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METHODS OF IDENTIFYNG MICROORGANISM

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. HANGING DROP C. FIXED STAINING.

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METHODS OF IDENTIFYNG MICROORGANISM

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  1. METHODS OF IDENTIFYNG MICROORGANISM • MOTILITY • HANGING-DROP METHOD • USE OF SEMI-SOLID MEDIA • STAINING OF FLAGELLA • SEROLOGICAL TEST • FLUORESCENT TEST • SWARMING PHENOMENA

  2. 2. MORPHOLOGY AND STAINING 3 GENERAL WAYS A. WET MOUNT B. HANGING DROP C. FIXED STAINING

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

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

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

  6. 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

  7. 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

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

  9. 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.

  10. 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

  11. 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.

  12. GRAM POSITIVE BACILLI

  13. GRAM NEGATIVE BACILLI

  14. 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.

  15. ACID FAST STAINING REAGENTS : • Carbolfuchsin • Acid Alcohol • Methylene Blue or Malachite Green

  16. 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

  17. ACID – FAST BACILLI

  18. NON-ACID FAST ORGANISMS

  19. 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 )

  20. 5. INDIA INK METHOD 6. MUIR METHOD 7. ANTHONY’S METHOD

  21. 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

  22. 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

  23. 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.

  24. 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.

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