Chapter 4 Dynamics of Prokaryotic Growth

Overview

This chapter describes how bacteria are cultivated in the laboratory. Bacterial growth is defined and

described. Methods to detect and measure bacterial growth are presented. Environmental factors that

influence microbial growth are identified and related to specific groups of bacteria. Nutritional factors

are presented. The growth curve and its component phases are discussed. The interactions of mixed

microbial communities in natural environments and biofilms are discussed.

Learning Objectives

After studying the material in this chapter, you should be able to:

1. Define pure culture and explain its significance.

2. Define colony.

3. Describe the streak plate method for the isolation of bacteria.

4. Define binary fission.

5. Explain generation or doubling time.

6. Describe how direct cell counts are done.

7. Describe how viable cell counts are done.

8. Describe the most probable number (MPN) method.

9. Explain how biomass is measured.

10. Explain how cell products can be used to measure bacterial growth.

11. List and describe the chemical and physical conditions necessary for bacterial growth.

12. Classify bacteria on the basis of temperature preference and tolerance.

13. Classify bacteria on the basis of oxygen utilization and tolerance.

14. Describe the roles of superoxide dismutase and catalase in oxygen utilization and/or tolerance.

15. Give the categories based on the oxygen requirements of bacteria that have

• Superoxide dismutase

• Catalase

16. List the nutritional factors that influence microbial growth.

17. Define growth factors.

18. List and describe the four nutritional groups of organisms based on energy source and carbon

sources. Indicate what kind of organisms are in each group.

19. Explain how bacterial growth requirements are provided in laboratory cultures.

20. List the basic types of media used in the bacteriological laboratory.

21. Differentiate between complex and chemically defined media.

22. Define, describe and give examples of the following kinds of media:

• Selective media

- • Differential media

23. Describe the use of the following equipment in the bacteriological laboratory:

• Candle jar

• Carbon dioxide incubator

• Anaerobe jar

• Anaerobe incubator

24. Describe logarithmic growth.

25. Draw and describe the bacterial growth curve identifying the events occurring in each section.

26. Explain how a bacterial culture can be sustained for a long period of time of time.

27. Define biofilm and explain its importance.

Key Concepts

1. Pure culture is a population of cells descended from a single cell.

2. Colony is a mass of bacterial cells that originated from one cell.

3. Isolation of bacteria refers to methods by which bacteria can be separated into pure cultures.

4. Binary fission is the type of asexual reproduction by which a bacterial cell splits and forms two

daughter cells.

5. Generation or doubling time is the amount of time that is necessary for a population of bacteria to

undergo one round of binary fission thereby doubling the number of bacteria present.

6. Measurement of microbial growth can be accomplished by direct microscopic counts, cell

counting instruments, plate counts, membrane filtration, most probable number method, turbidity,

total weight of a culture and the presence of certain cell constituents.

7. Spectrophotometers, Coulter counters and flow cytometers are instruments that can be used to

measure microbial growth.

8. Bacteria have both physical and chemical requirements for growth, which may include

temperature, oxygen levels, pH, water, required elements, organic growth factors and carbon and

energy sources.

9. Microorganisms can be separated into groups based on temperature, oxygen levels and pH

requirements.

10. Organisms can be separated into four groups based on their carbon and energy sources.

11. Growth in the laboratory requires that the needs of the bacteria be provided for in media.

12. Media is the material that provides nutrition for bacterial growth in culture.

13. In a closed system a population of bacterial will follow growth curve in which there is a lag

phase, a logarithmic growth phase followed by a stationary phase and a logarithmic decline

phase.

14. Bacteria may be kept in a continuous growth stage of growth by supplying nutrients using a

chemostat.

15. Bacterial growth in natural environments is generally more dynamic than under artificial

conditions resembling that of a continuous culture.

16. Biofilms are communities of bacteria with characteristic structure containing open channels

through which nutrients and waste products may pass.

