Sputum examination provides important diagnostic information. Sputum is collected through deep coughing into a sterile container and examined microscopically and through culture. Microscopic examination with stains like Gram stain and Ziehl-Neelsen stain looks for bacteria, fungi and acid-fast bacilli. Culture grows pathogens and identifies causative organisms of respiratory infections. Sputum analysis is useful for diagnosing conditions like tuberculosis, pneumonia and lung cancer. Proper collection and rapid testing are important for accurate results.

1. SPUTUM EXAMINATION
MITHUN VENUGOPAL

2. INTRODUCTION
Sputum is a highly specialized watery, colorless and odorless product of the respiratory
tract (bronchia, trachea & lungs).
Sputum is mainly composed of mucus and also certain cellular and noncellular
components of host origin.
During expectoration, sputum gets contaminated with normal bacterial flora and cells
from pharynx and mouth.
It is the most frequently received specimen from the respiratory tract.
Both its collection and examination are advantageous as samples are easily obtained
and its cellular content is representative of the entire respiratory tract.

3. INDICATIONS
Identification of causative agent or organism associated with a particular suspected
infection of the lower respiratory tract like,
• Suspected tuberculosis.
• Pneumonia especially if severe or in an immuno-compromised host.
• Pneumocystis carinii pneumonia in HIV-positive patients.
• Suspected fungal infection.
• Chronic disease like bronchiectasis.
Cytological examination for the investigation of viral infections (viral inclusions in
cytomegalovirus and herpes simplex infections), fungal infection, asbestosis and
malignant cells.

4. SPUTUM COLLECTION
Sputum sample is ideally collected in the morning (since secretions accumulate
overnight).
Sputum sample is collected in a sterile, clean, dry and wide-mouthed plastic container
with a securely fitting screw cap. The container should be of break resistant plastic and
leak-proof to prevent desiccation and aerosol formation, and should have the capacity of
about 30 ml.
The patient is advised to take a deep breath 2-3 times filling his/her lungs, coughs
deeply, and spit into the plastic container.
About 2-5 ml of sputum is collected.
Sample consisting only saliva (watery appearance, clear, and foamy) is not acceptable
for laboratory investigations; in such case, another sample should be collected.

5. SPUTUM COLLECTION
In those patients who cannot produce sputum spontaneously by deep coughing, a
specimen of sputum may be induced. This is done by inhalation of 15% NaCl spray or
propylene glycol for 20 minute which are aerosolized to stimulate sputum production.
The container, containing sputum sample, is caped securely and labelled properly.

6. TRANSPORT
For microbiological examination of sputum, sample should be sent to the laboratory
immediately.
If sputum is allowed to stand, rapid reproduction of contaminating bacterial flora from
the throat and oral cavity will occur leading to incorrect results.
Pathogenic organism especially Haemophilus influenza, do not survive for a long time
in the collected sample.
Sputum sample for bacterial culture should not be refrigerated.
If the sample is to be transported to a remote laboratory for mycobacterial culture,
sputum should be collected in 25 ml of the following solution:
• N-acetyl pyridinium chloride-5 gm
• Sodium chloride-10 gm
• Distilled water-1000 ml

7. SPUTUM CONCENTRATION METHOD
(PETROFF’S METHOD)
The sputum is transferred to a sterile test tube and equal amount of sterile 4% NaOH is
added.
The tube is incubated at 37’c for 30 minutes with vigorous shaking every 5 minutes.
The mixture is centrifuged at 3000 rpm for 30 minutes and supernatant poured off.
The deposit is neutralized by N/10 HCL using a drop of phenol red as indicator.

8. PHYSICAL EXAMINATION
1. QUANTITY:
Large amount of purulent sputum is coughed out In bronchiectasis in bronchiectasis
Large amount of watery sputum with pink tinge suggests pulmonary oedema.
2. APPEARANCE/COLOR:
White, viscid, mucoid: Asthma, tuberculosis.
Serous, clear, watery: Pulmonary oedema.
Frothy, pink, serous: Broncho-alveolar carcinoma.
Clear or mucoid, grey, glassy, tenacious: Chronic bronchitis.
Yellow due to pus/neutrophils: Acute lower respiratory tract (pulmonary) infections.
Green: Long standing infection (bronchiectasis, lung abscess), pseudomonas.

9. PHYSICAL EXAMINATION
Rusty due to lysis of red cells: Pneumonia (e.g. pneumococcal) and pulmonary
infarction.
Bright red due to fresh blood: Pulmonary tuberculosis, lung tumors, pulmonary
infarction.
Black due to coal dust: Coal workers, in heavy smokers.
Anchovy sauce (chocolate brown): Rupture of amoebic liver abscess into lung.
Blood tinged sputum: Mitral stenosis, pulmonary tuberculosis, carcinoma lung,
pulmonary infarction.

10. MICROSCOPIC EXAMINATION
Staining of sputum: Two to three smears are made on a clean dry glass slides
and are stained with:
Leishman's stain or Wright stain for differential count.
Gram’s stain for microorganisms
Ziehl-Neilsen stain for acid fast bacilli.
Special stains for fungi.
Papanicolaou stain for study of malignant cells.
Other stains (depends on the clinical/pathological features).

11. Gram’s stain for microorganisms
From the purulent portion of the sputum, a thin smear is made on the grease free sterile
glass slide with a clean stick.
The slide is air-dried, fixed and stained with Gram's stain.
Entirely watery, mucoid, white, or frothy samples often show squamous epithelial cells
covered with bunches of bacteria; this indicates that the sample consists mainly of
secretions from the mouth and the throat.
Such samples are not acceptable for bacteriological examination.
Culture is not carried out if polymorphonuclear neutrophils are less than 10 per epithelial
cell.

12. Gram’s stain for microorganisms
Pathogenic organisms found in sputum are;
 Gram-positive: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus
pneumoniae.
 Gram-negative: Klebsiella pneumoniae, Moraxella catarrhalis, Haemophilus
influenzae, Yersinia pestis, Pseudomonas aeruginosa

13. Ziehl-Neilsen stain for acid fast
bacilli
Ziehl-Neilsen-stained sputum smear is considered
as positive if 5000-10000 tubercle bacilli/ml are
present in the sputum.
Possibilities of detection of tubercle bacilli are
increased if multiple sputum samples are examined.
Mycobacteria appear as bright red straight or
slightly curved roads against a blue background.
Minimum 100 fields are examined before reporting
the smear as negative.

14. Parasites found in sputum
Larvae of Strongyloides stercoralis and roundworm.
Entamoeba histolytica: Cysts or the trophozoites may be found when an amoebic liver
abscess ruptures into the lungs.
Echinococcus granulosae: Scolices and hooklets of the larval form may be seen with
the rupture of the hydatid cyst of the lungs into the bronchus.

15. Leishman stain or Wright stain for
differential count
Normal sputum consists of a few
neutrophils, few lymphocytes, carbon laden
macrophages, occasional eosinophils and
red cells.

17. Papanicolaou stain for study of
malignant cells
Cytological examination of sputum is normally carried out for the diagnosis of
bronchogenic carcinoma.
For cytological examination, early morning sputum sample is preferred.
A thin sputum smear is prepared on clean, sterile, grease-free glass slide from a
yellowish, greyish, opaque, or blood-tinged portion, or from tissue fragments in sputum
and stained with Papanicolaou technique.

18. SPUTUM CULTURE
Culture media is inoculated with a floccule of the purulent portion of sputum for
absolute identification of microorganism.
Sputum sample is considered as unsuitable for the bacterial culture if it contains >25
squamous epithelial cells/low power field.
An ideal sputum sample for bacterial culture contains bronchial epithelial cells,
numerous neutrophils (>5/high power field), alveolar macrophages, and few squamous
epithelial cells (<10/high power field).
Saliva is washed away from sputum with sterile normal saline in order to reduce the
amount of contaminating normal bacterial flora in the inoculum.

19. SPUTUM CULTURE
Blood agar plate and chocolate agar are inoculated with the washed sputum. The
chocolate agar plate is incubated in an atmosphere of extra carbon dioxide (CO2) and
blood agar plate is incubated aerobically.
After the incubation for 18 hours, inoculated agar plates are examined for growth; if
growth is not sufficient, incubation for further 24 hours is indicated.
Lowenstein-Jensen medium is used for culturing Mycobacterium Tuberculosis.