Examination of sputum. Sputum . Sputum is a thick fluid produced in the lungs and in the adjacent airways A sputum culture is a test to detect and identify bacteria or fungi that infect the lungs or breathing passages. . Possible pathogens. BACTERIA Gram positive Streptococcus pneumoniae
causes of acute respiratory tract infections in tropical
bronchopneumonia in young children (especially when
malnourished), in those co-infected with HIV (major
HIV-related pathogen), the elderly, the bed-ridden and
other debilitated persons.
S. aureus, S. pyogenes, and H. influenzae are often secondary invaders in patients with influenza virus
H. influenzae is associated with acute andchronic bronchitis and chest infections in post-surgical patients and the elderly. S. aureus can produce a severe
pneumonia (with a tendency to form abscesses),
especially in children and following influenza.
chronic lung cavities or as a complication of treatment
with immunosuppressive drugs.
● K. pneumoniae may be found with E. coli and yeasts as a complication of antibiotic therapy.
● Moraxellacatarrhaliscan cause upper and lower respiratory tract infections, mostly in adults with pre-existing respiratory disease and those with immunodeficiency.
●Mycoplasmapneumoniae ( M. pneumoniae) causes primary atypical pneumonia.
● Legionellapneumophila (L. pneumophila) causes Legionnaire’s disease, a severe and often fatal form of pneumonia.
Sputum as it is being collected passes through the
pharynx and the mouth. It therefore becomes contaminated with small numbers of commensal
organisms from the upper respiratory tract and
1 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.
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.
When pneumonic plague is suspected: Deliver the sputum to the laboratory as soon as possible. Make sure the specimen is marked HIGH RISK.
1 Collect the sputum in a container supplied by the microbiology laboratory .Follow the technique and observe the precautions mentioned under the hospital collection of sputum.
2 To ensure the survival of pathogens such as S. pneumoniae and H. influenzae, transfer a purulent part of the sputum to a cotton-wool swab, and insert it in a container of Amies transport medium. Label the container using a lead pencil.
Amies medium will help the pathogens to survive and
avoid the overgrowth of fast-multiplying commensals.
3 Send the sputum specimen and swab with a request form to reach the microbiology laboratory within 6 hours.
sputum specimens should be examined in a biological safety cabinet
1- Describe the appearance of the specimen
Describe whether the sputum is:
covered with bacteria) are present and only a few or no pus or macrophage cells, this indicates that the specimen is unsuitable for culturing.
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 . The
● Gram positive cocci in groups that could be S. aureus , but not often seen.
● Gram negative rods and cocco-bacilli that
could be H. influenza, particularly when these are the predominant organisms.
● Gram negative capsulated rods that could be
K. pneumoniae, but not often seen.
● Gram negative diplococci in and between pus
cells that could be M. catarrhalis
with caution. Cocci, diplococci, streptococci, and rods
may be seen in normal sputum because these
organisms form part of the normal microbial flora of
the upper respiratory tract.
Note: When pus cells are present but no bacteria are
seen in a Gram stained smear, this may indicate the
presence of microorganisms such as M. tuberculosis,
Legionellapneumophilia or viruses.
theNaOC1 concentration technique is also safer for laboratory staff. Because NaOC1 kills M. tuberculosis,
Eosin preparation when an allergic condition requires investigation
Transfer a small amount of sputum to a slide. Add a small drop of alkaline eosin solution, mix, and cover with a cover glass. Using the
10 and 40 objectives with the condenser iris
closed sufficiently to give good contrast, examine the
preparation for eosinophils*.
*Eosinophils can be easily differentiated from pus cells
because they contain bright red-staining granules and a bilobed
nucleus. Free eosinophilic granules may be seen in the
preparation and occasionally elongated refractile Charcot
Leyden crystals (formed from the material of dead
Large numbers of eosinophils in sputum can also be found
when pneumonic plague is suspected
Fix the sputum smear with methanol for 5 minutes.
Stain using Giemsa technique or rapid Wayson’s technique Bipolar
Caution: Y. perstis is highly infectious (Hazard Group
3 pathogen), therefore handle specimens with great
care. Laboratory-acquired infections can occur following
accidental inoculation or inhalation of the
organisms. Minimize procedures that create aerosols
and whenever possible carry out procedures in a
To obtain as pure a culture as possible of a respiratory
pathogen it is necessary to reduce the number
of commensals inoculated. Ways of reducing commensal
numbers include washing the sputum free
from saliva or liquefying and diluting it. The technique
using saline-washed sputum is described. The
dilution technique requires a liquefying agent such
as dithiothreitol (Sputolysin, Sputasol ) which is
expensive and unstable.
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. For the total cell count, place a drop of the liquefied sputum in a haemocytometer for enumeration. For a differential cell count, fix a dried smear in methyl alcohol and stain with haematoxylin and eosin or with Lieshmann stain.
The working solution of Sputasol will remain stable for at least 48 hours stored at 2-8°C.An investigation into the survival of respiratory pathogens in specimens that had been stored for 48 hours at 4°C following homogenisation using Sputasol, showed that the organisms remained viable and, when necessary, treated specimens could be successfully re-cultured8.