MCB3020 - The Microbial Cell

Hooke

Micrographia in 1665 (describing things using compound microscope)

Pasteur, Sedillot, MacCormac

1850s-1880s, microorganism, microbe, and microbiology emerged during research during this time, namely by these three researchers

NIH microbe definition

"Microbes are tiny living things that are found all around us. Also known as microorganisms, they are too small to be seen by the naked eye"

ASM microbe definition

tiny living organism that is too small to be seen by the naked eye, including bacteria, fungi, archaea, protists, some algae

Microbiology textbook definition of microbe

living organism that requires a microscope to be seen

Acasta Gneiss of Slave craton

approx. 4 billion years old by radiometry

Stromatolites

fossilized microbes, some have been dated to 3.5b years old

Spontaneous generation

Aristotle's theory, 200 BC to mid 1800s AD, life came from nowhere

Pasteur

Worked on food spoilage in 1860s, challenged spontaneus generation

morphology

used by early taxonomists to assign nomenclature

Why is microbe morphology constrained

small size due to being single celled.

Why do bacteria aggregate?

nutrient acquisition, attachment, protection, communication, biofilm formation, multicellular-like behavior

How does aggregates form

cell division

agglutination

stains

organic, reactive small molecules to enhance contrast

methylene blue, safranin, crystal violet, acridine orange

Leeuwenhoek

1670s, first description of microorganisms, created single lens microscope, created 300 letters describing them (Animalcules) published in 1677 by Royal Society

Pasteur experiment

Two flasks with media and a long neck are set up, then heat is applied.

One flask has its neck broken, while another has it intact

The broken neck flask had bacteria grow, while the intact flask had no bacterial growth.

FUCA

could be a nonliving acellular progenote, described by Woese in 1977

Carl Woese

redefined tree of life based on primary DNA sequence data in the 1990s,

compared rRNA gene sequences, resolved 3 major domains from LUCA

Miller Experiment 1950s

in vitro formation of organic molecules from inorganic molecules

Gloeobacter

cyanobacteria, oldest living extant organism (approx. 3.5byo)

L. aurora

Horseshoe crab, approx. 450 million years old

H. sapiens

approx 300,000 years old

Pasteur’s food spoilage work

inferred a role of microbes in infectious disease and germ theory

History of food spoilage prevention

drying (smoking and sun) is >1 million years old, curing/brining is from 12,000 BC

virus-first hypothesis

Koonin, 2005, proposes viruses were FUCA

traits shared by all domains

b-form DNA

RNA polymerase

domains

cell membranes

same codons

Eukaryote unique traits

organelles and compartmentalization (endomembrane systems), which limit diffusion

only monocistrons in eukaryotes, linear DNA, can be multicellular

microbe classes

bacteria (most diverse with thousands of phyla), archaea, microalgae, microfungi, protists

number of microbe species

estimated 1 trillion species, less than 0.001% identified

microbe size

most are too small to see with eye (<0.2 mm)

Size variability between microbes

Prokaryotes like can be 0.4-20 micrometers, protists can be 2-2000 micrometers

Why are microbes small

diffusion limitations due to being single celled

why are prokaryotes generally smaller than eukaryotes

no compartmentalization

gigantism

thiomargarita namibiensis is a prokaryote that can be bigger than 0.5 mm

microbe shape

important for function, taxonomy, and evaluation

diversity in shape is constrained for single-celled organisms

shape conservation

determined by cytoskeleton, where prokaryotes are generally simpler shaped than eukaryotes

light microscopes and type examples

modulate light as it passes through objects

bright field, darkfield, fluorescence, phase-contrast

brightfield

commonplace

washed-out/translucent appearance of specimens, limitation of arrpxo. 1500x or 100-200 nm resolution, determined by light, contrast, and optical properties

simple staining

staining with one chemical ex. methylene blue

differential staining

applies multiple stains

ex. acid-fast staining

ex. gram reaction staining

fluorescence microscopy

fluorophore is used to bind to a specimen, which will absorb UV light and releasing visible light

Fluorophore specificity can be determined by:

chemical affinity

antibody-antigen interactions

dna hybridization

protein expression systems

how to improve specificity

labeled DNA probes or antibodies will be more selective than chemical affinity, which is generic

ex. DAPI affinity for AT-rich DNA

ex.x DNA hybridization of Cy5 labelled nirK DNA probe

Immunofluorescence

fluorophores which are on antibodies which will detect antigens and bind to them

darkfield light microscopy

Lister in 1830s

excludes light around specimen

microbes have light haloes on dark backgrounds.

