Transcript Classifying Microbes

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