PREPARATION AND EXAMINATION OF SPUTUM IN TUBERCULOSIS LABORATORY

Laboratory examination of sputum

 Description on the appearance of sputum

Purulent: green-looking, mostly pus

Mucopurulent: green looking with pus and mucus

Mucoid: mostly mucus

Mucosalivery: mucus with small small amount of saliva

Haemoptysis: containing blood

Preparation of sputum for AFB

In sputum preparation the container that contains the sample is check for proper labelling and also the nature of the sputum if it contains blood, pus or mucus to enable the proper identification of the organism. to prepare a smear, the purulent part of the sputum is selected and a wooden stick is use  to spread the area for about 2X3 cm, and the smear is avoided to be too thick at least if possible can read a newsprint through it. the smear is air dried in a safe place to protect it from dust and insect

Materials/ reagent  

Carbol fuchsin (filtered), Acid alcohol 3%v/v i.e.  3%v/v hydrochloric acid solution in 70%v/v alcohol

Filter paper,Water, Methylene blue, glass slide drainage rack immersion oil, timer, sputum container microscope, applicator stick, pencil

Ziel-neelson technique

Ziel-neelson use to stain mycobacterium species. Mycobacteria, unlike most other bacterial do not stain well by gram technique. They can how ever be stained with carbol fuchsin combined with phenol. The stain binds to the mycolic acid in mycobacteria cell wall after fix-heating After staining the acid decolorizing solution is applied, this removes the red dye from the background.  Cells, tissues fibres, and any organisms in the smear except mycobacterial which retain the dye and are there for referred to as acid fast bacilli. Following decolourization, the smear is counter stained with methylene blue, which stains the background material providing a contrast colour against which the AFB can be seen.

STEPS FOR THE PREPARATION

1.     after the smear have been prepared and air-dried as mention above

2.      the smear is heat-fix and dried

        Alcohol fixation is recommended when the smear has not been prepared from sodium hypochlorite(bleech) treated sputum and will not stain immediately. Mycobacterium tuberculosis is killed by bleech and during the staining process heat fixation of untreated sputum will not kill mycobacterium tuberculosis where as alcohol fixation is bactericidal.

3. The smear is covered with carbol fuchsin stain

4. heat is  applied until vapour begin to rise i.e. to about 60 degree to avoid overheating, the smear is allowed on the slide for 5minutes

5 the stain is washed off with clean water and

6 the smear is covered with 3%v/v acid alcohol for 5 minutes until the smear is sufficiently decolourized i.e. pink

7. Clean water is applied to wash off the any excess alcohol

8 the smear is then covered with methylene blue 5g/l for 1-2 minutes

9.wash off the stain

10 wipe the back of the slide and stand I to air dry in a draining slide rack.

EXAMINATION

The smear is examined microscopically using 100X oil immersion objective, when dispensing the oil, it is noted that the dispenser shouldn’t touch the smear reason been that there could be transfer of  Acid fast bacilli from one prep to another.

Result   

AFB: red straight or slightly curved, occurs singly or in small groups, may appear beaded

CELLS:  green

BACKGROUND MATERIAL: green

REPORTING OF SPUTUM SMEAR

When no AFB is seen after examining 300 filds, report the smear as ‘No AFB seen’

When very few AFB are i.e. when only 1 or two AFB are seen after examinating 100 fields, request a further specimen to examine (those AFB might have come from tap water or may be scratch of glass slide or by the use same piece of blotting paper while drying

When any red bacilli are seen, report the smear as AFB positive and give an indication of the number of bacteria present as follows( the greater the number the, the more infectious the patient)

a)      more than 10AFB/field at least in 20 fields report +++

b)      1-10AFB/field report as ++

c)      10-99AFB/100 report as +

d)     1-9AFB/100 fields report the exact number

PRECAUTIONS
1) smear for sputum is must notfix-heating.
2) over-heating is not advised
3) accuracy in timing is considered
4) ensure proper handling of chemicals such as ethanol, these is a flammable substance and must b handled with care

LIMITATION OF AFB MICROSCOPY

1.    Does no distinguish between viable and dead organism

2.    Limited sensitivity

3.    Limited specificity