MICROBIOLOGY – ALCAMO
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Transcript of MICROBIOLOGY – ALCAMO
1. INTRODUCTION
Why?
--- MOs are small and transparent
--- Cytoplasm of bacteria lacks color
--- Stains enhance visibility
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
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 SLIDE
2.DROP OF MOs ½ INCH BEFORE STAIN
3.WITH 2ND SLIDE HELD AT 45*, DRAW ACROSS MOs, THEN ACROSS STAIN
4.REVERSE DIRECTION, SMEAR FORWARD
SIMPLE STAIN − One color dye only
− EX: Crystal Violet, Methylene Blue
− Easy, fast stain method with good results
− TECHNIQUE:1. Add the MO to slide
2. Air dry the MO
3. Fix the MO – Put through flame 3X
4. Flood with stain
5. Rinse with water
6. 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)