Part II: Microscopy
acid-fast staining procedures

Mycobacteria retains the primary stain even after exposure to decolorising acid-alcohol, hence the term 'acid-fast'. A counter-stain is employed to highlight the stained organisms for easier recognition. There are several methods of determining the acid-fast nature of mycobacteria. In the carbolfuchsin (Ziehl-Neelsen) procedure, acid-fast organisms appear red against a blue background, while in the fluorochrome procedures (auramine-O, auramine-rhodamine), the acid-fast organisms appear as fluorescent rods, yellow to orange (the colour may vary with the filter system used) against a paler yellow or orange background.

Stain powders are often not pure and a corrected weight should be used to ensure proper staining. Most manufacturers print the % available dye content on the label. The corrected weight is determined by calculating a correction factor and multiplying the amount of dye required by this factor:

Example
75% available dye and 3g 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 3g = 3.99
• 3.99 of impure dye is required to obtain 3g usable dye

If powder with a dye content of 88% or more is used, no correction factor needs to be calculated.

Pure phenol crystals are colourless; brown-tinted crystals should not be used since they may cause unsatisfactory staining.

ZIEHL-NEELSEN STAINING
Reagents

Fuchsin

Basic fuchsin	3.0g
95% ethanol (technical grade)	100ml
Dissolve basic fuchsin in ethanol	Solution 1

Phenol

Phenol crystals	5g
Distilled water	100ml
Dissolve phenol crystals in distilled water  (gentle heat may be required)	Solution 2

Working solution

Combine 10ml of solution 1 with 90ml of solution 2 and store in an amber bottle. Label bottle with name of reagent as well as preparation and expiry dates. Store at room temperature for six to twelve months and filter before use.

Decolourising agent: 3% acid-alcohol

Concentrated hydrochloric acid (technical grade)	3ml
95% ethanol (technical grade)	97ml

Carefully add concentrated hydrochloric acid to 95% ethanol. Always add acid slowly to alcohol, not vice versa.The mixture will heat up. Store in an amber bottle. Label bottle with name of reagent and dates of preparation and expiry. Store at room temperature for six to twelve months.

In countries where the acquisition of alcohol may be problematic, a solution of 25% sulphuric acid may be used as decolourising agent. This is prepared as follows:

Decolourising agent: 25% sulphuric acid

Concentrated sulphuric acid (technical grade)	25ml
Sterile distilled water	100ml

Carefully add concentrated sulphuric acid to water. Always add acid slowly to water, not vice versa. The mixture will heat up. Store in an amber bottle. Label bottle with name of reagent and dates of preparation and expiry. Store at room temperature for six to twelve months.

Counterstain: Methylene blue

Methylene blue chloride	0.3g
Distilled water	100ml

Dissolve methylene blue chloride in distilled water and store in an amber bottle. Label bottle with name of reagent and dates of preparation and expiry. Store at room temperature for six to twelve months.

Procedure
Refer to Diagram 2.

FLUOROCHROME STAINING
Fluorescence microscopy uses illumination from either a quartz-halogen lamp or a high-pressure mercury vapour lamp. The advantage of fluorescence microscopy is that a low magnification objective is used to scan smears, allowing a much larger area of the smear to be seen and resulting in more rapid examination. However, one drawback in using a low magnification is the greater probability that artifacts may be mistaken for acid-fast bacilli. It is therefore strongly recommended that suspect bacilli be confirmed at higher magnification, and that positive fluorochrome stains be confirmed by Ziehl-Neelsen microscopy.

Much money can be wasted in purchasing very expensive and sophisticated fluorescence microscopes that are not necessary for tuberculosis microscopy. The following technical specifications will suffice:

• Halogen lamps are adequate. They are usually built-in and are much less expensive than mercury vapour lamps which also have a limited life span. Halogen lamps also warm up  immediately
• Good light-passing ability is required, including high numerical apertures for the  condenser and for two objectives. Three objectives are necessary, eg. 10x and 25x for scanning and 45x for morphological definition
• Objectives should be uncorrected since smears are examined without cover glasses
• Fluorescence oil should always be used because of its better refraction index

Reagents

Auramine O

Auramine	0.1g
95% ethanol (technical grade)	10ml
Dissolve auramine in ethanol	Solution 1

Auramine is carcinogenic and direct contact with the powder or the solution should be avoided.

Phenol

Phenol crystals	3.0g
Distilled water	87ml
Dissolve phenol crystals in water	Solution 2

Mix solutions 1 and 2 and store in a tightly stoppered amber bottle away from heat and light. Label bottle with the name of the reagent and dates of preparation and expiry. Store at room temperature for three months. Turbidity may develop on standing but this does not affect the staining reaction.

Decolourising solution

Concentrated hydrochloric acid	0.5ml
70% ethanol (technical grade)	100ml

Carefully add concentrated hydrochloric acid to the ethanol. Always add acid slowly to alcohol, not vice versa. Store in an amber bottle. Label bottle with name of reagent and dates of preparation and expiry. Store at room temperature for three months.

Counterstains
Either potassium permanganate or acridine orange may be used as counterstains.

Dissolve potassium permanganate in distilled water in a tightly stoppered amber bottle. Label bottle with name of reagent and dates of preparation and expiry. Store at room temperature for three months.

Acridine orange

Anhydrous dibasic sodium phosphate  (Na2HPO4)	0.01g
Distilled water	100ml
Acridine orange	0.01g

Dissolve sodium phosphate in distilled water. Add acridine orange and dissolve. Store in a tightly stoppered amber bottle away from heat and light. Label bottle with name of reagent and dates of preparation and expiry. Store at room temperature for three months.

Procedure
Refer to Diagram 3.

Precautions during staining

• Avoid under-decolourisation with acid-alcohol. Organisms that are truly acid-fast are difficult to over-decolourise
• Avoid making thick smears. This may interfere with proper  decolourisation, and counterstains may hide the presence of acid-fast bacilli. Additionally, thick smears may flake, resulting in loss of smear material and possible  transfer of material to other slides
• Strong counter-staining may mask the presence of acid-fast bacilli
• Smears that have been examined by fluorescence microscopy  may be re-stained by Ziehl-Neelsen staining to confirm observations (recommended). However, once smears have been stained by Ziehl-Neelsen staining they cannot be used for fluorescence microscopy