MICROBIOLOGY – ALCAMO LECTURE: SPECIMEN PREPARATION AND STAINING

1. INTRODUCTION Why? --- 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 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

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 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 2 nd 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 wallMore lipid in cell wall Peptidoglycan in cell wall No peptidoglycan Spore forming rodsMany intestinal rods Many cocciFew cocci Tolerant to dryingSusceptible to drying

DIFFERENTIAL STAIN −TECHNIQUE: 1.Stain with Crystal Violet (all MO’s are purple) 2.Cover with Gram’s iodine 3.Decolorize with alcohol 4.G+ stay purple 5.G- will lose the purple dye 6.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 proceduresbacteriastaining 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 retentionmycolic acidcell walls Mycobacteria

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. Ziehl-Neelsen stain

Acid Fast Stain of Mycobacterium tuberculosis

SPECIAL STAINS −Involve special complicated methods not for amateurs −Used to observe special structures: –ENDOSPORES –FLAGELLA –CAPSULES