Chapter 2 - Tools of Laboratory

Light microscopy

microscopes that use visible light to observe specimens

Types of light microscopy

• compound light microscopy

• darkfield microscopy

• phase-contract microscopy

• differential interference contrast (DIC) microscopy

• fluorescence microscopy

• confocal microscopy

Compound light microscope

• series of lenses for magnification

• uses visible light

• the image from the objective lens is magnified again by the ocular lens

• total magnification = objective lens x ocular lens

Resolution

the ability to distinguish between two points

• greater resolution allows finer detail and structure to be observed

Refractive index

a measure of the light-bending ability of a medium

• the path of light may change as it passes through different mediums

• if it bends or refracts too much, it will not enter the objective lens and resolution decreases

• some mediums cause light to refract more than others

Staining

coloring microorganisms with a dye that emphasizes certain structures

• before staining microbes must be smeared and then fixed to the glass slide

Smear

created when a thin film of material containing the microorganisms is spread over the slide

Fixing microbes

• fix = attach; causes them to stick to the slide so they do not wash off

• kills organisms

• preserves various structures for observation

3 common straining techniques

simple stain, differential stain, special stain

Simple stain

• use of a single dye

• highlights the entire microorganisms to visualize cell shapes and structures

• revels cell morphology: size, shape, and arrangement of bacterial cells

Differential stain

used to distinguish between different bacterial species

Special stain

used to color structures such as flagella and capsules and distinguish specific structures of microorganisms

Simple Stain Procedure

• stain applied to fixed smear for certain amount of time and then washed off

• slide is dried and specimen examined with a microscope

Typical dyes of simple stains

crystal violet, safranin, methylene blue

Most common types of differential stains

gram stain, acid-fast statin, endospore stain

Gram stain

• classifies bacteria into two large groups: gram-positive or gram-negative

• one of the most important techniques in medical microbiology (providing valuable information for the treatment of disease)

• results allow for the selection of the proper antibiotics to fight bacterial infections

Gram-negative bacteria

have a thin peptidoglycan cell wall and an outer membrane composed of lipopolysaccharides

Gram-positive bacteria

have a thick peptidoglycan cell wall

Acid-fast stain

• dye binds to bacteria that have a waxy material in their cell walls (cell wall is not decolorized by acid-alcohol)

• used to identify bacteria in the genera (plural of genus)

Endospore stain

• endospores are resistant, dormant structures located inside some cells

• protect bacteria from adverse environmental conditions

• cannot be stained by ordinary methods

Endospore procedure

• primary stain (green), usually with heat

• decolorize cells

• counterstain (pink)

• results: spores appear green within red or pink cells

Capsule stain

• bacterial capsules are a gelatinous outer covering (not found on all bacteria, contribute to the ability of the bacteria to cause disease, pathogenicity)

• are difficult to stain (water soluble and wash away easily, do not accept most dyes)

• is a form of negative staining meaning the background is stained instead of the cell

Flagella stain

• structures used for bacterial locomotion (too small to be seen alone using light microscopy)

• uses a mordant and dye to thicken appearance of flagella, making them visible under the light microscope

Culture

microbes growing in or on a culture medium

Inoculum

microbes introduced into a medium

Culture/growth media (singular—medium)

nutrients prepared for microbial growth

Culture media is classified based on…

physical state, chemical composition, or functional type (purpose)

Culture media - physical states

liquid, solid, semisolid

Agar

• a physical state

• solidifying agent for culture media

• complex polysaccharide isolated from marine algae

• typically, not metabolized by microbes

Plates (Petri plates)

shallow dishes with a lid to prevent contamination

• useful for examining colony morphology counting colonies, and preparing pure cultures

Slants

agar solidifies while the tube is at an angle

• increases the surface area for growth

Deeps

agar solidifies in a vertical tube

• used to examine gas requirements of microbes

Chemical composition

have two main categories - chemically defined and complex

Chemically defined media

the exact chemicals and the exact amounts of each are known

Complex media

extracts and digests of yeasts, meat, or plants; chemical composition varies batch to batch

General-purpose media

• used to grow a wide range of organisms

• nutrients are often provided in excess, usually a complex media

Differential media

designed to provide visual differences between organisms (i.e. size, colony color, etc.)

Selective media

• contains chemicals that inhibit the growth of some organisms but not others

• “selects” for growth of certain organisms

Reducing media

• contains chemicals that remove oxygen from media

• used to examine O2 requirements or to grow of anaerobes

Transport media

long-term storage and transport

Assay media

antimicrobial testing

Enumeration media

counting colonies

Differential media

distinguishes between different microbe colonies growing on the same plate

Selective media

• suppress unwanted microbes and encourage desired microbes

• contains inhibitors

Colony

a population of cells arising from a single cell or spore or from a group of attached cells

Pure culture

a culture containing only the microbe of interest

Inoculation

introduction of microbes into media for growth

Inoculum

the substance used for inoculation

Inoculate

verb form; for example, 5 mL of broth was inoculated with