Exam 3

Question 1

Define the term biotechnology and describe what fields it pertains to.

Answer 1

Biotechnology: The science of using living systems to benefit humankind.
Biotechnology can be used for industrial, medical, and agricultural applications with synthetic biology (assembling D N A pieces into new genetic elements)

Question 2

Define recombinant DNA technology (DNA cloning) and explain the example of using microorganism to make human insulin or the example in the biotechnology worksheet of cloning an anti-microbial fungal gene.

Answer 2

Recombinant DNA technology (also called DNA cloning): a piece of DNA is copied from one organism and pasted into a small piece of DNA called a plasmid.

The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation).

Question 3

Explain the steps of creating recombinant DNA (DNA cloning).

Answer 3

1. PCR makes multiple copies of GeneX
   THe polymerase chain reaction (PCR) is DNA replication invitro, multiplying segments of target DNA(a few kbp in length) up to the billionfold during amplification.
   PCR requires the following components which are mixed in a tube:
   Template DNA—contains the target DNA to be amplified
   Primers—short pieces of DNA that designate where the copying will begin and end
   DNA polymerase—enzyme that will replicate the DNA
   Buffer solution—provides the proper pH and salt concentration for the replication reaction to occur efficiently
   Steps in PCR:
2. Denaturation– separate strands of template D N A by heating at a high temperature
3. Annealing: lower temperature to allow primers to bind to target sequence
4. Extension: D N A polymerase extends primers using original D N A template
5. Restriction enzymes cut the PCR products and plasmid
6. Ligate the plasmid and PCR products together
7. Transform the cell

Question 4

Explain the purpose of PCR (polymerase chain reaction) and describe what happens in each step.

Answer 4

The P C R amplifies specific D N A sequences.
(a) Target D N A is heated to separate the strands, and a large excess of two oligonucleotide primers, one complementary to each strand, is added along with D N A polymerase.
(b) Following primer annealing, primer extension yields a copy of the original double-stranded D N A.
(c) Two additional P C R cycles yield four and eight copies, respectively, of the original D N A sequence.
(d) Effect of running 20 P C R cycles on a D N A preparation originally containing 1 copy of a target gene. Note that the plot is semilogarithmic.

Question 5

Describe restriction endonucleases, where they cut DNA (restriction sites), and how they cut (leave sticky ends).

Answer 5

Restriction endonucleases (restriction enzymes) are used to cut the DNA at specific sequences called restriction sites.
Why might bacteria have these naturally?
Protect the bacteria from invading bacteriophages (viruses)

• DNA fragments separated by gel electrophoresis

Question 6

Explain the job of ligase and what DNA pieces it can glue together.

Answer 6

Any DNA molecules cut with same restriction enzyme can be joined together by another enzyme called ligase because of sticky ends.

Question 7

Explain the purpose of gel electrophoresis, how it works, including what the charge of the DNA molecule is and the positioning of the electrodes.

Answer 7

Agarose gel acts like a maze of small holes

Small DNA fragments move quickly through the small pores

Larger fragments take more time to “wiggle” though.

DNA (negatively charged) moves through gel toward the positive end by adding an electrical current

Question 8

Define the term plasmid vector and describe the 3 essential features of plasmid vectors.

Answer 8

Plasmid vector: Carries the inserted piece of DNA into the bacterium where it gets replicated/expressed

Plasmid vectors usually contain:
1. Ori (origin of replication)
2. Antibiotic resistance markers, usually at least two
3. Known restriction sites often in the form of several grouped together called a multiple-cloning site or polylinker.

Question 9

Explain the three different types of horizontal gene transfer: transformation, transduction, and conjugation.

Answer 9

There are 3 different methods:

1. Transformation: competent cells take up DNA directly from their environment
2. Transduction: DNA transferred by bacteriophage (virus that infects bacteria)
3. Conjugation: One live bacterium transfers the plasmid to another live bacterium via a pilus.

