VMCB 121 Demonstartion of Bacteria - Stains

Advantage and Disadvantage of Unstained Bacteria

Adv: Bacteria is still alive

Disadv: Can be hard to detect with the light microscope

advantage and disadvantage of stained bacteria

adv: provides contrast in the structure

disadv: no mobility

classification: morphology and arrangement

what does fixation do

• kill the pathogenic bacteria

• attach bacteria firmly to the slide

• improve staining

two types of fixation

HEAT FIXATION

• gently flame heating an air-dried film of bacteria and adequately preserve overall morphology but not structures within the cells

CHEMICAL FIXATION

• used to protect fine cellular substructure and the morphology of larger and more delicate microorganisms

types of stain/dyes

• acidic dye

• basic dye

• neutral dye

acidic dye

anionic chromophore = negative charge

• stains basic components

basic dye

cationic chromophore = positive charge

• stains acidic component

neutral dye

• eosinate of methylene blue

types of stain

• simple stain = one dye = same color

• differential stain = two stains = diff microorganisms

• special stains = highlight specific cell structures

simple stain process

1. smear

2. fixing

3. stain

4. washing

5. air dry

6. observation

why does bacteria have an affinity to basic dyes?

due to the acidic nature if their protoplasm

differential stain process

1. smear

2. fixing

3. primary stain

4. decolorize

5. counter stain

6. air dry

7. observation

examples pf simple stai

methylene blue, crystal violet, safranin, carbol fuchsin

example of differential stain

gram stain, acid-fast stain

example of special stains

hansen’s, anthony’s, schaeffer-fulton

Gram Stain (by Hans Christian Gram 1884)

• differentiate 2 groups: gram positive and gram negative bacteria

• cell wall is composed of peptidoglycan

Gram + bacteria

• thick layer (50-90%)

• no/less lipid

• purple

Gram - bacteria

• thin layer (-10%)

• takes up safranin

• pink/red

Gram Stain Process

1. Crystal Violet - primary stain added to the smear (1min)

2. Iodine - mordant makes dye less soluble = adheres to the cell wall (1min)

3. Alcohol - decolorizer washes away stain from G-cell walls (10secs)

4. Safranin - counterstain allows dye adherence to G-cell walls (30secs)

Acid Fast

cells that retain a basic stain in the presence of acid-alcohol

other term for acid stain

ZN, Ziehl-Neelsen’s Stain

• used for those microorganisms that are not stained by simole or Gram staining methos

Myolic Acid

waxy material that composes the cell wall of acid fast materials. it does not allow the decolorizer into the cell wall

• Myobacterium

why do acid-fast bacteria retain the color of carbon fuchsin

because of mycolic acid

acid-fast staining process

1. carbol fuchsin: primary stain

2. heat: mordant

3. acid-alcohol: decolorizer

4. methylene blue: counterstain

special stain

staining procedures used to identify specific external or internal structures that are not found in all bacterial species

example or special stains

• capsule stain

• flagella stain

capsule stain

the capsule is synthesized in the cytoplasm and secreted outside the cell, where it surrounds the bacterium

methods of capsule stain

• india ink method

• anthony’s stain method

india ink method

• shows a halo around the cell

• colorless bacteria against a colored background

• cells are highly visible

why are the distortions of cell size and shape minimized in india ink method

because heat fixing is not necessary and the cells do not pick up stain

anthony’s stain method

uses specific reagents and avoids heat-fixing to properly highlight this structure

• capsule appears as a faint blue halo around a purple cell

reagents used by anthony’s stain method

• crystal violet: primary stain

• 20% of CuSO4: decolorizing agent and counterstain

endospore stain

• schaeffer-fulton method

• heat is required to drive the stain into the endospore

• spores

endospore stain process

1. stained with malachite green

2. heat for stain penetration

3. decolorized

4. counter stained with safranin

flagella staining

• demonstrates the number and arrangement of flagella, crucial for identifying motile bacteria

• coats the thin bacterial flagella with heavy metals or other compounds to make them visible in the light microscope

what is the use of silver nitrate in flagella staining

• to see bacterial flagella that are too slender

• makes flagella appear larger

• used to determine arrangement, location, number of flagella for identification