summer 2022: week 4

topic 7
INHIBITING THE GROWTH
OF MICROORGANISMS IN
VITRO
DR. NELYA N. CLETO
 INTRODUCTION
INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

In certain environments, it is necessary and/or desirable to inhibit

the growth of microbes. In hospitals, nursing homes, and other
healthcare institutions, for example, it is necessary to inhibit the
growth of pathogens so that they will not infect patients, staff
members, or visitors. Other environments where it is necessary,
and/or desirable to inhibit microbial growth include food and
beverage processing plants, restaurants, kitchens, and bathrooms.
DEFINITION OF TERMS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

Contamination - denotes contact of a sterile or aseptic item
with microorganisms. Medically aseptic items become
contaminated if they get contact with disease-producing
organisms. Sterile items become contaminated if they get in
contact with items that are not sterile.
DEFINITION OF TERMS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

Decontamination- The process where physical or chemical means
are used to remove inactivate, or destroy pathogens on surface or
item making them safe for handling or use and incapable of
transmitting infectious germs.
Degerming - The mechanical removal of microbes from a limited
area.
Sanitization - Lowering microbial counts to safe public health
levels and minimizing disease transfer from person to person.
 DEFINITION OF TERMS

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•Sterilization is the complete destruction of all microbes, including
cells, spores, and viruses.

–Accomplished by dry heat, autoclaving (steam under pressure), gas,

various chemicals, and certain types of radiation.
 DEFINITION OF TERMS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

Commercial Sterilization- Killing Clostridium botulinum endospores
from canned goods.
Disinfection is the destruction or removal of pathogens from non-living
objects by physical or chemical methods; pasteurization is an example
of a disinfection technique.
–Disinfectants are chemical substances that eliminate pathogens on
inanimate objects.
–Antiseptics are solutions used to disinfect skin and other living tissues.
 DEFINITION OF TERMS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

Antisepsis - destroying harmful microorganisms from living tissue.
-The prevention of infection.
Sepsis refers to bacterial contamination. The presence of
pathogens in blood or tissues, whereas asepsis means the absence
of pathogens.
Asepsis is the absence of significant contamination.
- Aseptic surgery techniques prevent the microbial contamination of
wounds .
 DEFINITION OF TERMS

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•The suffix –cide or –cidal refers to “killing.”

•Germicidal agents, biocidal agents, and microbicidal

agents are chemicals that kill microbes.
 DEFINITION OF TERMS

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•Bactericidal agents are chemicals that specifically kill bacteria,
but not necessarily bacterial endospores.

–Sporicidal agents kill bacterial endospores.

–Fungicidal agents kill fungi, including fungal spores.
–Algicidal agents kill algae.
–Viricidal agents destroy viruses.
DEFINITION OF TERMS

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A microbistatic agent is a drug or chemical that
inhibits growth and reproduction of microbes.
A bacteriostatic agent is one that specifically inhibits
the metabolism and reproduction of bacteria.
DEFINITION OF TERMS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

Personal Protective Equipment (PPE)
Specialized equipment and attire used
by healthcare workers to protect them
from infections. These includes gloves,
masks, gowns, and goggles.
 DEFINITION OF TERMS

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•Lyophilization is a process that combines dehydration (drying)
and freezing. This process is widely used in industry to preserve
foods, antibiotics, microorganisms, and other biologic materials.
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

HEAT
Two factors, temperature and time - determine the effectiveness of heat
for sterilization
.
The thermal death point (TDP) of any species is the lowest temperature
that will kill all of the organisms in a standardized pure culture within a
specified time.
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TYPES OF HEAT
DRY HEAT

e.g., oven, electrical incinerator,

or flame

DRY HEAT STERILIZATION

Kills by oxidation:
- Flaming
- Incineration
- Hot-air sterilization
INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