Question 10

For transformation: definite the term competent cells, understand the difference between artificial and natural competence, describe how the DNA gets inside the cell.

Answer 10

Transformation is when competent cells take up DNA from the environment.

Cells are either naturally competent or artificially competent
- Natural: Competence factors: surface proteins that allow bacteria
- Artificial: treatments can increase membrane permeability (CaCl2, electroporation )

Large (7,00-10,000 bp) DNA fragments can be accepted.

1. Donor DNA associates with DNA binding protein on the cell surface
2. One strand of the DNA is transported into the cell while the other strand is hydrolyzed by a nuclease.
3. At least part of the donor DNA can move into the cell’s chromosome by homologous recombination

Question 11

For transduction, describe the difference between virulent viruses that use the lytic cycle and temperate viruses that use the lysogenic cycle and lytic cycle. Explain the difference between general transduction and specific transduction.

Answer 11

Transduction: using a virus that infects bacteria to pass genes from one bacteria to another.

Viruses can infect using to different approaches
- Virulent viruses infect using the lytic cycle
- Temperate viruses infect using the lysogenic cycle

1. A bacteriophage injects its DNA into a bacterium
   - During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new phages, and destroys the cell
2. Phage DNA is replicated, and the bacterium’s chromosome is broken down. Viral proteins are synthesized
3. When new phage particles are assembled, some phage heads may get stuffed with bacterial rather than viral DNA
4. If that phage infects another bacterium, it can transfer genes from the lysed bacterium into a new cell.

Temperate phage can incorporate into the cell’s chromosome (lysogenic) but can be induced into a lytic cycle in which many infective phage particles are produced.

Transduction occurs when a bacteriophage transfers bacterial DNA from one bacterium to another during sequential infections.

There are two main types:

1. Generalized transduction: when the virus picks up a random piece of bacterial chromosome by mistake during the lytic cycle.
2. Specialized transduction: occurs at the end of the lysogenic cycle, when the prophage is excised, and the bacteriophage enters the lytic cycle.

Question 12

For conjugation, describe the process step by step.

Answer 12

Conjugation: when DNA is transferred between 2 liver bacteria.

• DNA is transferred between cells
• Requires physical contact between cells
• F plasmids; code for pilus

Question 13

Describe the purpose and process of using antibiotic resistance genes for selection.

Answer 13

We use antibiotic resistance and a reporter gene.

Each plasmid has an antibiotic resistance gene, so if we grow the bacteria on media containing that antibiotic, only bacteria that took up the plasmid will live.

Question 14

Explain the concept of using a reporter gene (blue-white screening) to check for a gene insert in a plasmid.

Answer 14

A reporter gene is another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion.

Blue/white screening
- Insertional inactivation of gene within lacZ (encodes the β-galactosidase enzyme) used to detect cloned D N A
- Transformants are plated on media containing ampicillin and X-gal which turns blue when broken down by β-galactosidase.
- Cells containing vector with insert are white because no β-galactosidase formed.

Question 15

Explain the purpose of the CRISPR-Cas9 in bacteria.

Answer 15

Genome editing: Use of C R I S P R/Cas9 system from Streptococcus pyogenes to alter eukaryotic genomes in living cells

Sequence Targeting by the Cas9 Protein
- Cas proteins of C R I S P R systems function as endonucleases when guided to nucleic acids by binding of C R I S P R R N A s (c r R N A s)
- Synthetic R N A (synthetic guide R N A [s g R N A]) can be designed to recruit Streptococcus Cas9 and bind to target D N A, enabling cutting in genome of almost any cell
- At cut site, D N A can be ligated or used to insert new D N A

Sequence Targeting by the Cas9 Protein
. Also requires protospacer adjacent motif (P A M) on target D N A for complete endonuclease activity
- Various methods of C R I P S R system delivery by injection
. Plasmid
. s g R N A and mR N A can be made in vitro
- Homologous recombination can be used to incorporate new D N A (insertion)
- Nonhomologous double-stranded D N A break repair pathway can ligate after deletion

Question 16

Describe in general terms how CRISPR-Cas9 can be used for genome editing.

