MCB2610 Fundamentals of Microbiology

Study Guide
Fall 2016, Exam #1
Chapter 1, Appendix B, and Chapter 6A (up to slide #52)
1) 5 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) Dr. Jonathan Klassen's presentation. Focus on general material. (4 questions)
Attine Ants; these ants feed a fungus, and they eat the fungus
Symbiotic relationships feature multiple reproductives
Maximizing reproduction is in the best interest of each organism
Leaf cutter ants are the most highly evolved fungus growing ants
Clonal propogation: the queen carries the fungus to start the new garden
A Huge problem is pathogen susceptibility
If the fungus dies, so does the ant colony
Clonal propagation limits genetic innovation
Access to diversity evolved in other lineages is restricted
But: Pathogenesis also can stabilize cooperation by changing the focus of selection
Escovopsis is a specific pathogen of the fungal cultivar
Solutions
The ants dump waste
They groom the fungus
They groom the leaves they feed to the fungus
Antimicrobial secretions
They switch cultivars
They use pseudonocardia; antibiotic producing actinobacteria
Pseudonocardia may compete with each other which is bad
3) Read the assigned paper on fungus-growing ants. Focus on general material (4
questions)
It was though that there were only two symbionts in the relationship between Attine
ants and their fungus
BUT actually, the fungus gardens are actually hosts to Escovopsis
The paper describes a third mutualist, a bacterium that produces antibiotics
specifically targeted to suppress the growth of the parasite Escovopsis
Powdery white crust coated on ants that is bacterium
It selectively inhibit the growth of the garden parasite Escovopsis
They studied 22 species of ants and fungus growing ants
The bacteria was associated with all 22 types of ants
They isolated this bacteria
Isolated, it wasn't able to produce antibiotics
But showed effects towards Escovopsis
The bacteria completely suppressed spore germination of Escovopsis
Ants provide nourishment for the growth of the bacteria

Lecture Material (12 questions will come from this material)

Chapter 1
1) Prokaryotes vs. eukaryotes
prokaryotic cells lack a true membrane-delimited nucleus, membrane bound
organelles
-This is not absolute
eukaryotic cells have a membrane-enclosed nucleus, are more complex
morphologically (membrane-enclosed organelles), and are usually larger than
prokaryotic cells
2) Definition of life
- Metabolism
- Growth
- Reproduction
- Genetic variation/evolution
- Response/adaptation to the external environment
- Homeostasis (maintaining internal organization and order, usually by expending
energy)
3) How the classification schemes have evolved over time
- two groups Aristotle
- five kingdom system Robert Whittaker 1960s
- three domain system developed due to advances in electron microscopy,
biochemistry and physiology, and ability to determine nucleic acid and protein
sequences
- Carl Woese et al (1990 paper) based on a comparison of small subunit (SSU)
ribosomal RNA gene sequences, divides microorganisms into:
Bacteria (true bacteria),
Archaea
Eukarya (eukaryotes)
4) Carl Woese and the 3 domain system of classification. Make a list of the features of
each of the 3 domains so you can easily compare them
1. Domain Bacteria:
- Usually single-celled
- Majority have cell walls with peptidoglycan
- Most lack a membrane-bound nucleus
- Ubiquitous and some live in extreme environments
- Some cause disease but most are beneficial
- Many recycle elements (e.g., Cyanobacteria produce amounts of
significant oxygen)
2. Domain Archaea:
- distinguished from Bacteria by unique rRNA sequences
- lack peptidoglycan in cell walls and have unique membrane lipids
- some have unusual metabolic characteristics (e.g., methanogens which
produce methane gas)

