Prepared from IASTD.

1. SIDE LAB INVESTIGATIONS
IN STD
Dr. SRINIVASAN. G
1st Yr
Dept of DermatoVenereoLeprology

2. 1. DARK FIELD MICROSCOPY
2. GRAM STAINING
3. GIEMSA STAINING
4. WET MOUNT
5. KOH WET MOUNT
6. ACETIC ACID TEST
7. WHIFF OR SNIFF TEST
8. TWO AND THREE GLASS TEST
9. BUBO ASPIRATION
CONTENT

3. DFM
1. DARK FIELD MICROSCOPY
• It is the only method to demonstrate Treponema pallidum.
• In this , the light rays hitting the organism in an oblique angle, enter the microscope
objective, to give a luminous appearance against a black background.
REQUIREMENTS
• Dark Field Microscope
• Thin glass slides.

4. DFM
Collection & Preparation
• T. pallidum can be identified in serous fluids from the lesions of Primary, Secondary and
Early Congenital Syphilis.
• Steps :
• Gently abrade the lesion with dry gauze,wipe off any blood stained serum and squeeze
the lesion to produce clear exudate.
• It is then transferred to slide either by pressing the cover slip directly on to exudate or by
pasteur pipette.
• Cover the slip and seal the edges of cover slip with petroleum jelly.
• Examine immediately.

5. DFM
Setting the microscope
• Bring down the condenser of the dark field microscope.
• Put a drop of liquid paraffin on the condenser.
• Place the slide on the microscope stage.
• Raise the condenser until there is good contact between oil and bottom of the slide.
• Avoid trapping of air bubbles in oil.

6. DFM
Reading
• T. pallidum appear white, illuminated on a dark background.
• It is identified by its typical morphology, size and movements
• Shape - Thin spiral organism - 6 - 14micron long.
• 8 -14 spirals which are regular & pronounced.
• They rotate relatively slowly on longitudinal axis ( cock screw ) accompanied by
sudden bending at acute angle.
• Ideally,
• Test should be done on three consecutive days or
• 2 DF smears should be collected , since DFM has sensitivity of 50% only.

7. 2. GRAM STAINING
Gram Stain
 Gonococcol & Non Gonococcol Urethritis.
 Mucopurulant Cervicitis,
 Chancroid,
 Bacterial Vaginosis,
 Candidal Infections.
Used to detect -

8. Gram Stain
GRAM STAINING - Requirements:
 Crystal Violet - methylrosanilinium chloride
 Iodine Solution
 Decolourizing solution - Acetone + 96% ethanol.
 Counter stain - Fuschsin / Safranin.

9. Gram Stain
Collection of Specimens
a. Sterile cotton or
b. Calcium alginate or
c. PET ( polyethylene terephthalate )
In India , Sterile cotton is commonly used.

10. Gram Stain
GS - GONORRHOEA
SITES :
The site depends on age , sex, sex practices and clinical symptoms.
Men:
Heterosexual Men - Uretha
Homosexual Men - Urethra, Rectum, Oropharynx
Women -
Primarily from - Endocervical Canal.
Secondary sites from - Urethra, Vagina, Rectum & Oropharynx

11. Gram Stain
Collection - Gonorrhoea
METHODS :
Urethra- Time - 1hr before urination.
In Men :
 Retract the prepuce , clean the tip with NS, Collect the pus directly into swab.
 If No discharge is seen, milk the urethra from root of the penis towards glans to express pus.
 If No pus is obtained even after milking, insert a thin sterile swab 2-3cm into the urethra and
gently scarpe the mucosa by rotating the swab for 5-10secs.
In Females :
 Massage the urethra against the pubic symphysis and use the same technique as for men.

12. Gram Stain
Collection - Gonorrhoea
In Females :
 Endocervical Specimen -
 Insert Vaginal Speculum
 Clean the exocervix with sterile cotton swab,
 Insert another sterile swab 2 cm into the cervical canal, rotate and move from side to
side for 5 -10 secs and withdraw.
 Vagina - Swab from Posterior Cervix .
 From prepubertal girls
 From women who had hysterectomy.

13. Gram Stain
Collection :
 NGU , Mucopurulant Cervicitis
Same as Gonorrhoea,
Samples are collected after holding urine for 3 - 4 hrs , since the collection is scanty in
these diseases.
 Bacterial Vaginosis
Specimen is collected from Posterior or Lateral wall of vagina with a sterile saline saoked
swab
 Chancroid
From the edges of the ulcer.
Clean the ulcer with saline soaked gauze, then roll a sterile swab in one direction on
the edge of undermined ulcer , then reroll the swab in reverse direction on a glass slide
and stain it. Organism can also be showed from intact bubo aspirate.