Summary Outline

I. Obtaining a pure culture

A. About one-tenth of one percent of bacteria can be cultured in the laboratory.

B. Cultivating bacteria on a solid medium

1. A single bacterial cell will multiply to form a visible colony.

2. Agar is used to solidify nutrient-containing broth.

C. The streak plate method is used to isolate bacteria in order to obtain a pure culture.

D. Maintaining stock cultures

1. Stock cultures can be used as an inoculum in later experiments.

2. Stock cultures can be stored on an agar slant in the refrigerator, frozen in a glycerol solution or lyophilized.

II. Principles of bacterial growth

A. Most bacteria multiply by binary fission.

B. Microbial growth is an increase in the number of cells in a population.

C. The time required for a population to double in number is the generation time.

III. Methods to detect and measure bacterial growth

A. Direct cell counts generally do not distinguish between living and dead cells.

1. Direct microscopic count

2. The Coulter counter and a flow cytometer count cells as they pass through a

minute aperture.

B. Viable cell counts

1. Plate counts are based on the fact that an isolated cell will form a single colony.

2. Membrane filtration concentrates bacteria by filtration.

3. The most probable number (MPN) method is a statistical assay based on the

theory of probability and is used to estimate cell numbers.

C. Measuring biomass

1. Turbidity of a culture is a rapid measurement that can be correlated to the number

of cells; a spectrophotometer is used to measure turbidity.

2. Wet weight and dry weight are proportional to the number of cells in a culture.

3. The quantity of a cell constituent such as nitrogen can be used to calculate

biomass.

D. Measuring cell products

1. pH indicators can be used to monitor acid production.

2. Gas production can be detected by pH changes or by using an inverted tube in

culture media to trap gas.

3. ATP is detected by employing luciferase.

IV. Environmental factors that influence microbial growth

A. Temperature requirements

1. Psychrophiles have an optimum between -5°C and 20°C.

2. Mesophiles have an optimum between 20°C and 45°C.

3. Thermophiles have an optimum between 45°C and 70°C.

4. Hyperthermophiles have an optimum between 70°C and 110°C.

5. Storage of foods at refrigeration temperatures retards spoilage because it limits the

growth of mesophiles.

6. Some microorganisms can inhabit certain parts of the body but not others because

of temperature differences.

B. Oxygen requirements

1. Obligate anaerobes cannot multiply if oxygen is present.

2. Facultative anaerobes can multiply if oxygen is present but can also grow without

it.

3. Microaerophiles require small amounts of oxygen but higher concentrations are

inhibitory.

4. Aerotolerant anaerobes are indifferent to oxygen.

5. Oxygen can be converted to superoxide and hydrogen peroxide, both of which

are toxic. Superoxide dismutase and catalase can break these down.

C. pH

1. Most bacteria live within the pH range of 5 to 8.

2. Acidophiles grow optimally at a pH below 5.5.

3. Alkaliphiles grow optimally at a pH above 8.5.

D. Water availability

1. All microorganisms require water for growth.

2. If the solute concentration is higher in the medium than in the cell, water diffases

out of the cell, causing plasmolysis.

3. Halophiles have adapted to live in high salt environments.

V. Nutritional factors that influence microbial growth

A. Required elements

1. The major elements make up cell constituents and include carbon, nitrogen,

sulfur and phosphorus.

2. Heterotrophs use organic carbon.

3. Autotrophs fix 002.

4. Trace elements are required in very minute amounts.

B. Growth factors are cell constituents such as amino acids and vitamins that the cell

cannot synthesize.

C. Nutritional diversity

1. Prokaryotes use diverse sources of carbon and energy.

2. Photoautotrophs use the energy of sunlight and the carbon in the atmosphere to

make organic compounds.

3. Chemolithoautotrophs use inorganic compounds for energy and derive their

carbon from 002.

4. Photoheterotrophs use the energy of sunlight and derive their carbon from organic

compounds.

