LABORATORY TECHNIQUES AND METHODS

Microscopy

use of light and electrons to magnify objects

magnification

the apparent increase in size of an object

a. thickness of the lens

b. curvature of the lends

c. speed of light

The image enlarged depends on the following

Resolution or Resolving Power

- ability to distinguish objects as close as 0.2 micrometer (200nm max)

- human eye can see objects as small as about 0.1mm (100um)

0.2 micrometer (200nm max)

ability to distinguish objects as close as

0.1mm (100um)

human eye can see objects as small as about

a. shorter wavelength

b. greater numerical aperture

Better resolution are due to

the ability to gather light

greater numerical aperture

Resolution Distance

= 0.61 X wavelength

NA

Resolution or Resolving power

Contrast

different intensity between two objects or its

background

Par focal

ability of the microscope to stay in focus even you shift from different objective

Working Distance

distance between the lens and the specimen

Total magnification

the number of times the object is enlarged from its original size

objective lens x ocular lens

Total magnification formula

Gamma rays

X-days

UV light

Infrared

Microwave

Radio waves and Television

Visible light

Ocular lens

Remagnifies the image formed by the objective lens

Body

Transmits the image from the objective lens to the ocular lens using prisms

Objective lenses

Primary lenses that magnify the specimen

Stage

Holds the microscope slide in position

Condenser

Focuses light through specimen

Diaphragm

Controls the amount of light entering the condenser

Illuminator

Light source

Coarse focusing knob

Move the stage up and down to focus the image

Light microscopes

Useful magnification 1x to 2000x; resolution

to 200nm

Light microscopes

Use visible light; shorter, blue wavelengths provide better resolution

Bright-Field

Colored or clear specimen against bright background

Bright- Field

Simple to use; relatively inexpensive; stained specimens often required

Dark- Field

Bright specimens against dark background

Dark- Field

Use a special filter in the condenser that prevents light from directly passing through a specimen; only light scattered by the specimen is visible

Bright- Field

Observation of killed stained specimens and naturally colored live ones; also used to count microorganisms

Dark- Field

Observation of living, colorless, unstained organisms

Phase- Contrast

Specimens has a light and dark areas

Phase- contrast

Use a special condenser that splits a polarized light b e a m into two beams, one of which passes through the specimen, and one of which bypasses the specimen; the beams are then rejoined before entering the oculars; contrast in the image results from the interactions of the two beams

Differential Interference Contrast (Nomarski)

Image appears three dimensional

Differential Interference Contrast ( Nomarski)

Use two separate b e a m s instead of a split beam; false color and a three - dimensional effect result from interactions of light b e a m s and lenses; no staining required

Differential Interference Contrast (Nomarski)

Observation of internal structures of living microbes

Fluorescent

Brightly colored fluorescent structures against dark background

Fluorescent

An ultraviolet light source causes fluorescent natural chemicals or dyes t o emit visible light

Fluorescent

Localization of specific chemicals or structures; used as an accurate and quick diagnostic tool for detection of pathogens

Confocal

Use a laser to fluoresce only one plane of the specimen at a time

Confocal

Detailed observation of structures of cells within communities

Bright- Field

Dark- Field

Phase- Contrast

Differential Interference Contrast (Nomarski)

Fluorescent

Confocal

Light microscope

Transmission

Scanning

Electron Microscopes

Electron Microscopes

Typical magnification 1000x to 100,000x;

resolution to 0 . 0 0 1 n m

Electron microscopes

Use electrons traveling as waves with short wavelengths; require specimen to be in a vacuum, so cannot be used to examine living microbes

Transmission

Monotone, two- dimensional, highly magnifies images; may be color- enhanced

Transmission

Produce two-dimensional image of ultrastructure of cells

Transmission

Observation of internal ultrastructural detail of cells and observations of viruses and small bacteria

Scanning

Monotone, three-dimensional, surface images; may be color enhanced

Scanning

Produce three-dimensional view of the surface of microbes and cellular structures

Scanning

Observation of the surface details of structures

Scanning Tunneling

Atomic Force

Probe Microscopes

Scanning Tunneling

Individual molecules and atoms visible

Scanning tunneling

Measures the flow of electrical current between the tip of a probe and the specimen to produce an image of the surface at atomic level

Scanning tunneling

Observation of the surface of objects; provide extremely fine detail , high magnification, and great resolution

Atomic force

Individual molecules and atoms visible

Atomic force

Measure the deflection of a laser beam aimed at the tip of a probe that travels across the surface of the specimen

Atomic Force

Observation of living specimens at the molecular and atomic levels

Working areas

should be disinfected before and after every activity.

mechanical pipettes

Use mechanical

Eating, drinking and applying cosmetics

are prohibited inside the laboratory.

