Viability of Hirsutella spp. strains preserved in mineral oil

Serious mycological work requires a reliable source of cultures that are maintained under safe long-term storage. In this study, 1186 clinical fungal isolates consisting of molds (20 species in 11 genera) and yeasts (21 species in seven genera) maintained in water, under mineral oil at room temperature and cryopreserved at -80°C for periods ranging from 1 to 12years, were evaluated for their viabilities and stabilities. The strains were subcultured onto either Sabouraud dextrose agar or potato dextrose agar to determine the viabilities and purities. The stabilities of the dermatophytes were investigated using urease test medium, the Trichophyton agar test and morphological examination. The stabilities of yeasts were evaluated by microscopic morphology and by determining the antifungal susceptibilities of random samples of yeasts (n=120). Additionally, 365 strains (dermatophytes, n=115; yeasts, n=250) were further characterized by "matrix-assisted laser desorption/ionization time-of-flight mass spectrometry." After 12years of preservation, the survival rates with the three different preservation techniques, i.e., in water, under mineral oil and by freezing, were assessed as 94.7, 82.0 and 97.4%, respectively. Viability was generally unrelated to the duration of storage. More stable and consistent growth was achieved after storage in water and freezing compared with mineral oil preservation. Our results demonstrate that the procedure for maintaining fungal cultures in water is a simple and inexpensive method, next to cryopreservation, and that both can be reliably used for the long-term preservation of most fungal isolates.

Influence of Culture Medium on the Sporulation and Viability of Aspergillus spp. and Talaromyces spp. Entomopathogenic Fungi. The purpose of this study was to determine the effect of three kinds of cultures media on the spore production and viability of Aspergillus spp. (AS1, 6, 7, 9) and Talaromyces spp. (AS2–5, 8, 10) entomopathogenic fungi. This study was arranged using Factorial-Completely Randomized Design (CRD) with 2 factors and 3 replications. The first factor was three kinds of cultures media (potato dextrose agar (PDA), corn meal agar (CMA), and sabouraud dextrose agar (SDA)) and the second one was isolates of Aspergillus spp. Or Talaromyces spp.. Data of spore production and spore viability were tested using ANOVA and if there was significantly difference, the data then further analyzed using Tukey‘s Honestly Significant Difference (HSD) test at 5% of significant level. The spore production of Aspergillus spp. were in the range of 0.58 - 14.27 x 108 spores mL-1 (PDA); 0.28 – 2.68 x 108 spores mL-1 (SDA) and 1.85 - 5.33 x 108 spores mL-1 (CMA). The highest spore production was achieved by AS1 isolate that was grown on PDA media. The spore produced by Talaromyces spp. were in the range of 2.15 – 28.62 x108 spores mL-1 (PDA); 0.28 – 29.43 x108 spores mL-1 (SDA); and 1.88 – 16.63 x108 spores mL-1 (CMA). The highest spore production was produced by AS8 isolate which were cultured on PDA. The spore viability among isolates of the two entomopathogenic fungi were not significantly different. The spore viability of Aspergillus spp. was in the range of 95.10 – 97.66% (PDA), 94.02 – 98.45% (SDA) and 92.86 – 98.20% (CMA). The spore viability of Talaromyces spp. was in the range of 95.83 – 100% (PDA), 85.83 – 100% (SDA), and 90.75 – 100% (CMA). Culture medium influenced spore production but not the spore viability. The best culture media used for spore production of both of the entomopathogenic fungi was PDA media.

Nutritional Influences on VolvariellaVolvacea Growth in Puerto Rico. II: Vitamins, Oils and pH

