Principles of

Microbiology
(BIOL 360)
Dr. Cyrus Taheri
Session 10

Microbial Growth Control

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 Learning Outcomes
• Compare and contrast different physical and chemical means of
microbial growth control and discuss their pros and cons
• Propose the best microbial growth controlling method (e.g.,
physical, chemical, etc.) in a given situation
• Compare and contrast different types of chemical antimicrobial
agents including sterilants, disinfectants, and antiseptics.
• Distinguish between the antimicrobial effects of –statics, -cidal,
and –lytic agents.
• Explain how the antimicrobial activity of chemical agents is
measured.
• Outline different classes of antimicrobial drugs
• Explain the mode of action and the spectrum of activity of
common antimicrobial drugs.
• Compare and contrast different methods of antimicrobial drug
susceptibility testing.
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 Microbial growth control
• Microbial growth control have significant practical
applications (e.g. sterilizing surgery tools)

Specific terminology is used to define different

microbial growth control strategies:

• Decontamination: means treating microbes

associated with an item or marital in a manner that
it is safe to handle.

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 Microbial growth control
• Decontamination includes:
• Sterilization: The killing or removal of all organisms
associated with the object/ area.
• Disinfection: means removing or killing, most but not of
all microbes. The goal here is to make the object safe to
handle.
• Pasteurization is the process of using moderate heat to
reduce the number of viable microbes found in heat-
sensitive liquids such as milk.

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 Microbial growth control
• Microbial growth control measures depend on different
factors most importantly:
• The environment in which unwanted microbes need to
be treated. For instance, we cannot use the same
measures to get rid of microbes residing on our skin (in-
vivo) and the ones inhabiting lab equipment (in-vitro).
• The type of microbe (e.g. eukaryotic, prokaryotic,
acellular) and their lifestyle.
• Microbial control measures fall into two categories:
• Physical control methods
• Chemical control measures

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 Physical methods
Physical methods:
• Heat
• Radiation
• Filtration
• Heat sterilization is the most common control
method and can be used in different forms:
• Dry heat sterilization
• Autoclave sterilization (wet heat sterilization)
• Incineration

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 Dry heat sterilization
• High temperature (e.g.
200 °C) is used to sterilize
lab tools and equipment
that can withstand high
temperatures (e.g. metal,
glassware). Heat oxidizes
microbial cell structures.

• Limitation: cannot be https://www.google.ca/search?q=STERILIZING+glasswares+using+oven&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjM9

used for liquids (e.g.

Nqd5-fRAhVCaxQKHbLFD-8Q_AUICCgB&biw=1920&bih=1055#imgrc=S5O58bQBwPGHkM%3A

culture media),
clothing, and plastics.

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 Wet heat sterilization (autoclave).
• The autoclave is a sealed device that uses steam under
pressure to kill microbes and their spores. Presence of water
vapor helps heat to penetrate deeper.
• High pressure (minimum 1 atmosphere) make it possible to
have water vapor temperature above the boiling point of
water (e.g. 121 °C).
• The time required to sterilize small amount of liquids is about
15 min, but more time may be required for bulky or large
volume loads.

• Certain pathogens (e.g.

prions) cannot be destroyed
by regular autoclave cycles.

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 Incineration
• High temperature sometimes close to 1000 °C is used
to burn and destroy contaminated materials.
• Commonly used in hospitals to ensure safe disposal of
medical waste.
• Some microbial contaminations such as prion
infections are extremely hard and even impossible to
be neutralized using routine disinfection methods
mentioned before. Usually a combination of different
techniques (harsh chemical treatment and heat) should
be used.
• A highly preferred method for treating prion infected
tools and tissue remaining would be incineration at
very high (e.g. 1000 °C) temperatures.
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 Radiation
• UV radiation between 220 and 300 nm damages DNA
and kills the microbe. UV seriously damages DNA and
causes mutations.
• UV is commonly used for sterilizing surfaces (work
surface under biosafety cabinet) and air.
• One of the most important
limitations of UV is its poor
penetrating power. Therefore it
cannot be used to disinfect
clothing and food.

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 Other types of radiation
• Other types of radiation such as X-ray and γ-rays
have sufficient penetration powers and are used to
kill microbes in bulk items such as medical supplies
(e.g. syringes, clothing, pipets, vaccines).
• Radiation is also a common technique in sterilizing
food.
• Killing spores usually need higher amount of
radiation. Killing a spore may need several times
more radiation than the radiation required to kill a
vegetative (i.e. normal) cell.

