MICROBIOLOGY – ALCAMO LECTURE: SPECIMEN PREPARATION AND STAINING
Feb 24, 2016
MICROBIOLOGY – ALCAMO
LECTURE: SPECIMEN PREPARATION AND STAINING
1. INTRODUCTIONWhy?
--- MOs are small and transparent
--- Cytoplasm of bacteria lacks color
--- Stains enhance visibility
2. WET SPECIMEN PREPARATIONS
ORGANISMS ARE NOT DRIED BEFORE HANDLING
WET MOUNT− Quick and easy− Since no stain is used only large
dense organisms are
visible− TECHNIQUE:
1. Place drop of specimen on clean slide
2. Place cover slip over it
Simple Staining– Positively and negatively charged
molecules are attracted to each other – MO’s cytoplasm has (-) charge– Basic stains have (+) charge
– Crystal Violet– Methylene Blue
– Therefore: Use (+) stains to color (-) MOs
Bacterial cocci stained with crystal violet
NEGATIVE STAIN− Easy, fast, good for size evaluation− Stain is acidic and negatively charged:
− Nigrosin (black dye)− Congo Red
− Stains the background, not the MO − No need for chemicals and heat fixing− Cells appear less shriveled and
distorted – more natural
−TECHNIQUE:1.PLACE DROP OF STAIN AT END
OF SLIDE2.DROP OF MOs ½ INCH BEFORE
STAIN3.WITH 2ND SLIDE HELD AT 45*,
DRAW ACROSS MOs, THEN ACROSS STAIN
4.REVERSE DIRECTION, SMEAR FORWARD
Bacterial cocci stained with nigrosin stain
3. DRY PREPARATIONS• MOs are dried and killed by “FIXING”
– To flame quickly 3X
Simple Differential
SIMPLE STAIN − One color dye only− EX: Crystal Violet, Methylene Blue− Easy, fast stain method with good results− TECHNIQUE:
1. Add the MO to slide2. Air dry the MO3. Fix the MO – Put through flame 3X4. Flood with stain5. Rinse with water6. Dry for microscopic examination
DIFFERENTIAL STAIN − GRAM staining differentiates bacteria into 2
groups based on the differences in cell walls − Use two different colored dyes− All bacteria absorb the first stain color− But some lose the color when rinsed with
alcohol and are stained with a 2nd color stain− Results are somewhat difficult and variable− Named for Christian Gram – Dutch physician
DIFFERENTIAL STAIN • GRAM (+) bacteria have peptidoglycan
in their cell walls and retain the initial purple stain
• GRAM (–) bacteria have more lipids in their cell wall and treatment with alcohol dissolves the lipids and the purple color leaks out
• The GRAM (-) bacteria are now colorless, so a 2nd stain is needed to color these MO’s
Gram Positive Bacteria
Gram Negative Bacteria
Less lipid in cell wall More lipid in cell wall
Peptidoglycan in cell wall
No peptidoglycan
Spore forming rods Many intestinal rods
Many cocci Few cocci
Tolerant to drying Susceptible to drying
DIFFERENTIAL STAIN − TECHNIQUE:
1. Stain with Crystal Violet (all MO’s are purple)2. Cover with Gram’s iodine3. Decolorize with alcohol4. G+ stay purple5. G- will lose the purple dye6. Stain with Safranin dye (G- MO now appear red)
Gram (-) Gram (+)
Acid Fast Stain• Acid-fastness is a physical property of
some bacteria referring to their resistance to de-colorization by acids during staining procedures
• The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria, is responsible for the staining pattern of poor absorption followed by high retention
Acid Fast Stain• The most common staining technique
used to identify acid-fast bacteria is the Ziehl-Neelsen stain, in which the acid fast bacilli are stained bright red and stand out clearly against a blue background.
Acid Fast Stain of Mycobacterium tuberculosis
SPECIAL STAINS − Involve special complicated methods
not for amateurs− Used to observe special structures:
– ENDOSPORES– FLAGELLA– CAPSULES