# Exercise: Preparation of reagents and their quality control

-   Introduction (on flipchart): 0.5 hr

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-   various stains used for ZN and fluorescence

> ZN hot, original:
>
> basic fuchsin 10 g
>
> phenol 50g
>
> alcohol (denatured or methanol) 150 ml
>
> dissolve fuchsin in heated phenol + alcohol
>
> water up to 1000 ml
>
> 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
>
> auramine 0.1%:
>
> auramine 1 g (1% stock)
>
> dissolve in in alcohol (denatured or methanol) 100 ml
>
> for 100 ml working solution
>
> stocksolution 10 ml
>
> \+ stocksolution phenol 33.3 g in water 1000 ml
>
> sulphuric acid \~20%
>
> water 800 ml (cooled)
>
> slowly add 200 ml sulphuric acid 96%
>
> acid alcohol 1% for auramine staining
>
> alcohol (denatured) 700 ml
>
> hydrochloric acid (fuming) 10 ml
>
> water 290 ml
>
> acid alcohol 3% for ZN
>
> use 1% acid alcohol, 500 ml
>
> add hydrochloric acid (fuming) 10 ml
>
> methylene blue 0.1%
>
> dissolve methylene blue 1 g in 1000 ml water
>
> Gabbett destaining and counterstaining solution
>
> alcohol (denatured) 150 ml
>
> dissolve methylene blue 5 g
>
> water 250 ml
>
> sulphuric acid 100 ml

permanganate 0.1%

stock 5g permangate in 1000 ml water

dilute to 0.1% same day

-   water quality

> distilled water not absolutely needed
>
> importance of bacteriologically pure water: ultra-filtration

-   labeling

> each bottle
>
> at least name of reagent and date of preparation

-   QC of stains and logbook

> as explained in modules

-   Prepare stains in 3 groups of 2 persons; 1.5 hr

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-   demonstration: prepare "saturated" stock basic fuchsin in alcohol

> [show amount of sediment left]{.underline}

-   first batches: carbolfuchsin Kinyoun (at least 100 ml)

> [compare sediment after dissolving in phenol + alcohol]{.underline}
>
> carbolfuchsin ZN 1 % (at least 1000 ml)
>
> [compare sediment after dissolving in phenol + alcohol]{.underline}
>
> auramin stock in alcohol: 100 ml

let stand in dark bottle

separately prepare stock phenol 3.33% in water

-   second batches: sulphuric acid \~20% (500 ml)

> Gabbett (500 ml)
>
> acid alcohol 1% (at least 1000 ml) + use half to make 3%
>
> methylene blue 0.1% (1000 ml)

-   QC of stains prepared: 1 hr

only staining, reading on day 3

each separately one set

[Group 1: hot Ziehl Neelsen]{.underline}

-   CF 1% 10 minutes + sulphuric acid 20% 5 minutes + MBlue 0.1% 1/2 to
    1 min

-   CF 1% 10 minutes + acid alcohol 3% 3 minutes + MBlue 0.1% 1/2 to 1
    min

[Group 2: cold staining]{.underline}

-   Kinyoun 3 minutes + Gabbett 1 minute (Tan Thiam Hok staining)

-   Kinyoun 5 minutes + acid alcohol 3% 3 minutes + MBlue 0.1% 1/2 to 1
    min

[Group 3: fluorescence staining, NO HEATING]{.underline}

-   auramin 15 minutes + acid alcohol 1% 3 minutes + MBlue 0.1% 1 minute

-   auramin 15 minutes + acid alcohol 1% 3 minutes + 0.1% permanganate 1
    minute

> each set 2 positive and 2 negative controls
>
> label slides: POS or NEG & STAINING TYPE
>
> examples: POS / ZN- Sulph
>
> NEG / KIN- Gab
>
> POS / AUR -Mbl
>
> stain POS controls only once
>
> 3 staining cycles for NEG controls
>
> put smears away in boxes

# Exercise: Use of the microscope (SEARO booklet)

-   Brief description of microscope types and how they work: 15 minutes

-   brightfield

-   fluorescence

-   brightfield microscope parts

-   Adjustment of a microscope with practice: 45 minutes

-   condenser

-   diaphragm

-   interpupillary distance

-   dioptric adjustment

-   changing objectives / slides

-   lighting (mirror) and filters

-   focusing

-   Demonstration of fluorescence microscopy: 0.5 hrs

-   high positive

-   low positive

-   negative

-   artefacts to be distinguished from AFB: characteristics of AFB in
    flu

-   Reading smears ordinary microscope (ZN): 1 hr

-   1 panel of smears per participants

-   for the more experienced: try to quantify, IUATLD/WHO scale