DISCLAIMER: This is a summary of all the exercises covered in the 1st half of the lab.

It doesn’t include every detail in the lab

manual. It is supplemental study material only.

BIOL 221 Lab Practical 2 Study Guide

Ex. 12 UV Radiation of Spore Formers
- Endospores are resistant to desiccation (drying out), extreme hot and cold,
chemicals (such as ethanol and other disinfectants), and UV radiation.
- When nutrients become available again, the endospore can germinate back into
a vegetative cell.
- Endospore formation in bacteria is for survival, NOT reproduction.
- Consists of a central core (containing an outer core wall, cell membrane, nuclear
region, and other cell components), a peptidoglycan cortex, and two protein
layers (a spore coat and an exosporium).

- UV is light that has wavelengths between 100 nm and 400 nm.

- DNA within the cells can absorb the energy in the UV light, with a maximum
absorption around 260 nm.

DNA damage caused by UV light: cytosine deamination and thymine dimer formation.
Cytosine Deamination
- Deamination (removal of amine group) converts cytosine to uracil.
- Next cycle of DNA replication will result in one normal copy of the DNA,
and one copy in which the U paired with an A.
- From then on, each subsequent generation of this mutated organism will
retain the mutation
Thymine Dimer Formation
- Covalent bond occurs between adjacent thymines on the same strand
- .DNA polymerase will stop when it reaches the thymine dimer and will not
be able to continue, potentially causing the cell to die.
DNA Damage Repair
- Translesion polymerase, a repair enzyme, can bypass the stalled DNA
polymerase, but this results in a lot of mutations at this site on the DNA… eh, not
great
- Many cells are able to repair the damage to DNA (without mutation) by using
excision repair or photoreactivation mechanisms.
Excision Repair
- Repair proteins use ATP as an energy source to remove damaged DNA
- Uses opposite strand as template to fill the part that was spliced out
Photoreactivation
- Uses visible light as an energy source to activate photolyase (enzyme) to break
the covalent bond between the thymine dimer.

Photoreactivation (left) and Excision (right)

Ex. 15a and 15b Antibiotic MIC and Disc Diffusion
- Organisms can be classified as resistant, intermediate, or sensitive to an
antimicrobial
- Even if there is slight inhibition of bacterial growth by an antibiotic, it may still be
considered resistant if it is not inhibited by a therapeutic concentration (sure,
that concentration would kill the bacteria… but it would also kill the patient if we
gave it to them).

Disc Diffusion Standardization Factors

1. Antimicrobial concentration 5. Incubation time
2. Antimicrobial molecule size 6. Temperature
3. Diffusion 7. Interactions
4. Number of cells

“Bacteriostatic” - inhibits growth

“Bactericidal” - prevents growth by killing

MIC - Minimum Inhibitory Concentration

- Lowest concentration of antibiotic needed to inhibit growth
MBC - Minimum Inhibitory Concentration
- Lowest concentration that prevents growth and induces cell death

Check Brightspace for MIC Calculation Practice

***If MBC is close to MIC, agent is bactericidal

***If MBC >> MIC, agent is bacteriostatic
Ex. 16 Antimicrobial Treatment
“Biofilm” - sticky material made up of polysaccharides, proteins, lipids, and DNA that
contains bacteria. Four stages:
1. Reversible attachment - planktonic cells (free, motile) bump into a surface and
can swim away.
2. Non-reversible attachment - increase in cell structures (pili and glycocalyx) and
cell proteins make cells stick to a surface.
3. Maturation - biofilm grows, cells lose their flagella (no need to move if they are
comfy in the biofilm)
4. Active dispersal - “this place is lame, let’s leave”

“Disinfectants” - Used on inanimate objects. Destroy the bacterial cell membrane and
interfere with metabolism. Can hurt living tissue.
“Antiseptics” - Used on living tissue. “Broadspectrum” - can affect living tissue at high
doses. Important to use “therapeutic concentration” - high enough to inhibit bacterial
growth, but low enough to avoid tissue damage.

