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Preparation of Reagents for FM Microscopy
Learning ObjectivesPreparation of Reagents for FM Microscopy
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
|
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