Physical and Chemical
Control of
Microorganisms
Controlling Microorganisms

Reduce or destroy undesirable microbes in a given area



Physical
Chemical
Mechanical
Relative Resistance of Microbes

Highest resistance


Moderate resistance





Bacterial endospores & prions
Pseudomonas sp.
Mycobacterium tuberculosis
Staphylococcus aureus
Protozoan cysts
Least resistance





most vegetative cells
fungal spores
enveloped viruses
yeast
protozoan trophozoites
Terms for Microbial Control

Sterile


Sterilization




destroy vegetative pathogens
not endospores
Sanitization


destroys all viable microbes
including viruses & endospores
Disinfection


Inanimate objects free of all life
cleansing technique that mechanically removes microbes
to safe levels
Degerming

removing organisms from an object’s surface
Terms for Microbial Control

Microbicidal agents


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
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
Causes microbial death
Bactericide
Sporocide
Fungicide
Viricide
Microbistasis

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Prevents microbial growth
Bacteriostatic
Fungistatic
Factors That Affect Death Rate
1.
2.
3.
4.
5.
6.
Number of microbes
Nature of microbes in the population
Temperature & pH of environment
Concentration or dosage of agent
Mode of action of the agent
Presence of solvents, organic matter, or
inhibitors
Cellular Targets of Control

Mode of action of
antimicrobials:




Cell wall
Cell membrane
Cellular synthetic
processes (DNA, RNA)
Proteins
Practical Concerns for Microbial Control






Does the application require sterilization?
Is the item to be reused?
Can the item withstand heat, pressure, radiation,
or chemicals?
Is the method suitable?
Will the agent penetrate to the necessary extent?
Is the method cost- and labor-efficient & is it
safe?
Methods of Physical Control
1.
2.
3.
4.
Heat – Moist verse Dry
Cold temperatures
Desiccation
Radiation
1. Heat (Moist)
Moist heat – use of hot
water or steam
 mode of action




denaturation of proteins
destruction of membranes
destruction of DNA
Sterilization


Steam under pressure
Autoclave


15psi/121oC/10-40 min
Steam must reach surface
of item being sterilized!
Autoclave Tape
1. Heat (Moist)

Intermittent sterilization


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
Disinfection



unpressurized steam
100oC 30-60 min for 3 days
spores, which are unaffected, germinate during
the intervening periods and are subsequently
destroyed
boiling at 100oC for 30 minutes
destroy non-spore-forming pathogens
Pasteurization
1. Heat (Moist)

Pasteurization



heat applied to kill potential agents of
infection and spoilage
without destroying the food flavor or value
63°C - 66°C for 30 minutes


71.6°C for 15 seconds


batch method
flash method
Not sterilization


kills non-spore-forming pathogens and lowers
overall microbe count
does not kill endospores or many nonpathogenic
microbes
1. Heat (Dry)
Dry heat - higher
temperatures than moist
heat

incineration

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
dry ovens




150-180oC
coagulate proteins
Bunsen burner


600-1200oC
combusts & dehydrates
cells
1870oC
Dehydrates cells and
removes water
Can also sterilize
Thermal Death Measurements

Thermal death time (TDT)


shortest length of time required to kill all
microbes at a specified temperature
Thermal death point (TDP)

lowest temperature required to kill all microbes
in a sample in 10 minutes
2. Cold Temperatures

Microbistatic

slows the growth of
microbes
refrigeration 0-15oC
 freezing <0oC
 used to preserve food,
media and cultures

3. Desiccation
gradual removal of water from cells
 leads to metabolic inhibition
 not effective microbial control


many cells retain ability to grow when water is
reintroduced
4. Radiation
1.
Ionizing radiation



2.
deep penetrating power breaks DNA
gamma rays, X-rays, cathode rays
used to sterilize medical supplies & food
products
Nonionizing radiation
little penetrating power to sterilize air, water &
solid surfaces
 UV light creates thymine pyrmidines

 interfere with replication
Chemical Agents in Microbial Control

Chemicals that sterilize


Chemicals that inhibit deterioration


Disinfectants, antiseptics, sterilants
Degermers, and preservatives
Desirable qualities of chemicals:
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rapid action in low concentration
solubility in water or alcohol, stable
broad spectrum, low toxicity
penetrating
noncorrosive and nonstaining
affordable and readily available
Levels of Chemical Decontamination

