Micro 1 MST

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Who published the first drawings of microorgnaisms?

Robert Hooke in 1655

Who first used a microscope to view microorganisms?

Stelluti in 1625-1630

What did Antony van leeuwenhoek do/his importance?

-developed a better/stronger microscope
-observed and drew bacteria

What was Louis Pasteur's contribution to microbiology?

-He helped prove that spontaneous generation of living organisms form non living matter can't occur
-He did this by filtering air through cotton wool. After doing so, he found microorganisms that he then was able to grow in a sterile medium

What was the Pasteur flask experiment?

-1861
-He took 2 swan neck flasks and put in a broth with microoransisms
-He boiled both flasks until they were sterile
-One flask's neck was broken. This allowed air and dust in. This flask showed microbial growth
-The other flask's neck remained intact. The airbourne/dust particles remained trapped at the base. The broth remained sterile

What are miasmas?

-poisonous vapours
-This is what people believed caused/spread disease

How did Semmelweiss support the germ theory?

-He noted increased incidence of death from puerperal fever in delivering women treated by students/physicians rather than midwives

How did John Snow support the germ theory?

-correlated incidence of cholera with living/drinking water from certain sources

How did Lister support the germ theory?

-used heat sterilization and phenol to reduce incidence of surgical infections

What 2 scientists proved the germ theory?

Pasteur and Koch

What was Koch's significance?

-showed specific microorganisms caused particular diseases
-showed bacillus anthracis caused anthrax in 1876 in cattle
-perfected methods for sterilization, disinfection, and filtration
-discovered causative agents in tuberculosis and cholera
-established postulates to link a particular microorganism with a particular disease

What were Koch's original postulates?

-the microorganism must be present in every case of disease but absent in healthy organisms
-the suspected microorganism must be isolated and grown in pure culture
-the sam disease must result when the isolated microorganism is inoculated into a healthy host
-the same microorganism must be isolated again from the diseased host

What was wrong with Koch's original postulates?

-some pathogens are part of the normal microbiota
-some pathogens can't be grown/cultured
-some pathogens are only found/cause disease in humans
-in some diseases more than one pathogen is the cause

What are Koch's molecular postulates?

Virulence gene (VG)
-VG must be found in pathogenic strains
-V must be expressed during infection/disease
-mutation/deletion of the VG decreases pathogenicity
-replacement/restoration of the VG mutation/deletion restores pathogenicity

What are the taxonomy groups?

-domain
-kingdom
-phylum
-class
-order
-family
-genus
-species

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What was the first way microbials were classified?

phenetic system
-based on phenotypic characteristics

What is used today to classify microorganisms?

-small subunit rRNA molecules

What are the 3 domains?

bacteria
archaea
eukarya

What is the centre of a phylogenetic tree called?

origin

What does LUCA stand for?

last universal common ancestor

What are some characteristics of archaea?

-similar shapes to bacteria
-often 1-10um
-cell wall is made of glycoprotein, not peptidoglycan
-L amino acids used in X-links
-membrane lipids are ether linked, not ester linked\= more stable in extreme pH and heat
-many are extremophiles: thermophiles, halophiles

What are some basic characteristics of bacteria?

- 1 to 5 micrometers in size
-have a plasma membrane
-the cell wall contains peptidoglycan
-have 70S ribosomes
-divide by binary fission

What are 2 common bacterial cell shapes?

-coccus (plural\=cocci)
-bacillus (plural\=bacillli)

What are some bacterial cell arrangements?

-single cells
-chains
-clusters

What does the bacterial cell envelope include?

-plasma membrane
-cell wall
-some have: outer membrane, capsule, slime layer, S-layer

What are some characteristics of a bacterial's plasma membrane?

-about 5-10nm thick
-selectively permeable (nutrients/waster)
-location of many metabolic processes
-encompasses the cytoplasm
-has a phospholipid bilayer

Describe the plasma membrane's lipid composition:

-amphipathic (hydrophobic and philic)
-structurally asymmetric
-lacks sterols (cholesterol)
-many contain hopanoids

In LOW temperatures are there more unsaturated fatty acids or saturated fatty acids in bacterial PMs?

there are more UNsaturated

In HIGH temperatures are there more saturated fatty acids or unsaturated fatty acids in bacterial PMs?

there are more SATURATED

Describe the cell wall:

-rigid
-just outside the plasma membrane
-strength is due to peptidoglycan
-is one of the main differences between gram positive and gram negative

What are 2 ways peptidoglycan helices join?

-direct linkages
-peptide interbridges

Is there more X-linking in the peptidoglycan in gram positive or in gram negative bacteria?