- many live in extreme environments

- Their role in causing diseases is unclear
3. Domain Eukarya:
- Plants
- Animals
- Multicellular organisms
- algae photosynthetic
- protozoa may be motile, hunters, grazers
- slime molds two life cycle stages (mold-like, protozoan-like)
- water molds protists that grow in moist environments - some cause
- devastating diseases in plants
- fungi (e.g. yeasts unicellular, molds multicellular)
- Primarily decomposers, some cause disease
5) Know where viruses fit in
- smallest of all microbes
- Very simple some consist of only proteins and nucleic acids
- requires host cell to replicate
- cause range of diseases, some cancers
- viroids and virusoids
- infectious agents composed of RNA
- prions infectious proteins
- Technically, viruses arent considered to be alive.
- They dont replicate outside of a host cell.
- They (usually) have little to no biochemical activity outside of a host cell.
- They are inert and nonreactive outside of a host cell.
- Microbiology still studies viruses, though, since they are too small to be seen with the
naked eye.
6) Stanley Miller and his experiment
- In the 1950s, a grad student named Stanley Miller formed organic molecules from a
primordial soup.
7) RNAS place in evolution
- ribozymes Discovered by Thomas Cech in 1981
- RNA molecules that form peptide bonds
- perform cellular work and replication
- earliest cells may have been RNA surrounded by liposomes
- Some RNA molecules have the ability to catalyze reactions (these are known as
ribozymes, a combination of ribonucleic acid and enzymes)
-This means RNA could serve the dual purpose of genetic information storage AND
catalyzing reactions!
8) Endosymbiotic theory and hypothesis
- So then, the basic idea of how microbial life arose on Earth is:
- Early conditions formed RNA and micelles.
- These came together into a primitive cell using RNA for storing genetic info
and coding.
- Primitive cells eventually changed from using RNA to DNA instead for storing
their genetic information.

- Endosymbiotic theory: Primitive prokaryotic microbes ingested other microbes,

starting a symbiotic relationship, forming the first basic eukaryotes.
-Ingested microbes that could use oxygen for a respiratory process to
produce chemical energy became mitochondria.
- Ingested microbes that could fix carbon dioxide into organic molecules
using light energy became chloroplasts.
-Endosymbiotic Hypothesis
- mitochondria, hydrogenosomes, and chloroplasts are all thought to have
evolved from bacterial cells that invaded or were ingested by early ancestors of
eukaryotic cells
- mitochondria and chloroplasts are very similar to extant bacteria and
- cyanobacteria, respectively
- Both mitochondria and chloroplasts contain bacterial DNA and
ribosomes
- Both are similar based on SSU rRNA gene sequences
9) Correct microorganism naming
- Bacteria and Archaea do not reproduce sexually and are referred to as strains
- A strain consists of descendents of a single, pure microbial culture share
stable properties
- may be biovars, serovars, morphovars, pathovars
- binomial nomenclature developed by Carolus Linnaeus (1750)
- genus and species epithet
- (e.g., Escherichia coli italics or underline)
- Can abbreviate after first use (e.g. E. coli)
10) Robert Hooke and his contribution to the cell theory
- Robert Hooke (1635-1703) developed a microscope that could magnify 30 X
- Looking at a thin slice of cork with a crude microscope
- Life consisted of little boxes cells
- Cell theory all living things are composed of cells
11) Antony van Leeuwenhoek and his contribution to microscopy
- Contemporary of Hooke and read his book Micrographia
- First person to observe and describe microorganisms accurately with a microscope
that could magnify 50 300 times
- Called microbes animalcules
- Studied his teeth scrapings and diarrhea
12) The theory of spontaneous generation and Redis experiment to discredit it
- spontaneous generation
- living organisms can develop from nonliving or decomposing matter
- Francesco Redi (1626-1697)
- discredited spontaneous generation
- showed that maggots on decaying meat came from fly eggs
- His experiment 3 containers with meat, one sealed, one covered with gauze,
one open

