Exam #1 Flashcards
Species
A group of organisms capable of interbreeding and producing fertile offspring.
Species: a group of closely related isolates or strains (microorganisms)
Strain/isolate: a subgroup within a species: operational taxonomic unit (OTU)
members of the microbial world
All living things can be classified into one of three groups, or domains
Bacteria
Archaea
Eukarya
Organisms in each domain share certain important properties
Two basic cell structures
Prokaryotes do not have a membrane-bound nucleus
Eukaryotes have a membrane-bound nucleus and organelles
domain Bacteria
Single-celled prokaryotes
= “prenucleus”
no membrane-bound nucleus
no other membrane-bound organelles
DNA in nucleoid
most have specific shapes (rod, spherical, spiral)
rigid cell wall contains peptidoglycan (unique to bacteria)
multiply via binary fission
many move using flagella
domain Archaea
like Bacteria, Archaea are prokaryotic
similar shapes, sizes, and appearances to Bacteria
multiply via binary fission
may move via flagella
rigid cell walls
However, major differences in chemical composition
cell walls lack peptidoglycan
ribosomal RNA sequences different
Many are extremophiles
high salt concentration, temperature
domain Eukarya
eukaryotes = “true nucleus”
membrane-bound nucleus and other organelles
more complex than prokaryotes
microbial members include fungi, algae, protozoa
algae and protozoa also termed protists
some multicellular parasites including helminths (roundworms, tapeworms)
Fungi
diverse group
single-celled (yeasts) or multicellular (molds, mushrooms)
energy from degradation of organic materials
primarily live on land
Algae
diverse group
single-celled or multicellular
photosynthetic
contain chloroplasts with
chlorophyll or other pigments
primarily live in water - rigid cell walls
many have flagella
cell walls, flagella distinct from those of prokaryotes
protozoa
diverse group
single-celled
complex, larger than prokaryotes
most ingest organic compounds as food sources
no rigid cell wall
most motile
Helminths
Parasitic helminths are worms that live at expense of a host
adult stage can be seen without magnification eggs and larvae - microscopic
helminths include roundworms, tapeworms, flukes.
acellular infectious agents
viruses, viroids, prions
not alive
not microorganisms, so general term microbe often used to include them
Viruses
nucleic acid packaged in protein coat
variety of shapes
infect living cells, termed hosts
multiply using host machinery, nutrients
inactive outside of hosts: obligate intracellular parasites
all forms of life can be infected by different types of viruses
Viroids
simpler than viruses
require host cell to replicate
single short piece of RNA
no protective protein coat
cause plant diseases
some scientists speculate they may cause diseases in humans
- no evidence yet
Prions
infectious proteins: misfolded versions of normal cellular proteins found in brain
misfolded version forces normal version to misfold
abnormal proteins bind to form fibrils
cells unable to function
cause several neurodegenerative
diseases in humans, animals
resistant to standard sterilization
procedures
Prion protein
PRNP gene encodes a protein called prion protein (PrP), which is active in the brain and several other tissues.
precise function of protein unknown
proposed roles in several important processes:
-transport of copper into cells
-protects brain cells (neurons) from injury (neuroprotection)
Theory of spontaneous generation
Theory of Spontaneous Generation
“organisms can arise from non-living matter”
Theory had its detractors
Francesco Redi
Louis Pasteur
John Tyndall
… each contributed to disproving the theory…
Francesco Redi
Italian biologist and physician
~1668 – demonstrated that worms found on rotting meat came from eggs of flies landing on meat
-proved this by placing rotting meat in jars:
-left one jar open
-covered one jar with fine gauze
-and another with parchment
Flies could only enter the uncovered jar, and in this, maggots appeared.
In the jar that was covered with gauze, maggots appeared on the gauze but did not survive.
No flies or maggots in the jar covered with parchment.
French chemist Louis Pasteur
Considered father of modern microbiology
~1860’s – demonstrated that air is filled with microorganisms
Proved this by filtering air in cotton plug
->Identified organisms in cotton as same organisms contaminating broths
Pasteur developed swan-necked flask
boiled infusions remained sterile despite opening to air
ended arguments that unheated air or broths contained “vital force” necessary for spontaneous generation
John Tyndall 1850’s
Irish physicist
Tyndall concluded different infusions
required different boiling times
-Some infusions were sterile after boiling five minutes…others not sterile after five hours of boiling
Attributed contamination to a heat-resistant life-form called endospore
Endospores
Bacterial genera that form endospores include Bacillus and Clostridium.
Robert Koch
Robert Koch - supporting the GERM THEORY OF DISEASE
Experimental support for the concept of infectious disease – in 1876:
Koch discovered formation of endospores in Bacillus anthracis
Koch’s work with anthrax notable for
being first to link a specific microorganism with a specific disease
rejecting idea of SPONTANEOUS GENERATION and the MIASMA THEORY
miasma - a noxious form of “bad air” also known as night air
Koch showed that Bacillus anthracis caused anthrax
The bacterium could be observed in the tissue of anthrax victims
He extracted bacterium from sheep which had died of anthrax, grew it , injected a mouse with it …..
The mouse developed the disease as well.
Koch repeated this process in over 20 generations of mice, then he announced in 1876 that he had proved this bacterium caused anthrax.
Anthrax is caused by Bacillus anthracis
Koch’s postulates
The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.
The microorganism must be isolated from a diseased organism and grown in pure culture.
The cultured organism should cause disease when introduced into a healthy organism.
The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.
golden age of microbiology
As spontaneous generation was disproved,
Golden Age of Microbiology was born
The principle that microorganisms cause diseases is known as Germ Theory of Disease.
most pathogenic bacteria identified (1875–1918)
work on viruses began
understanding that microscopic agents could cause disease led to control efforts
huge improvements in past century in human health
antibiotics to treat infectious diseases
vaccines to prevent diseases
applications of microbiology
biodegradation: degrade PCBs, DDT, trichloroethylene and others
help clean up oil spills
bioremediation: using microorganisms to hasten decay of pollutants
Cellulose
Plant cellulose, which makes up the cell walls of most plants, is tough and mesh-like; cellulose fibrils are primary architectural elements.
Bacterial cellulose has the same molecular formula as plant cellulose, but it has significantly different macromolecular properties and characteristics:
it is more chemically pure
higher water holding capacity
greater tensile strength resulting from more polymerization
ultrafine network architecture.
Bacteria synthesize valuable products
cellulose
hydroxybutyric acid (manufacture of disposable diapers and plastics)
ethanol (biofuel)
hydrogen gas (possible biofuel)
oil (possible biofuel)
insect toxins (insecticides)
antibiotics (treatment of disease)
amino acids (dietary supplements)
Insect toxins
Bacillus thuringiensis is closely related to B. cereus, a soil bacterium, and B. anthracis, cause of anthrax; the three organisms differ mainly in their plasmids. There are several dozen recognized subspecies of Bacillus thuringiensis.
Upon sporulation, B. thuringiensis forms crystals of δ-endotoxins (called crystal proteins or Cry proteins), which are encoded by cry genes. The cry genes are located on a plasmid.
B. thuringiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops.
When insects ingest toxin crystals, their alkaline digestive tracts denature the insoluble crystals, making them soluble, now able to be cut with proteases found in the insect gut, which frees the toxin. The Cry toxin then inserts into the insect gut cell membrane, forming a pore.
microorganisms as model organisms
excellent model organisms
metabolism, genetics similar to higher life-forms
all cells composed of same elements
synthesize structures in similar ways
replicate DNA
degrade foods via metabolic pathways
present and future challenges
Emerging diseases
pathogens can become resistant to antimicrobial medications (tuberculosis, malaria)
increased travel and immigration
many diseases eliminated from developed countries still exist in many parts of world (malaria, cholera, plague, yellow fever)
changes in population
weakened immune systems (elderly, HIV/AIDS)
chronic diseases may be caused by bacteria
-peptic ulcers caused by Helicobacter pylori
Compound light microscope
light is bent through lenses and enables a magnified view
view size, shape and motility of prokaryotes and unicellular eukaryotes
Iris diaphragm
controls the diameter of the light beam
condenser
focuses the light on the sample on your slide
refraction
Refraction is the bending of light as it passes from one medium to another.
air <-> glass: different refractive index
oil<-> glass: same refractive index
More light passing through your sample gives you better resolution.
Resolution
resolution: ability to clearly distinguish two objects that are close together together
light microscope resolution:~ 0.3µm
electron microscope resolution: ~0.3nm
resolution is affected by:
-quality and type of lens
-wavelength of light
-preparation of sample
Calculate magnifying power
I = 340/d
where I is the magnifying power
d is the diameter of the sphere expressed in mm.
For example with a sphere of 1,7 mm of diameter you will obtain about a magnification of 200 X.
Maintaining cell shape
Bacterial cell walls maintain cell shape and rigidity and protect the cells from bursting due to osmotic pressure (turgor pressure).
Bacterial cell walls are composed of peptidoglycan (but Archeal cell walls are not!).