Answer 16

C R I S P R Editing in Practice:
- Edit genomes of crops and farm animals
- Edit the human genome to treat genetic diseases
- Treat other diseases (for example, viral diseases)
- Diagnostic tool to detect pathogens

Ethical questions about use in humans:
https://www.npr.org/sections/health-shots/2023/03/06/1158705095/genome-summit-gene-editing-ethics-crspr

Question 17

Explain the fact the viruses are acellular, obligate, intracellular parasites.

Answer 17

acellular: not made of cells
Obligate intracellular parasites:
- Lack genes needed for successful reproduction
- Require a host cell to reproduce

obligate intracellular pathogen microorganism that cannot synthesize its own ATP and, therefore, must rely on a host cell for energy; behaves like a parasite when inside a host cell, but is metabolically inactive outside of a host cell

1. Infectious, acellular pathogens
2. Obligate intracellular parasites
   - Lack genes needed for successful reproduction
   - Require a host cell to reproduce
3. DNA or RNA genome
4. Genetic material is surrounded by a capsid (protein coat)

Question 18

Define the term virion.

Answer 18

virion: inert particle that is the reproductive form of a virus

Question 19

Describe what viruses are made of: a genome (RNA or DNA), a capsid, and some have an envelope, including its composition.

Answer 19

All viruses have genetic material and a capsid.
- Capsid(protective coat): made of capsomeres protein subunits
- Nucleic acid (DNA or RNA)

Some viruses have an envelope (enveloped) and others do not (naked or non-enveloped).
- Envelope (phospholipid membrane)
- Spike proteins (glycoproteins that help cell entry)

Question 20

Explain the concept of host range and why viruses normally have a limited host range.

Answer 20

Most viruses can only infect one kind of cells or a few species = HOST RANGE
- Having a wide host range is rare.
- Viruses that infect bacteria are called bacteriophages.

Why is host range so limited?
- Cells must have specific receptor sites that the virus can attach to
- Cell must contain the enzymes the virus needs to uncoat
- Cell must have the rest of the synthetic machinery

Question 21

Explain the concept of transmission, including what a vector is, both mechanical and biological.

Answer 21

Transmission: how a virus is passed from host to host.

Viruses can be passed by
1. Direct contact
2. Indirect contact through an object
3. Vector: which is an animal that passes the virus from one host to another
- Mechanical vector: virus travels outside vector
- Biological vector: virus travels inside

Question 22

Describe the three most common capsid shapes.

Answer 22

Viruses can vary in the shape of their capsids
The shape of the capsid is determined by their capsomeres.
Helical: rod-shaped
Polyhedral: many sided
Complex: have features of both

Question 23

Identify the two main groups that classify viruses.

Answer 23

Viruses are not classified under the three domains of life, but still need classification.

However, they can mutate so quickly that it’s difficult to label them with a genus and species.
There are two main systems of classification:
- International Committee on Taxonomy of Viruses (ICTV) classifies viruses into families and genera based on viral genetics, chemistry, morphology, and mechanism of multiplication.
- The Baltimore System classifies viruses according to their genomes.

Question 24

Know all the different types of genomes that viruses can have and explain what they mean (ssDNA, dsDNA, ssRNA + strand, ssRNA – strand, dsRNA, and RNA with reverse transcriptase.

