TY - JOUR
AU1 - Granger, Donald L
AU2 - Call, Donna M
AB - Abstract We found that a large inoculum of Cryptococcus gattii cells, when plated on Dulbecco's modified eagle's medium (DMEM) incorporated into agar, died within a few hours provided that DMEM agar plates had been stored in darkness for approximately 3 days after preparation. Standard conditions were developed for quantification of killing. The medium lost its fungicidal activity when exposed to visible light of wave length ∼400 nm. The amount of energy required was estimated at 5.8 × 104 joules @ 550 nm. Liquid DMEM conditioned by incubation over DMEM agar plates stored in darkness was fungicidal. We found that fungicidal activity was heat-stable (100°C). Dialysis tubing with MWC0 < 100 Daltons retained fungicidal activity. Neutral pH was required. Strains of Cryptococcus were uniformly sensitive, but some Candida species were resistant. Components of DMEM required for killing were pyridoxal and cystine. Micromolar amounts of iron shortened the time required for DMEM agar plates to become fungicidal when stored in the dark. Organic and inorganic compounds bearing reduced sulfur atoms at millimolar concentrations inhibited fungicidal activity. Our results point to a light-sensitive antifungal compound formed by reaction of pyridoxal with cystine possibly by Schiff base formation. cryptococcus, fungicidal, DMEM, agar, light Introduction Cultivation of pathogenic yeast under in vitro physiological conditions reveals phenotypic properties which may not be present when traditional mycology media are used.1 To observe colony morphology of Cryptococcus gattii under the prevailing conditions of mammalian extracellular fluid, we incorporated a cell culture medium, Dulbecco's modified eagle's medium (DMEM), into agar.2–4 While DMEM contains all nutrients required for C. gattii growth, we observed that large inocula (106 cells per 90 mm diameter dishes) failed to grow. We found that colony formation was restored when DMEM agar plates were exposed to room light prior to plating C. gattii cells. We considered this observation might yield an undiscovered antifungal compound. Moreover, knowledge of this property could prevent potential confounding effects for in vitro experiments on antifungal properties of host defense leukocytes or extracellular soluble factors.5,6 Thus, we further investigated the basis for DMEM light-sensitive inhibitory effect. Methods Fungi All strains of Cryptococcus species7 and other yeast species were human isolates obtained from the University of Utah clinical mycology laboratory (ARUP) courtesy of Ms. Helene Segal. Fungi were purified by repeated isolation on Sabouraud glucose agar (SGA) plates. Then they were grown in liquid yeast nitrogen base (YNB) supplemented with L-asparagine, 11.4 mM, and D-glucose, 25 mM, washed by centrifugation in phosphate-buffered saline (PBS), pH 7.4, and stored frozen (−80oC) in medium consisting of DMEM with 25% glycerol, 20% fetal bovine serum, and 10% dimethyl sulfoxide. Quantification of stocks before and after freezing showed high level of preservation of viability for all yeast species used. For experiments, SGA plates were streaked weekly from frozen stocks, and overnight liquid cultures (YNB) were diluted in PBS for seeding to test media. Unless noted, all experiments were performed using a strain of Cryptococcus gattii designated MG95, isolated from a case of meningoencephalitis managed at our hospital. Media Several lots of commercial DMEM powder were used (Gibco, Sigma, Flow Laboratories, North Andover, MA, USA) including preparations with and without pH indicator dye, phenol red (see reference [2] for recipe). DMEM was prepared using double distilled, deionized water (DDW) with the following modifications: bicarbonate deleted and replaced with 25 mM NaCl, phenol red deleted, glucose, 25 mM, final concentration, L-glutamine, 4 mM, MOPS buffer, 25 mM, pH 7.4, penicillin G, 80 μM, and gentamicin, 10 μM. No animal serum was added. DMEM powder was dissolved in DDW at 2× concentration; pH adjusted to 7.4 with NaOH; medium was filter sterilized (0.22 μ pore diameter) and then mixed with 2× (3%) sterilized Bacto agar (Difco) at 45oC. Liquid DMEM agar was poured into sterile plastic petri dishes (60 × 15 mm) in a darkened room and allowed to solidify in the dark. Then the plates were divided into two groups and stored at room temperature under a light box equipped with fluorescent lamps (Philips F15T8/D 15 watts) in humidified sealed clear plastic bags or in the same bags in complete darkness. Measurements of light intensity are given below and in the text. Thermometer readings assured that the different storage conditions did not vary significantly. For each batch of DMEM agar the pH was determined in a small solidified aliquot equilibrated at 35oC. For data presented in the figures and tables, day zero corresponds to the day on which the media was prepared. Reagents Unless otherwise stated, chemicals were obtained from Sigma. Amino acids and other constituents of DMEM were of “ultrapure” category. All media were prepared with water which had been distilled, then passed through deionization cartridges and then redistilled using a glass distillation apparatus (Corning Glass Works). Unless stated, agar was Bacto from Difco Co. Manipulations with light A standard light source was used. This consisted of an X-ray view box (light origin area equaled 43 × 36 cm) equipped with two 44-cm fluorescent lamps having 15-watt output per lamp (PhilipsF15T8/D). DMEM agar plates were positioned at 10 cm from the light source. At this distance, the effect of temperature was negligible. Illumination at 10 cm from the standard light source was measured with a calibrated Lutron Digital Lux Meter (LX-101, East Granby, CT, USA) and was consistently recorded at approximately 3400 lumens/meter2 (mean value) at randomly chosen loci under the light box. Variation from locus to locus was not significant. One lumen/meter2 = 1 lux, which is equivalent to an irradiance of approximately 5 watts/m2 at 550 nm. Intensity of illumination was varied in two ways. Agar plates were exposed to the standard light source for varying periods of time. Alternatively, plates were exposed for a constant time in blackened 13 × 13 × 5 cm cardboard boxes containing a circular port (55 mm diameter) covered with 7.5 × 7.5 cm Wratten Neutral Density Filters (Edmund Scientific, Barrington, NJ, USA) possessing calibrated transmissions of 1, 10, 25, 50, and 63%. The filters reduce light intensity across the visible spectrum without altering the spectral profile of light passing through the filters. Measurements with a calibrated light meter agreed with the manufacturer's (Kodak Co.) stated specifications. DMEM agar plates were secured within the blackened boxes beneath the Wratten filters. The quantity of light delivered to the agar plates (luminous energy) was expressed as kilolux for 1 hour (kilolux • hour). In other experiments agar plates were illuminated with different portions of the visible spectrum. High saturation dichroic color separation filters (55 mm diameter) were used (Edmund Scientific). Filters were fixed to circular ports in blackened boxes and used as above. Six filters possessing different spectral transmission