control of microbial growth - medicinebau.com · sterilization: a defined process used to render a...
TRANSCRIPT
Copyright © 2010 Pearson Education, Inc.
Control of Microbial growth Dr. Hala Al Daghistani
Copyright © 2010 Pearson Education, Inc.
Terminology
Sepsis: Characterized by the presence of pathogenic microbes in
living tissues or associated fluids.
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 (reduce the number of viable
microorganisms)
Antiseptic: used on living tissue, destroys or inhibits the growth of
microorganisms
Nosocomial Infection(Hospital Acquired Infection) an infection that is contracted from the environment or staff of a healthcare facility.
Copyright © 2010 Pearson Education, Inc.
Sterilization: A defined process used to render a surface or
product free from viable organisms, including bacterial spores.
Biocide: A chemical or physical agent, usually broad
spectrum, that inactivates microorganisms.
Chemical biocides include hydrogen peroxide, alcohols, bleach,
cycloheximide, and phenols
physical biocides include heat and radiation.
Fungicide, Sporicide, Germicide
Sanitization: reduces microbial numbers to safe levels
(e.g.: eating utensils)
Bacteriostatic: Inhibits bacterial reproduction
Bactericidal: Kills bacteria
Copyright © 2010 Pearson Education, Inc.
Preservation: The prevention of multiplication of M.O. in
formulated products, including pharmaceuticals and foods.
Antibiotics: Naturally occurring and synthetically derived
organic compounds that inhibit or destroy selective bacteria,
generally at low concentrations.
Copyright © 2010 Pearson Education, Inc.
Effectiveness of Antimicrobial Treatment Depends on
Time it takes to kill a microbial population is
proportional to number of microbes.
Microbial species and life cycle phases (e.g.: endospores) have different susceptibilities to
physical and chemical controls.
Organic matter may interfere with heat treatments
and chemical control agents.
Exposure time: Longer exposure to lower heat
produces same effect as shorter time at higher
heat.
Copyright © 2010 Pearson Education, Inc.
Actions of Microbial Control Agents
Disruption of the Cell Membrane or cell Wall
Damage to proteins (disruption of the tertiary structure
of a protein or protein denaturation)
Damage to nucleic acids (include ionizing radiations,
ultraviolet light, and DNA-reactive chemicals( e.g. alkylating
agents and other compounds that react covalently with purine
and pyrimidine bases). Ultraviolet light, induces cross-linking
between adjacent pyrimidines on one or the other of the two
polynucleotide strands, forming pyrimidine dimers
Copyright © 2010 Pearson Education, Inc.
Disruption of Free Sulfhydryl Groups
Enzymes and coenzyme containing cysteine have side
chains terminating in sulfhydryl groups. Such enzymes and
coenzymes cannot function unless the sulfhydryl groups
remain free and reduced. Oxidizing agents and heavy metals
do widespread damage.
Chemical Antagonism
The interference by a chemical agent with the normal reaction
between a specific enzyme and its substrate is known as
chemical antagonism.
The antagonist acts by combining with some part of the
holoenzyme (the protein apoenzyme, the mineral activator, or
the coenzyme), thereby preventing attachment of the normal
substrate.
Copyright © 2010 Pearson Education, Inc.
(e.g. carbon monoxide and cyanide combine with the iron atom in heme-
containing enzymes and prevent their function in respiration).
Copyright © 2010 Pearson Education, Inc.
Physical Methods of Microbial Control
Heat is very effective (fast and cheap).
Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min.
Thermal death time (TDT): Time to kill all cells in a culture
Decimal Reduction Time (DRT): Minutes to kill 90% of a population at a given T.
Copyright © 2010 Pearson Education, Inc.
A temperature of 100°C will kill all but not spore
forms of bacteria within 2–3 minutes in laboratory-
scale cultures.
a temperature of 121°C, pressure of 15 lb/sq
inches for 15 minutes is used to kill spores. Steam is
generally used, both because bacteria are more
quickly killed when moist and because steam
provides a means for distributing heat to all parts of
the sterilizing vessel.
Copyright © 2010 Pearson Education, Inc.
For sterilizing materials that must remain dry,
circulating hot air electric ovens are available.
because heat is less effective on dry material, it is
customary to apply a temperature of 160–170°C
for 1 hour or more.
Under these conditions ( excessive temperatures
applied for long periods of time), heat acts by
denaturing cell proteins and nucleic acids and by
disrupting cell membranes.
Copyright © 2010 Pearson Education, Inc.
Moist Heat Sterilization
Denatures proteins
Autoclave: Steam under pressure, Most dependable sterilization method
Steam must directly contact material to be sterilized.
