MICRO: Lab 2: Staining Techniques

Microbial Staining

•  giving colour to microbes

Microbes

• colourless and highly transparent structures

Staining

process in which microbes are stained

Stains/dyes

• organic compounds which carry either positive charges, negative charges, or both

Basic Stain

•  stain with +ve charge

Acidic Stain

• stain with -ve charge

Neutral Stain

•  stain with both charges

Simple staining

• only one dye used; differentiation among bacteria impossible

Differential staining

 more than one dye used; differentiation among bacteria possible

Gram staining, Acid fast staining

Examples of differential staining

Special staining

More than one dyes used; special structures seen

Capsule staining, Spore staining

Examples of Special staining

Basic Dye

• To stain -ve charged molecules of bacteria 

Basic stain

Mostly used because cell surface is -ve charge.

Acid Stain

• To stain +ve charged molecules of bacteria.

Acid stain

• Used to stain the bacterial capsules.

Inoculating loops

to transfer bacterial suspension to slide

Bunsen burner

•  to sterilise inoculating loops before and after smear preparation

Pencil marker

to mark central portion of slide where bacterial smear is applied

Simple Stain

Simple to perform – only one basic stain used

Hans Christian Gram 1880

Gram staining is developed by

Gram staining

Difference due to cell wall composition

Gram Staining

• Based on reaction, bacteria classified into Gram positive and Gram negative

Crystal violet, iodine, alcohol, safranine

Requirements – Staining Reagents

Crystal violet

Primary Stain

Gram’s iodine

1. mordant/fixative

Acetone (95%)

Decolorizer

Safranine/dilute carbol fuchsin

Counterstain

Crystal violet

1. all bacteria appear violet

Iodine

1.  forms Crystal Violet-Iodine (CV-I) complex

Acetone

bacteria with high lipid content lose CV-I complex (colourless); bacteria with less lipid retain CV-I (violet)

Safranine dilute carbol fuschin

 colourless bacteria take stain (pink)

Purple

• Gram positive

Pink

• Gram negative
  
  

Cocci

Spherical

Bacilli

Rod

Staphylococci

Cocci in clusters

Streptococci

Cocci in chains

Staphyloccosi

Gram positive cocci in clusters

Streptococci

Gram negative cocci in chains

Neisseria

Gram negative cocci

E. coli, K. pneumoniae

Gram negative bacilli

Ziehl neelsen staining

• Acid-fast mycobacteria contain mycolic acid in outer membrane → waxy, resistant to aqueous stains (e.g., Gram stain)

Carbol fuchsin

Primary stain of ziehl neelsen staining

Acid alcohol

Removes excess stain ziehl neelsen staing

Methylene blue

Secondary stain in ziehl neelsen staining

Mycolic acid

waxy, resistant to aqueous stains (e.g., Gram stain) for colorblind workers staining with ziehl neelsen

Picric acid

yields yellow background

Kinyoun modification

• Used for identification of acid-fast Mycobacterium spp. and parasites (Cryptosporidium, Isospora spp.)