properties were used individually. Further light separation was done by constructing a black metal machined 90o angle square tunnel (5 × 5 cm), each arm measuring 14 cm length. One arm contained a sealed port housing a 15 watt fluorescent white light source (Philips Prolight A54922). At the 90o angle, a 55 × 50 × 1.7 mm dichroic blue reflector (Edmund Scientific) was secured at 45o to the incident light. The apparatus reflected the desired light spectrum, a narrow band (350–430 nm) and an undesired broad band (480–750 nm). The second arm of the tunnel was fitted with a blue corrector filter (Edmond Scientific), which allowed transmission of the former reflected band and absorption of the latter band. Light entering the box passed through an ultraviolet filter (0% transmission at <370 nm) before illuminating the test DMEM agar plate. Thus, white light entering the apparatus was separated into an illuminating band with a spectral width of 370–430 nm, whose peak was approximately 380–400 nm. Experiments All operations were performed in a darkened room. Plates were seeded with 50 μL (100–200 yeast cells) from an overnight culture diluted 10−4 in PBS by spreading the liquid over the agar surface with four 6 mm sterile glass beads. A SGA plate was seeded likewise as a positive growth control. At 10−4 dilution there was no significant carry-over of constituents from YNB to test media. Plates were incubated at 35oC in a humidified air incubator admitting no light. Thus, once yeast cells were plated, all incubations were done under the same conditions in the absence of light. After suitable incubation (2–7 days) colonies were counted and expressed as the percent colony-forming-efficiency (CFE) relative to the control, which was calculated by: [(# colonies on test medium) ÷ (# colonies on SGA)] × 100. All plates giving no growth (zero CFE) were held at least 14 days to assure that late occurring growth did not take place. Statistical analysis The threshold for electromagnetic energy needed to abolish inhibitory activity of DMEM agar plates stored in the dark was determined by analyzing data statistically employing regression models on subsets of data with incrementally (steps of 0.1 kilolux • hour) decreasing upper bound on the range of kilolux • hour. We identified the minimum consistent effect as the smallest value of the upper bound for which the slope of this and all models with larger upper bounds were significantly greater than 0 (P-value < .05). The estimated limit for no effect was the minimum consistent effect value minus 0.1 kilolux • hour (the next smaller upper bound). Results Fungicidal activity of DMEM agar medium stored without light DMEM agar plates were separated into two groups and stored in complete darkness or under white fluorescent room light (380 lumens per meter2 measured using a calibrated light meter). Each day one plate from each group was seeded with 100–200 Cryptococcus gattii (designated MG95) cells and incubated at 35oC in the dark. Colonies were counted 2–4 days later and compared to the same inoculum plated on SGA plates (Fig. 1A). DMEM agar plates stored under light always gave high colony-forming efficiency (CFE). In numerous experiments DMEM agar plates stored in the dark always inhibited growth by 2–10 days after preparation. When plates stored in the dark were illuminated for at least 24 hours, they acquired high CFE, but when plates stored in light were placed in darkness, they continued to support high CFE (Fig. 1B). The inhibitory property of DMEM agar plates lasted at least 100 days (Fig. 1C). To determine fungistatic versus fungicidal activity, DMEM agar plates stored in the dark (2 weeks) were seeded with 200–300 MG95 cells. At various intervals, thereafter 24 mm circular agar slabs were excised and transferred to SGA plates for enumeration 3 days later. After 3–6 hours contact with DMEM agar plates stored in darkness, yeast cell rescue was lost (Fig. 1D). Large SBA plates (60-, 100-, 150-cm diameter) gave the same result, suggesting that failure to rescue living cells from the DMEM agar excised discs was not due to insufficient volume of diffusion for a putative inhibitor present in the transferred slabs. In addition, MG95 colonies never developed on dark-stored DMEM agar plates (provided an initial contact time had elapsed, i.e., ∼6 hours) incubated in the dark or light for up to 4 weeks after seeding. Figure 1. Open in new tabDownload slide Dulbecco's modified Eagle's medium agar plates stored in the dark inhibit growth and kill Cryptococcus gattii, strain MG95. A: On the days shown by the symbols, plates were removed from storage in room light illuminated at 380 lumens/meter2 (triangles) or the dark (circles) and plated with approximately 100–200 MG95 cells. After 3 days, colonies were counted. Data expressed as colony-forming efficiency (CFE) as defined in Materials and Methods section. B: On day 5 of storage, DMEM agar plates stored in the dark (circles) were switched to room light (380 lumens/m2) and vice versa (triangles). Then on the days shown they were seeded with 100–200 M95 cells. After 3 days colonies were counted and data expressed as CFE. C: The experimental protocol was identical to panel A, except that plating with M95 cells was continued at the times shown for > 100 days. Circles represent plates stored in the dark; triangles are for plates stored in light. D: MG95 cells (100–200 cfu) were plated on DMEM agar stored under the standard light source (3400 lumens/m2, triangles) or in complete darkness (circles). At the times shown identical circular agar slabs (24 mm diameter) were removed and transferred (plated side up) to SGA plates (90 mm diameter, 25 ml medium per plate). Colonies forming on the slabs were counted 3 days later. Figure 1. Open in new tabDownload slide Dulbecco's modified Eagle's medium agar plates stored in the dark inhibit growth and kill Cryptococcus gattii, strain MG95. A: On the days shown by the symbols, plates were removed from storage in room light illuminated at 380 lumens/meter2 (triangles) or the dark (circles) and plated with approximately 100–200 MG95 cells. After 3 days, colonies were counted. Data expressed as colony-forming efficiency (CFE) as defined in Materials and Methods section. B: On day 5 of storage, DMEM agar plates stored in the dark (circles) were switched to room light (380 lumens/m2) and vice versa (triangles). Then on the days shown they were seeded with 100–200 M95 cells. After 3 days colonies were counted and data expressed as CFE. C: The experimental protocol was identical to panel A, except that plating with M95 cells was continued at the times shown for > 100 days. Circles represent plates stored in the dark; triangles are for plates stored in light. D: MG95 cells (100–200 cfu) were plated on DMEM agar stored under the standard light source (3400 lumens/m2, triangles) or in complete darkness (circles). At the times shown identical circular agar slabs (24 mm diameter) were removed and transferred (plated side up) to SGA plates (90 mm diameter, 25 ml medium per plate). Colonies forming on the slabs were counted 3 days later. Inoculum size overcame inhibition of C. gattii colony formation at 107 cells per ml when 50 μl cell suspension was seeded to 60 mm diameter DMEM agar plates (Fig. 2). To test for satellite growth, a large slurry of washed concentrated Cryptococcus neoformans (strain H99) cells were placed as a 1-cm spot in the center of DMEM agar plates stored in the dark, which had just been inoculated with 1000–2000 cfu MG95 cells. After several days incubation, small colonies grew very close to the margin of the confluent mass of H99 cells. These colonies were picked the typed on CGB agar8. All colonies typed were C. gattii, indicating that they were satellites. No colonies formed beyond 5 mm from the concentrated patch of H99 cells. Figure 2. Open in new tabDownload slide Effect of inoculum size on growth of MG95 cells on DMEM agar plates stored in the dark. MG95 cell suspensions containing approximately the number of cfu/ml as shown in the photograph were plated in 50 μL on DMEM agar dishes which had been stored in the light or in the dark for 14 days. After 3 days incubation, the plates were photographed. Quantitative plate counts on SGA showed that the lowest dilution contained 7.8 × 107 cfu/ml. CFE on DMEM agar plates stored in the light was approximately 100% for countable inoculums. Figure 2. Open in new tabDownload slide Effect of inoculum size on growth of MG95 cells on DMEM agar plates stored in the dark. MG95 cell suspensions containing approximately the number of cfu/ml as shown in the photograph were plated in 50 μL on DMEM agar dishes which had been stored in the light or in the dark for 14 days. After 3 days incubation, the plates were photographed. Quantitative plate counts on SGA showed that the lowest dilution contained 7.8 × 107 cfu/ml. CFE on DMEM agar plates stored in the light was approximately 100% for countable inoculums. Electromagnetic energy required to reverse fungicidal activity of DMEM agar plates stored in the dark We determined the quantity of light required to permit colony formation on DMEM agar plates stored in the dark. Plates stored in darkness for 2–4 weeks were illuminated at 3400 lumens per meter2 (3400 lux) for intervals between 30 and 360 minutes, or plates were illuminated for 240 minutes under a series of calibrated filters, which attenuated incident light by known amount evenly across the visible spectrum. Calibrations were checked by measuring with a light meter the amount of light falling upon, and passing through, the filters. The relationship between illumination, expressed as kilolux for 1 hour, and CFE in 10 pooled experiments is shown in Figure 3. For all results combining variable time of exposure at constant light intensity, and constant time of exposure at variable light intensity, the limit for no effect was 8.8 kilolux • hour. At 8.9 kilolux • hour the P value = .0074, indicating the threshold for statistically significant effect of light. Expressed as total optical power this threshold was 13.5 watt • hour at 550 nm or 5.8 × 104 joules. Figure 3. Open in new tabDownload slide Quantification of electromagnetic energy required to convert DMEM agar plates to a growth-permissive state. DMEM agar plates stored in the dark for 2–4 days were illuminated under the standard light source (3400 lumens/m2) for various periods of time (circles) or for a constant time under various degrees of illumination (triangles) by using Wratten filters (for details see Methods section). The dishes were then plated with 100–200 MG95 cells and were incubated at 35oC. Colonies were counted 3–5 days later. The data are from 10 separate experiments in which exposure to light in kilolux • hour is plotted against CFE. The threshold for a statistically significant biological effect is 8.9 kilolux • hour (see text). Figure 3. Open in new tabDownload slide Quantification of electromagnetic energy required to convert DMEM agar plates to a growth-permissive state. DMEM agar plates stored in the dark for 2–4 days were illuminated under the standard light source (3400 lumens/m2) for various periods of time (circles) or for a constant time under various degrees of illumination (triangles) by using Wratten filters (for details see Methods section). The dishes were then plated with 100–200 MG95 cells and were incubated at 35oC. Colonies were counted 3–5 days later. The data are from 10 separate experiments in which exposure to light in kilolux • hour is plotted against CFE. The threshold for a statistically significant biological effect is 8.9 kilolux • hour (see text). We determined the window of light within the electromagnetic spectrum required to abolish fungicidal activity. We used filters of different reflective/transmissive properties interposed between a standard light source at zero-degree angle of incidence and DMEM plates stored in the dark (Table 1). After 7 days the plates were seeded with 100–200 MG95 cells. Neither ultraviolet (<370 nm) nor infrared (>700 nm) light was responsible. Dichroic filters narrowed the active wave length spectrum to 370–460 nm. To test this result filters were aligned in series (45 degree blue reflector followed by zero angle blue corrector plus a UV filter) to produce transmitted light between 370 and 430 nm. In agreement with the single filter results the incident light, monochromatic blue light, gave the same result as white light. Table 1. Testing segments of the electromagnetic spectrum using filters to block transmission. Filtera . Transmission wave lengths (nm) . Percent CFE (range)b . Infrared <700 63–95 Red >570 0 Blue <460 57–86 Green 510–600 0 Cyan <600 61–99 Magenta <530; 580–700 65–100 Yellow >500 0–8 45o Angle blue reflector plus perpendicular blue correctorc 370–430 27–86 UV >370 60–88 Filtera . Transmission wave lengths (nm) . Percent CFE (range)b . Infrared <700 63–95 Red >570 0 Blue <460 57–86 Green 510–600 0 Cyan <600 61–99 Magenta <530; 580–700 65–100 Yellow >500 0–8 45o Angle blue reflector plus perpendicular blue correctorc 370–430 27–86 UV >370 60–88 aHigh color saturation dichroic filters obtained from Edmund Scientific Co., Barrington, NJ, USA. bDMEM Agar plates stored in the dark were exposed to a constant white light source attenuated by filters placed at zero degrees angle of incidence. After 7 days the plates were seeded with 100–200 cryptococci and colonies were counted 4 days later. Data compiled from nine experiments. cSelects short wavelength visible monochromatic blue light from the white light source. See Methods for details. Open in new tab Table 1. Testing segments of the electromagnetic spectrum using filters to block transmission. Filtera . Transmission wave lengths (nm) . Percent CFE (range)b . Infrared <700 63–95 Red >570 0 Blue <460 57–86 Green 510–600 0 Cyan <600 61–99 Magenta <530; 580–700 65–100 Yellow >500 0–8 45o Angle blue reflector plus perpendicular blue correctorc 370–430 27–86 UV >370 60–88 Filtera . Transmission wave lengths (nm) . Percent CFE (range)b . Infrared <700 63–95 Red >570 0 Blue <460 57–86 Green 510–600 0 Cyan <600 61–99 Magenta <530; 580–700 65–100 Yellow >500 0–8 45o Angle blue reflector plus perpendicular blue correctorc 370–430 27–86 UV >370 60–88 aHigh color saturation dichroic filters obtained from Edmund Scientific Co., Barrington, NJ, USA. bDMEM Agar plates stored in the dark were exposed to a constant white light source attenuated by filters placed at zero degrees angle of incidence. After 7 days the plates were seeded with 