Bacterial Infection Practical Report: Purulent Urethral Discharge

Gonorrhea is the second most prevalent sexually transmitted disease. It is caused by Nesseria gonorrhea bacteria, which are gram negative, diplococcic, non-spore forming, and non-motile1. Additionally, the bacterium has type IV pili used for attachment to the columnar epithelial cell lining of the cervix and urethra and surface Opa protein for anti-immunity properties. The common clinical symptoms of the disease are urethitis, cervicitis, arthritis, salpingitis, bacteremia, and pelvic inflammatory disease when left untreated. Diagnosis of the disease is done by isolation of the bacteria from the endocervical, vaginal, urethral smears and urine specimens2. In this experimental study, the smears of a patient diagnosed with the disease were used to conduct Gram staining because of its high specificity and sensitivity, β-lactamase test to identify the resistant strain, and inoculation on CHA (selective) and GC (non-selective) media to examine the cultural properties of the bacteria3.

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The smear inoculums were cultured on the “Bi-plate” containing CHA and GC medium according to the guidelines in the ONPS1045 Practical Manual 2012 RMIT University p. 19. After the cultural period, a few colonies were extracted from the culture plate and used to conduct Gram staining according to the BIOL2310 Practical Manual 2012 RMIT University. Eventually, β-lactamase resistant strain was identified using the rapid method of cefinase test. CTA sugar test and rapid NH test were conducted in accordance with the instructions on the kit by manufacturers, to identify or confirm N. gonorrhea.


3.1. “Bi-plate” culture

“Bi-plate” Observation
GC plate Colonies: circular (about 1mm)



CHA plate Circular (1-2 mm)



Characteristics of the two plates, GC culture exhibited inhibited growth whereas overgrowth was observed on CHA plate.

3.2. Gram stain

Gram negative bacteria were observed. The morphology was coffee bean shaped diplococci.

3.3. Rapid β-lactamase test

No color change was observed – negative rapid β-lactamase test.

3.4. Identification tests

There was positive oxidase test. The N. gonorrhea was tested CTA sugar to study the ability to oxidize various carbohydrates – maltose, sucrose, lactose and glucose – positive on glucose only. Rapid NH test was also conducted on the isolated bacteria to confirm if it was indeed N. gonorrhea.


GC media contained specific antibiotics that selected the N. gonorrhea. The identification and confirmation was achieved through Gram staining, and positive oxidase test using CTA sugar (glucose), and positive rapid NH test3. The isolated strain was not fully confirmed to be susceptible to penicillin inspite of the negative β-lactamase test. This is because recent studies have found out that some strain showing negative β-lactamase test have developed resistance to penicillin since they produce β-lactamase or have altered their penicillin binding proteins5. In this regard, further experimental studies on resistance would be necessary such as multilocus sequence typing (MLST).



A young female patient was diagnosed with persistent vaginal discharge that was light, watery, light and unpleasant odor and occurs especially after sexual intercourse. The diagnostic assumption was Gardnerella vaginalis bacteria infection4. The bacterium is a Gram variable but exhibit Gram positive wall-type and is facultative anaerobe. V. vaginalis are normal floras of the vagina but causes bacterial vaginosis (BV) if they become dominant5. Severe infections are associated with infertility and pre-term labor.

In this experiment, smears from case patient discharge were cultured in different media – chromogenic agar, HBA, CHA, and HBA anO2. G. vaginalis has the ability to haemolyze human blood hence it grows massively on HBA both under aerobic and anaerobic conditions. Nevertheless, HBA is not entirely selective to G. vaginalis and will allow growth of other vaginal flora. The purpose of the chromogenic agar was to select Candida species. Identification testing involved Gram staining of isolated cultures to study cultural differences and disc susceptibility test with antibiotics Sulphonamide and Metrodazole.



Smear innoculum was cultured in different media – chromogenic agar, HBA, CHA, and HBA anO2 that were provided by demonstrators. The isolates were used to perfume Gram staining according to methods in the 2012 BIOL2310 practical manual 2012 RMIT University. Then the disc susceptibility test was done according to methods in 2012 ONPS1045 Practical Manual 2012 RMIT University, p18-20. Then the determination of MICs and measuring of inhibition zone was done according to the recommended CLSI standards.

    • Culture on different media

Table 2: Summary of morphological results of bacterial cultures on different media

Medium Morphologies of G. vaginalis
HBA Small, transparent, circular colonies

Some large colonies (>2mm)

Heamolysis around some colonies

No heamolysis+++

CHA Small, circular, convex, gray colonies

No heamolysis++

HBA anO2 Heamolysis around some colonies

Small, transparent, circular colonies

Some large colonies (>2mm)

No heamolysis++++

Chromogenic agar No growth detected



There was heavy growth of bacteria on HBA media in both aerobic and anaerobic conditions and detection of haemolysis around colonies. However, CHA media was strikingly different because it exhibited low cultural growth and no heamolysis occurred. No detectable growth occurred on chromogenic agar, which confirmed absence of fungus in the smear.

3.2 Gram stains on HBA and HBA anO2

Bacteria isolates of both aerobic and anaerobic HBA culture were similar (Coccibacilli). The Gram stains of the hemolytic positive colonies were either Gram positive or Gram variable, which pre-confirmed G.vaginalis, whereas those of the non-hemolytic colonies were large Gram positive bacilli, which suggested Lactobacillus bacteria.