Answer 24

[Genome, family, example virus, clinical features]

dsDNA; enveloped:
(Poxviridae, Orthopoxvirus, skin papules, pustules, and lesions), (Poxviridae, Parapoxvirus, skin lesions), and (Herpesviridae, Simplexvirus, Cold sores, genital herpes, sexually transmitted disease)

dsDNA, naked:
(Adenoviridae, Atadenovirus, Respiratory infection (common cold)), (Papillomaviridae, Papillomavirus, Genital warts, cervical, vulvar, or vaginal cancer), and (Reoviridae, Reovirus, Gastroenteritis severe diarrhea (stomach flu))

ssDNA, naked:
(Parvoviridae, Adeno-associated,dependoparvovirus A, Respiratory tract infection), (Parvoviridae, Adeno-associated dependoparvovirus B, Respiratory tract infection)

dsRNA, naked: (Reoviridae, Rotavirus, Gastroenteritis)

+ssRNA, naked:
(Picornaviridae, Enterovirus C, Poliomyelitis), (Picornaviridae, Rhinovirus, Upper respiratory tract infection (common cold)), and (Picornaviridae, Hepatovirus, Hepatitis)

+ssRNA, enveloped: (Togaviridae, Alphavirus, Encephalitis, hemorrhagic fever), (Togaviridae, Rubivirus, Rubella), and (Retroviridae, Lentivirus, Acquired immune deficiency syndrome (AIDS))

−ssRNA, enveloped:
(Filoviridae, Zaire Ebolavirus, Hemorrhagic fever), (Orthomyxoviridae, Influenzavirus A, B, C, Flu), and (Rhabdoviridae, Lyssavirus, Rabies)

Question 25

Know the six steps of a general viral life cycle.

Answer 25

1. Attachment
2. Penetration
3. Uncoating
4. Viral genome replication: mechanism depends on genome type
5. Maturation
6. Release

Question 26

Explain the 4 stages of the viral growth curve.

Answer 26

Viral growth curve: described by viral titer: number of virions per unit volume.

1. Inoculation: inoculum of virus binds to cells
2. Eclipse: virions penetrate the cells
3. Burst: host cells release many viral particles
4. Burst size: number of virions released per bacterium

Question 27

Explain the differences in the genomes and life cycle/replication of +ssRNA and -ssRNA viruses. Be able to explain the steps in the life cycle of each of these viral types including the function of RdRP.

Answer 27

Genomes of ssRNA viruses for positive(+) vs negative (-):
- If the genome sequence is the mRNA sequence it’s a positive (+) strand virus. (sense: 5’UGACCAUGGGA3’)
- If the genome sequence is complementary to the mRNA sequence it’s a negative (-) strand virus. (antisense: 3’ACUGGUACCCU5’)

+ssRNA virus:
1. Virions enter the cell by endocytosis and are uncoated to release the +ssRNA genome.

2. +ssRNA genome is translated by host cell’s ribosomes to produce viral proteins including RNA dependent RNA polymerase (RdRp).
3. RdRp synthesizes -ssRNA complementary copies of the +ssRNA genome.
4. RdRp synthesizes +ssRNA complementary genomic copies of this –ssRNA.
5. The newly synthesized copies of the genome and the viral proteins are assembled.
6. The virions exit the cell.

-ssRNA virus:
1. Virions enter the cell by endocytosis and are uncoated to release the -ssRNA genome and RNA dependent RNA polymerase (RdRp).

2. RdRp synthesizes +ssRNA complementary copies of the -ssRNA genome.
3. The +ssRNA complementary copies are translated by the host cell’s ribosomes to produce viral proteins including more RdRp.
4. RdRp synthesizes the -ssRNA genome using additional copies of the +ssRNA as the template.
5. The newly synthesized copies of the genome and the viral proteins are assembled.
6. The virions exit the cell.

Question 28

Explain the steps in a retroviral life cycle and the function of reverse transcriptase.

Answer 28

The flow of events during the life cycle:

1. Entry and uncoating of the retrovirus.
2. Reverse transcriptase activity, two steps.
3. Viral DNA enters nucleus and integrates into the host genome.
4. Transcription by host RNA polymerase forms viral mRNA and genome copies.
5. Translation of mRNA forms viral proteins; new nucleocapsids assembled and released through the host cytoplasmic membrane by budding.