High-level germicides

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
Intermediate-level

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
kill endospores
devices that are not heat sterilizable and intended to be
used in sterile environments (body tissue)
kill fungal spores (not endospores), tubercle bacillus, and
viruses
used to disinfect devices that will come in contact with
mucous membranes but are not invasive
Low-level


eliminate only vegetative bacteria, vegetative fungal cells,
and some viruses
clean surfaces that touch skin but not mucous membranes
Factors that Affect Germicidal
Activity of Chemicals
Nature of the material being treated
 Degree of contamination
 Time of exposure
 Strength and chemical action of the
germicide


Dilutions

Volume of liquid chemical diluted in larger volume
of solvent (water)
Chemical Control Of Microbial Agents
1.
2.
3.
4.
5.
6.
7.
8.
9.
Halogens
Phenolics
Chlorhexidine
Alcohols
Hydrogen peroxide
Detergents & soaps
Heavy metals
Aldehydes
Gases and aerosols
1. Halogens

Chlorine

Cl2, hypochlorites (chlorine bleach), chloramines



Denaturation of proteins by disrupting disulfide bonds
Can be sporicidal
Iodine

I2, iodophors (betadine)



Denature proteins
Can be sporicidal
Milder medical & dental degerming agents, disinfectants,
ointments
2. Phenolics

Disrupt cell
membranes &
precipitating proteins



bactericidal, fungicidal,
virucidal, not sporicidal
Lysol
Triclosan

antibacterial additive
to soaps
3. Chlorhexidine
A surfactant & protein denaturant with
broad microbicidal properties
 Not sporicidal
 Used as skin degerming agents for
preoperative scrubs, skin cleaning &
burns

4. Alcohols
Ethyl, isopropyl in solutions of 50-90%
 Act as surfactants


dissolve membrane lipids and coagulating
proteins of vegetative bacterial cells and fungi
Not sporicidal
 Good for enveloped viruses

5. Hydrogen Peroxide
Weak (3%) to strong (35%)
 Produce highly reactive hydroxyl-free
radicals that damage protein & DNA while
also decomposing to O2 gas



toxic to anaerobes
Strong solutions are sporicidal
in increasing concentrations
6. Detergents & Soaps

Ammonia compounds
act as surfactants



Soaps


alter membrane
permeability of some
bacteria & fungi
Not sporicidal
mechanically remove
soil and grease
containing microbes
Low concentrations

Only have microbistatic
effects
7. Heavy Metals




Solutions of silver &
mercury
kill vegetative cells in
low concentrations by
inactivating proteins
Oligodynamic action
Not sporicidal
8. Aldehydes
Glutaraldehyde & formaldehyde kill by
alkylating protein & DNA
 glutaraldehyde in 2% solution (Cidex)
used as sterilant for heat sensitive
instruments
 formaldehyde


disinfectant, preservative, toxicity limits use
9. Gases & Aerosols
Ethylene oxide, propylene oxide,
betapropiolactone & chlorine dioxide
 Strong alkylating agents, sporicidal

Mechanical Control

Filtration

physical removal of
microbes



passing a gas or liquid
through filter
organisms above a certain
size trapped in the pores
used to sterilize heat
sensitive liquids & air in
hospital isolation units &
industrial clean rooms

Air can be filtered using a
high-efficiency particulate air
(HEPA) filter
Antimicrobial Therapy
Origins of Antimicrobial Drugs

Antibiotics

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
Common metabolic products of aerobic sporeforming bacteria & fungi
bacteria in genera Streptomyces & Bacillus
molds in genera Penicillium & Cephalosporium
Inhibiting other microbes in the same
habitat

antibiotic producers have less competition for
nutrients & space
Ideal Antimicrobial Drug…..

Selectively toxic to microbe
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Not host cells
Microbicidal, not microbistatic
Soluble
Potent
No antimicrobial resistance
Remains active
Readily delivered to site of infection
Expense
Not allergen
Chemotherapy

Antimicrobial


Antibiotic

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
Produced by the natural
metabolic processes of
microorganisms
Can inhibit or destroy
other microorganisms
Semisynthetic


Control infection
Chemically modified drugs
in lab
Synthetic

Synthesized compounds in
lab
Chemotherapy

Narrow spectrum



Effective against limited microbial types
Target a specific cell component that is found only in
certain microbes
Broad spectrum


Effective against wide variety microbial types
Target cell components common to most pathogens
Selectively Toxic
Should kill or inhibit microbial cells without
simultaneously damaging host tissues
 Complete selective toxicity




