Sample collection, treatment and storage

From the Newcastle Infirmary site, 210 articulated individuals were recovered and prefixed with the code SK. Forty-two soil samples from either chest cavities or distal to 21 skeletons, labelled SK 1, 3-5, 12, 21, 27, 31-33, 40-42, 46-48, 52, 102, 103, 107 and 108, were selected from those collected from the Newcastle Infirmary site between November 1996 and March 1997. Soil samples were taken from the chest cavities of freshly exposed, articulated individuals, prior to any cleaning or lifting procedures and from the distal area around the skull. Soil samples were collected into polythene-capped sterile glass universal containers and autoclaved (121°C, 15 min.) prior to room temperature storage. During soil sampling, face masks and plastic disposable gloves were worn. Twenty-one mid-shaft rib samples were taken from the identified skeletons after osteological examination; disposable plastic gloves were worn during excavations, skeletal examination and subsequent handling. Bones were crushed, using a mortar and pestle, to a fine powder; soil samples were air-dried and powdered.

Mycolic acid extraction and derivatisation

Powdered bone and soil samples from the Newcastle Infirmary site were analysed for mycolic acids, using a modification of a method developed for the analysis of clinical material (Minnikin et al. 1993). Samples of bone (1.0g), soil (1.0g) and lyophilised M. tuberculosis Strain C (50mg) were heated with 1ml 15% aqueous tetrabutylammonium hydroxide at 100°C overnight in 8ml Pyrex tubes, sealed with a PTFE-lined screw cap. After cooling, 1ml water was added and, after centrifugation, the supernatants were mixed with 1ml 0.05% 9-chloromethyl-anthracene in dichloromethane and the tubes placed on a tube rotator for 1 hour at room temperature. The lower dichloromethane layer was washed successively with 1ml 10% aqueous hydrochloric acid and twice with 1ml water. Removal of the dichloromethane, under nitrogen flow, gave crude mycolic esters which were dissolved in toluene (25 µl) and the solutions adsorbed on toluene-prewashed 1ml BondElut C-18 reverse-phase columns. The columns were washed with acetonitrile (4.0ml), followed by acetonitrile:toluene (4:1) (4.0ml) and the washings discarded. The purified anthrylmethyl esters were eluted with acetonitrile:toluene (1:1) (3.0ml) and evaporated to dryness.

High Performance Liquid Chromatography (HPLC) of Mycolic Acids

Anthrylmethyl mycolates were analysed by HPLC, at 37°C with an eluent flow rate of 1.0ml -1, using a Merck Hitachi L6200 pump and a Gilson 122 fluorimeter using 252nm light for excitation and a 435nm emission filter. Hexane solutions of mycolic acid extracts were applied to a reverse-phase 5 µm Merck LiChrosorb RP 18 cartridge (150 x 4.6mm), eluted with acetonitrile:tetrahydrofuran (1:1). All data were collected using X-Chrom v. 2.04k (Labsystems, Fisons plc.) software data collection system. Fractions, corresponding to mycolic acid derivatives were collected, blown down to dryness, dissolved in hexane and applied to a normal phase 5 µm Merck LiChrospher Si-60 cartridge (150 x 4.6mm) for separation into component alpha-, methoxy-, and ketomycolate fractions using hexane:tetrahydrofuran (98:2). Individual fractions were analysed by further reverse phase chromatography, using the above conditions.


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Last updated: Tue Dec 1 1998