Microbiology Laboratory


Written by MicroDok

AIM: To detect the presence of pus cells and predominant bacteria in sputum specimens as an aid in the diagnosis of lower respiratory tract infections like pneumonia or bronchopneumonia.

MATERIAL/APPARATUS: Sputum specimen, glass slide, microscope, immersion oil, inoculating loop, Bunsen burner, piece of stick.


  1. Transfer a purulent part of the sputum specimen to a clean glass slide using a sterilized inoculating loop or a piece of clean stick.
  2. Make a thin smear of the purulent sputum on the glass slide.
  3. Place the slide in a safe place away from dust for it to dry.
  4. Heat fix the smear on the slide by passing it thrice over a Bunsen burner flame.
  5. Perform the Gram staining technique.
  6. Allow the Gram stained slide to dry.
  7. Add a drop of immersion oil on the slide.
  8. Examine/Observe the stained slide under the microscope using the ×100/oil immersion objective lens.
  9. Look out for pus cells and predominant bacteria in the smear.


Describe and report the appearance of the sputum specimen. The appearance of a sputum specimen can be described with any of the following parameters:

  • Purulent: Green – looking, mostly pus.
  • Mucopurulent: Green – looking with pus and mucus.
  • Mucoid: Mostly mucous.
  • Mucosalivary: mucous with a small amount of saliva.
  • Bloody: Sputum containing some amount of blood.

NOTE: The presence of blood in a sputum specimen can be due to Paragonimiasis caused by the parasite, Paragonimus westermani. P. westermani is a human lung fluke which affects the lungs.

Examine the slide carefully under the microscope and look especially for:

  • Capsulated Gram positive diplococcic (e.g. Streptococcus pneumoniae).
  • Gram positive cocci in groups (e.g. Staphylococcus aureus).
  • Capsulated Gram negative rods (e.g. Klebsiella pneumoniae).
  • Gram negative rods and cocco – bacilli (e.g. Haemophilus influenzae).
  • Gram negative diplococcic that are usually found in and between pus cells (e.g. Moraxella catarrhalis).

The result of a Gram stained smear of sputum specimen should be reported with utmost care and caution, as cocci, diplococcic, rods, and streptococci can also be seen in a normal sputum sample. This is because these organisms form part of the normal microbial flora of the human upper respiratory tract. The clinical presentation or diagnosis of the patient as given by the physician can also help in taking care of this.

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