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Microbial Growth Control: Physical & Chemical Methods

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CFU

What are some ways you could make water safe to drink?
Controlling
Microbial GrowthPhysical Measures
Oliver Yuan
BIOL 240
Learning Objectives

Be able to order vocabulary in terms of most clean to least

Calculate D-value from a microbial death curve
Vocabulary

Sepsis: microbial contamination

Asepsis: the absence of significant contamination

Aseptic surgery techniques prevent the microbial contamination of
wounds

Antisepsis: destroying harmful microorganisms from living tissue

Sanitization: lowering microbial counts to safe levels for public health

Commercial sterilization: removal of foodborne pathogens while
maintaining food quality

Killing Clostridium botulinum endospores from canned goods
How Clean Does it Need to Be?
Item
Cleaning Procedure
Critical item
Sterilization: removing and
destroying ALL microbial life,
including endospores and viruses
Semicritical item
Thorough disinfection: destruction
of most harmful microorganisms
from a fomite
Noncritical item
Degerming: mechanical removal
of microbes from a limited area
Example
Controlling Bacteria

Biocide (germicide): treatments that kill microbes

Bactericidal agents: Chemicals result in bacterial death in the
culture, no growth


Subculture: no bacteria grows
Bacteriostatic agents: Chemicals cause temporary inhibition of
growth, no growth

Subculture: bacteria grow
Effectiveness of Treatment

Depends on:

Number of microbes

Environment (organic matter, temperature, biofilms)

Time of exposure

Microbial characteristics
Microbial Death Curve
D-value (decimal
reduction time):
time it takes to
reduce microbe
population by
one magnitude
(90%)
CFU
What is the Dvalue of this
agent?
Physical Methods of Microbial
Control

Heat

Filtration

Low Temperatures

High pressure

Desiccation

Osmotic pressure
Heat

Thermal death time (TDT): shortest time during which all cells in a
culture are killed at a given temperature

Thermal death point (TDP): lowest temperature at which all cells in a
culture are killed in 10 min
Moist Heat Sterilization

Moist heat denatures proteins

Boiling

Autoclave: steam under pressure
that kills all organisms and
endospores

121°C at 15 psi for 20 min

Steam must contact the item's
surface
Pasteurization

Reduces spoilage organisms and
pathogens

Equivalent treatments

63°C for 30 min

High-temperature short-time (HTST):
72°C for 15 sec

Ultra-high-temperature (UHT): 138°C
for 2-4 sec
Dry Heat Sterilization

Kills by oxidation

Flaming

Incineration

Hot-air sterilization
Filtration

Passage of substance through a
screen-like material

Used for heat-sensitive materials

High-efficiency particulate air (HEPA)
filters: remove microbes >0.3 μm

Membrane filters remove microbes
>0.22 μm
Physical Methods of Microbial
Control

Low temperature inhibits microbial growth

Refrigeration

Deep-freezing

Lyophilization: freezing dry

High pressure denatures proteins

Desiccation prevents metabolism

Osmotic pressure causes plasmolysis

Radiation damages DNA
Controlling Microbial
Growth- Cleaning Agents
and Antimicrobial Drugs
Oliver Yuan
BIOL 240
Learning Objectives

Compare and contrast different kinds of cleaning agents, their
mechanisms, and their evaluation of effectiveness
Chemical Methods of Microbial
Control

Phenol and Phenolics

Bisphenols

Alcohols

Halogens

Heavy Metals

Surfactants
Phenol and Phenolic Compounds

Disrupt plasma membranes and denaturing proteins
a) Phenol b) o-Phenylphenol c) hexachlorophene (pHisoHex)

Triclosan: another bisphenol

Inhibits an enzyme needed for the biosynthesis of fatty acids

Effective against gram-positive, certain gram-negative and yeasts

FDA banned in 2016 from soaps and body washes
Alcohols

Denature proteins and disrupting membranes

No effect on endospores and nonenveloped viruses

70% works better than 100% since the latter coagulates proteins too
fast
Halogens and Heavy Metals

Halogens

Iodine: Alters protein synthesis and
membranes

Chlorine
 Bleach
 Oxidizing agents

Heavy Metals

Ag, Hg, and Cu

Bind to and denature proteins

Oligodynamic: very effective at very
low concentrations
For a disk diffusion assay, the zone of
inhibition is:
ⓘ Start presenting to display the poll results on this slide.
CFU
 For a disk diffusion assay, the zone of inhibition is:
A.
How far the antimicrobial agent spread into the agar
B.
The growth around a disk with an antimicrobial agent
C.
Measured in mm
D.
The same for all antimicrobial agents for a given species
CFU

Why is 70% ethanol a more effective antimicrobial agent than 100%
ethanol?
Surfactants

Soap: salts with hydrophobic tails; only degerming

Quaternary ammonium salts (quats): Disrupt membrane integrity

Effective against fungi, protozoans, and enveloped viruses, but
endospores are unaffected
Chemical Food Preservatives

Sulfur dioxide (SO2) prevents wine spoilage

Organic acids


Inhibit metabolism

Sorbic acid, benzoic acid, and calcium propionate prevent molds in
acidic foods
Nitrites and nitrates prevent endospore germination in processed
meat

Creates carcinogenic nitrosamines when heated
Evaluating a Disinfectant

Use-Dilution Tests

Metal cylinders are dipped in test
bacteria and dried

Cylinders are placed in disinfectant
for 10 min at 20°C

Cylinders are transferred to fresh
culture media without disinfectant to
determine whether the bacteria
survived treatment
Disk Diffusion Method

Filter paper disks are soaked in a chemical and placed on a culture

Look for zone of inhibition around disks
Antimicrobial Drugs

Compound produced by microorganisms that inhibit growth of
other microorganisms

Selective toxicity: specifically inhibits or kills microbial targets without
harm to the host

Actions of antimicrobial drugs

Alteration of membrane permeability

Damage to proteins (enzymes)

Damage to nucleic acids

Damage to cell walls

Alteration of metabolic pathways
Targets of Antibacterial Drugs
Evaluating Antibacterial Drugs

Kirby-Bauer Disk Diffusion Test

Filter paper disks are soaked in antimicrobial drug and placed on
Mueller-Hinton agar

Look for zone of inhibition around disks
Variability in Disk Diffusion Test

Water solubility of agent

Ability to diffuse into agar

Concentration of agent

Concentration of agar

Type of bacteria selected

Evenness of agar and of bacterial lawn
Other Antimicrobial Drugs


More difficult to treat, either because they are eukaryotes or because
they use much of the host’s own machinery
Antifungal


Antiprotozoan


Quinoline: chloroquine thought to disrupt heme breakdown in malaria and
Entamoeba histolytica
Antihelminth


Imidazole: Disrupts cell membrane by targeting ergosterol in Candida
vaginal yeast, athlete’s foot, etc.
Praziquantel: thought to cause influx of Ca2+ into tapeworms and flukes
(Schistostoma), paralyzing them
Antiviral

Remdesivir: adenosine analog that binds to RdRP and initiates termination;
hepatitis C, previous emergency treatment for COVID-19
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