Microscopy, Staining, And Classification

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simple microscope

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one lens

ie magnifying glass

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compound microscope

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two or more lenses that act in a series to cooperatively increase the magnification of the image

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ocular lens

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the lens closest to the eye

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objective lens

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the lens closest to the specimen

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specimen

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the object being examined

often mounted on a glass slide

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condenser

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a lens that focuses light on the specimen

usually includes an iris diaphragm which controls the amount of light illuminating the sample

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light source

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electric light bulb or a mirror that focus sunlight on the condenser

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body

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holds the lenses in place and any prisms or mirrors that are neede if the light path is not straight

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arm

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hols the body over the specimen

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focusing knob

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moves stage up and down to place the specimen at the point to produce a focused image

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stage

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holds specimen

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base

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keeps the instrument from toppling over

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magnification

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the ability of a microscope or lens to increase the apparent size of the image or object

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total magnification

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for a compound microscope the total magnification is the product of the magnifying abilities of all the lenses in the system

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resolution

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the ability to discern fine details in an image of an object

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resolving power

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aka resolution distance

measure of the smalles distance between to separate objects that can be seen to be two separate objects

smaller = ability to see finer details

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resolving power formula

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0.61 x wavelength/ numerical aperture

to get better resolution--increase numerical aperture or decrease the wavelength of light used to illuminate the object

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limits of resolution with light microscopy

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typical bacteria cells are 700-3000 nm in lenght

typical viruses are smaller than 100 nm in diameter

viruses are too small to see with a light microscope and bacteria are so small you can't see much detail

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electron microscope

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allows you to see viruses and subcellular structures such as ribosomes, because electrons have a shorter wavelength that light and thus smaller resolving power

can use higher magnifications without encountering the phenomena of "empty magnification"

up to 100000x magnificatino

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microscopy techniques

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bright-field microscopy

dark-field microscopy

phas contrast microscopy

fluorescence microscopy

electron microscopy

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bright-field microscopy

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light shines through the specimen and directly onto the objective lens and to the observer's eye creating a lighted or bright field of vision

bacteria and other microorgs are seen as dark objects on a white background

works best when bacteria are stained a bright color and immobilized

easy to use

works best with fixed stained microorgs (dead)

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dark-field microscopy

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light shines on the specimen from an oblique angle and does not go directly into the objective lens

background is dark

only light that gets to the observer's eye is the light that is scattered by the specimen

specimen does not have to be stained

works for live microorgs., but their movement makes it difficult to see

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phase contrast microscopy

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relies on a complicated optical trick to enhance the visual effect seen when light goes from a medium of lesser optical density (water) to a medium of greater optical density (cytoplasm)

beams of light are bent when they cross the boundary between two different transparent substances with different optical densities

allows you to see the phase boundary even if both phases are transparent

most useful for examining living protozoa and other eukaryotic cells

does not require staining

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fluorescent microscopy

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microbial specimen is illuminated with UV light which can cause some chemicals to emit visible light

UV is called black light because you can't see it

only visible light present is that which is produced by fluorescence of either naturally occuring compounds in the microorgs or special dyes that are used to stain the microorg.

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natural fluorescence

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some bacteria, ie Pseudomonas aeruginosa, make fluorescent pigment

photosynthetic pigments like chlorophyl are fluorescent

methanogenic archaea produce fluorescent pigment

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fluorescent dyes

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for DNA, acridine orange

for Bacillus anthracis, fluorescein isothiocyanate

for Mycobacterium tuberculosis, auramine O

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immunofluorescence microscopy

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fluorescent dye such as fluorescein (yellow-green) or rhodamine (red) is chemically joined to an antibody to make a specific probe

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general features of electron microscopy

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sample is bombarded with electrons

image is produced using a cathode ray tube

must be dehydrated

must be stained with heavy metal

must be placed in a evacuated chamber

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TEM--transmission electron microscopy

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thin sliced sample

used to see cellular organelles such as mitochondria, ER, chloroplasts, ribosomes, etc

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SEM--scanning electron microscopy

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sample is sputter coated with gold

used to look at the surface of objects

variant= freeze-fracture etching

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stains of microscopy

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simple stains - basic dyes

negative stains - acidic dyes

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different stains

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gram stain

schaeffer-fulton endospore stain

ziehl-neelsen acid-fast stain

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simple stains

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used to stain microorgs a bright color to make them more visible in bright-field microscopy

basic dyes or cationic dyes, are fairly complicated organic molecules that ionize water

for a basic dye, chromophore (colored portion) is a proton acceptor, is positively charged

many components of the bacterial cell wall and cytoplasmic membrane are acidic and will be deprotonated in water, this makes the cell envelope negatively charged

