52
3.
FIXATION
ing the lead of Sabatini, Bensch, and Barrnett (1963), we have had good
success using glutaraldehyde.
The fixative is prepared as follows:
Stock buffer salt solution:
Monosodium (monobasic) sodium phosphate
2.26%
Stock alkali solution:
Sodium hydroxide
2.52%
Fixative, 10% buffered formalin:
Monosodium phosphate solution
83 ml
Sodium hydroxide solution
17 ml
Choice of:
Formalin (methanol-free, 40% formaldehyde)
11 ml
or
Paraformaldehyde (powder, pH adjustment and
60°C necessary for solution)
4 gm
Adjust pH to 7.2-7.4 as necessary
If it is desired to make the approximate glutaraldehyde equivalent of
this buffered 10% formalin (4% formaldehyde), one must take into account that glutaraldehyde (and some other aldehydes) are supplied
by the manufacturer in 25% solution. Thus one would use 18 ml of such
a solution with 100 ml of the above vehicle.
The addition of small quantities of glucose to the above solutions is of
doubtful value, although originally this was done in exactly patterning
the vehicle after Millonig. Undoubtedly this fixative is hypertonic, but
it does not seem feasible to reduce the aldehyde content of any aldehyde
fixative to isotonicity.
The use of methanol-free formalin deserves special comment. Most
"reagent grade" formalin that is manufactured and sold in the United
States contains substantial amounts of methyl alcohol, added as a preservative. If one reads the small print on the manufacturer's labels, this
varies from 11 to 16%. Obviously, methyl alcohol is a fixative in its own
right, albeit a very crude and undesirable one. In retrospect, it is quite
likely that many unhappy experiences trying to use formalin as a refined
fixative have resulted from overlooking this hidden enemy. Grades of
formalin other than "reagent grade" are not required by United States
law to specify the presence of preservatives. In foreign countries also, it
may be difficult to find out whether or not alcohol has been added to
formalin. Thus, there is technical uncertainty about almost all published
3.
FIXATION
ing the lead of Sabatini, Bensch, and Barrnett (1963), we have had good
success using glutaraldehyde.
The fixative is prepared as follows:
Stock buffer salt solution:
Monosodium (monobasic) sodium phosphate
2.26%
Stock alkali solution:
Sodium hydroxide
2.52%
Fixative, 10% buffered formalin:
Monosodium phosphate solution
83 ml
Sodium hydroxide solution
17 ml
Choice of:
Formalin (methanol-free, 40% formaldehyde)
11 ml
or
Paraformaldehyde (powder, pH adjustment and
60°C necessary for solution)
4 gm
Adjust pH to 7.2-7.4 as necessary
If it is desired to make the approximate glutaraldehyde equivalent of
this buffered 10% formalin (4% formaldehyde), one must take into account that glutaraldehyde (and some other aldehydes) are supplied
by the manufacturer in 25% solution. Thus one would use 18 ml of such
a solution with 100 ml of the above vehicle.
The addition of small quantities of glucose to the above solutions is of
doubtful value, although originally this was done in exactly patterning
the vehicle after Millonig. Undoubtedly this fixative is hypertonic, but
it does not seem feasible to reduce the aldehyde content of any aldehyde
fixative to isotonicity.
The use of methanol-free formalin deserves special comment. Most
"reagent grade" formalin that is manufactured and sold in the United
States contains substantial amounts of methyl alcohol, added as a preservative. If one reads the small print on the manufacturer's labels, this
varies from 11 to 16%. Obviously, methyl alcohol is a fixative in its own
right, albeit a very crude and undesirable one. In retrospect, it is quite
likely that many unhappy experiences trying to use formalin as a refined
fixative have resulted from overlooking this hidden enemy. Grades of
formalin other than "reagent grade" are not required by United States
law to specify the presence of preservatives. In foreign countries also, it
may be difficult to find out whether or not alcohol has been added to
formalin. Thus, there is technical uncertainty about almost all published