5. Chemoorganoheterotrophs use organic compounds for energy and as a carbon source.

VI. Cultivating prokaryotes in the laboratory

A. General categories of culture media

1. Complex medium contains a variety of ingredients such as peptones and extracts.

(Examples: nutrient agar, blood agar and chocolate agar)

2. A chemically defined medium is composed of precise mixtures of pure chemicals;

an example is glucose-salts medium.

B. Special types of culture media

1. A selective medium inhibits organisms other than the one being sought

(Examples: Thayer Martin agar and MacConkey agar)

2. A differential medium contains a substance that certain bacteria change in a

recognizable way (Examples: Blood agar and MacConkey agar)

C. Providing appropriate atmospheric conditions

1. A candle jar provides increased 002, which enhances the growth of many

medically important bacteria.

2. Microaerophilic bacteria are incubated in a gas-tight jar along with atmospheric

oxygen to form water.

3. Anaerobes may be cultivated in either an anaerobe jar or a medium that

incorporates a reducing agent.

4. An enclosed chamber that maintains anaerobic conditions can also be used.

D. Enrichment cultures provide conditions in a broth that enhance the growth of one

particular organism in a mixed population.

VII. Growth characteristics of bacteria in the laboratory

A. Bacterial growth follows a growth curve when they are grown in a closed system.

1. Lag—number of cells does not increase

2. Log—cells divide at a constant rate

3. Stationary—a required nutrient is used up, oxygen is in short supply, or toxic

metabolites accumulate

4. Death—number of viable cells in the population decreases

B. Colony growth: The position of a single cell within a colony markedly determines its

environment; cells on the edge may be in log phase whereas those in the center may be in

the death phase.

C. Continuous cultures: Bacteria can be maintained in a state of continuous exponential

growth by using a chemostat.

VIII. Growth characteristics of bacteria in nature

A. Mixed populations: Bacteria often grow in close associations with other kinds of

organisms; the metabolic activities of one organism may facilitate the growth of another

organism.

B. Bioflims: Bacteria may live suspended in an aqueous environment but many attach to

surfaces and live as a biofilm, a polysaccharide-encased community.

Terms You Should Know

Aerotolerant anaerobes

Agar

Alpha-hemolysis

Aseptic techniques

Autotrophs

Beta-hemolysis

Binary fission

Biofilm

Blood agar

Capnophiles

Carbon fixation

Catalase

Chemically defined media

Chemoautotrophs

Chemoheterotrophs

Chemotrophs

Chocolate agar

Complex media

Death or decline phase

Differential media

Durham tube

Enrichment culture

Exponential or log phase

Facultative anaerobes

Growth curve

Growth factors

Hemolysin

Heterotrophs

Hyperthermophiles

Lag phase

Lyophilization

MacConkey agar

Mesophiles

Microaerophiles

Nitrogen fixation

Nutrient agar

Nutrient broth

Obligate aerobes

Obligate anaerobes

Petri dish

pH

pH indicator

Photoautotrophs

Photoheterotrophs

Phototrophs

Plasmolysis

Psychrophiles

Pure culture

Selective media

Stationary phase

Sterile

Superoxide

Superoxide dismutase

Thayer-Martin agar

Thermophiles

Trace elements

Turbidity

Chapter 5 Control of Microbial Growth

Overview

Control of microbial growth is essential to preventing infections, limiting the spread of disease and

preserving foodstuffs and goods. In this chapter the physical and chemical methods for the control of

microbial growth are presented. Situational considerations as well as the various techniques are

discussed. Characteristics of specific chemicals are presented. Selection and applications of the

appropriate physical or chemical methods based on the type of microorganism, the numbers of

microorganisms present, environmental conditions and potential risk of infection are discussed.

