Lecture 8

Question 1

What does PCR stand for and what is it used

for?

Answer 1

Polymerase chain reaction. Its used for

replicating a specific piece of DNA in a test

tube.

Question 2

What are some advantages and potential

applications of CPR?

Answer 2

Advantages of PCR:

Sensitive: Can amplify a single gene copy
Specific: Can be made to amplify a specific
gene in a complex mixture
Rapid: Can replicate target DNA from a few
copies to billions of copies in a few hours

Some important uses of PCR
• Identify the presence and abundance of a
specific gene in a sample
• E.g. for clinical diagnostics, or crime scene
investigation (CSI)
• Identify the presence and abundance of
microbes in a sample
• E.g. PCR targeting SSU rRNA genes
• Combined with sequencing, can be used to
identify microbes
• Isolate a specific gene from a sample for
molecular cloning

Question 3

What are the components in PCR?

Answer 3

1. Template DNA
2. Primers
3. DNA polymerase
4. dNTP

Question 4

Which components determines the specific

region of DNA that will be amplified?

Answer 4

TEMPLATE DNA

Question 5

Why is the TAQ DNA polymerase used?

Answer 5

“Taq” polymerase isolated from the

thermophile Thermus aquaticus • Remain

active at elevated temperatures used in PCR

Question 6

What are the 3 steps in PCR?

Answer 6

1. Denaturation
2. Priming
3. Extension

Question 7

How does repetition in the steps result in

exponential amplification of the specific

region of template DNA?

Answer 7

Amplified DNA from one step acts as

template in the next step, resulting in

exponential amplification

Question 8

What is sterilization, disinfection,

decontamination, sanitization, antisepsis,and degermination?

Answer 8

1. Sterilization: Process or chemical 
agent (sterilant) that destroys orremoves all viable microorganisms
(including viruses)
2. Disinfection: Physical process or a 
chemical agent (disinfectant) to 
destroy vegetative pathogens but not 
bacterial endospores
3. Decontamination/Sanitization: 
Cleansing technique that 
mechanically removes or inhibits 
microorganisms to reduce 
contamination to safe level
4. Antisepsis/Degermation: Reduces the 
number of microbes on the human 
skin

Question 9

Which can be used on the skin?

Answer 9

Decontamination/ sterilization

Antisepsis/ degermation

Question 10

Why is killing bacterial endospores

considered the gold standard of sterilization>

Answer 10

Bacterial endospores have traditionally been
considered the most resistant microbial
entities

Question 11

What is the relative resistance to
antimicrobial agents among bacterial 
endospores, vegetative Gram positives, 
vegetative Gram negative cells, and 
enveloped viruses?

Answer 11

1. Endospores are among the most
resistant 
2. Viruses are usually the most 
susceptible 
3. Gram negatives (outer membrane) 
are typically more resistant that Gram 
positives and fungi

Question 12

How is microbial death defined?

Answer 12

Permanent termination of an organism’s vital

processes

Question 13

What are factors that might affect the

amount of time a given sterilization

technique needs to be applied?

Answer 13

1. The nature of the microorganisms in

the population

2. State of microbes in the population
3. Temperature and pH of the

environment

4. The concentration (dose, intensity) of

the agent

5. The presence of solvents, interfering

organic matter, and inhibitors

Question 14

What is sepsis and asepsis?

Answer 14

Sepsis: The growth of microorganisms in theblood and other tissues

Asepsis: State of being free from disease-

causing contaminants (such as bacteria,

viruses, fungi, and parasites) or,

contact with microorganisms.

Question 15

What is the difference between microbicidal

and microbiostatic?

Answer 15

Have the suffix –cide or –cidal, meaning

“killing” or “to kill”

Stasis and static mean “to stand still” (used

as suffix –static)

Question 16

What are 4 targets of physical and chemical

agents for control of microbes?

Answer 16

1. Cell wall
2. Cell membrane
3. Cellular synthetic
4. Proteins

Question 17

What effects do these agents have on these

targets?

Answer 17

1. Cell wall

Chemical agents can damage the cell wall by:

• Blocking its synthesis
• Digesting the cell wall

2. Cell membrane

Agents physically bind to lipid layer of the cell

membrane, opening up the cell membrane

and allowing injurious chemicals to enter the

cell and important ions to exit the cell.

3. Cellular synthetic processes

Agents can interrupt the synthesis of proteins

via the ribosomes, inhibiting proteins needed

for growth and metabolism and preventing

multiplication. • Agents can cause mutation.
4. Proteins

Some agents are capable of denaturing

proteins, changing their structure and usually

inhibiting activity. • Agents may attach to the

active site of a protein, preventing it from

interacting with its chemical

Question 18

What is thermal death time?

Answer 18

Thermal death time (TDT): Shortest length of

time required to kill all test microbes at a

specified temperature

Question 19

What are typical treatment conditions for

sterilization used for sterilization by moist heat in an autoclave?

Answer 19

Done at 121°C under 15 psi pressure (30 psi

total pressure) for 10-40 minutes (20 minutestypical)

Question 20

Will boiling water be effective in killing

endospores?

Answer 20

yes

Question 21

What is tyndalization?

Answer 21

The procedure is designed to kill vegetative

cells and encourage spores to germinate

during the cooling periods so they can then

be killed in the next steaming step

Question 22

What are typical treatment conditions used

in flash and batch pasterurization?

