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Chapter 7
The Control of Microbial Growth
SLOs
Define sterilization, disinfection, antisepsis, sanitization, biocide, germicide, bacteriostasis, and asepsis.
Describe the microbial death curve.
Describe the effects of microbial control agents on cellular structures.
Compare effectiveness of moist heat (autoclaving, pasteurization) vs .dry heat.
Describe how filtration, low temperature, high pressure, desiccation, and osmotic pressure suppress microbial growth.
Explain how radiation kills cells.
List the factors related to effective disinfection.
Interpret results the disk-diffusion test.
Identify some methods of action and preferred uses of chemical disinfectant.
Differentiate between two halogens used as antiseptics and disinfectants.
List the advantage of glutaraldehyde and ethylene oxide over other chemical disinfectants.
Identify the method of sterilizing plastic labware.
Explain how microbial control is affected by the type of microbe.
How is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria?
Would a chemical microbial control agent that affected plasma membranes affect humans?
How is microbial growth in canned foods prevented?
What is the connection between the killing effect of radiation and hydroxyl radical forms of oxygen?
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?
Why is alcohol effective against some viruses and not others?
Is Betadine an antiseptic or a disinfectant when it is used on skin?
What chemicals are used to sterilize?
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 gram-positive bacteria?
SLOs cont.: Check Your Understanding
Terminology
Sepsis: microbial contamination.
Asepsis: absence of significant contamination.
Aseptic surgery techniques prevent microbial contamination of wounds.
Antimicrobial chemicals, expected to destroy pathogens but not to achieve sterilization
Disinfectant: used on objects
Antiseptic: used on living tissue
Nosocomial
see
. . . More Terminology
Sterilization: Removal of all microbial life (heat, filtration)
For food: Commercial sterilization to kill C. botulinum endospores
Sanitization: reduces microbial numbers to safe levels (e.g.: eating utensils)
Bacteriostatic: Inhibits bacterial reproduction
Bactericidal: Kills bacteria
Fungicide, sporicide, germicide, biocide
Single most effective measure!
Remember Semmelweis,
Pasteur, and Lister from
Ch 1
Rate of Microbial Death
Microbial Death Curve, plotted logarithmically, shows this constant death rate as a straight line.
Bacterial populations subjected to heat or antimicrobial chemicals die at a constant rate.
Rate: 90% / min
Foundation Fig 7.1
How is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria?
Foundation Fig 7.1 cont.
Effectiveness of Antimicrobial Treatment
Time it takes to kill a microbial population is to number of microbes.
Different microbial species and life cycle phases (e.g.:_____________) have different susceptibilities to physical and chemical controls.
Organic matter may interfere.
Temperature determines exposure time: Longer exposure to lower heat produces same effect as shorter time at higher heat.
Actions of Microbial Control Agents
Alteration of membrane permeability
Damage to proteins
Damage to nucleic acids
Check your understanding:
Would a chemical microbial agent that affects
plasma membranes affect humans?
Moist Heat Sterilization
___________proteins
Autoclave: Steam under pressure
Most dependable sterilization method
Steam must directly contact material to be sterilized.
Pressurized steam reaches higher temperatures.
Normal autoclave conditions: _____C for ___ min.
Prion destruction: 132C for 4.5 hours
Limitations of the autoclave Fig 7.2
Physical Methods of Microbial Control
Pasteurization
Significant number reduction (esp. spoilage and pathogenic organisms) does not sterilize!
Historical goal: destruction of M. tuberculosis
Classic holding method: 63C for 30 min
Flash pasteurization (HTST): 72C for 15 sec. Most common in US. Thermoduric organisms survive
Ultra High Temperature (UHT): 140C for < 1 sec. Technically not pasteurization because it sterilizes.
Hot-air Autoclave
Equivalent treatments
170˚C, 2 hr 121˚C, 15 min
Dry Heat Sterilization Kills by Oxidation
Flaming of loop
Incineration of carcasses Anthrax
Foot and mouth disease
Bird flu
Hot-air sterilization
Filtration
Air filtration using high efficiency particulate air (HEPA) filters. Effective to 0.3 m
Membrane filters for fluids.
Pore size for bacteria: 0.2 – 0.4 m
Pore size for viruses: 0.01 m
Compare to Fig 7.4
Low Temperature
Slows enzymatic reactions inhibits microbial growth Refrigeration (watch out for _______________!
