JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1979, p. 144-146 0095-1 137/79/08-0144/03$02.00/0
Vol. 10, No. 2
Merthiolate Treatment of Pathogenic Fungi FLORENCE J. DEIGHTON, NANCY K. HALL,t AND HOWARD W. LARSH* Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019 Received for publication 17 May 1979
The action of Merthiolate on the pathogenic yeasts Blastomyces dermatitidis, Histoplasma capsulatum, and Sporothrix schenckii was compared to the effect of treatment with formaldehyde. Concentrations of 1:10,000 and 1:5,000 Merthiolate for three exposure times (24, 48, and 72 h) at 4 and 25°C were tested on three media (brain heart infusion with and without blood, and modified Sabouraud agar). The effect of Merthiolate on these three yeasts was primarily fungistatic, with maximum effect using 1:5,000 Merthiolate at 25°C for at least 48 h. Mycelial suspensions of B. dermatitidis, H. capsulatum, S. schenckii, and the yeast phase of Cryptococcus neoformans were susceptible to the 1:5,000 Merthiolate concentration after 24 h of treatment. The antifungal effect of Merthiolate varies with species and growth phase of the fungus. Concentration, time of exposure, and temperature of incubation are important variables.
Merthiolate or thimerosal is routinely used as a fungicidal agent in the killing of pathogenic fungi for routine laboratory use, i.e., (i) in antigens, (ii) in preparation of organisms for animal sensitization, and (iii) as a preservative in skin test antigens and vaccines. An accidental laboratory inoculation with Merthiolate-treated Blastomyces dermatitidis yeast cells resulted in primary cutaneous blastomycosis (1). Therefore, the possibility of Merthiolate being only fungistatic was considered. This paper investigates the effects of Merthiolate on four pathogenic
tured on blood (BHI base), BHI, and MS agars with the exception of C. neoformans, which was cultured on MS agar only. Duplicate plates were placed at 37 and 25°C and observed for growth at 7, 14, and 21 days. The saprophytic phases of B. dermatitidis, H. capsulatum, and S. schenckii were grown on MS and glucose-yeast extract agars. Each culture was overlaid with a solution of 1:5,000 Merthiolate in boratebuffered saline. At 24, 48, and 72 h the Merthiolatetreated mycelium of each organism was scraped from the surface of the agar, washed three times with saline, and suspended in 5 ml of saline. Two 10-fold dilutions were plated on glucose-yeast extract and MS agar plates. Plates were incubated at 25°C, and growth was recorded at 7, 14, and 21 days.
fungi. MATERIALS AND METHODS Yeast cells of B. dermatitidis (ATCC 26199), Histoplasma capsulatum (Scritchfield), and Sporothrix schenckii (CDC-010) were grown in brain heart infusion (BHI) broth. Cryptococcus neoformans (184) was grown in modified Sabouraud (MS) broth. All cultures were grown at 37°C for 72 h on a gyratory shaker. A 1% solution of Merthiolate (Parke-Davis), in either saline or borate-buffered saline (final concentration 1: 5,000 = 0.02% or 1:10,000 = 0.01%), or formaldehyde (final concentration 0.2%) was added to separate logphase cultures. Identically treated flasks were placed at 4 and 25°C during the 72-h testing period. Control cultures for viability also were tested at the same temperatures and time periods. At 0, 24, 48, and 72 h after the addition of the two reagents, 25-ml samples were removed from each flask, washed three times with saline, and counted by hemacytometer. The washed cell pellet was adjusted to the sampling concentration by resuspending in 25 ml of saline. The adjusted cell concentrate and a series of 10-fold dilutions (through 10-7) were plated. The fungi were cult Present address: Department of Pathology, University of
Oklahoma Health Science Center, Oklahoma City, OK 73190.
RESULTS The Merthiolate-treated yeast forms of B. dermatitidis, H. capsulatum, and S. schenckii grew on all three media. However, blood agar was a better medium for growing the treated cells. The effects of Merthiolate on these three fungi after plating on blood agar are shown in Fig. 1. Direct plating of the treated yeast cells yielded no growth, or growth only around the edge of the inoculum. At 10-', however, good growth was observed. On BHI and MS agars, dilutions greater than 10-' were necessary to detect growth. Also shown in Fig. 1 is the C. neoformans Merthiolate-treated culture grown on MS agar. Similar results were obtained, i.e., good growth, at the 10-I dilution. The effect of treatment with 1:10,000 Merthiolate at 4 and 25°C is shown in Fig. 2 and 3. B. dermatitidis was the only fungus that showed any susceptibility at this concentration with a 2log reduction in growth. The 2-log decrease in
144
VOL. 10, 1979
MERTHIOLATE TREATMENT OF PATHOGENIC FUNGI
145
FIG. 1. Inhibitory effect of 1:5,000 Merthiolate on (A) H. capsulatum, (B) B. dermatitidis, and (C) S. schenckii on blood agar and (D) C. neoformans on MS agar. (Dilutions in A, C, and D, top to bottom: 10°, 10-1, lo-; in B: 100, 10-1) a. (n a (A
SPOROTHMIX ISTOPLASMA
1
z
Histoplasma
otOU
A
Sporothrix
J
a ASTOMYCES 53
-j
0
0
-
Blastomyces
Vol. 10, No. 2
Merthiolate Treatment of Pathogenic Fungi FLORENCE J. DEIGHTON, NANCY K. HALL,t AND HOWARD W. LARSH* Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019 Received for publication 17 May 1979
The action of Merthiolate on the pathogenic yeasts Blastomyces dermatitidis, Histoplasma capsulatum, and Sporothrix schenckii was compared to the effect of treatment with formaldehyde. Concentrations of 1:10,000 and 1:5,000 Merthiolate for three exposure times (24, 48, and 72 h) at 4 and 25°C were tested on three media (brain heart infusion with and without blood, and modified Sabouraud agar). The effect of Merthiolate on these three yeasts was primarily fungistatic, with maximum effect using 1:5,000 Merthiolate at 25°C for at least 48 h. Mycelial suspensions of B. dermatitidis, H. capsulatum, S. schenckii, and the yeast phase of Cryptococcus neoformans were susceptible to the 1:5,000 Merthiolate concentration after 24 h of treatment. The antifungal effect of Merthiolate varies with species and growth phase of the fungus. Concentration, time of exposure, and temperature of incubation are important variables.
