Microscopy and Culturing Microorganisms Techniques

Inoculation

Adding a small sample (the inoculum) into a container of medium.

Isolation

The separation of different microorganisms from each other.

Colonies

Visible masses of cells formed as microbes grow; if formed from a single cell, each cell in the colony will be genetically identical (pure culture).

Incubation

Keeping the sample between 20° and 40°C to allow growth.

Pure culture

A culture that has a single known species, also called axenic.

Mixed culture

A culture that has two or more identified species.

Contaminated cultures

Cultures that have unwanted microorganisms of uncertain identity (contaminants).

Streak Plate Method

A method involving serial dilution on a solid surface to obtain isolated colonies.

Spread Plate Method

A method where dilutions are performed beforehand and a small volume (e.g., 0.1 ml) is spread out on the plate surface.

Pour Plate Method

A method where cultures are diluted beforehand in liquefied agar at 50 to 55°C and poured into plates.

Types of Media

Media can be classified by physical state, chemical composition, and functional type.

Physical state of media

Can be liquid, solid, or semi-solid.

Defined Media

Media where the chemical composition is precisely known (synthetic), e.g., 0.1 M sodium phosphate, pH 7.0.

Complex Media

Media where one or more components is not chemically definable (nonsynthetic), e.g., 5% yeast extract.

General purpose media

Media designed to grow as broad a spectrum of microbes as possible, usually non-synthetic.

Enriched Media

Media that contain complex organic substances (growth factors) to support the growth of fastidious bacteria.

Selective media

Media that allows some organisms to grow but not others.

Differential media

Media that allow many microorganisms to grow but display visible differences (usually colors).

Inspection and Identification

Using appearance, metabolism (biochemical tests), and sometimes genetic analysis or immunologic testing to identify organisms in a culture.

Stock cultures

Cultures that can be maintained for future use.

Sterilization of cultures

The process of properly sterilizing and destroying cultures that are no longer in use.

Microscopy

The study of using microscopes to view small objects.

Resolution

Ability to distinguish detail and structure in microscopy.

Numerical Aperture

Lens's ability to gather light for better resolution.

Contrast

Difference in intensity between two objects in microscopy.

Compound Microscope

Microscope with ocular and objective lenses for magnification.

Total Magnification

Power of Ocular Lens × Power of Objective Lens.

Brightfield Microscopy

Standard microscopy illuminated by white light.

Darkfield Microscopy

Specimens appear light against a dark background.

Phase-Contrast Microscopy

Enhances light and dark areas of unstained specimens.

Fluorescence Microscopy

Uses UV light to observe fluorescing specimens.

Immunofluorescence

Uses antibodies with fluorescent dyes to stain organisms.

Confocal Microscopy

Uses laser light to create 3D images of specimens.

Transmission Electron Microscopy (TEM)

Uses electron beams to magnify specimens 10,000X to 100,000X.

Scanning Electron Microscopy (SEM)

Illuminates specimens with electron beams for surface views.

Staining

Adding color to specimens to enhance contrast.

Simple Stains

Use one dye or one step for staining.

Differential Stains

Use multiple dyes and steps to distinguish cell types.

Gram Stain

Differential stain distinguishing Gram + and Gram - bacteria.

Acidic Dyes

Negative ion chromophore, binds to positively charged surfaces.

Basic Dyes

Positive ion chromophore, binds to negatively charged structures.

Smear

Thin layer of cells spread on a slide.

Fixation

Method to kill and adhere cells to slides.

Chromophore

Colored ion in a dye that provides color.

Mordant

Substance that increases dye interaction with specimens.

Ameba

Single-celled organism, often studied in microscopy.

Colonial Alga (Pediastrum)

Microscopic plant-like organism forming colonies.