13) The conflicting results of Needham and Spallanzani and the very important
difference in their experimental designs
- John Needham (1713-1781)
- his experiment:
- mutton broth in flasks boiled sealed
- results: broth became cloudy and contained microorganisms
- Lazzaro Spallanzani (1729-1799)
- his experiment:
- broth in flasks sealed boiled
- results: no growth of microorganisms
14) Louis Pasteur and his famous swan-neck flask experiment as well as his other work.
- Settled the matter of spontaneous generation
- his experiments
- placed nutrient solution in flasks
- created flasks with long, curved necks
- boiled the solutions
- left flasks exposed to air
- results: no growth of microorganisms
- Biogenesis Life comes from life
- demonstrated that microorganisms carried out fermentations
- developed pasteurization low temperature heating to destroy unwanted microbes in
- wine and milk used to reduce spoilage bacteria and kill harmful bacteria heated
to 145F (63C) for 30 min. then cooled, now 161F (72C) for 15 sec
- showed that the pbrine disease of silkworms was caused by a protozoan
15) Tyndall, Cohn, Semmelweis, Lister, Snow, Chamberland, and Roux's, contributions
to microbiology
- John Tyndall (1820-1893)
- demonstrated that dust carries microorganisms
- showed that if dust was absent, nutrient broths remained sterile, even if directly
exposed to air
- also provided evidence for the existence of exceptionally heat-resistant forms
of bacteria
- Ferdinand Cohn (1828-1898)
- heat resistant bacteria could produce endospores
- Ignaz Semmelweis (1840s) the savior of mothers
- High incidence of puerperal fever in obstetrical patients could be reduced by
washing hands (autopsy source) (18% to 2%) before the age of germ theory
- Joseph Lister (1827-1912) considered the Father of Modern Surgery
- provided indirect evidence that microorganisms were the causal agents of
disease
- developed a system of surgery designed to prevent microorganisms from
- entering wounds as well as methods for treating instruments and surgical
dressings using phenol
- his patients had fewer postoperative infections
- John Snow 1st Epidemiologist 1854 Cholera outbreak

- Charles Chamberland (1851-1908)(worked with Pasteur)

- discovered chicken cholera vaccine by accident
- His ground work on sterilization lead to the discovery of the autoclave
- developed porcelain bacterial filters which they attached to water faucets
- The filters were used by Ivanoski and Beijerinck to study tobacco mosaic
disease
- determined that extracts from diseased plants had infectious agents present
which were smaller than bacteria and passed through the filters
- infectious agents were eventually shown to be viruses
- Called filterable particles
- Pasteur and Roux (1st micro class)
- discovered that incubation of cultures for long intervals between transfers
caused pathogens to lose their ability to cause disease could be used to
develop vaccines
16) Koch and his postulates.know them inside and out
- Robert Koch (1843-1910) - German
- established the relationship between Bacillus anthracis and anthrax
- Robert Koch determined Bacillus anthracis and Mycobacterium tuberculosis
were the causes of anthrax and tuberculosis (respectively).
- His work with anthrax helped sheep herders and cattle ranchers avoid costly
animal losses.
- Kochs Posulates:
- the microorganism must be present in every case of the disease but absent
from healthy individuals
- the suspected microorganism must be isolated and grown in a pure culture
- the same disease must result when the isolated microorganism is inoculated
into a healthy host
- the same microorganism must be isolated again from the diseased host
- Limitations to Postulates:
- Many organisms cannot be grown in pure culture (e.g. Mycobacterium leprae)
- using humans in completing the postulates is unethical (e.g. Ebola
hemorrhagic fever)
- molecular and genetic evidence may be used instead

17) Know some of the developments in microbiology that came from Kochs lab.
- Kochs work led to discovery or development of:
- Agar (Fanny Hess)
- Not broken down by most organisms
- Doesnt melt until it reaches 100C
- Doesnt solidify until it reaches 50C
- Produces a clear product
- Petri dish (Richard Petri)

