MICB 1560 1 .2: Sample Preparation & Staining

Wet mount

-Add a few drops of bacteria growing in broth culture to a slide
−Add a few drops of sterile water to a slide then transfer some bacteria growing in
colonies on a plate

Smear

Thin film of a sample containing microorganisms spread over a slide

Fixation

-kills the cells by stopping cell reactions and stabilizing cell structures
-attaches cells to slide, preventing them from moving around
-heat (used in lab) or certain chemicals (ethanol, methanol, formaldehyde, etc.)

unstained samples

have low contrast with the surrounding medium
− usually consist of live cells that have not been fixed:
• live cells are used to study cell behaviour
• use darkfield or phase contrast microscopy to view unstained, live cells

stained samples

− have high contrast with the surrounding medium because a colored stain/dye has been added
− cells can be alive or dead

stain

positive chromophore ion (color)

basic dye

negative chromophore ion (color)

acidic dye

Simple Stains

-Use a single dye (most commonly a basic dye)

-Stain the entire microorganism to
visualize cell shapes and structures

-Different bacteria of the same
morphology in a sample will all appear
the same

Differential Stains

-One or two stains used

-Differentiates different bacterial cells from each other based on:

cell wall biochemical characteristics (Gram stain ,Acid-fast stain)

presence or absence of certain bacterial structures (capsule stain, endospore stain, flagella stain)

Gram Stain

-Differential stain

-Classifies bacteria into gram-positive or gram-negative based on color after staining procedure

-Color difference is a result of cell wall differences between 2 types of bacteria

-2 stains crystal violet and safranin

Gram-positive bacteria

-Appear violet/purple/blue

-Contain thick layer of peptidoglycan with some teichoic acids

-Less lipid layers (no outer membrane)

-e.g., Staphylococcus aureus, Streptococcus
pyogenes, Bacillus cereus

Gram-negative bacteria

-Appear pink/red

-Contain thin layer of peptidoglycan with no teichoic acids

-More lipid layers (outer membrane present)

-e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium

Gram stain steps

1. Primary stain- crystal violet
2. Mordant (fixative)- iodine
3. Decolorizing agent- 95% ethanol (alcohol)
4. Counterstain- safranin

Wash with water between each step

Gram variable

Both pink and purple cells observed with a pure culture (one bacterial species)