Study Guide

MCB2610 Fundamentals of Microbiology

Exam 2 Fall 2016

1) Study your notes for the 5 new microbe minutes. Make tables that allow you to
compare specific features of the microbes (aka, Gram + vs. Gram -, spore former
versus non, flagella versus none, pathogenic versus non, morphology so
bacterial shape, cocci, rods etc.)(4 questions will come from this material)
2) Study your notes and slides from Dr. Hird's presentation. Focus on general
material. (4 questions will come from this material)
3) Read the assigned paper on Cowbird Gut Microbiota. Focus on general material
(4 questions will come from this material)
Lecture Material Chapter 6A (from slide #64)
(10 questions will come from this material)
1) Measuring microbial population growth: know the different methods, how they work,
think about what circumstances might make one method more suitable than another;
know when we measure in CFUs vs. # of cells and why.
Different methods:
Direct counts: Uses a special slide with an etched grid, a known volume
is loaded into the gride cells and counted under a light microscope.
PROS: Cheap and easy
CONS: living vs. dead cells undifferentiated
Viable Cell Counting:
Spread Plate: Sample is serially diluted, a small volume of the
dilution over a surface
Pour Plate: Sample is serially diluted, diluted mixture with liquid
agar, and poured onto a sterile dish
CONS: Mixtures could already be very diluted, might need a filter
CFU/mL calculation: count/dilution x volume plated

Most probable number method: Count how many positive tubes

you have and get the MPN
Turbidity: Using a spectrophotometer, turbidity means high
absorbance reading.
Flow Cytometry: Microbial suspension forced through small hole
with a laser beam, and the movement of the microbe through
hole impacts the electric current also flowing through the
Disruptions of current are counted.

the
hole.

Determine what the advantages and disadvantages are of each method of

enumeration.
Would the method you use vary depending on what you are testing?
E.g. what is the best method for a soil sample, a drinking water sample, a urine
specimen?

2) Know how to use counts to do calculations!! Example slide in our class

powerpoint (count/dilution x volume plated) (simple calculations, no calculator
required)
3) MPN (most probable number estimate) - I will NOT ask you to calculate MPN.
Most probable number method: Count how many positive tubes
you have and get the MPN
4) Growth and the growth curve in Batch culture - know what a standard curve looks
like, know all phases of the curve, know what is considered balance growth and what
happens during shift up and downshift experiments.

Growth refers to population growth rather and individual growth

Batch culture: culture incubated in a closed vessel with a single batch of
medium
Growth curve: plotted as logarithm of cell # vs time
Lag, log, stationary and death
Log Phase: population is most uniform in terms of chemical and physical
properties during this phase, balanced growth
o Shift-up: poor medium to rich medium
o Shift Down: rich medium to poor medium

Stationary Phase: nutrient limitation, limited oxygen availability, toxic

waste accumulation critical population density reached

5) Know what generation time, growth rate and growth yield are and how to determine
simple calculations (no calculator required).

Generation time: The time to double the population in the exponential

phase
Growth rate: Number of generations/unit of time (inverse of the

generation time)
Growth yield: The maximum population density and/or amount of cellular
material produced by the culture

6) Know the reasons we get into stationary phase, the hypotheses for senescence, and
some adaptations that allow cells to survive starvation (persister cells, VBNC).

Stationary Phase: nutrient limitation, limited oxygen availability, toxic

waste accumulation critical population density reached
Entry into stationary phase can also be from starvation and other stressful
conditions
o Morphological changes can occur; endospore formation
o Bacteria decrease in size
o RpoS protein assists RNA polymerase in transcribing genes for
starvation proteins
Producing starvation proteins makes it harder to kill cells
o Starvation proteins increase cell linking in cell wall
o Dps protein protects DNA
o Chaperone proteins prevent protein damage
persister cells
o cells that can survive for a long time and be virulent
o able to survive under adverse conditions
o dont form spores

7) Death and why growth doesnt just plummet during the death phase.

Number of viable cells declines exponentially

Two alternative hypotheses

o Cells are Viable But Not Culturable (VBNC)
cells are alive, but dormant
they are capable of new growth when conditions are right
o Programmed cell death
fraction of the population genetically programmed to die
(commit suicide)

8) Continuous vs batch culturing systems.

