09/03/2024

CONTROLLING MICROBIAL GROWTH

IN VITRO
CONTROLLING MICROBIAL
GROWTH
BURTON’S MICROBIOLOGY FOR THE HEALTH SCIENCES 1. Introduction
2. Factors that Affect Microbial Growth
3. Encouraging the Growth of Microbes in Vitro
4. Inhibiting the Growth of Microbes in Vitro

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FACTORS THAT AFFECT MICROBIAL GROWTH TEMPERATURE

• Availability of Nutrients Every organism has an optimum growth temperature.

– All living organisms require nutrients to sustain life.  Thermophiles are microorganisms that grow best at
high temperatures.
– Nutrients are energy sources.

– Organisms obtain energy by breaking chemical bonds.
• Moisture
– Water is essential for life.
– It is needed to carry out normal metabolic processes.
 Mesophiles are microbes that grow best at
moderate temperatures (e.g., 37o C).
 Psychrophiles prefer cold temperatures (like deep
ocean water).

– Certain microbial stages (e.g., bacterial endospores and

protozoal cysts) can survive a drying process.

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PH OSMOTIC PRESSURE AND SALINITY

• “pH” refers to the acidity or alkalinity of a solution.

• Most microorganisms prefer a neutral or slightly alkaline  Osmotic pressure is the pressure that is exerted on a cell
membrane by solutions both inside and outside the cell.
growth medium (pH 7.0 - 7.4)
 Osmosis is the movement of a solvent, through a
• Acidophiles prefer a pH of 2 to 5 permeable membrane, from a lower concentration of
solutes (dissolved substances) to a higher concentration
• Alkaliphiles prefer a pH > 8.5
of solutes.

 pH range

1 7 14

Acid Neutral Alkaline

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OSMOTIC PRESSURE AND SALINITY FACTORS THAT AFFECT MICROBIAL GROWTH,

CONT.

When the concentration of solutes in the external In hypertonic solution a cell shrink
environment of a cell is greater than that of solutes inside
the cell, the solution in which the cell is suspended is said
If a bacterial cell is placed into a hypotonic solution, Cells
swell up, and sometimes burst.
to be hypertonic.
When the concentration of solutes outside a cell is less than
In isotonic soln. the cell has normal turgor.
that of solutes inside a cell, the solution in which the cell is
suspended is said to be hypotonic.
In the case of erythrocytes, this bursting is called hemolysis
A solution is said to be isotonic when the concentration of
solutes outside a cell equals the concentration of solutes
inside the cell.

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CHANGES IN OSMOTIC PRESSURE FACTORS THAT AFFECT MICROBIAL GROWTH,

CONT.

– Organisms that prefer to live in salty environments

are called halophilic organisms.
– Microbes that can survive in high atmospheric
pressure are know as piezophiles.

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GASEOUS ATMOSPHERE

 Microorganisms vary with respect to the type of gaseous

atmosphere that they require.
 Obligate aerobes prefer the same atmosphere that humans do
(~20-21% O2 and 78-79% N2, other gases < 1%).
Encouraging the Growth
 Microaerophiles require reduced concentrations of oxygen
of Microbes in Vitro
(~5% O2).

 Obligate anaerobes are killed by the presence of oxygen.

 Facultative grow in presence or absence of oxygen

 Capnophiles require increased concentrations of CO2 (5-10%

CO2).
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BACTERIAL GROWTH

• Bacterial growth as an increase in the number of organisms

rather than an increase in their size. BINARY FISSION
• Bacteria divide by binary fission (one cell divides to become OF
two cells) when they reach their optimum size. STAPHYLOCOCCI.
• Binary fission continues through many generations until a
colony is produced on solid culture medium.

• Binary fission continues for as long as there is a sufficient

supply of nutrients, water, and space.

• The time it takes for one cell to become two cells is called the 13 14

generation time (e.g., E. coli = 20 minutes).

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CULTURE MEDIA CULTURE MEDIA

• Media are used in microbiology labs to culture (i.e.,
grow) bacteria.
• Culture media can be liquid or solid.
• An enriched medium is a broth or solid containing a
rich supply of special nutrients that promote the growth
of fastidious organisms.
• A selective medium has added inhibitors that
discourage growth of certain organisms while allowing
the growth of a desired organism.
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• A differential medium permits the differentiation of
organisms that grow on the medium.
• The various categories of media are not mutually
exclusive; e.g., blood agar is enriched and differential.
• Thioglycollate broth (THIO) is a popular liquid medium
in bacteriology labs; it supports the growth of all
categories of bacteria from obligate aerobes to obligate
anaerobes.
– There is a concentration gradient of dissolved
oxygen in the tube; organisms grow only in that part of
the broth where the oxygen concentration 16

meets their needs.

