Microbiology
Lecture 1:
A microbe is a living organism that can be seen by a microscope that range from millimeters
in size to 0.2 micrometers.
Link between infection and disease (bacteria):
1. The microbe is found in all cases of the disease but is absent from the healthy
individual.
2. The microbe is isolated from the diseased host and grown in pure culture.
3. When the microbe is introduced into a healthy, susceptible host, the host shows the
same disease.
4. The same strain of microbe is obtained from the newly diseased host. When cultured,
it shows the same characteristics as before.
Vaccines work against viruses, in that they utilize the virus infection mechanism to produce
the antibodies against them. – ineffective against live bacteria.
Antiseptics come in two forms: physical and chemical
- Physical antiseptics usually include heat (burner flame, autoclave)
- Chemical antiseptics are molecules kill not only bacteria, but also the host if ingested.
Antibiotics are chemicals that would kill the microbes but leave the host unharmed. Due to
host variability this has been adapted for killing >99.9% of microbes.
Supporting ecosystems
- Aerobic environments are abundant with oxygen
- Microaerophilic environments have reduced oxygen levels and may led to multiple
forms of metabolism.
Capnophilic (CO2 loving) environments are a form of microaerophilic
environments.
- Anaerobic environments are lacking oxygen.
- Strict anaerobic environments are completely void of oxygen (oxygen is lethal).
- Within a growing biofilm, any or all of these environments can exist simultaneously.
If you don’t have chloroplast, you have an animal cell.
Three kingdom scheme
- Eukarya (eukaryotes) – multi-celled organisms
- Bacteria (common bacteria) – single celled organisms, of which proteobacteria
(mitochondria) and cyanobacteria (chloroplasts) were derived.
- Archaea – (rare bacteria) including thermophiles, sulfur oxidizers, that are not
susceptible to antibiotics.
The main difference between gram positive and gram negative is that the gram positive has
the peptidoglycan layer. It does exist in gram negative, but it is much thinner.
,To grow and multiply, a microbial cell must obtain nutrients faster than its competitors (or
share them for mutual advantage, like symbiotes with plants).
Most bacteria share these 3 traits:
- Thick, complex outer envelope (cell wall or membrane, with or without thick
peptidoglycan layer).
- Compact genome.
- Tightly coordinated functions.
In contrast:
- Archaea have unique membrane and envelope structures to allow survival in
extremes.
- Eukaryotic cells have extensive membranous (internal) organelles.
Cell fractionation
- It is possible to separate components of a cell based on their mass.
- Ultra-centrifugation techniques (high speed) are used to pellet heavier components,
while leaving the lighter components in suspension.
- After removing the heavier pellet, the centrifuge speed is increased, and a lighter
component is next pelleted. (repeated for all interesting components).
- Protein separation in gels (SDS-PAGE) works in a similar manner (diffusion, rather
than centrifugation).
Membrane components
- Membrane lipids (phospholipids) form a bilayer that separates water environments
of the internal cell and surrounding exterior environment.
- Membrane proteins:
Provide structural support
Detect environmental signals
Secrete virulence factors and communication signals
Function with ion transport and energy storage
- Thick peptidoglycan structure is a target for several antibiotics (against gram positive
bacteria).
Methods of transport across the membrane:
- Passive diffusion – small, uncharged molecules
- Osmosis – concentration of water balance (internal and external) known as osmotic
pressure.
- Membrane permeant weak acids/bases – cross in uncharged form.
- Transmembrane ion gradients – require transfer proteins to move across membrane
Passive – move according to concentration gradient
Active – move against concentration gradient and requires energy
Nutrient supplies limit growth
- Bacteria have a simple purpose: survive and growth
- In the absence of nutrients, bacteria go into survival mode
- In the abundance of nutrients, bacteria go into a replication mode
- Heterotrophy uses nutrients to produce CO2
- Autotrophy uses CO2 to produce nutrients
,Bacteria are grown in culture medium
- Pure cultures are bacterial suspensions derived from a single bacterial colony in a
liquid growth medium, grown beyond the exponential phase.
- After growth, pure cultures are placed on solid growth medium (agar plates) to
confirm a single colony characteristic (purity of culture).
- To ensure single colonies are visible for analysis, dilution streaking technique is used.