Look on pages 161 and 162 Know Mode of Action and Target for Antibiotics

Ex. 17 Conjugation
“Vertical gene transfer” - parent to offspring by cell division, passing on the same
genetic material from one generation to another.
“Horizontal gene transfer” - from one organism to another but not by cell division, a
way for bacteria to pick up new genetic material.
“Conjugation” - direct cell to cell contact that mediates transfer of DNA
- Contact formed by F pilus (F = fertility factor)
- F+ (donor ♂) gives a single strand (ssDNA) of its fertility factor to F- (recipient ♀)
- F- will then use this ssDNA as a template to create dsDNA, and is now F+
- F- → F+ = transconjugant
“Plasmid” - small circular DNA molecule that replicates independently
Acquiring a Plasmid
- Pro: antibiotic resistance, virulence factors, and nutrient utilization.
- Con: replicating extra DNA slows growth rate
- The plasmid needs to be worth the extra work. It better offer something
beneficial or else it’s not worth it.
“R plasmids” - plasmids that carry more than 1 gene for antibiotic resistance.

Summary:
1. F+ has plasmid (named F’128) that has genes for lactose fermentation and
antibiotic resistance to chloramphenicol (Cat)
2. F- has kanamycin (Kan) resistance in its chromosome, not lactose fermenting
3. F+ gives its F-128 plasmid to F-, and now we have a cell with Kan and Cat
resistance and lactose fermentation.

MacConkey + F+ F- TC
antibiotics

Cat Growth, pink No growth *Growth, pink

Kan No growth Growth, yellow **Growth, yellow

and pink

Cat + Kan No growth No growth ***Growth, pink

*growth mostly F+ cells, very few TC (all pink)
**growth mostly F- cells (more yellow), very few TC (bits of pink)
***growth TC cells only (all pink)

Look at the GroupMe to see pictures of the F+, F-, and TC growth on the 3 different
MacConkey plates for a deeper explanation.
Ex. 18 Bacteriophage Typing
“Bacteriophage” - virus that infect bacteria
“Transduction” - genetic info carried by bacteriophages from one bacterium to another.
“Virion” - produced in host cells under viral control, vehicles for spreading viral genome
to other cells, allows virus to exist outside the cell in an infective yet nonliving state.

Lytic Cycle: bacteriophage introduces its genetic material and replicates so much that
the host cell explodes (lyses)
1. Attachment/Adsorption: weak bonds from between receptor and viral
attachment site
2. Penetration: phage makes enzymes to burrow a hole into the bacterial cell wall.
Binal phages like T4 stay outside the cell and use the hole to inject the dsDNA
from their head out of their tail. Reverse mosquito.
3. Biosynthesis: use host cell’s machinery to replicate viral genome. Freeloading.
4. Maturation: Lots of viral proteins and nucleic acid, new virions are assembled.
5. Release: viral enzymes (holins and lysins) create holes in the host cell’s
membrane and peptidoglycan so the virus can be released.

Lysogenic Cycle: bacteriophage introduces its genetic material and only replicates and
explodes (lyses) the host cell under stressful conditions.
1. Lysogeny - viral nucleic acid incorporates into the host’s genome which allows
viral nucleic acid to persist as a provirus/prophage.
2. Stress such as starvation, DNA damage caused by UV light, and extreme
temperature induces the prophage to be read and synthesize more
bacteriophage virions. The rest of the lytic cycle then occurs.
*** Stress puts the “S” in lysogenic. Requires stress to jump into the lytic cycle. If there’s
no stress, the prophage will remain dormant, watching, waiting, plotting…

“Temperate phage” - phage capable of initially integrating into the host genome
“Prophage” - the latent, non-infectious DNA form of the phage. Must be
useful/beneficial to the bacteriophage. If not, it will not be maintained.

Check Brightspace for Practice Bacteriophage Plaque Titer Questions

Just for Fun: https://www.youtube.com/watch?v=8Z_e9sPckNI

***Fact Check: Contrary to what Jimmy says, bacteriophages do NOT have
mitochondria… Mitochondria are about 0.5 to 3 µm and bacteriophages are 24-200
nm. Nickelodeon obviously never went to lecture.