100–200 cryptococci and colonies were counted 4 days later. Data compiled from nine experiments. cSelects short wavelength visible monochromatic blue light from the white light source. See Methods for details. Open in new tab Effect of pH We prepared DMEM agar plates at different pH using a series of organic buffers bearing a pKa at close to the desired pH. Plates at each pH were stored in dark or light environments and at frequent intervals, plated with MG95 cells to test CFE. Neutral pH was required; at or below pH 6.5 fungicidal activity was lost. At pH 8.5, colonies failed to develop on plates stored in either light or dark conditions (Fig. 4). Figure 4. Open in new tabDownload slide Effect of pH on inhibition of growth on DMEM agar medium stored in the dark. DMEM agar plates were prepared at pH 5.5 (squares), 6.5 (inverted triangles), 7.5 (upward triangles), and 8.5 (circles) using 25 mM MES, PIPES, MOPS, and TRIS organic buffers, respectively. The plates were then stored in the light at 3400 lumens/m2 (not shown), or in the dark, and at the times shown in the figure, were seeded with 100–200 MG95 cells. Colonies were counted 4 days later. Plates stored in the light at pH 5.5, 6.5, and 7.5 gave high CFE (65–100%) at all days tested. Plates at pH 8.5 stored in the light gave no growth at any time. Shown is a representative example of four experiments. Figure 4. Open in new tabDownload slide Effect of pH on inhibition of growth on DMEM agar medium stored in the dark. DMEM agar plates were prepared at pH 5.5 (squares), 6.5 (inverted triangles), 7.5 (upward triangles), and 8.5 (circles) using 25 mM MES, PIPES, MOPS, and TRIS organic buffers, respectively. The plates were then stored in the light at 3400 lumens/m2 (not shown), or in the dark, and at the times shown in the figure, were seeded with 100–200 MG95 cells. Colonies were counted 4 days later. Plates stored in the light at pH 5.5, 6.5, and 7.5 gave high CFE (65–100%) at all days tested. Plates at pH 8.5 stored in the light gave no growth at any time. Shown is a representative example of four experiments. Inhibitory activity in liquid DMEM When agar was deleted from DMEM and the liquid medium was stored under light or dark conditions, the effect on M95 growth was inconsistent. Yeast cells always grew in liquid medium stored in the light; however, the same medium stored in the dark inhibited cell growth with some batches of media but not in others. Inconsistency was neither a function of different commercial lots of DMEM nor commercial versus “laboratory-made” DMEM, nor was it due to variations in pH. We developed a method so that consistent inhibitory activity was obtained. Growth-permissive liquid DMEM (4.4 ml) was added to flasks (75 square cm) containing DMEM agar (40.0 ml), which had been stored in light or dark environments. After equilibration on a rocking platform (24 hours), the liquid media were removed and seeded with MG95 cells. Growth and death were monitored by turbidity (not shown) and quantitative plate counts (Fig. 5). Cell viability dropped off between 6–10 hours by > 2 Log10 (Fig. 5A). As with the previous experiments employing DMEM agar, fungicidal activity was always dark-dependent in the same fashion. Figure 5. Open in new tabDownload slide Characteristics of inhibitory factor(s) recovered from DMEM agar plates stored in the dark. Liquid DMEM medium (4.4 ml) without agar was added to flasks (75 square cm) containing 40.0 ml DMEM agar stored in either the dark or the light, and flasks were incubated at 20oC on a rocking platform in the dark for 24 hours (liquid DMEM: agar DMEM ratio = 1:10). The liquid medium samples were removed and seeded with MG95 cells for grown curve assays (panel A); or incubated at various temperatures as shown on the bar graph for 90 minutes (panel B), or subjected to dialysis (three 24-hour changes, panel C) against growth-permissive liquid DMEM using molecular weight cutoff (MWCO) tubing with pore sizes as shown. The liquid samples (neat, panel A; heated, panel B; or dialyzed, panel C) were seeded with approximately 104 MG95 cells/ml (final cell density) and colony counts were done at the times shown (panel A), or 3 days later (panels B and C). Panel A shows a representative of 10 experiments (medium from flasks stored in the dark, circles; medium from flasks stored in the light, triangles). Panel B shows results of one of three experiments giving the same results. Panel C shows data compiled from six experiments. Figure 5. Open in new tabDownload slide Characteristics of inhibitory factor(s) recovered from DMEM agar plates stored in the dark. Liquid DMEM medium (4.4 ml) without agar was added to flasks (75 square cm) containing 40.0 ml DMEM agar stored in either the dark or the light, and flasks were incubated at 20oC on a rocking platform in the dark for 24 hours (liquid DMEM: agar DMEM ratio = 1:10). The liquid medium samples were removed and seeded with MG95 cells for grown curve assays (panel A); or incubated at various temperatures as shown on the bar graph for 90 minutes (panel B), or subjected to dialysis (three 24-hour changes, panel C) against growth-permissive liquid DMEM using molecular weight cutoff (MWCO) tubing with pore sizes as shown. The liquid samples (neat, panel A; heated, panel B; or dialyzed, panel C) were seeded with approximately 104 MG95 cells/ml (final cell density) and colony counts were done at the times shown (panel A), or 3 days later (panels B and C). Panel A shows a representative of 10 experiments (medium from flasks stored in the dark, circles; medium from flasks stored in the light, triangles). Panel B shows results of one of three experiments giving the same results. Panel C shows data compiled from six experiments. The light-sensitive inhibitory activity of liquid DMEM afforded the opportunity to test additional properties. Heating up to 100oC for 90 minutes failed to affect MG95 growth-inhibition (Fig. 5B). Exhaustive dialysis of inhibitory liquid DMEM against large volumes of growth-permissive liquid DMEM showed that the inhibitor(s) was retained in tubing <100 Dalton pore size, but was partially dialyzed through 500 MWCO tubing, and completely dialyzed through 6–8 kilodalton tubing (Fig. 5C). Effect of light-sensitive DMEM agar inhibition on other yeast species Species and strains of four Genera (Cryptococcus, Candida, Saccharomyces, and Rhodotorula) were tested on DMEM agar plates stored in the light and dark. All species tested gave high CFE on plates stored in the light. We observed three types of results for plates stored in the dark (Table 2). The first was typical for the MG95 strain giving zero to very low CFE. This pattern was characteristic for Cryptococcus species. The second was typical for Candida species. Candida albicans strains gave high CFE on dark-stored DMEM plates. Not all Candida species conformed to this phenotype. Candida lusitaniae, C. kefyr, and C. zeylanoides were identical to the Cryptococcus phenotype. A third type occurred with Candida quilliermondii where we observed moderately low but consistent CFE. We selected eight colonies of C. quilliermondii survivors on DMEM agar stored in the dark, purified them separately through four passages under nonselecting conditions (SGA), and replated on DMEM agar stored in