HPLC Analysis with C18 Column and PAD Detector
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Plates were kept at 25°C until the mycelia of tested fungi were fully grown in the control plate. The inhibition ______ was calculated according to Ghazy and El-Nahrawy (2021). Inhibition ______ = C - T / C imes 100$ Eq: (A) Where, C is the radial growth of fungi in control, and T is the radial growth of the fungi in the presence of the bacterial isolate.

percentage

Whereas, the diameter of the inhibition zone (mm) around the well was measured to evaluate ______ activity of antimicrobial compound in cell-free supernatant of bacterial isolates (Jia et al., 2020).

antagonistic

One bacterial isolate was selected on the basis of antagonistic activity against B. cinerea BC21. Selected isolate was investigated for several indirect mechanisms that may inhibit ______, including the production of siderophores, hydrogen cyanide, exopolysaccharides, ammonia, extracellular enzymes, and biosurfactant.

phytopathogens

The ability of bacteria to produce ______ was qualitatively determined using Chrome Azurol S (CAS) assay (Pranaw et al., 2020).

<p>siderophores</p> Signup and view all the answers

the presence of ______ was indicated by an orange halo zone around the colony after 5 days of incubation at 30°C

<p>siderophores</p> Signup and view all the answers

the ______ of the orange halo zone was used to calculate the siderophores production index (SPI)

<p>diameter</p> Signup and view all the answers

a change in ______ from yellow to orange-brown confirmed HCN production after 4 days of incubation at 30°C

<p>filter paper color</p> Signup and view all the answers

______ production was determined by precipitating EPS from cultured bacteria using ethanol

<p>exopolysaccharides (EPS)</p> Signup and view all the answers

cellulose, protease, lipase, and chitinase production was evaluated using specific media and observing ______ around colonies

<p>clear zones or hydrolysis areas</p> Signup and view all the answers

the ______ of biosurfactants was measured using the height of the emulsion layer in a test tube

<p>emulsifying activity</p> Signup and view all the answers

Samples were analyzed using ______ equipped with a PAD detector and C18 reverse-phase column for separation. Each sample (20 μL) was injected and eluted with 1 ml min-1 using a step-by-step gradient to increase the amount of acetonitrile (ACN) in water. The gradient started at 40% of ACN for 4 min, then increased to 64% in 7.5 min, reached 75% in 16.5 min, and ended at 100% in 18.5 min (100% was maintained for 3 min before being reduced to 40% for 5 min)

<p>HPLC</p> Signup and view all the answers

Chromatograms were recorded at ______ nm, and the 2,4-DAPG was quantified according to standard curve with 2,4-DAPG

<p>270</p> Signup and view all the answers

The organic compounds were separated by gas chromatography Agilent ______ interfaced to an Agilent 5973c Network Mass Selective Detector and an Agilent 7683 Series Injector, Agilent Technologies, Santa Clara, CA, USA (GC-MS). A 5 μL CFS of the selected bacterial isolate was injected with a 1:5 split (sample; carrier gas) through 0.3% SD onto a HP-5MS column (30 m × 0.25 mm I.D., 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA, USA) using He as the carrier gas at 1 mL min-1

<p>7890A Series Gas Chromatograph</p> Signup and view all the answers

The ion source was maintained at ______; the GC oven was programmed with a temperature gradient starting at 40°C (for 5 min) and gradually increased to 300°C (for 5 min) at 5°C min-1

<p>250°C</p> Signup and view all the answers

Mass spectrometry analysis was performed in electron impact mode at an ionization potential of ______ eV. Mass spectra from m/z 40 to 800 were recorded

<p>70</p> Signup and view all the answers

Identification of organic compounds was performed using Agilent Technologies Enhanced ChemStation (______) and The Wiley Registry of Mass Spectral Data (version 3.2, Copyright 1988–2000 by Palisade Corporation with, 8th 213 Edition with Structures, Cop.

<p>G1701EA ver. E.02.00.493</p> Signup and view all the answers

A 5 μL CFS of the selected bacterial isolate was injected with a 1:5 split (sample; carrier gas) through 0.3% SD onto a HP-5MS column (30 m × 0.25 mm I.D., 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA, USA) using ______ as the carrier gas at 1 mL min-1

<p>He</p> Signup and view all the answers

Mass spectra from m/z 40 to ______ were recorded

<p>800</p> Signup and view all the answers

Study Notes

  • Bacterial cultures were grown and inoculated onto CAS agar medium for siderophore production assessment.
  • The presence of siderophores was indicated by an orange halo zone around the colony after 5 days of incubation at 30°C.
  • The diameter of the orange halo zone was used to calculate the siderophores production index (SPI).
  • Hydrogen cyanide (HCN) production was assessed using a method involving King’s B agar medium and picric acid.
  • A change in filter paper color from yellow to orange-brown confirmed HCN production after 4 days of incubation at 30°C.
  • Exopolysaccharides (EPS) production was determined by precipitating EPS from cultured bacteria using ethanol.
  • Ammonia production was assessed by adding K-Na tartrate and Nessler’s reagent to bacterial supernatants and observing a color change.
  • Cellulose, protease, lipase, and chitinase production was evaluated using specific media and observing clear zones or hydrolysis areas around colonies.
  • The emulsifying activity of biosurfactants was measured using the height of the emulsion layer in a test tube.
  • Volatile organic compounds (VOCs) were investigated for antifungal activities against B. cinerea using the double plate method.
  • The cell-free extract was obtained by centrifuging a bacterial culture and extracting the supernatant with ethyl acetate.
  • The TLC method was used to separate and detect phloroglucinol in the cell-free extract.
  • The HPLC method was employed to quantify 2,4-diacthylphloroglucinol (2,4-DAPG) in the cell-free supernatant of the selected isolate.

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Learn about the analysis of samples using HPLC equipped with a PAD detector and C18 reverse-phase column. Understand the step-by-step gradient used to separate compounds in the samples.

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