Tissue washing procedures

Inherent in all these methodologies, which measure either absolute Sr levels or strontium isotope ratios in mineralized tissue, is the assumption that diagenesis has not altered the signal since death. This has been a matter of some considerable debate (e.g., Nelson et al. 1986), but the consensus of current opinion amongst practitioners is that the repeated acid-washing procedures used remove any diagenetic mineral, because it has a higher... 

Despite the elaborate washing procedures usually employed, cell-wall preparations may also frequently be contaminated with cytoplasmic constituents that sediment with the walls after tissue homogenization. Starch grains and proteins are particularly difficult to remove in this respect. Both incubation with purified alpha amylase (EC 3.2.1.1)55,101 and extraction with chloral hydrate118 have been utilized for removal of starch from cell-wall preparations. Pronase57,101 has been used to remove proteins. 

Sucrose Absorption by Vascular Bundles. Sucrose uptake into 2-4 mm long pieces of vascular bundles isolated from the petiole of Cyclamen persicum was described in detail in (5). Briefly, the tissue material was preincubated (30 min) with brassinosteroids or related compounds, which were also present during the following incubation with 1 mM 1AC-labelled sucrose (30 min). All solutions contained 1 mM CaSO, 1 mM KC1 and 10 mM morpholinoethanesulfonic acid and were adjusted with NaOH to pH 5.0. After a 5-min-washing procedure and extraction in methanol the uptake rate for sucrose was determined analogously to the experiments with leaves. 

Both bile acids and pepsin are adsorbed and their damaging activity is reduced. More important, the gel has been shown to adhere with particular affinity to necrotic tissue, such as the cratered ulcers from which it is difficult to dislodge by washing procedures. The ability of sucrasulfate to complex with proteins such as fibrinogen and albumin may be part of the explanation. 

Lipid extracts from tissues, obtained in the above manner, tend to contain appreciable amounts of nonlipid contaminants, such as sugars, amino acids, urea and salts. These must be removed before the lipids are analysed. Most workers use a simple washing procedure, devised originally by Folch, Lees and Stanley [259], in which a chloroform-methanol (2 1, v/v) extract is shaken and equilibrated with one fourth its volume of a saline solution (i.e. 0.88% potassium chloride in water). The mixture partitions into two layers, of which the lower phase is composed of chloroform-methanol-water in the proportions 86 14 1 (by volume) and contains virtually all of the lipids, while the upper phase consists of the same solvents in the proportions of 3 48 47, respectively, and contains much of the non-lipid contaminants. It is important that the proportions of chloroform, methanol and water in the combined phases should be as close as possible to 8 4 3 (by volume), otherwise selective losses of lipids may occur. If a second wash of the lower phase is needed to remove any remaining contaminants, a mixture of roughly similar composition to that of the upper phase should be used, i.e. methanol-saline solution (1 1, v/v). 

The tissue (100 g) is homogenized with a solvent mixture consisting of chloroform and methanol (300 ml, 1 2 v/v), for 4 min. In case that a two-phase system is formed, more solvent is added until a single phase is obtained. After extraction, the mixture is filtered while the tissue residue is rehomogenized with more chloroform (100 ml) and filtered once more. The combined filtrates are shaken thoroughly with one fourth its volume of a potassium chloride solution (0.88% in water). Once the mixture is settled out, the upper aqueous layer is drawn off by aspiration, then one foiulh the lower layer of methanol—saline solution is added (1 1, v/v), to repeat the washing procedure. The bottom layer is finally filtered and the solvent evaporated to obtain the purified lipid fraction, which is stored in a small volume of nonpolar solvent at —20°C until analysis. 

Fig.2 demonstrates that the washing procedure did not in itself alter the responsix ess of the tissue to glucagon. Of two succesive incubations with glucagon (fig.2, g) the second one showed a slightly lowered lipolytic rate (glycerol release). However, if an incubation without hormone was interposed, the tissue seemed to recover its full responsiveness. It is further apparent that the incubations without hormone (control = c) did not differ significantly whether they initiated the incubation series of a tissue pool or followed an incubation with hormone. 

The results demonstrate that the hormone must have dissociated quickly from the tissue, i.e. within at least the 5 minutes used for the washing procedure. It also shows that the "activated state of those enzymes which lead to lipolysis is not maintained in the absence of hormone. These findings contrast with results recently reported for ACTH (11) with respect to binding. 

Keswick et al. [72] have observed different classifications of soaps in terms of mildness by using the flex wash test and the forearm wash test. They attribute the difference to a differential action of the soaps on the skin in the two tests. In the flex wash test, skin washing is done with a sponge having a slight abrasion effect under such conditions, the product exerts a deeper action in the skin than in the forearm wash procedure. In the forearm wash test, a nonabrasive tissue towel is used and the product exerts a more superflcial action on skin. The forearm wash test would be, according to the authors, more reliable to home-use conditions. 

Sonneveld, C. and P.A. van Dijk. 1982. The effectiveness of some washing procedures in the removal of contaminants from plant tissue samples of glasshouse crops. Commun.Soil Sci. Plant Anal. 13 487-496. 

An oxalate sensor by immobilizing spinach tissue as the source of oxalate oxidase was developed by Li [50], The sensor responds linearly to oxalate concentration in the range of 1.0-100 pM with a detection limit of 0.6 pM. The sensor was stable for 30 days when stored at 4°C and a complete analysis for the determination of oxalate could be performed in 1 min including sampling and washing. Considering the low cost of the plant tissue and simple procedure for plant tissue immobilization, this report is most valuable. 

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