darkness. CFE for these subclones was not different form the parent strain ruling out a subpopulation of resistant cells in the original strain. Table 2. Growth of fungal species on DMEM agar plates stored in the light and in the dark. Genus . Species . No. strains testeda . % CFE on DMEM agar (stored in the dark)b . Cryptococcus neoformans 14 0–40 var. neoformans neoformans 2 0 var. gattii albidus 1 0 laurentii 1 0 uniguttulatus 1 2 Candida albicans 3 90–100 parapsilosis 1 71 tropicalis 1 84 krusei 1 60 guilliermondii 1 28 lusitaniae 1 0 glabrata 1 99 kefyr 1 0 paratropicalis 1 100 zeylanoides 1 0 Saccharomyces cerevisiae 2 0–7 Rhodotorula rubra 2 86–100 Genus . Species . No. strains testeda . % CFE on DMEM agar (stored in the dark)b . Cryptococcus neoformans 14 0–40 var. neoformans neoformans 2 0 var. gattii albidus 1 0 laurentii 1 0 uniguttulatus 1 2 Candida albicans 3 90–100 parapsilosis 1 71 tropicalis 1 84 krusei 1 60 guilliermondii 1 28 lusitaniae 1 0 glabrata 1 99 kefyr 1 0 paratropicalis 1 100 zeylanoides 1 0 Saccharomyces cerevisiae 2 0–7 Rhodotorula rubra 2 86–100 aYeast strains grown in YNB at either 35oC or 20oC to stationary phase, diluted in PBS to 100–200 cfu/100 microliters and plated on DMEM agar stored in either light or dark environments. Colonies were counted 3–6 days later. All yeast strains gave approximately 100% CFE on DMEM agar stored in the light (3400 lumens/m2). bResults compiled from 15 experiments. Each species was tested at least 3 times. Open in new tab Table 2. Growth of fungal species on DMEM agar plates stored in the light and in the dark. Genus . Species . No. strains testeda . % CFE on DMEM agar (stored in the dark)b . Cryptococcus neoformans 14 0–40 var. neoformans neoformans 2 0 var. gattii albidus 1 0 laurentii 1 0 uniguttulatus 1 2 Candida albicans 3 90–100 parapsilosis 1 71 tropicalis 1 84 krusei 1 60 guilliermondii 1 28 lusitaniae 1 0 glabrata 1 99 kefyr 1 0 paratropicalis 1 100 zeylanoides 1 0 Saccharomyces cerevisiae 2 0–7 Rhodotorula rubra 2 86–100 Genus . Species . No. strains testeda . % CFE on DMEM agar (stored in the dark)b . Cryptococcus neoformans 14 0–40 var. neoformans neoformans 2 0 var. gattii albidus 1 0 laurentii 1 0 uniguttulatus 1 2 Candida albicans 3 90–100 parapsilosis 1 71 tropicalis 1 84 krusei 1 60 guilliermondii 1 28 lusitaniae 1 0 glabrata 1 99 kefyr 1 0 paratropicalis 1 100 zeylanoides 1 0 Saccharomyces cerevisiae 2 0–7 Rhodotorula rubra 2 86–100 aYeast strains grown in YNB at either 35oC or 20oC to stationary phase, diluted in PBS to 100–200 cfu/100 microliters and plated on DMEM agar stored in either light or dark environments. Colonies were counted 3–6 days later. All yeast strains gave approximately 100% CFE on DMEM agar stored in the light (3400 lumens/m2). bResults compiled from 15 experiments. Each species was tested at least 3 times. Open in new tab We observed differences between species for sensitivity to DMEM inhibition by plates stored in the dark. With2 hours illumination (6800 Lux • hr), C. kefyr cells grew with high CFE (63%), whereas 6 hours illumination (20400 Lux • hr) was required to give the same result for C. lusitaniae (71% CFE; data not shown). Vitamin component of DMEM affecting anti-fungal activity Our results indicating that visible light ranging from 370 to 430 nm inactivated the inhibitory activity of DMEM agar plates led us to explore components of the medium with light absorption within this range. Particular vitamins (e.g., riboflavin, thiamine, pyridoxal, folic acid, nicotinamide) and amino acids (e.g., L-tyrosine, L-tryptophan) fulfilled this criterion. Of these compounds thiamine is an essential nutrient for growth of Cryptococcus species. DMEM agar plates without vitamins supplemented with 12 μM thiamine·HCl and stored in the light or in the dark supported high CFE identical to light-exposed complete DMEM. Individual vitamins were added back to this medium. Vitamin B6, pyridoxal, at 20 μM (standard concentration in DMEM) reconstituted fungicidal activity, which was lost after light exposure for a minimum of 5 days. DMEM agar plates containing all 8 vitamins except pyridoxal and stored in darkness never inhibited MG95 cells. Biological systems employ four different forms of vitamin B6: pyridoxine, pyridoxal, pyridoxamine and pyridoxal 5-phosphate. In add-back experiments only the aldehyde forms of the vitamin (pyridoxal and pyridoxal 5-phosphate) supported antifungal activity (Fig. 6). In agreement with this result the published recipe for DMEM contains the aldehyde form, pyridoxal.2 By varying the concentration of pyridoxal, we found that 20 μM but not 2.0 μM was sufficient. Light exposure in these experiments required to support high CFE was significantly increased. This result raised the possibility that when DMEM was formulated by us using pyridoxal from a freshly prepared solution, greater antifungal activity was obtained compared to commercial sources of complete DMEM or laboratory-prepared DMEM supplemented with a commercial vitamin mixture. Figure 6. Open in new tabDownload slide Effect of vitamin B6 congeners on inhibition of MG95 cell growth on DMEM agar plates stored in the dark. DMEM agar plates containing no vitamin B6 (diamonds), pyridoxine (upward triangles), pyridoxal (circles), or pyridoxamine (inverted triangles), at 20 μM, were prepared and stored in the light (not shown) or in the dark (shown). On the days indicated one plate from the two storage conditions was seeded with 100–200 MG95 cells. After incubation at 35oC (4–7 days) colonies were counted and CFE was calculated. All plates stored in the light gave high CFE (70–110%). Figure 6. Open in new tabDownload slide Effect of vitamin B6 congeners on inhibition of MG95 cell growth on DMEM agar plates stored in the dark. DMEM agar plates containing no vitamin B6 (diamonds), pyridoxine (upward triangles), pyridoxal (circles), or pyridoxamine (inverted triangles), at 20 μM, were prepared and stored in the light (not shown) or in the dark (shown). On the days indicated one plate from the two storage conditions was seeded with 100–200 MG95 cells. After incubation at 35oC (4–7 days) colonies were counted and CFE was calculated. All plates stored in the light gave high CFE (70–110%). Amino acid component of DMEM required for antifungal activity We found that when L-cystine (DMEM standard concentration = 200 μM)2 was deleted, fungicidal activity was lost from DMEM stored in the dark. Increasing L-cystine concentration to 1.0 mM decreased the time required for dark-storage to produce antifungal activity (Fig. 7). In a sequence of photos (Fig. 8) DMEM agar plates without L-cystine stored in the dark showed tiny colonies after the first 2–3 days incubation (not easily seen in Fig. 8 but readily visible using a hand lens). As the incubation time was extended (days 4–5), large colonies developed at high CFE equal to DMEM stored in the light. The inhibitory effect of L-cystine-free DMEM plates stored in the dark seen on days 2 and 3 in Figure 8 is unexplained and was inconsistent (see Fig. 7, day 3). Possibly pyridoxal reacted with an endogenous disulfide leading to slowing for growth rate. Consistent with all previous experiments, L-cystine-free medium stored in the light supported CFE with the same efficiency and growth kinetics