Objective: The assessment ofthe vaginal lactobacillary flora helps to direct further diagnostic microbiologic investigations in genital infectious disease and seems to represent a powerful tool in predicting infectious morbidity and preterm labor during pregnancy. In the absence of a "gold standard," we studied the variations in assessing lactobacillary morphotypes according to the method used.Methods: The lactobacillary flora from 183 pregnant women was classified according to 3 groups: normal, intermediate, and abnormal. This grading of lactobacilli was appled to vaginal and cervical specimens by means of 1) immediate wet-smear microscopy, 2) Gram's stain on a fresh, air-dried specimen, and 3)delayed Gram's stain after specimen transportation in Stuart's growth medium for 3-6 h.Results: The assignment of intermediate or abnormal flora (grade II or grade III) showed high concordance rates among the different preparatory techniques, but the assignment ofgrade I (normal flora) did not. Fewer lactobacilli were found 2.6 times more often after Gram's stains of fresh specimens [Relative Risk (RR) 2.6, 95% confidence interval (CI) 1.7-4.1] and 6 times more often when the Gram's stain was performed in a delayed examination after transport than in a fresh wet-mount specimen (RR 6.2, 95% CI 2.5-15.6). Disturbed lactobacillary grades were also found more frequently in specimens from the cervix than those from the vagina (RR 4.0, 95% CI, 1.5-10.4).Conclusions: There are discrepancies in the diagnosis of lactobacillary grades between gramstained and fresh vaginal specimens. The evidence is ambiguous as to which of the 2 methods is responsible. If an evaluation is to be done on a gram-stained specimen, then the storage of the sample in Stuart transport medium before staining should be avoided. (C) 1996 Wiley-Liss, Inc.KEY WORDS Pap smear, lactobacillary grades, bacterial vaginosis ince its discovery by D/3derlein in 1892, the vaginal Lactobacillus has fascinated researchers and clinicians with its intriguing ability to protect women from a large number of pathogens entering the upper genital tract. Schrtider (1913) and Hunter (1945) divided the vaginal flora into 3 subtypes in order to link them to clinical vaginitis and vaginal pathogens. However, they were limited by the diagnostic facilities available at that time and by unknown microorganisms yet to be discovered.