Abstract:
Gram staining (GS) is widely used for rapid visualization and determination of
bacteria based on cell wall characteristics, classifying them as Gram-positive or
Gram-negative. The conventional slide-based technique requires multiple reagents,
including ammonium oxalate-crystal violet, Lugol’s iodine, acetone-iodine, and dilute
carbol fuchsin, often in relatively high volumes. This study aimed to introduce
a novel capillary tube-based GS method to significantly reduce reagent usage and
offer a cost-effective alternative method while maintaining accuracy. The capillary
tube method (CTM) and the conventional glass slide method (GSM) were
performed on selected control strains and clinical isolates. A small volume of bacterial
suspension was drawn into a capillary tube, heat-fixed, and Gram-stained
after natural cooling. The crystal violet and Gram’s iodine steps were extended
to one minute to optimize staining. Stained capillary tubes were evaluated using
eight scoring variables. Sensitivity, specificity, positive predictive value (PPV),
and negative predictive value (NPV) were calculated. A chi-square test was applied
for data analysis using SPSS. Cost and turnaround time per assay were
also estimated. There was no statistically significant difference about uniformity
of background staining between the two methods (p=0.825), with a sensitivity
of 88.9%, specificity of 20%, PPV of 90.9%, and NPV of 16.7%. Similarly, the
presence of staining artifacts was not statistically significant between methods
(p=0.064), with a sensitivity of 70%, specificity of 62.5%, PPV of 31.8%, and
NPV of 89%. Hence, no significant difference was observed between CTM and
GSM. The CTM was the most cost-effective method while GS required less processing
time. The CTM offers a cost-effective alternative to the conventional GSM
without compromising staining quality or diagnostic reliability, making it suitable
for resource-limited laboratory settings.