DRY HEAT
STERILIZATION
USING A BUNSEN BURNER FLAME USING AN ELECTRICAL HEATING DEVICE
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TYPES OF HEAT
MOIST HEAT
A. Boiling
B. Use of an Autoclave
MOIST HEAT STERILIZATION
Large containers require longer
sterilization times.
BOILING WATER
Test strips are used to indicate sterility.
Not all objects can withstand moist heat AUTOCLAVE MACHINE
VERTICAL 50L
sterilization
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THE AUTOCLAVE
INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

A large metal pressure cooker that uses

steam under pressure to completely destroy
all microbial life.
Increased pressure raises the temperature
above the temperature of boiling water
(above 100oC) and forces steam into
materials being sterilized.
Autoclaving at a pressure of 15 psi at
121.5oC for 20 minutes destroys vegetative
microorganisms, bacterial endospores, and
viruses.
Can use pressure-sensitive tape or spore
strips or solutions as a quality control
measure to ensure proper autoclaving.
A LARGE, BUILT-IN AUTOCLAVE.
 USING PHYSICAL METHODS TO INHIBIT MICROBIAL GROWTH
THE AUTOCLAVE
INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

TYPES OF HEAT
MOIST HEAT

STERILIZATION
CONTAINER SIZE LIQUID VOLUME
TIME (MIN)

Test tube:
10 ml 15
18 x 150 mm

Erlenmeyer flask:
95 ml 15
125 ml

Erlenmeyer flask:
1500 ml 30
2000 ml

Fermentation bottle:
9000 ml
6750 ml 70 REGULAR
STERILOMETER
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

PASTEURIZATION

Pasteurization reduces spoilage

organisms and pathogens

High-temperature short-time (HTST):

72°C for 15 sec
Thermoduric organisms survive.
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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 in
diameter.
Membrane filters removes microbes >
0.22 µm
-Pore sizes of as small as 0.01 µm are
available, which can filter out viruses and
large proteins. REPLACEABLE HEPA FILTER
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LOW TEMPERATURE

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LOW TEMPERATURE
Low temperature inhibits microbial growth by slowing down microbial
metabolism. Examples include refrigeration and freezing.

Freezing at -10°C stops microbial growth, but generally does not kill
microorganisms, and keeps food fresh for several months.
Refrigeration at 5° C slows the growth of microorganisms and keeps
food fresh for a few days.

High pressure denatures proteins

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LOW TEMPERATURE

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COLD STORAGE
low-temperature; freezing
inhibit growth - bacteriostatic
enzymic inactive
Psychrophiles can grow
- spoil the food in the refrigerator PSYCHROPHILES
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LOW TEMPERATURE

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

DESSICATION
many dried microorganisms remain viable, but they cannot reproduce.
absence of water prevents metabolism.

Osmotic Pressure uses high concentrations of salts and sugars to create

hypertonic environment; causes plasmolysis..
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LOW TEMPERATURE

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

DRYING
Draw water out of cells; dehydration
inhibit growth- bacteriostatic
drying food:
- desiccation (removing water) MEAT JERKIES
-Lyophilization (freeze-drying)
~coffee, milk, meats, vegetables
DRIED
Adding salt, sugar MANGOES

S. aureus can grow in high salt conditions

COFFEE
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LOW TEMPERATURE

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

RADIATION
Ionizing Radiation (X-rays, gamma rays, electron
beams)
- Ionizes water to create reactive hydroxyl radicals
-Damages DNA by causing lethal mutations.

Non-ionizing Radiation (ultraviolet, 260nm)

-Damages DNA by creating thymine dimers.

Microwaves kill by heat; not especially

antimicrobial.
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

GASEOUS ATMOSPHERE
- can be altered to inhibit growth
 INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO
USING CHEMICAL METHODS TO
INHIBIT MICROBIAL GROWTH

CHEMICAL DISINFECTION
-refers to the use of chemical agents to inhibit the growth of pathogens,
either temporarily or permanently.
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

ANTISEPTICS
–May safely be used on human tissues.
–Reduce the number of organisms on the
surface of the skin; do not penetrate pores and
hair follicles.