Continuous System: always adding nutrients and taking away waste, it

maintains cells log phase at a constant biomass concentration for a long
period of time
o This can be useful for studying minimal nutrient requirements to
mimic natural environment, used a lot in food/industrial microbio
o Microbes in nature dont exist in a closed system
o A chemostat flows in fresh medium and takes out old
medium/microbes to keep it fresh

9) Importance of dilution rate in the chemostat and how that can influence growth

Chemostat: rate of incoming medium = rate of removal of medium from

vessel
Dilution rate: the rate at which medium flows through vessel relative to

vessel size
Chemostat best operates at low dilution rate
At low dilution rates energy is limited and cells cant grow

10) chemostat vs. turbidostat

Chemostat: essential nutrient limited, low dilution rates

Turbidostat: maintains turbidity, dilution rate varies, operates best at high
dilution rates

Lecture Material Chapter 6B (all slides)

1) Difference between sanitization and sterilization
Sterilization: destruction or removal of all viable organisms
o Reduction in microbial levels to safe levels
Disinfection: Killing, inhibition, or removal of disease causing organisms
o Disinfectants
o Antiseptics: chemical agent that kills growth of microorganisms
when applied to tissue
2) Difference between antiseptics and disinfectants
Disinfectant: used on inanimate objects
Antiseptic: used on tissue
3) Difference between cidal and static agents
-cidal agents: kills pathogens and many non pathogens but not
necessarily endospores. Bactericides, fungicides, etc
-static agents: inhibits growth, ex. baceriostatic
4) Conditions that influence the effectiveness of antimicrobial agents - what they are
and why these specific conditions might change the effectiveness of an agent
Death occurs exponentially
Measurements of an agents killing efficiency
o decimal reduction time (D-value) time to kill 90% of
microorganisms and spores in a sample under specific conditions
o Z- value- temperature change needed to kill 90% of
microorganisms and spores in a sample under specific conditions
o must be sure persister cells (viable but nonculturable (VBNC))
are dead
once they recover they may regain the ability to reproduce

and cause infection

Conditions Influencing the effectiveness of antimicrobes
o Population size (longer time to kill bigger pop)
o Population composition (varies)
o Concentration or intensity of antimicrobial agent (moreis better)
o Contact time (more is better)
o Temperature (higher is better)
o Local environment (pH, biofilms)

5) Difference between physical and chemical control methods

Chemical methods of controlling microbes
o Many chemicals can kill microbes, or inhibit their growth.
o Disinfectants: Chemicals used on non-living surfaces to kill
potentially infectious microbes.
o Antiseptics: Chemicals that can be used on living tissue to kill
potentially infectious microbes (usually only used topically)
o PROS:
Should kill a wide-range of microbes
Shouldnt be corrosive or overly toxic
Shouldnt leave a residue
Shouldnt emit fumes or smell TOO bad
Should be cheap
Should be temperature stable

6) Filtering - how it works, pros and cons, types of things that can be filter sterilized
Filtration is an old way to purify liquids
New methods use nylon/Teflon filters with a very small pore size
Viruses can be removed from liquids by ultrafiltration methods (100 nm

pore)
CONS: large particles clog filters, ultrafiltration requires high pressure,

viscous liquids cant filter well

Membrane filters: removes microorganisms from heat sensitive liquids

(pharmaceuticals, antibiotics), have a defined pore size

Surgical masks are filters
High efficiency particulate air (HEPA) filters:

7) Moist Heat - know the mechanism of killing and the 4 processes involving moist heat
(boiling, pasteurization, steam sterilization, Tyndallization)
Moist Heat: destroys viruses, fungi, and bacteria
Boiling: will not destroy spores and does not sterilize

