Two‐step method for isolating Cryptococcus species complex from environmental material using a new selective medium

Abstract

Summary Cryptococcosis is an opportunistic infection caused by the Cryptococcus species complex. An outbreak of cryptococcosis caused by Cryptococcus gattii (AFLP6/VGII) in North America has indicated the need for studies of this organism and its environmental niche. Difficulties in isolating the Cryptococcus spp. because of the overgrowth of filamentous fungi onto culture media and its low fungal population size under natural conditions limit studies of these pathogenic yeasts. We designed a selective medium that inhibits the growth of environmental filamentous fungi but does not inhibit that of Cryptococcus cells. After enrichment in acidified YPD media and inoculation onto selective media, Cryptococcus cells in brown‐coloured colonies were isolated from environmental materials. This two‐step method is useful for isolating environmental members of the Cryptococcus species complex, which is essential for further studies involving diversity and the microbe–environment relationship of this yeast.

Molecular epidemiology and phylogenetic analyses of environmental and clinical isolates of Cryptococcus gattii sensu lato in Taiwan

“…l . can be effectively isolated using a two‐step method, 25 some isolates with a slower growth rate during enrichment may have been neglected by this isolation method. To minimise such possibility, re‐sampling without enrichment in locations with high C .…”

“…Samples of decayed wood swabbed with cotton sticks as well as the soil samples were processed 25 . Cotton sticks, rinsed in sterile water, were used to swab the decayed wood samples.…”

“…Cotton sticks, rinsed in sterile water, were used to swab the decayed wood samples. The swabs were then placed in centrifuge tubes, containing 10 mL of acidified yeast extract‐peptone‐dextrose (YPD) broth prepared as described previously 25 . Thereafter, the cotton sticks were broken down with heads left in the centrifuge tubes.…”

“…Each rhizosphere soil sample weighing 2 g was suspended in acidified YPD broth and mixed well by shaking. Subsequently, the broth was incubated at 25°C for 24 hours and spread on benomyl and eucalyptus oil in modified dichloran rose Bengal chloramphenicol (DRBC) (BEMD) medium 25 . Colony growth was monitored daily and the brown, yeast‐like colonies were selected for identification.…”

Antifungal susceptibility of the clinical and environmental strains of Cryptococcus gattii sensu lato in Taiwan

“…The environmental strains used in this study were collected from various sites in Taiwan (Figure S1) and identified using matrixassisted laser desorption/ionisation-time of flight mass spectrometry (Bruker, Daltonik, GmbH) with Biotyper software, version 3.1, with library version 5989, as previously reported. 25 For analyses of clinical strains, 21 stored clinical C. gattii s.l.…”