Ex. 19 Transformation
“Transformation” - Process of acquiring DNA from the surrounding environment
“Competent” - able to do transformation by using transport systems. Allows organisms
to adapt to surroundings, repair DNA damage, or degrade DNA to obtain growth
factors. Can occur naturally (some Bacillus) or can be chemically induced:
1. CaCl and ice helps free DNA stick to cell membrane.
2. Then heat shocked to loosen membranes to allow DNA to squeeze in.
3. Put on ice again to tighten up membrane and prevent DNA leaking back out.

***In lab, we chemically induced competence so E. coli could pick up the pGlo plasmid.
Again, plasmids have to offer some kind of benefit such as antibiotic resistance to be
used. The ampicillin resistance (bla) and regulatory protein (araC) genes are always
transcribed/synthesized. But the green fluorescent protein (gfp) gene is under the
control of the arabinose promoter (Pbad). Results:

Top 2 plates did not receive the plasmid, therefore they are not resistant to the
ampicillin that is in the plates.
Bottom 2 plates received the plasmid and can grow in the presence of ampicillin. Only
the plate with the arabinose (bottom right) will allow the bacteria to express the gfp
gene and let the colonies to glow.
Reading Summary: Role of Bacterial Biofilms in Antimicrobial Resistance
Intro
 - Majority of human infections are biofilm mediated
- Biofilms are associated with medical equipment (catheters) and tissue
infections
- “Polymicrobial” - multiple types of bacteria can make up a biofilm. Makes
it difficult to choose the best antibiotic for the job.
What are Biofilms?
- Biofilms are formed when bacteria colonize a surface and adhere to it by
producing EPS (extracellular polysaccharide matrix). EPS = glue.
- EPS makes a “channeled structured smart community” that shields the
maturing inner bacteria and provides a hidden network for nutrient
delivery/waste disposal.
- When the bacteria in the biofilm mature, they leave to make their own
biofilm or join a new one.
How Biofilms Help Perpetuate Antimicrobial Resistance
- Bacteria living in a biofilm can have a 10-1000x increase in antibiotic
resistance
- When free (planktonic) S. epidermidis cells were given vancomycin, they
all died. But when vancomycin was given to a S. epidermidis biofilm, only
25% died.
- “Recalcitrance” - biofilm surviving even in the presence of a high [ ] of
antibiotics
- Common antibiotic resistance mechanisms (point mutations, enzymes,
and efflux pumps) are NOT likely to be responsible for resistance that is
seen in biofilms. Biofilms use the methods described below:
1. Blocking at the surface - sticky EPS blocks and/or slows antibiotics
from reaching the bacteria.
2. Hostile microenvironment - Anaerobic inner environment can
hinder bactericidal action of tobramycin and ciprofloxacin.
Changes in pH inhibit aminoglycoside (class of antibiotic) action.
3. Persister cells - deep down, cells in a “spore-like” dormant state
survive in extreme conditions like antibiotics. When the stress is
taken away, they become active again (pre-persister). Their
resistance is NOT due to genetic changes.
- Greatest advantage of biofilm: cells living close together to share
resistance information and biofilm promoting factors through plasmid
stability and quorum sensing.
Diagnosis and Management of Biofilms on Medical Devices
 - Difficult to diagnose infections caused by biofilms
- Medical device associated infections are commonly caused by S.
epidermidis (80%) and S. aureus.
- “CAUTI” - Catheter Associated Urinary Tract Infection. Multidrug resistant
gram negative bacteria like E. coli and P. aeruginosa are primarily the
cause.
- Two Types of Biofilm Prevention:
1. Surface Coating/Elution - modify device surfaces using
antimicrobial agents.
2. Physical/Mechanical/Electrical/Biological Approaches - high
powered spray to remove biofilms.

Good Luck on Finals, and have a Great Summer!