•Antiseptic soaps and scrubbing are used by

BACTERIA healthcare personnel to remove organisms
UNDER A MICROSCOPE
lodged in pores or folds of the skin.
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A COMPARISON OF THE EFFECTIVENESS OF VARIOUS ANTISEPTICS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

Effect of germicidal activity on Microbes
CYTOPLASMIC
DNA PROTEINS
MEMBRANE

Alcohols
Biguanides Aldehydes
Ethylene oxides Halogens
Phenolics Metals
Aldehydes Ozone
Quats Peroxygens
Phenolics
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•DISINFECTANTS ARE AFFECTED BY:
–Prior cleaning of the object or surface

–The organic load (e.g., feces, blood, pus)

–The bioburden; types and numbers of microbes

–Concentration of the disinfectant

–Contact time

–Physical nature of the object being disinfected

–Temperature and pH
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

PRINCIPLES OF EFFECTIVE DISINFECTION
Concentration of disinfectant
Organic matter
pH
Time
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Impacts of pH on Chlorine Disinfection

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

pH impacts the form of Chlorine
Chlorine is the most effective between
pH 5.5 - 7.5

Water H 2O Hypochlorous acid HOCl

O O
H
H H Cl
pH dependent
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

CARCINOGENIC
ORGANIC DISINFECTION
MATTER BYPRODUCTS
 INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO
EXAMPLES OF DISINFECTANTS
INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO
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INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

THE DISK-DIFFUSION METHOD
Evaluates efficacy of chemical
agents.
Filter paper disks are soaked in
a chemical and placed on a
culture.
Look for zone of inhibition
around disks.
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PHENOL AND PHENOLICS
Injure lipids of plasma
membranes, causing leakage

OH OH

a.) PHENOL b.) O-PHENYLPHENOL

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BISPHENOLS

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Contain two phenol groups connected
by a bridge.
Hexachlorophene and triclosan.
Disrupt plasma membranes.
Cl Cl Cl Cl Cl OH
H

C Cl O Cl

H
Cl OH HO Cl

c.) Hexachlorophene (a bisphenol) d.) Triclosan (a bisphenol)

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BIGUANIDES
Chlorhexidine
Used in surgical hand scrubs
Disrupt plasma membranes
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HALOGENS

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IODINE
Tincture: Solution in aqueous alcohol.
Iodophor: Combined with organic
molecules.
Impairs protein synthesis and alters
membranes.

CHLORINE
Oxidizing agents; shut down
cellular enzyme systems
BETADINE CHLORINE
SOLUTION SOLUTION
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ALCOHOLS - DISINFECTANTS & ANTISEPTICS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

ALCOHOL
Denatures proteins and dissolves lipids
Effectively kills bacteria and fungi
No effect on endospores and non- HYDROXYL
enveloped viruses.

Ethanol and Isopropanol: Technically, an alcohol is just something

-Require water
-Most effective at 70%
The active ingredient in most hand
sanitizers is an alcohol.
with a hydroxyl (-OH) group.
There are several that are good at
breaking up viral and bacterial
membranes and denaturing (unfolding)
proteins.
USING CHEMICAL METHODS TO
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ALCOHOLS - DISINFECTANTS & ANTISEPTICS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

ETHANOL
ISOPROPANOL
This is what's in
"adult beverages" propan-1-ol
2-propanol

Commonly called
PROPANOL "rubbing alcohol"
or Isopropyl
propan-1-ol
Alcohol
1-propanol
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HEAVY METALS AND THEIR COMPOUNDS

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Oligodynamic action - very small
amounts exert antimicrobial activity.

