A sputum culture test identifies bacteria and fungi affecting the lungs, with specific organisms listed for both gram-positive and gram-negative categories. The document outlines collection, examination procedures, and staining techniques needed for accurate diagnosis, emphasizing the importance of timely sample delivery to the laboratory. Additionally, methods for reducing commensal organisms and culturing techniques on various agar types are described to ensure pure cultures of respiratory pathogens.

3.  .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.

4.  BACTERIA
Gram Positive
Streptococcus pneumoniae
Strephylococcus Aureus
Streptococcus pyogenes
Gram Negative
Haemophilus influenzae
Klebsiella pneumoniae
Pseudomonas aeruginosa
Proteus Species
Yersina pestis

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

6.  Production of sputum
 Collection of sample
 Examination
 Macroscopic
 Microscopic

7. 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.

8. 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.

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

11. 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

12. 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

13. Wash with water and dry
Mount under oil immersion

15. 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.

16.  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.

17. 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.

18. 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

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

21. 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.

22. 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

23. 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 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.

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