We investigated the effect of nutritional and environmental factors on Ophiocordyceps longissima mycelial growth. The longest colony diameter was observed on Schizophyllum (mushroom) genetics complete medium plus yeast extract, Schizophyllum (mushroom) genetics minimal medium, and Sabouraud dextrose agar (SDA); however, malt-extract yeast-extract agar, SDA plus yeast extract, yeast-extract malt-extract peptone dextrose agar, SDA, oatmeal agar, and potato dextrose agar showed higher mycelia density. A temperature of 25°C was optimum and 7.0 was the optimum pH for mycelial growth. Colony diameter was similar under light and dark conditions. Maltose and yeast extract showed the highest mycelial growth among carbon and nitrogen sources respectively. The effect of mineral salts was less obvious; however, K3PO4 showed slightly better growth than that of the other mineral salts tested. Among all nutrition sources tested, complex organic nitrogen sources such as yeast extract, peptone, and tryptone were best for mycelial growth of O. longissima. Ophiocordyceps longissima composite medium, formulated by adding maltose (2% w/v), yeast extract (1% w/v), and K3PO4 (0.05% w/v) resulted in slightly longer colony diameter. In vitro mycelial O. longissima growth was sustainable and the production of fruiting bodies could be used for commercial purposes in the future.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMFungus collection is an essential approach to allow future studies, including molecular, and epidemiological characteristics, virulence factors, susceptibility profile, and others. Ideal methods should keep the isolates viable for long periods of storage. The mineral oil method is theoretically a secure and efficient method for the storage of filamentous and dimorphic fungi, including Sporothrix spp., for long periods.ObjectiveWe aimed to evaluate the recovery rate of isolates of Sporothrix spp. maintained for years through the mineral oil technique in a fungal collection.MethodsSporothrix spp. isolates were originally cultivated in tubes with potato dextrose agar, incubated at 30°C for at least 7 days, and covered by mineral oil, until around 1 cm above the surface, after the growth and sporulation of the colonies. Tubes were maintained at room temperature (∼25°C). A total of 238 isolates of Sporothrix spp. (mostly S. brasiliensis) maintained for 4-108 months (mean of 49.7 months) were included in the study, being 191 isolates from cats (80%), 31 from humans (13%), 14 from dogs (6%), and 2 (1%) from the environment. To evaluate the rate of recovery, a fragment of the colonies was cultivated in duplicate in plates with Sabouraud dextrose agar and incubated at 25°C until the fungal growth or for 30 days. To analyze the results, frequency, mean, and standard deviation (SD) were calculated, as well and the Kruskal-Wallis test was performed, using the SPSS statistical program.ResultsOnly 14% (n = 34) of the fungal isolates were recovered from oil minerals after a mean of 64 months of storage (SD = 18.4), being 82% (n = 28) of the recovered isolates from cats, 12% (n = 4) from humans, and 6% (n = 2) from dogs (n = 1), or environment (n = 1). The 204 isolates that could not be recovered, were stored for a mean of 47 months (SD = 25.5) (P ˃ 0.05).ConclusionDisagreeing with previous reports, mineral oil was a failure method to keep Sporothrix spp. isolates viable independently of the period of storage. Therefore, optimization of this methodology is necessary, and/or other methods must be implemented to guarantee the preservation of Sporothrix spp. isolates in fungal collections.

Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF)

ABSTRACTThe effect of four routine methods of preservation of bacteria (in Dorset medium, under mineral oil, freeze-drying, cryoconservation in liquid nitrogen) on the biochemical characteristics of 11 E. coli strains from the collection of NBIMCC, was followed up. After 1 and 3 years of preservation, half of the strains changed their biochemical activity, 33 % of the parameters examined having turned into negative. The biochemical characteristics of the strains studied turned out to best preserved in Dorset medium and under mineral oil. Freeze-drying is appropriate for long-term preservation but it requires additional following up of the biochemical parameters.

Viability, purity, and genetic stability of entomopathogenic fungi species using different preservation methods

EUCAST Definitive Document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts: Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)∗

Biosurfactant production by Pseudomonas guguanensis D30 was reported using mineral oil in submerged condition. Twelve medium components were tested at two levels by Plackett–Burman design, among them, mineral oil, yeast extract, peptone, MgSO4, and CaCl2 found significant on the basis of emulsification index. These five significant components were further optimized through central composite design (CCD). The experimental design was successfully used for regression analysis and the significant model suggested the solution of 10% (v/v) mineral oil, 3.0 g/L (w/v) yeast extract and 0.2 g/L (w/v) peptone for 13.14 g/L predicted biosurfactant production. We kept the suggested concentrations of medium components and got 13.34 ± 0.08 g/L biosurfactant production, which is almost double the conventional one-factor-at-a-time production (7.126 ± 0.12 g/L). It reduced the surface tension of the medium up to 28 ± 1.2 mN/m. We found ethyl acetate a suitable solvent for biosurfactant extraction amongst methanol, chloroform, and methanol:chloroform. The partially purified biosurfactant was chemically characterized as lipopeptide by Fourier transform infrared spectroscopy (FT-IR).