- nutrient broth and nutrient agar

- methods for isolating microorganisms
- Aseptic technique
- Charles Chamberland (1851-1908)(worked with Pasteur)
-discovered chicken cholera vaccine by accident
-His ground work on sterilization lead to the discovery of the autoclave
-developed porcelain bacterial filters which they attached to water
faucets
-The filters were used by Ivanoski and Beijerinck to study tobacco mosaic
disease
- determined that extracts from diseased plants had infectious
agents present which were smaller than bacteria and passed
through the filters
- infectious agents were eventually shown to be viruses
- Called filterable particles
- Pasteur and his coworkers
- developed vaccines for chicken cholera, anthrax, and rabies
18) Jenner, Ehrlich, Fleming, Rettgers contribution to microbiology
Edward Jenner (ca. 1798)
- used a vaccination procedure to protect individuals from small pox
- Milkmaid with cowpox was immune
- Tested on 8 year old volunteer
- NOTE: this preceded the work establishing the role of microorganisms in
disease
Paul Ehrlich (1910) German Physician (worked for a time in Kochs lab)
- 1st chemotherapy, synthetic drug salvarsan, arsenic derivative, offered salvation
from syphilis
Alexander Fleming discovered the first antibiotic.
- He observed that Penicillium fungus made an antibiotic, penicillin, that killed
Staphylococcus aureus.
- 1940s: Penicillin was tested clinically and mass produced.
Leo F. Rettger
- Contemporary of Koch, Pasteur, and Lister
- Taught at Yale did research on poultry at UCONN in the summers
- 1st to teach Microbiology course (bacteriology)
- 18th President of the American Society of Microbiology
- 1st President of the CT Valley Branch of the ASM

Appendix B Microscopy (12 questions will come from this material)

19) Resolution-its importance as well as all of the factors that can influence resolution
(ex: wavelength, numerical aperature, immersion oil, working distance etc.)
- Resolution (or resolving power) - ability of a lens to separate or distinguish small
objects that are close together
- The wavelength of light used is a major factor in resolution
shorter wavelength greater resolution

(blue light 450 500 nm can not resolve structures smaller than 0.2 um)
20) Determining total magnification of a microscope.
- There are issues if you magnify too much, 30x ocular would be too much, total mag of 3,000
21) Microscopes: what each scope is used to visualize, which can give you 3D vs. 2D
images. I will not have you label microscope diagrams.
The Differential interference Contrast Microscope
Can view live, 3D organisms,
Similar to phase-contrast - creates image by detecting differences in refractive indices
and thickness of different parts of specimen
Uses two beams of polarized light to create a
excellent way to observe living cells
live, unstained cells appear brightly colored and three-dimensional
cell walls, endospores, granules, vacuoles, and nuclei are clearly visible
The Dark-Feld Microscope
Image is formed by light reflected or refracted by specimen
produces a bright image of the object against a dark background
used to observe living, unstained preparations
has been used to observe internal structures in eukaryotic microorganisms
has been used to identify bacteria such as Treponema pallidum, the causative agent
of syphilis
The Phase-Contrast Microscope
converts slight differences in refractive index and cell density into easily detected
variations in light intensity.
Uses a hollow cone of light
The hollow cone of light passes through a specimen and is retarded (out of phase).
The light then passes through a phase plate which advances it and brings the light
back into phase.
Produces an image where cells are dark and the background is light
excellent way to observe unstained, living cells
studying microbial motility
detecting bacterial structures such as endospores and inclusion bodies that have
refractive indices different from that of water
The Fluorescence Microscope
exposes specimen to ultraviolet, violet, or blue light
specimens usually stained with fluorochromes (fluorescent dyes)
shows a bright image of the object resulting from the fluorescent light emitted by the
specimen
has applications in medical microbiology and microbial ecology studies
Other uses:
Photosynthetic organisms naturally fluoresce when excited with specific
wavelengths.
Can also be used to localize specific proteins within cells
One method is to fuse the gene of the protein of interest to the jellyfish
(Aequorea) protein, green fluorescent protein (GFP)
Immuno-Flourescence:
Type of fluorescent microscopy
essential tool in microbiology

 fluorochrome-labeled probes, such as antibodies, or fluorochromes tag specific cell