Freezing forms ice crystals that damage microbial cells
Deep freezing and lyophilization
Various Other Methods
Desiccation prevents metabolism
Osmotic pressure causes plasmolysis
Ionizing Radiation
X-rays, -rays have short wave length dislodge e- from atoms production of free radicals and other highly reactive molecules
Used for sterilization of heat sensitive materials: drugs, vitamins, herbs, suture material
Also as “cold pasteurization” of food Consumer fears!?
Effect: thymine dimers
Actively dividing organisms are more sensitive because thymine dimers cause ______________?
Used to fight air and surface contamination. Only kills at close range and directly exposed microbial agents
E.g.: germicidal lamps in OR, cafeteria, and our lab ??
Nonionizing Radiation: UV light
Heats H2O
Indirectly kills bacteria. How ?
Solid food heats unevenly. Why?
Nonionizing Radiation: Microwave
Fig 7.5
Few chemical agents achieve sterility.
Disinfectants regulated by EPA
Antiseptics regulated by FDA
Evaluating Disinfectants:
Use-dilution test
Disk-diffusion method
Chemical Methods of Microbial Control
Fig 7.6
Types of Antibacterial Chemicals
Phenol = carbolic acid (historic importance)
Who used first?
Many derivatives today:
Phenolics, e.g.: Lysol
Bisphenols, e.g.:
Hexachlorophene (in pHisoHex used in hospitals)
Triclosan (toothpaste, antibacerial soaps, etc.)
Phenol and derivatives disrupt plasma membranes (lipids!) and lipid rich cell walls (??)
Remain active in presence of organic compounds
Fig 7.7
Halogens
Chlorine Oxidizing agent
Widely used as disinfectant
Forms bleach (hypochlorous acid) when added to water.
Broad spectrum, not sporicidal (pools, drinking water)
Iodine
More reactive, more germicidal. Alters protein synthesis and membranes.
Tincture of iodine (solution with alcohol) wound antiseptic
Iodophors: Iodine plus organic molecule. E.g.: complexed with detergent: Betadine®. Occasional skin sensitivity.
Cl I
Ethyl (60 – 80% solutions) and isopropyl alcohol
Denature proteins, dissolve lipids
No activity against spores and poorly effective against viruses and fungi
Easily inactivated by organic debris
Also used in hand sanitizers and cosmetics
Alcohols
Heavy Metals
Oligodynamic action: toxic effect due to metal ions combining with sulfhydryl (—SH) and other functinal groups proteins are denatured.
Silver (1% AgNO3): Antiseptic for eyes of newborns
Copper against chlorophyll containing organisms Algicides; also X-gel hand sanitizer
Zinc (ZnCl2) in mouthwashes, ZnO as antifungal in paint
Soaps and Detergents
Major purpose of soap: Mechanical removal and use as wetting agent
Definition of detergents Acidic-Anionic detergents Anion reacts with plasma membrane.
Nontoxic, non-corrosive, and fast acting. Laundry soap, dairy industry.
Cationic detergents Quarternary ammonium compounds (Quats). Strongly bactericidal against wide range, but esp. Gram+ bacteria
Surface Acting Ingredients / Surfactants
Soap Degerming
Acid-anionic detergents Sanitizing
Quarternary ammonium compounds (cationic detergents)
Strongly bactericidal, denature proteins, disrupt plasma membrane
Sodium nitrate and nitrite prevent endospore Prevents ES germination. Used in meats. Conversion to nitrosamines: Carcinogenic!
Organic acids Inhibit metabolism E.g.: Sorbic acid, benzoic acid, etc. In foods and cosmetics
Sulfur dioxide wine
Chemical Food Preservatives
Aldehydes (alkylating agents)
Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, –SH)
Formaldehyde: Embalming Formalin
Virus inactivation for vaccines
Glutaraldehyde: Liquid Sterilant for delicate surgical instruments (Kills S. aureus in 5, M. tuberculosis in 10 min, ES in 3 – 10h)
Ethylene oxide: Gaseous Sterilant
Aldehydes and Chemical Sterilants
Hydrogen Peroxide: Oxidizing agent
Inactivated by catalase
Not good for open wounds
Good for inanimate objects; packaging for food industry (containers etc.)
3% solution (higher conc. available)
Especially effective against anaerobic bacteria (e.g.:
Effervescent action, may be useful for wound cleansing through removal of tissue debris
Microbial Characteristics and Microbial Control
Fig 7.11