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A Stain in the Neck

Clinician's Brief (Capsule)

Clinical Pathology

|October 2015

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The most commonly used cytological stain in small animal practice is a modified Romanowsky stain. Multiple specimens are typically dipped directly in the solutions, which might result in contamination with organic material and bacteria. There are anecdotal reports of bacterial contamination of stains, especially with Pseudomonas aeruginosa, which could lead to misdiagnoses. This study evaluated whether or not P aeruginosa could contaminate stains and, if so, the viability and visibility of the organisms. P aeruginosa was inoculated into clean fixative, eosin, and methylene blue. Stock broth solution was used as a positive control and sodium hypochlorite as a negative control. Viable bacteria were isolated from the eosin and methylene blue at 1-hour post-inoculation but not at 24 hours. When the experiment was repeated after first contaminating the solutions with hair and skin, viable bacteria survived in methylene blue for 1 hour and in eosin for at least 14 days. Compared to the broth control that supported continued bacterial growth over the 2-week study period, the stains maintained steady bacterial counts. There was no growth in the fixative or sodium hypochlorite solution at any time. When viable bacteria were present, they could be detected by microscopic examination. The authors note that despite these results, there is a low likelihood of clinically significant contamination of the eosin and methylene blue because slides are usually first immersed in the fixative, which was found to be bactericidal in this study. Contamination is more likely if fixative is not used (eg, with acetate tape).


Cytology hygiene is critical in a point-of-care laboratory. Simple steps will improve specimen quality. To prevent streaking and stain artifact, change the stains and their containers weekly and use distilled water. In my practice, we use urine collection cups as containers. If glass jars are used, clean them thoroughly. Change the rinse water daily. Cover the stains so dust and debris cannot accumulate. Gently blot the slide on tissue paper between stains to minimize carryover. Allow slides to dry thoroughly before evaluating. Heat fixation is not necessary. To prevent bacterial contamination, this study encourages using fixative, avoiding introduction of large amounts of gross debris into the stains, changing stains frequently, and using clean jars disinfected with alcohol. If unexpected findings are encountered, check the stain. With acetate tape preparations, skip the fixative step because it will dissolve the adhesive and may fog the specimen.—Karen A. Moriello, DVM, DACVD


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