as complete DMEM agar medium stored in the light. Figure 7. Open in new tabDownload slide Effect of L-cystine concentration on fungicidal activity of DMEM agar plates stored in the dark. DMEM agar plates were prepared without L-cystine (inverted triangles), with 200 μM L-cystine (upward triangles), or with 1.0 mM L-cystine (circles) and then stored in the light (not shown) or the dark (shown). On the days indicated one plate from each storage condition was seeded with 100–200 MG95 cells and incubated at 35°C in the dark. Plates giving no growth were incubated for 14 days total and then 24-mm agar slabs from the plates were transferred to SGA plates. No colonies were recovered. All plates stored in the light gave high CFE (40–110%). Figure 7. Open in new tabDownload slide Effect of L-cystine concentration on fungicidal activity of DMEM agar plates stored in the dark. DMEM agar plates were prepared without L-cystine (inverted triangles), with 200 μM L-cystine (upward triangles), or with 1.0 mM L-cystine (circles) and then stored in the light (not shown) or the dark (shown). On the days indicated one plate from each storage condition was seeded with 100–200 MG95 cells and incubated at 35°C in the dark. Plates giving no growth were incubated for 14 days total and then 24-mm agar slabs from the plates were transferred to SGA plates. No colonies were recovered. All plates stored in the light gave high CFE (40–110%). Figure 8. Open in new tabDownload slide L-cystine is required for fungicidal, but not fungistatic activity of DMEM agar plates stored in the dark. Photographs of the time course (days shown) of colony development on DMEM agar plates with (left-sided plates in each panel), and without (right-sided plates of each panel), L-cystine. Plates were stored in light (3400 lumens/m2, top plates of each panel) or dark (bottom plates of each panel) for 9 days. Then they were seeded with 100–200 MG95 cells on day 0 and incubated at 35oC. On the days shown the plates were removed briefly (< 3 minutes) for photography. Although they grew more slowly, as the sequence shows, DMEM agar plates lacking L-cystine stored in the dark eventually supported colony formation with high efficiency. Figure 8. Open in new tabDownload slide L-cystine is required for fungicidal, but not fungistatic activity of DMEM agar plates stored in the dark. Photographs of the time course (days shown) of colony development on DMEM agar plates with (left-sided plates in each panel), and without (right-sided plates of each panel), L-cystine. Plates were stored in light (3400 lumens/m2, top plates of each panel) or dark (bottom plates of each panel) for 9 days. Then they were seeded with 100–200 MG95 cells on day 0 and incubated at 35oC. On the days shown the plates were removed briefly (< 3 minutes) for photography. Although they grew more slowly, as the sequence shows, DMEM agar plates lacking L-cystine stored in the dark eventually supported colony formation with high efficiency. Effect of sulfhydryl compounds added to DMEM agar plates stored in darkness We found that sulfhydryl compounds, when added to DMEM agar plates stored in the dark, inhibited fungicidal activity. Moreover inorganic compounds in the reduced oxidation state of sulfur also inhibited this activity (Table 3). None of the added sulfhydryl compounds at the concentrations shown affected CFE of DMEM agar plates stored in the light (data not shown in Table 3). Table 3. Effect of sulfur compounds added to DMEM medium.a Additive . Concentration (mM) . % CFE on DMEM agarb . L-cysteine 100 63 10 57 1.0 0 Glutathione (reduced) 100 92 10 58 1.0 0 L-homocysteine 1000 82 Djenkolic acid 10 0 Sulfinic acid 10 0 2-Mercaptoethanol 1000 92 100 60 10 20 1.0 0 Dithiothreitol 1000 83 100 0 Na2SO4 10 0 Na2S2O3 10 0 Na2SO3 10 82 1.0 98 0.1 0 Additive . Concentration (mM) . % CFE on DMEM agarb . L-cysteine 100 63 10 57 1.0 0 Glutathione (reduced) 100 92 10 58 1.0 0 L-homocysteine 1000 82 Djenkolic acid 10 0 Sulfinic acid 10 0 2-Mercaptoethanol 1000 92 100 60 10 20 1.0 0 Dithiothreitol 1000 83 100 0 Na2SO4 10 0 Na2S2O3 10 0 Na2SO3 10 82 1.0 98 0.1 0 aCompounds added to plates in concentrations shown and incubated on a rocker over night at 20oC. Ratio of volume of additive to volume of agar medium = 1:10. bCryptococci gave approximately 100% CFE on DMEM agar stored in the light and treated with the listed additives. Before plating MG95 cells, excess additive solution removed by suction. Open in new tab Table 3. Effect of sulfur compounds added to DMEM medium.a Additive . Concentration (mM) . % CFE on DMEM agarb . L-cysteine 100 63 10 57 1.0 0 Glutathione (reduced) 100 92 10 58 1.0 0 L-homocysteine 1000 82 Djenkolic acid 10 0 Sulfinic acid 10 0 2-Mercaptoethanol 1000 92 100 60 10 20 1.0 0 Dithiothreitol 1000 83 100 0 Na2SO4 10 0 Na2S2O3 10 0 Na2SO3 10 82 1.0 98 0.1 0 Additive . Concentration (mM) . % CFE on DMEM agarb . L-cysteine 100 63 10 57 1.0 0 Glutathione (reduced) 100 92 10 58 1.0 0 L-homocysteine 1000 82 Djenkolic acid 10 0 Sulfinic acid 10 0 2-Mercaptoethanol 1000 92 100 60 10 20 1.0 0 Dithiothreitol 1000 83 100 0 Na2SO4 10 0 Na2S2O3 10 0 Na2SO3 10 82 1.0 98 0.1 0 aCompounds added to plates in concentrations shown and incubated on a rocker over night at 20oC. Ratio of volume of additive to volume of agar medium = 1:10. bCryptococci gave approximately 100% CFE on DMEM agar stored in the light and treated with the listed additives. Before plating MG95 cells, excess additive solution removed by suction. Open in new tab Effect of DMEM iron concentration on agar plates stored in the dark We prepared DMEM agar plates in the laboratory from individual components without added iron. Only traces of iron are needed for MG95 cell growth, hence DMEM agar plates stored in the light with no added iron supported colony formation like plates prepared with commercial DMEM. We found that the concentration of iron affected the time it took for DMEM agar plates stored in the dark to become fungicidal. The greater the concentration of added iron, the faster the plates stored in darkness became inhibitory to yeast cell colony formation (Table 4). Variation in iron concentration did not affect fungicidal activity. That is once the plates with no added iron became inhibitory, colonies never developed despite prolonged incubation or transfer of agar slabs to SGA plates as per the protocol described in Figure 1D. Table 4. Colony-forming-efficiency of cryptococci on DMEM agar plates stored in the dark: effect of the iron concentration. Number of experiments . Iron concentration (μM)a . Time required for inhibition of colony formation (days)b . 6 0 13 ± 1.9 1 0.0025 12 5 0.25 9.6 ± 1.4 5 2.5 7.4 ± 1.2 4 25.0 4.8 ± 1.8 Number of experiments . Iron concentration (μM)a . Time required for inhibition of colony formation (days)b . 