|# 11: Basic cell wall histochemistry|
|The preservation of structural details of cells and tissues is influenced by the condition of the material at the time of collection and by the subsequent preparative steps used for killing and fixing the material. In other words, if you wish to prepare sections in which the structural details are well preserved one should select healthy plant material. Exceptions to this rule apply only when the researcher is interested in observing the effects of disease, fungal infection, insect damage etc. on the normal structure of the plant material being examined.|
"There is only one reliable criterion by which we can determine whether the image that we see with the microscope is a good representation of what existed in life, and that criterion is comparison with living cells".
from: Baker, J.R. 1966: Cytological Technique (5th Ed.) Methuen: London.
The use of dyes to make structures within sections of plant material visible, has been used for an extensive period of time. Dye substances may be used to differentiate between primary and secondary wall structures, show the presence or absence of lignin within the walls, show the presence or absence of other specialized chemical substances within cell walls, and may stain nuclei, or chromosomes within living cells. These substances rely on the cationic or anionic nature of the substances within the cell walls, or within the cytoplasm of the cells themselves. By carefully selecting dyes and relatively simple chemical reactions, one may undertake histochemical analysis of the wall, and the cytoplasm. These simple tests are thus invaluable if we are to determine the nature of living plant cells.
The histochemical tests described below, are useful aids in determining cell wall structure in plants. Please remember, these are relatively simple ones, but, like all tests, they do not always work!
Mount the section in phloroglucinol (1% in 95% alcohol). Run in a drop of 25% HCl at the side of the coverslip. Lignified walls will turn pink. Carried out in this way the test is sensitive, but note that it is also positive for hemicellulose pentosans.
(b) Ferric chloride.
Tannin in sections mounted in FeCl3 (10% in methyl alcohol), imparts a blue-black colour. The reaction is not specific for phenolics but it is the most generally useful test.
(c) Sudan IV
Use saturated, alcoholic mixtures). The Sudan dyes depend for their action on a partition in favour of tissue lipids. Stain the section in a watch glass for 5 minutes. Wash rapidly in absolute alcohol and mount in 25% glycerol. Cutinized or suberized walls and any other lipid inclusions, stain yellow.
(d) Toluidine Blue
(0.05%aq.). Cut thin sections and wash in tap water for 1min. Stain for 1 min. and mount in tap water. Toluidine blue is a cationic dye which binds to tissue anions, but being metachromatic, gives two sets of colour reactions, pink-purple and blue-green. Thus: Carboxylated polysaccharides = pink, e.g. uronic acids. PO4- on macromolecules = purple-greenish blue, e.g. nucleic acids. Also by a different reaction, Polyphenols = Green-blue, e.g. lignin's, tannins. Thus most primary walls stain pinkish and lignified secondary walls stain blue. Hydroxylated polysaccharides, such as starch and cellulose walls are usually unstained.
(e) Hydroxylamine - Ferric chloride.
For localization of methyl esters of pectin. An alkaline solution of hydroxylamine will react with methyl esters of pectin present in sections of plant material to produce hydroxamic acids which, after acidification with HCl, yield a red colour upon addition of ferric chloride solution. Soak sections in freshly prepared hydroxylamine solution. (Stocks are 14% sodium hydroxide in water, and 14% hydroxylamine-hydrochloride in water). The reagent must contain absolutely equal proportions of both reagents.) Soaking is best achieved on a slide or in a watch glass. Stain for at least 5 mins. Add an equal volume of 33% hydrochloric acid, tilting the slide to make sure that all parts of the section become acidified. Allow 1-2 minutes for the reaction to take place. Drain off excess reagent and replace with a 10% aqueous solution of ferric chloride in 0.1N HCl. Cover the specimen with a coverslip and observe under the microscope. Pectin-containing areas should stain red. Note, if staining intensity is weak, try prolonging reaction time in hydroxylamine and/or a 20% ferric chloride in 0.1N HCl.
(f) Ruthenium red
Specific staining of pectic substances. Place section in 0.05% (in distilled water) ruthenium red for 1-5 mins., mount in water and observe under the microscope. Calcium pectate should absorb the stain readily. This method is very reliable. Best seen in cross-sections of collenchyma
Mount the section in aqueous. I2/KI. Note the distribution of starch. Blot off excess iodine, replace by 72% H2SO4 and immediately examine under the microscope. Cellulose structures swell and turn bright blue due to hydrolysis to a lower polymer, forming hydrocellulose, which forms a blue adsorption complex with iodine. Carefully look for pits and plasmodesmata. Lignified walls remain yellowish, but nevertheless the distribution of cellulose may still be seen, for example in the middle lamella, where the colours may mix to a greenish tinge. Cutin also stains yellow.
(h) Potassium hydroxide.
(30% aqueous.). Suberized walls when heated in this solution, turn dark yellow and then exude oily droplets of potassium phellonate. Cutin does not give this reaction. Note that cutin turns yellow in the iodine-sulphuric test.
WARNING! Many of the chemical and procedures referred to here are potentially harmful. Please exercise caution when handling chemicals in the laboratory environment.