Spheroplasts
Disruption of the cell wall of rod-shaped Bacillis species or Escherichia coli with lysozyme or penicillin results in formation of round, osmotically sensitive cells (SPHEROPLASTS)
after microbe’s cell wall digested, membrane tension causes cell to acquire spherical shape
spheroplasts - osmotically fragile, lyse in hypotonic solution
cytoskeletal elements and cell shape
FtsZ –forms part of ring in the middle of dividing cell required for constriction of cell membrane and cell envelope to yield two daughter cells. FtsZ can polymerise; bacterial homolog of Eukaryotic cytoskeletal component tubulin
MreB - bacterial homolog of the Eukaryotic cytoskeletal component actin
Crescentin is an intermediate-filament-like protein with an essential role in the curved-rod shape of Caulobacter crescentus
dyes
basic dyes (positive charge)
attracted to negatively charged cellular components
acidic dyes (negative charge)
negative staining:
cells repel,
so dye colors background
bacterial stains
simple staining - involves one dye
differential staining - used to distinguish between different groups of bacteria
Gram stain is a differential stain that distinguishes between
Gram-positive bacteria and Gram-negative bacteria
special stains (capsule, endospore, flagella) also useful
Gram stains
Gram stain reflects fundamental difference in cell wall structure
separates most bacteria into two groups:
Gram stain distinguishes between Gram-positive bacteria (purple)
& Gram-negative bacteria (pink)
Steps in gram staining
- CRYSTAL VIOLET (PRIMARY STAIN)
Cells stain purple - IODINE (MORDANT)
Cells remain purple - ALCOHOL (DECOLORIZER)
Gram positive:purple
Gram negative: colorless - SAFRANIN (COUNTERSTAIN)
Gram positive: purple
Gram negative: pink
differential stain: acid fast stain
Mycobacterium genus: cell wall has mycolic acid – waxy fatty acid prevents uptake of dyes then resists decolorization.
tuberculosis
Mycobacterium tuberculosis:
leprosy - chronic infectious disease caused by Mycobacterium leprae, an acid-fast, rod-shaped bacillus. Leprosy curable – treatment in early stages averts disability.
multi-cellular arrangements
Plane refers to the orientation of the septum during division.
If division occurs on one plane a chain is formed (strepto- Greek: twisted chain)
Perpendicular division planes cause packets to form: tetrads and sarcinae
Random division planes causes clusters to form (staphylo- Greek: grapelike cluster)
Prokaryotic cells divide by binary fission
Origin of replication (OriC) is replicated
oriC and newly synthesized DNA move to opposite ends of cell
Plasma membrane and cell wall form septum
(a partition separating two chambers)
2 identical daughter cells are formed
Surface-volume ratio of a cell
Surface area is the area around the cell membrane
Volume is the space inside the cell
The ratio is the surface area divided by the volume
As radius of cell increases 1x to 3x,
surface area increases from 1x to 9x
volume increases from 1x to 27x
As radius of cell increases 1x to 3x, surface area increases from 1x to 9x, volume increases from 1x to 27x
The smaller the cell, the greater the surface-to-volume ratio.
As the cell gets bigger, you have less surface area per unit of volume.
.
typical structures of a prokaryotic cell
cytoplasmic membrane
phospholipid bilayer: essential part of cytoplasmic membrane - separates internal contents of cell from outside environment
embedded integral membrane proteins - communication, transport
cytoplasmic membrane lipids
—>Bacterial, Eukaryotic cell membrane: fatty acids linked to glycerol by ester linkage
Bacteria (and Eukaryotes):
glycerol moiety is ester-linked to glycerol-3-phosphate backbone
Archeal membrane
Archeal cytoplasmic membrane:
Hydrocarbons linked to glycerol by ether linkage
Archaeal cell membranes are chemically different from all other living things, including a “backwards” glycerol molecule and isoprene derivatives in place of fatty acids.
Archea: isoprenoid side chains are ether-linked to an glycerol-1-phosphate moiety
b. monolayer-forming tetra-ether lipids:
glycophospholipid from thermoacidophilic Thermoplasma acidophilum - heat resistant
c. bilayer formed of archaeal diether lipids, found in order Halobacteriales
Membrane proteins
Within phospholipid bilayer
some membrane proteins function as selective gates and/or sensors of environmental conditions
plasma membrane: a selectively permeable barrier
cytoplasmic membrane and energy transformation
electron transport chain
uses energy from electrons to move protons out of cell
creates electrochemical gradient across membrane
energy called proton motive force
harvested to drive cellular processes including
ATP synthesis, some forms of transport, and motility
protons outside
hydroxide ions inside
Proton motive force
ejection of protons creates electro-chemical gradient
used to synthesize ATP, power transporters and flagella
Plasma membrane
a selectively permeable barrier – directed movement through selective gates
Highly specific transport system - carriers transport a certain molecule type.
directed movement of molecules across cytoplasmic membrane
facilitated diffusion: form of passive transport
movement down gradient; no energy required
not typically useful in low-nutrient environments
active transport: requires energy*
movement against gradient
two main mechanisms:
transporters use proton motive force
transporters use ATP (ABC transporter) ATP Binding Cassette
group translocation chemically alter compound phosphorylation common glucose
symport
Green circles moving against their concentration gradient through a transport protein (requires energy)
Yellow circles move down their concentration gradient (releases energy). The movement is coupled.
antiport
Blue circles moving against their concentration gradient through a transport protein (requires energy)
Yellow circles move down their concentration gradient (releases energy).
The movement is coupled.
Antiporters
Many different antiporters support bacterial pH homeostasis, nutrient uptake, motility and ATP synthesis.
Both Escherichia coli and Bacillus subtilis establish a proton motive force, (PMF).
The PMF is used to energize solute transport, motility and ATP synthesis.
ABC transporters
Use the energy of ATP binding and hydrolysis to transport substances across cell membranes.
ABC transporters consist of trans-membrane domains which determine specificity of the transporter, and cytoplasmic ATP-binding domains.
Can export and import substances across the cytoplasmic membrane.
Importers also have a high-affinity binding protein that recognizes the substrate in the periplasm and delivers it to the transporter.
Prokaryotic cell structure
Cell membranes
Cell wall
peptidoglycan
Outer structures:
Capsule
Flagella and bacterial motility
Pili
Internal structures
Nucleoid
Plasmids
Ribosomes
Storage granules
Endospores
Cell wall-peptidoglycan
Only bacteria have a peptidoglycan cell wall
peptidoglycan structure
Lysozyme hydrolyses the glycosidic bonds that link NAM and NAG
peptidoglycan structure
inhibition of cell wall biosynthesis by antibiotics
Penicillin inhibits formation of crosslinks in peptidogycan wall
binds enzyme: transpeptidase (penicillin binding protein)
transpeptidase forms tetra-peptide crosslinks between adjacent glycan chains
Penicillin becomes covalently linked to the enzyme’s active site - inhibits it, irreversibly
Why does penicillin kill only actively multiplying cells, while lysozyme kills cells in any stage of growth?
Penicillin will kill only cells that are actively synthesizing peptidoglycan (cells that are growing) because it interferes with peptidoglycan synthesis.
Lysozyme breaks the bonds that join the subunits, thereby weakening the existing structure.
Gram-positive cell wall
Gram-positive cells
thick (20-80 nm) cell wall peptidoglycan layer outside plasma membrane
Gram-positive cell walls contain
teichoic acids
negatively charged; give Gram-positive bacteria a negative exterior charge
PAMP – recognition by immune system cells
lipopolysaccharide (LPS)
structure and function
LPS consists of:
O side chain - vary sugar composition in response to antibodies
lipid A portion (part of outer membrane lipid bilayer)
LPS slows entry of antibiotics and other toxins
Lipid A
toxic (endotoxin) to humans - part of LPS molecule recognized by our host defenses.
When large amounts accumulate (such as in a bloodstream infection), response by defense system itself can be deadly.
Cell wall in archaea
Archaea have several different types of cell wall. Some contain a structure reminiscent of
peptidoglycan called pseudomurein. Other microbes will have a surface layer (S-layer) composed of repeating units
of one or a few proteins, glycoproteins or sugar. These crystal lattices serve to protect the cell.
S-layers
S-layers in Archaea: glycoprotein lattices : wall component composed of subunits with pillar-like, hydrophobic trans-membrane domains, or lipid-modified glycoprotein subunits.
Some Archaea have a rigid wall layer (pseudomurein in methanogens) as intermediate layer between plasma membrane and S-layer.
In Gram-positive bacteria S-layer proteins are bound to rigid peptidoglycan-containing layer via secondary cell wall polymers.
In Gram-negative bacteria S-layer closely associated with lipopolysaccharide of outer membrane.
capsules and slime layers
Bacteria with capsules attaching to intestinal cells(TEM)
Bactria adhering to each other in a layer of slime (SEM)
glycocalyx: extracellular polymer of glycoprotein (polysaccharide)
protective outer layer
not all bacteria have one
if thick and sturdy, a capsule.
if thin and diffuse, a slime layer
Capsules
capsule considered virulence factor - enhance ability of pathogenic bacteria to
evade phagocytosis
attach to surfaces
be protected from toxins, detergents, bacteriophages
Example: Streptococcus pneumoniae capsule
assembly of capsule - steps in Streptococcus pneumoniae capsule biosynthesis
glycosyltransferases assemble oligosaccharide repeats on cytoplasmic face of membrane
Wzx flippase transports repeat units to external surface of membrane.
repeat units polymerized by Wzy capsular polymerase to form high-molecular-weight capsular polysaccharides, which are then ligated to cell wall
Flagella
MONOTRICHOUS: single flagellum at one end
Example: Caulobacter crescentus Vibrio cholerae, Pseudomonas aeruginosa, Isiomarina loihiensis
LOPHOTRICHOUS: flagella lined up at one end
Example: Vibrio fischeri, Helicobacter pylori
PERITRICHOUS: flagella are distributed all over the cell E. coli, Example: Salmonella typhimurium
SPIROCHETES: specialized flagella inside periplasm causes corkscrew motion
Example: Borerelia, Treponema and Leptospira
bacterial flagella are powered by the proton motive force, but Archea use ATP for energy
bacterial chemotaxis
A cell moves via a series of runs and tumbles.
