Examination of sputum

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1. EXAMINATION OF SPUTUM Made By:- Atifa Ambreen

2. SPUTUM CULTURE .A sputum culture is test to be detect and identify bacteria or fungi that infect the lungs or breathing passages & cause infections. Sputum is a thick fluid produced in the lungs and in the adjacent airways.

3. POSSIBLE PATHOGENS BACTERIA Gram Positive Streptococcus pneumoniae Strephylococcus Aureus Streptococcus pyogenes Gram Negative Haemophilus influenzae Klebsiella pneumoniae Pseudomonas aeruginosa Proteus Species Yersina pestis

4. Mycobacterium tuberclosis Mycoplasma pneumoniae & Legionella Pneumophila FUNGI & ACTINOMYCETES Pneumosystis carinii Blastomyces dermatidis Histoplasma capsulatum Aspergillus species Candida Albicans Nocardia species PARASITES Paragonimus Species

5. OBJECTIVE Production of sputum Collection of sample Examination Macroscopic Microscopic

6. COLLECTION OF SAMPLE OF SPUTUM (Day 1) 1.Before collecting sputum the mouth should be prerinsed and this removes contaminants from oral cavity. 2.Give the patient a clean dry wide necked leak-proof container and request him or her to cough deeply to produce a sputum specimen. 3.For best results early morning freshly expectorated sputum specimen should be collected.

7. 4.Label the container, and complete the request form. 5.When Pneumonia or Bronchopneumonia is suspected, deliver the sputum to the laboratory with a little delay as soon as possible because organisms such as S. pneumoniae & H. influenzae require culturing as soon as possible. Specimen for the isolation for S. Pneumoniae & H. Influenzae must never be refrigerated.

8. EXAMINATION VOLUME:- . A 24 hr volume of sputum is measured in patient with chronic bronchitis, lung abscess or bronchial asthma. A rising volume indicates worsening & decreasing volume indicates improvement

9. Appearance:- Purulent: Green Looking, mostly pus Mucopurulent : Green Looking with pus & mucus. Mucoid : Mostly mucus Mucoslivary : Mucus with small amount of sliva .

10. Microscopic:- After macroscopic examination transfer material to a clean slides. Smears made on clear slides should be air dried, fixed over flame and stained.   Different stains used:- 1:- Gram’s stain 2:- Ziehl-Neelsen stain for AFB

11. GRAM STAIN Smear the sputum Stained by Gentian violet stain Keep for 1 min. Bacteria get violet color Pour Gram’s Iodine For 1 min. Wash with Alcohol

12. Wash with water and dry Mount under oil immersion

13. GRAM STAIN

14. GRAM SMEAR Examine the smear for pus cells and predominat bacteria. Look specially among the pus cells for:- Gram positive Diplococci (capsulated) that could be S. Pneumoniae ,. Gram positive cocci in groups that could be S. Aureus . Gram negative Rods & cocco -bacilli that could be H. influenza. Gram negative Diplococci in and between pus cells that could be M. catarrhalis .

15. ZEIL-NEELSON SMEAR TO DETECT AFB Chance of detecting AFB in sputum smears are significantly increased if the organisms are first concentrated by centrifugation sodium hypochlorite(Bleach) is recommended for liquefying the sputum because it kills M. Tuberculosis. NaOC1 centrifugation technique to concentrate AFB 1:Transfer 1-2 ml of sputum to a screw cap universal bottle. 2:Add an equal volume of concentrated NaOC1 solution and mix well.

16. 3:Leave at room temperature for 10-15 min shaking at interval to break down the mucus in the sputum. 4:Add about 8ml of distilled water mix well. 5:Centrifuge at 3000g for 15 min. 6:Using a glass pasture pipette to remove and discard the supernatant fluid. Mix the sediment.Transfer a drop of well mixed sediment to a clean glass slide. Make a thin smear & Allow to air dry.

17. ZEIL-NEELSON STAIN Smear the sputum Fixed by Heating Pour carbol fuchin and heat it from below Keep for 5 min. Wash with water Decolorize with 20%H 2 SO 4 Wash with Malachite green/ methyene blue for 1 min. Wash & dry Mount under oil immersion

18. RESULT AFB  Red, Straight or slightly curved rods, occurring singly or in a small groups, may appear beaded. Cells  Green Background material  Green

19. CULTURE OF THE SPECIMEN To obtain as pure a culture as possible of a respiratory pathogens it is necessary to reduce the number of commensals inoculated. Ways of reducing commensal number include washing the sputum free from sliva or liquefying and diluting it. The technique using saline-washed sputum described. The dilution technique requires a liquid agent such as dithiothreitol ( Sputolysin , Sputasol ) which is expensive and unstable.

20. Each specimen received for culture should be plated on agar. Different agars:- 1: Blood agar 2: Chocolate agar 3: MacConkey’s agar  4: Thioglycollate broth

21. BLOOD AGAR AND CHOCOLATE AGAR 1: Wash a purulent part of sputum in about 5ml of sterile physiological saline. 2: Inoculate the washed sputum on plate of: Blood agar Chocolate agar 3: Add an aptochin disc to the blood plate within the area of 2 nd spread. This will help to identify S. pneumoniae . 4: Incubate the blood agar plate aerobically and the chocolate agar plate in a carbon dioxide enriched atmosphere.

22. DAY 2 & ONWARD Examine and report the cultures Blood agar and chocolate agar cultures:- Look especially for a significant growth of: streptococcus Pneumoniae Haemophilus influenzae Stephlococcus aureus