INTRODUCTION
*STAINED PREPARATIONS ARE NEEDED TO EXAMINE MICRO-ORGANISMS
MICROSCOPICALLY IN ORDER TO STUDY THEIR MORPHOLOGY AND OBSERVE
THEIR CELLULAR CONSTITUENTS.
*SMEARS (OR TISSUE SECTIONS) ARE MADE AND STAINED BY ANY ONE OF THE
REQUIRED STAINING METHODS.
*SMEARS CAN BE MADE FROM LIQUID OR SOLID CULTURES OR FROM THE
CLINICAL SPECIMEN
INTRODUCTION
•THE STAINING RESULT IS DEPENDENT ON THE QUALITY OF THE SMEAR.
•FROM THE LIQUID CULTURE, SMEARS SHOULD BE EVENLY SPREAD ON
THE SLIDE AND ALLOW TO DRY IN AIR.
•SMEARS FROM SOLID CULTURE SHOULD NOT BE THICK
TYPES OF STAINING:
•INDIRECT STAINING- THIS IS WHEN AN ORGANISM IS STAINED ONLY IN
THE PRESENCE OF A MORDANT.
• E.G. GRAM'S STAIN.
•THE DIRECT STAINING- THIS A SIMPLE ONE-STEP STAINING
PROCEDURE IN WHICH THE PRESENCE AND MORPHOLOGY OF
BACTERIA ARE DEMONSTRATED.
TYPES OF STAINING:
•NEGATIVE STAINING - THIS IS WHEN THE
ORGANISM REMAINS UNSTAINED AGAINST A
STAINED BACKGROUND. THIS IS ONE OF THE FEW
METHODS WHERE ACID STAINS SUCH AS
NIGROSIN, ARE USED.
TYPES OF STAINING:
•METACHROMATIC STAINING - IN THIS STAINING
METHOD THE ORGANISM OR PART OF THE
ORGANISM IS STAINED A DIFFERENT SHADE OF
COLOUR FROM THAT OF THE STAIN.
METHODS OF STAINING:
1. SIMPLE STAINS: THIS MAKES USE OF THE DIRECT STAINING METHOD.
2. DIFFERENTIAL STAINS: THIS STAINING METHOD DIVIDES BACTERIA INTO
TWO GROUPS
3. SPECIAL STAINS: THESE ARE SPECIALIZED STAINING METHODS TO
DEMONSTRATE CERTAIN BACTERIAL COMPONENTS, E.G. SPORE.
1. SIMPLE STAINS
•REQUIRE ONLY A SINGLE DYE
•EXAMPLES INCLUDE MALACHITE GREEN, CRYSTAL
VIOLET, BASIC FUCHSIN, AND SAFRANIN
•ALL CELLS APPEAR THE SAME COLOR BUT CAN
REVEAL SHAPE, SIZE, AND ARRANGEMENT
Figure 3.25
Figure 3.25
2. DIFFERENTIAL STAINS
•USE TWO DIFFERENTLY COLORED DYES, THE PRIMARY
DYE AND THE COUNTERSTAIN
•DISTINGUISHES BETWEEN CELL TYPES OR PARTS
•EXAMPLES INCLUDE GRAM, ACID-FAST, AND ENDOSPORE
STAINS
GRAM STAINING
•IN 1884, CHRISTIAN GRAM, A DANISH BACTERIOLOGIST,
DESCRIBED THIS STAINING METHOD WHICH IS THE MOST
IMPORTANT STAIN IN ROUTINE BACTERIOLOGY.
•THE MOST UNIVERSAL DIAGNOSTIC STAINING TECHNIQUE FOR
BACTERIA
•IT DIVIDES BACTERIA INTO TWO GROUPS - THE GRAM POSITIVE
(PURPLE) AND GRAM NEGATIVE (RED) .
GRAM STAINING
• THE GRAM POSITIVE ORGANISM MUST HAVE AN INTACT CELL WALL. A DAMAGED CELL WALL
INEVITABLY RESULTS IN GRAM NEGATIVE REACTION.
