Most micro-organisms can’t be observed properly
under the light microscope because they are transparent
& appear colourless .
We must prepare them for the observation. One of the
ways it can be done is by staining.
*Acidic stain:- They have negatively charged groups and stain the
cytoplasmic components of the cells which have positive charge.
e.g:- picric acid, rose bengal, acid fuchsin, eosin.
* Basic stain:- They have +vely charged groups and stain cytoplasmic
components of the cells which have negative charge (nucleus).
Eg:- methylene blue, crystal violet, safranine.
* Neutral stain:- They are formed by mixing together aquous solution of
certain acidic & basic dyes. The colouring matter in them is both
negatively & positively charged components.
Staining solution are prepared by dissolving a
particular stain in either distilled water or alcohol.
The stain is applied to smear for a definite time then
washed dried & examine under the microscope.
Depending upon how the stain reacts with the specimen,
staining are of 2 types :-
Positive staining :- it involves the sticking of the stain to
the specimen and giving it a color.
Negative staining :- they stain background and doesnot
stain the specimen.
Negative staining technique is advantageous for 2 reasons;
* Microbial cells appear less shriveled or distorted because heat
fixation is not carried out,
* Capsulated bacteria that are difficult to stain can be
observed by this technique.
The most commonly used endospore stain is the Schaeffer-fulton
Malachite green, the primary stain is applied to the heat fixed smear
& heated to steaming for about 5 minutes. The heat helps the stain to
penetrate the endospore wall.
The preparation is washed for about 30 sec with water to remove the
Then safranine , a counter stain is applied to the smear to stain the
portions of the cell other than the endospore.
The endospore appear green within red or pink cell.
Generally all the algal cells contain pigments which specify
them widely therefore they donot require any specific stain.
They are generally mounted with glycerine.
Sometimes safranine is used to stain algal cells in order to
observe the clarity of the cells.
- Microbiology :- Presscott , Harley , Klein .
-Principlesof Microbiology :- Ronald M. Atlas .
- Basic & Applied Microbiology :- K. R. Aneja .
- Wikipedia .