The effects of temperature and mycological media on mycelial growth and estimates of spore production of an indigenous entomopathogenic fungus, Isaria sp., found during natural epizootics on whiteflies in the Lower Rio Grande Valley of Texas, were investigated. The radial growth (mm/day) of Isaria sp. as a function of temperature fits a linear model; with faster growth on Sabouraud dextrose agar with yeast extract, SDAY slopes (0.23) than on Sabouraud maltose agar, SMA slopes (0.14) from 20 to 30 degrees C, with an optimal temperature of 30 degrees C (SDAY: 4.1 mm, SMA: 3.1 mm). Moderate growth occurred at 25 degrees C (SDAY: 3.4 mm, SMA: 2.7 mm). Growth was lowest at 20 degrees C (SDAY: 1.9 mm, SMA: 1.8 mm). No fungal growth was observed at 35 degrees C and 40 degrees C. However, when Isaria sp. was exposed to 35 degrees C for the first 7 days, it could recover and grow when transferred to 25 degrees C (SDAY: 3.5 mm, SMA: 2.8 mm). No recovery or growth occurred after transfer from 40 degrees C to 25 degrees C. The average conidial production on SDAY after 20 days incubation at 25 degrees C and a photoperiod of 14:10 h light: dark was 1.2 x 10(8) conidia/cm(2) with 100% spore viability. When compared on SDAY at 25 degrees C, the radial growth rate of I. javanica ex type CBS 134.22 (5.1 mm/day) was greater than seven Isaria isolates including Isaria sp.; but maximum growth rates were similar among all related Isaria isolates (90-97%). The Isaria sp. fungus tolerates high temperatures (35 degrees C), suggesting that it is naturally selected for the subtropical semi-arid environment, where it could serve as an important natural control agent of the sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B, one of the most invasive and economically damaging insects to agriculture.

The aims of the study are to identify an effective and versatile fungal strain for bioengineering mycelium composites. The influence of temperature and four different growth media on mycelium growth of two white rot fungi, Pleurotus ostreatus (Winter Oyster) and Ganoderma lucidum (Reishi) were investigated in laboratory conditions. The results of the experiment indicated that potato dextrose agar (PDA) was the most suitable growth media for mycelium growth of fungal strains, P. ostreatus and G. lucidum. However, P. ostreatus was the better performing strain, with highest mean mycelium growth of 23.28cm, compared to G. lucidum at 9.03cm on PDA after 12 days of inoculation. Potato dextrose agar (23.28cm) and potato dextrose agar supplemented with yeast extract (14.74cm) were more favorable for mean radial mycelium growth of P. ostreatus, followed by sabouraud dextrose agar (9.85cm) and iron sulphite agar (8.35cm). The fungal strain, P. ostreatus obtained improved mycelium morphology on potato dextrose agar (PDA) supplemented with yeast extract and obtained cottony textured mycelium with good density and growth on potato dextrose agar (PDA) and sabouraud dextrose agar (SDA). Iron sulphite agar (ISA) was least favourable growth media for mean radial mycelium growth and mycelium morphology. Fungal strain, G. lucidum only mycelium growth was obtained on potato dextrose agar (PDA) as a result of this study, was the least favourable fungal strain studied. Optimal temperature for mycelium growth for both fungal strains, P. ostreatus and G. lucidum was obtained at 22 °C.

