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MICROBIOLOGYfor the Health Sciences

. العمرين : ابو فريد أ اعداد

Chapter 8,9.

Controlling Microbial Growth

Chapter 8. Controlling Microbial Growth in Vitro

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Chapter 8 Outline

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

•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.

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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).

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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

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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.

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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.

Osmotic Pressure and Salinity

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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

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Changes in Osmotic Pressure

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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%).

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).

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Encouraging the Growth of Microbes in Vitro

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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).

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Binary fission of staphylococci.

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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.

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A Thioglycollate (THIO) Broth Tube

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Bacterial colonies on MacConkey agar (a selective & differential medium)

S. aureus on mannitol-salt agar (a selective & differential medium)

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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

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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.

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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)

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Bacterial Population Growth Curve

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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 during

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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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.

A population growth curve of living organisms.

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Inhibiting the Growth of Microbes in Vitro

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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.

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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.

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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.

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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.

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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.

•Gaseous atmosphere; can be altered to inhibit growth.

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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.

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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.

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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.

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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 liquids using filters having appropriate pore sizes.

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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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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

– Temperature and pH

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Using Chemical Agents to Inhibit Microbial Growth, cont.

• Should have a broad

antimicrobial spectrum

• Fast acting

• Not affected by the presence of

organic matter

• Nontoxic to human tissues and

noncorrosive

• Should leave a residual

antimicrobial film on surface

• Soluble in water and easy to

apply

• Inexpensive and easy to

prepare

• Stable as both a concentrate

and a working solution

• Odorless

Characteristics of an ideal chemical antimicrobial agent:

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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.

Chapter 9.Controlling Microbial Growth in Vivo

Using Antimicrobial Agents