SPUTUM EXAMINATION

• 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
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
- for malignant cells
- Looking for viral inclusions
- 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. influenza do not survive for long

• Donot refrigerate in any case

 SAMPLE ANALYSIS
• Physical appearance

Bloody (hemoptysis) Pulmonary TB

Lung abcess
Bronchiectasis
Bronchogenic carcinoma
Mitral stenosis
Pulmonary infarction

Rusty Pneumococcal lobar pneumonia

Physical appearance

Red currant jelly Klebsiella

Green Pseudomonas
Purulent Pneumonia, lung abcess
Pink and frothy Pulmonary edema
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 • GRAM NEGATIVE
1. staphylococcus aureus and 1. neiserria
epidermidis 2. H. influenzae
2. streptococcus viridans and 3. fusobacterium
pneumonia 4. coliforms
3. diphtheroids 5. m. catarrhalis
4. enterococci
5. micrococci
6. lactobacilli
7. yeasts (candida)
Analysis
Organisms Morphology (Gram stain)
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

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

AFB
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
Reporting of sputum smear
• When no AFB are seen after examining 300 fields, report the smear as ‘No AFB seen’.
• When very few AFB are seen i.e. when only 1 or 2 AFB are seen after examining 100 fields,
request a further specimen to examine (Those AFB might have came from tap water
(saprophytic mycobacteria), or it may be scratch of glass slide or by the use of 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 more infectious the patient):
• More than 10 AFB/field at least in 20 fields: report as + + +
• 1-10 AFB/field at least in 50 fields: report as + +
• 10-99 AFB/ 100 fields: report as +
• 1-9 AFB/100 fields: report the exact number
CULTURE
• 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
2. FLUORESCENCE MICROSCOPY
- Slides are stained with fluorescent auramine-rhodamine or
auramine O
- Observed under fluorescent microscope – mycobacteria appear
bright yellow against green background
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
CULTURE
• Advantage:
1. sensitivity 80-85%
2. can detect as low as 10-100 bacteria/ml

• Drawbacks:
1. expensive
2. requires 6 weeks for results

- COMMERCIAL AUTOMATED CULTURE METHODS (BACTEC)

1. Can give results in 2 weeks
2. Mycobacteria are inoculated in a broth containing 14C palmitate
3. Mycobacteria metabolise 14C palmitate and release 14CO2 which is detected by the instrument
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
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
 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
• Ideal sample should contains:
a. Alveolar macrophages
b. numerous Neutrophils (>5/Hpf)
c. Bronchial epithelial cells (<10/Hpf)
d. Few Squamous cells (<10/Hpf)
Dittrich’s plugs
• These plugs are seen most
commonly in chronic bronchitis,
bronchiectasis & bronchial
asthma
When expectorated, they are
usually solitary of a variable size.
When crushed, they are found to
be made of cellular debris, fatty
acid crystals, fat globules &
bacteria
Parasites present in sputum
a. Ascaris Lumbricoides
b. Echinococcus granulosus
c. Toxo caracaris
d. Paragonimus westermani
Curschmann’s Spirals
• Curschmann’s Spirals are characteristic
of bronchial asthma sputum but may
be seen in other respiratory disorders.
• Macroscopically, they can sometimes
be recognized by naked eye & appear
as yellow, white, mucoid, wavy
threads frequently coiled into little
balls. Their length may exceed 1-5cm.
• Microscopically, they show a central
thread around which mucus is
wrapped, supported by a fibril
network.
Charcot Leyden crystals
• Charcot Leyden crystals are seen
in sputum of bronchial asthma
cases.
• The crystals are colorless,
pointed hexagons & variable in
size (may look needle shaped).
• These are derived from
disintegration of eosinophils
hence they stain strongly with
eosin.
Significance of sputum in heart
disease
a. In acute Pulmonary oedema: Sputum is a abundant, frothy & pink
(upto 1 liter a day) Microscopically, it shows numerous RBC’S & large
hyaline masses

b. Mitral heart disease: Sputum is tenacious & blood is present, either

in streaks or in dark masses mixed with mucus

c. In chronic congestive heart failure: Sputum is frothy & rust colored

Microscopically, presence of RBC’s & heart failure cells
kind of inclusions seen in different viruses of
sputum sample

Parainfluenza & amp;measles virus Eosinophilic intracytoplasmic inclusion

Respiratory syncytial cytomegalovirus Basophilic intracytoplasmic inclusion