6 0 13 ± 1.9 1 0.0025 12 5 0.25 9.6 ± 1.4 5 2.5 7.4 ± 1.2 4 25.0 4.8 ± 1.8 aDMEM agar plates were prepared from individual components without added iron. Ferric chloride was added to the final concentrations shown. Because colonies developed on media without added iron, there must be some contaminating iron in the agar and chemical reagents used to prepare DMEM which is sufficient to support colony formation of the yeast cells. The standard concentration of iron in DMEM is 250 nanomolar. bNumber of days of storage in the dark after preparing the media required for complete inhibition of colony formation. Plates stored in the light gave approximately 100% CFE at all time points tested. Values are the means ± SEM for the number of experiments shown. Open in new tab Table 4. Colony-forming-efficiency of cryptococci on DMEM agar plates stored in the dark: effect of the iron concentration. Number of experiments . Iron concentration (μM)a . Time required for inhibition of colony formation (days)b . 6 0 13 ± 1.9 1 0.0025 12 5 0.25 9.6 ± 1.4 5 2.5 7.4 ± 1.2 4 25.0 4.8 ± 1.8 Number of experiments . Iron concentration (μM)a . Time required for inhibition of colony formation (days)b . 6 0 13 ± 1.9 1 0.0025 12 5 0.25 9.6 ± 1.4 5 2.5 7.4 ± 1.2 4 25.0 4.8 ± 1.8 aDMEM agar plates were prepared from individual components without added iron. Ferric chloride was added to the final concentrations shown. Because colonies developed on media without added iron, there must be some contaminating iron in the agar and chemical reagents used to prepare DMEM which is sufficient to support colony formation of the yeast cells. The standard concentration of iron in DMEM is 250 nanomolar. bNumber of days of storage in the dark after preparing the media required for complete inhibition of colony formation. Plates stored in the light gave approximately 100% CFE at all time points tested. Values are the means ± SEM for the number of experiments shown. Open in new tab Effect of other components of DMEM We tested all other amino acids, vitamins, pyruvic acid, pH indicator (phenol red), antibiotics (penicillin, gentamicin), and organic buffer (MOPS) in add-back and deletion experiments for fungicidal activity of DMEM agar plates stored in the dark. None of these had any effect. The inorganic components (other than iron) of DMEM were not tested. Discussion To select Cryptococcus sp. colony phenotypes varying in capsule production under physiological conditions, we incorporated a commonly used cell culture medium into agar.1 To our frustration, we found that plating efficiency for viable yeast cells varied from day to day. We traced this anomaly to one variable: plates stored in darkness before seeding inhibited colony growth while exposure of agar plates to incandescent light led to ∼ 100% plating efficiency (relative to the control: colony-forming units of the same inoculum on SGA agar plates). We found that inhibition of colony formation was due to fungicidal activity of DMEM agar plates stored in the dark. All components of DMEM plates are defined (except agar) and without exception are nutrients, unlikely to cause death of fungi. Because of its unique property and because elucidation thereof might yield a non-toxic therapeutic prototype, we sought to define and identify the fungicidal activity of DMEM. To do so we developed standard conditions for quantification. We searched the literature for reports describing this phenomenon. We used search words: “antifungal,” “fungistatic,” “fungicidal,” “inhibition of growth,” “Dulbecco's modified eagle's medium,” “tissue culture medium,” “cell culture medium,” singly or in combinations. Our search failed to reveal any publications describing or resembling data from this report. On the contrary several publications cite inhibitory activities of medium exposed to light due to generation of toxic products from light-induced photodynamic reactions of medium constituents.9–16 By employing filters downstream of incident incandescent light, we determined that a band of visible light approximately 370–430 nm was responsible for destroying fungicidal activity of DMEM agar plates stored in darkness. In a series of 10 experiments we varied the time of white light exposure or the amount of white light passing through attenuating filters at a constant time of exposure and found a threshold required to reverse fungicidal activity. This was approximately 8.9 kilolux for 1 hour at 550 nm. The inhibitory effect was pH-dependent requiring physiological H+ concentration. Fungicidal activity was diffusible into liquid DMEM kept in darkness. The activity was heat-stable (100oF) and dialyzable down to 0.5 Kd but retained within MWCO tubing of <0.1 Kd. Testing a panoply of pathogenic yeast species showed that some were completely inhibited while others were unaffected. Candida quilliermondii gave populations of resistant and susceptible cells. We traced fungicidal activity to two components of the chemically-defined DMEM recipe. These were: pyridoxal (vitamin B6) and L-cystine (the oxidized disulfide form of the non-essential amino acid). We found that iron, in presumably catalytic concentrations, lessened the time of dark-storage of DMEM agar plates to develop their fungicidal effect. We found that a variety of sulfhydryl compounds bearing reduced sulfur moiety inhibited the fungicidal activity. We speculate that Shiff base formation between the aldehyde group of pyridoxal and the α-amino groups of L-cystine or the sulfur atoms of cystine's disulfide linkage form an inhibitory (and fungicidal) molecule. This product absorbs visible light in the blue range of the spectrum in a photodynamic reaction which destroys it biological activity. The inhibitory compound is unstable in liquid DMEM, stored in darkness, but in some way, incorporation of DMEM into agar results in a stabilizing effect once the pyridoxal-cystine reaction proceeds in the dark. Fungicidal activity of the complex may be destroyed by electrochemical reduction with electrons supplied from sulfhydryl compounds or reduced inorganic sulfur in a fashion similar to the photochemical reaction occurring with exposure to light. We believe that the findings reported here may have two ramifications for medical mycology research. First, DMEM may be the culture medium employed for studies on interaction of mammalian host defense cells and/or fluid phase mediators with cryptococci in vitro.17–20 This may also apply to studies on environmental cues which regulate virulence factors such as capsule synthesis.21–23 The question arises: Could the inhibitory factor described here confound results of cytotoxicity experiments carried out with medium never exposed to light? We think that this is unlikely because of the instability of the putative pyridoxal-cystine complex in liquid DMEM as opposed to DMEM incorporated into agar. However sub-inhibitory concentrations possibly present in dark-stored liquid DMEM might conceivably perturb experiments on the induction of virulence factors by various stimuli, e.g. CO2, Fe+3. In this context it is worth noting that RPMI cell culture medium is used by some groups for studying phagocyte-cryptococcus interactions in vitro.24–29 RPMI medium contains cystine but vitamin B6 is supplied as the alcohol form, pyridoxine. Consequently, from the data shown here, we would predict that RPMI incorporated into agar would lack fungistatic/fungicidal activity under any storage conditions. Second, albeit unstable, the putative pyridoxal-cystine complex could provide a prototype molecule, which when stabilized, might lead to discovery of a class of anti-fungal compounds useful for medical or agricultural purposes. Acknowledgments Ms. Helene Segal provided species of fungi from ARUP Clinical Mycology Laboratory of the University of Utah. C. Andrew Lloyd (Eastman Kodak Co.) kindly provided guidance for light-attenuating and light-partitioning filters. Dr. John Perfect (Duke University) provided H99 strain of Cryptococcus neoformans. We gratefully acknowledge Dr. Molly Leecaster for statistical analysis of data presented in Figure 3. Drs. John Hibbs, Jr., Neil Bastian, and Paul Shami provided valuable advice. Drs. Zell McGee and George G. Jackson generously reviewed the manuscript and gave cogent suggestions. Funding support The authors thank the Willard L. Eccles Charitable Foundation (#647385) for providing funds to pursue this project. A Technology Innovation Grant (#51000914) from the University of Utah is also gratefully acknowledged. