Gram Stain Evaluation With Sputum Culture Reflex

Expectorated sputum

5 to 10 mL sputum

1 to 2 mL sputum

Sterile screw-cap container

The patient should be instructed to remove dentures, rinse mouth, and gargle with water. The patient should then be instructed to cough deeply and expectorate sputum into proper container. Be sure the patient understands the difference between sputum and saliva.

Evaluate the acceptability of a sputum specimen for aerobic bacterial culture and to determine the extent of culture work-up

An adequate sputum specimen should contain many neutrophils (the exception being in the immunocompromised patient) and few to no squamous epithelial cells. A sputum specimen showing ≥25 squamous epithelial cells/lpf, regardless of the number of white blood cells, is indicative that the specimen is contaminated with saliva and is of poor quality, and culture results therefore cannot be properly interpreted. An additional sputum specimen should be collected and submitted to the laboratory, if clinically indicated, if the sputum is found to be of poor quality.

Results obtained by culture without evaluation for contamination may be noncontributory or misleading.¹ A Gram stain from a carefully collected specimen, with neutrophils and lancet-shaped diplococci staining gram-positive, which are intracellular or encapsulated, can provide strong support to the clinical diagnosis of pneumococcal pneumonia; however, culture of expectorated sputum in subjects with bacteremia and pneumococcal pneumonia does not always identify the pathogen.²

Microscopy and culture

The client will be notified of an unacceptable specimen. If the specimen is of good quality, a culture is performed for the isolation, identification, and, if necessary, antimicrobial susceptibility testing, of aerobic bacteria considered pathogenic in the lower respiratory tract.

Potential pathogens recovered by usual sputum culture methods include Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus parainfluenzae, Pseudomonas aeruginosa, Escherichia coli, Proteus sp, Moraxella catarrhalis, and rarely many other organisms. Haemophilus sp and Neisseria sp may not be routinely isolated and identified. Thus, if their presence is clinically suspected, they should be specifically requested.

Agents such as Bordetella pertussis, Chlamydia pneumoniae Corynebacterium diphtheriae, Legionella pneumophila, Mycoplasma pneumoniae, and Mycobacterium tuberculosis require special laboratory measures for isolation. Clinical suspicion of involvement by these agents should be communicated to the laboratory.