constituents for identification of unknown pathogens
localization of specific proteins in cells
Can also be used to localize specific proteins within cells
One method is to fuse the gene of the protein of interest to the jellyfish
(Aequorea) protein, green fluorescent protein (GFP)
Confocal Microscopy:
Type of fluorescent microscopy
Uses a computer
confocal scanning laser microscopy (CLSM) creates sharp, composite 3-D image of
specimens by using laser beam, aperture to eliminate stray light, and computer
interface
Specimen is usually fluorescently stained
22) The major similarities and differences between the various light and electron
microscopes.
**What are the advantages of bright field, dark field, phase contrast, DIC, fluorescence and
confocal microscopy?
23) Remember for any type of fluorescent imaging your specimen needs to either be
naturally fluorescent, or labeled with a fluorescent dye or fluorescently labeled antibody
understand the concept of immunofluorescence
Some organisms are natural fluorescent
Photosynthetic organisms
Jellyfish and GFP
23) Why we stain plus different types of fixations pros and cons
increases visibility of specimen
accentuates specific morphological features
preserves specimens
Fixation:
preserves internal and external structures and fixes them in position
organisms usually killed and firmly attached to microscope slide
heat fixation done with a benson burner, routinely used with bacteria and archaea
preserves overall morphology but not internal structures, inactivates enzymes and can
destroy some proteins
chemical fixation used with larger, more delicate organisms
protects fine cellular substructure and morphology
Inactivates, makes insoluble and immobilizes, proteins and lipids (e.g., ethanol, acetic
acid, formaldehyde and glutaraldehyde).

24) Dyes, simple staining, Gram stain (know inside and out), Acid Fast, endospore,
capsule and flagella staining
Dyes:
make internal and external structures of cell more visible by increasing contrast with
background

have two common features

chromophore groups
chemical groups with conjugated double bonds
give dye its color
ability to bind cells
Dyes
ionizable dyes have charged groups
basic dyes; have positive charges that bind to negatively charged molecules
(e.g., nucleic acids, many proteins, and cell surfaces) methylene blue, basic
fuchsin, crystal violet, safranin, malachite green
acid dyes; have negative charges that bind to positively charged cell structures
but are repelled by the cell surface (produces negative staining, nigrosin, good
for seeing capsules) eosin, rose bengal acid fuchsin
Simple Stains
a single stain is used (easy and quick)
can determine size, shape, and arrangement of bacteria
Differential Staining
divides microorganisms into groups based on their staining properties
e.g., Gram stain
e.g., acid-fast stain
differential stain also used to detect presence or absence of structures
endospores, flagella, capsules
Gram Staining:
Most widely used differential staining procedure
Developed by Christian Gram, 1884
Divides bacteria (but not archaea) into two groups, Gram positive and Gram negative,
based on differences in cell wall structure
Gram positive cells are purple, Gram negative cells are pink
STEPS:
1) Make a smear of a bacterial suspension on a glass slide
2) Heat fix by passing through a flame
3) Stain with primary stain Crystal Violet, let stand for 1 minute, rinse with water
(all cells stain)
4) Treat with iodine for 1 minute (a mordant which binds with the CV to make a
large crystal inside the cell), rinse with water
5) Decolorize with alcohol to remove the stain if it is a Gram neg. organism.
Stain cant be removed in a Gram pos. organism due to thick cell wall
peptidoglycan
6) Counter stain with Safranin

**Decolorize step is the most important, cannot decolorize for too long or the
Crystal Violet will be washed out as well. Very critical step.
**Can you leave a step out? The last step, you can still see the difference between
gram negative and positive
Acid-Fast Stain:
particularly useful for staining members of the genus Mycobacterium (which dont
stain well with the Gram stain)
ex. Mycobacterium tuberculosis causes tuberculosis
ex. Mycobacterium leprae causes leprosy
high lipid content in cell walls (mycolic acid) prevents dyes from readily binding

 Uses high heat and phenol to drive basic fuchsin into the cells
Differential Staining of Specific Structures:
endospore staining exceptionally resistant to staining (e.g. Bacillus sp. and
Clostridium sp.)
heated, double staining technique
bacterial endospore is one color and vegetative cell is a different color
capsule stain used to visualize capsules surrounding bacteria (India ink or nigrosin)
negative stain - capsules may be colorless against a stained background
flagella staining very thin and can only be seen with an electron microscope
Uses a mordant and a stain. The mordant is used to increase thickness of flagella
**What is a mordent? Binds with stain to form crystal.
**Staining that involves mordents? Gram stains (iodine) and flagella stains.

