Microbio Test 1

what is a microbe/ microorganism?

small organism, can’t be seen with naked eye, usually unicellular

when was the earliest evidence of life?

a rock that is 3.8B years old showed earliest evidence

how old is the earth?

4.5B years old

stelly pool formations

australia, cell lumens had carbonaceous walls enriched with nitrogen

why are the strelley pool formations important?

scientists were arguing that other evidence of life in early rocks could be replicated through geothermal processes

eukaryotes

membrane bound nucleus, predators for existing biomass(prokarytotes)

prokaryotes

no membrane bound nucleus, specialists for rapid growth and maximizing nutrients for increasing mass

common misconception about eukaryotes vs, prokaryotes

both eukaryotes and prokaryotes can have membrane bound organelles, so when differentiating them, the only real difference is the membrane bound nucleus

microbiota

microbial taxa associated with microflora

microbiome

microbiota+ surrounding environmental conditions

metagenome

collection of ONLY genes/genome

examples of things that can change your microbiome

colorectal cancer, ptsd, artificial sweeteners, age, IBS

tree of life

almost entirely microbial

one of the biggest driving forces for diversity

prokaryotes and their ability to inhabit an immense range of habitats

prokaryotes can catalyze…?

transformation of chemical compounds, essential to sustainability of life

reactions catalyzed by prokaryotes that are generally absent from eukaryotes

denitrification of N₂, fixation of N₂ into organic nitrogen

why is nitrogen so important?

present in every single amino acid, as well as all nucleic acids

what are microbes mostly composed of?

water, ~70%

dry weight of microbes mostly consists of..?

macromolecules + lipids

macromolecules

very large, polymeric structure

lipids

much smaller than macromolecules, not polymeric, hydrophobic

proteins

largest and most diverse category of macromolecules

cytoplasm

dense suspension of ribosomes

small molecules of a cell

constantly changing, being used/regenerated

average e.coli

has 2-4 million proteins, can divide in ~20 mins.

microbio was founded on 2 principles

microscopy and pure culture technique

“seeing is believing”

range of microbial morphology depends on improvements of microscope design

leeuwenhoek

credited with seeing living things under simple single lens microscope, discovered microbes

simple microscopes

offered higher quality images due to less distortion

advances in light microscopy…?

largely confined to improving methods of enhancing contrast

advances made to light microscopy in 20th century

phase contrast microscopy, darkfield microscopy

most significant advance in 20th century

development of electron microscopy

where did people think life came from for most of history?

sexual + asexual processes, and from non-living matter (spontaneous generation)

Francesco Redi

experiment: meat-n-maggots

• open jar- flies could come in freely, maggits appeared

• closed jar- no flies/air could enter, no maggots appeared

• jar w/ gauze: air can pass, no maggots but fly eggs on gauze

what did francesco redi’s experiment challenge?

planted 1st seed of doubt against spontaneous generation

John Needham

experiment: “life gravy”

• briefly boiled a broth, thinking this killed all life forms, covered with corks, let sit for days and observed that broth became cloudy with microbes

conclusion of John needhams experiment

there must be a life force that causes inanimate objects to come to life

Lazzaro Spallazani

repeated needhams experiment, boiled for one hour, sealed jar tight by melting openings

Lazzaro Spallazanis conclusions

Needham didnt boil long enough/ used poorly sealed jars, microorganisms exist in air, SG does not appear to be inherent to ‘matter’

why did critics fault Spallazani’s work?

argued that lack of air and/or boiling killed ‘life force’

Louis Pasteur

experiment: swan-necked flasks

convinced scientific community SG was incorrect

controversy over spontaneous generation

required development of sterilization methods

John Tyndall

discovered heat resistance of spores, kill them through repeated boiling

What did Pasteur also develop?

first rabies vaccine

3 forms of anthrax

cutaneous, inhalation, GI

cutaneous anthrax

most common, w/o treatment 20% fatality, if treated almost all survive

inhalation anthrax

w/o treatment almost always fatal, if treated ~55% survival rate

GI anthrax

w/o treatment ½ die, w/ treatment 60% survive

hydroscopic

loves/attracts water

Robert Koch

drop of blood from sheep with anthrax into a mouse → mouse dies next day

what is pure culture technique necessary for?

show that different diseases were caused by different microbes

Koch was likely first to realize that…?

solid media (agar) could be used to separate different kinds of microbes, can produce localized growth (colonies)

Koch’s postulates

define what is logically necessary and sufficient to prove microbial etiology of a disease

Koch’s first postulate

microbe must be present wherever disease is, but absent from healthy organisms

Koch’s second postulate

microbe must be isolated from the diseased host organism and obtained in a pure culture

Koch’s third postulate

microbe must be inoculated back into a healthy susceptible host, which must then become ill with same disease

Koch’s fourth postulate

same microbe must be found in the experimentally infected and diseased host

exceptions to Koch’s postulates

asymptomatic carriers, inability to grow pathogens in pure culture (viruses, causative agent of leprosy), single pathogen can cause multiple diseases (GAS, varicella zoster virus)

enrichment culture

allows isolation of microbes that are present in the source material in very low numbers

model systems for study of cell bio, molecular genetics/development

Escherichia coli, Bacillus subtilis, Caulobacter crescentus, Saccharomyces cerevisiae

resolution

smallest distance between2 separate points that can be distinguished a 2 separate objects

limit of resolution(distance) is related to:

1. wavelength of light being used

2. numerical aperture of the objective lens

3. d= 0.5(λ) / NA

numerical aperture

range of angles over which a system can accept/emit light

working aperture

sum of aperture angles of the objective and condenser

oil immersion lenses

oil helps the light not bend as much, lowers amount of refraction so more light can enter objective lens

what is bright field microscopy used for?

observing stained cells

dark field microscopy

can resolve very small objects, lights scatters against a dark backgrounds

iris diaphragm

limits light scattering that enters the objective, can make it easier to see clear samples like live cells

Phase contrast is routinely used for….?

observing live microbial cells, deals with light rays out of phase

what differentiates a phase contrast microscope from a regular one?

phase plate and a darkstop

downside of phase contrast

can add a halo to images, which can get too thick and hide details

Differentiation Interference Contrast AKA Nomarski Microscopy

reference light is polarized, useful for transparent samples, sharper images

what does it mean when light is polarized?

any light rays not going in a specific direction are blocked

why use PC?

cheaper, halo artifact, limitation of sample thickness

why use DIC?

doesn't work with plastic, some natural substances interfere w/ contrast, much more costly

fluorescent microscopy

can locate specific molecules within cells

fluorescence

electrons jump up an energy/orbital level then go back down, and during that process, energy and light is emitted

fluorophore

fluorescent chemical compound that can re-emit light when excited by a specific wavelength of light

what are most fluorescence microscopes constructed around?

filters and a dichroic mirror

confucal microscopy

takes a picture w green/blue/red fluorescence, theyre stacked together to make a high quality sharp image

mitosis

nuclear division

electron microscope

uses electromagnetic lenses to bend a beam of electrons

how does an x-ray work?

bone slows x-rays down, rays can travel easier through soft tissue