There is more X linking in gram POSITIVE. This is because gram positive is less porous

Describe a Gram positive cell envelope:

- the peptidoglycan is 20-80nm thick
- doesn't have an outer membrane
-has a small periplasmic space
-Contains teichoic acids
-Cells retain crystal violet dye during gram stain

Describe a Gram negative cell envelope:

-the peptidoglycan is 2-7nm thick (smaller than GP)
-there IS an outer membrane
-the periplasmic space is large
-Contains LPS
-cells don't retain crystal violet dry during gram stain

Describe capsules:

-well organised polysaccharide material
-not easily washed off
-aids in cell adhesion
-protects against dehydration, phagocytosis, bacteriophage attack, toxic stuff
-can be seen in negative staining

Describe Slime (outer layer from cell wall)

-diffuse unorganised polysaccharide material
-easily washed off
-not easy to visualize
-facilitates motility
-commonly produced by gliding bacteria

Describe S-layers (outer layer from cell wall)

-regular arrays of protein or glycoprotein
-found in GP, GN, archaea
-protects against the environment
-helps in cell shape, rigidity, adhesion

What is the importance of ribosomes?

they are the site of protein synthesis: translation

What is the size difference between eukaryotic ribosomes and bacterial ribosomes?

bacterial: 70S (50S and 30S) (smaller)
Eukaryote: 80S (60S and 40S) (larger)

What is the purpose of inclusions in bacteria?

they are mainly used for storage
-they are aggregates of organic and inorganic material

What makes up the bacterial genome?

nucleoid: single chromosome of closed circular dsDNA

Plasmids:
-there may be more than one
-they replicate independently of the chromosome

What is a flagella and what does it do?

-a long, thin, hollow, rigid structure
-important in motility

How does a flagella work (motion)?

propeller like manner

What is fimbriae (pili) and what does it do?

-short, fine, hair-like appendages
-they are used to attach to surfaces
-type IV is used for motility and uptake of DNA in transformation
-mostly found on GN

What are 3 types of bacterial motility?

-flagellar motility (propeller like)
-twitching
-gliding

Do bacteria move aimlessly?

-nope
-chemotaxis (towards attractants, away from toxic stuff)

How do most bacteria grow?

binary fission

Describe the process of binary fission:

-parent cell enlarges and duplicates chromosome
-septum growth starts
-chromosome and cytosolic components move to opposite ends
-septum and new wal synthesis completes
-daughter cells divides (in some species they remain attached)

What are the 3 phases in the bacterial cell cycle?

-period of cell growth
-DNA replication and partitioning
-cytokinesis

Where does the replication of bacterial chromosome start and end?

-starts at the origin of replication (ori) at a single point
-ends directly opposite the origin (terminus ter)

What is a replisome?

group of proteins required for replication
-it assembles at the origin

What direction does replication of bacterial chromosome go?

it proceeds in both directions of the ori (circular chromosome)

What is cytokinesis?

-processes that apportion the cytoplasm and organelles, synthesize a septum, and divide a cell into 2 daughter cells

what is septation?

process of forming a cross-wall (spetum) between 2 daughter cells

How is the Z ring assembled?

-FtsZ proteins polymerise to form filaments
-portions are constantly being exchanged with the newly formed FtsZ polymers\=dynamic assembly

What is FtsZ?

tubulin like protein

What is a divisome?

a collection of proteins that aggregate at the region in a dividing cell where a septum will form

How does the divisome form?

-anchoring proteins link the Z ring to the plasma membrane
-synthesis of pqeptiodglyan and other cell wall components
-constriction of the Z ring
-invagination of the cell membrane and synthesis of the septum wall

Why is the cell wall so important?

-maintains cell shape
-gives the cell strength
-constrains turgor pressure exerted by the cytoplasm which prevents osmotic lysis

How is a new cell wall made during binary fission without compromising the cell's integrity?

-there is a balance between degradation and synthesis/insertion of peptidogylcan

How is peptidoglycan synthesised?

PBPs are involved:
-they link strands of peptidoglycan together
-it controls degradation of the peptidoglycan layer to allow insertion of new units

It grows due to the FtsZ placement

What are degradative PBPs called?

autolysins

What is a batch culture?

closed culture vessel containing a single batch of fresh medium

What are the bacterial growth curve phases?

lag phase
exponential log phase
stationary phase
death phase

What is occurring during the lag phase

-the cells are metabolically active but not increasing in number
-cells are adapting to the new environment
-synthesis of enzymes, ribosomes, etc
-large production of energy

What is occurring in the log phase?

