Development of a standardized Gram stain procedure for bacteria and inflammatory cells using an automated staining instrument

Abstract

Gram stain is a subjective and poorly controlled test, and the resultant errors often perplex laboratory scientists. To reduce errors and make Gram stain a precisely controllable and meritorious test, a standardized Gram stain procedure for bacteria and inflammatory cells was developed using an automated staining instrument in this study. Freshly expectorated sputum specimens, used as the optimized targets, were smeared on slides by laboratory technicians, defining each slide loaded with uniform matrix and monolayer cell. And then, the staining and decolorizing time, as well as the stain and decolorant volume, were optimized as 15, 105, 1, and 25 s and 1.1, 1.4, 0.3, and 0.7 ml, respectively. Culture‐positive blood specimens and original purulent fluids were used for confirming the developed standardized Gram stain procedure. Distinct tinctures of bacteria and inflammatory cells adhered to slide uniformly in a monolayer were observed, and the obtained staining results of these samples were highly consistent with their cultured results. Furthermore, according to the staining results under different staining conditions, an updated molecular mechanism of Gram stain for bacteria and the probable staining mechanism for inflammatory cells were also proposed in this study.

Molecular identification of thermophilic bacteria with antimicrobial activity isolated from hot springs in North Sumatra, Indonesia

“…A total of 2-3 drops of safranin was added, allowed to stand for 10 sec, rinsed with running water, and dried. The stained bacteria were observed under a microscope with 100x magnification with the addition of immersion oil (Li et al 2020).…”

Isolation and Identification of Bacterial and Fungal Spoilage Organisms in Branded and Unbranded Milk; Consumer Perception of Safety Hazard for Milk

“…After that, the slides were placed in a draining rack to air dry the smear. After drying, a drop of immersion oil was applied to the smear, which was then examined under the microscope with oil immersion objectives [18].…”

Identification of lipase producing bacteria from palm oil sewage sludge processing plant at Malimping, Banten, Indonesia

“…Gram staining was performed by preparing smearing sample on the object-glass. This method is based on Li et al (2020). The liquid culture isolates on less than 24 hour period were used, using 14 hours liquid culture isolate.…”

“…Bacterial identification using morphological observation by Li et al (2020) methods. Morphological observation was performed by dropping 10 µL of liquid culture isolate on 14 hour period on the object-glass and observing its morphology and motility under the light microscope.…”