The cell moves randomly when there is no concentration gradient of attractant or repellent.
When a cell senses it is moving toward an attractant, it tumbles (T) less frequently, resulting in longer runs (R).
What mechanism causes a cell to tumble?
flagellum bacterial propulsion-
Driven by a transmembrane proton gradient, rotates both CCW and CW
filament is helical and converts torque into thrust. The motor consists of stators or Mot complexes and a rotor or C ring, which also serves as the CCW⇄CW switch.
Archaea flagellin
Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins.
The Archaellum is a rotating Type IV pilus
After the pre-archaellin has been processed, the motor complex assembles the filament. The motor complex is formed by the ring-forming scaffold protein FlaX
The dimeric soluble domain of FlaF interacts with the S-layer.
protein appendages
pilli, fimbriae and adhesins
overview of type IV pili system
dynamic adhesive structures, major virulence determinants in several human pathogens
pilus fiber composed of pilin subunits made by prepilin peptidase
cleaved by prepilin peptidase PilD
for proper pilus assembly and function
pilin translocated across inner membrane where it forms dynamic multimeric filament
secreted via pore-forming secretin PilQ to bacterial surface
PilC - transmembrane protein on inner membrane
ATPases (PilB and PilT) mediate pilus extension and retraction
horizontal gene transfer by transformation of exogenous DNA.
DNA in the environment can be entangled by retracting type IV pili and introduced into the cell through the outer (OM) and inner membranes (IM). In the cytoplasm, the incoming DNA is integrated into the genome by homologous recombination
Pilus
structure used by bacteria during conjugation (direct contact)
transfer of genetic material between a donor and a recipient cell
Plasmid DNA forms a mating bridge
Pili works in attaching to naked DNA, other cells, and cellulose (to move around).
Pili also retract and extend
adhesins
cell-surface components on pili that facilitate adhesion to other surfaces
Nucleoid
The nucleoid is a chromatin-dense area within the cytoplasm and contains the bacterial DNA, associated proteins and RNA that are responsible for controlling the bacteria’s activity and reproduction
Binary fission
The bacterial chromosome is packed tightly
Although there are no histones in prokaryotes, other bacterial proteins condense the bacterial chromosome within the cell.
Ribosomes
Site of protein synthesis
Prokaryotic ribosome: 70S ribosome composed of 30S and 50S subunits
Eukaryotic ribosome: 80S - composed of 40S and 60S subunits
Ribosomes in translation
consist of RNA and protein
differences between bacterial and eukaryotic ribosomes exploited to create antibiotics that specifically target bacterial ribosomes
similarities and differences between DNA sequences that encode small subunit of ribosomal RNA are used to identify organisms and to create phylogenies
16S rRNA genes are sequenced and compared in Bacteria and Archea
18S rRNA genes are sequenced and compared in Eukaryotes.
Storage granules
accumulations of polymers synthesized from a nutrient a cell has in excess.
Endospores
dormant cell type formed by species of Bacillus and Clostridium
(anthrax, botulism, food poisoning)
resist: high temperatures (including boiling), most disinfectants, low energy radiation, desication, UV light
can survive many years until an environmental stimulus triggers germination
germinating endospores exit dormant stage to become typical multiplying cell (vegetative cell).
Antibiotics
attack essential molecular
machines in bacteria, stopping
or slowing their action, ultimately
slowing growth or killing the cell.
IN CELL WALL
BETA-LACTAM ANTIBIOTICS
such as penicillin and
methicillin, contain an extremely
reactive beta-lactam ring that
attacks BPs (penicillin-binding
proteins) that build the cell wall.
VANCOMYCIN
sequesters the building
blocks of the cell wall so
that they can no longer
be crosslinked to form a
tough protective layer.
IN CYTOPLASM
MACROLIDES and
AMINOGLYCOSIDES
attack ribosomes, blocking
manufacture of new proteins.
FUSIDIC ACID glues
elongation factor G
(EF-G) to ribosomes,
stalling protein synthesis.
RIFAMPICIN, QUINOLINES
and ANTIFOLATES attack
essential enzymes
in bacteria.
endocytosis in Eukaryotic cells
Phagocytosis: pseudopodium is made when cell membrane pinches to trap a solid particle and a phagosome is made (food vacuole)
Pinocytosis: membrane pinches to make a vesicle that traps extracellular fluid
Receptor mediated endocytosis: receptors trap particles and a coated vesicle is made
Endosymbiont theory
Endosymbiont theory
states that the ancestors of mitochondria as well as chloroplasts were bacteria residing within other cells in a mutually beneficial partnership.
The intracellular bacterium in such a partnership is called an endosymbiont. As time went on, the endosymbiont lost key features sucha s a cell wall amd the ability to replicate independently.
Mitochondria multiply by elongating and then dividing (binary fission).
Plastids (bacteria) also became plants (eukaryotes)
Phagocytosis
the process by which a cell engulfs a solid particle to form an internal vesicle (phagosome)
phagocytosis - specific form of endocytosis involving vesicular internalization of bacteria
In immune system, a major mechanism used to remove pathogens.
prokaryotic cells divide by binary fission
oriC is the region where replication is initiated
septum is the division site - division is initiated by assembly of the tubulin homologue FtsZ into a membrane‐tethered ring‐like structure
- Origin of replication (OriC) is replicated
- oriC and newly synthesized DNA move to opposite ends of cell
- Plasma membrane and cell wall form septum
(a partition separating two chambers) - 2 identical daughter cells are formed
Bacillus subtilis has two alternative life cycles
a | The vegetative life cycle. In favorable conditions Bacillus subtilis elongates, replicates its chromosome and divides by binary fission.
b | Bacillus subtilis can develop a highly resistant, dormant cell to survive harsh environmental conditions.
When conditions improve the endospore germinates and B. subtilis re-enters vegetative life cycle.
Growth
exponential growth: population doubles each division
generation time - time it takes for population to double
varies among species
environmental conditions
growth can be calculated:
Nt = N0 x 2n
Nt = number of cells in population at time t
N0 = initial number of cells
n = number of generations at that point
example: pathogen in potato salad at picnic in sun
assume 10 cells with 20 minute generation time
N0 = 10 cells in original population
n = 12 (3 divisions per hour for 4 hours)
Nt = N0 x 2n = 10 x 212
Nt = 10 x 4,096
Nt = 40,960 cells of pathogen in 4 hours!
Power of exponential growth
rapid generation time with optimal conditions can yield huge populations quickly
remember that generation time depends on species and growth conditions
Pure culture
pure culture is defined as a population of cells derived from a single cell
pure culture obtained using aseptic technique
minimizes potential contamination
cells grown on culture medium
contains nutrients dissolved in water
broth (liquid) or solid gel
growing microorganisms on solid medium
…need culture medium, container, aseptic conditions, method to separate individual cells
with correct conditions, single cell will multiply
form visible colony (~1 million cells easily visible)
agar used to solidify
not destroyed by high temperatures and can be sterilized
liquifies above 95°C
solidifies below 45°C
few microbes can degrade
growth in Petri dish
allows air
excludes contaminants
culture medium in
a Petri dish called a plate
Colony
mass of cells arising from a single cell by binary fission to produce clonal copies of that cell
Pure culture obtained using aseptic technique - minimizes potential contamination
-bacterial sample diluted on the plate by process called “streaking”
-wire loop, sterilized by heating used to take sample, and make a streak on the agar dish
-repeated second, third, and sometimes a fourth time
result: individual bacterial cells isolated on plate, which then divide and grow into single “clonal” bacterial colonies
Pure culture is defined as a population of cells derived from a single cell
prokaryotic growth in laboratory conditions
prokaryotes grown on agar plates or in tubes or flasks of broth
closed systems
nutrients not renewed; wastes not removed
termed batch cultures
yields characteristic growth curve
Lag phase
number of cells does not increase
begin synthesizing enzymes required for growth
delay depends on conditions
Log phase
cells divide at constant rate
generation time can be measured during active division
most sensitive to antibiotics
production of primary metabolites
Stationary phase
variable length, depends on species and environment
nutrient levels too low to sustain growth
total numbers remain constant
some die, release contents; others grow
secondary metabolite production occurs - nutrients depleted and wastes accumulate
Metabolites
Primary metabolites are directly involved in the normal growth, development, and reproduction of the microoganism.
Secondary metabolites (late log/stationary phase) are not involved in normal growth, but are organic compounds that may have a more ecological or relational function. Streptomycin (Streptomyces sp.) and penicillin (fungi) are examples of important secondary metabolites.