Learning Objectives

After studying the material in this chapter, you should be able to:

1. List the conditions that influence the selection of a particular antimicrobial procedure.

2. List and describe the physical methods of microbial control and give their applications.

3. Describe the general action ofmicrobial agents.

4. Differentiate between antiseptics and disinfectants.

5. Describe the factors that should be considered in selection of an appropriate antimicrobial chemical.

6. List and describe the chemical methods ofmicrobial control (antiseptics/disinfectants) and give

their applications.

7. Describe how food products and other products can be preserved by preventing the growth of

microbes.

Key Concepts

1. Sterilization is the process by which all microorganisms are killed.

2. Disinfection is the process in which the number of microbes is reduced to a level where they are no

longer a problem.

3. Both physical and chemical methods can be used to sterilize or disinfect.

4. Physical methods of control include both moist and dry heat treatment, irradiation, filtration and

mechanical removal.

5. Methods of control used depend on the situation and the degree of control required.

6. The type of microbe, the numbers present, environmental conditions and the potential risk of

infection must be considered in selecting the appropriate method of sterilization or disinfection.

7. Moist heat such as boiling destroys vegetative bacterial cells and many viruses.

8. Pasteurization does not kill all microorganisms present, but significantly reduces the numbers of

heat-sensitive organisms.

9. Autoclaves use live steam under pressure to microbes, viruses and endospores.

10. Dry heat also kills microorganisms, but requires a significantly greater length of time.

11. The canning process is specifically designed to destroy the endospores of Clostridium botulinum.

12. Antimicrobial chemicals, which can be used to disinfect and, under some circumstances, sterilize, are less reliable than heat.

13. Bacterial endospores of Bacillus and Clostridium, Mycobacterium species, Pseudomonas species

and naked viruses are resistant to antimicrobial treatment.

14. Microorganisms and viruses can be removed from liquids and air by filtration.

15. Gamma irradiation can be used to sterilize materials and to decrease the number of microorganisms

in foods. Ultraviolet light is not very penetrating, but can be used to disinfect surfaces.

16. Preservation of foodstuffs to delay spoilage may be accomplished by slowing or stopping the

growth of the microbes.

Summary Outtine

I. The methods used to destroy or remove microorganisms and viruses can be:

A. Physical such as heat treatment, irradiation and filtration or Chemical

B. Principles of control

1. Sterilization destroys all microorganisms and viruses.

2. Disinfection eliminates most disease-causing bacteria or viruses.

3. Disinfectants are chemicals used for disinfecting inanimate objects

4. Antiseptics are chemicals formulated for use on skin.

5. Pasteurization uses heat treatment to reduce the number of spoilage organisms or kill

disease-causing microbes.

C. Situational considerations

1. Hospitals must be scrupulous in controlling microorganisms because of the danger of

nosocomial infections.

2. Microbiology laboratories must use aseptic technique to avoid contaminating cultures

with extraneous microbes and to protect workers and the environment from

contamination.

3. Foods and other perishable products retain their quality and safety when the growth ol

contaminating microorganisms is prevented.

II. Selection of an antimicrobial procedure

A. Type of microorganism

1. Type of microbial population present.

2. The endospores of Bacillus and Clostridium are most resistant.

3. The waxy cell wall of mycobacteria makes them resistant.

4. Pseudomonas are common environmental organisms are very resistant.

5. Viruses that lack a lipid envelope are more resistant to disinfectants than are

enveloped viruses.

B. Numbers of microorganisms initially present

C. Environmental conditions affect death rate of microorganisms

1. pH

2. Presence of fats and other organic compounds

D. Potential risk of infection

III. Using heat to destroy microorganisms and viruses

A. Moist heat—Moist heat, such as boiling water and pressurized steam, destroys

microorganisms by causing the irreversible coagulation of their proteins.

B. Dry heat—Dry heat, such as in direct flaming and ovens destroy microorganisms by

oxidizing cells to ashes or irreversibly denaturing their proteins.

