MICROBIOLOGY

for the Health Sciences

Chapter 8,9.
Controlling Microbial Growth
1
Controlling Microbial Growth in Vitro
 1. Introduction
2. Factors that Affect Microbial Growth
3. Encouraging the Growth of Microbes in Vitro
4. Inhibiting the Growth of Microbes in Vitro

3
Factors That Affect Microbial Growth

• Availability of Nutrients
– All living organisms require nutrients to sustain life.

– 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.

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

protozoal cysts) can survive a drying process.

4
 Temperature

Every organism has an optimum growth temperature.

 Thermophiles are microorganisms that grow best at
high temperatures.
 Mesophiles are microbes that grow best at moderate
temperatures (e.g., 37o C).
 Psychrophiles prefer cold temperatures (like deep
ocean water).

5
 pH

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

• Most microorganisms prefer a neutral or slightly alkaline
growth medium (pH 7.0 - 7.4)

• Acidophiles prefer a pH of 2 to 5
• Alkaliphiles prefer a pH > 8.5

 pH range

1 7 14

Acid Neutral Alkaline

6
 Osmotic Pressure and Salinity

 Osmotic pressure is the pressure that is exerted on a cell

membrane by solutions both inside and outside the cell.
 Osmosis is the movement of a solvent, through a
permeable membrane, from a lower concentration of
solutes (dissolved substances) to a higher concentration
of solutes.

7
 Osmotic Pressure and Salinity

When the concentration of solutes in the external

environment of a cell is greater than that of solutes inside
the cell, the solution in which the cell is suspended is said
to be hypertonic.
When the concentration of solutes outside a cell is less than
that of solutes inside a cell, the solution in which the cell is
suspended is said to be hypotonic.
A solution is said to be isotonic when the concentration of
solutes outside a cell equals the concentration of solutes
inside the cell.

8
Factors That Affect Microbial Growth,
cont.

In hypertonic solution a cell shrink

If a bacterial cell is placed into a hypotonic solution, Cells
swell up, and sometimes burst.
In isotonic soln. the cell has normal turgor.
In the case of erythrocytes, this bursting is called hemolysis

9
 Changes in Osmotic Pressure

10
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.

11
 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% O2and 78-79% N2, other gases < 1%).

 Microaerophiles require reduced concentrations of oxygen

(~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).

12
 Encouraging the Growth
of Microbes in Vitro

13
 Bacterial Growth

• Bacterial growth as an increase in the number of organisms

rather than an increase in their size.

• Bacteria divide by binary fission (one cell divides to become

two cells) when they reach their optimum size.

• 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
generation time (e.g., E. coli = 20 minutes).
14
Binary fission
of
staphylococci.

15
 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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 Culture Media

• 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
meets their needs.
17
A Thioglycollate (THIO)
Broth Tube

18
 Bacterial colonies on S. aureus on mannitol-
MacConkey agar (a salt agar (a selective &
selective & differential differential medium)
medium)

19
 Colonies of a β-hemolytic Streptococcus species on a
blood agar plate (in this case, the blood agar is both
enriched and differential)

Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

20
 Inoculation of Culture Media

• 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
using a sterile inoculating
loop.
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 Importance of Using “Aseptic Technique”

Aseptic technique is practiced to prevent

• (a) microbiology professionals from becoming infected,
• (b) contamination of their work environment.
• (c) contamination of clinical specimens, cultures, and
subcultures.

22
 Incubation

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

which will maintain the appropriate atmosphere, temperature, and
moisture level; the process is known as incubation.

• 3 types of incubators are used in clinical microbiology

laboratories:

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

– A non-CO2 incubator (contains room air)

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

23
 Bacterial Population Growth Curve

24
 A population growth curve of living 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.

25
 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.
 Death phase or decline phase
 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.

26
 Inhibiting the Growth of
Microbes in Vitro

27
Definition of Terms

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.

28
 Definition of Terms, cont.

• 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.

29
 Definition of Terms (cont.)

• A static 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.
• 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.
• Sepsis refers to the presence of pathogens in blood or
tissues, whereas asepsis means the absence of
pathogens.
• Antisepsis is the prevention of infection.
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 Physical Methods to Inhibit
Microbial Growth

• Heat
 Heat is the most practical, efficient, and inexpensive method of
sterilization of those inanimate objects and materials that can
withstand high temperatures.
 Because of these advantages, it is the means most frequently
used.

• Types of Heat

– Dry heat – e.g., oven, electrical incinerator, or flame

– Moist heat – boiling or use of an autoclave

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 Dry Heat Sterilization

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

glassware, some powders, oils, and waxes.
 These items must be at:
1. 160°C to 165°C for 2 hours
2. 170°C to 180°C for 1 hour.
 The effectiveness of dry-heat sterilization depends on how deeply
the heat penetrates throughout the material, and the items to be
baked must be positioned so that the hot air circulates freely among
them.
 Incineration (burning) is an effective means of destroying
contaminated disposable materials.