Question 29

Define latent vs chronic infections.

Answer 29

• Latent: viruses that hide or stay dormant in the cell after an acute infection but can reemerge. (example: varicella-zoster virus [chicken pox/ shingles])
• Chronic: virus that cause persistent symptoms over time; if body can’t eliminate virus, the virus can persist in tissues for years before symptoms. (example: HIV)

Question 30

Differentiate between virulent and temperate phages.

Answer 30

Two main types of viruses that infect prokaryotes:

Virulent phages:
- Lead to cell death through the lytic cycle.
- Use the lytic cycle only

Temperate phages:
- Become part of the host genome = prophage
- Are replicated with host genome and passed to offspring
- Can be induced into lytic phase
- Use the lysogenic cycle and the lytic cycle

Question 31

Explain the lytic and lysogenic cycles of bacteriophage replication.

Answer 31

• Lytic cycle results in the death of the bacteria
• Lysogenic cycle results in the viral genome combining with the host until induction into the lytic cycle.

Question 32

Compare and contrast plant vs animal viruses.

Answer 32

Plant viruses are more like animal viruses than they are like bacteriophage.

• May be envelope or non-enveloped
• May have a DNA or RNA genome
• May have a broad or narrow host range
• How do you think they are transmitted?
  > Mechanical vectors (insects)
  > Through wounds from pruning or damage

Question 33

Identify the major attributes of the viral families: Picornaviridae, Retroviridae, Paramyxoviridae, and Coronaviridae.

Answer 33

Picornaviridae:
large family containing 47 genera and 110 species; virion: non-enveloped, 30-32 nm (small), icosahedral; genome: +ssRNA; host range: vertebrates; examples: enterovirus: polio and rhinovirus: common cold

Retreoiridae:
large family of viruses that reverse transcribe their RNA to DNA = RETROVIRUSES; virion: enveloped, 80-100 nm (medium), spherical capsid; genome: dimer(2) +ssRNA; host range: human and animal vertebrates; examples: Human Immunodeficiency Virus (HIV), which is the virus that causes AIDS

Paramyxoviridae:
Many members of this family cause human diseases such as measles, mumps, and some parainfluenzas; virion: enveloped, 300-500 nm (large), mostly spherical; genome: -ssRNA; Host range: mammals, birds, fish, and reptiles; spread by physical contact or airborne transmission

Coronaviridae:
Virion: enveloped, 120-160 nm (medium-large) helical capsid; genome: -ssRNA with 7-8 segments; host range: mammals, birds, and fish; examples: influenza (Hemagglutinin and Neuraminidase in envelope) and Coronavirus

Question 34

Compare and contrast viroids, virusoids, and prions.

Answer 34

1. Viriods: composed of a short strand of circular RNA that can self replicate
2. Virusoids: ssRNA that’s not self-replicating and needs a “helper virus” to cause disease
3. Prions: infectious protein particles, no DNA or RNA required

Question 35

Define the term fomite and explain how we decide how clean a surface needs to be.

Answer 35

Fomites: various items that humans interact that might harbor microbes; examples: doorknobs, toys, cell phones, etc.

To decide how to clean a surface, you need to ask 2 questions:
1. What’s the application for which that item is used? If the item is inserted in the body, then it must be much cleaner.
2. How resistant are the potential pathogens to antimicrobial treatment? Because C.botulinum can produce endospores that can survive harsh conditions, it must be killed by extreme temperature, etc.