All microorganisms even spore forming bacteria are killed at 121.5C for 15 min.
Prion destruction: 132C for 4.5 hours
Copyright © 2010 Pearson Education, Inc.
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 method. - Thermoduric organisms survive
Ultra High Temperature (UHT): 140C for < 1 sec. Technically not pasteurization because it sterilizes.
Copyright © 2010 Pearson Education, Inc.
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
Copyright © 2010 Pearson Education, Inc.
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
Fig 7.4
Copyright © 2010 Pearson Education, Inc.
Low Temperature
Slows enzymatic reactions inhibits microbial growth
Freezing forms ice crystals that damage microbial cells
lyophilization
Various Other Methods
High pressure in liquids denatures bacterial proteins
and preserves flavor
Desiccation prevents metabolism
Osmotic pressure causes plasmolysis
Copyright © 2010 Pearson Education, Inc.
Ionizing Radiation
X-rays, -rays, electron beams production of free radicals and other highly reactive molecules
Commonly used Cobalt-60 radioisotope
Salmonella and Pseudomonas are particularly sensitive
Sterilization of heat sensitive materials: drugs, vitamins, herbs, suture material
Copyright © 2010 Pearson Education, Inc.
Most effective wave legnth
~ 260 nm
Effect: thymine dimers
Actively dividing organisms are more sensitive
because thymine dimers cause . . . .?
Used to limit air and surface contamination. Use at
close range to directly exposed microorganisms
E.g.: germicidal lamps in our lab
Nonionizing Radiation: UV light
Copyright © 2010 Pearson Education, Inc.
Chemical Methods of Microbial Control
Few chemical agents achieve sterility.
Consider presence of organic matter, degree of contact
with microorganisms, and temperature
Copyright © 2010 Pearson Education, Inc.
Disk-diffusion Method
Disk of filter paper is soaked with a chemical and placed on an inoculated agar plate; a zone of inhibition indicates effectiveness.
Fig 7.6
Copyright © 2010 Pearson Education, Inc.
Types of Disinfectants
Phenol = carbolic acid
(historic importance)
Phenolics: Cresols (Lysol)
- disinfectant
Bisphenols
Hexachlorophene
(pHisoHex, prescription),
hospitals, surgeries,
nurseries
Triclosan (toothpaste,
antibacerial soaps, etc.)
Phenol and derivatives disrupt plasma membranes (lipids!) and lipid rich cell walls (??)
Remain active in presence of organic compoundsP
Fig 7.7
Copyright © 2010 Pearson Education, Inc.
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 combined with an organic molecule iodine detergent complex (e.g. Betadine®). Occasional skin sensitivity, partially inactivated by organic debris, poor sporicidal activity.
Halogens
Copyright © 2010 Pearson Education, Inc.
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
Table 7.6
Alcohols
Copyright © 2010 Pearson Education, Inc.
Heavy Metals
Oligodynamic action: toxic effect due to metal ions
combining with sulfhydryl (—SH) and other groups
proteins are denatured.
Mercury (HgCl2, Greeks & Romans
for skin lesions); Thimerosal
Copper against chlorophyll containing organisms
Algicides
Silver (AgNO3): Antiseptic for eyes of newborns
Zinc (ZnCl2) in mouthwashes, ZnO in antifungal in
paint
Copyright © 2010 Pearson Education, Inc.
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 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
Copyright © 2010 Pearson Education, Inc.
Sulfur dioxide
wine
Organic acids
Inhibit metabolism
Sorbic acid, benzoic acid, and calcium propionate
Control molds and bacteria in foods and cosmetics
Sodium nitrate and nitrite prevents endospore germination. In meats. Conversion to nitrosamine (carcinogenic)
Chemical Food Preservatives
Copyright © 2010 Pearson Education, Inc.
Aldehydes (alkylating agents)
Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, –SH)
Glutaraldehyde: Sterilant for delicate surgical instruments (Kills S. aureus in 5, M. tuberculosis in 10 min)
Formaldehyde: Virus inactivation for vaccines
Chemical Sterilants for heat sensive material
Denature proteins
Ethylene oxide
Aldehydes and Chemical Sterilants
Copyright © 2010 Pearson Education, Inc.
Plasma
Luminous gas with free radicals that destroy
microbes
Use: Tubular instruments, hands, etc.
Copyright © 2010 Pearson Education, Inc.
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)
Esp. effective against anaerobic bacteria (e.g.:
Effervescent action, may be useful for wound cleansing through removal of tissue debris
Copyright © 2010 Pearson Education, Inc.
Microbial Characteristics and Microbial Control
Fig 7.11