Cover-Glass Test Differentiates Bacteria

A simple and cost-effective cover-glass test differentiates between micrococci and pathogenic staphylococci in the clinical laboratory.

A cover-glass placed on a heavily inoculated culture plate clearly differentiates facultative anaerobic staphylococci growing underneath the cover-glass after overnight incubation from nongrowing aerobic micrococci.

Medical microbiologists at the Charles University (Prague, Czech Republic) used a study set that included 18 strains of Micrococcus luteus as the most common micrococcal species isolated from clinical specimens and indoor air, 15 strains of Staphylococcus aureus as a significant pathogen, and 20 strains of other staphylococcal species to test the cover-glass method.

To carry out the cover-glass test, a sterile cover-glass was placed with a pair of sterile tweezers onto a heavily inoculated area of the Columbia blood agar plate (Merck; Darmstadt, Germany) with a clinical specimen or bacterial culture and after overnight incubation at 37 °C, the plate was checked for pigmented colonies or bacterial biomass underneath the cover glass.

The result was classified as strongly positive if an unaffected growth was observed beneath the cover glass, moderately positive if the growth was affected and weakly positive if a fine growth occurred. A negative result meant that the strain was not able to grow beneath the coverslip. Each strain was also tested for antibiotic resistance. All S. aureus strains analyzed were positive in the cover-glass test in contrast to the strains from the genus Micrococcus or related genera such as Dermacoccus, Kytococcus, and Kocuria, that remained negative, except for one Kocuria kristinae strain. Seven of 35 staphylococcal strains tested were cover-glass negative.

The cover-glass test proved rapid, highly specific, easy to perform, and easy to interpret. It should be used in combination with the furazolidone and bacitracin disks in both primary and secondary culture as a useful tool for differentiating S. aureus and other staphylococci of human origin from Micrococcus species or related genera. Additional screening tests such as Gram-staining and plasma-coagulase test, and biochemical or matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry identification approaches may need to be performed to identify clinical isolates of catalase-positive cocci to the species level. The study was published in the June 2012 issue of the Journal of Microbiological Methods.

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Charles University
Merck