control of microbial growth - كلية الطبsterilization: a defined process used to render a...

Copyright © 2010 Pearson Education, Inc.

Control of Microbial growth Dr. Hala Al Daghistani


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.

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 (kill) microorganisms.

Chemical biocides include hydrogen peroxide, alcohols,

bleach, cycloheximide, and phenols

physical biocides include heat and radiation.

Fungicide, Virucide, Germicide, bactericide

Sanitization: Lowering of microbial counts to prevent

transmission in public setting (e.g., restaurants & public

rest rooms)

Antibiotics: Naturally occurring and synthetically derived organic compounds that inhibit or destroy selective bacteria, generally at low concentrations.

Bacteriostatic: Inhibits bacterial reproduction

Bactericidal: Kills bacteria

Factors affect the antimicrobial treatment

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.(heat treatment is efficient

at low pH)

Exposure time: Longer exposure to lower heat

produces same effect as shorter time at higher

heat.

Actions of Microbial Control Agents

Disruption of the Cell Membrane permeability(damage

protein or lipids of PM)

Disruption of the cell Wall synthesis(either by inhibit the

enzyme involved in cell wall synthesis or interfere with CW building block

synthesis). E.g. penicillin interfere with transpeptidase and prevent

the assembly of PG layer.

Damage to proteins (break down H- bonds, destruction of three

dimensional structure of a protein , inhibition of translation and transcription

of genetic material).

Damage to nucleic acids (include ionizing radiations,

ultraviolet light, and DNA-reactive chemicals. Ultraviolet light,

induces cross-linking between adjacent pyrimidines on one or

the other of the two DNA strands, forming thiamine dimers

Antimetabolite groups(sulfa drugs interfere with folic acid

synthesis). Antimetabolites are substances that interfere with the

normal metabolism of an organism, thereby causing its death.

Evolution of drug resistance:

* spontaneous mutation

* gene transfer

http://science.jrank.org/pages/4253/Metabolism.html

http://science.jrank.org/pages/4906/Organism.html


Antibiotic Resistance

Mechanisms of antibiotic resistance

1. Enzymatic destruction of drug

2. Prevention of penetration of drug

3. Alteration of drug's target site

4. Rapid ejection of the drug

Resistance genes are often on plasmids or

transposons that can be transferred between bacteria.


Resistance to Antibiotics

Fig 20.20

The disk-diffusion method Antibiotic Susceptibility test

KIRBY-BAUER TEST DETERMINES SUSCEPTIBILITY OF AN ORGANISM TO

A SERIES OF ANTIBIOTICS OR CHEMICALS Disk-diffusion method is used in teaching laboratories to evaluate the efficacy of a chemical agent. A disk of filter paper is soaked with a chemical and placed on an agar plate that has been previously inoculated and incubated

with the test organism

After incubation, if the chemical is effective, a clear zone representing inhibition of growth can be seen around the

disk (Inhibition Zone)


Physical Methods of Microbial Control

A) Heat (used for media, food, glass wares)

Two types (moist and dry heat)

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): Min Time to kill all cells in a culture at a given temp.

Decimal Reduction Time (DRT): Time in minutes in which 90% of a bacterial population are killed at a given Temp.

Moist heat

A temperature of 100°C (Boiling) will kill bacteria, viruses,

and fungi spores, but not spore forms of bacteria within 10

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 heat are

used, and because steam provides a means for distributing

heat to all parts of the sterilizing vessel. Autoclave: Steam

under pressure, Most dependable sterilization method

Prions (infectious proteins) need 134°C and NaOH solution

for 4-5 h.

Moist heat kill M.O. by denaturing of proteins(breakdown

H bonds that hold 3-dimentional structures).


Pasteurization Moist heat method

Significant number reduction (esp. spoilage and pathogenic organisms) does not sterilize

Denaturation of proteins by breaking H-bonds

Historical goal(LOUIS PASTEUR): destruction of M. tuberculosis

Classic pasterization method: 63C for 30 min

Flash pasteurization (HTST): 72C for 15 sec. Most common method (milk pasteurization). Thermoduric(heat resistant) organisms survive

Ultra High Temperature (UHT):140C for 4 sec. (liquid is sprayed with high T –steam under pressure then rapidly cooled).