25) Disadvantages and advantages of TEM, SEM, Scanning Probe, etc.

Electron Microscopy
The best light microscope has a resolving limit of 0.2um (max. mag of 1500X)
In the electron microscope electrons replace visible light as the illuminating beam
(resolution of 0.5 nm, max mag of 100,000X)
Wavelength of electron beam is much shorter than visible light, resulting in much
higher resolution
Allows for study of microbial morphology in great detail
Transition Electron Microscope
Uses an electromagnet to focus an electron beam
electrons scatter when they pass through thin sections of a specimen
transmitted electrons are under vacuum which reduces scatter and are used to
produce clear image
specimens are chemically fixed and stained with electron dense materials, such as
heavy metals, that differentially scatter electrons
denser regions in specimen, scatter more electrons and appear darker
Disadvantages of TEM:
Electrons can only penetrate very thin specimens
Usually gives only 2D image
Specimens must be viewed under high vacuum
Specimens are dead
Because of harsh treatment (dehydration, etc) specimens are shrunken and
distorted (artifacts)
**Why do electron microscopes have better resolution than light? Electron beams have a
shorter wavelength of light.
Scanning Electron Microscope:
Uses electrons reflected from the surface of a specimen to create detailed image
Produces a realistic 3D image of specimens surface features
Can determine actual in situ location of microorganisms in ecological niches (e.g.
human skin, gut, etc) samples must be dehydrated and coated with a thin film of
metal
Electron Cryotomography

 since 1990s, rapid freezing technique has provided way to preserve native state of
structures examined in vacuum
images are recorded from many different directions to create 3-D structures
provides extremely high resolution of
cytoskeletal elements, magnetosomes, inclusion bodies, flagellar motors, viral
structures
2 Types of Scanning Probe Microscopy
Scanning tunneling microscope (1980)
magnification 100 million times ca view atoms on surface of a solid
steady current (tunneling current) maintained between microscope probe and
specimen)
up and down movement of probe as it maintains constant current is detected
Atomic force microscope
sharp probe moves over surface of specimen at constant distance
can see down to the molecular level
**What type of microscope has the lowest magnification? Any microscope that uses light.
Ex. brightfield uses blue light, the wavelength of light is much longer than the wavelength of
electrons
**What type of microscope has the highest magnification? Electron microscopy

Chapter 6A Up to slide #63

Chapter Outline:
Nutritional requirements of microorganisms
Factors affecting microbial growth
Growing microorganisms in the laboratory
Measuring microbial population growth
Eliminating microbes and preventing their growth
26) Nutritional requirements - macronutrients, micronutrients
What do microorganisms generally need to grow?
Macronutrients - All cells need access to large amounts of carbon, nitrogen,
phosphorus, sulfur, and oxygen to build macromolecules. Microorganisms need a lot
of these.
Micronutrients - are also required by microbes:
Includes several metal ions (Na+, Mg2+, Mn2+, etc.)
Often required for protein structure/activity, biosynthesis of ATP by electron
transport-related processes
Trace elements
often supplied in water or in media components

 ubiquitous in nature
serve as part of enzymes and cofactors
Aid in catalysis of reactions and maintenance of protein structure
Some organisms also require unique substances such as silicic acid used to construct
the silica walls of diatoms (diatoms)
No matter what an organisms nutrient requirement they require a balanced mix
If one nutrient is limited or in short supply the organism will have limited growth
27) Naming an organism based on its energy source, electron source and carbon
source IN THAT ORDER (they are always named energy, electron, carbon even if our
information is given in a different order)
Acquisition of nutrients
Autotrophs assimilate carbon from inorganic sources.
Heterotrophs assimilate carbon in preexisting organic form.

