5.

Toluidine blue – A basic thiazine metachromatic dye with high affinity for acidic tissue components,
thereby staining tissues rich in DNA and RNA.

Principle: It has found wide applications both as vital staining in living tissues and as a special stain
owing to its metachromatic property. Toluidine blue has been used in vivo to identify dysplasia and
carcinoma of the oral cavity. Use of toluidine blue in tissue sections is done with the aim to highlight
components, such as mast cells granules, mucins, and cartilage.

Expected result: The result of toluidine blue staining showed that 16 (94.12%) out of 17 potentially
malignant lesions stained positive while one result was negative (5.88%) to toluidine blue staining.

Methylene blue-Methylene blue stain is a general biological stain used to highlight parts of animal,
bacteria, and blood tissue specimens. Methylene blue stains acidic cell parts (like the nucleus) blue and
is a good counterstain with Eosin Y. It can be substituted for Janus Green B stain or Carmine stain.

Principle: The presence of negatively charged molecules in the cell causes the staining phenomenon, as
the positively charged dye is attracted to negatively charged particles, such as polyphosphates like DNA
and RNA

Expected result: Solutions of this substance are blue when in an oxidizing environment, but will turn
colorless if exposed to a reducing agent.

6. silver stains-a very sensitive method for detecting small amounts of proteins and low-molecular-
weight nucleic acids in polyacrylamide gels.

Principle: the gel is saturated with silver ions. The less tightly bound metal ions are washed out of the gel
and those bound to the proteins are reduced to form metallic silver. The sensitivity of silver stain is at
least 10-fold greater than that of Coomassie blue staining Because silver staining methods can be
complex and need to be stopped at some time point to avoid overdeveloping the gel.

Expected result: In silver-ammonia staining, the solution cannot be prepared because it makes a brown
to black precipitate. In silver nitrate staining, the developer turns black and makes a deposit on the gel.
A “black cloud” should appear when the gel is dipped in the developer.

7.background staining or relief staining or negative staining- Use to study the morphological shape, size
and arrangement of the bacteria cells that is difficult to stain. eg: Spirilla. It can also be used to stain cells
that are too delicate to be heat-fixed.

Principle: Negative staining requires an acidic dye such as India Ink or Nigrosin.
India Ink or Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion (proton)
and the chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is
negatively charged, the cell surface repels the stain.

Expected result: In this technique, the background is stained, leaving the actual specimen untouched,
and thus visible. This contrasts with 'positive staining', in which the actual specimen is stained.
8. Hiss method- a type of differential stain which uses acidic and basic dyes to stain background &
bacterial cells respectively so that presence of capsule is easily visualized.

Principle: Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces.
Therefore, the best way to visualize them is to stain the background using an acidic stain (e.g., Nigrosine,
congo red) and to stain the cell itself using a basic stain. (E.g. Crystal violet, safaris, basic fuchsin and
methylene blue)

Expected result: Capsule staining can be done in two ways they are

Positive staining method – In positive staining method only capsule is stained.

Negative staining method – Here capsule is not stained but it is made visible by colorless area of capsule
in between colored cells and colored background.

REWFERENCES:

https://www.homesciencetools.com/product/methylene-blue-30-ml/
https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MethyleneBlueLoefflers.htm
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/silver-staining
https://microbiologyinfo.com/negative-staining-principle-reagents-procedure-and-result/
https://microbeonline.com/capsule-stain-principle-procedure-results/

RESEARCHER: ALPHA ABOLI