Question 36

Understand how different BSL are determined and what precautions are taken at each level (BSL-1, BSL-2, BSL-3, and BSL-4)

Answer 36

BSL-1: microbes are not known to cause disease in healthy hosts and pose minimal risk to workers and the environment. (nonpathogenic strains of E.coli)

BSL-2: Microbes are typically indigenous and are associated with disease of varying severity. They pose moderate risk to workers and the environment. (S.aureus)

BSL-3: Microbes are indigenous or exotic and cause serious of potentially lethal disease through respiratory transmission. (M.tuberculosis)

BSL-4: Microbes are dangerous and exotic, posing a high risk of aerosol-transmitted infections, which are frequently fatal without treatment or vaccines. Few labs are at this level. (Ebola and Marburg viruses)

Question 37

Compare and contrast the processes of sterilization, disinfection, and sanitization.

Answer 37

1. Sterilization: complete removal or killing of all vegetative cells, endospores, and viruses.
   - Most extreme measure
   - Used in laboratories, medical, and food industry
   - Can be done through physical means, chemical means, or by filtering
   -Chemicals that achieve sterilization are called sterilants.
2. Disinfection: inactivates most of the microbes on a fomite by chemicals or heat.
   - Surface isn’t sterile after disinfection because endospores remain.
   - Should be fast acting, cheap, and easy to used.
   - Examples: vinegar, bleach
3. Sanitization: the cleansing of fomites to a level deemed safe for public health.
   - Done by application of heat or chemicals
   - Example: commercial dishwashing or cleaning public restrooms

Question 38

Define the process of aseptic technique, antiseptics, and degerming, and give examples.

Answer 38

Aseptic technique: prevents contamination of sterile surfaces.
- Collection of protocols that maintain sterility (asepsis)
- If not done correctly in a clinical setting, can cause sepsis in the patient, which is a systemic inflammatory response that can lead to death.
- Medical procedures with risk of infection must be done in a sterile field, a designated area to remain free of all metabolically active microbes, endospores, and viruses

Antiseptics: antimicrobial chemicals safe for use on living skin or tissues.
- Disinfection on surfaces is done using this.
- They must kill bacteria, but not damage tissue. (examples: hydrogen peroxide and isopropyl alcohol)

Degerming: reducing microbial number by gentle scrubbing and using a mild chemical. (examples: hand washing, alcohol swabs)

Question 39

Explain how we can use the microbial death curve to describe the effectiveness of a protocol and define the decimal reduction time.

Answer 39

Microbial Death Curve: describes the effectiveness of a certain protocol.
- The percentage of microbes killed is the most useful information.
- The amount of time it takes for the population to decrease 10-fold is the decimal reduction time (DRT) or D value

Question 40

Describe the 3 factors that determine how effective a protocol is for reducing microbe number.

Answer 40

1. Length of time of exposure
2. Susceptibility of the organism to that disinfectant or protocol
3. The concentration of the disinfectant or intensity of exposure

Question 41

Explain how physical methods for controlling microbe growth actually affect the microbes.

Answer 41

Physical methods for controlling microbe growth include heat, cold, pressure, desiccation(drying), and radiation.

How do they damage microbe?
- Disrupting membranes
- Changing membrane permeability
- Damaging proteins or nucleic acids
- Degradation
- Chemical modification

Question 42

Describe the physical methods: heat (dry-heat and moist-heat, the details of autoclaving and pasteurization), cold (refrigeration and freezing), pressure, desiccation (drying), and radiation (Ionizing, non-ionizing, and UV rays).

Answer 42

Heat:
Thermal death point (TDP) is the temperature at which a microbe is killed after a 10-minute exposure.
Thermal death time is the length of time needed to kill a microbe at a certain temperature.
Boiling: oldest form and quite effective on bacteria, but not on endospores (can withstand 20 hours of boiling).
- Dry-heat: is direct application of high temperatures like a Bunsen burner.
> Oxidizing molecules
> Often involves open flame
- Moist-heat: uses steam and is generally more effective because it penetrates cells better.
- Denatures proteins and nucleic acids.

