How will the cell surface of the bacteria react to a negatively charged
dye?
Repels it (negative charged cell surface)
Differential Staining:
Negative, Endospore, Capsule, Flagella
Negative Staining
Uses a dye solution in which the chromogen is acidic and carries a negative charge!
Negative Stain Procedure
- Label slides with organism’s name!
- Add a drop of nigrosin stain to one
end of slide - Aseptically add bacteria with loop
– Sterilize loop following - Take a second slide and place it on
surface of first - Draw slide back to the drop
- Get liquid to flows across width of
spreader slide - Push the spreader slide to other end
- Allow to air dry (Do NOT heat!)
Acid-Fast Staining
Used to identify bacteria that contain mycolic acids in their cell walls
Mycolic acid
Mycolic acid is a waxy substance
– Gives cells a higher affinity for primary stain
– Gives resistance to decolonization by an acid-
alcohol solution
– Repels water-based stains (only weakly Gram +)
Acid-Fast Stain Colors
- Acid-fast cells: Fuchsia
- Non acid-fast cells: Green or blue
Capsule Stain
- Capsule production increases the virulence in some microbes
– Makes them less vulnerable to phagocytosis - Capsules are composed of mucoid
polysaccharides or polypeptides (extra outer layer) that repel most stains - the capsule stain technique stains around the cell
Typically use two stain types:
* Acidic Stain (background): Congo red or nigrosin
* Basic Stain (colorizes cell proper)
Bacterial Flagella
- Flagella are used by bacteria for mobility (rotate like oars of a rowboat)
- Flagella vary in number and arrangement depending on the bacteria
- Monotrichous ( 1 polar)
- Amphitrichous (1 each end)
- Lophotrichous (multiple from one or - both end)
Peritrichous (from all surfaces)
Endospores
- An endospore is a dormant form of a bacterium
– Allows it to survive in harsh environmental conditions
– Resistant to heat and chemical staining from outer covering comprised of the protein keratin
Endospore morphology
Location: Terminal (end), Subterminal (close to end), Central (middle)
Shape: Spherical, Elliptical (oval)
Size: Deforming (cell can look swollen)
Endospore Stain
- Uses a primary stain called malachite green
– Steam bacterial emulsion to force into spore
– Leave on slide for 15+ minutes - Malachite green is a water-soluble dye
– Has a low affinity for cellular material
– Vegetative and spore mother cells can be decolorized with water (spore retains color) and counter stained (safranin)
Cell Morphology
Coccus (plural: cocci) – Spherical or round shape
Examples: Staphylococcus aureus, Streptococcus pneumoniae
Bacillus (plural: bacilli) – Rod-shaped
Examples: Escherichia coli, Bacillus subtilis
Vibrio – Comma-shaped or curved rod
Example: Vibrio cholerae
Spirillum (plural: spirilla) – Rigid spiral shape
Example: Spirillum volutans
Spirochete – Flexible, corkscrew-like shape
Example: Treponema pallidum (causes syphilis)
Coccobacillus – Short, oval rods (between cocci and bacilli)
Example: Haemophilus influenzae
Cell Arrangement
treptococci – chains of cocci (e.g., Streptococcus pyogenes)
Staphylococci – clusters of cocci (like grapes; e.g., Staphylococcus aureus)
Diplobacilli – pairs of rods
Streptobacilli – chains of rods
Palisades – rods lined up side-by-side like a fence