Choosing the proper medium
- Minimum defined medium provides the bare minimum to reproduce
- Complex medium provides many building blocks bacteria would have needed to
produce themselves (more varieties may grow)
- Selective medium limits growth of bacteria not wanted by the researcher (can
contain antibiotics)
- Differential medium provides visual cues that differ between colonies to allow for
visual identification.
Quantifying bacterial counts
- Under a microscope counts live and dead bacteria per unit area
- Dilution plating counts viable colonies, assumed to be from a single bacteria –
designated by colony forming units (CFU) per volume or per unit area
- Optical density (OD) should be used with a standard curve of that bacterial
suspension in the growth medium provided. McFarland standards are used as an
estimated concentration.
- Biochemical assays (quantify DNA, ATP, etc.) to then translate to counts.
A biofilm is a community of bacteria embedded in a matric of extracellular polymers
associated with a surface.
Lecture 2:
, The green line represents the linear rate constant.
If the environment conditions are favorable, then they will go in replication mode and when
they are unfavorable they go into survival mode.
Physical, chemical and biological control of microbes:
- Sterilization: all living cells, spores and viruses are destroyed on an object.
- Disinfection: killing or removal of disease producing organisms from inanimate
surfaces.
- Antisepsis: killing or removal of pathogens from living tissues.
- Sanitation: reducing the microbial population to safe levels (cleaning and disinfecting,
typical)
Bacteriostatic – inhibits growth (number of viable cells remains near constant)
Bactericidal – kill (number of viable cells reduced)
Germicidal – kill pathogens, but not spores
Physical agents that kill microbes:
- High temperature and pressure (autoclave)
Boiling water reaches 100 degrees
With high pressure, autoclaves reach 121 degrees at 15 psi. 20 minutes under
these conditions are thought to kill all spores except those of some
thermophiles (but thermophiles are not pathogenic).
- Pasteurization (heating of product)
Used in dairy – long enough to kill cause of Q fever, but not all.
- Cold – slow growth and preserves strains (ultra-low cold storage at -80 degrees).
- Filtration – microbes will not be able to pass through < 0.2 micrometer pores (but not
viruses.
- Irradiation – UV light exposure will damage bacterial DNA.
Chemical agents to kill microbes:
- The choice of chemical agent requires knowledge of factors:
Presence of organic matter
Lecture 1:
A microbe is a living organism that can be seen by a microscope that range from millimeters
in size to 0.2 micrometers.
Link between infection and disease (bacteria):
1. The microbe is found in all cases of the disease but is absent from the healthy
individual.
2. The microbe is isolated from the diseased host and grown in pure culture.
3. When the microbe is introduced into a healthy, susceptible host, the host shows the
same disease.
4. The same strain of microbe is obtained from the newly diseased host. When cultured,
it shows the same characteristics as before.
Vaccines work against viruses, in that they utilize the virus infection mechanism to produce
the antibodies against them. – ineffective against live bacteria.
Antiseptics come in two forms: physical and chemical
- Physical antiseptics usually include heat (burner flame, autoclave)
- Chemical antiseptics are molecules kill not only bacteria, but also the host if ingested.
Antibiotics are chemicals that would kill the microbes but leave the host unharmed. Due to
host variability this has been adapted for killing >99.9% of microbes.
Supporting ecosystems
- Aerobic environments are abundant with oxygen
- Microaerophilic environments have reduced oxygen levels and may led to multiple
forms of metabolism.
Capnophilic (CO2 loving) environments are a form of microaerophilic
environments.
- Anaerobic environments are lacking oxygen.
- Strict anaerobic environments are completely void of oxygen (oxygen is lethal).
- Within a growing biofilm, any or all of these environments can exist simultaneously.
If you don’t have chloroplast, you have an animal cell.
Three kingdom scheme
- Eukarya (eukaryotes) – multi-celled organisms
- Bacteria (common bacteria) – single celled organisms, of which proteobacteria
(mitochondria) and cyanobacteria (chloroplasts) were derived.
- Archaea – (rare bacteria) including thermophiles, sulfur oxidizers, that are not
susceptible to antibiotics.
The main difference between gram positive and gram negative is that the gram positive has
the peptidoglycan layer. It does exist in gram negative, but it is much thinner.
,To grow and multiply, a microbial cell must obtain nutrients faster than its competitors (or
share them for mutual advantage, like symbiotes with plants).
Most bacteria share these 3 traits:
- Thick, complex outer envelope (cell wall or membrane, with or without thick
peptidoglycan layer).