IV. Using chemicals to destroy microorganisms and viruses

A. Germicidal chemicals can be used to disinfect and, in some cases, sterilize, but they are

less reliable than heat. Most chemical germicides react irreversibly with vital enzymes and

other proteins, the cytoplasmic membrane, or viral envelopes.

B. Potency ofgermicidal chemical formulations

1. Sterilants

2. High-level disinfectants

3. Intermediate-level disinfectants

4. Low-level disinfectants

C. Selection factors for the appropriate germicidal chemical

1. Toxicity

2. Residue

3. Activity in the presence of organic matter

4. Compatibility with the material being treated

5. Cost and availability

6. Storage and stability

7. Ease of disposal

D. Classes of germicidal chemicals

1. Ethyl or isopropyl alcohol (60-80% solution) in water rapidly kills vegetative

bacteria and fungi by coagulating enzymes and other essential proteins, and by

damaging lipid membranes.

2. Gluteraldehyde and formaldehyde destroy microorganisms and viruses by

inactivating proteins and nucleic acids. A 20% solution of alkaline gluteraldehyde is

one of the most widely used chemical sterilants.

3. Chlorhexidine is a biguanide extensively used in antiseptic products.

4. Ethylene oxide is a gaseous sterilizing agent that penetrates well and destroys

microorganisms and viruses by reacting with proteins.

5. Sodium hypochlorite (liquid bleach) is one of the least expensive and most readily

available forms of chlorine. Chlorine dioxide is used as a sterilant and disinfectant.

lodophores are iodine-releasing compounds used as antiseptics.

6. Metals interfere with protein function. Silver-containing compounds are used to

prevent wound infections.

7. Ozone is used as an alternative to chlorine disinfection of drinking water and

wastewater.

8. Peroxide and peracetic acid are both strong oxidizing agents that can be used alone

or in combination as sterilants.

9. Phenolics destroy cytoplasmic membranes and denature proteins.

10. Quaternary ammonium compounds are cationic detergents that are non-toxic

enough to be used to disinfect food preparation surfaces.

V. Removal of microorganisms by filtration

A. Filtration of fluids

1. Depth filters have complex, tortuous passages that retain microorganisms while

letting the suspending fluid pass through the small holes.

2. Membrane filters are produced With graded pore sizes extending below the

dimensions of the smallest known viruses.

B. Filtration of air

1. High efficiency particulate air (HEPA) filters remove nearly all microorganisms.

2. HEPA filters are used in specialized hospital rooms to protect patients, biological

safety cabinets and laminar flow hoods.

VI. Using radiation to destroy microorganisms and viruses

A. Gamma irradiation cause biological damage by producing superoxide and hydroxyl free

radicals. Irradiation can be used to:

1. Sterilize heat-sensitive materials

2. Decrease the numbers of microorganisms in foods.

B. Ultraviolet light is used to disinfect surfaces by damaging nucleic acids by causing the formation ofcovalent bonds between adjacent thymine molecules in DNA, creating thymine dimers.

C. Microwaves do not effect microorganisms directly but they can kill microorganisms by the heat they generate in a product.

VII. Preservation of perishable products by techniques that slow or halt the growth of microorganisms to delay spoilage.

A. Chemical preservatives

1. Organic acids such as benzoic, sorbic and propionic acids

2. Nitrate and nitrite

B. Low temperature storage

1. Low temperatures above freezing inhibit microbial growth.

2. Freezing essentially stops all microbial growth.

C. Reducing the available water by addition of sugars and salts

D. Lyophilization is used for preserving food

Terms You Should Know

Antiseptic

Aseptic technique

Autoclave

Bactericidal

Bacteriostatic

Decimal reduction time

Decontamination

Disinfectant

Disinfection

Fungicide

Germicide

HEPA filter

Laminar flow hoods

Lyophilization

Normal flora

Nosocomial infections

Pasteurization

Preservation

Sanitize

Sterile

Sterilization

Viricide