Chapter 7

The Control of Microbial Growth

Copyright 2010 Pearson Education, Inc.

Lectures prepared by Christine L. Case

The Terminology of Microbial Control

Learning Objective 7-1 Define the following key terms related to microbial control: sterilization, disinfection, antisepsis, degerming, sanitization, biocide, germicide, bacteriostasis, and asepsis.

Copyright 2010 Pearson Education, Inc.

The Terminology of Microbial Control

Sepsis refers to microbial contamination Asepsis is the absence of significant contamination Aseptic surgery techniques prevent microbial contamination of wounds

Copyright 2010 Pearson Education, Inc.

The Terminology of Microbial Control

Sterilization: Removing all microbial life Commercial sterilization: Killing C. botulinum endospores Disinfection: Removing pathogens Antisepsis: Removing pathogens from living tissue

Copyright 2010 Pearson Education, Inc.

The Terminology of Microbial Control

Degerming: Removing microbes from a limited area Sanitization: Lowering microbial counts on eating utensils Biocide/germicide: Kills microbes Bacteriostasis: Inhibiting, not killing, microbes

Copyright 2010 Pearson Education, Inc.

Check Your Understanding The usual definition of sterilization is the removal or destruction of all forms of microbial life; how could there be practical exceptions to this simple definition? 7-1

Copyright 2010 Pearson Education, Inc.

The Rate of Microbial Death

Learning Objective 7-2 Describe the patterns of microbial death caused by treatments with microbial control agents.

Copyright 2010 Pearson Education, Inc.

Population Death Rate Is Constant

Table 7.2
Copyright 2010 Pearson Education, Inc.

A Microbial Death Curve

Figure 7.1a
Copyright 2010 Pearson Education, Inc.

Effectiveness of Treatment
Depends on
Number of microbes Environment (organic matter, temperature, biofilms) Time of exposure Microbial characteristics

Copyright 2010 Pearson Education, Inc.

A Microbial Death Curve

Figure 7.1b
Copyright 2010 Pearson Education, Inc.

Check Your Understanding How is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria? 7-2

Copyright 2010 Pearson Education, Inc.

Actions of Microbial Control Agents

Learning Objective 7-3 Describe the effects of microbial control agents on cellular structures.

Copyright 2010 Pearson Education, Inc.

Actions of Microbial Control Agents

Alteration of membrane permeability Damage to proteins Damage to nucleic acids

Copyright 2010 Pearson Education, Inc.

Check Your Understanding Would a chemical microbial control agent that affects plasma membranes affect humans? 7-3

Copyright 2010 Pearson Education, Inc.

Physical Methods of Microbial Control

Learning Objectives 7-4 Compare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat. 7-5 Describe how filtration, low temperatures, high pressure, desiccation, and osmotic pressure suppress microbial growth. 7-6 Explain how radiation kills cells.

Copyright 2010 Pearson Education, Inc.

Heat
Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min Thermal death time (TDT): Time during which all cells in a culture are killed

Copyright 2010 Pearson Education, Inc.

Decimal Reduction Time (DRT)

Minutes to kill 90% of a population at a given temperature

Table 7.2
Copyright 2010 Pearson Education, Inc.

A Microbial Death Curve

Figure 7.1b
Copyright 2010 Pearson Education, Inc.

Moist Heat Sterilization

Moist heat denatures proteins Autoclave: Steam under pressure

Copyright 2010 Pearson Education, Inc.

An Autoclave

Figure 7.2
Copyright 2010 Pearson Education, Inc.

Steam Sterilization
Steam must contact items surface

Figure 7.3
Copyright 2010 Pearson Education, Inc.

Pasteurization
Reduces spoilage organisms and pathogens Equivalent treatments
63C for 30 min High-temperature short-time: 72C for 15 sec Ultra-high-temperature: 140C for <1 sec Thermoduric organisms survive

Copyright 2010 Pearson Education, Inc.

Dry Heat Sterilization

Kills by oxidation
Dry heat Flaming Incineration Hot-air sterilization

Hot-Air Equivalent Treatments 170C, 2 hr

Autoclave 121C, 15 min

Copyright 2010 Pearson Education, Inc.

Filtration
HEPA removes microbes >0.3 m Membrane filtration removes microbes >0.22 m

Figure 7.4
Copyright 2010 Pearson Education, Inc.

Q&A
In what situation is filtration the only practical way to eliminate undesirable microbes?

Copyright 2010 Pearson Education, Inc.

Physical Methods of Microbial Control

Low temperature inhibits microbial growth
Refrigeration Deep-freezing Lyophilization

High pressure denatures proteins Desiccation prevents metabolism Osmotic pressure causes plasmolysis

Copyright 2010 Pearson Education, Inc.

Radiation

Figure 7.5
Copyright 2010 Pearson Education, Inc.

Radiation
Ionizing radiation (X rays, gamma rays, electron beams)
Ionizes water to release OH Damages DNA

Nonionizing radiation (UV, 260 nm)

Damages DNA

Microwaves kill by heat; not especially antimicrobial

Copyright 2010 Pearson Education, Inc.

Check Your Understanding

How is microbial growth in canned foods prevented? 7-4 Why would a can of pork take longer to sterilize at a given temperature than a can of soup that also contained pieces of pork? 7-5 What is the connection between the killing effect of radiation and hydroxyl radical forms of oxygen? 7-6

Copyright 2010 Pearson Education, Inc.