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Bacterial colonies on
MacConkey agar S. AUREUS ON MANNITOL-
SALT AGAR (A SELECTIVE &
(a selective and differential
DIFFERENTIAL MEDIUM)
A THIOGLYCOLLATE medium)
(THIO) BROTH TUBE

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COLONIES OF A Β-HEMOLYTIC STREPTOCOCCUS

SPECIES ON A BLOOD AGAR PLATE (IN THIS CASE, THE INOCULATION OF CULTURE MEDIA
BLOOD AGAR IS BOTH ENRICHED AND DIFFERENTIAL)
• Culture media are inoculated
with clinical specimens (i.e.,
specimens collected from
patients with a suspected
infectious disease).

• Inoculation involves adding a

portion of a specimen to the
medium.

• Inoculation is accomplished
Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins 19
using a sterile inoculating

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loop.

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IMPORTANCE OF USING “ASEPTIC TECHNIQUE” INCUBATION

• After media are inoculated, they must be placed into an incubator

Aseptic technique is practiced to prevent which will maintain the appropriate atmosphere, temperature, and
•(a) microbiology professionals from becoming infected, moisture level; the process is known as incubation.

•(b) contamination of their work environment. • 3 types of incubators are used in clinical microbiology
•(c) contamination of clinical specimens, cultures, and laboratories:

subcultures. – A CO2 incubator (contains 5-10% CO2)

– A non-CO2 incubator (contains room air)

– An anaerobic incubator (the atmosphere is devoid of oxygen)

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A POPULATION GROWTH CURVE OF LIVING

BACTERIAL POPULATION GROWTH CURVE ORGANISMS.

 Lag phase during which the bacteria absorb

nutrients, synthesize enzymes, and prepare for cell
division.
• The bacteria do not increase in number

 Logarithmic growth phase (log phase)

• the bacteria multiply so rapidly that the number of organisms
doubles with each generation time.
• Growth rate is the greatest during the log phase.
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A POPULATION GROWTH CURVE OF LIVING

ORGANISMS.

 Stationary phase
 As the nutrients are used up and the concentration of toxic
waste products build up, the rate of division slows, such that
the number of bacteria that are dividing equals the number
that are dying. Inhibiting the Growth of
 Death phase or decline phase Microbes in Vitro
 As overcrowding occurs, the concentration of toxic waste
products continues to increase and the nutrient supply
decreases.
 The microorganisms then die at a rapid rate.
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DEFINITION OF TERMS DEFINITION OF TERMS, CONT.

In Vitro: In an artificial environment, as in a laboratory
setting; used in reference to what occurs outside an
organism.
In Vivo: Used in reference to what occurs within a living
organism.
• Sterilization is the complete destruction of all microbes,
including cells, spores, and viruses.
• Disinfection is the destruction or removal of pathogens
from nonliving objects.
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• The suffix –cide or –cidal refers to “killing.”
Germicidal agents, biocidal agents, and microbicidal
agents are chemicals that kill microbes.
• Bactericidal agents are chemicals that specifically
kill bacteria.
• Sporicidal agents kill bacterial endospores.
– Fungicidal agents kill fungi, including fungal spores.
– Algicidal agents kill algae.
– Viricidal agents destroy viruses.
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DEFINITION OF TERMS (CONT.) PHYSICAL METHODS TO INHIBIT

MICROBIAL GROWTH
• Heat
• A static agent is a drug or chemical that inhibits growth and  Heat is the most practical, efficient, and inexpensive method of
reproduction of microbes. sterilization of those inanimate objects and materials that can
• A bacteriostatic agent is one that specifically inhibits the withstand high temperatures.
metabolism and reproduction of bacteria.  Because of these advantages, it is the means most frequently
• Lyophilization is a process that combines dehydration (drying) used.
and freezing. • Types of Heat
• This process is widely used in industry to preserve foods,
antibiotics, microorganisms, and other biologic materials. – Dry heat – e.g., oven, electrical incinerator, flame, use of an
• Sepsis refers to the presence of pathogens in blood or tissues, autoclave
whereas asepsis means the absence of pathogens.
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– Moist heat – boiling 30
• Antisepsis is the prevention of infection.

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DRY HEAT STERILIZATION THE AUTOCLAVE

 Dry Heat. Dry-heat oven provides effective sterilization of metals,

A large metal pressure machine that uses steam
under pressure to completely destroy all microbial
glassware, some powders, oils, and waxes.
life.
 These items must be at:  Increased pressure raises the temperature above the
1. 160°C to 165°C for 2 hours temperature of boiling water (above 100oC) and forces
2. 170°C to 180°C for 1 hour. steam into materials being sterilized.
 The effectiveness of dry-heat sterilization depends on how deeply
Autoclaving at:
the heat penetrates throughout the material, and the items to be
1. a pressure of 15 psi
baked must be positioned so that the hot air circulates freely among
them. 2. 121 oC
 Incineration (burning) is an effective means of destroying 3. for 20 minutes.
contaminated disposable materials. 31 To destroys vegetative microorganisms, bacterial 32

endospores, and viruses.

