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

Detection of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum by multiplex PCR in semen sample of infertile men

The aim of this study was to detect Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum from semen samples of infertile men by Multiplex PCR and investigation of influence of bacteriospermia on semen parameters. Semen samples of 200 infertile men were evaluated by Multiplex PCR. In addition, analysis of semen parameters was performed according to the WHO guidelines. All the patients were without clinical symptoms of urogenital tract infection. Thirty three percent of cases showed at least one bacterium. We found a noticeable relation between the presence of bacteriospermia and the rate of non motile and morphologically abnormal sperms [P< 0.0001]. In addition, sperm concentration was lower in positive cases [P< 0.04]. There was no relation between leukocytospermia and bacteriospermia [P> 0.05]. Asymptomatic existence of Chlamydia and Mycoplasmas in urogenital tracts might play an important role in sperm impairment due to infertility. Bacteriospermia can influence sperm's motility, morphology and concentration

Detection of Mycoplasma DNA from the sperm specimens of infertile men by PCR

Infections in accessory sex glands are considered as potential hazards to male fertility. These infections can affect different sites of the male reproductive tract such as the testis, epididymis and male accessory sex glands. Transmission of these infections to female partners causes genital infection, infertility and abortion. The aim of this study was to detect Mycoplasma, as one of the causatives, from the semen of infertile males using PCR assay. The survey considered 100 infertile men who referred to clinics and had not used antibiotics for 7 days prior to sampling. The infertility of cases was confirmed by a physician specialist. The sperm specimens were collected in sterile condition and sent to the laboratory rapidly. Specimens were examined for presence of Ureaplasma urealiticum and Mycoplasma hominis by PCR. Meanwhile, the history of vaginal infections and abortion in the female sexual partners was investigated. The results of multiplex PCR were compared with spermogram. All patients had no symptoms of genital infection. Of 100 infertile men, 33 [33%] were positive for CMU organisms [Chlamydia, Mycoplasma and Unreaplasma]. Uureaplasma urealiticum and Mycoplasma hominis were detected in 17 and 3 of patients respectively. Due to some problems in culturing of CMU organisms, PCR can be used as a diagnostic technique to detect such pathogens from seminal fluid of infertile men that leads to choose appropriate therapy in a shortest time

[Detecting legionella pneumophila with sputum culture and urinary antigen test in patients with acute pulmonary infection]

Community acquired pneumonia [CAP] is a common health problem and one of the main mortality factors worldwide. Legionella pneumophila is one of the most common responsible microorganisms for CAP and may lead to severe complications if left untreated. The present study was conducted to determine the frequency of this organism in patients with CAP.We enrolled 118 patients with CAP, COPD and asthma in Masihdaneshvari Medical center during 2004-2005. For microbiological purposes sputum culture and legionella urinary antigen measurement were achieved. The study population included 32 females and 86 males, their age range 58-77 years. The most frequently isolated respiratory microorganism were: streptococcus pneumonia [88%], candida spp. [76.2%], streptococcus beta-hemolytic [61.8%], staphylococcus [40.6%], klebsiella spp. [27.1%], fungi [16.1%], E.coli [8.4%], pseudomonas spp. [5.1%]. Dyspnea, cough, sputum production, and fever were the most common findings. Smoking was the most commonly found risk factor. Atypical pathogens are responsible for 40% of community-acquired pneumonia and several studies have ranked legionella pneumophila among the three most common microbial cause of CAP in patients admitted to the hospitals. So this is important to notice that urinary antigen test is particularly useful, simple and rapid test for legionella positive cases, because it is often easier to obtain urine in ill patients and the results can be available within hours and also reliable to commence treatment