Preparation of reagents
Reagent 1: 45g of NaOH was added to 150 µL of methanol and150 mL of distilled water. The reaction
mixture was mixed thoroughly.
Reagent 2: 325 mL of 6N HCl and
275 mL of methyl alcohol was mixed thoroughly.
Reagent 3: 200 mL of hexane and 200 mL of
methyl-tert-butyl-ether was mixed.
Reagent 4: 10.8 g of NaOH was
dissolved in 900 mL of distilled water.
For the fatty acid analysis, biomass was
harvested from strain and reference strains in logarithmic
growth phase on R2A plate after incubation at appropriate temperature. Fatty acids were
saponified, methylated, and extracted using the standard protocol of MIDI
(Sherlock Microbial Identification System, version 6.0B). The fatty acids were
analysed with a gas chromatograph (HP 6890 series GC system; Hewlett Packard)
and identified using the TSBA6 database of the Microbial Identification System
(Sasser, 1990).
The detailed processes:
- Harvesting:
A 4 mm loop was used to harvest about 40 mg of bacterial cells from the third
quadrant of the quadrant streaked plate. The cells were put in a clean dry
13x100 culture tube.
- Saponification:
1 mL of Reagent 1 was added to each tube containing cells. The tubes
were securely sealed with telfon lined caps, vortexed briefly and heated in a
boiling water bath for 5 min, and again the tubes were vigorously vortexed for
5-10 seconds and returned to the water bath to complete 30 min heating.
- Methylation:
The cooled tubes are uncapped and 2 mL of reagent 2 was added. The tubes were
capped and vortexed briefly. After proper vortexing, the tubes were heated for
10 ±1 min at 80°C ± 1°C.
- Extraction: 1.25 mL of Reagent
3 was added to the cooled tubes followed by recapping and gentle
tumbling on a clinical rotator for about 15 min. Bottom phase were removed and
top phase were saved.
- Base wash: About 3 mL of Reagent
4 was added to the organic phase remaining in the tubes, tubes were
rotated for 5 min, about 5mL of saturated NaCl was added and tubes were rotated
for 2 min. About 2/3 of the top phase was transferred to GC vile for
analysis.
Naming of the pick
and profile comparison to database were carried out by gas chromatography.
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