(2) MICROBIO: Laboratory Techniques and Methods

microscopy

- use of light and electrons to magnify objects

magnification

- the apparent increase in size of an object

- ex. scanner = 4x

- the image enlarged depends of the following:

a. thickness of the lens

b. curvature of the lens

c. speed of light

a. thickness of the lens

b. curvature of the lens

c. speed of light

- the magnification of an object depends on these

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 0.1 mm (100um)

- better if there is:

a. shorter wavelength

b. greater numerical aperture (the ability to gather light)

a. shorter wavelength

b. greater numerical aperture (ability to gather light)

- better resolution are due to these

resolution distance

- the smallest distance between two points or objects that can be distinguished as separate entities by an imaging system

- (0.61 x wavelength) / NA or numerical aperture

lambda (wavelength) / 2 x NA (numerical aperture)

formula for resolution or resolving power

contrast

- different intensity between two objects or its background

- in microbiology, it is staining

par focal

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

working distance

- distance between the lens and the specimen

total magnification

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

- actual size

formula: objective lens x ocular lens

- ex. scanner = 40x

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

fine focusing know

- used for making small, precise adjustments to the focus after the initial coarse focus has been achieved

arm

- supporting the microscope head (or body) and acting as a handle for carrying the instrument

base

- provides stability and support for the entire instrument

light microscope

- useful magnification 1x to 2000x

- resolution to 200 nm

- use visible light

- shorter, blue wavelengths provide better resolution

bright field microscope

- simple to use

- bright background

- relatively inexpensive

- stained specimens often required

- observation of killed stained specimens and naturally colored live ones

- also used to count microorganisms

dark field microscope

- dark background

- use a special filter in the condenser that prevents light from directly passing through a specimen

- only light scattered by the specimen is visible

- observation of living, colorless, unstained organisms

phase contrast microscope

- specimen has dark and light areas

- use a special condenser that splits a polarized light beam 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

- observations of internal structures of living microbes

Differential Interference Contrast (nomarski) Microscope

- image appears thee-dimensional

- use of two separate beams instead of a split beam

- false color and a three dimensional effect result from interactions of light beams and lenses

- no staining required

- observation of internal structures of living microbes

fluorescent microscope

- brightly colored fluorescent structures against dark background

- an ultraviolet light source causes fluorescent natural chemicals or dyes to emit visible light

- localization of specific chemicals or structures

- used as an accurate and quick detection of pathogens

Confocal Microscope

- single plane of structures or cells that have been specifically stained with fluorescent dyes

- use a laser to fluorescence only one plane of the specimen at a time

- detailed observation of structures of cells within communities

electron microscope

- typical magnification 1000x to 10,000x

- resolution to 0.001nm

- use electrons travelling as waves with short wavelengths

- require specimens to be in a vacuum, so cannot be used to examine living microbes

transmission microscope

- monotone, two-dimensional, highly magnified images

- may be colored-enhanced

- produce two-dimensional image of ultrastructure of cells

- observation of internal ultrastructural detail of cells and observation of viruses and small bacteria

scanning microscope

- monotone, three-dimensional, surface images

- may be colored-enhanced

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

- observation of the surface details of structures

probe microscopes

- magnification greater than 100,000,000x with resolving power greater than that of electron microscopes

- use microscopic probes that move over the surface of a specimen

scanning tunneling

- individual molecules and atoms visible

- 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

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

atomic force microscope

- individual molecules and atoms visible

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

- observation of living specimens at the molecular and atomic levels

staining

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

- to have contrast

- functions:

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

simple stain

- uses a single dye

- ex. crystal violet, methylene blue

- result: uniform purple stain, uniform blue stain

- used to highlight or reveals size, morphology, and arrangement of cells

- aqueous or alcohol solutions of a single basic dye (sometimes a mordant is added to intensify the stain)

differential stain

- use two or more dyes to differentiate between cells or structures

- ex. gram stain, zielh-neelsen acid-fast stain, schaeffer-fulton endospore stain

gram stain

- differential stain

- result: may be purple or pink

- differentiates between bacterias, which is typically the first step in their identification

Ziehl-Neelsen acid-fast stain

- differential stain

- results: can be pink to red and blue

- distinguishes the genera Mycobaterium and Nocardia from other bacteria

purple

color of gram positive cells

colorless to pink

color of gram negative cells

pink to red

color of acid fast cells

blue

color of non acid fast cells

Schaeffer-Fulton endospore stain

- differential stain

- result: green endospore and pink to red vegetative cells

- highlights the presence of endospores produced by species in the genera Bacillus and Clostridium

- 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/pink) is applied, it stains the remainder of the cells red or pink

special stains

- ex. negative stain for capsules, flagellar stain

- used to color and isolate various structures, such as capsules, endospores, and flagella

- sometimes used as a diagnostic aid

negative stain for capsules

- special stain

- background is dark, cells unstained or stained with simple stain with halos around bacterial cells

- reveals bacterial capsules

flagellar stain

- special stain

- bacterial flagella become visible

- allows determination of number and location of bacterial flagella

- a mordant is used to build up the diameters of flagella until they become visible microscopically when stained with carbolfuchsin

MgRNA

- gram + contains this which when combined to crystal violet and gram's iodine with form an insoluble compound

- gram - does not contain this, thus easily decolorized

Benian

- gram + are less permeable to the decolorizer (has thick cell wall)

- gram - are more permeable to the decolorizer (thinner cell wall)

stearn-steam

- gram + have lower isoelectric pH, thus they are more acidic

- gram - have higher isoelectric pH, thus, it does not easily combine with the basic dye (CV)

neisseria, veillonella, moraxella (branhamella)

- all cocci are gram (+) except these

Mycobacterium, Corynebacteria, Bacillus, Clostridium, Lactobacillus, Listeria monocytogenes, Erysipelotrix insidiosa

- all bacilli are gram (-) except these

spiral organisms

- are hard to stain but once stained, they are gram (-) even though they take up the primary stain

0.5 nm - 10 nm

scanning tunneling microscope (STM)

1 nm - 10 nm

atomic force microscope (AFM)

10 nm - 100 micrometer

transmission electron micrsocope (TEM)

10 nm - 1mm

scanning electron microscope (SEM)

200 nm - 10 mm

compound light microscope (LM)

200 micrometer-

unaided human eye