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For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark Chapter 26
Microbial Growth
Lectures by
John Zamora
Middle Tennessee State University
2012 Pearson Education, Inc.
Microbial Growth Control • Sterilization – The killing or removal of all viable organisms within a growth medium • Inhibition – Effectively limiting microbial growth – The treatment of an object to make it safe to • Disinfection – Directly targets the removal of all pathogens, not necessarily all microorganisms 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Microbial Growth Control • Disinfectant – Specialized chemical or physical agents called disinfectants can kill microorganisms or inhibit microbial growth. – e.g. chlorine gas and sodium hypochlorite solution are used to disinfect drinking water.
2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
I. Physical Antimicrobial Control • 26.1 Heat Sterilization• 26.2 Radiation Sterilization• 26.3 Filter Sterilization 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

26.1 Heat Sterilization • Heat sterilization is the most widely used method of controlling microbial growth (Figure 26.1) – High temperatures denature macromolecules– Amount of time required to reduce viability tenfold is called the decimal reduction time (Figure 26.2) • Some bacteria produce resistant cells called – Can survive heat that would rapidly kill vegetative 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 26.1 The effect of temperature over time on the viability of a mesophilic bacterium
Time (min)
The decimal reduction time, D, is the time at which only 10% of the original population of organisms remains viable at a given temperature. For 70oC, D = 3 min; for 60oC, D = 12 min; for 50oC, D = 42 min.
2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

Figure 26.2 The relationship between temperature and the rate of killing in mesophiles and thermophiles
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ni
m( emit noi
Data were obtained for decimal reduction times, D, at several different temperatures. For organism A, a typical mesophile, exposure to 110oC for less than 20 sec resulted in a decimal reduction, while for organism B, a thermophile, 10 min was required to achieve a decimal reduction.
2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
26.1 Heat Sterilization • The autoclave is a sealed device that uses steam under pressure (Figure 26.3) – Allows temperature of water to get above 100C– Not the pressure that kills things, but the high • Pasteurization is the process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids – Does not kill all organisms, so it is different than 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

Figure 26.3 The autoclave and moist heat sterilization
panel. The steam inlet and exhaust fittings are on the right and valve
Air exits through vent
Steam enters here
The flow of steam through an autoclave A modern research autoclave The temperature of the object rises and falls more slowly than the temperature of the temperature of the object must reach the target temperature and be held for 10–15 minutes to ensure of object being of autoclave
sterilized
sterility, regardless of the temperature and time recorded in the Total cycle time (min)
A typical autoclave cycle 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
26.2 Radiation Sterilization • Microwaves, UV, X-rays, gamma rays, and electrons can reduce microbial growth • UV has sufficient energy to cause modifications and breaks in DNA – UV is useful for decontamination of surfaces – Cannot penetrate solid, opaque, or light-absorbing 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

Figure 26.4 A laminar flow hood
An UV light source prevents contamination of the hood when it is not in use. When in use, air is drawn into the cabinet through a HEPA filter. The filtered air inside the cabinet is exhausted out of the cabinet, preventing contamination of the inside of the hood. The cabinet provides a contaminant-free workspace for microbial and tissue culture manipulations.
2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
26.2 Radiation Sterilization • Ionizing radiation – Electromagnetic radiation that produce ions and other reactive molecules – Generates electrons, hydroxyl radicals, and – Some microorganisms are more resistant to radiation than others • Amount of energy required to reduce viability tenfold is analogous to D value (Figure 26.5) 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

Figure 26.5 Relationship between the survival fraction and the radiation dose of a microorganism
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elac sgol( noit D10 10% survival
The D10, or decimal reduction dose, can be interpolated from the data as shown.
2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
26.2 Radiation Sterilization • Sources of radiation include cathode ray tubes, X-rays, and radioactive nuclides • Radiation is used for sterilization in the medical field and food industry – Radiation is approved by the WHO and is used in the USA for decontamination of foods particularly susceptible to microbial contamination • Hamburger, chicken, spices may all be irradiated 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

26.3 Filter Sterilization • Filtration avoids the use of heat on sensitive liquids and gases – Pores of filter are too small for organisms to pass – Pores allow liquid or gas to pass through • Depth filters – HEPA filters (Figure 26.6a) • Membrane filters – Function more like a sieve (Figure 26.6b and c) 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 26.6 Microbiological filters
Scanning electron micrograph showing the structure of (a) a depth filter, (b) a conventional membrane filter, and (c) a nucleopore filter 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI

26.3 Filter Sterilization • Membrane filters (cont'd) – Filtration can be accomplished by syringe, pump, or vacuum (Figure 26.7) – A type of membrane filter is the nucleation track (nucleopore) filter (Figure 26.8) 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 26.7 Membrane filters
Disposable, presterilized, and assembled membrane filter units. Left: a filter system designed for small volumes. Right: a filter system designed for larger volumes 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 26.8 Scanning electron micrographs of bacteria trapped on nucleopore membrane filters
(a) Aquatic bacteria and algae. The poresize is 5 µm. (b) Leptospira interrogans. The bacterium is about 0.1 µm in diameter and up to 20 µm in length. The pore size of the filter is 0.2 µm.
2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
II. Chemical Antimicrobial Control • 26.4 Chemical Growth Control• 26.5 Chemical Antimicrobial Agents for 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
26.4 Chemical Growth Control • Antimicrobial agents can be classified as bacteriostatic, bacteriocidal, and bacteriolytic(Figure 26.9) 2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 26.9a Bacteriostatic, bacteriocidal, and bacteriolytic antimicrobial agents
• Bacteriostatic agents Total cell count
are frequently inhibitors of protein synthesis and act by binding to Viable cell count
An antimicrobial • If the concentration of agent is added to the agent is lowered, growing culture. the agent is released from the ribosome and The turbidity of each culture, coupled with viable • Many antibiotics work plate counts, establishes the relationship by this mechanism.
between viable and total cell counts.
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Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 26.9b Bacteriostatic, bacteriocidal, and bacteriolytic antimicrobial agents
• Bacteriocidal agents Total cell count
bind tightly to their cellular targets, are not removed by dilution, and kill the cell. An antimicrobial cell count
• The dead cells, agent is added to growing culture. destroyed, and total cell numbers, reflected by the turbidity of the The turbidity of each culture, coupled with viable plate counts, establishes the relationship between viable and total cell counts.
2012 Pearson Education, Inc.
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Figure 26.9c Bacteriostatic, bacteriocidal, and bacteriolytic antimicrobial agents
• Some -cidal agents are also -lytic agents, killing by cell lysis and release of cytoplasmic Total cell count
An antimicrobial • Lysis decreases the agent is added to cell count
viable cell number and growing culture. also the total cell number, shown by a decrease in culture The turbidity of each culture, coupled with viable plate counts, establishes the relationshipbetween viable and total cell counts.
• Bacteriolytic agents include antibiotics that inhibit cell wall synthesis, such as penicillin, and chemicals such as detergents that rupture the cytoplasmic membrane.
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26.4 Chemical Growth Control • Minimum inhibitory concentration (MIC) is the smallest amount of an agent needed to inhibit growth of a microorganism (Figure 26.10) – Varies with the organism used, inoculum size, temp, pH, etc.
• Disc diffusion assay – Antimicrobial agent added to filter paper disc– MIC is reached at some distance • Zone of inhibition – Area of no growth around disc 2012 Pearson Education, Inc.
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Figure 26.10 Antimicrobial agent susceptibility assay using dilution methods
The assay defines the minimum inhibitory concentration (MIC). A series of increasing concentrations of antimicrobial agent is prepared in the culture medium. Each tube is inoculated with a specific concentration of a test organism, followed by a defined incubation period. Growth, measured as turbidity, occurs in those tubes with antimicrobial agent concentrations below the MIC.
2012 Pearson Education, Inc.
Marmara University – Enve303 Env. Eng. Microbiology – Prof. BARIŞ ÇALLI
Figure 26.11 Antimicrobial agent susceptibility assay using diffusion methods
agar plate
with a liquid
culture of a test
organism
Discs containing
antimicrobial
agents are placed
on surface
Incubate for 24–48 h
Test organism shows
The antimicrobial agent
susceptibility to some
diffuses from disks into
agents, indicated by
the surrounding agar,
inhibition of bacterial
inhibiting growth of test
growth around discs
(zones of inhibition)
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26.5 Chemical Antimicrobial Agents for • These antimicrobial agents can be divided into two – Products used to control microorganisms in commercial and industrial applications • Examples: chemicals in foods, air-conditioning cooling towers, textile and paper products, fuel tanks – Products designed to prevent growth of human pathogens in inanimate environments and on external body surfaces • Sterilants, disinfectants, sanitizers, and antiseptics 2012 Pearson Education, Inc.
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26.5 Chemical Antimicrobial Agents for • Sterilants: Chemical sterilants, also called sterilizers or sporicides, destroy all forms of microbial life, including endospores. – Chemical sterilants are used in situations where it is impractical to use heat or radiation for decontamination or sterilization.
– Liquid sterilants such as a sodium hypochlorite solution or amylphenol are used for instruments that cannot withstand high temperatures or gas.
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26.5 Chemical Antimicrobial Agents for • Hospitals and laboratories, must be able to decontaminate and sterilize heat-sensitive materials, such as; – thermometers– lensed instruments– polyethylene tubing and – reusable medical equipment.
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26.5 Chemical Antimicrobial Agents for • Disinfectants: are chemicals that kill microorganisms, but not necessarily endosporesand are used on inanimate objects. – Disinfectants such as ethanol and cationic detergents are used to disinfect floors, tables, bench tops, walls, etc. and important for infection control in hospitals. – General disinfectants are used in households, swimming pools and water purification systems 2012 Pearson Education, Inc.