Merthiolate or thimerosal is routinely used as a fungicidal agent in the killing of pathogenic fungi for routine laboratory use, i.e., (i) in antigens, (ii) in preparation of organisms for animal sensitization, and (iii) as a preservative in skin test antigens and vaccines. An accidental laboratory inoculation with Merthiolate-treated Blastomyces dermatitidis yeast cells resulted in primary cutaneous blastomycosis (1). Therefore, the possibility of Merthiolate being only fungistatic was considered. This paper investigates the effects of Merthiolate on four pathogenic
tured on blood (BHI base), BHI, and MS agars with the exception of C. neoformans, which was cultured on MS agar only. Duplicate plates were placed at 37 and 25°C and observed for growth at 7, 14, and 21 days. The saprophytic phases of B. dermatitidis, H. capsulatum, and S. schenckii were grown on MS and glucose-yeast extract agars. Each culture was overlaid with a solution of 1:5,000 Merthiolate in boratebuffered saline. At 24, 48, and 72 h the Merthiolatetreated mycelium of each organism was scraped from the surface of the agar, washed three times with saline, and suspended in 5 ml of saline. Two 10-fold dilutions were plated on glucose-yeast extract and MS agar plates. Plates were incubated at 25°C, and growth was recorded at 7, 14, and 21 days.
fungi. MATERIALS AND METHODS Yeast cells of B. dermatitidis (ATCC 26199), Histoplasma capsulatum (Scritchfield), and Sporothrix schenckii (CDC-010) were grown in brain heart infusion (BHI) broth. Cryptococcus neoformans (184) was grown in modified Sabouraud (MS) broth. All cultures were grown at 37°C for 72 h on a gyratory shaker. A 1% solution of Merthiolate (Parke-Davis), in either saline or borate-buffered saline (final concentration 1: 5,000 = 0.02% or 1:10,000 = 0.01%), or formaldehyde (final concentration 0.2%) was added to separate logphase cultures. Identically treated flasks were placed at 4 and 25°C during the 72-h testing period. Control cultures for viability also were tested at the same temperatures and time periods. At 0, 24, 48, and 72 h after the addition of the two reagents, 25-ml samples were removed from each flask, washed three times with saline, and counted by hemacytometer. The washed cell pellet was adjusted to the sampling concentration by resuspending in 25 ml of saline. The adjusted cell concentrate and a series of 10-fold dilutions (through 10-7) were plated. The fungi were cult Present address: Department of Pathology, University of
Oklahoma Health Science Center, Oklahoma City, OK 73190.
RESULTS The Merthiolate-treated yeast forms of B. dermatitidis, H. capsulatum, and S. schenckii grew on all three media. However, blood agar was a better medium for growing the treated cells. The effects of Merthiolate on these three fungi after plating on blood agar are shown in Fig. 1. Direct plating of the treated yeast cells yielded no growth, or growth only around the edge of the inoculum. At 10-', however, good growth was observed. On BHI and MS agars, dilutions greater than 10-' were necessary to detect growth. Also shown in Fig. 1 is the C. neoformans Merthiolate-treated culture grown on MS agar. Similar results were obtained, i.e., good growth, at the 10-I dilution. The effect of treatment with 1:10,000 Merthiolate at 4 and 25°C is shown in Fig. 2 and 3. B. dermatitidis was the only fungus that showed any susceptibility at this concentration with a 2log reduction in growth. The 2-log decrease in
144
VOL. 10, 1979
MERTHIOLATE TREATMENT OF PATHOGENIC FUNGI
145
FIG. 1. Inhibitory effect of 1:5,000 Merthiolate on (A) H. capsulatum, (B) B. dermatitidis, and (C) S. schenckii on blood agar and (D) C. neoformans on MS agar. (Dilutions in A, C, and D, top to bottom: 10°, 10-1, lo-; in B: 100, 10-1) a. (n a (A
SPOROTHMIX ISTOPLASMA
1
z
Histoplasma
otOU
A
Sporothrix
J
a ASTOMYCES 53
-j
0
0
-
Blastomyces
