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Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:

OVERVIEW 1. 2. 3. 4. Indications Collection of sputum Sample transport 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

Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:

* 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

Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:


(i) Physical appearance

Bloody (hemoptysis) Pulmonary TB Lung abcess Bronchiectasis Bronchogenic carcinoma Mitral stenosis Pulmonary infarction Pneumococcal lobar pneumonia Klebsiella Pseudomonas Pneumonia, lung abcess Pulmonary edema

Rusty Red currant jelly Green Purulent Pink and frothy

(ii) Microbiological examination

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

Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:

Analysis Streptococcus Pneumoniae Staphylococcus aureus Candida H. influenzae M. catarrhalis Actinomyces Gram positive diplococci with surrounding clear space Gram positive cocci in grape like clusters Gram positive yeast cells with budding yeasts and pseudohyphae Gram negative coccobacilli Gram negative diplococci both intra and extracellular Large granules with center gram negative and periphery gram positive

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

Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:


!. 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 m in length and 0.2 to 0.5 m 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
Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:

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 14C palmitate 3. mycobacteria metabolise 14C palmitate and release 14CO2 which is detected by the instrument 5. MOLECULAR METHODS (PCR) 1. 2. 3. 4. DNA sequences identified in MTB genome by PCR can detect bacteria as low as 10-100 organisms / ml of sputum direct sputum sample or culture samples can be used laboratory cross contamination is an important issue here
Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:

#EXAMINATION OF OTHER ORGANISMS ON SMEAR P. carinii Yersinia Fungus Histoplasma Aspergillus Paragonimus SPECIFIC INVESTIGATIONS Use BAL and stain with silver stain and giemsa Giemsa stain SDA/KOH mount Giemsa KOH Saline wet mount of sputum for eggs

Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website:

(iv) Cytological examination

Prerequisites: 1. 2. 3. 4. fresh morning sample transport without delay for suspected lung Ca collect sample for 5 consecutive days if delay anticipated, prefix with Saccomanos 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

Notes on sputum examinationBy Dr. Ashish V. Jawarkar Contact: Website: