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Examination of Gram Stains of Sputum Send Print

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Created: Tuesday, 06 February 2007
Last update: Tuesday, 17 August 2010
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This work is from the original published atlas: Tenover, F. C., and J. V. Hirschmann. 1990. Interpretation of Gram stains and other common microbiologic slide preparations. The UpJohn Company, Kalamazoo, Mich. Permission granted to the ASM MicrobeLibrary by Pfizer Inc.
 
This atlas was written to help clinicians, microbiologists, and laboratory personnel identify organisms in infected materials stained by techniques commonly used in most clinical laboratories. Please refer to the atlas' main page for more information and a guide to all of the images.
 
Expectorated sputum is necessarily contaminated by upper respiratory secretions as it passes through the oropharynx and mouth. When examining the Gram stain of such a specimen, the examiner should determine whether it is mostly purulent material from the lung or mainly saliva. Since squamous epithelial cells line the upper airway but not the tracheo-bronchial tree, their presence indicates that upper airway secretions have contaminated the specimen to some degree. Several criteria have been proposed for determining that a sputum specimen is not excessively contaminated with oropharyngeal flora and is therefore acceptable for culture.
 
For example:
- fewer than 10 squamous epithelial cells per field when viewed through a low-power (10x) lens (total magnification 100x) and more than 25 neutrophils per low-power field, or
- a ratio of neutrophils to epithelial cells greater than 5:1

Even if the specimen conforms to those standards, the patient doesn’t necessarily have pneumonia. That diagnosis depends upon clinical and radiographic information. Patients with viral bronchitis, chronic obstructive lung disease, or tracheal intubation, for example, can have purulent sputum with a predominant organism in the absence of infection of the lung parenchyma.  

In some clearly satisfactory sputum specimens collected from patients with pneumonia due to a single organism (e.g., Streptococcus pneumoniae), not only the infecting organism is visible with Gram stain, but often small numbers of other types of bacteria are also present. A common mistake is to conclude that the Gram stain shows “mixed flora” and therefore does not help establish a diagnosis. To prevent that error, the type of organism that predominates numerically must be determined. Usually, the organism present in the greatest number is the cause of the pneumonia. If many bacteria are present but none clearly predominates, the pneumonia may be a polymicrobial infection, especially one due to a mixture of oral flora (aerobes and anaerobes) or aspirated in a large volume of oropharyngeal contents (aspiration pneumonia). 
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