1.
MOTILITY
A.
B.
C.
D.
E.
F.
HANGING-DROP METHOD
USE OF SEMI-SOLID MEDIA
STAINING OF FLAGELLA
SEROLOGICAL TEST
FLUORESCENT TEST
SWARMING PHENOMENA
2. MORPHOLOGY AND STAINING
3 GENERAL WAYS
A. WET MOUNT
B. HANGING DROP
C. FIXED STAINING
1.
CLASSIFICATION ACCORDING TO
SHAPE:
A. BACILLI : ROD – SHAPED
B. COCCI : ROUND OR SPHERICAL
C. SPIRILLA : SMALL, COMMA-SHAPED OR
SPIRAL ORGANISM
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
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VIBRIO
SPIRILLUM
SPIROCHETE
2. CLASSIFICATION ACCORDING TO
ARRANGEMENT:
A. STAPHYLO
B. STREPTO
C. SARCINAE
D. TETRAD
E. DIPLO
F. PALLISADE

PROCESS OF ARTIFICIAL COLORING OF
MICROORGANISM WITH DYES OR REAGENTS
IN ORDER TO FACILITATE MORPHOLOGICAL
IDENTITY AND ARRANGEMENT OF BACTERIA
UNDER THE MICROSCOPE.
1.
SIMPLE STAIN
-
ONE PARTICULAR STAIN OR DYE RESULTING
INTO ONE COLOR
ALSO KNOWN AS DIRECT STAINING
AN AQUEOUS OR ALCOHOLIC SOLUTION OF A
SINGLE DYE.
EX:
 LOFFLER’S METHYLENE BLUE
 GENTIAN VIOLET
 CARBOL FUCHSIN
 SAFRANIN
2. DIFFERENTIAL STAIN
◦ TWO OR MORE DYES OR STAINS ARE
USUALLY EMPLOYED
COMPOSITION :
1. PRIMARY STAIN
2. MORDANT
3. DECOLORIZER
4. SECONDARY OR COUNTER STAIN
EX. : GRAM’S STAIN, ACID FAST STAIN
REAGENTS :
1.
2.
3.
4.
CRYSTAL VIOLET
GRAM’S IODINE
ALCOHOL AND /OR ACETONE
SAFRANIN



THE DIFFERENCE IN THE COMPOSITION OF
BACTERIAL CELL WALL COMPOSITION
Gram positive organisms: with thick
peptidoglycan layer and teichoic acid cross
linkages.
Gram negative organisms : thin layer of
peptidoglycan.
GENERAL RULES :
1. ALL COCCI ARE GRAM POSITIVE EXCEPT
Neisseria, Veillonella, and Branhamella
2.
3.
4.
ALL BACILLI ARE GRAM NEGATIVE EXCEPT
Bacillus, Clostridium, Mycobacterium,
Corynebacterium,Nocardia, Erysipelothrix,
Lactobacillus, and Listeria.
ALL COCCI ARE NON-MOTILE AND NONSPORE-FORMER
ALL ENCAPSULATED ORGANISMS ARE NONMOTILE
5. Bacillus and Clostridium ARE SPORE
FORMING ORGANISMS
6. THE HIGHER FORMS OF ORGANISM
INCLUDING Actinomyces, Streptomyces,
yeast and mold ARE GRAM POSITIVE
7. SPIRAL ORGANISMS ARE NOT STAINABLE
EXCEPT FOR SOME WHICH ARE GRAM
NEGATIVE.
GRAM POSITIVE BACILLI
GRAM NEGATIVE BACILLI



PRINCIPLE : USED FOR SUBSET OF BACTERIA
WHOSE CELL WALL CONTAINS LONG CHAIN
FATTY ACID.
ACID FAST : ORGANISMS WITH MYCOLIC ACID
IN THEIR CELL WALL.
NON-ACID FAST : ORGANISMS WITHOUT
MYCOLIC ACID IN THEIR CELL WALL.
REAGENTS :
1. Carbol fuchsin
2. Acid Alcohol
3. Methylene Blue or Malachite Green
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




METHODS :
ZIEHL – NEELSEN METHOD
KINYOUN METHOD (phenol)
PAPPENHEIM’S METHOD ( alcohol/Rosolic acid)
BAUMGARTEN’S METHOD (diluted alc.fuchsin)
RHODAMINE – AURAMINE METHOD
ACID – FAST BACILLI
NON-ACID FAST ORGANISMS
A.
CAPSULAR STAINS
1. HISS’S COPPER SULFATE METHOD
- CAPSULATED ORGANISMS APPEAR AS DARK PURPLE
BODY WITH A FAINT BLUE CAPSULE AROUND IT
2. GIN’S METHOD
- BACTERIA WILL BE STAINED BUT THE CAPSULE
IS UNSTAINED WITH THEIR MARGIN DELINEATED
BY THE INK.
3. WELCH’S METHOD
- CAPSULE STAINS PALE VIOLET
4. WADSWORTH’S METHOD
-BACTERIA ( BLUE ), Capsule ( PINKISH )
5. INDIA INK METHOD
6. MUIR METHOD
7. ANTHONY’S METHOD
LOEFFLER’S ALKALINE METHYLENE BLUE
( LAMB )
2. NEISSER STAIN
- BACILLI APPEAR ENTIRELY BROWN OR
SHOW DARK BLUE ROUND BODY AT BOTH
ENDS.
3. ALBERT’S METHOD
- GRANULES APPEAR BLUE-BLACK
4. LJUBINSKY METHOD
1.
1.
HEAT AND ACETIC ACID
2.
3.
4.
5.
-BACTERIA (BLUE), SPORES (RED)
DORNER METHOD
-BACTERIA ( ALMOST COLORLESS), SPORES (RED)
WITH DARK GRAY BACKGROUND
WIRTZ – CONKLIN METHOD
-SPORES ARE SEEN AS GREEN SPHERULES IN RED
STAINED RODS OR DEBRIS.
10% NIGROSIN AND CARBOL FUCHSIN
SCHAEFFER AND FULTON
- SPORES APPEAR AS LIGHT GREEN
1.
LEIFSON METHOD
2.
3.
4.
5.
6.
- FLAGELLA ( RED ), BACTERIAL CELL ( BLUE )
SILVER STAIN FOR FLAGELLA
FISHER – CONN
CASARE’S – GIL’S METHOD
LOFFLERS METHOD
VAN ERMENGEN’S METHOD
* FLAGELLAR STAIN USED A SPECIAL MORDANT SUCH
AS TANNIC ACID THAT SWELLS, COATS, FORMS A
PRECIPITATE WITH THE FLAGELLA.
1.
BURRI’S INDIA INK METHOD
- SPIROCHETES/BACTERIA (WHITE IN
DARK FIELD)
-USED FOR ORGANISMS WITH POOR
STAINING PROPERTIES
2. NIGROSIN
- USED FOR STUDYING SPIROCHETES
3. RELIEF STAIN ( DORNER )
- CELLS ARE UNSTAINED AGAINST THE
DARK BACKGROUND.