Death phase
total number of viable cells decreases
cells die at constant rate
exponential, but usually much slower than cell growth
phase of prolonged decline
some fraction may survive
adapted to tolerate worsened conditions
colony growth
colonies and liquid cultures share similarities
important differences based on location
-position of single cell determines its environment
-edge of colony has O2, nutrients
center of colony has depleted O2, nutrients
-accumulation of potentially toxic wastes including acids
colony may range from exponential growth at edges to death phase in center
continuous culture
open system required to maintain continuous growth
-termed continuous culture
-nutrients added, wastes removed continuously
chemostats can maintain continuous growth
continually drips fresh medium into culture in chamber
equivalent volume removed
-contains cells, wastes, spent medium
nutrient content and speed of addition can be controlled
-achieve constant growth rate and cell density
What would the growth curve of bacteria grown in a chemostat look like?
Nutrients added, wastes removed continuously
Nutrient content and speed of addition can be controlled
Achieves constant growth rate and cell density
steady state is reached ~ 3.7 hours after start up in this example.
steady state equilibrium - point where cells will not grow any faster in the system.
control factors:
pH level, temperature, dissolved oxygen level, dilution rate, and agitation speed.
microbial growth in nature
in nature, organisms grow as members of mixed communities and biofilms
nutrients usually in short supply
direct analysis of DNA from environmental samples provides more information than culture
prokaryotes regularly grow in close association, many different species together
their interaction can be cooperative
metabolic waste of one can serve as nutrient for other
or, their interaction can be competitive
some bacteria synthesize toxins to inhibit competitors
rate of multiplication in a community
Because of logarithmic growth, small differences in generation times quickly produce very large differences in total numbers of cells
The ability of a microbe to compete successfully for a habitat is often related to the rate at which it multiplies
Antagonism also helps determine community makeup
bacteriocins
bacteriocins kill closely related strains:
They are ribosomally synthesized antimicrobial peptides produced by bacterial species to inhibit the growth of other closely related bacteria. The producer strain also encodes an immunity protein to protect itself from its own bacteriocin.1
microbial growth in nature
microorganisms historically studied in laboratory but dynamic, complex conditions in nature have profound effect on microbial growth, behavior
cells sense changes, adjust to surroundings, synthesize compounds useful for growth
bacteria in low-nutrient environments common in nature; include lakes, rivers, streams
microorganisms that can grow in dilute aqueous solutions are widespread; most grow in biofilms polysaccharide-encased communities
extract trace nutrients absorbed by water from air or absorbed onto the biofilm
Biofilms cause slipperiness of
rocks, in sink drains, scum in
toilet bowls, dental plaque
biofilms
biofilms have characteristic architecture
channels through which nutrients and wastes pass
*cells communicate by synthesizing chemical signals
biofilms have important implications
dental plaque leads to tooth decay, gum disease
most infections (ear infections, cystic fibrosis)
industrial concerns: accumulations in pipes, drains
biofilm structure shields microbes growing within
may be hundreds of times more resistant to disinfectants
biofilms can also be helpful
bioremediation, wastewater treatment
Biofilm
bacterial cells living within a biofilm:
growth of aerobic organisms can deplete O2, create microzones where obligate anaerobes can grow
fermenters can produce organic acids that may be metabolized by other organisms; various growth factors can also be transferred between organisms
microbes unexpected in a macroenvironment might thrive in the microenvironment
Living organisms interact with each other in symbiotic relationships: commensalism, mutualism, parasitism
environmental factors that influence microbial growth
Prokaryotes inhabit nearly all environments
some live in comfortable habitats favored by humans
some live in harsh environments
termed extremophiles; most are Archaea
Major conditions that influence growth:
temperature
atmosphere
pH
water availability
each species has well-defined temperature range
optimum growth usually close to upper end of range
psychrophiles: –5° to 15°C
found in Arctic and Antarctic
psychrotrophs: 15° to 30°C
important in food spoilage
mesophiles: 25° to 45°C
pathogens 35° to 40°C
thermophiles: 45° to 70°C
common in hot springs
hyperthermophiles: 70° to 110°C
usually members of Archaea
found in hydrothermal vents
temperature requirements
proteins of thermophiles resist denaturing
thermostability comes from amino acid sequence
number and position of bonds which determine structure
temperature and food preservation
refrigeration (~4°C) slows spoilage by limiting growth of fast-growing mesophiles
psychrophiles, psychrotrophs can still grow, but slowly
freezing preserves food; not effective at killing microbes
temperature and disease
temperature of different parts of human body differs
some microbes cause disease in certain parts
Hansen’s disease (leprosy) in coolest regions (ears, hands, feet, fingers) due to preference of M. leprae
Obligate aerobe
Grows only when O2 is available
Requires O2 for respiration
Produces superoxide dismutase and catalase
Facultative anaerobe
Grows best when O2 is available, but also grows without it.
Uses O2 for respiration, if available.
Produces superoxide dismutase and catalase
Obligate anaerobe
Cannot grow when O2 is present
Does not use O2
Does not produces superoxide dismutase and catalase
Microaerophile
Grows only if small amounts of O, are available.
Requires O, for respiration.
Produces some superoxide dismutase and catalase.
Aerotolerant anaerobe
Grows equally well with or without O2
Does not use O2.
Produces superoxide dismutase but not catalase.
Oxygen as an electron acceptor
Oxygen, aerobic respiration, electron transfer chain, oxygen as final electron receptor
(series of redox reactions - gets reduced to water)
oxygen requirements
reactive oxygen species
using O2 in aerobic respiration produces harmful reactive oxygen species (ROS) as by-products
includes superoxide (O2–) and hydrogen peroxide
damaging to cellular components
cells must have mechanisms to protect
obligate anaerobes typically do not
almost all organisms growing in presence of oxygen produce enzyme:
superoxide dismutase - inactivates superoxide by converting to O2 and H2O2
almost all also produce catalase –
converts H2O2 🡪 O2 + H2O
exception is aerotolerant anaerobes; makes for useful test
production of catalase distinguishes
Staphylococcus species (catalase +)
from
Streptococcus species (catalase -)
pH
bacteria survive a range of pH; have optimum
most maintain constant internal pH, typically near neutral
-pump out protons if in acidic environment
-bring in protons if in alkaline environment
most microbes are neutrophiles
range of pH 5 to 8; optimum near pH 7
food can be preserved by increasing acidity
H. pylori grows in stomach; produces urease to split urea into CO2 and ammonia to decrease acidity of surroundings
acidophiles grow optimally at pH below 5.5
Picrophilus oshimae has optimum pH of less than 1!
alkaliphiles grow optimally at pH above 8.5
Microorganisms and environmental changes
environmental changes often alter communities
organisms adapted to live in one environment likely not well suited
to a different one
growth, metabolism of organisms changes
nutrients become depleted, wastes accumulate
can bring about succession of bacterial species
succession in raw milk is example…
Production of acid causes souring and encourages growth of yeasts and molds.
Eventually, bacteria digest the proteins, causing putrefaction.
water availability
all microorganisms require water for growth
dissolved salts, sugars make water unavailable to cell
If solute concentration is higher outside of cell, water diffuses out (osmosis)
some microbes withstand or even require high salt
halotolerant: withstand up to 10% (Staphylococcus)
halophiles: require high salt concentrations
marine bacteria ~3%
extreme halophiles ≥ 9%
(Dead Sea, Utah’s salt flats)
nutritional factors that influence microbial growth
prokaryotes have remarkable metabolic diversity
require nutrients to synthesize cell components
lipid membranes, cell walls, proteins, nucleic acids
made from subunits:
phospholipids, sugars, amino acids, nucleotides
major elements like carbon, hydrogen, oxygen, nitrogen,
sulfur, phosphorus, potassium, magnesium, calcium, and iron make up cell components
carbon source distinguishes different groups:
->heterotrophs use organic molecules as carbon source
->autotrophs use inorganic carbon: CO2 (carbon fixation)
nitrogen required for amino acids, nucleic acids
many use ammonia (some convert nitrate to ammonia)
nitrogen fixation important
iron, phosphorus often limiting
trace elements usually available
(cobalt, zinc, copper, molybdenum, manganese)
nutritional requirement for iron
Bacterial iron sources include host proteins (transferrin, lactoferrin, heme, siderophores)
These iron sources are transported into a Gram-negative cell via outer membrane receptors,
periplasmic binding proteins (PBP),
ATP-binding cassette (ABC) transporters
Growth factors
some microbes can’t synthesize certain molecules
fastidious: have complicated nutritional requirements
amino acids, vitamins, purines, pyrimidines
only grow if these growth factors are available
reflects biosynthetic capabilities
-E. coli synthesizes all cellular components from glucose, has wide metabolic capabilities
-Neisseria unable to synthesize many, requires numerous growth factors
Energy sources
energy sources:
sunlight, chemical compounds
phototrophs obtain energy from sunlight
plants, algae, photosynthetic bacteria
chemotrophs extract energy from chemical compounds
mammalian cells, fungi, many types of prokaryotes
sugars, amino acids, fatty acids common sources
chemolithotrophs use inorganic chemicals
such as hydrogen sulfide (H2S), hydrogen gas (H2)
Photoautotroph
Energy source: sunlight
Carbon source: CO2
Photoheterotroph
Energy source: sunlight
Carbon source: organic compounds
Chemolithoautotroph
Energy source: Inorganic chemicals (Ha, NH3, NO,”, Fe?*, HyS)
Carbon source: CO2
Chemoorganoheterotroph
Energy source: Organic compounds (sugars, amino acids, etc.)