Chemical Methods of Microbial Control

Learning Objectives 7-7 List the factors related to effective disinfection. 7-8 Interpret the results of use-dilution tests and the disk-diffusion method.

Copyright 2010 Pearson Education, Inc.

Principles of Effective Disinfection

Concentration of disinfectant Organic matter pH Time

Copyright 2010 Pearson Education, Inc.

Use-Dilution Test
Metal rings dipped in test bacteria are dried Dried cultures are placed in disinfectant for 10 min at 20C Rings are transferred to culture media to determine whether bacteria survived treatment

Copyright 2010 Pearson Education, Inc.

Disk-Diffusion Method

Figure 7.6
Copyright 2010 Pearson Education, Inc.

Clinical Focus
Which preparation is more effective?

Clinical Focus, p. 201

Copyright 2010 Pearson Education, Inc.

Check Your Understanding

If you wanted to disinfect a surface contaminated by vomit and a surface contaminated by a sneeze, why would your choice of disinfectant make a difference? 7-7 Which is more likely to be used in a medical clinic laboratory, a use-dilution test or a disk-diffusion test? 7-8

Copyright 2010 Pearson Education, Inc.

Chemical Methods of Microbial Control

Learning Objectives
7-9 7-10 Identify the methods of action and preferred uses of chemical disinfectants. Differentiate halogens used as antiseptics from halogens used as disinfectants. Identify the appropriate uses for surface-active agents. List the advantages of glutaraldehyde over other chemical disinfectants. Identify chemical sterilizers.

7-11 7-12
7-13

Copyright 2010 Pearson Education, Inc.

Phenol & Phenolics

Disrupt plasma membranes

Figure 7.7a, b
Copyright 2010 Pearson Education, Inc.

Bisphenols
Hexacholorphene, triclosan
Disrupt plasma membranes

Figure 7.7c, d
Copyright 2010 Pearson Education, Inc.

Biguanides
Chlorhexidine
Disrupt plasma membranes

Copyright 2010 Pearson Education, Inc.

Halogens
Iodine
Tinctures: In aqueous alcohol Iodophors: In organic molecules Alter protein synthesis and membranes

Chlorine
Bleach: Hypochlorous acid (HOCl) Chloramine: Chlorine + ammonia Oxidizing agents

Copyright 2010 Pearson Education, Inc.

Alcohols
Ethanol, isopropanol
Denature proteins, dissolve lipids Require water

Table 7.6
Copyright 2010 Pearson Education, Inc.

Heavy Metals
Ag, Hg, and Cu
Silver nitrate may be used to prevent gonorrheal ophthalmia neonatorum Silver sulfadiazine used as a topical cream on burns Copper sulfate is an algicide

Oligodynamic action
Denature proteins

Copyright 2010 Pearson Education, Inc.

Surface-Active Agents, or Surfactants

Soap
Acid-anionic detergents Quarternary ammonium compounds (cationic detergents)

Degerming
Sanitizing

Bactericidal, denature proteins, disrupt plasma membrane

Copyright 2010 Pearson Education, Inc.

Chemical Food Preservatives

Organic acids
Inhibit metabolism Sorbic acid, benzoic acid, and calcium propionate Control molds and bacteria in foods and cosmetics

Nitrite prevents endospore germination Antibiotics

Nisin and natamycin prevent spoilage of cheese

Copyright 2010 Pearson Education, Inc.

Aldehydes
Inactivate proteins by cross-linking with functional groups (NH2, OH, COOH, SH) Use: Medical equipment
Glutaraldehyde, formaldehyde, and ortho-phthalaldehyde

Copyright 2010 Pearson Education, Inc.

Gaseous Sterilants
Denature proteins Use: Heat-sensitive material
Ethylene oxide

Copyright 2010 Pearson Education, Inc.

Plasma
Free radicals destroy microbes Use: Tubular instruments

Copyright 2010 Pearson Education, Inc.

Supercritical Fluids
CO2 with gaseous and liquid properties Use: Medical implants

Copyright 2010 Pearson Education, Inc.

Peroxygens
Oxidizing agents Use: Contaminated surfaces
O3, H2O2, peracetic acid

Copyright 2010 Pearson Education, Inc.

Check Your Understanding Why is alcohol effective against some viruses and not others? 7-9 Is Betadine an antiseptic or a disinfectant when it is used on skin? 7-10 What characteristics make surface-active agents attractive to the dairy industry? 7-11 What chemical disinfectants can be considered sporicides? 7-12 What chemicals are used to sterilize? 7-13

Copyright 2010 Pearson Education, Inc.

Microbial Characteristics and Microbial Control

Learning Objective 7-14 Explain how the type of microbe affects the control of microbial growth.

Copyright 2010 Pearson Education, Inc.

Microbial Characteristics

Figure 7.11
Copyright 2010 Pearson Education, Inc.

Endospores and Mycobacteria

Table 7.7
Copyright 2010 Pearson Education, Inc.

Check Your Understanding

The presence or absence of endospores has an obvious effect on microbial control, but why are gram-negative bacteria more resistant to chemical biocides than grampositive bacteria? 7-14

Copyright 2010 Pearson Education, Inc.