Moist-heat:
> Autoclaves: use high temperature, steam, and pressure
- 121oC for 20 min
- Slow exhaust
- Materials must be loosely wrapped and have indicator tape.
> Pasteurization: used when boiling or autoclaving would ruin the food (like milk) but does not sterilize.
- Reduces microbe number to prolong time before spoilage
- high-temperature short-time (HTST) pasteurization, exposes milk to a temperature of 72 °C for 15 seconds
- ultra-high-temperature (UHT) pasteurization, in which the milk is exposed to a temperature of 138 °C for 2 or more seconds. Doesn’t need refrigeration, but changes taste.

Cold:
- Refrigeration and freezing is also effective at controlling microbe growth.
> Refrigeration (0-7 degree C) slows growth.
> Freezing (-2) may kill microbes or halt growth, but when thawed growth can continue.
> Long term storage is best at -70 degrees C or in liquid nitrogen.

Pressure and Desiccation (drying):
> Pressure: exposure to high pressure often kills many microbes (but not endospores).
- Often used in the food industry to maintain food quality and extend shelf life.
- Pressure between 100 and 800 Mpa
> Hyperbaric oxygen therapy: can be used to treat infections in clinical settings
- Patient breaths pure oxygen at higher-than-normal pressure
- Increases oxygen saturation in tissues that have become hypoxic due to infection
- Increased oxygen saturation also increases efficiency of immune system cells and antibiotics
- Disadvantages: rarely causes oxygen toxicity or can damage delicate tissue due to increased air pressure

Desiccation (drying): has been used to preserve food for a long time.
- Cells need water to survive, so drying controls their growth.
- Can be dried by the sun or freeze-dried (lyophilization)
- The water activity (the water content) can also be decreased by addition of high concentrations of salt or sugar
Most bacteria can’t grow in high osmotic pressure

Radiation:
Radiation is the use of high energy radiation or sunlight is used to kill microbes or slow growth.
- Ionizing radiation: permeates the cell, damages DNA and forms peroxides which are strongly oxidizing
Penetrates packaging to sterile lab supplies that cannot be autoclaved
- Non-ionizing radiation: not powerful enough to permeate packaging
UV rays can even be used at home
- Microwaves
Not effective against spores since they contain little water

Question 43

Explain how chemical methods for controlling microbe growth affect the microbes and give examples of each.

Answer 43

Chemicals can be used to control microbial growth by denaturing proteins or disrupting the membrane.
> How can we choose the proper disinfectant or antiseptic?; Consider:
- What kind of microbe are we trying to control?
- How clean does the item need to be?
- Is the chemical toxic to people, animals, or plants?
- How costly is it?
- How easy is it to use?

Phenolics inhibit microbes by denaturing proteins and disrupting membranes.

Heavy Metals kill microbe by acting an inhibitors of enzymes.
- Effective in very low concentrations.
- Disadvantage: toxicity not limited to microbes (can accumulate in human cells and be toxic)
- Examples:
> Mercury: has been used for many years to treat syphilis but is not banned in US for toxicity
> Silver: lined water jugs with silver in ancient times
- Sometimes combined with antibiotics to make them more effective
> Copper, Nickel, and Zinc
- Copper used in swimming pools as an algicide, becoming more popular
- Zinc chloride is very safe, found in mouthwashes

Halogens (iodine, chlorine, and fluorine) Oxidize cellular components, destabilizing them
> Betadine: an iodine containing liquid that’s used to prep skin before surgery
> Chlorine gas: used to clean drinking water but, exposure to people working at treatment plants must be minimized.
- Also used in swimming pools
- Doesn’t kill everything, sometimes boiling’s required.
> Fluoride: added to drinking water for dental health.

