Presentation for Examination of Sputum for Laboratory Faculty
Sputum is a thick fluid produced in the lungs and in the adjacent airways Sputum culture is a test to detect and identify bacteria or fungi that infect the lungs or breathing passages
2. Sputum
• Sputum is a thick fluid produced in the lungs and in the adjacent
airwaysA sputum culture is a test to detect and identify bacteria
or fungi that infect the lungs or breathing passages.
2
3. Possible pathogens BACTERIA
• Possible pathogens BACTERIA
• Gram positive Streptococcus pneumoniae
Staphylococcus aureus
• Streptococcus pyogenes
• Also Mycobacterium tuberculosis
• Gram negative
• Haemophilus influenzae
• Klebsiella pneumoniae
• Pseudomonas aeruginosa
• Proteus species
• Yersina pestis
• Moraxella catarrhalis
• Also Mycoplasma pneumoniae, and Legionella pneumophila.
3
4. COLLECTION AND TRANSPORT OF SPUTUM
• Sputum for microbiological investigation is collected and transported
as follows:In a hospital with a microbiology laboratory1 Give the
patient a clean (need not be sterile), dry, wide-necked, leak-proof
container, and request him or her to cough deeply to produce a
sputum specimen.
4
5. COLLECTION AND TRANSPORT OF SPUTUM
• Caution: When a sputum specimen is being collected, adequate safety
precautions must be taken to prevent the spread of infectious organisms
and to avoid contaminating the outside of the container. Use a phenol-
containing disinfectant to wipe the outside of the container after collecting
the specimen.Important: The specimen must be sputum, not saliva.
Sputum is best collected in the morning soon after the patient wakes and
before any mouth-wash is used.When pulmonary tuberculosis is suspected,
up to three specimens may need to be examined to detect AFB.Mucopus
aspirated from the nasopharynxWhen it is not possible to get sputum from
childrenwith suspected pneumonia or bronchopneumonia,pathogens can
often be isolated from mucopus aspiratedfrom the nasopharynx.
5
6. COLLECTION AND TRANSPORT OF SPUTUM
• 2-Label the container, and complete a request
• 3- When pneumonia or bronchopneumonia is suspected, deliver the
sputum to the laboratory with as little delay as possible because
organisms such as S. pneumoniae and H. influenzae require culturing
as soon as possible.Note: Specimens for the isolation of S
pneumoniae and H. influenzae must never be refrigerated.
• When pneumonic plague is suspected: Deliver the sputum to the
laboratory as soon as possible. Make sure the specimen is marked
HIGH RISK.
6
7. LABORATORY EXAMINATION OF SPUTUM
• sputum specimens should be examined in a biological safety cabinet
• 1- Describe the appearance of the specimen
• Describe whether the sputum is:
• Purulent: Green-looking, mostly pus
• Mucopurulent: Green-looking with pus and mucus
• Mucoid: Mostly mucus
• Mucosalivary: Mucus with a small amount of saliva
• When the sputum contains blood, this must also bereported.
7
8. LABORATORY EXAMINATION OF SPUTUM
• When the sputum is mostly saliva, report the specimen as ‘Unsuitable for
microbiological investigation’ and request another specimen.
• Note: Before culturing sputum, many laboratories examine a wet preparation or
Field’s stained smear microscopically for cells.
• When large numbers of squamous epithelial cells (oftencovered with bacteria)
are present and only a few or no pus or macrophage cells, this indicates that the
specimen is unsuitable for culturing.
• When the sputum is mostly saliva, report the specimen as ‘Unsuitable for
microbiological investigation’ and request another specimen.
• Note: Before culturing sputum, many laboratories examine a wet preparation or
Field’s stained smear microscopically for cells.
• When large numbers of squamous epithelial cells (oftencovered with bacteria)
are present and only a few or no pus or macrophage cells, this indicates that the
specimen is unsuitable for culturing.
8
9. LABORATORY EXAMINATION OF SPUTUM
• 2- Examine the specimen microscopically Gram smear Using a piece
of stick, transfer a purulent part of the sputum to a glass slide, and
make a thin smear. Allow the smear to air-dry in a safe place. Fix
Examine the smear for pus cells and predominant bacteria. Look
especially among the pus cells for:
• Gram positive diplococci (capsulated) that could be S. pneumoniae
. Ziehl-Neelsen smear to detect AFB
9
10. LABORATORY EXAMINATION OF SPUTUM
• Culture the specimenTo obtain as pure a culture as possible of a
respiratorypathogen it is necessary to reduce the numberof
commensals inoculated. Ways of reducing commensalnumbers
include washing the sputum freefrom saliva or liquefying and diluting
it. The techniqueusing saline-washed sputum is described.
Thedilution technique requires a liquefying agent suchas
dithiothreitol (Sputolysin, Sputasol ) which isexpensive and unstable.
10
11. LABORATORY EXAMINATION OF SPUTUM
• The procedure for the routine liquefaction of sputum is as follows:
The sputum is expectorated into a sterile Universal container or other
wide mouthed screw-capped bottle Add approximately 5 times the
volume of 0.85% saline and agitate to free the sputum from adherent
saliva. Remove the saline with a sterile Pasteur pipette. To the washed
sputum, add an equal volume of Sputasol solution Shake the mixture
well, place in a 37°C water bath and incubate, with periodic shaking,
until liquifaction is complete Inoculate on to a suitable culture
medium.
11
12. Blood agar and chocolate agar
• Wash a purulent part of the sputum in about5 ml of sterile
physiological saline.
• Inoculate the washed sputum on plates of:
• ● Blood agar
• ● Chocolate (heated blood) agar, Inoculation technique to reduce
commensal numbers
• Flame the loop in between eachspread. This will help to obtain a pure
growth of thepathogen in the areas of the 3rd and 4th spread.– Add
an optochin disc to the blood agar platewithin the area of 2nd spread.
This will help toidentify S. pneumoniae.
12
