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