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26.5 Chemical Antimicrobial Agents for • Sanitizers: are agents that reduce, but may not eliminate, microbial numbers to levels considered to be safe. – Food contact sanitizers are widely used in the food industry to treat surfaces such as mixing and cooking equipment, dishes and utensils.
– Non–food contact sanitizers are used to treat surfaces such as counters, floors, walls, carpets, and laundry.
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26.5 Chemical Antimicrobial Agents for • Antiseptics and germicides are chemical agents that kill or inhibit growth of microorganisms and that are nontoxic enough to be applied to living tissues. – Most of the compounds in this category are used for handwashing or for treating surface wounds.
– Certain antiseptics are also effective disinfectants; they are effective antimicrobial agents when applied to inanimate surfaces.
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Figure 2 Handwashing
Handwashing is the easiest and one of the most important interventions to prevent pathogen spread in healthcare, home and laboratory settings. This handwash station is in a clinical laboratory.
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26.5 Chemical Antimicrobial Agents for • Ethanol, is categorized as an antiseptic, but can also be a disinfectant. • This depends on the concentration of ethanol used and the exposure time, with disinfection generally requiring higher ethanol concentrationsand exposure times of several minutes.
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26.5 Chemical Antimicrobial Agents for • Antimicrobial Efficacy – Several factors affect the efficacy of chemical antimicrobial agents. – For example, many disinfectants are neutralized by organic material. – These materials reduce effective disinfectant concentrations and microbial killing capacity.
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26.5 Chemical Antimicrobial Agents for • Antimicrobial Efficacy (cont'd) – Pathogens are often encased in particles or grow in large numbers as biofilms, covering surfaces of tissue or medical devices with several layers of microbial cells.
– Biofilms may slow or even completely prevent penetration of antimicrobial agents, reducing or negating their effectiveness.
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26.5 Chemical Antimicrobial Agents for • Antimicrobial Efficacy (cont'd) – Only sterilants are effective against bacterial – Endospores are much more resistant to other agents than are vegetative cells because of their low water availability and reduced metabolism.
– Mycobacterium tuberculosis, the causal agent of tuberculosis, are resistant to disinfectants because of the waxy nature of their cell wall.
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26.5 Chemical Antimicrobial Agents for 2012 Pearson Education, Inc.
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26.5 Chemical Antimicrobial Agents for 2012 Pearson Education, Inc.
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III. Antimicrobial Agents Used In Vivo • 26.6 Synthetic Antimicrobial Drugs• 26.7 Naturally Occurring Antimicrobial Drugs: • 26.8 -Lactam Antibiotics: Penicillins and • 26.9 Antibiotics from Prokaryotes 2012 Pearson Education, Inc.
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III. Antimicrobial Agents Used In Vivo • Antimicrobial drugs are classified on the basis of – Molecular structure – Mechanism of action (Figure 26.12)– Spectrum of antimicrobial activity (Figure 26.13) 2012 Pearson Education, Inc.
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Figure 26.12 Mode of action of some major antimicrobial agents
Cell wall synthesis
DNA gyrase
DNA-directed RNA polymerase
Folic acid metabolism
structure and function
Cytoplasmic Cell wall
Agents are classified according to their target structures in the bacterial cell.
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26.6 Synthetic Antimicrobial Drugs • Paul Ehrlich studied selective toxicity in the early – Selective toxicity is ability to inhibit or kill a pathogen without affecting the host – Salvarsan – one of the first antimicrobial drugs • Growth factor analogs are structurally similar to growth factors but do not function in the cell – Analogs similar to vitamins, amino acids, and 2012 Pearson Education, Inc.
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Figure 26.14 Annual worldwide production and use of antibiotics
Each year an estimated 10,000 metric tons of antimicrobial agents are manufactured worldwide. The β-lactam antibiotics include cephahalosporins (30%), penicillins (7%), and other β-lactams (15%). "Others" includes tetracyclines, aminoglycosides, and all other antimicrobial drugs 2012 Pearson Education, Inc.
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26.7 Naturally Occurring Antimicrobial Drugs: Antibiotics • Antibiotics are naturally produced antimicrobial – Less than 1% of known antibiotics are clinically • Can be modified to enhance efficacy (semisynthetic) • The susceptibility of microbes to different antibiotics – Gram-positive and gram-negative bacteria vary in their sensitivity to antibiotics – Broad-spectrum antibiotics are effective against both groups of bacteria 2012 Pearson Education, Inc.
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26.8 -Lactam Antibiotics: Penicillins and Cephalosporins • -Lactam antibiotics are one of the most important groups of antibiotics of all time – Include penicillins, cephalosporins, and – Over half of all antibiotics used worldwide • Penicillins – Discovered by Alexander Fleming– Primarily effective against gram-positive bacteria– Some synthetic forms are effective against some gram-negative bacteria – Target cell wall synthesis 2012 Pearson Education, Inc.
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26.9 Antibiotics from Prokaryotes • Many antibiotics effective against Bacteria are also produced by Bacteria • These antibiotics include: – Aminoglycosides– Macrolides– Tetracyclines– Daptomycin– Platensimycin • They have major clinical applications 2012 Pearson Education, Inc.
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Source: http://mebig.marmara.edu.tr/Enve303/Chapter26.pdf