- Compact genome.
- Tightly coordinated functions.
In contrast:
- Archaea have unique membrane and envelope structures to allow survival in
extremes.
- Eukaryotic cells have extensive membranous (internal) organelles.
Cell fractionation
- It is possible to separate components of a cell based on their mass.
- Ultra-centrifugation techniques (high speed) are used to pellet heavier components,
while leaving the lighter components in suspension.
- After removing the heavier pellet, the centrifuge speed is increased, and a lighter
component is next pelleted. (repeated for all interesting components).
- Protein separation in gels (SDS-PAGE) works in a similar manner (diffusion, rather
than centrifugation).
Membrane components
- Membrane lipids (phospholipids) form a bilayer that separates water environments
of the internal cell and surrounding exterior environment.
- Membrane proteins:
Provide structural support
Detect environmental signals
Secrete virulence factors and communication signals
Function with ion transport and energy storage
- Thick peptidoglycan structure is a target for several antibiotics (against gram positive
bacteria).
Methods of transport across the membrane:
- Passive diffusion – small, uncharged molecules
- Osmosis – concentration of water balance (internal and external) known as osmotic
pressure.
- Membrane permeant weak acids/bases – cross in uncharged form.
- Transmembrane ion gradients – require transfer proteins to move across membrane
Passive – move according to concentration gradient
Active – move against concentration gradient and requires energy
Nutrient supplies limit growth
- Bacteria have a simple purpose: survive and growth
- In the absence of nutrients, bacteria go into survival mode
- In the abundance of nutrients, bacteria go into a replication mode
- Heterotrophy uses nutrients to produce CO2
- Autotrophy uses CO2 to produce nutrients
,Bacteria are grown in culture medium
- Pure cultures are bacterial suspensions derived from a single bacterial colony in a
liquid growth medium, grown beyond the exponential phase.
- After growth, pure cultures are placed on solid growth medium (agar plates) to
confirm a single colony characteristic (purity of culture).
- To ensure single colonies are visible for analysis, dilution streaking technique is used.
Choosing the proper medium
- Minimum defined medium provides the bare minimum to reproduce
- Complex medium provides many building blocks bacteria would have needed to
produce themselves (more varieties may grow)
- Selective medium limits growth of bacteria not wanted by the researcher (can
contain antibiotics)
- Differential medium provides visual cues that differ between colonies to allow for
visual identification.
Quantifying bacterial counts
- Under a microscope counts live and dead bacteria per unit area
- Dilution plating counts viable colonies, assumed to be from a single bacteria –
designated by colony forming units (CFU) per volume or per unit area
- Optical density (OD) should be used with a standard curve of that bacterial
suspension in the growth medium provided. McFarland standards are used as an
estimated concentration.
- Biochemical assays (quantify DNA, ATP, etc.) to then translate to counts.
A biofilm is a community of bacteria embedded in a matric of extracellular polymers
associated with a surface.
Lecture 2:
, The green line represents the linear rate constant.
If the environment conditions are favorable, then they will go in replication mode and when
they are unfavorable they go into survival mode.
Physical, chemical and biological control of microbes:
- Sterilization: all living cells, spores and viruses are destroyed on an object.
- Disinfection: killing or removal of disease producing organisms from inanimate
surfaces.
- Antisepsis: killing or removal of pathogens from living tissues.
- Sanitation: reducing the microbial population to safe levels (cleaning and disinfecting,
typical)
Bacteriostatic – inhibits growth (number of viable cells remains near constant)
Bactericidal – kill (number of viable cells reduced)
Germicidal – kill pathogens, but not spores
Physical agents that kill microbes:
- High temperature and pressure (autoclave)
Boiling water reaches 100 degrees
With high pressure, autoclaves reach 121 degrees at 15 psi. 20 minutes under
these conditions are thought to kill all spores except those of some
thermophiles (but thermophiles are not pathogenic).
- Pasteurization (heating of product)
Used in dairy – long enough to kill cause of Q fever, but not all.
- Cold – slow growth and preserves strains (ultra-low cold storage at -80 degrees).
- Filtration – microbes will not be able to pass through < 0.2 micrometer pores (but not
viruses.
- Irradiation – UV light exposure will damage bacterial DNA.
Chemical agents to kill microbes:
- The choice of chemical agent requires knowledge of factors:
Presence of organic matter
