Microbiology Lab 11: Staining of Microorganisms

smear

thin dry film of organisms

process of staining

smear
air dry
heat fixation or any fixation
staining

solid culture, agar culture inoculum

add water

why should the sample be thoroughly dried before fixation?

might boil and destroy the cell

purpose of fixation

kills organisms
binds to the slide

heat fixation

pass through2-3x
do not scorch
should not burn
film side up

chemical fixation

smear with 95% ethyl

milk samples and alcohol treatment

95% alcohol smear, 1 minute then drain

milk curdling

sign of poor smearing

effect of coagulation of protoplasm

cell appears bigger and thicker, visually more appalling

stain reveal what

chemical nature ++ other structures

Basic Dyes

cations
attracts negatively charged cells

Examples of Basic Dyes

Methylene blue
Basic Fuschin
Crystal Violet
Safranin
Malachite Green

Acidic Dyes (Negative Staining)

anions
binds with the background

Examples of Acidic Dyes (Negative Staining)

Nigrosin
India Ink

Kinds of Staining

Simple Staining
Differential Staining
Structural Staining

different types of strucutral staining

Endospore
Flagella
capsule

Simple Staining

to visualize morphology
Positive ang Negative Staining

Differential Staining

More than 1 dye is used
differentiates cells based on cell wall composition and chemical nature

process of gram staining

Crystal Violet --> primary staining
Iodine --> mordant
Decolorizer --> Alcohol (20 seconds only)
Safranin --> Counter staining

different colors of cells during gram staining

Violet Vilolet
Violet Violet
Colorless Violet
pink Violent

mordant

causes the primary staining to PENETRATE and ADHERE to the organism

Iodine solution

adheres the crystal violet , traps the dye
PARTICLES GET BIGGER

What does Decolorizer do?

dissolves primary staing for G-

why does G+ resist discoloration?

because of its thick peptidoglycan layer

E.coli

used mostly for gram staining

osmotic lysis

bursting of the cell membrane
prevented by the peptidoglycan layer

charge of the peptidoglycan layer

negative

G+ cell wall

has thicker peptidoglycan layers
has no outer membrane

Peptidoglycan

rigid envelope surrounding the cytoplasmic membrane of most species
it preserves and prtects the morphoology

2 parts of Peptidoglycan G+ bacteira

Teichoic Acid (anionic, attract cations)
Lipoteichoic acid (provides protection against cationic antimicrobial particles)

G- bacteria

has thinner peptidoglycan
has outer membrane called Lipolyssacharides

endospores

resistant, dormant spore formed, cannot be penetrated by staining

resistant to heat, chemicals and radiation
dormant stage of bacterial life cycle

stains involved in endospore staining

Malachite green (endospore)
Safranin (Vegetative part)

process of endospore staining

malachite green
Heat (mordant)
water (decolorizer)
Safranin 0.5%

how is malachite green forced into the cell

steaming bacterial emulsion
5 mins

flagella

20 nm thick hollow tubes
enables bacteria to overcome topographies
very fragile and sensitive to chemicals
uses mordant to increase in diameter --> used before staining

what does it mean when cells have heavy capsules?

virulent (capable of carrying diseases)
it protects bacteria from phagocytotic activities of the cell

why does capsule staining doesn't allow heat fixation

may cause shrinkage and cause a halo might be mistaken for the capsule

Capsule staining

acidic background
basic dyes for the cell

Anthony's method

Capsule staining with no mordant
halo purple cell

process of Anthony's method

1. crystal violet primary stain 1%
2. copper sulfate decolorizing agents and secondary stain 20%

Tannic Acid

mordant for flagella staining