THE APPLICATION of the mineral oil conservation method to maintaining large collections of fungous cultures has been described in the preceding paper (Buell and Weston, 1947). In connection with that study it seemed desirable to determine to what extent respiratory changes were brought about in the cultures under oil. We have been unable to find any reports dealing with respiratory studies of bacteria and fungi covered with oil. Several investigations have been reported on the viability of fungous cultures preserved over a period of time by means of paraffin or mineral oil (see Buell and Weston, 1947, for bibliography) and of bacterial cultures so treated (Morton and Pulaski, 1938; Rahn and Richardson, 1941). Studies have also been made of the diffusion of oxygen through mineral and vegetable oils of various densities (Sawaya, 1945) and through various bacterial media (Rahn and Richardson, 1941). Although not bearing directly upon the present problem, it should be noted that an interesting discussion of the breakdown and synthesis of oils by marine bacteria is presented in a recent book by Zobell (1946). In the present series of experiments we have applied a micro-respirometric technique to the study of the oxygen consumption of normal and oil-covered cultures of Sordaria fimicola (Rob.) Ces. and de Not. The data so obtained provide a physiological basis for the mineral oil conservation method. MATERIAL AND METHODS.-The fungus used in this study was a strain of Sordaria fimicola, an Ascomycete, originally obtained as one of the organisms isolated from a textile exposure test conducted in Panama. A modification of the method originally described by Hansen for isolation of pure yeast (Prescott and Dunn, 1940) was used under aseptic conditions to obtain the single spore isolations with which the stock cultures were inoculated. A dilute spore suspension of Sordaria fimicola was micropipetted onto the area of potato maltose agar which had been divided into minute squares with a razor. The spores germinated in about 4-6 hours. Shortly after germination occurred the slide was removed from the chamber and placed under the microscope. A square of agar with one spore was then selected, removed from the slide by means of a sterile needle and transferred to a slant of nutrient agar. The mature perithecia of the resulting colony were used as inocula for the experimental flasks. The flasks each contained 10 cc. of potato maltose agar and were incubated at 260C. following inoculation. The oxygen consumption was measured by means

Coconut eriophyid mite Aceria guerreronis Keifer has emerged as a major pest of coconut in the country since 1998. Among the various biocontrol agents, Hirsutella thompsonii is a promising fungal pathogen used in field biosuppression of the pest. Influence of various synthetic and laboratory-derived growth media, viz., Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), Glucose Yeast Extract Agar (GYA), and Carrot Agar (CA), was compared with a locally available inexpensive substrate, coconut water on growth, and sporulation of H. thompsonii. Fungal growth characters, viz., radial growth, conidial production, and micromorphometry of the fungal mycelium and spore, were recorded. Results indicated comparable growth in coconut water media (1.91 cm/20 days) to that of PDA (1.94 cm/20 days). In solid media, laboratory-derived coconut water agar showed significantly higher spore count (12.9 × 104spores/cm3) followed by laboratory-derived PDA (11.38 × 104spores/cm3). Regarding conidial production in broth culture, maximum spore production of H. thompsonii was observed in Sabouraud Dextrose broth (51.2 × 104spores/cm3). Coconut water showed comparable spore count (18.2 × 104spores/cm3) to that of Potato Dextrose broth (19.6 × 104spores/cm3) and Glucose Yeast Extract broth (18.6 × 104spores/cm3). There was no significant difference in micromorphometric characters among the media tested. The results suggest the potential of using coconut water for mass production of H. thompsonii in the laboratory.

The genus Nocardia is a cause of infection in the lungs, skin, brain, cerebrospinal fluid, eyes, joints and kidneys. Nocardia isolation from polymicrobial specimens is difficult due to its slow growth. Several methods have been reported for Nocardia isolation from clinical samples. In the current study, we used three methods: paraffin baiting technique, paraffin agar, and conventional media for Nocardia isolation from various clinical specimens from Iranian patients. In this study, we examined 517 samples from various clinical specimens such as: sputum of patients with suspected tuberculosis, bronchoalveolar lavage, sputum of patients with cystic fibrosis, tracheal aspirate, cutaneous and subcutaneous abscesses, cerebrospinal fluid, dental abscess, mycetoma, wound, bone marrow biopsy, and gastric lavage. All collected specimens were cultured on carbon-free broth tubes (paraffin baiting technique), paraffin agar, Sabouraud dextrose agar, and Sabouraud dextrose agar with cycloheximide and were incubated at 35°C for one month. Seven Nocardia spp. were isolated with paraffin baiting technique, compared with 5 positive results with the paraffin agar technique and 3 positive results with Sabouraud dextrose agar with and without cycloheximide. The prevalence of nocardial infections in our specimens was 5.28%. In the present study, the use of the paraffin baiting technique appeared to be more effective than other methods for Nocardia isolation from various clinical specimens.