To study the colony morphology, sporulation of the pure cultures was done by transferring a mycelial plug to 2% water agar (20g/L dH2O
) mixed with sterile host material (citrus), branch sections (Mehl et al., 2011) and leaves (Inderbitzin et al., 2010).
Separation was performed using a gradient elution with mobile phase A consisting of acetonitrile, and mobile phase B consisting of DH2O
at a flow-rate of 1.0 ml/min.
Aliquots of 10X PTZ stock solution (75 mM) were prepared by dissolving via 60 s vortex 0.311 g of PTZ (Sigma) into 30 mL dH2O
. Stock solutions were stored overnight at -20 [degrees]C and used the next day.
The slides were washed with dH2O
. Hematoxylin restaining, dehydration, transparent, sheet sealing, and microscopic examination were performed.
The volume was made up with double distilled water (dH2O
1 medium for actinomycetes, and Martin's Rose Bengal medium for fungi (Martin, 1950), and an improved selective medium (K2HPO4 1 g, KCl 0.5 g, MgSO4 0.5 g, Na-EDTA 0.01 g, pentachloronitrobenzene 0.05 g, L-asparagine 2 g, bile salt from ox 0.5 g, L-sorbose 2 g, sodium tetraborate 1 g, dH2O
1000 mL, pH 5.4, and 1% streptomycin stock solution 0.3 mL after autoclaving sterilization when the mixture cooled to 55degC) for V.
The supernatant was removed by pipette; the pellets were then suspended with 4 mL of dH2O
. After that, 10 mL of methanol and 5 mL of chloroform were added in sequence, resulting in a 10:5:4 ratio of methanol: chloroform: water.
cDNA (1 [micro]L) was added in a 25 [micro]L reaction mixture which contained 12.5 [micro]L SYBR Premix Ex Taq II (Tli RNaseH Plus) (2x), 10 [micro]M each primer, 2 [micro]L cDNA and 8.5 [micro]L dH2O
. The program setting was 30 s at 95[degrees]C and then 40 cycles of 95[degrees]C for 5 s, 60[degrees]C to 62[degrees]C (depending on the primers used) for 30 s, and 72[degrees]C for 30 s.
Each tray was containing 100 third-instar larvae in 1000 ml dH2O
infused with various concentrations of the bacterial extract according to Rey et al.
Reagent Quantity 5x PrimeScript Buffer (for real time) 2.0 [micro]L PrimeScript RT Enzyme Mix I 0.5 [micro]L Oligo dT Primer (50 [micro]M) 0.5 [micro]L Random 6 mers (100 [micro]M) 0.5 [micro]L Total RNA X RNase Free dH2O
UP TO 10 [micro]L TABLE 2: mRNA primer design for the key metabolic enzymes in liver.
Amplification was performed in a total volume of 20 [micro]L containing 0.4 [micro]L of PCR forward primer and reverse primer, 0.4 [micro]L of ROX reference dye II, 10 [micro]L of SYBR Premix Ex Taq, 6.8 [micro]L of dH2O
, and 2.0 [micro]L of DNA.
The 3% polyacrylamide gel was made with 15 mL 5x TBE, 120 mL dH2O
, 11 mL 40% bisacrylamide, 110 [micro]L TEMED, and 1 mL 10% APS.