[13] Lipids of purple membrane from extreme halophiles and of methanogenic bacteria

This chapter presents a procedure for the extraction of lipids of purple membrane from extreme halophiles and of methanogenic bacteria.

Abstract

This chapter presents a procedure for the extraction of lipids of purple membrane from extreme halophiles and of methanogenic bacteria. For lipid extraction, cells of Halobacterium cutirubrum or Halobacterium halobiurn are first grown aerobically at 37° and whole cells are extracted. A suspension of purple membrane (PM) is diluted with methanol and stirred in the dark. The mixture is centrifuged and the supernatant is collected. After centrifugation the combined supernatants are diluted with CHCl3 and KCI with gentle mixing and the phases are allowed to separate in a separating funnel for several hours. The lower chloroform phase is removed, diluted with benzene and ethanol, and brought to dryness on a rotary evaporator. The residual total lipids are dissolved in chloroform to a known volume and subjected to separation and analysis. To extract lipids of Methanospirillurn hungatei, a suspension of cells is diluted with methanol and chloroform and the mixture stirred. The mixture is then centrifuged and the supernatant is collected. The pellet is suspended in NaC1 and extracted then supernatants are diluted with chloroform and distilled water in a 4-liter separatory funnel, mixed gently, and left at room temperature for several hours to allow the phases to separate. The chloroform phase is then removed, diluted with benzene and 99% ethanol, and brought to dryness on a rotary evaporator at 30–35 °. The residual lipids are dissolved in chloroform and stored at –20 °.