Table 3: Table summary of Gram stain of different colonies on HBA under aerobic and anaerobic condition


Small and haemolytic (green) colonies


Gram-variable coccibacilli

Gram-positive coccibacilli Abundant clue cells


Gram-variable coccibacilli

Gram-positive coccibacilli Abundant clue cells

Large and non-haemolytic colonies Gram-positive bacilli Gram-positive bacilli


  • Disc susceptibility test

The microorganism was susceptible to Sulphonamide and resistant to Metronidazole.


The observation of hemolytic positive colonies of either Gram positive or Gram variable coccibacilli in both aerobic and anaerobic HBA cultures, susceptibility to Sulphonamide, and resistance to Metronidazole helped to confirm that the microorganism was G. vaginalis5. Isolation of pure G. vaginalis cultures is almost impossible because they share most of the physiological characteristic with other normal vaginal flora. Since Gram stain is the most cost effective and highly sensitive in which it confirms G. vaginalis, it is the most recommended technique. Additionally, susceptibility test may be used to determine G. vaginalis etiology of BV.



This study is about a 30 year-old woman that was 25 weeks pregnant and screened was screened for group B streptococcus in the vaginal carriage. Streptococcus agalactiae (group B streptococcus, GBS) cause perinatal and neonatal infection – epidemiologic data puts it at 1.8 per 1000 birth lives per year6. Pregnant women are recommended to undergo screening and use of antimicrobial treatment such as penicillin and ampicillin on positive diagnosis to reduce mother to child transmission7. Centers for Disease Control and Prevention (CDC) recommend the use of selective enriched medium to isolate GBS as the most effective method7. Therefore, screening should involve the detection of GBS is done by culturing patients samples in HBA, neomycin HBA, and/or Granada medium (GM). GBS is detected on GM by identification of distinctive orange pigmented colonies (granadae) under anaerobic conditions8. HBA and neomycin HBA identifies GBS under different cultural conditions. Moreover, confirmatory test such as agglutination test should be done in order to identify the isolate as GBS.


Vaginal swab from patient were cultured on HBA, neomycin HBA and GM and were provided by demonstrators for identification of group B streptococcus (GBS). An agglutination test was used to identify GBS and was conducted according to manufacturer manual.

    • Colonies on different media

Large, rounded, and non-pigmented colonies were observed on both HBA and neomycin HBA cultures. The colonies produced β-hemolytic zone on the immediate media environment. The colonies detected on GM culture were round and most were pigmented orange and some had white colonies. Agglutination test was done to confirm GBS.

Table 4: Table summary of bacterial colonies on HBA, neomycin HBA and GM media

Media Descriptions
HBA Large up to 2mm, circular, convex and clear colonies

Β-haemolytic zones around colonies

Some smaller (~1mm), white, clear edges colonies ++++


Neomycin HBA Large up to 2mm, circular, convex and clear colonies

Β-haemolytic zones around colonies

Some smaller (~1mm), white, clear edges colonies +++

GM Small (less than 1mm), circular, convex and orange colonies

Some smaller (~1mm), white, clear edges colonies +++



  • Identification of GBS

Agglutination test was positive, which confirmed the organism was group B streptococcus.


Culturing the swabs on anaerobic HBA condition inhibited microbial growth other than GBS alone. The smaller colonies in GM culture made it more sensitive for detecting GBS and is the most suitable and recommended method8. Moreover, agglutination was conducted to confirm GBS. Therefore, the patient was successfully diagnosed with GBS using HBA and/or GM culturing and agglutination test.



  1. Kampmeier RH: Identification of the gonococcus by Albert Neisser. 1879, Sexually transmitted diseases 1978, 5:71-72
  2. Fenton KA, Lowndes CM: Recent trends in the epidemiology of sexually transmitted infections in the European Union, Sexually transmitted infections 2004, 80:255-263
  3. Boslego JW, Tramont EC, Takafuji ET, Diniega BM, Mitchell BS, Small JW, Khan WN, Stein DC: Effect of spectinomycin use on the prevalence of spectinomycin-resistant and of penicillinase-producing Neisseria gonorrhoeae, The New England journal of medicine 1987, 317:272-278
  4. Yeoman CJ, Yildirim S, Thomas SM, Durkin AS, Torralba M, Sutton G, Buhay CJ, Ding Y, Dugan-Rocha SP, Muzny DM, Qin X, Gibbs RA, Leigh SR, Stumpf R, White BA, Highlander SK, Nelson KE, Wilson BA: Comparative genomics of Gardnerella vaginalis strains reveals substantial differences in metabolic and virulence potential, PloS one 2010, 5:e12411
  5. Mania-Pramanik J, Kerkar SC, Salvi VS: Bacterial vaginosis: a cause of infertility?, International journal of STD & AIDS 2009, 20:778-781
  6. Prevention of perinatal group B streptococcal disease: a public health perspective. Centers for Disease Control and Prevention, MMWR Recommendations and reports : Morbidity and mortality weekly report Recommendations and reports / Centers for Disease Control 1996, 45:1-24
  7. El Aila NA, Tency I, Claeys G, Saerens B, Cools P, Verstraelen H, Temmerman M, Verhelst R, Vaneechoutte M: Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women, BMC infectious diseases 2010, 10:285
  8. Rosa-Fraile M, Rodriguez-Granger J, Haidour-Benamin A, Cuerva JM, Sampedro A: Granadaene: proposed structure of the group B Streptococcus polyenic pigment, Applied and environmental microbiology 2006, 72:6367-6370




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