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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.
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
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