Carbon source: Organic compounds
cultivating microorganisms in the laboratory
complex media contain a variety of ingredients
exact composition highly variable
often a digest of proteins
chemically defined media
composed of exact amounts of pure chemicals
used for specific research experiments
usually buffered
cultivating prokaryotes in the laboratory
special types of culture media
useful for isolating and identifying a specific species
selective media inhibit growth of
certain species
differential media contain substance that microbes change in identifiable way
providing appropriate atmospheric conditions
aerobic
most obligate aerobes and facultative anaerobes can be incubated in air (~20% O2)
-broth cultures shaken to provide maximum aeration many medically important bacteria (Neisseria, Haemophilus) grow best with increased CO2
some are capnophiles, meaning require increased CO2
one method is to incubate in candle jar
microaerophilic
require lower O2 concentrations than achieved by candle jar
can incubate in gas-tight container with chemical packet
chemical reaction reduces O2 to 5–15%
Anaerobic atmosphere conditions
anaerobic: obligate anaerobes sensitive to O2
anaerobic containers useful if microbe can tolerate brief O2 exposures; can also use semisolid culture medium containing reducing agent (sodium thioglycolate)
reduces O2 to water
anaerobic chamber provides more stringent approach
enrichment cultures
enrichment cultures used to isolate organism that constitutes small fraction of mixed population
provide conditions promoting growth of particular species
specific carbon source
Types of cell count
direct cell counts: total numbers (living plus dead)
direct microscope count
cell-counting instruments (Coulter counter, flow cytometer)
viable cell counts: cells capable of multiplying
can use selective, differential media for particular species
plate counts: single cell gives rise to colony
plate out dilution series: 30–300 colonies ideal
plate counts determine colony-forming units (CFUs)
Colony forming unit
a colony-forming unit (CFU) is a unit used to estimate the number of viable bacteria (or fungal) cells in a sample.
viable – has the ability to multiply via binary fission under the controlled conditions. … DOESN’T INCLUDE CELLS IN SUPENSION THAT ARE DEAD, UNABLE TO FORM COLONIES ON PLATE.
concentration of bacteria in a liquid suspension is expressed as
cells/ml
methods to detect and measure microbial growth
membrane filtration
concentrates microbes by filtration
filter is incubated on appropriate agar medium
measuring biomass
turbidity is proportional to concentration of cells measured with spectrophotometer
measuring biomass
total weight can be measured
typically only used for filamentous organisms that do not readily separate into individual cells for valid plate counts
cells in liquid culture centrifuged; pellet is weighed
dry weight can be determined by heating pellet in oven
detecting cell products
pH indicators
Durham tubes (inverted tubes) to trap gas
CO2 production
ATP production using enzyme luciferase to produce light
nutritional factors that influence microbial growth
required elements
major elements make up cell components
carbon source distinguishes different groups
heterotrophs use organic molecules as carbon source
autotrophs use inorganic carbon as CO2 (carbon fixation)
nitrogen required for amino acids, nucleic acids
many use ammonia (some convert nitrate to ammonia)
nitrogen fixation important
iron, phosphorus often limiting
trace elements usually
available (cobalt, zinc, copper
molybdenum, manganese)
Organic and inorganic molecules
Organic molecules are the molecules of life and are built around chains of carbon atoms that are often quite long. There are four main groups of organic molecules that combine to build cells and their parts: carbohydrates, proteins, lipids, and nucleic acids.
Inorganic molecules: molecules other than organic molecules - generally simple and not normally found in living things. Although all organic substances contain carbon, some substances containing carbon, (ex: diamonds) are considered inorganic.
CO2 – as source of available carbon in the carbon cycle, atmospheric carbon dioxide is the primary carbon source for life on Earth
nutritional factors that influence microbial growth
energy sources:
sunlight, chemical compounds
phototrophs obtain energy from sunlight
plants, algae, photosynthetic bacteria
chemotrophs extract energy from chemical compounds
mammalian cells, fungi, many types of prokaryotes
sugars, amino acids, fatty acids common sources
CHEMOSYNTHESIS->some prokaryotes use inorganic chemicals such as hydrogen sulfide, hydrogen gas
History of microbial infection
Until late 19th century, patients undergoing even minor surgeries were at great risk of developing fatal infections
physicians didn’t know their hands could pass diseases from one patient to the next
did not understand airborne microbes could infect open wounds
During the 1800s, puerperal fever was widespread in Europe and a common cause of maternal death.
Oliver Wendel Holmes
In 1842 Oliver Wendel Holmes, Boston physician, spent a year researching puerperal fever by going through case reports and other medical literature in Boston.
1843 - published “The Contagiousness of Puerperal Fever,” in The New England Quarterly Journal of Medicine and Surgery
Puerperal fever is spread through physicians and midwives who make contact with the disease and carry it from patient to patient.
Physicians who plan on attending to pregnant women should not take part in autopsies on patients who died of puerperal fever.
If they do attend an autopsy, they should properly clean themselves and wait a full day before attending to pregnant patients.
If a physician has three closely connected puerperal fever cases, then that physician should be regarded as the reason for the spread of the disease.
Widespread cases of puerperal fever under any physician should not be seen as a misfortune but as crime.
Ignaz Semmelweis
Ignaz Semmelweis July 1818 – 13 August 1865 Hungarian physician/scientist
Hospital at University of Vienna 1847 - noticed women giving birth with midwives had much lower incidence of childbed fever than those giving birth in doctor’s maternity ward…
Germ theory of disease
1861 Louis Pasteur published germ theory of disease: some diseases are caused by microorganisms
Joseph lister
Joseph Lister (1827-1912) British surgeon - introduced methods to prevent infection of wounds
Impressed with Pasteur’s work, he wondered if ‘minute organisms’ might be responsible for infections
-applied carbolic acid (phenol) directly onto damaged tissues, where it prevented infections
-improved methods further by sterilizing instruments and maintaining clean operating environment
approaches to control
aseptic technique: procedures that minimize the chance of unwanted microbes being accidentally introduced
sterilization: removal of all microorganisms
sterile item is free of microbes including endospores and viruses (but does not consider prions)
disinfection: elimination of most or all pathogens
some viable microbes may remain
disinfectants used on inanimate objects: biocides, germicides, bactericides
antiseptics used on living tissues
pasteurization: brief heating to reduce number of spoilage organisms, destroy pathogens
foods, inanimate objects
decontamination: reduce pathogens to levels considered safe to handle
sanitized: substantially reduced microbial population that meets accepted health standards-not a specific level of control
preservation: process of delaying spoilage of foods and perishable products
-adjust conditions
-add bacteriostatic (growth-inhibiting) preservatives
Sterilization
the destruction or removal of all microbes through physical or chemical means
filtration
heat
irradiation
certain chemicals – a sterilant is a chemical that destroys all microbes
Disinfectant
disinfectant – a chemical that destroys many microbes.
Disinfectants are substances that are applied to non-living objects to destroy microorganisms living on the objects.
antiseptics used on living tissues
An antiseptic is a disinfectant non-toxic enough to be used on skin
pasteurization
brief heating to reduce number of spoilage organisms, destroy pathogens foods, inanimate objects
Wine has been briefly heated in China since the 1100’s. Pasteurization slows microbial growth.
1864 Louis Pasteur originally used heating to prevent spoilage of wine and beer.
Later, used for milk.
Flash pasteurization: 71ºC for 15 seconds
decontamination
reduce pathogens to levels considered safe to handle
sanitized
substantially reduced microbial population that meets accepted health standards
-not a specific level of control
make clean and hygienic
“new chemicals for sanitizing a pool”
Bacteria static and bactericidal in preservations
BACTERIOSTATIC – prevents the growth of, but does not kill, bacteria
BACTERICIDAL – kills bacteria
approaches to control
situational considerations:
microbial control methods depend upon situation and level of control required
daily life
washing and scrubbing with soaps and detergents achieves routine control
soap aids in mechanical removal of organisms
beneficial skin microbiota reside deeper on underlying layers of skin, hair follicles
-not adversely affected by regular use
Hand washing with soap and water most important step in stopping spread of many infectious diseases!
Mask wearing during COVID 19 pandemic
Hospitals
minimizing microbial population very important
danger of healthcare-associated infections (HAIs)
patients more susceptible to infection
may undergo invasive procedures (surgery)
pathogens more likely found in hospital setting
feces, urine, respiratory droplets, bodily secretions
instruments must be sterilized to avoid introducing infection to deep tissues
prions relatively new concern; difficult to destroy
Four reservoirs of infectious agents in healthcare settings:
- other patients
- healthcare environment (Pseudomonas aeruginosa)
- healthcare workers (Clostridium difficile, Staphylococcus aureus)
- patient’s own microbiota
microbiology laboratories
routinely work with microbial cultures
use rigorous methods of control
must eliminate microbial contamination to both experimental samples and environment
careful treatment both before (sterile media) and after (sterilize cultures, waste)
aseptic techniques used to prevent contamination of samples, self, laboratory
CDC guidelines for labs working with microbes
Biosafety levels range from BSL-1 (microbes not known to cause disease) to BSL-4 (lethal pathogens for which no vaccine or treatment exists)
primary risks that determine levels of containment:
infectivity, severity of disease, transmissibility, and the nature of the work conducted.
Origin of the microbe, or agent in question, and route of exposure also important.
Biosafety
the application of safety precautions that reduce a lab worker’s risk of exposure to a potentially infectious microbe and limit contamination of the work environment and, ultimately, the community.