Dry heat sterilization

For sterilizing materials that must remain dry,

circulating hot air electric ovens are available.

because heat is less effective on dry material, we

use to apply a temperature of 160–170°C for 2 h. Flaming of loop

Incineration of carcasses (Anthrax, Foot and mouth disease, Bird flu)

Hot-air sterilization

Dry heat kill M.O. by oxidation


Filtration Used for heat sensitive materials(vaccines, antibiotics)

Air filtration using high efficiency particulate air (HEPA) filters. Effective to 0.3 m

Membrane filters for fluids.

Pore size for bacteria: 0.22 – 0.45 m

Pore size for viruses: 0.01 m

Low Temperature

Refrigeration(0-7C) (reduce metabolic rate and prevent toxin

production).Freezing forms ice crystals that damage microbial

cells

Deep freezing(-50,-95C) bacteriostatic effect

Lyophilization( removing water at low T)

Three methods used for food and drugs)

Various Other Methods

High pressure in liquids: denatures bacterial proteins

and CH2O and inactivation of the cells, preserves flavor

Desiccation prevents metabolism

Osmotic pressure causes plasmolysis (hypertonic

solution). Molds and yeast can grow in high osmotic pressure

environment.


Radiation

Ionizing radiation (X-rays, -rays, electron beams)

- have short wavelength, penetrate deeply, ionize water forming OH¯ radicals and other highly reactive molecules

- Salmonella and Pseudomonas are particularly sensitive

Used for plastic syringes, surgical gloves, suturing materials, and

catheters.

Non ionizing radiation ( UV light)

Most effective wave legnth ~ 260 nm, Effect: thymine dimers (inhibit

correct replication)

Used to limit air and surface contamination. Use at close range to directly

exposed microorganisms. e.g.: germicidal lamps in the lab

used for sterilization of heat sensitive materials: drugs, vitamins, herbs, suture material


Chemical Methods of Microbial Control

Few chemical agents achieve sterility.

We should considered the followings:

The presence of organic matter

The degree of contact with microorganisms

The temperature

Types of Disinfectants

Phenol = carbolic acid

(historic importance)

Phenolics: Cresols (Lysol)

- disinfectant

Bisphenols

Hexachlorophene

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


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. Occasional skin sensitivity, partially inactivated by organic debris, poor sporicidal activity.

Halogens


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: Oligodynamic action: the ability of very

small amounts of heavy metals to exert antimicrobial activity

toxic effect due to metal ions combining with sulfhydryl (—SH)

and other groups proteins are denatured.

Mercury (HgCl2, used for skin lesions)

Copper against chlorophyll containing organisms

Algicides

Silver (AgNO3): Antiseptic for eyes of newborns

Zinc (ZnCl2) in mouthwashes


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 Quaternary ammonium compounds. Strongly bactericidal against a 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


Aldehydes (alkylating agents)

Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, –SH)

Glutaraldehyde: Sterilant for delicate surgical instruments Formaldehyde: Virus inactivation, use for vaccines


Plasma

The four state of matter in which a gas is excited by

electromagnetic field to make a mixture of nuclei with

assorted electrical charges and free electrons.

used for metal or plastic surgical instruments (Tubular

instruments).

Free radicals in plasma gases are used to sterilize plastic

instruments and destroy microbes

Supercritical Fluids

Supercritical fluids, which have properties of liquid and gas,

can sterilize at low temperatures.

Peroxygens and Other Forms of Oxygen

Hydrogen peroxide, peracetic acid, benzoyl peroxide, and

ozone exert their antimicrobial effect by oxidizing

molecules inside cells.


Hydrogen Peroxide: Oxidizing agent

Inactivated by catalase

Not good for open wounds

Good for inanimate objects; packaging

for food industry (containers etc.)

3% solution is used

Effective against anaerobic bacteria


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control of microbial growth - كلية الطبsterilization: a defined process used to render a...

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