visualising microorganisms

the human eye cannot see structures smaller than…

100um

morphology of pseudomonas aeurogenosa

rod/bacillus, 1 × 2-5um

prokaryotic microorganisms

archaea, bacteria

eukaryotic microorganisms

protozoa, algae, fungi

eukaryotic microorganisms range between what sizes ?

800nm - 4cm

diameter of RBC

8um (4x E.coli)

spherical

cocci

rod

bacilli

oval

coccobacilli

comma

vibrio

spiral

spirilla

corkscrew

spirochetes

variable

pleomorphic

example of microorganism with cocci shape

staphylococcus aureus

what shape are E.coli ?

bacillus (rod)

syphilis is caused by what microorganism ?

treponema pallidum

magnification

increase in apparent size of image

resolution

ability to distinguish detail between 2 distinct objects

what kind of sample would you visualise using a dark field microscope ?

thin, transparent or unstained

what kind of microscopy is used for visualising live cells and why ?

phase contrast - doesnt require stain

sarcina

cocci grouping of 8+ cells in cuboidal shape

example of coccobacilli shaped microorganism

haemophilus influenzae

why are vibrio curved ?

asymmetric cell wall growth

what is the advantage of a spirilla shape ?

helps microorganism navigate through environment

how has helicobacter pylori evolved ?

spirilla shape helps survive extreme low ph of gastric acid in stomach

what is the advantage of a spirochete shape ?

useful for burrowing through viscous environments

why do pleomorphic microorganisms not have a defined shape ?

absent cell wall

example of a pleomorphic microorganism

mycoplasm pneumoniae

subcellular structures can be seen with what type of microscope ?

electron

what is the fundamental resolution limit for light microscopy ?

400nm

what is the wavelength of visible light ?

400 to 750 nm

refraction

slowing down and bending of light due to change in medium and thus refractive index

refractive index

measure of how much light changes direction when moving between 2 different materials

higher refractive index means light is moving…

slower - more bend

microscopes utilise refraction through the means of…

parabolic glass lenses

what is the refractive index of a parabolic glass lens ?

1.5

“empty” magnification is said to lack…

resolution

resolution of light microscope

0.2um

total mag =

ocular x objective lens

what is a compound microscope ?

system of multiple low powered lenses that correct for aberrations

aberrations

distortions

what is meant by compound microscopes are parafocal ?

maintain focus when switching between objective lenses

purpose of oil immersion

enhances resolution by reducing light refraction at glass-slide interface

how does oil immersion improve resolution ?

refractive index of oil similar to that of microscope slide

when using bright field microscopes specimens appear…

dark against light background

when using dark field microscopes specimens appear…

light against dark background

phase contrast microscopy

enhances contrast in transparent, unstained specimens by exploiting difference in refractive index

how do fluorescent microscopes work ?

fluorophores absorb energy, move to higher energy state, when returning back to lower state emit light at longer wavelength, producing visible fluorescence

electron wavelength is what compared to that of visible light ?

100,000x shorter

what must be done to samples for electron microscopy to work ?

coated in heavy metals to allow for absorption of electrons

what is the purpose of sample prep ?

increases contrast, detection and resolution

fixation

cells immobilised on slide

what are the 2 methods of fixation ?

heat and chemical

what is the main issue with heat fixation ?

distorts morphology - more so than chemical

simple staining

single dye uniformly colours cells increasing visibility eg. methylene blue

differential staining

1 or more dyes to distinguish between cell types or structures eg. gram stain

methylene blue binds to…

negatively charged components via electrostatic interaction

what % v/v is methylene blue ?

1

what are the 2 subtypes of gram stain ?

crystal violet (+) safranin (-)

what is the difference between gram pos and neg ?

gram pos lack an outer mem but thick layer of peptidoglycan, gram neg has 2 cell walls thinner peptidoglycan

what % v/v of crystal violet and safranin are used ?

0.5

crystal violet is fixed with what and how ?

iodine - CVI complex trapped in thick peptidoglycan layer of gram pos

why can crystal violet not penetrate all bacterial cells ?

water based - some bacteria have waxy lipid rich (hydrophobic) cell walls

what type of bacteria does acid fast identify ?

those with waxy mycolic acid-rich cell walls

spore differentially stains…

spores and vegetative cells

vegetative cells

any cells of body except those which take part in production of gametes

subtypes of light microscopy

bright / darkfield

phase contrast

fluorescence

which has a higher resolution SEM or TEM ?

TEM - 0.1nm

SEM only 1 - 10

fluorophore

fluorescent dyes or proteins that absorb light at specific (excitation) wavelengths and emit it at longer ones (emission)

role of iris diaphragm in light microscopes

allows for control of light level and angle

differential interface contrast (DIC) microscopy

contrast of sample increased by identifying steep changes in refractive index

no staining required

produces 3D image

light source in fluorescent microscopy

lasers or xenon arc lamps (as opposed to halogen lamp)

SEM samples are fixed and preserved using…

glutaraldehyde and osmium tetroxide