Phase microscopy

Zernicke, 1930s

transforms intrinsic differences in refractive index of cell to differences in image brightness/contrast

Miller Urey Experiment

forming organic molecules from inorganic molecules in 1950s in vitro

relief staining

background staining ex. negative staining where india ink doesn't stain capsulated bacteria

Botulism, typhoid, and dysentery

Pre-1900, outbreaks were common due to no food hygiene

biogenesis

life requires common descent, proved by pasteur experiment

how can microbe contamination be prevented?

heat, pasteurization (Pasteur, 1864), pressure canning (USDA 1917), and preservatives

preservatives

boric acid, formaldehyde, copper sulfate etc.,

Gloeobacter

cyanobacteria, oldest living extant organism at 3 billion years old (living fossil)

L. aurora

horseshoe crab, approx. 450m years old

H. sapiens age

300,000 years old

history of food spoilage prevention

drying (smoking + sun) is older than 1 million years

curing/brining is from 12,000 BC

Virus-first hypothesis

Koonin, ca. 2005, viruses were FUCA

prokaryotic traits

small with less volume, and more diverse metabolism, circular chromosomes, monocistronic and polycistronic

protozoa, algae, fungi

eukaryotic, not prokaryotic (algae and fungi can be unicellular)

advances in molecular DNA analysis

ex. high-throughput DNA sequencing

Most diverse domain

bacteria, thousands of phyla

size of microbes

most invisible to unaided eye (<0.2 mm), but size can differ from order of magnitude.

Size examples

prokaryotes 0.4-20 micrometes, while protists can be 2-2000 micrometers

Gigantism

thiomargarita namibiensis is a prokaryote that can be greather than 0.5 mm in size

Shape conservation

cell shape is determined by cytoskeleton, which is important for function, taxonomy, and evaluation

shape variations

palisade aggregate, stalking, clubbed, vibrio, diplococci, staphylococci

Other affecting factors on cell shape

cytoplasm viscosity, membrane rigidity (biochemistry), and environmental factors

eukaryotic microbes shape variety

cocci, ovoid, cuboidal, cylindrical, planar, lenticular, fusiform, discoidal, crescent, ring stellate, polygonal, amorphous

what does microbe observation require

magnification and cultivation

what does cultivation require? and examples

specialized training, equipment, time ex. aseptic technique, anaerobic culture, autoclaves (sterilization), incubators

methods for obtaining culture examples

processing abscess or product testing

Non-cultivable microbes

only 1% of microbes are culturable by traditonal techniques

microscopy requirements

specialized training, equipment,microbe culture/specimen

most common form of microscopy

uses light and electric fields

brightfield magnification limits

magnification limits 1500x (100-200 nm), determined by light, cotnrast, and optical properites

specialized staining

targets microbial structures ex. spore staining where malachite green binds to spores

Electron microscopy

Knoll and Ruska in 1930s

magnification can exceed 1 million times of cells and intracellular structures

uses electron beams used with tungsten guns to create high resolution (<1 nm) images

transmission EM

electron fields transmitted through a specimen

scanning EM

electron fields scan across surface of specimen to make a 3D image

Microbe culturing creation

Pasteur did this first in the 1860s

Microbe culturing methodology by Pasteur

1) artificial media

2) sterilization

3) preventing contamination

Pure cultures limitations

cannot be done in liquid media

Pure culture technique invention

Robert Koch (physician, bacteriologist, 1860s)

Julius Petri (physician, bacteriologist, 1850s)

Pure-culture technique

solid media technique was done in the 1880s, with the addition of agar in liquid media

types of growth media

1) complex

2) synthetic/minimal

3) enriched

4) selective

5) differential

complex media

nutrient rich with cell extracts

poorly defined

ex. nutrient agar and broth

synthetic/minimum media

only essential nutrients, clearly defined

very selective when it comes to microbes,

selective media

uses selector to inhibit microbe growth

often combined with differential media

differential media

differentions microbes based on chromogenic reactions

ex. thiocitrate bile salts

often combined with selective media

Enrichment media

cultivation of fastidious microbes

molecular approaches to observation

PCR, DNA sequencing, DNA restriction analysis, immunoassays

prokaryote-eukaryote dichotomy

prokaryotes are not compartmentalized by organelles

Endogenous structure

prokaryotes and eukaryotes both have this

prokaryote endogenous structure

nucleoids (no limiting membrane)

eukaryote endogenous structure

nucleus

intracytoplasmic membranes

formed by membrane infolding

compartmentalizes the cell

prokaryotes: contiguous with cell membrane

most function in metabolism and energy production

thylakoids

discovered by Schimper and Kunkel

stacks (grana) of ICM used for photosynthesis

can be 50-70% of total membrane

thylakoid composition

packed with photosystems (protein-pigment complexes) and ATP synthases

eukaryotic ICM

endomembrane

unique to eukaryotes

discontiguous

Endomembrane system

compartmentalized system of membrane bound organelles

fully autonomous organelles