Biosafety Level 4 Organisms
viruses known to cause viral hemorrhagic fever such as Marburg virus, Ebola virus, Lassa virus, Crimean-Congo hemorrhagic fever.
food and food production facilities
perishables retain quality longer when contaminating microbes destroyed, removed, inhibited
heat treatment most common and reliable mechanism
can alter flavor, appearance of products
irradiation approved to treat certain foods
chemical additives can prevent spoilage
FDA regulates because of risk of toxicity
facilities must keep surfaces clean and relatively free of microbes
pharmaceuticals, cosmetics, deodorants must not carry microbial contamination
water treatment facilities
ensure drinking water free of pathogens
chlorine traditionally used to disinfect water
can react with naturally occurring chemicals
form disinfection by-products (DBPs)
some DBPs linked to long-term health risks
some organisms resistant to chemical disinfectants
Cryptosporidium parvum (causes diarrhea)
selection of an antimicrobial procedure
Selection of effective procedure complicated
ideal method does not exist
each has drawbacks and procedural parameters
Choice depends on numerous factors:
1. type of organisms
2. number of organisms
3. environmental conditions
4. risk of infection
5. composition of infected item
type of organism
multiple highly resistant microbes
bacterial endospores: most resistant, only extreme heat or chemical treatment destroys them
protozoan cysts and oocysts: resistant to disinfectants; excreted in feces; causes diarrheal disease if ingested
Mycobacterium species: waxy cell walls makes resistant to many chemical treatments
Pseudomonas species: resistant to and can actually grow in some disinfectants
non-enveloped viruses: lack lipid envelope; more resistant to disinfectants
Pseudomonas aeruginosa infections
opportunistic pathogen; major cause of healthcare-associated infection, important in lung infections and wound infections, especially thermal burns
Forms biofilms
signs and symptoms:
chills, fever, skin lesions, shock
pigments (fluorescent yellow pyoverdin and blue pyocyanin) yield characteristic green color
Gram-negative rod with polar flagellum
found in soils, water
aerobic; respires anaerobically in absence of O2
if nitrate is present
Naked viruses
lack lipid envelope; more resistant to disinfectants
Adenoviruses
Papovaviruses
Parvoviruses
Rotaviruses
Rhinoviruses
Polioviruses
Noroviruses
Astroviruses
number of microorganisms
time for heat, chemicals to kill affected by population size
fraction of population dies during given time interval
large population = more time
decimal reduction time
gauges
commercial effectiveness
D value:
time required to kill 90% of
population under specific
conditions
environmental conditions
dirt, grease, body fluids can interfere with heat penetration, action of chemicals
important to thoroughly clean
microorganisms in biofilm are more resistant
pH, temperature can influence effectiveness
sodium hypochlorite (household bleach) solution can kill suspension of M. tuberculosis at 55°C in half the time as at 50°C
even more effective at low pH
risk for infection
medical instruments categorized according to risk for transmitting infectious agents
critical items come in contact with body tissues
must be sterile
include needles and scalpels
semicritical instruments contact mucous membranes but do not penetrate body tissues
must be free of viruses and vegetative bacteria
few endospores blocked by mucous membranes
includes endoscopes and endotracheal tubes
non-critical instruments contact unbroken skin only
low risk of transmission
countertops, stethoscopes, blood pressure cuffs
composition of item
some sterilization and disinfection methods inappropriate for certain items
heat inappropriate for plastics and other sensitive items
irradiation provides alternative, but damages some types of plastic
moist heat, liquid chemical disinfectants cannot be used to treat moisture-sensitive material
Using HEAT to destroy microorganisms and viruses
heat treatment useful for microbial control
reliable, safe, relatively fast, inexpensive, non-toxic
can be used to sterilize or disinfect
sterilization using pressurized steam
autoclave used to sterilize using pressurized steam
increased pressure raises temperature; kills endospores
sterilization
longer for larger volumes
flash sterilization at higher
temperature can be used
Moist heat
irreversibly denatures proteins
boiling destroys most microorganisms and viruses
does not sterilize: endospores can survive
pasteurization destroys heat-sensitive pathogens, spoilage organisms
high-temperature–short-time (HTST): most products
milk: 72°C for 15 s; ice cream: 82°C for 20 s
ultra-high-temperature (UHT): shelf-stable boxed juice and milk; known as “ultra-pasteurization”
milk: 140°C for a few seconds, then rapidly cooled
Commercial canning process
uses industrial-sized autoclave called retort
designed to destroy Clostridium botulinum endospores
-virtually impossible to have so many endospores
critical because otherwise endospores can germinate in canned foods
cells grow in low-acid anaerobic conditions and produce botulinum toxin
canned food commercially sterile
Dry heat
less effective than moist heat; longer times, higher temperatures necessary
200°C for 90 minutes vs. 121°C for 15 minutes
hot air ovens oxidize cell components, denature proteins
used for glass, powders, oils, dry materials
incineration a method of dry heat sterilization
oxidizes cell to ashes
used to destroy medical waste and animal carcasses
laboratory inoculation loop sterilized by flaming
Filtration
some materials can’t withstand heat treatment
filtration retains bacteria
filtration of fluids used extensively
membrane filters small pore size (0.2 µm) to remove bacteria thin
depth filters thick porous filtration material (cellulose)
larger pores
electrical charges trap cells
filtration of air
high-efficiency particulate air (HEPA) filters remove nearly all microbes
using other physical methods to remove or destroy microbes
Irradiation can induce genetic damage and chemical changes in key biological macromolecules
electromagnetic radiation: radio waves, microwaves, visible and ultraviolet light, X rays, and gamma rays
energy travels in waves; no mass
ionizing radiation can remove electrons from atoms
gamma rays and X rays important forms
destroys DNA
damages cytoplasmic membranes
reacts with O2 to produce reactive oxygen species
high energy gamma-rays
used to sterilize heat-sensitive materials
generally used after packing
approved for use on foods, although consumer resistance has limited use
ultraviolet radiation destroys microbes directly
damages DNA
used to destroy microbes in air, water, and on surfaces
poor penetrating power
thin films or coverings can limit effect
cannot kill microbes in solids or turbid liquids
most glass and plastic block
must be carefully used since damaging to skin, eyes
microwaves kill by generated heat, not directly
microwave ovens heat food unevenly, so cells can survive
Physical method: High pressure
used in pasteurization of commercial foods
-guacamole-
avoids problems with high temperature pasteurization
employs high pressure up to 130,000 psi (pound force/sq. inch)
destroys microbes by denaturing proteins, altering cell permeability
products maintain color, flavor associated with fresh food
using chemicals to destroy microorganisms and viruses
potency of germicidal chemical formulations
sterilants destroy all microorganisms - also called sporocides
heat-sensitive critical instruments
high-level disinfectants destroy viruses, vegetative cells
do not reliably kill endospores
semi-critical instruments
intermediate-level disinfectants destroy vegetative bacteria, mycobacteria, fungi, and most viruses
disinfect non-critical instruments
low-level disinfectants destroy fungi, vegetative bacteria except mycobacteria, and enveloped viruses
do not kill endospores, non-enveloped viruses
disinfect furniture, floors, walls
selecting the appropriate germicidal chemical
toxicity: benefits must be weighed against risk of use
activity in presence of organic material
many germicides inactivated
compatibility with material being treated
liquids cannot be used on electrical equipment
residues: can be toxic or corrosive
cost and availability
storage and stability
concentrated stock decreases storage space
environmental risk
agent may need to be neutralized before disposal
classes of germicidal chemicals
alcohols
aldehydes
biguanides: extensive use as antiseptics
ethylene oxide: gaseous sterilant
halogens: oxidize proteins, cellular components
metal compounds
ozone (O3)
peroxygens
phenolic compounds (phenolics)
quaternary ammonium compounds (quats)
preservation of perishable products
chemical preservatives
food preservatives must be non-toxic for safe ingestion
weak organic acids (benzoic, sorbic, propionic)
ltaer cell membrane function
control molds and bacteria in foods
reducing available water
accomplished by salting, adding sugar, or drying food
addition of salt, sugar increases environmental solutes
causes cellular plasmolysis (water exits bacterial cells)
some bacteria grow in high salt environments
Staphylococcus aureus
drying often supplemented by salting
lyophilization (freeze drying) foods
coffee, milk, meats, fruits, vegetables
drying stops microbial growth but does not reliably kill
Many cases of salmonellosis from dried eggs
nitrate and nitrite used in processed meats
inhibit endospore germination and vegetative cell growth
stops growth of Clostridium botulinum
higher concentrations give meats pink color
shown to be carcinogenic—form nitrosamines
low-temperature storage
refrigeration inhibits growth of pathogens and spoilage organisms by slowing or stopping enzyme reactions
psychrotrophs, psychrophilic organisms can still grow
freezing preserves by stopping all microbial growth
Some microbial cells killed by ice crystal formation, but many survive and can grow once thawed
Metabolism
Metabolism is the sum total of chemical reactions for energy generation and biosynthetic processes within a cell.
During metabolism, cells take energy stored in nutrients such as glucose and redistribute that energy to other molecules, building more complex cellular structures.
A set of ordered reactions is required to extract and redistribute the energy stored in a molecule of glucose.