-adaptation to the environment is complete
-growth is occurring in a constant exponential rate
-cells are doubling in number at a regular interval
-doubling time is used as a measure of growth rate

What is occurring during the stationary phase?

-the total number of living cells remains constant
-new cells are made at the same rate as old cells die
-nutrients become more limited, wastes accumulate, etc
-critical cell population is reached

What is occurring in the death phase?

-cells lose the ability to divide and die
-total viable cell number decreases exponentially
-conditions aren't as supportive of cell division

What is the viable count method?

-count colonies produced on a SOLID media as an indication of cell number

-indicates total viable cells

What is the spectrophotometry reading method?

-measure of TURBIDITY of a LIQUID culture as an indication of cell number
-indicates total cells (living and dead)

What does a high turbidity mean?

-there is a higher cell number
-there is a lower transmittance of light through the culture
-the higher the absorbance of light (less light reaches sensor), the higher the cell number

What is growth rate?

number of generations per unit of time

What effects growth rate?

-temperature
-ion composition
-ion concentration
-amount of oxygen
-nutrient concentrations
-restraints (predation, antibiosis)
-energy available
-amount of nitrogen
-pH
-water and solutes
-pressure
-radiation

Is bacterial growth faster in nature or in the lab

faster in the lab

What are some sources of carbon/energy in media?

glucose/carbohydrate
peptones

What are some sources of nitrogen in media?

-peptones
-protein hydrolysates

What are some sources of inorganic elements in media?

-phosphates
-sulphates
-usually don't have to be added because they are normally present as contaminants

What are some sources of growth factors in media?

-blood
-serum
-vitamins
-they are usually heat labile substances

What is a basal media?

-it is a general purpose base media
-used to grow non-fastidious microbes

what is an enriched media?

-used for the fastidious microbes

What is the difference between an enrichment medium and an enriched media

A bacterial growth medium that favours the growth of a particular bacterial species over others in a mixed sample is called an enrichment medium
An enrichment medium does favour the growth of a particular bacterial species over others in a mixed sample; an enriched media does not.

When do you use a selective media?

when you want to encourage the growth of some microbes but suppress the growth of others

When do you use an indicator/differential media?

when you want to see a change when the microbe grows
-there is usually a component that causes an observable change

What is nutrient agar called when made without agar?

nutrient broth

When is nutrient agar used?

to culture/store non fastidous bacteria

What is HBA?

-horse blood agar
-its an enriched medium
-5% blood added after sterile NA cooled to 50C
-used for fastidious bacteria
-indicator of haemolysis

What is chocolate agar? (CHA)

-enriched medium
-7% blood added to sterile NA and held at 80C, then cooled and plates poured
-used for very fastidious bacteria
-often incubated in CO2

What is MacConkey agar? (MAC)

-it is a selective indicator medium
-contains bile salts, lactose, neutral red
-used in isolation and enumeration of coliforms and intestinal bacterial pathogens

Why do people use enriched cultures?

-they chemically inhibit other bacteria
-they nutritionally favour the desired bacterium
-its a manipulated environment to suit the desired bacterium

Describe anaerobes:

-can't use oxygen
-obligate (strict) anaerobes are killed by oxygen

Describe obligate aerobes

they require oxygen

describe facultative anaerobes/facultative aerobes

can grow without oxygen but grow better with it

describe microaerophiles:

they need oxygen, but at lower than atmospheric concentration

does oxygen diffuse faster in air or in water?

oxygen diffusion is 10^4 greater in air than in water
-it can take years for it to diffuse in water depending on how deep it is

What are some issues with adding oxygen?

-ROS is created
-ROS can oxidize sulphydryl groups in proteins which denatures enzymes
-ROS also damage lipids and nucleic acids

How do bacteria protect themselves against ROS?

-they possess enzymes and pigments that detoxify ROS
-many anaerobes don't have these mechanisms, so they can only live where there isn't oxygen

Describe biofilms:

-the are attached microbes in complex, slime-encased communities
-ubiquitous in nature
-dynamic and heterogeneous in composition

List the biofilm formation steps:

1-substratum precondition by ambient molecules
2-cell deposition
3-cell absorption
4-desorption
5-cell to cell signalling and onset of exopolymer production
6-convective and diffusive transport of O2 and nutrients
7-replication and growth
8-secretion of polysaccharide matrix
9-detachment, erosion, and sloughing

How do microbes communicate with each other?

-molecular signals in a density dependent manner

How do you obtain a pure culture?

-appropriate sterile culture medium in a sterile container
-appropriate incubation conditions
-good aseptic technique
-a method for selecting a single cell