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THE AUTOCLAVE QUALITY-CONTROL PHYSICAL METHODS TO INHIBIT

MICROBIAL GROWTH
• Pressure-sensitive autoclave tape and commercially
available strips or solutions containing bacterial spores • Cold; most microorganisms are not killed, but their metabolic
activities are slowed.
can be used as quality-control measures to ensure that
• Desiccation; For many centuries, foods have been
preserved by
autoclaves are functioning properly. drying. Many dried microorganisms remain viable, but they
cannot reproduce.
• autoclaving, the spores are tested to see whether
After
• Radiation; an ultra-violet (UV) lamp is useful for reducing the
they were killed. number of microbes in the air.
• Ultrasonic waves; used in hospitals and medical and dental
clinics to clean equipment.
• Filters; used to separate cells/microbes from liquids or gases.
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33 • Gaseous atmosphere; can be altered to inhibit growth.

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RADIATION X-RAYS AND GAMMA AND BETA RAYS

 UV lamp is useful for reducing the number of

microorganisms in the air.
 Sterility may also be maintained by having a UV lamp
placed in a hood or cabinet containing instruments,
paper and cloth equipment, liquid, and other inanimate
articles.
 Many biologic materials, such as sera, antisera, toxins,
and vaccines, are sterilized with UV rays.
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 Radiation may be lethal or cause mutations in microorganisms and
tissue cells because they damage DNA and proteins within those
cells.
 Studies performed in radiation research laboratories have
demonstrated that these radiations can be used for the prevention
of food spoilage, sterilization of heat-sensitive surgical equipment,
preparation of vaccines, and treatment of some chronic diseases
such as cancer, all of which are very practical applications for
laboratory research.
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ULTRASONICWAVES
 In hospitals, medical clinics, and dental clinics,
ultrasonic waves are a frequently used means of
cleaning delicate equipment.
 Ultrasonic cleaners consist of tanks filled with liquid
solvent (usually water); the short sound waves are then
passed through the liquid.
 The sound waves mechanically dislodge organic debris
on instruments and glassware.
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GASEOUS ATMOSPHERE
 In limited situations, it is possible to inhibit growth of microorganisms by
altering the atmosphere in which they are located. Because aerobes and
microaerophiles require oxygen, they can be killed by placing them into an
atmosphere devoid of oxygen or by removing oxygen from the
environment in which they are living.
 Conversely, obligate anaerobes can be killed by placing them into an
atmosphere containing oxygen or by adding oxygen to the environment in
which they are living.
 For instance, wounds likely to contain anaerobes are lanced (opened) to
expose them to oxygen. Another example is gas gangrene, a deep wound
infection that causes rapid destruction of tissues.
 Gas gangrene is caused by various anaerobes in the genus Clostridium.
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FILTRATION
 Filters of various pore sizes are used to filter or separate cells,
larger viruses, bacteria, and certain other microorganisms from
the liquids
or gases in which they are suspended.
 Filters with tiny pore sizes (called micropore filters) are used in
laboratories to filter bacteria and viruses out of liquids.
 The variety of filters is large and includes sintered glass (in which
uniform particles of glass are fused), plastic films, unglazed
porcelain, asbestos, diatomaceous earth, and cellulose
membrane filters.
 Small quantities of liquid can be filtered through a filter-containing
syringe, but large quantities require larger apparatuses.
 Microbes, even those as small as viruses, can be removed from 38
liquids using filters having appropriate pore sizes.
DISINFECTION
• Chemical disinfection refers to the use of chemical
agents to inhibit the growth of pathogens, either
temporarily or permanently.
• 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
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– Temperature and pH
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USING CHEMICAL AGENTS TO INHIBIT USING CHEMICAL AGENTS TO INHIBIT

MICROBIAL GROWTH, CONT. MICROBIAL GROWTH (CONT.)
Characteristics of an ideal chemical antimicrobial agent: • Antiseptics
• Soluble in water and easy to
• Should have a broad – May safely be used on human tissues.
apply
antimicrobial spectrum – Reduce the number of organisms on the surface of the skin; do not
• Inexpensive and easy to penetrate pores and hair follicles.
• Fast acting prepare
• Antiseptic soaps and scrubbing are used by healthcare personnel to
• Not affected by the presence
• Stable as both a concentrate remove organisms lodged in pores or folds of the skin.
of organic matter
and a working solution • Antimicrobial chemical agents that can safely be applied to
• Nontoxic to human tissues skin are called antiseptics.

and noncorrosive
• Odorless
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• Should leave a residual
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INTRODUCTION,
INTRODUCTION CONT.

• Drugs used to treat

• Chemotherapy is the use of any bacterial diseases are
chemical (drug) to treat any called antibacterial agents;
disease or condition. those used to treat
• fungal diseases, antifungal
• A chemotherapeutic agent is any
agents;
drug used to treat any condition or
• protozoal diseases,
disease.
antiprotozoal agents;
• An antimicrobial agent is any
• viral diseases, antiviral agents.
chemical (drug) used to treat an
• An antibiotic is a
infectious disease, either by substance produced by a
inhibiting or killing pathogens in microorganism that kills or
vivo. Some antimicrobial agents inhibits growth of other
are antibiotics. microorganisms.

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