1. MOTILITYA. HANGING-DROP METHODB. USE OF SEMI-SOLID MEDIAC. STAINING OF FLAGELLAD. SEROLOGICAL TESTE. FLUORESCENT TESTF. SWARMING PHENOMENA

METHODS OF IDENTIFYNG MICROORGANISM

2. MORPHOLOGY AND STAINING3 GENERAL WAYS

A. WET MOUNTB. HANGING DROPC. FIXED STAINING

1. CLASSIFICATION ACCORDING TO SHAPE:

A. BACILLI : ROD – SHAPEDB. COCCI : ROUND OR SPHERICALC. SPIRILLA : SMALL, COMMA-SHAPED OR

SPIRAL ORGANISM VIBRIO SPIRILLUM SPIROCHETE

MORPHOLOGY OF BACTERIA

2. CLASSIFICATION ACCORDING TO ARRANGEMENT:A. STAPHYLOB. STREPTOC. SARCINAED. TETRADE. DIPLOF. PALLISADE

PROCESS OF ARTIFICIAL COLORING OF MICROORGANISM WITH DYES OR REAGENTS IN ORDER TO FACILITATE MORPHOLOGICAL IDENTITY AND ARRANGEMENT OF BACTERIA UNDER THE MICROSCOPE.

STAINING

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

TYPES OF BACTERIOLOGICAL STAINS

2. DIFFERENTIAL STAIN◦TWO OR MORE DYES OR STAINS ARE

USUALLY EMPLOYED COMPOSITION :1. PRIMARY STAIN2. MORDANT3. DECOLORIZER4. SECONDARY OR COUNTER STAIN

EX. : GRAM’S STAIN, ACID FAST STAIN

REAGENTS :

1. CRYSTAL VIOLET2. GRAM’S IODINE3. ALCOHOL AND /OR ACETONE4. SAFRANIN

GRAM STAINING

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.

PRINCIPLE

GENERAL RULES :1. ALL COCCI ARE GRAM POSITIVE EXCEPT

Neisseria, Veillonella, and Branhamella2. ALL BACILLI ARE GRAM NEGATIVE EXCEPT

Bacillus, Clostridium, Mycobacterium, Corynebacterium,Nocardia, Erysipelothrix, Lactobacillus, and Listeria.

3. ALL COCCI ARE NON-MOTILE AND NON-SPORE-FORMER

4. ALL ENCAPSULATED ORGANISMS ARE NON-MOTILE

GRAM STAINING

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.

ACID - FAST STAINING

REAGENTS :1. Carbol fuchsin2. Acid Alcohol3. Methylene Blue or Malachite Green

ACID FAST STAINING

METHODS :

ZIEHL – NEELSEN METHOD KINYOUN METHOD (phenol) PAPPENHEIM’S METHOD ( alcohol/Rosolic

acid) BAUMGARTEN’S METHOD (diluted

alc.fuchsin) RHODAMINE – AURAMINE METHOD

ACID FAST STAINING

ACID – FAST BACILLI

NON-ACID FAST ORGANISMS

A. CAPSULAR STAINS1. 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 )

SPECIAL STAIN OR SELECTIVE STAIN

5. INDIA INK METHOD6. MUIR METHOD7. ANTHONY’S METHOD

1. 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

METACHROMATIC GRANULES

1. HEAT AND ACETIC ACID-BACTERIA (BLUE), SPORES (RED)

2. DORNER METHOD-BACTERIA ( ALMOST COLORLESS), SPORES (RED)

WITH DARK GRAY BACKGROUND3. WIRTZ – CONKLIN METHOD

-SPORES ARE SEEN AS GREEN SPHERULES IN RED STAINED RODS OR DEBRIS.

4. 10% NIGROSIN AND CARBOL FUCHSIN5. SCHAEFFER AND FULTON

- SPORES APPEAR AS LIGHT GREEN

SPORES STAIN

1. LEIFSON METHOD- FLAGELLA ( RED ), BACTERIAL CELL ( BLUE )

2. SILVER STAIN FOR FLAGELLA3. FISHER – CONN4. CASARE’S – GIL’S METHOD5. LOFFLERS METHOD6. VAN ERMENGEN’S METHOD

* FLAGELLAR STAIN USED A SPECIAL MORDANT SUCH AS TANNIC ACID THAT SWELLS, COATS, FORMS A PRECIPITATE WITH THE FLAGELLA.

FLAGELLAR STAIN

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.

INDIRECT/RELIEF OR NEGATIVE STAINING