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. References 1. Granger DL , Perfect JR, Durack DT. Virulence of Cryptococcus neoformans: regulation of capsule synthesis by carbon dioxide . J Clin Invest . 1985 ; 76 : 508 – 516 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Committee TCS . Tissue culture media . In Vitro . 1970 ; 6 : 273 – 277 . Google Scholar OpenURL Placeholder Text WorldCat 3. Eagle H . Nutrition needs of mammalian cells in tissue culture . Science . 1955 ; 122 : 501 – 514 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Smith JD , Freeman G, Vogt M et al. The nucleic acid of polyoma virus . Virology . 1960 ; 12 : 185 – 196 . Google Scholar Crossref Search ADS WorldCat 5. Granger DL , Perfect JR, Durack DT. Macrophage-mediated fungistasis in vitro: requirements for intracellular and extracellular cytotoxicity . J Immunol . 1986 ; 136 : 672 – 680 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 6. Granger DL , Perfect JR, Durack DT. Macrophage-mediated fungistasis: requirement for a macromolecular component in serum . J Immunol . 1986 ; 137 : 693 – 701 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 7. Kwon-Chung KJ , Bennett JE, Wickes BL et al. The case for adopting the “species complex” nomenclature for the etiologic agents of cryptococcosis . mSphere . 2017 ; 2 : e00357 – 168 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Kwon-Chung KJ , Polacheck I, Bennett JE. Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C) . J Clin Microbiol . 1982 ; 15 : 535 – 537 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 9. Wang RJ . Effect of room fluorescent light on the deterioration of tissue culture medium . In Vitro . 1976 ; 12 : 19 – 22 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Wang RJ , Nixon BR. Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with “daylight” fluorescent light . In Vitro . 1978 ; 14 : 715 – 722 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Hoffman PS , Pine L, Bell S. Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal . Appl Environ Microbiol . 1983 ; 45 : 784 – 791 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 12. Pine L , Hoffman PS, Malcolm GB et al. Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species . J Clin Microbiol . 1986 ; 23 : 33 – 42 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 13. Grzelak A , Rychlik B, Bartosz G. Light-dependent generation of reactive oxygen species in cell culture media . Free Radic Biol Med . 2001 ; 30 : 1418 – 1425 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Lewinska A , Wnuk M, Slota E et al. Total anti-oxidant capacity of cell culture media . Clin Exp Pharmacol Physiol . 2007 ; 34 : 781 – 786 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Smijs TG , Bouwstra JA, Schuitmaker HJ et al. A novel ex vivo skin model to study the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment in different growth phases . J Antimicrob Chemother . 2007 ; 59 : 433 – 440 . Google Scholar Crossref Search ADS PubMed WorldCat 16. Waterworth PM . The action of light on culture medium . J Clin Pathol . 1969 ; 22 : 273 – 277 . Google Scholar Crossref Search ADS PubMed WorldCat 17. O’Meara TR , Alspaugh JA. The Cryptococcus neoformans capsule: a sword and a shield . Clin Microbiol Rev . 2012 ; 25 : 387 – 408 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Pascon RC , Ganous TM, Kingsbury JM et al. Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence . Microbiology . 2004 ; 150 : 3013 – 3023 . Google Scholar Crossref Search ADS PubMed WorldCat 19. Coelho C , Tesfa L, Zhang J et al. Analysis of cell cycle and replication of mouse macrophages after in vivo and in vitro Cryptococcus neoformans infection using laser scanning cytometry . Infect Immun . 2012 ; 80 : 1467 – 1478 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Samantaray S , Correia JN, Garelnabi M et al. Novel cell-based in vitro screen to identify small-molecule inhibitors against intracellular replication of Cryptococcus neoformans in macrophages . Int J Antimicrob Agents . 2016 ; 48 : 69 – 77 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Coenjaerts FE , Hoepelman AI, Scharringa J et al. The Skn7 response regulator of Cryptococcus neoformans is involved in oxidative stress signalling and augments intracellular survival in endothelium . FEMS Yeast Res . 2006 ; 6 : 652 – 661 . Google Scholar Crossref Search ADS PubMed WorldCat 22. McFadden DC , Fries BC, Wang F et al. Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans . Eukaryot Cell . 2007 ; 6 : 1464 – 1473 . Google Scholar Crossref Search ADS PubMed WorldCat 23. Zaragoza O , Fries BC, Casadevall A. Induction of capsule growth in Cryptococcus neoformans by mammalian serum and CO(2) . Infect Immun . 2003 ; 71 : 6155 – 6164 . Google Scholar Crossref Search ADS PubMed WorldCat 24. Ballou D , Palmer G, Massey V. Direct demonstration of superoxide anion production during the oxidation of reduced flavin and of its catalytic decomposition by erythrocuprein . Biochem Biophys Res Commun . 1969 ; 36 : 898 – 904 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Davis MJ , Eastman AJ, Qiu Y et al. Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence . J Immunol . 2015 ; 194 : 2219 – 2231 . Google Scholar Crossref Search ADS PubMed WorldCat 26. Ghosn EE , Russo M, Almeida SR. Nitric oxide-dependent killing of Cryptococcus neoformans by B-1-derived mononuclear phagocyte . J Leukoc Biol . 2006 ; 80 : 36 – 44 . Google Scholar Crossref Search ADS PubMed WorldCat 27. Leopold Wager CM , Hole CR, Wozniak KL et al. STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans . Infect Immun . 2015 ; 83 : 4513 – 4527 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Monari C , Pericolini E, Bistoni G et al. Cryptococcus neoformans capsular glucuronoxylomannan induces expression of fas ligand in macrophages . J Immunol . 2005 ; 174 : 3461 – 3468 . Google Scholar Crossref Search ADS PubMed WorldCat 29. Murphy JW , Hidore MR, Wong SC. Direct interactions of human lymphocytes with the yeast-like organism , Cryptococcus neoformans. J Clin Invest. 1993 ; 91 : 1553 – 1566 . Google Scholar Crossref Search ADS WorldCat Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology 2018.
TI - Combination of nutrients in a mammalian cell culture medium kills cryptococci
JF - Medical Mycology
DO - 10.1093/mmy/myy040
DA - 2019-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/combination-of-nutrients-in-a-mammalian-cell-culture-medium-kills-dwnTXaiN1m
SP - 374
EP - 383
VL - 57
IS - 3
DP - DeepDyve
ER -