Technical variations in Koelle’s histochemical method for demonstrating cholinesterase activity

Koelle's histochemical method for demonstrating cholinesterase activity and the literature on its subsequent modifications have been reviewed. Experiments were carried out on the effect on the cholinesterase reaction of formaldehyde fixation, cold storage of tissues, pH of incubation solution, and progressive increase of incubation time. A series of experiments was also carried out in testing the specificity of substrates and selective inhibitors used in the Koelle method. Enzyme reaction was visualized by the ammonium sulphide method. As a result of these experiments the following technical desiderata have been established: 1. Fixation of tissues for 3 h in neutral formaldehyde solution at 4° C preserved the morphology of the tissues without appreciably affecting the histochemical results. Fixation for more than 6 h produced definitive inhibition of cholinesterases, especially AChE, in most tissues. 2. Periods of up to 24 h of cold storage before fixation had no appreciable effect on the cholinesterase reaction. 3. Incubation at pH values between 5.0 and 6.0 produced neither significant diffusion artifacts nor loss of enzyme activity. Below pH 5 the AChE reaction was affected to a varying extent according to the tissues used. 4. B.W. 284 at a concentration of 5 x 10-5 M and ethopropazine hydrochloride at a concentration of 1 x 10-4 M were found to be suitable selective inhibitors for AChE and ChE respectively. 5. Visualization of results by means of ammonium sulphide method was found to be preferable to phase-contrast microscopy.