Strains MUSC 135T and MUSC 137 were isolated in
December 2012 from different soil samples collected at
sites MUSC-TLS3 (3u 489 11.10 N 103u 269 6.90 E) and
MUSC-TLS4 (3u 489 21.30 N 103u 209 3.30 E) located in the
Tanjung Lumpur mangrove forest, State of Pahang,
Malaysia. Topsoil samples of the upper 20 cm layer (after
removing the top 2–3 cm) were collected and sampled into
sterile plastic bags using an aseptic metal trowel and stored
at 220 uC. Air-dried soil samples were ground with a
sterilized mortar and pestle. Selective pretreatment of soil
samples was performed using wet heat in sterilized water
(15 min at 50 uC) (Takahashi et al., 1996). Five grams
pretreated air-dried soil was mixed with 45 ml sterilized
water, ground in a mill and then spread onto the isolation
medium ISP2 (Shirling & Gottlieb, 1966) supplemented
with cycloheximide (25 mg ml 21) and nystatin (10 mg
ml21) and incubated at 28 uC for 7–14 days. Pure cultures
of isolates were maintained on slants of ISP2 medium at
4 uC for short-term storage and as glycerol suspensions
(20 %, v/v) at 220 uC for long-term preservation.