Difficult to achieve
Characteristics of the infectious agent become
more similar to the vertebrate host cell
More side effects are seen
Selective toxicity

toxic dose of a drug


therapeutic dose


The concentration causing harm to the host
the concentration eliminating pathogens in the host
Together, the toxic and therapeutic doses are used
to formulate the chemotherapeutic index
Targets of Antimicrobial Drugs
1.
2.
3.
4.
Inhibition of cell wall
synthesis
Inhibition of nucleic
acid synthesis,
structure or function
Inhibition of protein
synthesis
Disruption of cell
membrane structure
or function
1. Bacterial Cell Wall


Most bacterial cell walls
contain peptidoglycan
Penicillin and
cephalosporin block
synthesis of peptidoglycan


Penicillins do not penetrate
the outer membrane


Causes the cell wall to lyse
less effective against
gram-negative bacteria
Broad spectrum penicillins
and cephalosporins

cross the cell walls of
gram-negative bacteria
2. Inhibit Nucleic Acid Synthesis


May block synthesis of
nucleotides, inhibit
replication, or stop
transcription
sulfonamides and
trimethoprim

block enzymes required
for tetrahydrofolate
synthesis

needed for DNA & RNA
synthesis
3. Drugs that Block Protein Synthesis

Ribosomes


Aminoglycosides
(streptomycin,
gentamicin)


eukaryotes differ in size
and structure from
prokaryotes
insert on sites cause
misreading of mRNA
Tetracyclines

block attachment of
tRNA and stop further
synthesis
4. Disrupt Cell Membrane Function

Damaged membrane


These drugs have
specificity for a particular
microbial group


based on differences in
types of lipids in their cell
membranes
Polymyxins



dies from disruption in
metabolism or lysis
interact with phospholipids
cause leakage, particularly
in gram-negative bacteria
Amphotericin B and
nystatin


form complexes with
sterols on fungal
membranes
causes leakage
Drug Resistance

Microorganisms begin to tolerate an
amount of drug that would ordinarily be
inhibitory


due to genetic versatility or variation
intrinsic and acquired
Intrinsic verse Acquired

Intrinsic resistance


Microbe must be resistant to antibiotic that
they produce
Acquired resistance:
1. spontaneous mutations in critical
chromosomal genes
2. acquisition of new genes or sets of genes via
transfer from another species

originates from resistance factors (plasmids) encoded
with drug resistance, transposons
Mechanisms of Drug Resistance




Drug inactivation by
acquired enzymatic activity
 penicillinases
Decreased permeability to
drug or increased
elimination of drug from
cell
 acquired or mutation
Change in drug receptors
 mutation or acquisition
Change in metabolic
patterns
 mutation of original
enzyme
Antibiotic Resistance in Medical Community

Improper or excessive use of antibiotics
causes antibiotic resistance

Unnecessarily large antibiotic doses



Antibiotics are available over the counter in
developing countries


Allow resistant strains to overgrow susceptible ones
If resistant strains spread to other patients, a
superinfection occurs
allows for overuse and incorrect use
Antibiotic use is widespread in livestock feeds

can be transmitted to humans through meat
consumption
Side Effects of Drugs
5% of all persons
taking antimicrobials
will experience a
serious adverse
reaction to the drug




Toxicity to organs
Allergic responses
Suppression and
alteration of
microflora
Considerations in Selecting an
Antimicrobial Drug
1. Identify the microorganism causing the
infection

Specimens should be taken before
antimicrobials initiated
2. Test the microorganism’s susceptibility
(sensitivity) to various drugs in vitro
when indicated

(Next slide)
3. Overall medical condition of the patient
Testing for Drug Susceptibility


Essential for groups of
bacteria commonly showing
resistance
Kirby-Bauer disk diffusion test
Dilution tests

Minimum inhibitory
concentration (MIC)


In vitro activity of a drug is not
always correlated with in vivo
effect


smallest concentration of drug that
visibly inhibits growth
If therapy fails, a different drug,
combination of drugs, or different
administration must be considered
Best to chose a drug with
highest level of selectivity but
lowest level toxicity
What about viruses?!?!?
Do not destroy their target pathogen
 Instead they inhibit their development


Inhibit virus before enters cell




Viral-associated proteins
Stop it from entering the cell
Stop it from reproducing
Prevent from exiting the cell