Crystal Violet in Culture Media for Diagnosis of Neisseria Species

Neisseria species are gram negative diplococci an important characteristic of these bacteria is resistance against crystal violet. This study was done to investigate the effect of crystal violet on the growth of Neisseria, to observe the outcome of adding this substance in culture media for isolating these organisms, and finally to make a specific medium for isolation of the Nisseria species.

The study was done in 3 phases: 1) Initially urethral discharge from 106 male patients with urethritis was cultured on NYC, chocolate agar and chocolate violet agar with various concentrations of violet from 1:100000 to 1:250000. We also made direct smears for gram stains. 2) Pharyngeal secretions from 230 healthy persons were cultured on chocolate agar, Muller Hinton agar, and Thayer Martin agar with different concentrations of violet between 1:50000 to 1:500000. Also direct smears were made for gram stains. 3) The standard strain of gonococcus (ATCC) were cultured on the three media. Concurrently we added various concentrations of violet from 1:50000 to 1:200000 to the above media and studied the effect of adding crystal violet on the growth of the standard strain.

In first step, 69 out of 106 patients with urethritis were suspicious of gonorrhea, with positive culture of gonococcus on NYC medium from 64 patients. On chocolate agar only 54 positive cultures, (with 84% sensitivity against NCY medium), were seen together with a growth of normal flora. Chocolate agar plus violet in concentration 1:150000, showed 58 positive cultures, (with 91% sensitivity against NYC medium), with minimal growth of normal flora. In second step, 228 out of 230 healthy persons had positive culture of Neisseria, these organisms grew in different concentrations of crystal violet between 1:500000 and 1:50000. However, with minimal concentration of violet, there was a dense growth of normal flora and with gradual increase in concentration, normal flora grew sparsely. In direct exam, 228 cases of gram negative Neisseria like diplococci were observed. In third step, result of growth of the standard Neisseria gonorrhea in chocolate agar, Muller-Hinton agar and Thayer-Martin agar with and without different concentrations of crystal violet are as follow: In all media without crystal violet, the growth of the bacteria was perfect and abundant while in media containing crystal violet, minimum colony count was observed in concentrations of 1:50000 and maximum colony count occurred at concentrations of 1:200000

To isolate pathogenic species of Neisseria, for e.g. gonococcus, we can use a specific chromogen medium like chocolate-violet agar 1:150000, or Thayer-Martin-violet agar or Muller-Hinton-violet agar with a concentration of 1:200000. Although nonpathogenic Neisseria have high resistance to crystal violet and were isolated from cultures with 1:50000 dilutions of this substance, but growth become sparse with higher concentrations. We can make chromogen media of varying strengths by adding different amounts of crystal violet in various media to get the desired results.