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	The standard stock solution (15 mg mL-1) of TOL was prepared in methanol. The standard Trp stock solution (15 mg mL-1) was prepared in water. Series (n = 5) of working solutions for each standard were prepared (0.0625 - 125 μg/ml)by appropriate dilution of the stock solution with methanol. To determine the recovery of the studied indolic compounds from bacterial broth during sample preparation, standards were spiked into sterile LB broth  at five different concentrations. Bacterial broth was processed with the sample preparation procedure described below.		The standard stock solution (15 mg mL-1) of TOL was prepared in methanol. The standard Trp stock solution (15 mg mL-1) was prepared in water. Series (n = 5) of working solutions for each standard were prepared (0.0625 - 125 μg/ml)by appropriate dilution of the stock solution with methanol. To determine the recovery of the studied indolic compounds from bacterial broth during sample preparation, standards were spiked into sterile LB broth  at five different concentrations. Bacterial broth was processed with the sample preparation procedure described below.
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	1. The bacteria were cultivated for 72 h in LB broth with 0.5, 1.0, 2.0, 3.5 and 5.0 mM Trp supplementation		1. The bacteria were cultivated for 72 h in LB broth with 0.5, 1.0, 2.0, 3.5 and 5.0 mM Trp supplementation
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Revision as of 15:04, 1 November 2017

Sample Preparation

The standard stock solution (15 mg mL-1) of TOL was prepared in methanol. The standard Trp stock solution (15 mg mL-1) was prepared in water. Series (n = 5) of working solutions for each standard were prepared (0.0625 - 125 μg/ml)by appropriate dilution of the stock solution with methanol. To determine the recovery of the studied indolic compounds from bacterial broth during sample preparation, standards were spiked into sterile LB broth at five different concentrations. Bacterial broth was processed with the sample preparation procedure described below.

1. The bacteria were cultivated for 72 h in LB broth with 0.5, 1.0, 2.0, 3.5 and 5.0 mM Trp supplementation
2. Bacterial cultures were then centrifuged, and the bacterial culture supernatants were processed
3. Sample preparation consisted of a single centrifugal filtration step using 3-kDa cut-off membrane centrifugal filters.
4. 0.5 mL of bacterial culture supernatants or spiked sterile bacterial broths were transferred to the sample chamber of a 0.5 mL centrifugal filter tube and centrifuged at 14,0009*g at 4 C for 30 min.
5. The filtrates were directly analyzed by HPLC.

HPLC Conditions

Analysis

Initial Standard Run of Tryptophan

MW of compounds:
Tryptophan MM: 204.225 g/mol
Indole-3-pyruvic acid MM: 203.197 g/mol
Indole-3-acetaldehyde MM: 159.188 g/mol
Tryptophol MM: 161.20 g/mol

Tryptophan and Indole-3-pyruvic acid

Tryptophan and indole-3-pyruvic acid are too close in MM that unintended reactions during the run would make it difficult to differentiate the two.

References

King ED, Ward MK, Raney DE, 1954. Two simple media for the demonstration of pyocyanin and fluorescin. Journal of Laboratory and Clinical Medicine 44, 301–7.