METHODS OF IDENTIFYNG MICROORGANISM MOTILITY 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 2. MORPHOLOGY AND STAINING 3 GENERAL WAYS A. WET MOUNT B. HANGING DROP C. FIXED STAINING

MORPHOLOGY OF BACTERIA CLASSIFICATION ACCORDING TO SHAPE: BACILLI : ROD – SHAPED COCCI : ROUND OR SPHERICAL SPIRILLA : SMALL, COMMA-SHAPED OR SPIRAL ORGANISM VIBRIO SPIRILLUM SPIROCHETE

2. CLASSIFICATION ACCORDING TO ARRANGEMENT: STAPHYLO STREPTO SARCINAE TETRAD DIPLO PALLISADE

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

TYPES OF BACTERIOLOGICAL STAINS 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 : PRIMARY STAIN MORDANT DECOLORIZER SECONDARY OR COUNTER STAIN EX. : GRAM’S STAIN, ACID FAST STAIN

GRAM STAINING REAGENTS : CRYSTAL VIOLET GRAM’S IODINE ALCOHOL AND /OR ACETONE SAFRANIN

PRINCIPLE 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.

GRAM STAINING GENERAL RULES : ALL COCCI ARE GRAM POSITIVE EXCEPT Neisseria, Veillonella, and Branhamella ALL BACILLI ARE GRAM NEGATIVE EXCEPT Bacillus, Clostridium, Mycobacterium, Corynebacterium,Nocardia, Erysipelothrix, Lactobacillus, and Listeria. ALL COCCI ARE NON-MOTILE AND NON- SPORE-FORMER ALL ENCAPSULATED ORGANISMS ARE NON- MOTILE

5. Bacillus and Clostridium ARE SPORE FORMING ORGANISMS 6 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

ACID - FAST STAINING 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 : Carbol fuchsin Acid Alcohol 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 BACILLI

NON-ACID FAST ORGANISMS

SPECIAL STAIN OR SELECTIVE STAIN CAPSULAR STAINS 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

METACHROMATIC GRANULES 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

SPORES STAIN HEAT AND ACETIC ACID -BACTERIA (BLUE), SPORES (RED) 2. DORNER METHOD -BACTERIA ( ALMOST COLORLESS), SPORES (RED) WITH DARK GRAY BACKGROUND 3. WIRTZ – CONKLIN METHOD -SPORES ARE SEEN AS GREEN SPHERULES IN RED STAINED RODS OR DEBRIS. 4. 10% NIGROSIN AND CARBOL FUCHSIN 5. SCHAEFFER AND FULTON - SPORES APPEAR AS LIGHT GREEN

FLAGELLAR STAIN LEIFSON METHOD - FLAGELLA ( RED ), BACTERIAL CELL ( BLUE ) 2. SILVER STAIN FOR FLAGELLA 3. FISHER – CONN 4. CASARE’S – GIL’S METHOD 5. LOFFLERS METHOD 6. VAN ERMENGEN’S METHOD * FLAGELLAR STAIN USED A SPECIAL MORDANT SUCH AS TANNIC ACID THAT SWELLS, COATS, FORMS A PRECIPITATE WITH THE FLAGELLA.

INDIRECT/RELIEF OR NEGATIVE STAINING 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.