32
The autoclave

A large metal pressure machine 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:
1. a pressure of 15 psi
2. 121 oC
3. for 20 minutes.
 To destroys vegetative microorganisms, bacterial
endospores, and viruses.
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The autoclave quality-control

• Pressure-sensitive autoclave tape and commercially

available strips or solutions containing bacterial spores
can be used as quality-control measures to ensure that
autoclaves are functioning properly.

• After autoclaving, the spores are tested to see whether

they were killed.

34
 Physical Methods to Inhibit
Microbial Growth

• Cold; most microorganisms are not killed, but their

metabolic activities are slowed.
• Desiccation; For many centuries, foods have been
preserved by drying. Many dried microorganisms remain
viable, but they cannot reproduce.
• Radiation; an ultra-violet (UV) lamp is useful for
reducing the 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.
35
Gaseous atmosphere; can be altered to inhibit growth.
 Radiation

 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.

36
 X-rays and gamma and beta rays

 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.

37
 Ultrasonic Waves

 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.

38
 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
39 from liquids using filters having appropriate pore sizes.
 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.

40
 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

41
– Temperature and pH
42
43
Using Chemical Agents to Inhibit
Microbial Growth, cont.

Characteristics of an ideal chemical antimicrobial agent:

• Should have a broad • Soluble in water and easy to

apply
antimicrobial spectrum
• Inexpensive and easy to
• Fast acting
prepare
• Not affected by the presence
of organic matter
• Stable as both a concentrate
and a working solution
• Nontoxic to human tissues
and noncorrosive • Odorless

• Should leave a residual

44 antimicrobial film on surface
 Using Chemical Agents to Inhibit
Microbial Growth (cont.)

• 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 healthcare
personnel to remove organisms lodged in pores or folds of
the skin.

• Antimicrobial chemical agents that can safely be applied to

skin are called antiseptics.

45
 Introduction

• Chemotherapy is the use of any

chemical (drug) to treat any disease
or condition.
• A chemotherapeutic agent is any
drug used to treat any condition or
disease.
• An antimicrobial agent is any
chemical (drug) used to treat an
infectious disease, either by
inhibiting or killing pathogens in
vivo. Some antimicrobial agents are
antibiotics.
 Introduction, cont.

• Drugs used to treat

bacterial diseases are
called antibacterial agents;
those used to treat
• fungal diseases, antifungal
agents;
• protozoal diseases,
antiprotozoal agents;
• viral diseases, antiviral
agents.
• An antibiotic is a substance
produced by a
microorganism that kills or
inhibits growth of other
microorganisms.
Penicillin

The discovery of penicillin

by Alexander Fleming.

(A)Colonies of
Staphylococcus aureus are
growing well in this area of
the plate.
(B)Colonies are poorly
developed in this area of the
plate because of an
antibiotic (penicillin) being
produced by a colony of
Penicillium notatum (a
mould), shown at C.
 Characteristics of an Ideal
Antimicrobial Agent

• The ideal antimicrobial agent

should:
– Kill or inhibit the growth of
pathogens
– Cause no damage to the
host
– Cause no allergic reaction in
the host
– Be stable when stored in
solid or liquid form
– Remain in specific tissues in
the body long enough to be
effective
– Kill the pathogens before
they mutate and become
resistant to it
 How Antimicrobial Agents Work

• The 5 most common

mechanisms of action of
antimicrobial agents are:
– Inhibition of cell wall
synthesis
– Damage to cell membranes
– Inhibition of nucleic acid
synthesis (either DNA or
RNA synthesis)
– Inhibition of protein
synthesis
– Inhibition of enzyme
activity
 Antibacterial Agents

• Bacteriostatic drugs inhibit growth of bacteria, whereas bactericidal drugs kill

bacteria.
• Sulfonamide drugs inhibit production of folic acid (a vitamin) in those bacteria
that require p-aminobenzoic acid to synthesize folic acid; without folic acid
bacteria cannot produce certain essential proteins and die.
– Sulfa drugs are competitive inhibitors; they are bacteriostatic.
 Antibacterial
Agents
• Bacteriostatic drugs inhibit growth of
bacteria, whereas bactericidal drugs
kill bacteria.
• In most Gram-positive bacteria,
penicillin interferes with the synthesis
and cross-linking of peptidoglycan, a
component of cell walls. By inhibiting
cell wall synthesis, penicillin destroys
the bacteria.
 Antibacterial Agents, cont.

