Two -methods are generally used, (wet method) and (dry and fix method ) for Studying Microbes with a Compound Microscope

Wet Method: There are two primary methods generally used for studying microorganisms in wet conditions

wet mount method

hanging drop method.

The wet mount

A drop of fluid containing microorganisms.

The fluid spreads out in a thin layer between coverslip and slide.

The hanging Drop Method

It is used to observe the motility, germination, or fission of microorganisms.

Dry and fix method

Microorganisms, particularly bacteria, being too small need their permanent preparation be made by drying and fixing them on clean slide with or without staining.

For preparing a dry mount:

A drop of distilled water with a small amount of culture is spread as a thin smear on a clean slide. The smear is allowed to dry and it is then ‘fixed’ by passing it through a flame two to three times with the smeared slide away from the flame. If desired, this dried and fixed amount may be stained and the preparation dried again for observation under the microscope.

Bacterial Shapes :

Bacteria have three basic shapes:

Round cells (Cocci) , Rod-shaped cells are bacilli , spiral-shaped cells

Types of stain

Chromophore

Positive: basic stain

Attaches to cell wall and bacterium appears colored

Direct Stain

Negative: acidic stain

Repelled from cell wall; stains background

Negative Stain

Simple stains

To observe basic external structures of cell with brightfield scope (cellular morphology)

Reagent: Methylene Blue

Smears

Heat Fixing

Kill

Stops autolysis

Adherence to slide

Air dry first to prevent lysis (boiling)

Use of a single basic dye is called a simple stain.

A mordant may be used to hold the stain or coat the specimen to enlarge it.

The Gram stain classifies bacteria into gram-positive and gram-negative.

Gram-positive bacteria tend to be killed by penicillin and detergents.

Gram-negative bacteria are more resistant to antibiotics.

Gram Stain

Purpose

To view cellular morphology

Diagnostic purposes: What type of cell wall a bacterium has

Gram Positive

Thicker peptidoglycan layer and no outer membrane

Gram Negative

Thinner Peptidoglycan and an out membrane

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