Microbiology Lab Handout-1 Smear Preparation, Staining

Smear: a sample of bacteria placed on a microscope slide and air dried.

Heat fixing: treatment of smear which kills cells by denaturing proteins, and adhering the cells to the slide, such that the cells will not be washed from the slide during staining procedures.

Bacterial cells have a net negative charge Why? Smear preparation kills the cells and renders the entire cell permeable to anything applied to the smear. All those bacterial “guts” have a lot of negatively charged molecules (especially DNA and RNA)

Chromophore: the color producing compound of the stain. Can be prepared at different pH to create direct (basic) or negative (acidic) stain.

If the dye used in the staining procedure has a positive charge (basic stain) it will color the cell and is called a DIRECT STAIN.

If the dye used has a negative charge (acidic stain) it will be ionically repulsed from the cell, will stain the background only, and is called a NEGATIVE STAIN.

Simple stain: one stain only

Differential Stain: a staining procedure which allows classification of different types of bacteria, or stains different structures within a single cell.

Gram stain: a differential stain

Steps in the Gram stain:

1. Primary stain = crystal violet

all bacteria stained purple by this basic stain

2. Mordant = Gram’s iodine

Iodine (I) combines with crystal violet (CV) to form a water insoluble CV-I complex

3. Decolorizing = alcohol

Alcohol strips the outer membrane (G-) and shrinks the peptidoglycan layer. If the peptidoglycan layer is thick (G+), CV-I is trapped inside. If it is thin (G-), CV-I is washed away

4. Counterstain = safranin

All bacteria stained pink/red by this basic stain. Only G- cells appear red, since the CV-I has washed away. G+ cells still appear dark blue/purple from the CV-I.

For consistency….use fresh cells from a broth! Why? Old cultures have dead cells which have degraded peptidoglycan layers. Whereas a live Gram + cell will be colored purple/blue, a dead one may stain pink/red instead.

Other differential/compound stains: (compound is more than one stain, as compared to a simple stain.)

Endospore stain: Heating with malachite green drives stain into endospores, and is not washed away. Vegetative cells are counterstained with a red basic stain.

Acid fast stain: Mycobacterium (example, Mycobacterium tuberculosis, the causative agent of tuberculosis) has a waxy cell envelope. The acid fast stain is a differential stain similar to the Gram stain, as cells with waxy envelopes will resist decolorization by acid-alcohol.