This document provides an overview of sputum examination, including indications, sample collection and transport, and various analysis methods. Key points include: sputum examination can identify causative organisms in respiratory infections or detect malignant cells; samples should be collected in the morning and transported in preservative solution; analysis includes physical examination of appearance, microbiological tests like Gram stain and culture, and examination for acid-fast bacilli via staining or molecular methods. Cytological examination of sputum can detect lung cancer but has only 65% sensitivity.

1. 1
Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
SPUTUM
EXAMINATION

2. 2
Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
OVERVIEW
1. Indications
2. Collection of sputum
3. Sample transport
4. Sample analysis
a. Physical examination
b. Microbiological examination
1. Gram stain
2. Culture and sensitivity
3. Examination for acid fast bacilli
a. Zn stain
b. Fluorescent stain
c. Culture on conventional media
d. Commercial automated culture system
e. Molecular methods
4. Examination for other specific organisms
c. Cytological examination

3. 3
Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
* Indications
1. Smear and culture
Identification of causative organism in a suspected infection - like pneumonia, TB,
fungal infection, P. carinii in HIV, bronchiectasis
2. Cytological examination
1. for malignant cells
2. Looking for viral inclusions
3. asbestosis
* Collection of sputum
1. Early morning deep cough sample is preferred
2. If unable to cough, induction of sputum can be done by
a.15% NaCl aerosol spray & propylene glycol for 20 min or
b.Nebulized hypertonic saline and distilled water
Collected in:
1. dry wide mouthed container with 25 ml capacity
2. leak proof – to prevent aerosols
3. break resistant – to prevent dessication
* Sample transport
1. samples should be immediately transported to laboratory as such if nearby
2. if distant laboratory, transport in 25 ml of the following solution
N acetyl pyridinium chloride 5g
Sodium chloride 10g
Distilled water 1 lt
If sputum is allowed to stand without medium –
a. rapid proliferation of contaminating bacterial flora from oral cavity and throat
b. H. influenzae donot survive for long
Donot refrigerate in any case

4. 4
Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
* SAMPLE ANALYSIS
(i) Physical appearance
Bloody (hemoptysis) Pulmonary TB
Lung abcess
Bronchiectasis
Bronchogenic carcinoma
Mitral stenosis
Pulmonary infarction
Rusty Pneumococcal lobar pneumonia
Red currant jelly Klebsiella
Green Pseudomonas
Purulent Pneumonia, lung abcess
Pink and frothy Pulmonary edema
(ii) Microbiological examination
#GRAM STAIN:
Prerequisites
1. There should not be squamous cells covered with masses of bacteria – indicates sample
is mostly from mouth or throat
2. If PMNs are <10 per epithelial cell – no need for culture
3. Knowledge of flora of mouth and pharynx necessary before analyzing
Normal flora of oral cavity and pharynx
GRAM POSITIVE
1. staphylococcus aureus and epidermidis
2. streptococcus viridans and pneumonia
3. diphtheroids
4. enterococci
5. micrococci
6. lactobacilli
7. yeasts (candida)
GRAM NEGATIVE
1. neiserria
2. H. influenzae
3. fusobacterium
4. coliforms
5. m. catarrhalis

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Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
Analysis
Streptococcus Pneumoniae Gram positive diplococci with surrounding clear space
Staphylococcus aureus Gram positive cocci in grape like clusters
Candida Gram positive yeast cells with budding yeasts and pseudohyphae
H. influenzae Gram negative coccobacilli
M. catarrhalis Gram negative diplococci both intra and extracellular
Actinomyces Large granules with center gram negative and periphery gram
positive
#CULTURE:
Ideal sample for culture
1. should contain <25 squamous cells per low power filed or <10 squamous cells per high
power field
2. sample should contain alveolar macrophages
3. neutrophils should be >10 per epithelial cell or >5 per high power field
4. bronchial epithelial cells present
5. sample should be washed with normal saline to wash the saliva
Method:
Inoculate the sample on blood agar and chocolate agar
Incubate in an atmosphere of extra CO2
Inspect plates after 18 hours
If growth is significant, antibiotic sensitivity testing is carried out

