Bill McManus enquired about triple staining in foods.


<I am trying to triple label protein, fat, and starch in  dairy products.
<I have access to a  BioRad MRC 1024 with a Krypton/Argon Laser. Does anyone
<has ideas on the best ( or any) way to do this?



I don't know that there is a simple single answer to this as the
stain will depend on circumstance - e.g. is the stach gelatinised or
not, is there any helpful or nuisance autofluorescence? It also depends
on how specific you want your staining to be.  The choice will also
depend on the amount of preparation of the sample - if you can get
cryostat sections then you may simplify life by just using
a compound microscope.  If on the other hand you are trying to look
at a really awkward  sample where you are using a 'bulk' sample then
 you will limit some of your confocal options as well (e.g. the use of the
transmission detector).  Staining a fat continuous product may
require a different approach to a fat disperse one.
However I will put forward some ideas.  I'd also like to hear
other people's thoughts.

Proteins - lots of choice here, FITC has already been mentioned and
works well, eosin also works well for me as does saffranin (although
this appears metachromatic and may stain other components
differentially or non-specifically), acridine orange also favours
proteins (but again can stain other components and depending on pH
may act as a double stain for some carbohydrates and proteins).  If
you want better than these then you are probably into specific
labels.

Fat - the straightforward option here would be nile red (as has
previously been mentioned) you may choose to use it in the less pure
nile blue form.  Note that this can also act as a starch stain and that
differentiation may be difficult in the confocal though (not by
conventional light microscopy)  Alternatively if the fat is
crystalline you could use polarised light or use a dark stain (osmium
tetroxide or sudan black B) and if the preparation is thin enough use
the transmission detector.

Starch - congo red would be one choice but if the starch
grains are intact then it will not penetrate the grains may also get
a little fat staining.  Nile blue would be better but can also stain fat.
Periodic acid schiff (or similar) staining would stain starch and other
1,2 glycol containing carbohydrates quite well and should be differentiable
from FITC stained protein to give a dougle stain. Iodine would give very dark
areas and the transmission detector could be used and (as previously
mentioned) ungelatinised starch grains will be identifiable with polarised light
(and the transmission detector).  Previous correspondence mentioned
the use of labelled lectins that could be used.

I hope that this gives you some ideas although I'm sorry that I can't
provide an instant answer.

Regards

Dave Lewis




Dr Dave Lewis

Head of Food Science & Technology
SAC, Auchincruive
Ayr  KA6 5HW
Scotland
Tel 44 1292 525069; Fax 44 1292 525071
e-mail [log in to unmask]

Cassandra was right, OK?