• THIS SHOWS THE IMPORTANCE OF CELL WALL IN GRAM STAIN REACTION.
• THE GRAM'S STAIN REACTION IS BASED ON THE ABILITY OF THE ORGANISM TO RESIST
DECOLOURISATION WITH ACETONE, ALCOHOL OR ANILINE OIL AFTER THE INITIAL STAINING WITH
ONE OF THE ROSANILINE BASIC DYES AND THEN TREATING WITH A MORDANT.
E.G. THE ROSANILINE DYES COMMONLY USED ARE CRYSTAL VIOLET, METHYL VIOLET AND GENTIAN
VIOLET.
E.G. IODINE IS THE MORDANT USED.
Figure 3.25
ACID-FAST STAINING
•IMPORTANT DIAGNOSTIC STAIN
•DIFFERENTIATES ACID-FAST BACTERIA (PINK)
FROM NON-ACID-FAST BACTERIA (BLUE)
•IMPORTANT IN MEDICAL MICROBIOLOGY
ACID-FAST STAINING
• ACID-FAST STAINING IS ANOTHER EXAMPLE OF A DIFFERENTIAL STAIN USED IN
BACTERIOLOGY.
• IT DIVIDES BACTERIA INTO TWO GROUPS, ACID FAST-AND NON ACID FAST.
MEMBERS OF THE GENUS MYCOBACTERIUM ARE ACID-FAST IN NATURE. LIKE THE
GRAM REACTION, THE ACID-FASTNESS IS ALSO CALLED CELL WALL DEPENDENT.
• MYCOBACTERIA HAVE A HIGH LIPID CONTENT, ESPECIALLY MYCOLIC ACID, IN THEIR
CELL WALL. THE ORDINARY ANILINE DYE SOLUTIONS CANNOT PENETRATE THE
MYCOBACTERIAL CELL WALL.
Figure 3.25
ENDOSPORE STAIN
•DYE IS FORCED BY HEAT INTO RESISTANT BODIES CALLED
SPORES OR ENDOSPORES
•DISTINGUISHES BETWEEN THE STORES AND THE CELLS
THEY COME FROM (THE VEGETATIVE CELLS)
•SIGNIFICANT IN MEDICAL MICROBIOLOGY
Figure 3.25
3. SPECIAL STAINS
•USED TO EMPHASIZE CERTAIN CELL PARTS THAT
AREN’T REVEALED BY CONVENTIONAL STAINING
METHODS
•EXAMPLES: CAPSULE STAINING, FLAGELLAR
STAINING
Figure 3.25
Figure 3.25
FIXED, STAINED SMEARS
•SMEAR TECHNIQUE DEVELOPED BY ROBERT KOCH
•SPREAD A THIN FILM MADE FROM A LIQUID SUSPENSION OF CELLS AND
AIR-DRYING IT
•HEAT THE DRIED SMEAR BY A PROCESS CALLED HEAT FIXATION
•SOME CELLS ARE FIXED USING CHEMICALS
•STAINING CREATES CONTRAST AND ALLOWS FEATURES OF THE CELLS TO
STAND OUT
•APPLIES COLORED CHEMICALS TO SPECIMENS
•DYES BECOME AFFIXED TO THE CELLS THROUGH A CHEMICAL REACTION
•DYES ARE BASIC (CATIONIC) DYES OR ACIDIC (ANIONIC) DYES.
POSITIVE AND NEGATIVE STAINING
•POSITIVE STAINING: THE DYE STICKS TO THE SPECIMEN TO GIVE
IT COLOR
•NEGATIVE STAINING: THE DYE DOES NOT STICK TO THE SPECIMEN,
INSTEAD SETTLES AROUND ITS BOUNDARIES, CREATING A
SILHOUETTE.
•NIGROSINE AND INDIA’S INK ARE COMMONLY USED
•HEAT FIXATION IS NOT REQUIRED, SO THERE IS LESS SHRINKAGE OR
DISTORTION OF CELLS
•ALSO USED TO ACCENTUATE THE CAPSULE SURROUNDING CERTAIN
BACTERIA AND YEASTS
