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 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 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 Cytoplasmic membrane

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 3 layers A 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+

Why Gram Stain? to prescribe treatment with an appropriate antibiotic while waiting for more specific tests, such as a culture Absence or presence of white blood cells white blood cells are frequently present with infection determine type of bacteria and what other tests may need to be performed to definitively identify the cause of infection

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

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

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

B. Arrangements 1.Staph: Irregular or clusters *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..