Alcohols quickly denature proteins and disrupt membranes.
> Effective concentration: 70-100%[hand sanitizer]
> Mostly bactericidal and fungicidal, but only kill viruses that are enveloped

Surfactants are amphipathic and wash away microbes
> Not considered antiseptics or disinfectants, but may effectively reduce microbes on surfaces by degerming

Peroxygens: Hydrogen peroxide oxidized cellular components
Superchemical Gases: Carbon dioxide lowers intracellular pH
Natural and Chemical Food Preservatives: decrease pH and inhibit enzymes

Question 44

Describe antibiotics in terms of: their effect on the target (bactericidal vs bacteriostatic) and their spectrum of activity (broad vs narrow).

Answer 44

Effect on target
> Bacteriostatic: agent causes a reversible inhibition of growth
> Bactericidal: agent kills target.

Spectrum of Activity
> Broad spectrum kills many different types (overuse may lead to resistant infections)
> Narrow spectrum kills only a few types

Question 45

Understand why good antibiotics display selective toxicity, know the common targets of antibiotics on bacteria.

Answer 45

Good antimicrobial agents exhibit selective toxicity.
> Selectively kills microbial targets while sparing host.
> Most antimicrobials are antibiotics because prokaryotes have more unique targets than fungi or viruses.
> Common targets:
- Cell Wall
- Plasma Membrane
- Ribosomes
- Metabolic Pathways
- DNA synthesis

Question 46

Describe how antibiotic resistance happens and what it means.

Answer 46

Antibiotic resistance happens when germs like bacteria and fungi develop the ability to defeat the drugs designed to kill them; the germs that are not killed, will keep growing.

1. Lots of germs. A few are drug resistant
2. Antibiotics kill bacteria causing the illnesses, as well as good bacteria protecting the body from infection
3. The drug-resistant bacteria is now allowed to grow and take over
4. Some bacteria give their drug-resistance other than bacteria, causing more problems

Question 47

Understand the mechanisms of antibiotic resistance and the example of MRSA.

Answer 47

Mechanisms of Antibiotic resistance

• Efflux pump:
  > Fluoroquinolones
  > Aminoglycosides
  > Tetracyclines
  > Beta-lactams
  > Macrolides
• Blocked penetration:
  > Beta-lactams
  > Tetracyclines
  > Fluoroquinolones
• Inactivation of enzymes:
  > Beta-lactams
  > Aminoglycosides
  > Macrolides
  > Rifamycins
• Target modifications:
  > Fluroquinolones
  > Rifamycins
  > Vancomycin
  > Beta-lactams
  > Macrolides
  > Aminoglycosides

MRSA:
- Hospital acquired (HA-)
- Community acquired (CA -)
- Resistance to Beta-lactam antibiotics
- These strains may carry toxin genes like Panton-Valentine leucocidin (PVL) that make them highly virulent

Beta-lactam antibiotics permanently inactivate PBP enzymes (penicillin-binding protein-involved in peptidoglycan biosynthesis), which are essential for bacterial life, by permanently binding to their active sites. MRSA, however, expresses a PBP that does not allow the antibiotic into its active site.

Question 48

Understand how the effectiveness of an anti-microbial agent can be tested (Kirby-Bauer and dilution tests).

Answer 48

Kirby-Bauer Disc Diffusion Test:
> Starts with a lawn of bacteria
> Apply filter paper discs infused with suspected antibiotic
> Measure the zone of inhibition
> Disadvantage: Can’t directly compare because of confounding variables (ie. Diffusion rate, etc

Dilution Tests:
> Used to determine a drugs minimal inhibitory concentration (MIC) and its minimal bactericidal concentration (MBC)
> MIC determined by turbidity in tubes.
> MBC determined by inoculating clear tubes to see if they grow on agar

Question 49

Describe what an anti-viral is and how they function.

Answer 49

Antiviral: a substance that fights against viruses and inhibits their growth.

• Purine and pyrimidine analogs
  > Ribavirin (Virazole) and Acyclovir (Zovirax) are guanine analogs
  > AZT interferes with reverse transcriptase
• Protease inhibitors; interferes with protein processing
• Interferon
• Immunoenhancers