Pasteurization
o Controlled heating at temperatures below boiling (55-60C)
o Milk, beer, wine
o Process does not sterilize, but it kills pathogens present and slows
the spoiling process by reducing the total number of organisms

present
o 1889
Steam sterilization
o Must be carried out above 100C
o Carried out using autoclave
o Effective against all types of microorganisms including spores
o Quality control
Tyndallization
o Intermittent sterilization
o 30-60 mins of steam repeated 3 times
o Used to kill spores spores germinate and are then killed by the

steam treatment
8) Dry Heat - know the mechanism of killing, how it differs from wet heat, and when dry
heat is appropriate
Less effective that moist heat sterilization
It requires higher temperature and longer exposure time
2-3 hours at 170C
It oxidizes cell constituents and denatures proteins
Bench top incinerators used to sterilize loops in lab

9) UV/ionizing radiation-what situations each might be appropriate for

UV radiation of 260 to 280 nm wavelengths can damage DNA, forming

thymine dimers
This can be exploited to control microbial growth and non-living surfaces in

water
Ionizing radiation: gamma radiation penetrates deep into objects
It destroys bacterial endospores but not always effective against viruses

10)Classes of chemical agents (phenolics etc.), knowledge of their mechanism of killing

and examples of each
Phenolics:
o Act by denaturing proteins and disrupting cell membranes
o Bad odor and causes skin irritation
o Long lasting
o Effective in presence of organic material
Aldehydes
o Formaldehyde and glutaraldehyde
o Highly reactive
o Sporicidal
Alcohols
o Ethanol and isopropanol
o Bactericidal, fungicidal, but not sporicidal
o Can inactivate some viruses
o They denature proteins and possibly dissolve membrane lipids
Halogens
o F, Cl, Br, I,
o Important antimicrobial agents
o Iodine: skin antiseptic
It oxidizes cell constituents and iodinates proteins
High concentraitions can kill spores
Ex. Wescodyne
Iodophore: iodine compled with organic carrier, released
slowly to minimize skin burns
o Chlorine: oxidizes cell constituents
Destroys vegetative bacteria and fungi
Chlorine gas is sporicidal
Can react with organic matter to form carcinogenic
compounds
EX. Bleach is highly effective
o Heavy Metals: silver, arsenic, zinc and copper
Effective but usually toxic
o Quarternary Ammonium Compounds:
detergents that have antimicrobial activity and are effective
disinfectants

act as wetting agents and emulsifiers

Disrupt membranes and denature proteins
kill most bacteria, but not Mycobacterium tuberculosis or
endospores

11)Disk Diffusion assays for evaluating a disinfectant

The larger the clearing zone the more effective the disinfectant

12)Sterilizing gases (Ex. Ethylene oxide) mechanism of killing, appropriate

circumstances for use. Know which are methods of sterilization/sanitization and
when each method would be appropriate
used to sterilize heat-sensitive materials like plastics
microbiocidal and sporicidal
ethylene oxide sterilization is carried out in equipment resembling an
autoclave - rapidly penetrates packing materials, even plastic wraps
(5-8 hrs at 38C or 3-4 hrs at 54C at 40-50% RH) very toxic to

humans
betapropiolactone and vaporized hydrogen peroxide
combine with and inactivate DNA and proteins

Lecture Material Chapter 2 & 4 (all slides)

1) Bacterial morphology size and shape

Spherical
Rod-Shaped
Comma-shaped
Spiral
Bacteria can assume multicellular organizations:
o Hyphae (branching filaments)
o Mycelia (tufts of hyphae)
o Trichomes
Bacteria are often 0.5-5 micrometer in length
Large bacteria: thiomargarita namibiensis, epulopiscium
Why is it important to clump, chain together (especially for pathogens)? It
increases resistance, survive phagocytosis.
2) S/V ratio What is it? Why is it important?
Surface to volume ratio
As the S/V ratio increases, nutrient uptake and diffusion of molecules

become more efficient

The smaller the cell, the more the surface area compared to the volume of

the cell
Large size may be protective mechanism against predation
Rod shaped cells have higher S/V

