PREPARATION OF A SMEAR GRAM STAIN ACID FAST STAIN LAB: 3 PREPARATION OF A SMEAR GRAM STAIN ACID FAST STAIN

BACTERIAL SMEARS Before bacteria can be stained a smear must be made and fixed. A bacterial smear is made by evenly spreading bacteria on a clean slide in a drop of liquid (sterile water or broth).

BACTERIAL SMEARS Allow the slide to air dry and then: Heat or Chemically fix the sample to the slide: 1. Heat fix: Pass it over the incinerator to gently heat fix 2. Chemically fix: by flooding the slide with methanol and allow to evaporate (Denatures bacterial enzymes and enhances adherence of bacteria to the slide) THE SLIDE IS NOW READY TO STAIN

GRAM STAIN Developed by Hans Christian Gram in 1884 when he was studying bacteria from different respiratory diseases. The Gram Stain is a differential stain used to classify bacteria as Gram positive or Gram negative. The single most important technique in the Microbiology Laboratory.

The uptake and retaining of the basic stains depends on the cell wall: The Gram Stain The uptake and retaining of the basic stains depends on the cell wall: *Gram positive bacteria have many layers of peptidoglycan in their cell wall making it easier to hold onto the CV-I complex. *Gram negative bacteria have one layer of peptidoglycan. Thus the CV-I complex is not retained in the cell.

GRAM STAIN REAGENTS 1. Crystal violet (CV): primary basic stain 2. Grams Iodine (I): not a stain; it forms a crystal violet—iodine complex (CV-I) inside the cell wall. 3. Decolorizer: ethyl alcohol, used to remove the primary stain. It removes the CV-I complex from cells without teichoic acid. 4. Safranin: secondary (counter) basic stain

SET UP YOUR SLIDE FOR STAINING

GRAM STAIN SUCCESS DO NOT USE TOO MUCH WATER OR BROTH. A SMALL LOOPFUL IS ENOUGH. DO NOT USE A HEAVY INOCULUM (AMOUNT OF BACTERIA). MAKE AN EVEN, THIN SUSPENSION ON THE SLIDE. THIS WILL ALLOW FOR PROPER STAINING AND ESPECIALLY PROPER DECOLORIZATION.

GRAM STAIN PROCEDURE 1. Flood with crystal violet for 1 minute. Rinse 2.Flood with iodine for 1 minute. Rinse 3.Decolorize with alcohol until no more purple comes off the slide. Rinse 4. Flood with safranin. Rinse and dry.

RESULTS Gram positive Organisms will stain purple. Gram negative stain pink.

RESULTS WHAT WOULD HAPPEN IF WE: 1.) OVER – DECOLORIZE 2.) UNDER – DECOLORIZE

WHY DO WE NEED TO STAIN MICROORGANISMS???

ACID FAST STAIN Developed by Paul Ehrlich in 1882 Kinyoun published ‘cold-staining’ method in 1915, which is commonly used today Acid fast positive bacteria have fatty acid in their cell wall (mycolic acid) that makes them ‘acid fast’, i.e. do not decolorize with acid alcohol Acid Fast Stain is a differential stain used to classify Acid fast positive or Acid Fast negative microorganisms

ACID FAST STAIN The group of microorganisms that are differentiated by the acid fast stain are the mycobacteria. There are many species of mycobacteria, but the most important organism in this group is Mycobacterium tuberculosis, the causative agent of tuberculosis.

ACID FAST STAIN REAGENTS Carbol fuchsin: fuchsin is the red basic stain and carbol is the acid (carbolic acid) Acid alcohol: decolorizer (Hydrochloric acid and 95% ethanol) Methylene blue: a blue counter stain

For Acid-Fast Stain Lab your slide should look like this.

PROCEDURE – ACID FAST STAIN COLD KINYOUN METHOD ACID FAST STAIN STEPS Color of Acidfast + cells Acidfast – cells 1. Primary stain: Carbol fuchsin RED 2.Decolorizing agent: Acid alcohol COLORLESS 3.Counterstain: Methylene Blue BLUE

Acid-Fast Staining: Kinyoun Method ACID FAST POSITIVE - RED ACID FAST NEGATIVE - BLUE

Acid-Fast Staining: Kinyoun Method ACID FAST POSITIVE – RED ACID FAST NEGATIVE – BLUE

RESULTS ??? OVER – DECOLORIZE ???UNDER – DECOLORIZE

WHY DO WE USE THE ACID FAST STAIN????