Classifying and identifying Microbes

Slide preparations for bacteria A. Living preparations *Hanging drop method ** see Bacteria alive in their environment. – This also allows us to determine if specimen is motile or nonmotile.

B. Fixed Stained Smears 1.Smear – spread thin film of bacteria on slide -Air dry -Then gentle heat “fixes” bacteria to slide – heat kills bacteria *Creating smears of “fixed” bacteria cells is the first step in staining.

2. Staining – adding dyes to smears *Types of Staining Techniques a. Negative staining- dyes stick to boundary around cell forming a silhouette.

b. Positive Staining – dyes stick to cells and colors them *Types of Positive Staining 1) Special Stains- These emphasize certain cell parts (Flagella Stain)

2)Simple Stains- require dye only -All cell types are the same color. Examples: Crystal Violet, Safranin, Methylene blue & Carbolfuchsin

3. Differential Stains – Use two different dyes. -Primary dye and Counterstain *Different bacteria groups stain different colors **Examples: Gram Stain, Acid-Fast, and Spore stain

***The Gram Stain (Differential) a)Hans C. Gram did this in 1884 b) 4 Step stain technique that stains bacteria pink or purple. c)Pink= Gram Negative G-) d) Purple= Gram Positive G+) e) The 2 different color result from 2 different type of bacterial cell walls (different structures)

Gram Positives and Gram Negatives: Key Differences G+ have simpler, thicker walls, large amount of peptidoglycan G- are thinner and have less peptidoglycan but more complex in structure An outer membrane on the Gram negative cell wall contains lipopolysaccharides (LPS) – toxic substances responsible for making Gram negative organisms more threatening than Gram positives

f) Gram + (Purple) 1. Cell wall has thick Peptidoglycan Layer (PG) 2. Hold primary stain (purple) during alcohol rinse (decolorization phase)

Gram Positives Two layers: Outer cell wall - thick peptidoglycan layer, composed of complex cross-linked peptidoglycan, teichoic acid, polysaccharides and other proteins Cytoplasmic membrane - contains proteins that span the lipid bilayer Special Components of Gram Positive Bacteria Teichoic Acids – anchor the outer cell wall to the cytoplasmic membrane by attaching to glycolipids – also act as antigenic determinants (important for ID of Gram+ organisms)

Gram + Diagram Gram - Diagram

g) Gram – (Pink) 1. Cell wall has thin PG layer + big space between PG –CM (Cell membrane) 2.PG layer breaks up during alcohol rinse, Leaking primary stain out 3.Picks up Pink Counter stain

Gram Negatives The Gram negative cell envelope has 3 layers (not including the periplasmic space): A unique outer membrane A thin peptidoglycan layer Cytoplasmic membrane Special Components of Gram negative Bacteria Lipopolysaccharide (LPS) – major toxins of pathogenic Gram negative bacteria – When the cell dies, LPS are released and can cause problems with organs or tissues

h) Gram stain is important in Bacteria taxonomy

i) Some antibiotics work against Gram- other antibiotics work against Gram+

Bacterial Shapes and Arrangements A.Shapes 1.Coccus – Spherical shape -Cocci = plural 2. Bacillus – rod-shaped

3. Vibrio – Curved rods (Cholera) 4. Spirillium – corkscrew shape, rigid 5.Spirochete – Flexible, like a slinky 6. Neisseriae – Bean shape in pairs - Like in stoma

Coccus Coccobacillus Bacillus Vibrio Spirillum Spirochete Pleomorphic Flexible Stiff Slightly curved rods

B. Arrangements *Bacteria vary in the way they detach(Cleave) after cell division 1.Staph: Irregular or clusters - Example: Staphlococcus 2. Strep: Chains – Example: Streptobacillus

3. Diplo: Pairs – Example: Diplococci 4. Tetrads – Groups of 4 5. Sarcina – cubes of 8, 16..