Denature proteins Ag, Hg, Cu, Zn

OPHTHALMIA
1. Silver Nitrate is used to prevent NEONATORUM

ophthalmia neonatorum
2. Mercuric Chloride prevents mildew in
paint.
3. Copper Sulfate is an algicide,
4. Zinc Chloride is found in mouth wash
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EFFECTIVENESS OF CHEMICAL ANTIMICROBIALS AGAINST

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

ENDOSPORES AND MYCOBACTERIA
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DECREASING ORDER OF RESISTANCE OF

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

MICROORGANISMS TO CHEMICAL BIOCIDES
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ALDEHYDES

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Inactive proteins by cross-linking with
functional groups.
Used for preserving specimens and in
medical equipment:
1. Formaldehyde and
orthophthalaldehyde.
2. Glutaraldehyde is one of the few
liquid chemical sterilizing agents
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SURFACE-ACTIVE AGENTS (SURFACTANTS AND DETERGENTS)

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

SOAP Degerming; emulsificiation

Anions react with plasma

ACID-ANIONIC SANITIZERS
membrane

Cations are bactericidal,

QUARTERY AMMONIUM
denature proteins, disrupt
COMPOUNDS (QUATS)
plasma membrane.
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SURFACE-ACTIVE AGENTS (SURFACTANTS AND DETERGENTS)

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

CLEANING YOUR CLOTHES WITH CHEMISTRY
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ANTIBIOTICS
Proteins produced by one
bacterium that inhibits another.

Nisin and Natamycin (Pimaricin)

prevent spoilage of cheese.
ANTIBIOTICS
UNDER A MICROSCOPE
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ANTIBIOTICS

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ZONE OF INHIBITION
Area around antibiotic disk with no
bacterial growth.
Bacteriocidal- no bacteria living in ZOI
Bacteriostatic - bacteria are not
reproducing in ZOI.
note: the Lawn of Bacteria
ZONE OF INHIBITION (MM) OF
STREPTOCOCCUS MUTANS
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CHEMICAL FOOD PRESERVATIVES

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Sulfur dioxide prevents wine spoilage.

Organic acids:
- inhibit metabolism
- Sorbic acid, benzoic acid, and calcium
propionate prevent molds in acidic foods.

Nitrites and Nitrates prevent

FOOD ADDITIVES
endospore germination.
INHIBITING THE GROWTH
OF PATHOGENS IN
OUR KITCHENS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

INHIBITING THE GROWTH OF
PATHOGENS IN OUR KITCHENS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

•Many foods brought into our kitchens are contaminated with pathogens
For example, gastrointestinal pathogens such as; E. coli O157:H7, Salmonella
and Campylobacter are often present on poultry, ground beef, and other meat
products.

•Problems arise when handling foods before cooking.

•Remain aware of pathogens when preparing foods.

INHIBITING THE GROWTH OF
PATHOGENS IN OUR KITCHENS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

•Wash hands frequently.

•Thoroughly clean plates and counter tops that have had poultry or meat

on them with hot soapy water

•The use of antibacterial kitchen sprays is controversial.

CONTROVERSIES RELATING TO THE
USE OF ANTI-MICROBIAL AGENTS
IN ANIMAL FEED AND
HOUSEHOLD PRODUCTS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

CONTROVERSIES RELATING TO THE USE OF ANTI-MICROBIAL
AGENTS IN ANIMAL FEED AND HOUSEHOLD PRODUCTS

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

40% of the antibiotics manufactured in the U.S. are used in
animal feed; microorganisms resistant to these antibiotics
survive!

–Drug resistant organisms are transmitted in animal feces and in food

products.
–Efforts are underway to eliminate or reduce the practice of adding
antibiotics to animal feed.
CONTROVERSIES RELATING TO THE USE OF ANTI-MICROBIAL
AGENTS IN ANIMAL FEED AND HOUSEHOLD PRODUCTS
•Use of antimicrobial agents is widespread in toys, cutting boards, in

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO

hand soaps, and many other household products; resistant
microorganisms survive!

•Controversy: Should children be exposed to all sorts of

microorganisms for their immune systems to develop
properly?
INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO
THANK YOU!

INHIBITING THE GROWTH OF MICROORGANISMS IN VITRO