The use of malachite green during fixation of plant tissues for the preservation of lipids

SUMMARY Osmiophilic droplets are shown in chloroplasts of leaves of ryegrass after the addition of malachite green to glutaraldehyde during primary fixation. Similar droplets are shown in leaves of bean and maize but not in the roots of these plants. These droplets are not shown in tissue fixed in glutaraldehyde in the absence of malachite green. Evidence is presented which indicates that these droplets contain a high proportion of lipids, particularly phospholipids. This evidence includes the use of other fixation methods which retain lipids, e.g. formal calcium, potassium dichromate. The droplets give a positive reaction with the silver hydroxylamine reaction for ester linkages. Tests for aldehydes by treatment with thiocarbohydrazide and silver proteinate, and proteins by digestion with protease were ambiguous. Digestion with solvents before fixation indicate that the droplets are lipids. TLC of chloroform: methanol extracts of fresh and fixed tissue and of the fixative solvents support the idea that malachite green aids retention of lipids during fixation. Phospholipids are also demonstrated by silver hydroxylamine in plant cell nuclei, myelin of rat brain and vesicles in bean root tips.