Gram Stain and Nugent Scoring System

Nugent scoring is a golden standard test for BV and vaginal swabs were used to prepare smears on clean, grease-free slides. The smear was allowed to air-dry and then fixed with 95% acetone alcohol. Then, the smear was Gram stained using Gram stain protocol and read by an experienced microbiologist under a light microscope using the oil immersion objective (×100). Slides were read according to Nugent’s score as follows: Lactobacillus morphotypes were reported as large Gram-positive bacillus, Gardnerella were reported as small Gram variable bacilli. Using the Nugent scoring technique, score ranged from 0–4 for Lactobacillus; 0 indicating that ≥ 30 organisms were found and 4 indicated that no organism were found. In contrast, for Gardnerella a score 0 indicated that no organism were found and 4 indicated that ≥ 30 organisms were found. A total of numerical score was calculated ranging from 0–10 for each sample by summing the scores for each of the three morphotypes. The scores were classified into one of the three ranges to define a case of BV; a score in the range 0–3 indicated normal flora, 4–6 indicated mixed vaginal flora, which is not constituted BV, and 7–10 was constituted of BV.^33,34

Gram-stained slides were also examined for Candida species and polymorph nuclear cell (PMN) count for diagnosis of candidiasis and other Gram-positive and Gram-negative bacteria.