PRAKTIKUM

MIKRO
ACID FAST BACILI

BACTERIAL STAINING
Most initial observations of microorganisms are
made with stained preparations
Staining simply means coloring the
microorganisms with a dye that emphasizes certain
structures
Bacterial structures that play important role in the
staining are : cell wall and cytoplasmic
membrane
Staining of living bacteria difficult
Living state examination wet mount method,
hanging drop preparation
Usually staining of bacteria fixed state

STAINS
Stains are salt composed of a positive and a
negative ion, one of which is colored and is known
as the chromophore
The color of basic dyes is in the positive ion
(crystal violet, safranin, methylene blue)
The color of acidic dyes is in the negative ion
(acid fuchsin, nigrosin, eosin)
Bacteria are slightly negative charged at pH 7.
Thus,the colored positive ion in a basic dye is
attracted to the negatively charged bacterial cell
Acidic dyes color the background negative
staining

PREPARING SMEARS FOR STAINING

Clean object glass/slide
Wet/liquid specimens or clinical
specimens directly apply to the slide
smear
Colonies from culture suspension
smear on slide
Fixation several times passing over
fire or by applying methyl alcohol for 1
minute

PURPOSE OF FIXATION
Attaches the specimen to the slide
Kills the microorganisms without any
changes of the morphology
Easier to stain
To keep the slides

KINDS OF STAINING TECHNIQUES

Simple Stains :
- methylene blue
- safranin
Differential Stains :
GRAM Gram (+) / Gram ( - )
Acid Fast Acid Fast Bacilli
Special Stains :
- Metachromatic staining
- Spore staining
- Capsule staining
- Flagella staining

ACID FAST STAIN

Identify two groups of bacteria : acid fast bacteria and non-acid
fast bacteria
Acid fast bacteria have a waxy material in their cell wall (mycolic
acid) specify the acid fast characteristic
Acid fast bacteria are not easy to be stained once its can be
stained, the dye will be retained, even when decolorized with
hydrochloric acid in alcohol
Primary stain fuchsin red
Counterstain methylene blue
Chemical intensifier carbol
Physical intensifier heat
Kinds of acid fast staining :
- Ziehl Neelsen
- Tan Thiam Hok
- Kinjoun carbolfuchsin
Bacteria : Mycobacterium tuberculosis,
Mycobacterium leprae

PROCEDURE OF ACID FAST

STAINING
(ZIEHL NEELSEN)
Make smear on slide fixed
Apply carbolfuchsin gently heated for five
minutes
Washed with water
Decolorized with acid-alcohol 5-10 seconds
Washed with water
Apply methylene blue (counterstain) 30
seconds
Washed with water dried

 Buatlah bahan pemeriksaan sputum pd gelas

obyek. Sputum sudah basah, maka untuk
pembuatan sediaan tidak perlu akudes steril.
Biarkan kering di udara fiksasi
Tuang sediaan dng Carbol fuchsin
Panasin 5 menit ga boleh mendidih&kering
Buang sisa carbol fuchsin bilas air
Tuang Acid alkohol sampe warna cat luntur
Buang sisa alkohol bilas air
Tuang Methylene Blue slm menit
Buang sisa methylene blue bilas air
Keringkan
Liat di mikroskop perbesaran 100x