EXAMINATION OF SPUTUM

Made By:- Atifa Ambreen

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.

POSSIBLE PATHOGENS

BACTERIAGram PositiveStreptococcus pneumoniaeStrephylococcus AureusStreptococcus pyogenes

Gram NegativeHaemophilus influenzaeKlebsiella pneumoniaePseudomonas aeruginosaProteus SpeciesYersina pestis

Mycobacterium tuberclosisMycoplasma pneumoniae & Legionella Pneumophila

FUNGI & ACTINOMYCETESPneumosystis cariniiBlastomyces dermatidisHistoplasma capsulatumAspergillus speciesCandida AlbicansNocardia species

PARASITESParagonimus Species

OBJECTIVE

Production of sputum Collection of sample Examination Macroscopic Microscopic

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.

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.

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

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

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

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

Wash with water and dry

Mount under oil immersion

GRAM STAIN

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.

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.

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.

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%H2SO4

Wash with

Malachite green/methyene blue for 1 min.

Wash & dry

Mount under oil immersion

RESULT

AFB Red, Straight or slightly curved rods, occurring singly or in a small groups, may appear beaded.

Cells Green

Background material Green

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.

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

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 agarChocolate agar

3: Add an aptochin disc to the blood plate within the area of 2nd 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.

DAY 2 & ONWARD

Examine and report the cultures

Blood agar and chocolate agar cultures:-Look especially for a significant growth of: streptococcus PneumoniaeHaemophilus influenzae

Stephlococcus aureus