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Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
#EXAMINATION FOR ACID FAST BACILLI
!. ZEIHL NEELSON STAINING (AFB stain)
Sample:
1. According to RNTCP guidelines, 2 samples are collected, one stat and one early next
morning sample. It should be deep cough sputum sample
2. for children, gastric aspirate can be used as they often swallow sputum
Preparation:
smear is prepared with blood tinged/opaque/grayish/yellowish portion of the sputum
stained with ZN stain
Examined under microscope
Reporting guidelines (RNTCP):
1. Mycobacteria appear as bright red, slightly curved or red beaded rods, 2-4 µm in length
and 0.2 to 0.5 µm wide, against a blue green background.
2. Atleast 100 fields should be examined before declaring negative.
Drawbacks:
1. sensitivity 60-80%
2. minimum 5000-10000 bacilli / ml should be present for smear to be positive
Bleaching technique:
1. A solution of sodium hypochlorite is added to sputum sample – it leads to liquefaction
of mucous and killing of microbes
2. smears are prepared from sediment and stained with ZN stain

7. 7
Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
2. FLUORESCENCE MICROSCOPY
1. Slides are stained with fluorescent auramine-rhodamine or auramine O
2. Observed under fluorescent microscope – mycobacteria appear bright yellow against
green background
3. CULTURE ON CONVENTIONAL MEDIA
Indications:
1. drug susceptibility testing
2. species identification if other than M. tuberculosis suspected
3. sputum smear negative and strong clinical suspicion
Prerequisites:
1. 4% NaOH should be added before inoculation
2. this is because sputum samples are contaminated with normal flora, which grow and
digest the media before MTB can grow
3. 4% NaOH kills this flora
Media used:
1. solid media – LJ media (egg based) or Middle brook (agar based)
2. Liquid media – middle brook, TH9, TH 12
Advantage:
1. sensitivity 80-85%
2. can detect as low as 10-100 bacteria/ml
Drawbacks:
1. expensive
2. requires 6 weeks for results
4. COMMERCIAL AUTOMATED CULTURE METHODS (BACTEC)
1. Can give results in 2 weeks
2. mycobacteria are inoculated in a broth containing 14
C palmitate
3. mycobacteria metabolise 14
C palmitate and release 14
CO2 which is detected by the
instrument
5. MOLECULAR METHODS (PCR)
1. DNA sequences identified in MTB genome by PCR
2. can detect bacteria as low as 10-100 organisms / ml of sputum
3. direct sputum sample or culture samples can be used
4. laboratory cross contamination is an important issue here

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Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
#EXAMINATION OF OTHER ORGANISMS
ON SMEAR SPECIFIC INVESTIGATIONS
P. carinii Use BAL and stain with silver stain and giemsa
Yersinia Giemsa stain
Fungus SDA/KOH mount
Histoplasma Giemsa
Aspergillus KOH
Paragonimus Saline wet mount of sputum for eggs

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Notes on sputum examination…By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail.com Website: pathologybasics.wix.com/notes
(iv) Cytological examination
Prerequisites:
1. fresh morning sample
2. transport without delay
3. for suspected lung Ca collect sample for 5 consecutive days
4. if delay anticipated, prefix with Saccomano’s fixative (50% ethyl alcohol and 2%
carbowax)
Method:
1. smears are made from blood tinged portion or tissue fragments and stained with pap
stain
2. to be adequate, bronchial epithelial cells and alveolar macrophages must be seen
Drawback:
Sensitivity is only 65%
This sensitivity is more if –
1. smears are examined from multiple samples
2. lesion is located centrally
3. larger tumor size
4. histologic type is SCC rather than adenocarcinoma