14. Gram Stain
Procedure
 Preparation of smear
a. Take a galss slide , pass it through flame twice or thrice and wipe it to clean.
b. Draw 2 vertical lines 2.5cm apart with marker on the central part of slide.
c. Roll the swab with specimen over the marked area.
d. Label the smear.
 Fixing the smear - done to kill the organism, prevent changes and make it permeable to dye.
a. Hold the slide with smear facing upwards.
b. Pass the slide over the flame of a Bunsen's burner or spirit lamp twice or thrice.
c. Allow the fixed smear to cool.

15. Gram Stain
Procedure
 Staining
a. Cover the fixed smear with crystal violet for 1 minute and then rinse it with tap water.
b. Flood the slide with Gram's Iodine solution for 1 min. Drain off the solution and gently rinse
with running water.
c. Decolourize with acetone-ethanol until the drops falling off the slide are no longer blue. It
usually takes 10 - 20secs. Rinse quickly uner running water to stop decolourisation.
d. Counter stain with safranin or fuschsin for 1 min. Gently wash in running water and blot the
slide with absorbent paper.

16. Gram Stain
Smear Reading
 Examine under light microscope.
 Scan and focus on a good field.
 Put a drop of Liquid paraffin without air trapping and examine under 100x oil immersion
objective.
 Push the condenser up and open the iris diaphgram to allow max light to pass through.

17. Gram Stain
Smear Reading
 Intracellular kidney or coffee Bean shaped diplococci - Gonorrhoea
 Bipolar stained gram neg bacilli - Acinetobacter.
 5 or more Polymorphonuclear leucocytes ( PMN ) in abscence of diplococci - Non gonococcal
urethriris.
 Cervical Gram stain - with 30 PMN/hpf in women of menstrauating age - Mucopurulent cervicitis.
 “School of fish” or “rail road track” or gram neg bacilli in chains - Hemophilus ducreyi -
Chancroid.
 “Clue cells” - Gardenerella vaginalis - Bacterial vaginosis.
 Gram positive budding yeast looking like “fig of 8” and yeast hyphae - Vulvovaginal candidiasis /
Balanoposthitis.

19. Giemsa Stain
3. GIEMSA STAIN
Used to detect -
 Genital Herpes
 Molluscum contagiosum
 Donovanosis
 Chancroid.
 T. vaginalis and Chlamydia species can also be seen, but not used routinely.

20. Giemsa Stain
Requirments
 Giemsa Stock
• It is prepared by dissolving 4g Giemsa powder in 250ml glycerol at 60℃ and then adding
250ml methanol.
• 4ml of stock solution is added to 96ml of distilled water or buffered water at pH 7.0 -7.2.

21. Giemsa Stain
COLLECTION OF SPECIMEN
 Herpes Progenitalis
• The intact roof of the vesicle is opened along one side and folded back.
• scrape the under surface of the roof of vesicle and the floor of ulcer with a curette or scalpel
and smear the obtained material on a glass slide.
 Donovanosis
• A small piece of tissue is removed from the edge of the granlomatous ulcer after cleaning
the area with sterile saline soaked gauze or sraped tissue by a scalpel.
• Place it in a slide,then crush it with another slide from above , spread and allow it to dry.
 Molluscum Contagiosum
 The central semisolid core is removed and stained.

22. Giemsa Stain
Procedure
 Smear is fixed in methanol - dip the slide for 5 mins in coplin jar containing methanol.
 Allow the smear to dry
 Dilute the giemsa stain 10 times or 1ml Giemsa stain is mixed with 9ml of distilled water.
 Cover the slide with diluted Giemsa stain and leave it to stand for 20-30 mins.
 Wash the slide with distlled water.
 Allow the smear to dry.

23. Giemsa Stain
Smear Reading
Smear is first examined under a low power objective and then under oil immersion objective.
 Herpes progenitalis - shows multinucleated giant keratinocytes, nuclei will show blurring of
chromatin pattern and loss of staining.
 Donovanosis - Intracellular donovan bodies, Bluish purple colour and resemble safety pin.
 Molluscum contagiosum - Handerson - Patterson bodies / Molluscum bodies - appear as ovoid ,
deeply basophilic bodies with hyaline homogenous structure surrounded by a membrane.

