1. Classifying and identifying Microbes

2. 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.

3. 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.

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

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

6. 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

7. 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

8. ***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)

9. 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

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

11. Gram Positives Two layers: Outer cell wall - thick peptidoglycan layer
Cytoplasmic membrane

12. 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

13. 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

17. h) Gram stain is important in Bacteria taxonomy

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

19. 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

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

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

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

25. 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

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