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examples of basic dyes

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crystal violet

safranin

methylene blue

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negative stains - acidic dyes

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for acidic dye, chromophore is a proton donor when the dye dissolves in water, makes color portion negatively charged

dye does not stain organism but glass

is a procedure that is used to visualize the bacterial capsule

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negative capsule stain

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background stained with acidic dye congo red

cells are counterstained with the basic dye safranin

clear zones around the cells show where the capsular polysaccharide layer prevented the staining the staining of the slide glass

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examples of acidic dyes

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nigrosin

india ink

congo red

eosin

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differential stains

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uses two different dys to distinguish between different types of cells or different parts of cells

first dye usually called primary dye

primary dye usually followed by some sort of wash

then the second dye or counterstain is applied

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gram stain

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used to divide bacteria into two groups based on the thickness of the cell wall

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gram positive

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thich cell walls

appear purple in gram stain

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gram negative

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thin cell walls

appear pink in gram stain

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examples of gram positive bacteria

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Streptococcus pneumonia

Clostridium tetani

Corynebacterium diphtheria

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examples of gram negative bacteria

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Esherichia coli

Vibrio cholera

Yersinia pestis

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step 1 of gram stain

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primary stain

streptococcus and e. coli stained with crystal violet

iodin is added as a mordant to make the stain better

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step 2 of gram stain

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the wash

slide is washed with 95% ethanol for four seconds

bacteria of thin cell walls are decolorized

thick cell walls stay purple

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step 3 of gram stain

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the counterstain

sample is stained with safranin

cells that are purple remain purple

cells that were decolorized turn pink

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schaeffer-fulton endospore stain

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used to identify spor forming bacteria in the gram positive genera Bacillus and clostridium

especially useful for identifying:
-clostridium botulinum
-clostridium tetani
-clostridium perfringens
-bacillus anthracis

cell wall of an endospore is highly mineralized with dipicolinic acid and calcium

endospore stains with the mineral dye malachite green

most bacterial cells do not stain well with malachite green

red dye safranin is used as a counterstain to see the vegatative (non-spore) cells

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step 1 of S-F endospore stain

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primary stain with malachite green

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step 2 of s-f endospore stain

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decolorize wash with water

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step 3 of s-f endospore stain

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counterstain with safranin

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ziehl-neelsen acid-fast stain

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used to identify bacteria in the genera Mycobacteria and Nocardia, including the clinically very significant bacterium Mycobacterium tuberculosis

cell walls of the mycobacterium contain a lot of hydrophobic waxy mycolic acids

dye carbol fuchsin sticks tightly to the waxy mycolic acids

decolorizatin with acidified alcohol removes the carbol fuchsin from other types of bacteria

counterstain is typically methylene blue

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classification and identification of microorgs.

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mid 1700s-linnaea system (2 kingdoms)

1950s-whitaker's scheme (5 kingdoms)

1978-woese's tree of life (3 domains)

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sevel layers of classification in the linnaean system

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kingdom

phylum

class

order

family

genus

species

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species

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group of organisms that are capable of mating and producing viable offspring

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genus

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group of related species, several distinct species that share many common traits and are closely related from an evolutionary standpoint with a relatively recent common ancestor

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family

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group of closely related genera

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physical characteristics

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microscopic examination of cell morphology

shape, size, arrangement

staining characteristics

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biochemical tests

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check for the presence of specific enzymes and metabolic pathways
-carbohydrate fermentation
-utilization of specific amino acids or citric acid
-production of specific waste products

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serological tests

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recognition of the bacteria by specific antibodies

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phage typing

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differentiates strains on the basis of bacteriophage host range

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molecular analysis

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uses computer programs to calculate phylogenetic relationships

ribosomal RNA sequence comparisons

other gene sequences

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carbohydrate fermentation tests

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uninoculated tube of carbohydrate fermentation broth is red and translucent, not cloudy

medium is inoculated with the bacterium and incubated

growth of the bacterium will turn the medium cloudy

if bacteria can ferment the carbohydrate, medium turns yellow from acid production

if bacterium cannot ferment the carbohydrate the medium with turn red

commonly used carbohydrates: sucrose, lactose, mannitol, xylose, arabinose

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indole test

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looks for presence of the enzyme tryptophanase, which catalyzes the conversion of tryptophan to indole

media used is tryptone broth, which contains amino acids but no sugar

indole production is detected by adding kovac's reagent to a 1-2 day old culture

red layer forms at the top if the bacterium is able to make indole