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
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
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.

13. GRAM POSITIVE BACILLI

14. GRAM NEGATIVE BACILLI

15. 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.

16. ACID FAST STAINING REAGENTS : Carbol fuchsin Acid Alcohol
Methylene Blue or Malachite Green

17. 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

18. ACID – FAST BACILLI

19. NON-ACID FAST ORGANISMS

20. 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 )

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

22. 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

23. 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

24. 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.

25. 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.