Two pathways for the anaerobic breakdown of pyruvate
FERMENTATION LEADING TO EXCRETION OF LACTATE
FERMENTATION LEADING TO EXCRETION OF ALCOHOL AND CO2
microbial metabolism
All cells need to accomplish two fundamental tasks
harvest energy to power reactions
synthesize new parts
cell walls, membranes, ribosomes, nucleic acids
sum total of chemical reactions in cell called metabolism
Two parts of metabolism
catabolism
processes that degrade compounds to release energy cells capture to make ATP
anabolism
biosynthetic processes assemble subunits of macromolecules
use ATP to drive reactions
processes intimately linked
energy
energy is the capacity to do work
two types of energy
potential: stored energy (chemical bonds, rock on hill)
kinetic: energy of movement
Cells convert potential energy, usually in the form of C-C covalent bonds or ATP molecules, into kinetic energy to accomplish cell division, growth, biosynthesis, and active transport
Energy and organisms
photosynthetic organisms harvest energy of sunlight
convert kinetic energy of photons to potential energy of chemical bonds
powers synthesis of organic compounds from CO2
CO2 + H2O => C6 H12O6 + O2
chemoorganotrophs obtain energy from chemical bonds in organic compounds
C6 H12O6 + O2 => CO2 + H2O
- they depend on activities of photosynthetic organisms
…or in certain environments like thermal vents they depend on
organic compounds produced by chemolithoautotrophs
that obtain energy by oxidizing inorganic compounds.
prokaryotes can be categorized by how they obtain energy and carbon
phototrophs get energy from light
autotrophs use CO2 as a carbon source
heterotrophs use an organic nutrient to make organic compounds
Free energy
free energy is energy available to do work
energy released when chemical bond is broken
compare free energy of reactants, products:
exergonic reactions: reactants have more free energy
energy is released in reaction
energy released from exergonic reactions powers endergonic reactions
free energy is energy available to do work
endergonic reactions: products have more free energy
reaction requires input of energy
change in free energy is same regardless of number of steps involved (converting glucose to CO2 + H2O)
cells use multiple steps when degrading compounds
role of enzymes
biological catalysts: speed up conversion of substrate into product by lowering activation energy
metabolic pathways
series of chemical reactions that convert starting compound to end product
may be linear, branched, cyclical
role of ATP
adenosine triphospate (ATP) is energy currency of the cell
adenosine diphospate (ADP) acceptor of free energy
cells produce ATP by adding Pi to ADP using energy
release energy from ATP to yield ADP and Pi
adenosine triphosphate
composed of ribose, adenine,
three phosphate groups
three negatively charged phosphate groups repel
bonds inherently unstable, easily broken
releases energy to drive cellular processes
high energy phosphate bonds denoted by ~
ATP 🡪 ADP + Pi
three processes to generate ATP
substrate-level phosphorylation
exergonic reaction
oxidative phosphorylation
proton motive force
photophosphorylation
sunlight used to create
proton motive force
role of chemical energy source and terminal electron acceptor
When electrons move from a molecule that has a relatively low electron affinity (tends to give up electrons) to one that has a higher electron affinity (tends to accept electrons), ENERGY IS RELEASED.
(glucose to O2)
Energy transfer increases energy content of one system while decreasing energy content of other system by same amount.
Transfer characterized by quantity of energy transferred: an atom with higher electronegativity is better able to attract electrons.
Transfer occurs in a process that changes state of each system.
WHEN ELECTRONS MOVE FROM A MOLECULE THAT HAS A LOW ELECTRON AFFINITY TO ONE THAT HAS A HIGHER ELECTRON AFFINITY, ENERGY IS RELEASED.
More energy is released when the difference in electronegativity is greater
Electron donor: energy source
Acceptor: terminal electron acceptor
OXIDATION-REDUCTION REACTIONS:
The molecule that looses one or more electrons is OXIDIZED by the reaction; the one that gains those electrons is REDUCED.
substance loses electrons - oxidized
substance gains electrons - reduced
electron-proton pair or
hydrogen actually moves
dehydrogenation = oxidation
hydrogenation = reduction
prokaryotes remarkably diverse in using energy sources and terminal electron acceptors
organic, inorganic compounds used as energy source
O2 or other molecules used as terminal electron acceptor
electrons removed through series of oxidation-reduction reactions (redox reactions)
Electron carriers
CELLS INITIALLY TRANSFER THE ELCTRONS TO
ELECTRON CARRIERS
NAD+/NADH, FAD/FADH2 and NADP+/NADPH
Reduced electron carriers represent reducing power because they can transfer their electrons to another chemical that has a higher affinity for electrons.
NADH and FADH2 transfer their electrons to the electron transport chain, which uses the energy to drive a proton motive force
coenzyme cofactors: electron carriers
role of electron carriers
energy harvested in stepwise process
electrons transferred to electron carriers, which represent reducing power (easily transfer electrons to chemicals with higher affinity for electrons)
raise energy level of recipient molecule
NAD+/NADH, NADP+/NADPH, and FAD/FADH2
NAD+/NADH
oxidized and reduced forms of nicotinamide adenine dinucleotide
FAD/FADH2
Oxidized and reduced forms of flavin adenine dinucleotide
NADP+/NADPH
Oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate
How does the fate of electrons carried by NADPH differ from the fate of electrons carried by NADH?
Electrons carried by NADPH are used in biosynthesis whereas those carried by NADH are passed to the electron transport chain.
Precursor metabolites
precursor metabolites are intermediates of catabolism that can be used in anabolism
serve as carbon skeletons for building macromolecules
pyruvate can be converted into amino acids: alanine, leucine, or valine
E. coli can grow in glucose-salts medium
contains just glucose, inorganic salts
glucose is energy source
glucose is starting point for all
cellular components
including proteins, lipids,
carbohydrates, nucleic acids
some glucose molecules
completely oxidized for energy;
others used in biosynthesis
overview of catabolism
three central metabolic pathways
oxidize glucose to CO2
catabolic and precursor metabolites as well as reducing power can be diverted for use in biosynthesis
amphibolic - dual role
glycolysis
splits glucose (6C) to two pyruvates (3C)
generates modest ATP, reducing power, precursors
pentose phosphate pathway
primary role is production precursor metabolites, NADPH
tricarboxylic acid cycle
oxidizes pyruvates from glycolysis
generates reducing power, precursor metabolites, ATP
Substrate-level phosphorylation
directly phosphorylating ADP with a phosphate and energy provided from a coupled reaction.
Oxidative phosphorylation
when ATP is generated from the oxidation of NADH and FADH2 and the subsequent transfer of electrons and pumping of protons. That process generates an electrochemical gradient, which is required to power ATP synthase.
overview of catabolism
respiration transfers electrons from glucose to electron transport chain
electron transport chain generates proton motive force
harvested to make ATP via oxidative phosphorylation
*aerobic respiration
O2 is terminal electron acceptor
*anaerobic respiration
molecule other than O2 as terminal electron acceptor
also use modified version of TCA cycle
fermentation
if cells can’t respire, run out of carriers available to accept electrons
glycolysis will stop
fermentation uses pyruvate or derivative as terminal electron acceptor to regenerate NAD+
so glycolysis can continue
enzymes
enzymes are biological catalysts
name reflects function; ends in –ase
has active site to which substrate(s) bind(s) weakly
causes enzyme shape to change slightly, induced fit
existing substrate bonds destabilized, new ones form
enzymes are highly specific for substrate(s)
cofactors assist some enzymes
cofactors can assist different enzymes; include magnesium, zinc, copper, other trace elements
Coenzymes
coenzymes are organic cofactors
include electron carriers FAD, NAD+, NADP+, fewer types needed
derived from vitamins (B vitamins)
Coenzymes are organic molecules that transfer atoms from one molecule to another,
may bind to a number of different enzymes,
and are synthesized from vitamins.
environmental factors influencing enzyme activity
enzymes have narrow range of optimal conditions
temperature, pH, salt concentration
10°C increase doubles speed of enzymatic reaction up until maximum
proteins denature at higher temperatures
low salt, neutral pH usually optimal
allosteric regulation
enzyme activity controlled by binding to allosteric site
distorts enzyme shape, prevents or enhances binding
regulatory molecule is usually end product
allows feedback inhibition
Feedback inhibition
The activity of the first enzyme of the pathway is inhibited by the end product, thus controlling production of end product.
Enzyme inhibition
site to which inhibitor binds determines type
competitive inhibitor binds to active site of enzyme
chemical structure usually similar to substrate
concentration dependent; blocks substrate
example is sulfa drugs blocking folic acid synthesis
non-competitive inhibitor binds to a different site than active site
allosteric inhibitors are one example; action is reversible
some non-competitive inhibitors are not reversible
mercury oxidizes the S—H groups of amino acid cysteine, converts to cystine
cystine cannot form important disulfide bond (S—S)
enzyme changes shape, becomes nonfunctional
Glycolysis
converts: 1 glucose to 2 pyruvates
yields:
net 2 ATP (2 in 4 out)
reducing power: 2 NADH + 2H+
precursor metabolites:
end product, pyruvate
investment phase:
2 phosphate groups added
glucose split to two 3-carbon molecules
Intermediates:
glucose-6-phosphate
fructose6-phosphate
dihydroxyacetone phosphate
pay-off phase:
2 3-carbon molecules converted to 2 pyruvates
Intermediates:
3-phosphoglycerate
phosphoenolpyruvate
generates 4 ATP, 2NADH total
converts 1 glucose
to 2 pyruvates; yields
net 2 ATP (substrate level phosphorylation)
2 NADH
transition step from glycolysis to TCA cycle
A carboxyl group is removed from
pyruvate, releasing carbon dioxide.
