Ziehl-Neelsen Technique

 Ziehl-Neelsen technique for M. tuberculosis

and M. ulcerans

.

The Ziehl-Neelsen (Zn) technique is used to stain Mycobacterium species including M. tuberculosis,

M. ulcerans, and M. leprae. Mycobacteria, unlike most other bacteria, do not stain well by the Gram

technique. They can however be stained with carbol fuchsin combined with phenol. The stain binds to

the mycolic acid in the mycobacterial cell wall. After staining, an acid decolorizing solution is applied.

This removes the red dye from the background cells, tissue fibres, and any organisms in the smear except mycobacteria which retain (hold fast to) the dye and

are therefore referred to as acid fast bacilli, or simply AFB. Following decolorization, the smear is counterstained with malachite green or methylene blue which stains the background material, providing a

contrast colour against which the red AFB can be seen.

Note: Some actinomycetes, corynebacteria, and bacterial

endospores are also acid fast.(For these organism use 0.5 percent of H2SO4)

Differences between the acid fastness of Mycobacterium species

– M. tuberculosis and M. ulcerans are strongly acid fast. When staining specimens for these species,

a 3% v/v acid solution is used to decolorize the smears (as described in the following

technique).

– M. leprae is only weakly acid fast. A 1% v/v acid decolorizing solution is therefore used for

M. leprae smears and also different staining and decolorizing times as described in subunit

 ‘Acid and alcohol fast bacilli’: The acid decolorizing reagents used in the Zn staining technique also contain alcohol

(ethanol). It is not true, however, that mycobacteria can be differentiated by whether they are acid fast or acid and

alcohol fast. As Collins et al remark, ‘There is no basis for the old story that tubercle bacilli are acid and alcohol fast while

other bacteria are only acid fast. Acid-fastness varies with the physiological state of the organisms. The alcohol in the decolorizing

solution merely gives a cleaner stained smear’.1

Hot and cold Zn techniques In the ‘hot’ Zn technique), the phenolic-carbol fuchsin stain is heated to

enable the dye to penetrate the waxy mycobacterial cell wall. Techniques which do not heat the stain are

referred to as ‘cold’ techniques. In these, penetration of the stain is usually achieved by increasing the concentrations of basic fuchsin and phenol and incorporating a ‘wetting agent’ chemical. Comparisons

between the ‘hot’ and ‘cold’ methods have shown that both M. leprae and M. tuberculosis stain

less well by the ‘cold’ method and stained smears fade rapidly.2

Note: In the paper of Ridley, MJ and Ridley, DS.3 the

authors report that after staining smears for leprosy bacilli at

room temperature, examination was always difficult because

of pallor. Where bacilli were few, some were missed

altogether.

Ziehl-Neelsen technique for M. tuberculosis

and M. ulcerans

The preparation of sputum smears for the detection of M. tuberculosis is described in subunit 7.6, and

cerebrospinal fluid preparation in subunit. In HIV-infected patients, AFB may be detected in buffy

coat smears prepared from EDTA anticoagulated blood

Required

– Carbol fuchsin stain (filtered ) Reagent No. 21

– Acid alcohol, 3% v/v Reagent No. 4

– Malachite green, 5 g/l Reagent No. 55

(0.5% w/v)*

*If preferred, methylene blue, 5 g/l may be used instead of

malachite green.

Method

1 Heat-fix the dried smear as described in subunit

7.3.2.

Alcohol-fixation: This is recommended when the smear has not been prepared from sodium hypochlorite

(bleach) treated sputum and will not be stained immediately. M. tuberculosis is killed by bleach and during the

staining process. Heat-fixation of untreated sputum will not kill M. tuberculosis whereas alcohol-fixation is

bactericidal.

2 Cover the smear with carbol fuchsin stain.

3 Heat the stain until vapour just begins to rise (i.e. about 60 _C). Do not overheat. Allow the

heated stain to remain on the slide for 5 minutes.

Heating the stain: Great care must be taken when heating the carbol fuchsin especially if staining is carried out over a tray or other container in which highly flammable chemicals have collected from previous staining. Only a small flame should be applied under the slides

using an ignited swab previously dampened with a few drops of acid alcohol or 70% v/v ethanol or methanol.

Do not use a large ethanol soaked swab because this is a

fire risk.

4 Wash off the stain with clean water.

Note: When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater.

5 Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized,

i.e. pale pink.

Caution: Acid alcohol is flammable, therefore use it with care well away from an open flame.

6 Wash well with clean water.

7 Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the

smear is thin.

8 Wash off the stain with clean water.

9 Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not

blot dry).

10 Examine the smear microscopically, using the 100X oil immersion objective. When available,

use 7x eyepieces because these will give a brighter image. Scan the smear systematically

Note: Do not touch the smear with the end of the oil dispenser because this could transfer

AFB from one preparation to another. After examining a positive smear, the oil must be

wiped from the objective.

Results

AFB . . . . . . . . . . . . . . . . . . . . Red, straight or slightly

curved rods, occurring singly

or in small groups,

may appear beaded.

Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Green

Background material . . . . . . . . . . . . . . . . . . . . Green

enable the dye to penetrate the waxy mycobacterial cell wall. Techniques which do not heat the stain are

referred to as ‘cold’ techniques. In these, penetration of the stain is usually achieved by increasing the concentrations of basic fuchsin and phenol and incorporating a ‘wetting agent’ chemical. Comparisons

between the ‘hot’ and ‘cold’ methods have shown that both M. leprae and M. tuberculosis stain

less well by the ‘cold’ method and stained smears fade rapidly.2

Note: In the paper of Ridley, MJ and Ridley, DS.3 the authors report that after staining smears for leprosy bacilli at

room temperature, examination was always difficult because of pallor. Where bacilli were few, some were missed

altogether.

Reporting of sputum smears

When any definite red bacilli are seen, report the smear as ‘AFB positive’, and give an indication of the

number of bacteria present as follows:

More than 10 AFB/field . . . . . . . . . . . report +++

1–10 AFB/field . . . . . . . . report ++

10–100 AFB/100 fields . . report +

1–9 AFB/100 fields . . . . . report the exact number

When very few AFB are seen: e.g. when only one or two AFB are seen, request a further specimen to

examine. Tap water and deionized water (using ‘old’ resin) sometimes contain AFB that resemble tubercle bacilli, and occasionally stained scratches on a slide can be mistaken for AFB although these tend to be in a different focal plane from the smear. Occasionally AFB can be transferred from one

smear to another when the same piece of blotting paper is used to dry several smears.

When no AFB are seen after examining 100 fields: Report the smear as ‘No AFB seen’. Do not report

‘Negative’ because organisms may be present but not seen in those fields examined. Up to three specimens (one collected as an early morning specimen) may need to be examined to detect M. tuberculosis in sputum.

Quality control

At regular intervals, and always when a new batch of stain is started, two sputum smears of known high

and low AFB positivity should be stained with the routine smears to check that the carbol fuchsin,

staining method, and the microscopical examination of smears are satisfactory.