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 Filtration
• Filtration prevents microbes and their spores from
entering target environment (e.g. workspace or culture
medium).
• Advantage: perfect to sterilize heat on sensitive compounds
(e.g. some of vitamins and antibiotics). Pores of filter are too
small for organisms to pass through, but liquid or gas pass
through.
Examples:
• Filters used in biosafety cabinets
• Filters used to prevent the microbes from entering culture
medium

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Figure 4.38 Membrane Filters

http://www.argos-tech.com/syringe-filters-2.html

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 Microbes trapped on filter
© 2015 Pearson Education, Inc. Figure 5.34
 1- Assume that you are responsible for
decontaminating materials in a large hospital. How
would you sterilize each of the following? Briefly
justify your answers.
a. a bed sheet used by a plague patient

b. intravenous glucose-saline solutions

c. used disposable syringes

d. tissues taken from a vCJD (Prion) patients

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 3- Ozone can be used as disinfectant, can you explain
how it can kill a microorganism?

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 Chemical microbial control methods
• In many cases, physical control of microbial growth
is not possible. For instance, to remove microbes
from human skin there is no practical physical growth
control approach.
• Chemical antimicrobial agents damage microbial
cellular structures such as the membrane, cell wall or
other cell structures.

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 Chemical microbial control methods

• Sterilant (= sterilizer): A chemical used that

destroys all microorganisms, including endospores
(e.g. Formaldehyde). It is used to sterilize heat-
sensitive items such as thermometers and lensed
instruments. Many known sterilants cannot neutralize
prions.

• Disinfectant: Kills most vegetative cells but not

necessarily endospores. Is usually used on surfaces
or medical equipment/devices which results in
disinfection of the surface or equipment/device.

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 Other microbial growth control agents

• Antiseptics or germicides (Sanitizers): are a

more moderate version of disinfectants that are
safe to be applied on the skin (lower
concentrations of ethanol).

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 Chemical microbial control methods
• Cold sterilization: uses gases
such as formaldehyde and ozone
to sterilize objects within an
enclosed device. It resembles an
autoclave (chemical gas instead
of water vapor).

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 Heavy metals and self-disinfecting surfaces
• Some hospitals have started creating “self-disinfecting
surfaces” by using heavy metals (copper or silver) coats
on objects like doorknobs. Silver binds to cell wall
structures and damages them. Copper is suggested to
create reactive oxygen radicals that can damage DNA and
proteins [1].

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 Chemical Control of Microbial Growth
Antimicrobial agents can be classified as:

• Bacteriostatic: Inhibits microbial growth (i.e.

no new cell are produced and the cell number
remain the same.
• Bacteriocidal: kills microbial cells, but dead
cells remains intact.
• Bacteriolytic: kills cells by degrading cells
(e.g. detergents).

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Figure 4.39 Different Types of Antimicrobial Agents

Similar terminology is used for other groups of microbes, for

instance:
• Fungistatic
• Fungicidal
• Fungilytic

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 Antimicrobial Drug and Chemotherapy

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https://upload.wikimedia.org/wikipedia/commons/b/bf/Synthetic_Production_of_Penicillin_TR1468.jpg
 Antimicrobial Drug and Chemotherapy
• Antimicrobial drugs are compounds that kill or inhibits
the growth of microorganisms in a host (in vivo).
• The term antibiotic is usually used for antimicrobial
drugs used against bacteria and antimicrobial drugs
used against other microbes usually have a specific
term for instance:
• Antiviral
• Antifungal
• Annually 10,000 tons of different antimicrobial drugs
are used worldwide.

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 Antimicrobial Drug and Chemotherapy

• Effective microbial drugs show selective toxicity it

means they inhibit or kill pathogen but do not
adversely affect the host.

• Usually, each antimicrobial drug is effective against a

limited group of microorganisms.

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 Classification of Antimicrobial Drugs
Antimicrobial drugs are usually classified on the
basis of:
• Molecular structure

• Mechanism of action (Figure 27.20) (inhibiting cell

wall synthesis, translation or transcription).

• The spectrum of antimicrobial activity (broad

spectrum vs. narrow spectrum (Figure 27.21)).

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© 2015 Pearson Education, Inc. Figure 27.29 Ed. 14
Figure 28.12 Mechanisms of Action of
Major Antibacterial Agents

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 Antimicrobial drugs also can be classified on the basis of
the spectrum of antimicrobial activity (broad spectrum vs.
narrow spectrum (Figure 27.21).