Answer 22

Flash pasteurization: 71.6°C for 15 sec

Batch pasteurization: 63-66°C for 30 min

Question 23

Are low temp and dessiciations microbicidal

or microbiostatic?

Answer 23

MICROBIOSTATIC

Question 24

What is lyophilization?

Answer 24

A combination of desiccation and freezing

(freeze-drying) that is used as a long-term

preservation technique for microbes

Question 25

What types of radiation can be used for sterilization?

Answer 25

Gamma and x-rays

Question 26

What types of radiation can be used for disinfection?

Answer 26

UV radiation

Question 27

What size pore would be used in filtration to remove microbial cells?

Answer 27

0.2 micrometers

Question 28

What size might be used to remove virus particles?

Answer 28

0.2 micrometers

Question 29

Does filtration effectively remove microbial toxins?

Answer 29

NO

Question 30

What does HEPA stand for? liquid or gas?

Answer 30

High Efficiency Particulate Air (liquid)

Question 31

Can osmotic pressure be used as sterilization?

Answer 31

NO

Question 32

What is an aqueous solution and a tincture?

Answer 32

Aqueous solutions: Chemicals dissolved in pure water as the solvent Tinctures: Chemicals dissolved in pure alcohol or water-alcohol mixtures

Question 33

What are factors that affect the activity of various germicidal chemicals?

Answer 33

1. Nature of microraganisms and materials used 2. Degree of contamination 3. Time of exposure 4. Strength of chemical action of germicide

Question 34

What is the difference between high,intermediate, and low level of germicides?

Answer 34

1. high-level germicides kill endosporesand can be used as sterilants 2. intermediate-level germicides kill fungal, but not bacterial spores, resistant pathogens, and viruses 3. low-level germicides eliminate only vegetative bacteria, vegetative fungal cells, and some viruses

Question 35

For each of the following chemical germicides, indicate 1) whether they are high-level germicides or not, 2) some examples of each, 3) their mode(s) of action, and 4) some limitations. Chemical germicides: Chlorine, iodine, hydrogen peroxide, aldehydes, gaseous sterilants, phenol and other phenolics, chlorhexidine, alcohols, detergents, and heavy metal compounds.

Answer 35

``` 1. Chlorine (a halogen) • Examples: Bleach, chlorine gas. • Mode of action: Denatures proteins, disrupts a variety of cellular processes. Limitations: Effectivity is lower under certain conditions (high pH, organic matter). ``` ``` 2. Iodine (a halogen) • Examples: Iodine solutions, iodophores (iodine-alcohol complexes). • Mode of action: Affects protein stability by interfering with H- , disulfide bonding; other effects. • Limitations: Rather high toxicity to humans, irritant; long exposure times required to kill endospores. • ``` ``` 3. Hydrogen peroxide • Examples: 3% solution (common in drug store) is disinfectant; 35% for sterilization. Mode of action: Forms free radicals that are highly reactive and damage many cellular components. Especially effective against anaerobic microorganisms. • Limitations: Overall, a good germicide. Requires high concentrations (e.g. 35%) to sterilize. ``` ``` 4. Aldehydes • Examples: formaldehyde (gas or solution) and glutaraldehyde (liquid). • Mode of action: Chemical modification of proteins, nucleic acids. • Limitations: Unstable, can be highly toxic to humans. ``` ``` 5. Gaseous sterilants • Example: Ethylene oxide gas. • Mode of action: Chemical modification of proteins, nucleic acids. • Limitations: Toxic, carcinogenic; gas treatment may not be effective on certain materials. ``` ``` 6. Phenol (carbolic acid); other phenolics • Examples/forms: Composed of one or more aromatic rings; Triclosan is a common example. • Mode of action: In high concentrations, they disrupt cell membrane/wall and proteins; at lower concentrations, they can inhibit some enzymes. • Limitations: Carbolic acid is relatively toxic; other phenolics like triclosan are less so and used more. ``` ``` 7. Chlorhexidine • Examples/forms: Compounds with 2 aromatic rings, chlorine atoms. • Mode of action: Denatures proteins; disrupts membranes, causing loss of selective permeability. • Limitations: Effects on viruses and fungi are variable. • ``` ``` 8. Alcohols • Examples/forms: Hydrocarbons with –OH group; ethyl and isopropyl alcohol are common (drug store). • Mode of action: >50% concentrations denature proteins, can disrupt lipid membrane integrity. • (note: most sources other than textbook indicate protein denaturation is main mode of action) • Typically used at 70% in aqueous solution. Very high concentrations may not be as effective. • Limitations: Rapidly evaporate, mild toxicity to humans. • ``` ``` 9. Detergents • Examples/forms: Polar molecules that act as surfactants (see Fig 9.3); cationic detergents such as quaternary ammonium compounds (“quats”) are more effective than anionic detergents. • Mode of action: Interacts with cell envelope proteins and membrane, disrupting cytoplasmic membrane • Limitations: Ineffective against some important pathogens; high organic matter content reduces activity ``` ``` 10. Heavy metal compounds • Example: Mercury tinctures; silver nitrate • Mode of action: Bind to functional groups/active sites, replace cofactors of proteins and inactive them • Limitations: Relatively easy for microbes to develop resistance; relatively toxic to humans. ```