Wash hands

Wash _ before and after the activity

Routine disinfection

(liquid/powdered soaps)

Rapid disinfection

(50-70% alcohol for 20-30 seconds; soap scrub for 10-15 sec.)

50-70% alcohol

20-30 secs

contaminated materials

Disinfect all

chlorox or 0.5 % hypochlorite or household bleach

Decontaminate laboratory coats with

Staining

artificially coloring the organism with the use of different dyes and reagents

a. to appreciate more of the appearance and morphology of the organism

b. to differentiate one group of organism to another group of organism

c. to identify the organism by staining their special structures

Functions of staining:

simple stain

uses a single dye

Crystal violet

Methylene blue

Examples of simple stain

Uniform purple stain

Uniform blue stain

Results in simple stain

Simple stain

Reveals size, morphology, and arrangement of cells

Differential stains

(use two or more dyes to differentiate between cells or structures)

Gram stain

Ziehl-Neelsen acid- fast stain

Schaeffer-Fulton endospore stain

Examples of differential stain

Gram- positive cells are purple

Gram- negative cells are pink

Results in differential stain

Differential stains

Differentiates between Gram-positive Gram- negative bacteria, which is typically the first step in their identification

Pink tor red acid-fast cells and

blue non-acid- fast cells

Results in Ziehl-Neelsen acid- fast stain

Ziehl-Neelsen acid- fast stain

Distinguishes the genera Mycobacterium and Nocardia from other bacteria

Schaeffer-Fulton endoscope stain

Green endoscopes and pink to red vegetative cells

Schaeffer-Fulton endoscope stain

Highlights the presence of endoscopes produced by species in the genera Bacillus and Clostridium

Negative stains for capsules

Background is dark, cells unstained or stained with simple stain

Negative stain for capsules

Reveals bacterial capsule

Flagellar stain

Bacterial flagella become visible

Flagellar stain

Allows determination of number and location of bacterial flagella

Simple

(methylene blue, carbolfuchsin, crystal violet, safranin)

Simple

Aqueous or alcohol solution of a single basic dye. (Sometimes a mordant is added to intensify the stain.) Used to highlight microorganisms to determine cellular shapes a n d arrangements.

Gram

Acid- fast

Differential

Gram

React differently with different kinds of bacteria in order to distinguish among them. Classifies bacteria into two large groups: gram-positive and gram-negative. Gram-positive bacteria retain the crystal violet stain and appear purple. Gram-negative bacteria do not retain the crystal violet stain and remain colorless until counterstained with safranin and then appear pink.

Acid-fast

Used to distinguish Mycobacterium species and some species of Nocardia. Acid-fast bacteria, once stained with carbolfuchsin and treated with acid-alcohol, remain red because they retain the carbolfuchsin stain. Non-acid-fast bacteria, when stained a n d treated the same way and then stained with methylene blue, appear blue because they lose the carbolfuchsin stain and are then able to accept the methylene blue stain.

Gram-positive bacteria

retain the crystal violet stain and appear purple.

Gram-negative bacteria

do not retain the crystal violet stain and remain colorless until counterstained with safranin and then apear pink.

Acid- fast bacteria

once stained with carbolfuchsin and treated with acid-alcohol, remain red because they retain the carbolfuchsin stain.

Non-acid-fast bacteria

when stained a n d treated the same way and then stained with methylene blue, appear blue because they lose the carbolfuchsin stain and are then able to accept the methylene blue stain.

Special

Used to color and isolate various structures, such as capsules, endospores, and flagella; sometimes used as a diagnostic aid.

Negative

Used to demonstrate the presence of capsules. Because capsules do not accept most stains, the capsules apear as unstained halos around bacterial cells and stand out against a dark background.

Endospore

Used to detect the presence of endospores in bacteria. When malachite green is applied to a heat-fixed smear of bacterial cells, the stain penetrates the endospores and stains them green. When safranin (red) is then applied, it stains the remainder of the cells red or pink.

malachite green

When _ is applied to a heat-fixed smear of bacterial cells, the stain penetrates the endospores and stains them green.

safranin (red)

When _ is then applied, it stains the remainder of the cells red or pink.