Microscopy Flashcards
Time for holding urine before slide
2 hours
overnight if nil seen on microscopy but symptoms
Equipment for urethral slide
5 microlitre loop
clean microscopy slide, frosted side up
gonococcal culture medium (or Stuart’s Amies transport medium)
How to take urethral sample
inset 1-2cm into meatus and press on each side to get discharge collected in loop
Types of urine sample
FPU- to get urethral infection
MSU- to get urine infection without urethral contamination
retract foreskin if present
Vaginal sampling
5 microlitre loop from vaginal wall, rub over pH paper
pH normal <4.5
-tests for candida (or Sabouraud’s plate) and BV via gram stain
posterior fornis- onto saline
Cervical sampling
Insert loop into cervical os
Eye sampling
NAAT/culture
evert lower lid and rub swab over inner surface
rectal sample
symptoms: proctoscope and jelly, then swabs from mucosa
no symptoms- NAAT 2-3cm inside rectum
HSV sample
Ulcer base or rectal mucosa
if blister intact, puncture with sterile needle then swab
DFM
Chancre, balanitis of follman’s or oral lesion away from gum margin (must be cleansed with saline due to commensal treponemes)
1) choose a clean fresh ulcer
2) cleanse ulcer with saline, using gauze gently abrade ulcer to encourage ooze (and squeeze if nil comes)
3) Collect serum from the ulcer with the edge of the cover slip and press onto a glass slide
4) Press slide and cover slip between tow pieces of blotting paper to ensure a thin film of specimen
-collect at least 3 specimens
- can also scrape off surface of condyloma lata or get tissue from lymph nodes
LGV sample
ulcer base/rectal mucosa
swab vigorously with NAAT
Chancroid sample
remove pus from ulcer base
swab edge of ulcer with NAAT
Donovanosis sample
Donovan bodies in cellular material
cleanse and anaesthetise area
remove a piece of tissue and crush between two slides
-Giemsa/Wright’s Leishmann staining
Self sampling
Rectum= 2-3cm and 5-10 seconds
Vagina- 5cm and 10-20 seconds
How to examine slide
ensure smear is facing up
focus under lower power to find adequate area (individual cells without overlaps)
Zig sag left to right or up and down across sample
at least 20 high power fields
How to set up bright field microscopy
1) Get comfortable, clean microscope, rack down stage and turn light on minimum
2) use 10x lens, rack up stage, turn up light until comfortable
3) flip in auxiliary condenser, open aperture diaphragm and close field diaphragm
4) move condenser up and down until see spot of light
5) Move spot of light into centre of field
gram positive
purple
PMNC meaning
polymorphonuclear cells (pus/neutrophils)
1+ 5-10 per HPF
2+ 10-20 per HPF
3+ >20
NGU
5 or more PMNCs per HPF
over at least 5 HPF
No gram -ve intracellular diplococci (5% will have +ve GC culture)
GC
Gram negative intracellular diplococci
-if extracellular cannot confirm GC
GC cervix
30-50% detection by slide (95% in men)
Amsel’s criteria
3 of 4
thin homogenous discharge
pH >4.5
release of odour when 10% KOH added
clue cells on wet mount
Ison Hay
0- epithelial cells only
1- gram +ve rods only (lactobacilli)
2- mixed flora, reduced lactobacilli but also others
3- no lactobacilli, mix of gram +ve and -ve, clue cells, mobiluncus
4- gram +ve cocci
Wet mount
40x magnification (no oil)
Gram stained smear
100x with immersion oil
Principles of gram stain
gram +ve= lots of peptidoglycan in cell wall
gram -ve= less peptidoglycan so no purple stain, takes up counterstain
How to gram stain
1) Fix smear by heat (<50 degrees ~1 min) or 95% methanol for 2min
2) Flood with crystal violet for 30 seconds
-lodges in cell wall
3)Flood with iodine 30 seconds
-binds with crystal violet to form insoluble matrix
4) decolourise with acetone, wash with water immediately
5) Flood with red counterstain 1 minute
6) wash and blot dry
What can’t be seen on gram stain?
Mycoplasma- no cell wall
Chlamydia- too small
Mycobacterium- too difficult
Methylene Blue staining
In resource-poor setting to look for GC (presumptive dx)
1)Fix smear
2) flood with methylene blue for 3 minutes
3) wash off with water
4) blot
blue bacteria and blue/pink neutrophils. See blue gram -ve intracellular diplococci
