1. L/O/G/O بسم الله الرحمن الرحيم Diagnostic Medical Microbiology-Laboratory Manual

2. Genital Culture Genital culture  Is the use of enrichment and selective media to isolate and identify organisms that cause genital infections such as Urethritis, Cervicitis, Endometritis, Prostatitis, Vaginitis and salpingitis (Fallopian tubes infection).  A genital culture allows the organisms present in the genital to grow to levels enabling identification. Type of specimen: Swab of vagina, cervix discharge, aspirated endocervical, endometrial, prostatic fluid, or urethral discharge.

3. Pathogenic bacteriaCommensals bacteria Neisseria gonorrheaecoagulase negative Staph Group B StreptococciCorynebacterium spp. Gardnerella vaginalisE.coli and other coliform Enterococcus spp.Many species of anaerobic Certain anaerobes including Actinomyces spp. Haemophilus ducreyi Treponema pallidum Mycoplasma spp. Enterobacteriaceae Chlamydia trachomatis Fungi Candida albicans Parasite Trichomonas vaginalis Viruses Herpes simplex virus Human papilloma virus

4. Pre specimen processing Who is authorized to order the test Physician. Time relapse before processing the sample According to the type of swab ( recommended within 30 min). Storage Maintain specimen swab at room temperature. Do not refrigerate. Quantity of specimen Sufficient amount on swab or aspiration.

5. Specimen processing for Genital specimen

6. VaginitisVaginitis  Inflammation of the vaginal mucosa called vaginitis.  Women who present with vaginal symptoms often complain of an abnormal discharge and possibly other symptoms such as offensive odor and itching.  The three most common cause of vaginitis in premenopausal women are vaginal candidiasis, bacterial vaginosis and trichomoniasis. Bacterial vaginosis BV is caused by an imbalance of naturally occurring bacterial flora of vagina.

7. Specimen processing for Vaginal swab

8.  Non-cultural diagnosis can be made by detection at least three of the following four characteristics: I.Thin, homogeneous discharge adhering to the vaginal wall. II.pH greater than 4.5. III.odor intensified on addition of 10% potassium hydroxide. IV.Presence of clue cells by microscopic examination of the discharge. Bacterial Vaginosis

9.  A Whiff test Several drops of 10 % a potassium hydroxide (KOH) solution may also be added to a sample of the vaginal discharge to test for any resultant strong fishy (amine) odor from the mix, which would indicate bacterial vaginosis.  pH measuring by using litmus papers, Normal vaginal pH is between 3.8-4.5 pH.

10. At least 10–20 high power (1000× oil immersion) fields are counted and an average determined.

11. Clue cells  Clue cells are vaginal squamous epithelial cells coated with coccobacilli gram variable Gardnerella vaginalis adhering to their surface and sometimes obscuring their borders.  Clue cells indicate bacterial vaginosis. I.Mix a drop of vaginal fluid with a drop of saline on a glass slide. II.Place a coverslip over the suspension and examine the preparation microscopically at x 400 magnification. III.Clue cells are squamous epithelial cells covered with many small coccobacillary organisms,giving a stippled, granular aspect the edges of these epithelia cells are not clearly defined, owing to the large number of bacteria present.

12. Clue cells Wet mount

13. Clue cells Gram Stain

14. Normal Vaginal Gram Stain

15. Bacterial Vaginosis Garm Stain

16. Candidiasis or Candida Vaginitis  Diagnosis is confirmed either by a simple wet mount, or better, a 10% potassium hydroxide wet mount microscopy and culture.  Candida albicans is the major cause accounting for 85% of the isolates.

17. Wet mount and KOH smear KOH slide A sample of the vaginal discharge is placed on a slide and mixed with a solution of 10% potassium hydroxide (KOH). The KOH kills bacteria and cells from the vagina, leaving only yeast for easier detection of a yeast infection.

18. Sabouraud Dextrose agar Ingredients: I.Digest of casein. II.Digest of animal tissue. III.Dextrose. 4 % IV.Agar 1.5 % Final pH 5.6 ± 0.2 at 25 ° C Sabouraud Dextrose agar is used for the isolation, cultivation, and maintenance of saprophytic and pathogenic fungi. It supplies peptone as the protein source and dextrose as the carbohydrate source for nourishment. Bacterial suppression occur due to the low pH (5.6) pH. Note: chloramphenicol may use to inhibit growth of bacteria.

19. Candida albicans on Sab. agar Candida appears as large, round, white or cream (albicans is from latin meaning 'whitish') colonies with a yeasty odor on agar plates.

20. Trichomoniasis or Trichomonas Vaginitis Trichomonas vaginalis

21. Thayer - Martin agar  Chocolate agar has been modified to be selective for Neisseria gonorrhoeae and Neisseria meningitidis by the addition of antibiotics, (V-C-N inhibitor) including:  Colistin: to inhibit most gram negative bacteria other than Neiseria.  Vancomycin: to inhibit most gram positive bacteria.  Nystatin or anisomycin :to inhibit yeast.  Modified Thayer - Martain agar includes trimethoprim to inhibit Proteus.

22. Neisseria gonorrhoeae Typical colonial morphology on Thayer-Martin Selective Agar of Neisseria gonorrhoeae.... Small, grayish-white to colorless, mucoid

23. Jembec plate In the JEMBEC system, a tablet consisting of a mixture of citric acid and sodium bicarbonate is placed in a well within the plate and is activated by the moisture (humidity) produced by the culture medium within the sealed plastic bag. The CO2 levels generated are sufficient for the growth of Neisseria gonorrhoeae on the selective medium provided with the system

24. Neisseria gonorrhoeae Gram stain

25. Direct Immuno fluorescence (DIF) Antibody labeling for Neisseria gonorrhoeae

26. Direct Immuno fluorescence (DIF) Antibody labeling for Chlamydia trachomatis

27. With Iodine stain With Giema stain Chlamydia trachomatis Giemsa stain of Chlamydia inclusion bodies (purple "caps" on epithelial cell).

28. Interfering factors:  Patient on antibiotic therapy.  Improper sample collection. Result reporting:  Report Gram stain finding as an initial report.  Report the isolated and its sensitivity pattern as a final report. Turn around time:  Gram stain result should be available half hour after specimen receipt.  Isolation of a possible pathogen can be expected after 2-3 days.  Negative culture will be reported out 1-2 days after the receipt of the specimen. Post specimen processing

29. End of Lecture