Lecture 8 Flashcards
What does PCR stand for and what is it used
for?
Polymerase chain reaction. Its used for
replicating a specific piece of DNA in a test
tube.
What are some advantages and potential
applications of CPR?
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
What are the components in PCR?
- Template DNA
- Primers
- DNA polymerase
- dNTP
Which components determines the specific
region of DNA that will be amplified?
TEMPLATE DNA
Why is the TAQ DNA polymerase used?
“Taq” polymerase isolated from the
thermophile Thermus aquaticus • Remain
active at elevated temperatures used in PCR
What are the 3 steps in PCR?
- Denaturation
- Priming
- Extension
How does repetition in the steps result in
exponential amplification of the specific
region of template DNA?
Amplified DNA from one step acts as
template in the next step, resulting in
exponential amplification
What is sterilization, disinfection,
decontamination, sanitization, antisepsis,and degermination?
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
Which can be used on the skin?
Decontamination/ sterilization
Antisepsis/ degermation
Why is killing bacterial endospores
considered the gold standard of sterilization>
Bacterial endospores have traditionally been
considered the most resistant microbial
entities
What is the relative resistance to antimicrobial agents among bacterial endospores, vegetative Gram positives, vegetative Gram negative cells, and enveloped viruses?
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
How is microbial death defined?
Permanent termination of an organism’s vital
processes
What are factors that might affect the
amount of time a given sterilization
technique needs to be applied?
- The nature of the microorganisms in
the population
- State of microbes in the population
- Temperature and pH of the
environment
- The concentration (dose, intensity) of
the agent
- The presence of solvents, interfering
organic matter, and inhibitors
What is sepsis and asepsis?
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.
What is the difference between microbicidal
and microbiostatic?
Have the suffix –cide or –cidal, meaning
“killing” or “to kill”
Stasis and static mean “to stand still” (used
as suffix –static)
What are 4 targets of physical and chemical
agents for control of microbes?
- Cell wall
- Cell membrane
- Cellular synthetic
- Proteins
What effects do these agents have on these
targets?
- Cell wall
Chemical agents can damage the cell wall by:
- Blocking its synthesis
- Digesting the cell wall
- 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.
- 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
What is thermal death time?
Thermal death time (TDT): Shortest length of
time required to kill all test microbes at a
specified temperature
What are typical treatment conditions for
sterilization used for sterilization by moist heat in an autoclave?
Done at 121°C under 15 psi pressure (30 psi
total pressure) for 10-40 minutes (20 minutestypical)
Will boiling water be effective in killing
endospores?
yes
What is tyndalization?
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
What are typical treatment conditions used
in flash and batch pasterurization?
Flash pasteurization: 71.6°C for 15 sec
Batch pasteurization: 63-66°C for 30 min
Are low temp and dessiciations microbicidal
or microbiostatic?
MICROBIOSTATIC
What is lyophilization?
A combination of desiccation and freezing
(freeze-drying) that is used as a long-term
preservation technique for microbes
What types of radiation can be used for
sterilization?
Gamma and x-rays
What types of radiation can be used for
disinfection?
UV radiation
What size pore would be used in filtration to
remove microbial cells?
0.2 micrometers
What size might be used to remove virus
particles?
0.2 micrometers
Does filtration effectively remove microbial
toxins?
NO
What does HEPA stand for? liquid or gas?
High Efficiency Particulate Air (liquid)
Can osmotic pressure be used as
sterilization?
NO
What is an aqueous solution and a tincture?
Aqueous solutions: Chemicals dissolved in
pure water as the solvent
Tinctures: Chemicals dissolved in pure
alcohol or water-alcohol mixtures
What are factors that affect the activity of
various germicidal chemicals?
- Nature of microraganisms and
materials used
- Degree of contamination
- Time of exposure
- Strength of chemical action of
germicide
What is the difference between high,intermediate, and low level of germicides?
- high-level germicides kill endosporesand can be used as sterilants
- intermediate-level germicides kill
fungal, but not bacterial spores,
resistant pathogens, and viruses
- low-level germicides eliminate only
vegetative bacteria, vegetative fungal
cells, and some viruses
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.
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.