3) Bacterial cell structure

A. Cytoplasm nucleoid, plasmids, ribosomes, inclusion bodies

Nucleoid region: Usually not membrane bound, location of chromosome

and associated proteins
o Supercoiling; produces dense central core with loops
o Chromosome; a single close circular loop of double stranded DNA
o Nucleoid proteins aid in folding
Plasmids: small, closed circular DNA molecules
o Exist and replicate independently of chromosome
o Found in bacteria, archea and some fungi
o Episomes; plamids that can integrate into the cell chromosome

o
o
o
o
o

Contain few genes that are non-essential

may exist in many copies in cell
inherited stably during cell division
Sometimes plasmids can be loss during cell division called curing
classification of plasmids based on mode of existence, spread, and

function
Ribosomes
o Complex structures consisting of protein and RNA
o Bacterial and archaea ribosome = 70S
o Eukaryotic = 80S (bigger)
o S = Svedburg unit has to do with size and shape
o bacterial and archaeal ribosomal RNA
o 16S rRNA and SSU proteins - small subunit = 30S
o 23S rRNA, 5S rRNA and LSU proteins - large subunit = 50S
o archaea have additional 5.8S in large subunit (also seen in
eukaryotic large subunit)
o proteins vary
o archaea more similar to eukarya than to bacteria
Inclusion Bodies:
o Common in all cells
o Granules of organic or inorganic material that are stockpiled for
later use by the cell
o Storage inclusions: storage of nutrients, metabolic end products,

energy, building blocks, glycogen storage, carbon storage

Other things in cytoplasm:
o tRNA, rRNA, mRNA, proteins, etc.
o Inclusion bodies may also be present.
o Polyhydroxybutyrate granules - carbon storage
o Sulfur globules - sulfur storage
o Gas vesicles - buoyancy control, aquatic bacteria
o Carboxysomes - location of carbon fixation reactions
o Magnetosomes allow cells to align with magnetic field,
organelles associated with direction finding, found in aquatic
bacteria

B. Cytoskeleton FtzS, MamK, MreB/Mbl, CreS, Par M and R What are

they? What do they do?
The cytoskeleton is a series of internal proteins that assist in keeping
everything in the right location in cells
FtsZ
o Involved with cell wall synthesis during cell division
o Divides the cell in half
Other cytoskeletal proteins are involved in moving internal items
MamK
MreB: forms rollercoaster like scaffolding in the cells
CreS
o Causes cell to cause crescent shape
ParM
R
C. Plasma Membrane - function, important features, fluid mosaic model,
membrane proteins, lipids

Function
o Interface between inside and outside
o Encompasses the cytoplasm
o Selectively permeable barrier
o Interacts with external environment (receptors, transport systems,
metabolic process)
o Can be used for capturing energy (proton motive force)
o Sensory systems (environmental changes, can detect when to alter
gene expression)
Fluid Mosaic Model
o Membranes are lipid bilayers with floating proteins
o Amphipathic lipids; asymmetric lipids that have polar ends and nonpolar tails
Membrane proteins
o Peripheral proteins; loosely connected to the membrane
o Integral proteins; imbedded within membrane, amphipathic,
carbohydrates often attached, transport electron transport, can
move laterally
Lipids

o Very dynamic
o Membrane lipid saturation reflects environmental conditions
o Stabilizing molecule is hopanoids (cholesterol stabilizes
eukaryotes)

4) Movement of materials into cells - passive diffusion, facilitated diffusion, active

transport (ABC and MFS transporters), PTS system, siderophores for iron uptake

How do items cross the PM?

o O2 and CO2 are small and can diffuse across readily.
o H2O is helped across by aquaporin protein channels.
o Osmosis is the flow of water across the PM toward the side with a
higher solute (particle) concentration.
o Osmosis can cause a cell to swell with water or shrivel as water
leaves, but a strong cell wall can help keep a bacterial cell alive

during these hardships.

How do organisms take up nutrients?
o facilitated diffusion all microorganisms
o active transport all microorganisms
o group translocation Bacteria and Archaea
o endocytosis Eukarya only
Passive diffusion:
o molecules move from a region of higher concentration to one of
lower concentration between the cells interior and the exterior
o Not energy dependent
o The rate of diffusion depends on the concentration gradient conc.

