7.3.
ALKALINE LEAD HYDROXIDE STAINS
225
and accumulated relatively high levels of carbon dioxide. In the latter
instance the troubles vanished when we used the total immersion method
of stain application described below in considering the handling of
Watson's stain.
For some purposes, and particularly after aldehyde fixation, it may be
desirable to reduce the intensity of the stain. Reynolds suggests that his
stain can be diluted 5 to as much as 1000 times with 0.01 N sodium
hydroxide to achieve delicate staining effects. He also indicates that
digestion and leaching of tissue components have not been observed
with 30 minute staining employing full strength solutions.
Watson's (1958) lead hydroxide.
Alkaline lead hydroxide, prepared in
a particular manner, was introduced by Watson (1958) as a most vigorous
general purpose stain. He dissolved 8.26 gm of lead acetate in 15 ml of
distilled water to produce a nearly saturated solution. The lead salt has
to be ground under water. This can be done with a glass rod in a 50 ml
centrifuge tube to be used later, or it may be done more easily in a
mortar with a pestle, and then transferred into a 50 ml centrifuge tube.
Then 3.2 ml of 40% sodium hydroxide are blown rapidly into this
solution from a pipette. As this is stirred an extremely dense precipitate
is formed which subsequently is sedimented by centrifugal force. The
supernatant is decanted and discarded, and the precipitate is washed by
resuspending it in approximately the original volume (or less) of fluid,
recentrifuging, and decanting a second time. Much scum will be discarded
at this time, although it will not be possible to eliminate this entirely.
The sediment is resuspended once again. This time the centrifuge tube
can be nearly filled and, after centrifuging, the supernatant becomes the
staining solution. It is almost impossible to transfer this staining solution
to a storage bottle without also transferring a good deal of scum unless
this is done through a filter. As will become apparent later, this is
acceptable. The staining solution is reasonably stable if stored at room
temperatures, but refrigeration should not be attempted.
The precipitate which is left over from the above operations still
contains much useful staining salt so that it can be resuspended and
centrifuged to yield a second quantity of stain (and even a third batch)
that is likely to be fully as effective as the first sample. Thus, at least
100 ml of usable staining solution can be collected.
It may also be noted that the stain can be taken up and used in
20-50% alcohol. In our own laboratory we used alcoholic stains for about
a year, partly because they penetrated some plastic embedments better
ALKALINE LEAD HYDROXIDE STAINS
225
and accumulated relatively high levels of carbon dioxide. In the latter
instance the troubles vanished when we used the total immersion method
of stain application described below in considering the handling of
Watson's stain.
For some purposes, and particularly after aldehyde fixation, it may be
desirable to reduce the intensity of the stain. Reynolds suggests that his
stain can be diluted 5 to as much as 1000 times with 0.01 N sodium
hydroxide to achieve delicate staining effects. He also indicates that
digestion and leaching of tissue components have not been observed
with 30 minute staining employing full strength solutions.
Watson's (1958) lead hydroxide.
Alkaline lead hydroxide, prepared in
a particular manner, was introduced by Watson (1958) as a most vigorous
general purpose stain. He dissolved 8.26 gm of lead acetate in 15 ml of
distilled water to produce a nearly saturated solution. The lead salt has
to be ground under water. This can be done with a glass rod in a 50 ml
centrifuge tube to be used later, or it may be done more easily in a
mortar with a pestle, and then transferred into a 50 ml centrifuge tube.
Then 3.2 ml of 40% sodium hydroxide are blown rapidly into this
solution from a pipette. As this is stirred an extremely dense precipitate
is formed which subsequently is sedimented by centrifugal force. The
supernatant is decanted and discarded, and the precipitate is washed by
resuspending it in approximately the original volume (or less) of fluid,
recentrifuging, and decanting a second time. Much scum will be discarded
at this time, although it will not be possible to eliminate this entirely.
The sediment is resuspended once again. This time the centrifuge tube
can be nearly filled and, after centrifuging, the supernatant becomes the
staining solution. It is almost impossible to transfer this staining solution
to a storage bottle without also transferring a good deal of scum unless
this is done through a filter. As will become apparent later, this is
acceptable. The staining solution is reasonably stable if stored at room
temperatures, but refrigeration should not be attempted.
The precipitate which is left over from the above operations still
contains much useful staining salt so that it can be resuspended and
centrifuged to yield a second quantity of stain (and even a third batch)
that is likely to be fully as effective as the first sample. Thus, at least
100 ml of usable staining solution can be collected.
It may also be noted that the stain can be taken up and used in
20-50% alcohol. In our own laboratory we used alcoholic stains for about
a year, partly because they penetrated some plastic embedments better