NAD+ is reduced to NADH.
An acetyl group is transferred to
coenzyme A, resulting in acetyl
COA.
Acetyl CoA
brings carbon atoms from glycolysis (pyruvate) to the citric acid cycle to be oxidized for energy production
All genomes sequenced to date encode enzymes that use CoA as a substrate.
TCA cycle output
For I molecule of glucose
TCA cycle produces:
4 ATP
6 NADH
2 FADH2
precursor metabolites
The release of carbon dioxide is coupled with the reduction of NAD+ to NADH
Respiration
Uses reducing power (NADH, FADH2) generated by glycolysis, transition step, and TCA cycle to synthesize ATP
electron transport chain generates proton motive force
drives synthesis of ATP by ATP synthase
now called chemiosmotic theory
electron transport chain:
membrane-embedded electron carriers
pass electrons sequentially, eject protons in process
prokaryotes: in cytoplasmic membrane
eukaryotes: in inner mitochondrial membrane
ATP synthase
ATP synthase—harvesting the proton motive force to synthesize ATP
energy required to establish gradient
released when gradient is eased
ATP synthase allows protons to flow down gradient in controlled manner
uses energy to add phosphate group to ADP
1 ATP formed from entry of ~3 protons
Oxidase test
oxidase test chromogenic reducing agent changes color when oxidized. If the test organism produces cytochrome c oxidase, the oxidase reagent will turn blue or purple within 15 seconds.
OX+
bacterium contains cytochrome c oxidase and can therefore use oxygen for energy production with an electron transport chain
Neisseria, Pseudomonas, Campylobacter, Helicobacer pylori
Legionella pneumophila
OX-
bacterium does not contain cytochrome c oxidase and, therefore, either cannot use oxygen for energy production with an electron transport chain, or employs a different enzyme for transferring electrons to oxygen.
Enerobactericeae (family includes many genera among which is E. coli)
general mechanisms of proton ejection
some carriers accept only hydrogen atoms (proton-electron pairs), others only electrons
spatial arrangement in membrane shuttles protons to outside of membrane
when hydrogen carrier accepts electron from electron carrier, it picks up proton from inside cell
or mitochondrial matrix
when hydrogen carrier passes electrons to electron carrier, protons released to outside of cell
or intermembrane space of mitochondria
net effect is movement of protons across membrane
establishes concentration gradient
driven by energy released during electron transfer
electron transport chain of mitochondria
Complex I (NADH dehydrogenase complex)
accepts electrons from NADH, transfers to ubiquinone
pumps 4 protons
Complex II (succinate dehydrogenase complex)
accepts electrons from TCA cycle via FADH2, “downstream” of those carried by NADH
transfers electrons to ubiquinone
Complex III (cytochrome bc1 complex)
accepts electrons from ubiquinone from Complex I or II
4 protons pumped; electrons transferred to cytochrome c
Complex IV (cytochrome c oxidase complex)
accepts electrons from cytochrome c, pumps 2 protons
terminal oxidoreductase, meaning transfers electrons to terminal electron acceptor (O2)
calculating theoretical maximum yields
in prokaryotes:
glycolysis: 2 NADH🡪 6 ATP
transition step: 2 NADH 🡪 6 ATP
TCA cycle: 6 NADH 🡪 18 ATP; 2 FADH2 🡪 4 ATP
total maximum oxidative phosphorylation yield = 34 ATP
slightly less in eukaryotic cells
NADH from glycolysis in cytoplasm transported across mitochondrial membrane to enter electron transport chain
requires ~1 ATP per NADH generated
Why is the overall ATP yield in aerobic respiration only a theoretical number?
Different prokaryotes have different components in their electron transport chain, and proton motive force can be used for purposes other than ATP generation.
Which generates more reducing power—glycolysis or the TCA cycle?
The TCA cycle:
glycolysis: 2 NADH🡪 6 ATP
TCA cycle: 6 NADH 🡪 18 ATP
2 FADH2 🡪 4 ATP
Why would a cell ferment rather than respire?
Cells ferment if a suitable terminal electron acceptor is not available, or if they lack an electron transport chain.
Why is it important for cells to have a mechanism to oxidize NADH?
In order to be able to re-enter glycolysis with NAD+.
…the cells must oxidize NADH, otherwise they would run out of NAD+ and glycolysis would come to a halt.
Lactic acid bacteria
Lactic acid bacteria create ATP without oxygen.
…to produce NAD+ which can re-enter glycolysis.
example: Streptococcus sp.
catabolism of organic compounds other than glucose
microbes can use variety of compounds
excrete hydrolytic enzymes; transport subunits into cell
degrade further to appropriate precursor metabolites
polysaccharides and disaccharides
amylases digest starch; cellulases digest cellulose
disaccharides hydrolyzed by specific disaccharidases
lipids
fats hydrolyzed by lipases; glycerol converted to dihydroxyacetone phosphate, enters glycolysis
fatty acids degraded by β-oxidation to enter TCA cycle
proteins
hydrolyzed by proteases; amino group deaminated
carbon skeletons converted into precursor molecules
A difference between chemolithotrophs and chemoorganotrophs
chemolithotrophs can directly provide electrons to the electron transport chain,
while chemoorganotrophs must generate their own cellular reducing power by oxidizing reduced organic compounds (such as glucose)
Chemolithotrophs bypass this by obtaining their reducing power directly from the inorganic substrate or by the reverse electron transport reaction.
chemolithotrophs
prokaryotes unique in ability to use reduced inorganic compounds as energy sources
inorganic molecules (sulfate, nitrate) serving as terminal electron acceptors to produce hydrogen sulfide (H2S), ammonia (NH3) by anaerobic respiration
important example of nutrient cycling
four general groups
- hydrogen bacteria oxidize hydrogen gas (H2) - use hydrogen as an electron donor), - has high redox potential and can couple with oxygen or carbon dioxide or sulfate.
Facultative autotrophs; include both aerobes and anaerobes
mixotrophic – some aerobes also can have heterotrophic growth and use organic compounds for energy
- sulfur bacteria oxidize hydrogen sulfide (H2S)
Sulfur (non-photosynthetic) bacteria - some live at low pH (sulfuric acid – reduced product)
- iron bacteria oxidize reduced forms of iron (Fe2+)
- nitrifying bacteria:
2 groups - one oxidizes ammonia (NH3), forming nitritethe other oxidizes nitrite (NO2), forming nitrate.
photosynthesis
plants, algae, several groups of bacteria
can be considered in two distinct stages
1. light reactions (light-dependent reactions)
capture energy and convert it to ATP
- light-independent reactions (dark reactions)
use ATP to synthesize organic compounds
involves carbon fixationreaction-center pigments donate excited electrons to electron transport chain
*chlorophyll a (plants, algae, cyanobacteria)
*bacteriochlorophylls (anoxygenic bacteria)
cyanobacteria: photosystems in membranes of stacked structures inside cell - termed thylakoids
plants, algae: thylakoids in stroma of chloroplast-endosymbiotic theory explains
purple bacteria (anoxygenic): in cytoplasmic membrane, extensive infoldings
green bacteria (anoxygenic): specialized
chlorosomes attached to
cytoplasmic membrane
light-dependent reactions in cyanobacteria & photosynthetic eukaryotes
two distinct photosystems (I and II)
cyclic photophosphorylation
photosystem I alone produces ATP
reaction-center chlorophyll is terminal electron acceptor
non-cyclic photophosphorylation
used when cells need both ATP and reducing power
electrons from photosystem II drive photophosphorylation
are then donated to photosystem I
photosystem II replenishes electrons by splitting water
generates oxygen (process is oxygenic)
electrons from photosystem I reduce NADP+ to NADPH
electrons from photosystem II drive photophosphorylation, water used as an electron donor, oxygen produced
electrons from photosystem I reduce NADP+ to NADPH
carbon fixation
Chemolithoautotrophs and photoautotrophs use CO2 to synthesize organic compounds: carbon fixation
in photosynthetic organisms: light-independent reactions
consumes lots of ATP, reducing power
Calvin cycle most commonly used
three essential stages
-incorporation of CO2 into organic compounds
-reduction of resulting molecule
-regeneration of starting compound
six “turns” of cycle: net gain of one fructose-6-phosphate
consumes 18 ATP, 12 NADPH per fructose molecule
prokaryotes remarkably similar in biosynthesis
synthesize subunits using central metabolic pathways
if enzymes lacking, end product must be supplied
fastidious bacteria require growth factors
lipid synthesis requires fatty acids and glycerol
fatty acids: 2-carbon units added to acetyl group from acetyl-CoA
glycerol: dihydroxyacetone phosphate from glycolysis
nucleotide synthesis
DNA, RNA initially synthesized as ribonucleotides
purines: atoms added to ribose 5-phosphate to form ring
pyrimidines: ring made, then attached to ribose 5-phosphate
can be converted to other nucleobases of same type
light-dependent reactions
in anoxygenic photosynthetic bacteria
each has single photosystem
cannot use water as electron donor, so anoxygenic
use electron donors such as hydrogen gas (H2), hydrogen sulfide (H2S), organic compounds
purple bacteria: photosystem similar to photosystem II
energy of electrons insufficient to reduce NAD+
instead expend ATP to use reversed electron transport
green bacteria: photosystem similar to photosystem I
electrons can generate proton motive force or reduce NAD+