must be higher outside than inside

Facilitated diffusion:
o No energy required
o uses membrane bound specific carrier molecules (permease
proteins embedded in the cell membrane) which greatly increases
the rate of diffusion

o smaller concentration gradient is required for significant uptake of

molecules
o effectively transports glycerol, sugars, and amino acids
o carrier can become saturated
o lower concentrations = more rapid diffusion
Active transport
o energy-dependent process
ATP or proton motive force used
o move molecules against a gradient (ie., from low conc to high conc)
o concentrates molecules inside cell
o involves specific carrier proteins (permeases)
o carrier saturation effect is observed at high solute concentrations
2 types of Active transport:
o Primary Active Transporters (ABC Transporters) use energy
provided by ATP hydrolysis to move substances against a
conc gradient
Uniport; transports one molecule at a time
o Secondary Active transporters (MFS Transporters) uses ion
gradients to co-transport substances
Antiport; pumping something in and pumping something out,

opposite directions
Symport: pumping two things into the cell same direction
Phosphotransferase System (PTS)
o The PTS transports sugars (such as glucose, mannose, and
mannitol) into the cell.
o Found in many facultatively anaerobic bacteria and in some

obligate anaerobes but not found in aerobes

Iron Uptake
o microorganisms require iron
o ferric iron is very insoluble so uptake is difficult
o microorganisms secrete siderophores to aid uptake
o siderophore complexes with ferric ion
o complex is then transported into cell
o

5) Movement of materials out of cells Sec System, TAT system, Type 4, and Type
3 secretion systems and relationship to flagella and bacterial conjugation.

Sec System,

TAT system,

Type 4,

Type 3 looks like a syringe

6) Bacterial cell structure

Cell Wall - morphology, how is it made, effect of lysozyme, lysostaphin, B
lactam antibiotics
Cell Wall Structure
Peptidoglycan structure: meshlike polymer of two identical sugar
subunits
Each subunit is NAM and NAG, made up of amino acid side chain
and sugar ends
NAM stereoisomer is D
o How does the cell wall form?
NAM is made in cytoplasm
NAM is linked to peptide chain
NAG is added to NAM
It is exported to the outside of cell
And attached to outer membrane and linked together
o Can it be degraded?
Yes, penicillin and Beta lactam structures weaken the cross links of
the peptidoglycans
Naturally, lysozyme can cleave the peptidoglycans
As a result, the cell cant resist pressure changes and will burst

Difference between Gram negative and Gram positive cell envelopes (LPS, teichoic
acids) and mechanism of the Gram stain reactions make sure you know this well
o Gram Positive
A thick outer layer of peptidoglycan
A very narrow periplasmic space
Teichoic acids in the peptidoglycan (negatively charged)
Lipoteichoic acid
The Gram-positive peptidoglycan layer has large pores throughout
its matrix.
o Gram Negative
A varying width periplasmic space containing a very thin layer of

peptidoglycan
An outer membrane composed of lipopolysaccharide (LPS)
LPS: Consists of lipid A, core polysaccharide, O side chain,
contributes to negative charge on cell surface, stabilizes outer
membrane, creates barrier to bile salts, antibiotics, protection from

host defenses
Outer membrane more permeable than plasma membrane due to

presence of porin proteins and transporter proteins

porin proteins form channels through which small molecules
o How can nutrients get through the cell walls?
Peptidoglycan layer has pores
Then, molecules move into the periplasmic space.
Once there, active transport mechanisms can move the molecule
into the cytoplasm.
o How can molecules get out of gram negative periplasmic space?
Some move from the periplasm to outside directly (these are known

as autotransporters and are rare).

Some use single-step (never entering the periplasm) transport

systems
o Mechanism of Gram Stain Reaction
Cell wall characteristics can help explain how the Gram-stain works.
The alcohol decolorization step shrinks the large pores in the Grampositive cell, helping to lock the crystal violet stain in.

The alcohol also may strip away some of the outer membrane lipids
in the Gram-negative cells, making them more likely to lose the
initial crystal violet stain.

