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Author: Rebecca Buxton​

Citation: Rebecca Buxton. 2007. Sputum–unacceptable specimens and staining artifacts.​

Publication Date: February 2007​

​Sputum Specimen (Enlarged view)​
Slide 1. Unacceptable specimen ​
​This low-power (100x) view of a sputum specimen shows many squamous cells, each of which has a single nucleus surrounded by a large volume of cytoplasm. When squamous epithelial cells are numerous, oropharyngeal contamination is substantial. Numerous gram-positive organisms from the normal flora are visible, and many of them adhere to the epithelial cells. This specimen, even if some areas contain neutrophils, would not yield reliable culture information.​

Salivary Flora (Enlarged view)​
Slide 2. Normal salivary flora​
​Photographed through an oil-immersion lens, this unacceptable specimen contains epithelial cells and a few neutrophils. These two kinds of cells can be differentiated by their size and the structure of their nuclei. The slide shows the wide variety of bacteria normally present in saliva: gram-positive cocci singly and in pairs, chains, and clusters; gram-positive bacilli (probably "diphtheroids"); and gram-negative bacilli of varying sizes and shapes (possibly oral anaerobes).​

Gram-positive diplococci (Enlarged view)​
Slide 3. Epithelial cells​
​Gram-positive diplococci adhere to two large epithelial cells with abundant cytoplasm. A few such epithelial cells do not invalidate satisfactory ​sputum specimens. However, in determining the cause of pneumonia, the examiner should disregard bacteria adherent to epithelial cells. The ​bacteria are oral flora, not pathogens expectorated from the lungs, where no squamous epithelial cells exist.​

Artifact (Enlarged view)​
Slide 4. Artifact ​​Precipitated crystal violet stain may resemble cocci. The clues are the dense clumping, the small size of the gram-positive dots, and the paucity of ​organisms elsewhere in the field.​

​Artifact (Enlarged view)​
Slide 5. Artifact​
​Precipitated crystal violet can produce masses of needle-like material that can resemble gram-positive bacilli. The dense clumping of the stain is ​characteristic.​

Neutrophils (Enlarged view)​
Slide 6. Underdecolorized stain​
​The multilobed nuclei of the neutrophils are stained purple rather than red. This indicates that decolorization and counterstaining should be repeated.​

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