Gram Stain

 

Sample Preparation

 

Sputum

Sputum should be free of squamous epithelial cells (<5 per high-power field) and should contain neutrophils (10-15 per field) to confirm it is from the lower respiratory tract and is not contaminated with oropharyngeal secretions.

If possible, rinse mouth before collection. Cough deeply and spit directly into a sterile cup, avoiding contact with saliva. Bronchoscopy specimens may be used, though anesthetics can inhibit bacterial growth.

 

Fluids

Bodily fluids such as blood, pus, or centrifuged urine can be plated and dried on a slide.

 

 

Staining Protocol

 

Fix bacteria and cells on the slide with 95% methanol for 1 minute, or with heat. Allow methanol to run off as the slide airdries.

 

Apply the primary crystal violet stain for 30-120 seconds without allowing the slide to dry. Rinse with tap water, shaking off the excess.

 

Flood the slide with iodine for 30-120 seconds to fix the crystal violet to the bacterial cell wall. Keep the iodine on the slide for twice as long as the crystal violet.

 

Flood the slide with decolorizer (alcohol/acetone) to remove the stain from gram negative bacteria and immediately rinse with tap water. Repeat until blue dye no longer runs off the slide.

 

Flood the slide with safranin counterstain for 30-60 seconds. Rinse with tap water and pat dry or airdry.

 

Gram-stained specimens should be examined using various lenses up to x1000 (oil-immersion).

Gram-stained sputum smears

 

Presence of bacterial cells, Gram reactions, morphologies, and arrangements, all provide diagnostic information.

The presence of a predominant organism, particularly if found within leukocytes, suggests it is the likely pathogen.

If inflammatory cells but no pathogens are seen, a large number of possibilities exist, many of which are nonbacterial.