25. Wet Mount
4. WET MOUNT
( DIRECT MICROSCOPY WITHOUT STAINING )
Used for diagnosis of -
 Trichomoniasis
 Bacterial vaginosis
 Candidiasis

26. Wet Mount
WET MOUNT
 Collection of specimen
 Vaginal discharge is obtained with cotton tipped swab from the posterior fornix.
 In Men , the sample is collected by inserting a sterile swab into the urethra 2-3 cm deep.
 Procedure
 Sample is placed in a galss slide,
 Diluted with a drop of saline then cover slip is placed.
 Examined under a light microscope immediaty under 40x lens with reduced illumination.

27. Wet Mount
WET MOUNT
Slide Reading :
 Trichomoniasis -
 Clear pear shaped organism about a size of pus cell with 4 anterior flagellae and an
axostyle that traverse through body end to end.
 They show typical jerky movements.
 Bacterial vaginosis - Clue cells may be seen.
 Candiasis - Yeast cells can also be seen.

28. KOH Mount
5. KOH WET MOUNT
Used for diagnosis of -
 Candidal infection in genital tract.
 Requirements:
 10% or 20% KOH.
 Collection of specimen:
 Females - discharge is collected from posterior fornix.
 Males - a swab moistened in saline is rubbed against the glans penis.

29. KOH Mount
Procedure
 Take a clean microscope slide.
 Place the specimen on the slide.
 Add 2 drops of 10% KOH .
 Put a coverslip over the specimen, ensuring no air bubbles is trapped under the coverslip.
 Then examined under light microscope under 10x then using 40x.

30. KOH Mount
Reading
 Identification of yeast confirms the candidiasis.
 Yeast are round to ovoid cells ,showing typical budding (blastoconidia) and pseudohyphae

31. AA Test
6. Acetic Acid Test
 It is used to detect subclinical Genital Human Papilloma Virus ( HPV ) - associated infections.
 Requirments:
 Acetic Acid - 3 to 5 %
 Swab sticks.

32. AA Test
Procedure
 Clean the area with gauze soaked in saline and then with dry gauze.
 Apply acetic acid with the swab.
 Wait for 2 to 3 mins for lesions of vulva and penis.
 In Uterine cervix , the reaction appears in 1 min.

33. AA Test
Reading
 In Men - Affected area looks Red because of hypervascularization.
 In Females - Affected area became whitish, distinctle demrcated and thickened, sometimes
with elevated borders and centrally located epithelial fissures.
 The whtish appearance is due to over expression of cytokeratin 10 in HPV infected suprabasal
cells.
 The epithelial cells of HPV infected cells contain large nuclei and their protein content is very
high. Acetic acid application causes denaturation of these proteins, which results in whitish
appearance.

34. Whiff test
7. WHIFF TEST OR SNIFF TEST
 Used to diagnose - Bacterial vaginosis
 Procedure -
 Addition of 1 or 2 drops of 10% KOH solution to the specimen of vaginal discharge on a
glass slide causes enhancement of typical Fishy smell.

35. Two glass test
8. TWO AND THREE GLASS TEST
TWO GLASS TEST
 It is used to differentiate anterior urethritis from posterior urethritis or infection of bladder.
 Procedure -
 Pt is asked to pass urine in two seperate glasses.
 Reading -
 In Ant. Urethritis - 1st specimen if cloudy or hazy with pus / mucus thread. 2nd glass is
clear.
 In Post. Urethritis - 2nd glass will be hazy.
 In Bacterial Cystitis - Both glasess will be hazy.

36. Three Glass Test
Three Glass Test
 In this , anterior urethre is irrigated with colourless antiseptic solution until washing contained in
the first glass seem to be clear.
 the pt is then asked to pass urine in two glass containers.
 If the 1st glass contain pus - Post Urethra is infected.
 If the 2nd glass contain pus - Bladder is also is infected.

37. Asp of Bubo
9. ASPIRATION OF BUBO
 Used to detect -
 Chancroid
 LGV
in a fluctuating bubo.

38. Asp of Bubo
Procedure
 The pt is made to lie on a couch in supine position.
 Skin is sterilized with iodine and bubo is aspirated using 16 - 18G needle with 10 or 20 ml
syringe.
 Aspiration is done from non dependent fluctuant part of the bubo and continued till all parts of
the swelling are reduced.
 The material is sent for H.ducreyi, chlamydia and anaerobe cultures; smear for gram stain and
direct uorescene for chlamydia.

39. THANK YOU ALL !