Gram Staining and Bacterial Isolation

a fundamental microbiology technique used to obtain pure cultures of bacteria from a mixed sample

Bacterial Isolation

what is the goal of Bacterial Isolation?

separate out individual bacteria species

a thick, uniform layer of bacterial growth that covers the surface of an agar plate

a lawn of bacteria

when does a lawn of bacteria occur?

when a high concentration of bacteria is spread evenly across the plate

what color will the lawn of bacteria be?

cloudy

what materials are needed for Bacteria Isolation?

media, bacterial sample, inoculating loop, bunsen burner, incubator, permanent marker, parafilm

these are food sources used to grow the bacteria on (agar plate is most commonly used)

media

it transfers bacteria from source to plate

inoculating loop

sterilizes your loop using high heat

bunsen burner

Why do we use a bunsen burner for Gram staining?

to heat-fix the bacterial smear to the slide

where plates are kept to facilitate growth

incubator

this marks plates

permanent marker

helps seal plates

parafilm

essential for preventing contamination of both the sample and the surrounding environment

clam shell technique

a technique that allows you to distinguish between different types of microorganisms or different parts of a cell based on how they absorb stains

differential stain

thick peptidoglycan cell wall, stain purple, and are generally treatable with antibiotics

gram positive cells

a substance composed of carbohydrates and protein subunits

peptidoglycan

thin peptidoglycan cell wall + outer LPS layer, stain pink, and are more difficult to treat with antibiotics

gram negative cells

it makes the species difficult to treat with antibiotics, released as a toxin when gram negative cells die

LPS

what color is gram negative bacteria?

pink

a type of chemical substance, either naturally produced by microorganisms or synthetically made, that is used to kill or inhibit the growth of bacteria

antibiotic

it enters the peptidoglycan of all bacteria, and gives the bacteria a purple color. Primary stain

crystal violet

this combines with the crystal violet to make a big complex in the peptidoglycan wall

gram’s iodine

this is the most important step. a decolorizer, it removes the crystal violet/iodine complex from the thin peptidoglycan layers of gram negative cell walls but not thick layers from gram positive

Ethanol

what color is gram positive bacteria?

purple

this dye floods both cells. the gram positive cells remain purple since it is darker than the pink. counterstain

safranin

What are the 4 main steps of Gram staining?

Applying a primary stain (crystal violet). Adding a mordant (Gram's iodine). Rapid decolorization with ethanol. Counterstaining with safranin

What is “clam-shell” technique, and how does it help avoid contamination?

its when you open the dish like a clam to avoid contamination by shielding it