BACTE LAB - AFS Flashcards
Slides must be
new clean free from grease free from fingerprints unscratched
what to write on the frosted end of the slide
laboratory register serial number
order number of the sputum specimen
type of pencil to use on unfrosted slides
diamond pencil, left end of the slide
what part of the sputum must be taken
yellowish purulent part
smear shape
oval
smear size
2 cm wide x 3 cm length
the smear size corresponds to how many visual fields
150, to be counted in one length
if wire loop was used instead of applicator stick, dip it in ___
alcohol washed-sand bottle
clean the wire loop in a flask containing ___ before sterilizing in a flame
70% - 90% alcohol with glass beads or sand
for accidental contamination with sputum,
hand:
wash with soap and water
for accidental contamination with sputum,
cloth:
spray with 5% phenol OR
Soak in disinfectant OR
Autoclave OR
Boil
for accidental contamination with sputum,
Surface of container,
wear rubber gloves before handling it
for accidental contamination with sputum,
bench of floor
spray with 5% phenol or Lysol
How to dry the smears
air dry, not in direct sunlight or over a flame
Elements of Quality Smear
Quality of sputum size of smear thickness of smear evenness of smear staining technique cleanness of slide
What will happen if during fixation the slide was heated for too long or kept stationary
smear will be scorched
a good stained smear by ZN method shows ___
strong red AFB, against weak blue background
It is not possible to decolorize if _____ are used
cold watery acids
To prepare 0.3% Ziehl’s carbol fuchsin working solution, mix
10 ml solution A and 90 ml solution B
Solution A of Ziehl’s carbol fuchsin
Fuchsin Alcoholic Stock Solution
Solution A of Ziehl’s carbol fuchsin components
3g basic fuchsin
95% alcohol UP to 100 mL
Solution A of Ziehl’s carbol fuchsin NAME
Aqueous Phenol Solution
Solution A of Ziehl’s carbol fuchsin components
Phenol crystals 5g
Distilled water up to 90 mL
Before adding water, liquefy the phenol crystals by
gentle heating
Decolorizing Agent Solutions
Acid-alcohol solution
25% Aqueous Sulfuric Acid Solution
Acid-alcohol solution components
95% alcohol — 970 mL
35% HCl — 30 mL
25% Aqueous Sulfuric Acid Solution components
distilled water 300 ml
conc. sulfuric acid 100 ml
0.3% Methylene Blue Counterstaining Solution components
0.3 g methylene blue chloride
distilled water up to 100 mL
ZN method requires staining for ___ min
10
ZN method requires decolorizing for ___ min
approx 3 min
ZN method requires counterstaining for ___ min
not more than 1 min
what replaced carbon fuchsin in fluorescence staining
fluorescent dye
examples of fluorescent dye
auramine-O
rhodamine
auramine-rhodamine
acridine orange
counterstain in fluorescence staining
potassium permanganate
describe the decolorize in fluorescence staining
milder
with auramine staining, the bacilli appears as (color)
slender bright yellow luminous rods against a dark background
identification of mycobacteria with auramine O is due to the
affinity of mycolic acid in the cell walls for fluorochromes
light produced by a substance when it is stimulated by another light
fluorescence
fluorescence called cold light because
does not come from a hot source like an incandescent light bulb
dyes which have the property of absorbing light rays of shorter wavelength and emitting light rays of longer wavelength
fluorescent dye such as auramine O
source of light
mercury vapour lamp
by meas of ____
only light rays of shorter wave lengths are allowed to emerge
rays of ___ wavelengths used for fluorescence microscopy
shorter
Heat slides until
Vapor starts to rise and metallic sheen appears
Heat slides until
Vapor starts to rise and metallic sheen appeara
Let the carbol fuchsin staim stand for —min
10 min
Applying time for counterstaining reagent depends on (3)
Quality of specimen
Thickness of smear
Concentration of reagent
Poor quality smears can lead to —-
False results
If the smear is dark blue, this means that,
The smear is too thick
Employed to highlight the stained organisms for easier recognition
Counterstain
Examples of fluorescent dye
Auramine-O
Rhodamine
Auramine-rhodamine
Acridine orange
Counterstain in fluorescence microscopy
Potassium permanganate
Useful to quench background fluorescene
Counterstain (potassium permanganate)
Fluorescent dyes are also called
Fluorochromes
Principle lf auramine staining
Affinity of mycolic acids for fluorochromes
After smearing sputum, let the slide air dry for —- min
15-20 min
T or F
Wet slides can produce aerosols if disturbed
T
When is Bsc required
Large volume of positive sputum sx to be handled
In cultures
In drug susceptibility testing
Specimens can be discarded by (2)
Burying after chemical disinfection or autoclaving
Safe commercial waste collection services
Discard waste in a deep pit of at least — m depth
1.5
Limit the number of slides in each staining batch to a maximum of —
12