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 Synthetic Antimicrobial Drugs

• Growth factor analogs are structurally similar to

growth factors but do not function in the cell
• Analogs similar to vitamins, amino acids, and other
compounds

• Isoniazid is a growth factor analog effective only

against Mycobacterium
• Interferes with the synthesis of Mycobacterium cell wall

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 Natural Antimicrobial Drugs
• Natural antimicrobial drugs are naturally produced
antimicrobial agents
• Thousands of natural antimicrobial drugs are known but,
less than 1% of them are clinically useful because of
problems with host toxicity or lack of uptake by the host
cell. They can be modified to enhance efficacy
(semisynthetic).
• The susceptibility of microbes to different
antibiotics varies greatly
• Gram-positive and gram-negative bacteria vary in their
sensitivity to antibiotics
• Broad-spectrum antibiotics are effective against both
groups of bacteria

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 Common antibiotics
• Penicillins (Figure 27.25)
• Discovered by Alexander Fleming
• One of the most commonly used antibiotics
• Effective primarily against gram-positive bacteria
• Some synthetic forms are effective against some
gram-negative bacteria
• Target cell wall synthesis
• Tetracyclines (Figure 27.29)
• Widespread medical use in humans and animals
• Broad-spectrum inhibition of protein synthesis
• Inhibits functioning of 30S ribosomal subunit

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 Antiviral Drugs
• Most antiviral drugs also target host structures,
resulting in toxicity
• Most successful and commonly used antivirals are
the nucleoside analogs (e.g., AZT)
• Block production of the viral genome (nucleic acids).

Enzyme inhibitors: for example protease inhibitors

inhibit the processing of large viral proteins into smaller
components.

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 Antiviral Drugs
• Interferons are small proteins that prevent viral
multiplication by stimulating antiviral proteins in
uninfected cells

• Fusion inhibitors prevent viruses from successfully

fusing with the host cell
• One category of Fusion inhibitors drugs successfully limits
HIV infection.

• Neuraminidase inhibitors (e.g. Tamiflu)

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 Antiviral Drugs

Photo from: Anne Moscona, M.D., 2005 Neuraminidase Inhibitors for

© 2015 Pearson Education, Inc. Influenza. N Engl J Med 2005; 353:1363-1373September 29, 2005
Antifungal Drugs
• Fungi pose special problems for chemotherapy
because they are eukaryotic (Figure 28.32)
• Much of the cellular machinery is the same as that of
animals and humans.
• A few drugs target unique metabolic processes unique to
fungi. Ergosterol inhibitors target the unique fungal plasma
membrane component ergosterol.

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Figure 28.17 Targets of Some
Antifungal Agents

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 Antifungal Drugs

• Echinocandins
• Inhibit cell wall synthase

• Used to treat Candida infections

• Antifungal drugs are increasingly becoming more
important since:
• Fungal infections in immunocompromised
individuals become more prevalent.
• Antifungal-resistant fungi are emerging

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 Chemical Control of Microbial Growth

• Minimum inhibitory concentration (MIC) is the smallest

amount of an agent needed to inhibit growth of a
microorganism (Figure 4.40)
• Varies with the organism used, inoculum size, temperature,
pH, etc.

• Disc diffusion assay uses solid media (Figure 4.41)

• Antimicrobial agent added to filter paper disc
• MIC is reached at some distance
• Zone of inhibition
• Area of no growth around disc
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Figure 4.40 Antimicrobial Agent Susceptibility Assay Using Dilution Methods

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Figure 29.11a Antibiotic Susceptibility Testing

An empty 96 plate
https://www.edgebio.com/products/96-well-treated-microplates-u-bottom-50063

• 12 rows each contains

one antibiotics.
• The concentration of
antibiotics reduces
from left to right
• Clear wells are the
ones with no microbial
growth
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Figure 4.41 Antimicrobial Agent Susceptibility Assay Using Diffusion Methods

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 2- In the plate shown below each row contains one
antibiotic. Circle two antibiotics that cannot control
the microbe that is cultured in below 96 well plate
even when they are used at their highest
concentration.

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 Antimicrobial Drug Susceptibility Testing

• Disc diffusion test

• Standard procedure for assessing antimicrobial activity

• Agar media is spread evenly with liquid culture of

bacteria (Figure 27.8b, c, and d)

• Inhibition zones (Figure 27.8e and f)

• Used to determine an organism's susceptibility to an
antimicrobial agent

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© 2015 Pearson Education, Inc. Figure 27.8f 14 ed.
© 2015 Pearson Education, Inc. Figure 27.8e 14 ed.
 3- Antibiotics molecules are different regarding their
chemical and physical properties. What limitation(s)
do you think this can cause for disc diffusion test.

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 Antimicrobial Drug Susceptibility Testing

• Etest is a non-diffusion-based technique that

employs a preformed and predefined gradient of
an antimicrobial agent immobilized on a plastic
strip (Figure 27.8g)

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© 2015 Pearson Education, Inc. Figure 27.8g 14 ed.
 4- Your grandma says garlic cures sore throats, as a
microbiology student you want to test this
hypothesis, what do you do?

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 Additional sources:
1- Boyce, M. J. et al. (2016) Modern technologies for
improving cleaning and disinfection of environmental
surfaces in hospitals. Antimicrob Resist Infect
Control 5(10).

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