**To what major nutritional group do humans belong to? Chemoorganoheterotroph

28) Growth factors-what are they and why are they important
Some organisms can synthesize all organic molecules from a single carbon source and
inorganic salts, but some require growth factors to support growth
Growth factors are organic compounds that cannot be synthesized by an organism but are
essential for growth
Auxotroph; organism that cannot make amino acids, cell cant grow
Must be supplied by the environment if the cell is to survive and reproduce
amino acids
needed for protein synthesis
purines and pyrimidines
needed for nucleic acid synthesis
vitamins
Small organic molecules that function as enzyme cofactors, needed in very small
amounts
Humans need vitamin C
Other less common growth factors include heme (cytochromes, Haemophilus influenzae)
and cholesterol (required by some mycoplasmas)

** Why is it so difficult to grow some organisms in a laboratory culture? It is difficult to know

the growth factor requirements
29) How does nutrient concentration relate to growth?
Nutrient concentration
Growth rate will depend on the amounts of nutrients in the environment.
One key nutrient, available in the lowest amount, will dictate how much growth can
occur over time (i.e., it will be a limiting factor)

30) Why is oxygen important in growth?

Effects of oxygen on microbial growth
Aerobes grow in the presence of oxygen.
Obligate aerobes REQUIRE oxygen.
Microaerophiles grow best when there is less oxygen than normal.
Anaerobic growth occurs without oxygen.
Killed by exposure to oxygen
Aerotolerant anaerobes arent harmed by oxygen but dont use it, either.
Obligate anaerobes cannot grow when oxygen is present.
Facultative anaerobes CAN use oxygen but can also grow in the absence of
oxygen.
Does not require oxygen, but if oxygen is present it will not die
Microaerophile
Has a requirement for oxygen, but only a little bit

31) Anaerobes vs. aerobes and everybody in-between

**Need certain enzymes to detoxify oxygen
Factors Affecting Microbial Growth:
Effects of oxygen on microbial growth
Often determined by what defenses are available against oxygens negative effects in
the cell

32) ROS- how do cells deal with this?

The byproducts of oxygen are toxic and are taken care of by enzymes
Enzymes that protect against toxic Oxygen Products:

Superoxide dismutase
Catalase
Peroxidase

 All strict anaerobic microorganisms lack or have very low quantities of

superoxide dismutase
catalase
these microbes cannot tolerate O2
anaerobes must be grown without O2
work station with incubator
gaspak anaerobic system

33) Role of pH and temperature in growth

Effects of pH on microbial growth
pH affects macromolecule structures and transmembrane electrochemical gradients.
Each microbe will have an optimal pH range for growth.
Acidophiles = pH < 5.5
Neutrophiles = pH 5.5 to 8.5
Alkalophiles = pH > 8.5
34) Role of solutes and water availability in growth
changes in osmotic concentrations in the environment may affect microbial cells
hypotonic solution (lower osmotic concentration outside)
water enters the cell
cell swells may burst
hypertonic (higher osmotic concentration outside)
water leaves the cell
membrane shrinks from the cell wall (plasmolysis) may occur
Water Activity
water activity (aw)
amount of water available to organisms
reduced by interaction with solute molecules (osmotic effect)
higher [solute] lower aw
reduced by adsorption to surfaces (matric effect)
Aw of distilled water is 1.0
Aw of milk is 0.97
Aw of dried fruits is 0.5
Osmotolerant microbes can grow over wide ranges of water activity (e.g.,
Staphylococcus aureus 3M NaCl)
Effects of osmotic pressure and water availability on microbial growth
Different solute concentrations can result in influx of water into or efflux from the cell.
This can cause stress to the cell, causing it to either swell or shrink.
Water must also be available for biochemical reactions (measured in terms of water
availability or aw).
Interactions with solutes can decrease aw values.
Pure water aw = 1.0; seawater aw = 0.98; honey aw = 0.6
Most bacteria require an aw > 0.9 (revisited in Chapter 16).
*** Why is it difficult for organisms to grow at aw? There is water in the air, they need
water, and it takes energy to take in water

