Reagent Preparation

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Reagent Preparation
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Weigh balance

• Fragile and precise instrument
• Handle with the care
• Consult manual for operating instructions
• Keep balance and weights clean and dry to protect
  from corrosion

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Weigh balance contd

• Changes in the surface of the balance parts will
  affect accuracy
• Do not place material to be weighed directly on
  the pan-always-use a container or weighing paper
• Subtract weigh paper from the combined weight of
  the container and material being weighed

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Weigh balance contd

• To prevent contamination of stock materials do
  not return unused chemical to the stock bottle
• Protect the balance from drafts of air
• Air moving cross the pans will cause an
  inaccurate reading
• Some balances have compartments which are to be
  closed during reading

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Weighing the correct amount of dye

• If powder with a dye content of 88 or more is
  used, no correction factor needs to be
  calculated.
• 75 available dye and 3 g of dye needed
• Divide 1 by the decimal equivalent of the
  available dye to obtain the correction factor
• Decimal equivalent of 75 0.75
• 1/0.75 1.33 (correction factor)
• Multiply 1.33 by 3 g 3.99
• 3.99 of impure dye is required to obtain 3 g
  usable dye

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Weigh Scale Bottle of Methylene Blue
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Ziehl Neelsen Hot- Original

• Basic Fuchsin 10 g
• Phenol 50 g
• Alcohol denatured or methanol- 150 ml
• Dissolve fuchsin in heated phenol alcohol water
  up to 1000ml

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ZN cold, Kinyoun Stain

• Basic fuchsin 25 g
• Phenol 50 g
• Alcohol (denatured or methanol) 125 ml
• dissolve fuchsin in heated phenol alcohol
• water 625 ml
• ZN 10 saturated basic fuchsin stock in alcohol
• Basic fuchsin 10 g
• Dissolve in alcohol 100 ml

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Auramine 0.1

• Auramine 1 g (1 stock)
• Dissolve in in alcohol (denatured or methanol)
  100 ml for 100 ml working solution
• Stock solution 10 ml
• stock solution phenol 33.3 g in water 1000 ml

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Sulphuric acid 20

• Water 800 ml (cooled)
• slowly add 200 ml sulphuric acid 96

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Acid alcohol 1 for Auramine stain

• Alcohol (denatured) 700 ml
• Hydrochloric acid (fuming) 10 ml
• water 290 ml

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Acid alcohol 3 for ZN

• Use 1 acid alcohol, 500 ml
• Add hydrochloric acid (fuming) 10 ml

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Methylene blue 0.1

• Methylene blue 0.1
• Dissolve methylene blue 1 g in 1000 ml water

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Gabbett Destain/counter-staining

• Solution
• Alcohol (denatured) 150 ml
• Dissolve methylene blue 5 g
• Water 250 ml
• Sulphuric acid 100 ml

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Potassium Permanganate 0.1

• Stock 5g permangate in 1000 ml water
• Dilute to 0.1 same day

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Water Quality

• Distilled water not absolutely needed
• Importance of bacteriologically pure water
  ultra-filtration

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Reagent Labeling

• Each bottle
• At least name of reagent and date of preparation

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Quality Control Records for AFB Microscopy
Reagents

• As explained in modules
• Record results in logbook
• Primary stains
• Carbol fuchsin, ZN and Kinyouns
• Auramine
• Decolorizer
• Counter-stains
• Methylene blue
• Permanganate