• Colistin and nalidixic acid

destroy only Gram-negative
bacteria; they are referred to as
narrow-spectrum antibiotics.
• Antibiotics that are destructive
to both Gram-positive and
Gram-negative bacteria are
called broad-spectrum
antibiotics (examples:
ampicillin, chloramphenicol and
tetracycline).
• Multidrug therapy
– Sometimes one drug is not
sufficient; 2 or more drugs may
be used simultaneously, as in
the treatment of tuberculosis.
Some Major Categories of
Antibacterial Agents
• Penicillins: bactericidal; interfere with cell wall
synthesis
• Cephalosporins: bactericidal; interfere with cell wall
synthesis
• Tetracyclines: bacteriostatic; inhibit protein synthesis
• Aminoglycosides: bactericidal; inhibit protein synthesis
• Macrolides: bacteriostatic at lower doses; bactericidal
at higher doses; inhibit protein synthesis
• Fluoroquinolones: bactericidal; inhibit DNA synthesis
 Antibacterial Agents, cont.
• Synergism Versus Antagonism
– Synergism is when 2 antimicrobial agents are used together
to produce a degree of pathogen killing that is greater than
that achieved by either drug alone. Synergism is a good
thing!
– 2+2=6
– Antagonism is when 2 drugs actually work against each
other. The extent of pathogen killing is less than that
achieved by either drug alone. Antagonism is a bad thing!
– 2+2=1
Antiviral Agents
• Antiviral agents are the newest weapons in
antimicrobial methodology.
• Difficult to develop these agents because viruses
are produced within host cells.
• Some drugs have been developed that are effective
in certain viral infections, but not others; they work
by inhibiting viral replication within cells.
• Antiviral agent “cocktails” (several drugs that are
administered simultaneously) are being used to treat
HIV infection.
 Drug Resistance
“Superbugs”

• Superbugs are microbes (mainly bacteria) that have

become resistant to one or more antimicrobial agent.
Infections caused by superbugs are difficult to treat!
• Bacterial superbugs include:
– methicillin-resistant Staphylococcus aureus (MRSA)
– vancomycin-resistant Enterococcus spp. (VRE)
– multidrug-resistant Mycobacterium tuberculosis (MDRTB)
– multidrug-resistant strains of Acinetobacter, Burkholderia,
E. coli, Klebsiella, Pseudomonas, Stenotrophomonas,
Salmonella, Shigella. and N. gonorrhoeae;
– β–lactamase-producing strains of Streptococcus
pneumoniae
– Haemophilus influenzae; carbapenemase-producing
Klebsiella pneumoniae.
Drug Resistance
How Bacteria Become Resistant to Drugs

• Some bacteria are naturally resistant =

intrinsic resistance
• They lack the specific target site for the
drug
• The drug is unable to cross the
organism’s cell wall or cell membrane
and thus, cannot reach its site of action.
• Acquired resistance = bacteria that were
once susceptible to a particular drug
become resistant.
• Before a drug enters a bacterial cell it
must first bind to proteins on the surface
of the cell; these proteins are called
drug-binding sites. A chromosomal
mutation that affects the structure of a
drug-binding site can prevent the drug
from binding, resulting in drug
resistance.
 Drug Resistance
How Bacteria Become Resistant to Drugs, cont.

• To enter a bacterial cell, a drug must be

able to pass through the cell wall and
cell membrane
• Chromosomal mutations may alter the
structure of the cell membrane, thus
preventing the drug from entering the cell;
this results in drug resistance.
• Bacteria can develop the ability to
produce an enzyme that destroys or
inactivates a drug.
– Many bacteria have become resistant to
penicillin because they have acquired the
gene for penicillinase production during
conjugation.
• A plasmid that contains multiple genes
for drug resistance is known as a
resistance factor (R-factor).
 Some Strategies in the War
Against Drug Resistance

• Education of healthcare professionals and patients

• Patients should stop demanding antibiotics every
time they are, or their child is, sick
• Physicians should not be pressured by patients
and should prescribe drugs only when warranted
• Clinicians should prescribe a narrow-spectrum
drug if lab results indicate that it kills the pathogen

• Patients should destroy any excess or

out-dated medications

• Antibiotics should not be used in a

prophylactic manner

• Healthcare professionals should

practice good infection control

• Patients should take drugs in manner

prescribed
Undesirable Effects of
Antimicrobial Agents
• Reasons why antimicrobial agents should not be used
indiscriminately:
– Organisms susceptible to the agent will die, but resistant
ones will survive; this is known as selecting for resistant
organisms.
– The patient may become allergic to the agent.
– Many agents are toxic to humans and some are very toxic.
– With prolonged use, a broad-spectrum antibiotic may
destroy the normal flora, resulting in an overgrowth of
bacteria known as a superinfection.