35) Adaptations to high solute concentrations and high temps

halophiles; loves salt
grow optimally in the presence of NaCl or other salts at a concentration above about
0.2M
extreme halophiles
require salt concentrations of 2M and 6.2M
extremely high concentrations of potassium
cell wall, proteins, and plasma membrane require high salt to maintain stability and
activity (e.g., Halobacterium in Dead Sea)
36) Difference between complex and synthetic media
- Can be either complex (unknown chemical composition) or defined/synthetic
(precisely defined chemical composition)
37) Important complex media components and why they are important
- 3 common examples
-Nutrient broth
-Tryptic soy broth (functional,supportive)
-MacConkey Agar (selective)
- Components
- peptones
- protein hydrolysates prepared by partial digestion of various protein
sources
- extracts
- aqueous extracts, usually of beef or yeast
- agar
- sulfated polysaccharide used to solidify liquid media; most
microorganisms cannot degrade it usually extracted from red algae
38) Functional media types
- Supportive
- Support growth of many microorganisms
- Enriched
- General purpose media supplemented by blood or other special nutrients
- Selective
- Favor the growth of some microorganisms and inhibit growth of others
- Differential
- distinguish between different groups of microorganisms based on their
biological characteristics

39) Understanding differences between selective and differential and enriched

40) How can we identify organisms that we can't grow on artificial media?
- Cultivation independent methods
- DNA from unculturable bacteria can be amplified and sequenced by PCR.
- Sequences can be used to produce fluorescent probes that will bind to
complementary DNA (fluorescent in situ hybridization or FISH).
41) Why is it difficult to grow some organisms?
- Some microbes may just be too accustomed to growing with their friends and
neighbors to be isolated.

Microbe Minutes:

#1.
#2.
#3. September 8, 2016
Staphylococcus aureus
S. aureus
SEM (photo in the slide is purple grape-like clumps)
Grape-like clumps of coccus shapes
Common part of normal skin and nose microflora
Approx. 20% of human population are carriers
Isolated by Alexander Ogston in 1880 from pus from a surgical wound
Opportunistic pathogen - not normally a pathogen, but will take advantage of a bad
situation
Causes both minor and serious infection )ex. pimples, abscess, meningitis, and TSS)
Also causes food poisoning
One of the 5 most common causes of nosocomial infections (hospital-acquired)
Spread through human contact
Also shown to be spread by pets
Sensitive to ethanol which is a good topical sanitizer
Hand washing is the best preventative measure
Golden colors colonies
Only about 2% of staph infections are sensitive to penicillin (killed by penicillin)
MRSA- methicillin resistant S. aureus
1st found in 1961 in England, now is endemic in hospitals, penicillin was the
preferred antibiotic, now resistant to penicillin, cephalosporins, kanamycin,
gentamycin, and streptomycin
VRSA - Vancomycin resistant S. aureus
1st found in Japan in 1996 - Vancomycin is currently the preferred antibiotic for
serious Staph infections
Gram positive (blue/purple)
Facultative anaerobe (grows with or without oxygen)
Non-spore forming
Non-motile
Doesn't form a capsule
Approx. 1.0 um in diameter
Grow optimally at 37C

 Grow on high salt

#4 September 13, 2016

Clostridium difficile
C. Difficile
Long irregular-shaped rods, single or in short chains, often drumstick with a buldhe in their
terminal end
Found everywhere in nature especially in soil
2-5% of the population have it in their intestines- kept under control by normal flora
Motile with peritrichous flagella
First isolated from infant stool and named Bacillus difficile by Hall and Otoole
Renamed in 1970
First associated with disease in 1978
Resistant to most antibiotics that kill other microbes
Alcohol based hand rubs are ineffective
Hand washing with 0.55% bleach is best
Releases toxins causing diarrhea
Common nosocomial disease
Predominately caused by antibiotic treatment which kills the normal microflora
Approx 20% of patients acquire during their hospital stay
CDC tracking determined that there were over 0.5 million infections and 29,000 deaths in
2011 due to D.diff
Transferred via fecal/oral route
Can be treated with metronidiazole or vancomycin
Probiotics are used to help restore the normal gut flora
stool transplant has also been found to be successful
Gram positive (blue/purple)
Strict anaerobe
Spore-forming - can survive for up to 2 years on inanimate objects
Produces a capsule
Grows optimally at 37C ( on blood agar without O2)
#5 September 20, 2016