. 1 When fresh human serum (not older than 3 hours) was used in a like capacity, at least twice the amount was necessary to insure a fatal outcome. Thirty-six hours were considered a logical time over which to read results, especially since animals which did not die within that time survived. We observed that human serum is protective toward this toxic product. It is questioned that this is due to the presence of a developed resistance on the part of the body.

Toxicity for White Rats of Elements Implanted into their Subcutaneous Tissues.

With the view of observing the host response to various elements, the author has implanted such substances over the ribs of white rats anteriorly. The animals were anaesthetized with ether, a half inch incision made, the skin dissected up for a short distance, the element placed in position and the skin edges secured by skin clips, The whole procedure required less than 10 minutes and the clips were very effective. The following elements were used: aluminum, antimony, arsenic, barium, beryllium, bismuth, boron, bromine, cadmium, calcium, carbon, cerium, chromium, cobalt, columbium, copper, erbium, gold, iodine, iron, lead, lithium, manganese, magnesium, mercury, molybdenum, nickel, phosphorus, potassium, selenium, silicon, silver, sodium, sulphur, tantalum, tellurium, thallium, tungsten, uranium, vanadium, zinc and zirconium. Of the above elements only 2 proved fatal for the white rat—thallium and phosphorus, death occurring within 48 hours. This is in accord with the characteristics of the latter 2 elements, both thallium and the white phosphorus being poisonous. On autopsy, however, some of the phosphorus still glowed on being exposed to the air, showing that the absorption of the phosphorus was not rapid. The rats which survived were examined 7 and 30 days after implantation, and portions of tissue adjacent to the implantation were taken for histological examination. No marked reactions were noted around the implantations of aluminum, barium, beryllium, bismuth, boron, calcium, carbon, cerium, columbium, erbium, gold, iron, lead, magnesium, mercury, molybdenum, selenium, silicon, silver, tantalum, tellurium, tin, tungsten, uranium, vanadium, zinc and zirconium. A large ulcer was noted in the case of titanium and a small ulcer in the case of bromine and potassium. Induration around the implantation was noted in the case of sulphur, antimony, arsenic, chromium, cadmium, sodium and lithium.

Effects of Ethyl Alcohol on Red Blood Cells.

The observations present the microscopic changes occurring in human red blood cells when subjected to different dilutions of alcohol in saline. Approximately 50 specimens of blood from various patients were studied and the results obtained conform as a whole. Studies were made by the hanging drop method. Cells deposited in the serum of clotted blood were examined within 4 hours after withdrawal and whole blood was examined immediately following withdrawal. The red cells were diluted so that the suspension contained approximately 4000 per cu. mm. When the red blood cells were found to be in rouleux formation the alcohol dilutions overcame this formation more quickly than when normal saline was added, and the rapidity of this change in all cases depended upon the concentration of the alcohol. In one series, various iso-osmotic solutions of alcohol and sodium chloride were employed. In 0.1% alcohol the cells were more rounded and regular and this was more apparent in 0.5% alcohol. In the 1% alcohol, granules were extruded from the cells, coincidently they lost their pigment, became swollen and faded away as shadows. In the other series, alcohol was diluted with normal saline. In 0.2% (approximately 1.0016 X iso-osmotic) alcohol a cupped appearance was noted and a few of the cells buckled in and bent upon themselves; the inner edges of the cups showed in a few instances serrations and the margins of the cups were more refractile and apparent and the centers clearer. In 0.5% (approximately 1.003 X iso-osmotic) alcohol the irregularity of the cells was more marked. In this concentration numerous cells possessed very clear centers shaped like triangles and slits, while a few appeared small and round and no longer presented the cup effect.