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Comparison of Methylene Blue/Gentian Violet Stain to Gram's Stain for the Rapid Diagnosis of Gonococcal Urethritis in Men

Taylor, Stephanie N. MD; DiCarlo, Richard P. MD; Martin, David H. MD

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doi: 10.1097/OLQ.0b013e318225f7c2
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Available modalities for the diagnosis of gonorrhea include culture and nucleic acid amplification tests. Isolation of Neisseria gonorrhoeae in culture and nucleic acid amplification tests results are not immediately available in a clinic setting. Gram stain remains a reliable test and results are available immediately. This test has a sensitivity of greater than 90% and a specificity of greater than 95% for urethral swabs from symptomatic men.1,2 These findings cannot be extrapolated to include other anatomical sites or asymptomatic men. Other staining procedures such as methylene blue probably have sensitivity and specificity that are equal to those of Gram stain, but published data on the use of this stain are limited. Pattison presented a brief letter in 1983 describing the equivalence of the methylene blue stain and Gram stain but detailed data were not provided.3 This method requires heat fixation and heat drying.

The Delgado Personal Health Center (City of New Orleans STD Clinic) has used a mixture of methylene blue and gentian violet (MB/GV) for the detection of gonococcal organisms in male urethral swab specimens for decades. It is a simple procedure that has the advantage over the methylene blue stain of not requiring heat fixation, but there are no published data verifying the accuracy of the method. To maintain certification under the Clinical Laboratory Improvement Act in 1996, we performed this study to formally compare MB/GV to the more established Gram stain.

This study was conducted between April and September of 1996 at the Delgado STD Clinic. Approximately 50 patients were seen each day and 70% were men. Two urethral swabs were routinely collected from all men attending the clinic at that time. The first swab was used to prepare a slide for direct microscopic evaluation using the MB/GV stain and the second swab was used for a DNA hybridization test for gonorrhea and chlamydia (Gen-Probe PACE 2 Assay). On 2 days of each month, the first swab collected was also used for N. gonorrhoeae culture as part of the Center for Disease Control and Prevention Gonococcal Isolate Surveillance Program.4 Men who were tested on those days were included in this evaluation to be able to use the culture data as the reference for comparison to direct microscopy. On these days, the first swab was used to prepare 2 slides for staining instead of only one before inoculating the agar plate for culture.

Alternating whether the first or second slide was used, one slide was stained using the MB/GV stain and the other slide was stained using Gram stain. The MB/GV stain was read by a single reader at the time of the clinic visit. The Gram stain slide was labeled and read at the Infectious Diseases Research laboratory in a masked fashion by a single reader.

The MB/GV staining procedure uses a mixture of these 2 stains (see below). The stain was then applied to the air-dried slide for 5 to 10 seconds and washed off with water. The slide was read after being blotted dry. A MB/GV stain was considered positive if white blood cells with intracellular diplococci were seen. Gram's staining procedure has been well described. These smears were considered positive if Gram-negative diplococci were seen within the white blood cells. If extracellular diplococci alone were seen on either smear, it was considered an equivocal result. The results of these smears were compared with one another and with the results of N. gonorrhoeae cultures.

The MB/GV stain was made by mixing 4 parts methylene blue (Loeffler Formula) with 1 part gentian violet (Hucker Formula). Both were manufactured by the Ticca Chemical Company, Arlington, TX. All stains and the decolorizer used to prepare the Gram-stained specimens were from BBL GRAM Kit (Becton Dickinson Microbiology Systems, Cockeysville, MD). Cultures were plated on fresh Modified Thayer Martin (MTM II) agar (Becton Dickinson Microbiology Systems, Cockeysville, MD). Organisms that grew on this medium were Gram stained and had the oxidase test performed. Organisms that were oxidase positive and demonstrated typical Gram-negative diplococci were frozen and shipped to the regional laboratory for sensitivity testing. Further confirmation testing was not performed at the regional laboratory. Pure cultures were used for sensitivity testing. The DNA hybridization test was performed using the PACE 2 Gen-Probe Assay (Gen-Probe Inc, San Diego, CA).

Three hundred thirty-two men were enrolled during the study period. The mean age was 27.7 years (range: 14–68) and 97.8% were black. The results of culture were available for 307 of the 332 men enrolled. Seventy-five (24.4%) had a positive culture. The mean age of those with a positive culture was 25.3 years (range: 17–50), slightly lower that the overall study group with a mean age of 27.7 years. Clinical and microbiologic data comparing culture positive and culture negative men are shown in Table 1.

Clinical and Microbiologic Findings in 307 Patients Stratified by Results of Culture for Neisseria gonorrhoeae

Examples of positive Gram's and MB/GV stains are shown in Figure 1. Intracellular diplococci appear pink in the Gram stain and purple in the MB/GV stain. Note that the polymorphonuclear leukocyte cell membrane is equally well delineated by both smears. The sensitivity of both Gram stain and MB/GV stain was 97.3% (73/75). Among the 232 men who were culture negative for gonococcal urethritis, the specificity of both Gram stain and MB/GV stain was 99.6%. There was 100% correlation between the 2 staining methods.

Figure 1.:
Top panel - Gram stain versus bottom panel - methylene blue/gentian violet stain.

In recent years, there have been many advances in the laboratory diagnosis of gonorrhea in men from both urethral swab and urine specimens. However, the ability to make a rapid diagnosis at the point-of-care remains useful. Gram stain of male urethral swab specimens has been considered as the most sensitive of the rapid testing methods.1,2 The MB/GV staining procedure involves only a single step after air drying, and is therefore quicker than the Gram stain which involves 3 staining steps and a decolorizing step after heat fixation. The MB/GV stain only takes 10 to 15 seconds compared with 4 minutes for the Gram stain. The gonococcal organisms appear dark purple on a MB/GV-stained smear and they appear red or pink on a Gram-stained smear. The morphology of the organisms, however, as well as their location within white blood cells is the same. As there should be no other organisms, and especially not kidney bean shaped, present in the white blood cells of urethral smears, color differentiation is not necessary. Regarding the use of methylene blue alone for staining urethral smears,3 the addition of gentian violet allows better differentiation of the cytoplasm and the cell membranes of the leukocytes. Therefore, with methylene blue it is more difficult to tell whether the organisms are intracellular or not. These data establish that a simple, one-step staining procedure using MB/GV is as sensitive and specific as Gram stain for the diagnosis of gonococcal urethritis in men. Busy STD clinics currently using Gram stains for point-of-care diagnosis of urethritis may wish to consider the MB/GV stain as an alternative. The MB/GV staining procedure will save time without loss of accuracy.


1. D'Angelo LJ, Mohla C, Sneed J, et al. Diagnosing gonorrhea: A comparison of standard and rapid techniques. J Adolesc Health Care 1987; 8:344–348.
2. Luciano AA, Grubis L. Gonorrhea screening: Comparison of three techniques. JAMA 1980; 243:680–681.
3. Pattison FLM. In defense of methylene blue in diagnosing gonorrhea. Can Med Assoc J 1983; 128:506.
4. Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance 2009 Supplement: Gonococcal Isolate Surveillance Project (GISP) Annual Report - 2009. US Department of Health and Human Services, Public Health Service. Atlanta, GA: Centers for Disease Control and Prevention.
© Copyright 2011 American Sexually Transmitted Diseases Association