Content Status

Type

Linked Node

H5Content
Content

Reagent preparation is an important activity and it’s essential to use certified chemicals and reagents. One should always check the potency of the chemicals used and calculate the amount to be weighed accordingly.

 

The following are the steps for reagent (primary stain and counter stain) preparation for Florescence Microscopy:

 

0.1% Auramine–O (1 L), the primary stain

  • Weigh 1 gm Auramine–O and transfer to conical flask.
  • Add 30 gm Phenolic crystals (99.5% purity) and mix well.
  • Add 100 ml absolute Ethanol (98-100% purity) and mix well.
  • Add 870 ml distilled water to make up final volume of 1 L.
  • Filter and transfer to amber bottle.
  • Label the bottle as 0.1% Auramine–O.
  • Date of preparation, date of expiry, batch no. and name of the Senior TB Lab Supervisor (STLS) who has prepared the stain should be clearly mentioned on the bottle.

 

1% Acid Alcohol (1 L) to decolorize

  • Dissolve 5 gm Sodium chloride in 250 ml distilled water.
  • Add 5 ml concentrated hydrochloric acid.
  • Mix with 750 ml absolute alcohol.
  • Always add acid slowly to alcohol, not vice-versa.
  • Store in amber coloured bottle.
  • Label the bottle as 1% Acid Alcohol.
  • Date of preparation, date of expiry, batch no. and name of the STLS who has prepared the stain should be clearly mentioned on the bottle.

 

0.5% Potassium Permanganate, KMnO₄ (1 L), the counter stain

  • Weigh 5 gm of Potassium permanganate (99.5% purity).
  • Transfer to a conical flask.
  • Take 1 L of distilled water.
  • Add small amounts of distilled water tothe  conical flask containing KMnO₄ and mix well.
  • Add the remaining distilled water to make up final volume of 1 L.
  • Filter and transfer to a bottle.
  • Label the bottle as 0.5% Potassium permanganate.
  • Date of preparation, date of expiry, batch no. and name of the STLS who has prepared the stain should be clearly mentioned on the bottle.

 

Table: Preparation of Different Volumes of Stains for FM Microscopy

AURAMINE METHOD QUANTITY OF REAGENT FOR 5 L QUANTITY OF REAGENT FOR 1 L QUANTITY OF REAGENT FOR 500 ML QUANTITY OF REAGENT FOR 100 ML

Auramine

 

Ethanol

 

Phenol

 

Distilled water

 

0.1% Auramine

5.0 g

 

500 ml

 

150.0 g

 

To make final volume 5000 ml

 

1.0 g

 

100 ml

 

30.0 g

 

To make final volume 1000 ml

 

0.5 g

 

50 ml

 

15.0 g

 

To make final volume 500 ml

 

0.1 g

 

10 ml

 

3.0 g

 

To make final volume 100 ml

 

Hydrochloric acid

 

Sodium chloride

 

Ethanol

 

Distilled water

 

1% Acid–alcohol

25 ml

 

25.0 g

 

3750 ml

 

1250 ml

 

 

5 ml

 

5.0 g

 

750 ml

 

250 ml

 

 

2.5 ml

 

2.5 g

 

375 ml

 

125 ml

 

 

0.5 ml

 

0.5 g

 

75 ml

 

25 ml

 

 

Potassium permanganate

 

Distilled water

 

0.1% Potassium permanganate

25.0 g

 

5000 ml

 

 

 

5.0 g

 

1000 ml

 

 

 

2.5 g

 

500 ml

 

 

 

0.5 g

 

100 ml

 

 

 

Video file

Resources

 

 

Kindly provide your valuable feedback on the page to the link provided HERE

Content Creator

Reviewer