Protección del cultivo Manejo integrado
NOMBRE O LOGO DEL
The work presented here was undertaken with a view to achieving the following objectives % -
1. Solubilisation, using synthetic detergent, of a caroten old-containing membrane component from the membranes of S . flava, and the Investigation of Its properties, in an attempt to determine the way in which the pigment is incorporated into the general
architecture of the membrane.
2. Further characterisation of the polar carotenoid fraction from flava as Isolated by Thlrkell jgjb (1967) to determine the nature of the substituents and/or other molecules attached to the carotenoid which are responsible for Its extreme polarity.
3. Elucidation of the composition and properties of a water soluble pigment fraction from morrhuae.
4. Determination of the effect of age of bacterial culture on the chemical composition of the total membrane fraction from A. flava,
3. Electron microscopic examination of Sj^ morrhuae and S , flava, with particular emphasis on the mode of cell division In,
23.
A. MAINTENANCE AND LARGE SCALE CULTURE OF SARCINA FLAVA AND SARCINA MORRHUAE
1. Maintenance culture of S. flava
S , flava, (N.C.T.C. 7503)» was grown on nutrient agar
(Oxold Ltd.), enriched with 1% glucose, at 37^. A single colony was transferred to a fresh agar plate every 5 days, and a new maintenance culture thus prepared by streak culture.
2. Large scale culture of S . flava
a) Cells were grown in a Now Brunswick MF ll4 Microform
bacterial fermenter at 37^ in 12 litres of nutrient broth (Oxold Ltd.), enriched with 1% glucose. The cultures were vigorously agitated throughout the fermentation and aerated at a rate of 12
l./min. Harvesting was by centrifugation at 1500 g. and the timei for which different batches were grown is indicated at the
appropriate points In this section.
b) Cells were grown (at Imperial College, London), In 400 I nutrient broth (Oxold Ltd.) enriched with glucose as above.
Vigorous agitation and an air flow rate of 400 l./raln. were
employed throughout. Two batches of cells were grown at Imperial College, one being harvested after 48 hours growth, the other bein^ divided into two 200 1. portions, one of which was harvested after 24 hours, the other being allowed to continue growth for 57 hours before harvesting. In each case, harvesting was carried out on a Sharpies continuous flow centrifuge, and the cells deep-frozen for transport.
The approximate yield of flava cells grown In liquid culture under these conditions Is 7*5 g. (wet weight)/I.
3. The effect of light on the growth and pigmentation of S. flava A series of universal bottles, each containing 20 mis.
of nutrient broth (Oxoid Ltd.), enriched with 1^ glucose were each inoculated with 0.5 ml. of a 24 hour culture of flava. Half of the bottles were made light proof using aluminium foil, the
remainder being left uncovered. All were rotated on a rollor- shaker at 34^ with overhead illumination from a fluorescent strip light. At daily intervals for up to 7 days, aliquots were with drawn sterilely from each bottle, suitably diluted where necessary, and monitored for bacterial numbers by turbimetrie measurement at
6lO nm. When peak numbers had been reached, the remaining cells were harvested by centrifugation, and the light and dark grown |
cells pooled separately. The carotenoid pigment was then extractec by ultrasonication in a known volume of methanol, and estimated using a theoretical extinction coefficient (^ 3000) first used by Strang (1968) for the pigments from this organism. The amount of pigment present in the light and dark grown cells was related t< the dry weight of the bacteria.
4. Culture of S. morrhuae
The extreme halophile, Sj^ morrhuae, was grown on 2.5ÿ agar No. 3 ( Oxoid Ltd.), to which was added t 25^ NaCl ; 1^ MgSOj|^. 7H2O | 0.02^ CaClg.6HgO; 0.5^ KClj 1% yeast extract (Oxoid Ltd.); and
0.5^ tryptone (Oxoid Ltd.). (All percentages here are w/v). The agar plates were streak cultured with the microorganism and sealed polythene bags in order to minimise moisture loss and subsequent crystallisation of salt on the surface of the solid medium. The
25
plates were Incubated at 34^ for three weeks after which time the cells were harvested by careful scraping with a spatula.
B. PREPARATION OF MEMBRANES AND PROTOPLASTS FROM TOOLK CELLS OF 5. FLAVA
It Preparation of the total membrane fraction
Membranes were prepared from cells grown for 24, 48, 57»
72 and 91 hours, by the method of Salton ejb (1963b). Tris buffer was used throughout the preparation since these workers found that the use of phosphate buffer caused a greater degree of membrane instability. The method is depicted in the flow scheme
shown in Fig. M.l. (overleaf)4 The purified membrane fraction thus prepared was either used Immediately (as in Section C), or lyophilised and stored at -40^ for later work (as in section F.). 2. Estimation of yield of membrane fraction
Membranes were prepared by the above method, from
duplicate 5OO mg. samples of lyophilised whole bacteria, harvested at 24, 57 and 9I hours. The weights of lyophilised purified
membranes thus obtained were determined, and the yield of the total membrane fraction for each age of cell estimated.
3. Preparation of protoplasts
Protoplasts were prepared by the method of Baird-Parker and Voodroffe (1967), which is stated to be suitable for both B. megaterium and lysodeiktlcus. 100 ug./ml. of egg white
lysozyme (Sigma Ltd.) were added to a suspension of cells (20 mg. wet weight/ml.) in a medium containing! 0.01 M Sorensen double phosphate buffer, pH 7*0| 0.005M MgCl^l
O.OlM
NaCl and 0.75M sucrose. Despite the contention in the original method that150ug. egg white lysozyme
(Sigma Ltd.) per ml. suspensio] added. Stirred Ihr. at 25^. 30^g.
DNAase
(Sigma Ltd.) per ml. suspension added. Stirrei30 mine,
at
25^* Centrifuged Pellet of cell walldebris A unaffected whole bacteria. (Discarded) at 500 g. ^ Supernatant! suspension of crude membranes - (yellow) Centrifuged at 35*000 g for 4o mine, at 4
Pellet of membranes
(yellow) Supernatant (colourless or faintly yellow, discarded)
Washed 3 times on the centrifuge (as above) with O.O5 M Tris buffer to remove "soluble** protein and nucleic acid. Supernatants disc arded .