Flagella and pilli structure, mechanism of movement, chemotaxis, other

mechanisms of movement
o The Cell Envelope:
Flagella self-assemble, it takes no energy
Flagellar Movement
flagellum is 2 part motor producing torque-associated with

the basal body-functions like an electric motor

rotor
o C (FliG protein) ring and MS ring turn and interact with
stator
stator (electromagnet)- Mot A and Mot B proteins
o form channel through plasma membrane
o protons move through Mot A and Mot B channels and
produce energy through proton motive force (PMF)
created by the electron transport chain (ETC) located

in the plasma membrane

torque powers rotation of the basal body and filament
Flagella Distribution
monotrichous one flagellum
polar flagellum flagellum at end of cell
amphitrichous one flagellum at each end of cell
lophotrichous cluster of flagella at one or both ends
peritrichous spread over entire surface of cell
Parts of Flagella: it moves counterclockwise like a propeller
Filament of multiple flagellin proteins, 510 m long, hollow,

rigid cylinder
Hook protein portion that connects filament to basal body
Basal body: disk-like structure that produces torque on

filament to turn it like a propeller

Flagella made with Type 3 secretion system

Chemotaxis
movement toward a chemical attractant or away from a

chemical repellent
changing concentrations of chemical attractants and
chemical repellents bind chemoreceptors of chemosensing

system
Non flagellar motility
Gliding and twitching
Polymerization of actin in host cells for propulsion of bacteria
into adjacent cells (Shigella dysenteriae, Listeria

monocytogenes)
Adherence molecules to stick to surfaces
Mediated by pili (s. pilus), fibers of pilin protein possess other

proteins on their tips for sticking.

A sex pilus is a different structure used for conjugation
(sending a DNA plasmid from one cell to another).

Glycocalyx capsules, slime layers, s layers

o Glycocalyx; general term for slime or capsule
o Capsules; helps pathogens resist phagocytosis, protect against drying,
adhesion exclude viruses and detergents
o slime layers;
o S layers; formed by proteins that self assemble, protect from
environmental stressors, maintains shape and rigidity, adhesion,
crystalline array of proteins, acts as armor
in gram-negative bacteria the S layer adheres to outer membrane
in gram-positive bacteria it is associated with the peptidoglycan
surface

Endospores Sporulation and Germination what makes them so resistant?

Bacterial Endospore
o complex, dormant structure formed by some bacteria survival
mechanism commences when growth ceases
o Predominately associated with Gram positive cells (Bacillus,
Clostridium)
o various locations within the cell
o resistant to numerous environmental conditions
heat
radiation
chemicals
drying
o spore surrounded by thin covering called exosporium
o thick layers of protein form the spore coat
o cortex, beneath the coat, thick peptidoglycan
o core has nucleoid and ribosomes
o What makes endospore resistant? Calcium, SASPs, dehydrated core,
spore coat
Germination
o activation
prepares spores for germination
often results from treatments like heating
o germination
environmental nutrients are detected
spore swelling and rupture of absorption of spore coat
loss of resistance
increased metabolic activity
o outgrowth - emergence of vegetative cell

7) Archaeal morphology size and shape

Structure
o Size is usually 0.55 m in diameter.
o Similar shapes to Bacteria/Eukarya
o Both Archaea and Bacteria usually possess singular, circular
chromosomes and lack a membrane-bound nucleus.
o Archaeal DNA is complexed with histones (like Eukarya).
o Many of the DNA replication enzymes of Archaea look like those of
Eukarya.
o The Archaea plasma membrane structure is unique to this domain.

8) Archaeal cell structure what makes them a unique domain how do they
compare with bacterial structure
A. Cytoplasm
Histones form structures that DNA wraps around.
Histone structure/wrapping is different in Archaea from Eukarya.
Inclusion bodies such as gas vacuoles have been observed in some Archaea.

B. Cytoskeleton
Cytoskeletal homologues are found in both Bacteria and Archaea.

C. Plasma Membrane - important features

The plasma membrane can even be a monolayer instead of a bilayer.

D. Cell Wall morphology

The cell wall provides physical and osmotic protection.
In Archaea, it may be composed of pseudomurein (slightly different from

peptidoglycan structure).
Some Archaea lack a cell wall (cytoskeleton?)

E. Flagella structure, mechanism of movement

thinner (1014 nm vs. 2024 nm)
usually composed of two or more different versions of flagellin protein
likely growing from BASE rather than from TIP
G. S layers, cannulae

Some use an S-layer (single layer of many identical armorlike subunits) to protect

against predation/viruses and to mediate adhesion.

Some form cannulae, hollow glycoprotein tubes that link cells together to form a
complex network.