3. Lab Techniques

complex media

media where we don’t know the exact composition of it

chemically defined media

we know exactly each component of the media

selective & differential media

MSA

Numerical apeture

the light collecting ability of an optical system

brightfield microcope

white background, commonly/widely used. Compatible with different staining but difficult to see colorless sample.

darkfield microscope

dark background. Does not require staining (live samples). Limited visualization of internal cellular structure.

phase-contrast microscope

Neutral background color. Does not require staining and used for visualizing internal cellular structure. May not be compatible with staining

Basic dyes

dyes that are positive at ph 7 ex. methylene blue

acidic dyes

dyes that are negative at ph 7 ex Nigrosin

simple staining

use of a single basic dye

negative staining

use of a single acidic dye that stains the background. It does not affect shape because does need a heat fix.

Steps for Gram staining

Primary dye: Crystal violet
Mordant: Gram’s Iodine
Decolorizing: 95% etoh
Water Rinse
Counter stain: safranin
results in purple gram positive and pink gram negative cells

gram positive cell

a cell with a single layer of plasma membrane and a very thick cell wall

gram negative cell

a cell with two plasma membrane and a thin cell wall

Differential staining

staining that is used to differential parts or organisms

Endospore staining

differential stain that highlights endospore from the rest of bacterial cell

moist heat

a type of mordant that is used to push dye into cells through hot vapor

Endospore staining steps

primary dye: malachite green
mordant: moist heat
Decolorizer: water
counterstain: safranin

Acid fast staining

a differential stain that is used to identify bacteria that mycolic acid because the waxy cell wall prevents dyes from binding

Acid Fast staining steps

primary dye: carbol Fuchsin (purple)
mordant: moist heat
decolorizer: acid alcohol
counterstain: methylene blue

Structural Staining

Staining used to identify the structure of the cell

Capsule stain

A structural stain that combines simple stain and negative stain

steps of capsule stain

Primary Dye: nigrosin
Air dry and heat fix
counter stain: crystal violet as simple stain

cultural morphology

shape of culture growth

Flagella staining

assessing the presence of flagella

semisold media

Used to identify if a cell has flagella by testing motility