APOYO FAMILIAR Y CARACTERISTICAS SOCIOECONOMICOS DEL

2.3.1 Growth conditions

1 ml of 20% glycerol-spore preparation was used to inoculate a 500 ml flask containing

100 ml of liquid medium. The flask was aerated and agitated by the inclusion of a stainless steel spring as an internal baffle, forming a collar round the inside of the base of the flask. The cultures were incubated at 30°C for 24 h, in an orbital incubator rotating at 250 rpm. After this time, the contents of the flask were transferred to one of two types of vessel. 2 L flasks, baffled with stainless steel springs and containing a total of 450 ml media, were incubated in the orbital shaker, rotating at 275 rpm. The second type were

C h a p te r ! . M a te ria ls a n d m eth o d s

1 L vortex aerated bottles, which were used with a final volume 800 ml. The medium was agitated with a magnetic stirrer bar at a rate such that the vortex extended to the bottom of the bottle. All three types of flask were incubated at 30°C and the cells harvested in aU stages of growth as appropriate. CeU broth was centrifuged in 200 ml lots at 20,000g for 30 minutes, at 4°C, in a Sorvall RC-5B centrifuge. The pelleted cells were washed first with 0.05% Triton X-100 solution and then with HEPES buffer (20mM, pH 7.4). The cells were frozen at -20°C before being prepared for enzyme assays.

2.3.2 Monitoring of growth

Growth of bacterial cultures was monitored by the dry weight of cells and total protein concentrations. Optical density could not be used as a measure of growth since the cells grew in pellets rather than in dispersed form. Duplicate aliquots of 10 ml were removed from the culture medium, which was agitated to ensure that the pellets were evenly suspended in the broth. These were then filtered through pre-dried and weighed AP25 MiUipore pre-füters, under vacuum and washed with successive 20 ml volumes of 50% (v/v) ethanol;5% (w/v) Tween-80 solution, 50% (v/v) ethanol solution and distilled water. The filters were then dried to constant weight at 80°C. Where non-soluble substrates were used as the carbon source, the mass of a sample filtered at the time of inoculation was used to provide a zero point, which was taken into account in all subsequent samples. Total cellular protein was obtained to provide a separate method of determining cell

quantity. 1 0 ml aliquots of fermentation broth were sampled as above, spun down in a

bench-top centrifuge (MSE centaur 2) and the cell pellets resuspended in 1 ml of distilled water. To these was added 1 ml of IM NaOH, the mixture vortexed and heated to 100°C for 20 mins. The samples were microfuged and Bradford assays performed (Section 2.7.1).

2.3.3 Monitoring of actinorhodin production

Actinorhodin synthesis and secretion was followed by the appearance of the coloured

pigment in the liquid media. 1 ml samples were removed from the broth and centrifuged

for 5 min in a Microfuge II to remove cells and debris. Since the lipid caused the broth to

appear cloudy, a few of drops of chloroform/methanol (2 :1, v/v) were added and the

mixture vortexed, allowing the lipid to enter the solvent phase. The absorbance of the lower, aqueous layer could then be determined spectorphotometrically at 640 nm. A sample of media taken before inoculation was used as a blank.

Internal pigmentation was released from cells that had been washed with O.IM HCl by

sonication, in 10 s bursts with 20 s intervals, a total of 6 times. The debris was then

C h a p te r ! . M a te ria ls a n d m eth o d s 2.3.4 Media lipid content - vanillin assay

This assay is based on the Boehringer Mannheim (Lewes, UK) test kit (no. 124 303) which is no longer produced. The reaction of the lipid with sulphuric acid and vanillin creates a pink complex which can be quantitated spectrophotometrically.

The samples of media were heated to 40°C and mixed vigorously. A 1 ml Gilson pipette tip was cut to produce a bore of 3mm; it was then used to extract triplicate samples of 250

|il of the supernatant, which were placed in Pyrex tubes. 2 ml of 18.3M H2SO4 was then

added, the tubes vortexed, covered with a glass teardrop and heated in a boiling water

bath for 10 min. The tubes were allowed to cool, after which 100 |li1 samples were

transferred to clean tubes and mixed with 2.5 ml of vanillin reagent. This consisted of 1.98g of vanillin dissolved in 40 ml of ethanol, made up to 100 ml with distilled water, the volume then increased to 1 L with 14.7M orthophosphoric acid. The tubes were allowed to stand at room temperature for 30 min, with occasional mixing. The samples were transferred to cuvettes and read spectrophotometrically at 536 nm. A blank of 2 ml 18M sulphuric acid and a standard of known lipid concentration were also assayed. The amount of lipid present in each sample was determined as follows:

Lipid concentration = absorbance of sample absorbance of standard

The vanillin reagent was photoreactive and stored in a light-proof container.