Chapter 11

Chapter 11

Physical and Chemical Control of Microbes

The purpose of controlling microbial growth – To stop spreading the diseases or food spoilage

Methods:

• Physical agents

– Heat

– Radiation

• Chemical Agents

– Gases

– Liquids

• Mechanical removal

– Filtration

• Air

• Liquids

Methods of Microbial Control

• Sterilization - Destruction of all forms of microbes including endospores (by steam under pressure or ethylene oxide)

• Disinfection -Destruction of vegetative cells of pathogenic microorganisms (by chemicals or physical methods)

• Pasteurization - Application of high temperature (72⁰ C) for short period of time (15 sec) with the purpose of reducing the number of microbes

• Antiseptic - Antimicrobial agent that is sufficiently non-toxic to be applied on living tissue

• Sanitization - Lowering the number of microbes on eating and drinking utensils (by heat or chemical disinfectant)

• Decontamination – Mechanical removal of microbes from organisms or non-living objects

Terminology

• Bactericidal (germicidal, microbicidal)- agent that destroys or kills bacteria (suffix cide - kill)

• Bacteriostatic - agent that inhibits bacterial growth (stasis - to stop)

What is Microbial Death?

• Permanent loss of reproductive capabilities

• The cell structures become dysfunctional

• Antimicrobial treatment leads to killing of microbial population at the constant rate

Factors that affect death rate:

• Time of exposure (lower temp. can be compensated with longer exposure)

• The number of microbes

• Microbial characteristics (endospore, vegetative cells)

• Agent used

• Environmental influences (suspending medium, pH)

The Mode of Action of Antimicrobial Agents

• Plasma membrane - when damaged, cell content leaks into the surrounding medium

• Proteins- enzyme active sites inactivated

– Complete denaturation

– Different shape

– Blocking the active sites

• Nucleic Acid - radiation or some chemicals lethally damage the DNA or RNA (microbes can no longer replicate)

– UV radiation causes formation of dimmers between two thymine bases

Physical methods of microbial control

Heat

• Moist heat and dry heat

• Mechanism: denaturing the enzymes

• Most commonly used method of killing the microbes

• Thermal death point - the lowest temp at which all the microbes are killed in 10 min

• Thermal death time – the minimal length of time needed to kill all bacteria at given temperature

Moist heat – nonpressurized steam

• Mechanism: coagulation of proteins

• Boiling (100⁰C) for 10 min kills vegetative cells of bacteria, viruses, and fungi

• Hepatitis virus can survive up to 30 min of boiling; some bacterial spores can survive more than 20 h.

Tyndalization – boiling the medium for 60 min repeatedly for 3 day

Autoclaves – steam under pressure

• Provide high temp. and high pressure (Pressure: 1 atm, temp.: 121⁰ C)

• All microbes are killed in 15 min

• Steam should contact all surfaces

• Time is different for larger volumes

• Used for sterilization of:

– Culture media

– Equipment

– Biological waste

Pasteurization

• Original pasteurization: 63⁰ C for 30 min

• Today’s pasteurization – high temperature short-time pasteurization: 72⁰ C for 15 sec. or

• Ultra-high-temperature treatment - Exposure to 134⁰ C for 3 sec. then rapidly cooled

Dry heat sterilization

• Mechanism: oxidation

• Flaming – inoculating loops

• Hot-air sterilization

– Oven - 170⁰ C for 2h

Desiccation

• In the absence of water microbes cannot grow but can survive

• Bacterial spores can survive for centuries

• Survival depends on microbial type and organism’s environment (embedded in mucus - better survival)

– Mycobacterium tuberculosis – long survival

– Neisseria gonorrhoeae – dies after a few hours of air drying

Low temperatures

• Effect depends on the microbial type

• Ordinary refrigeration (0-7⁰ C) - bacteriostatic effect

• Psychotrophs grow slowly

• Pathogenic bacteria will not grow

• Rapid freezing – microbes become dormant

– Lyophilization – frozen samples (bacterial cultures) dried in vacuum

Slow freezing – more harmful

Can Microbes Survive Millions of Years Traveling in Space? Experts Say "Yes"

Radiation

· Ionizing radiation (gamma rays, X rays) -radiation ejects electrons – ions are formed

· Non-ionizing radiation (UV light)

Ionizing radiation

• Short wavelength, high energy

• Emitted by radioactive elements (Co)

• Mechanism of action: ionization of water which forms hydroxyl radicals which react with DNA

• Used for sterilization of

– Medical supplies (plastic syringes, Petri plates etc.)

– Certain food (spices, meat, vegetables)

Nonionizing radiation

• UV light, germicidal light – 260 nm – used for disinfection

• Mechanism of action:

– damage of DNA – formation of thymine dimmers

– Toxic free radicals are formed

• Sterilization of the air (hospital rooms, operating rooms, cafeteria)

• Disadvantage

– Poor penetration

– Harmful for human eyes, skin

Filtration

• Removal of microbes from a solution

• Membrane filters (pore size 0.2 or 0.45 um)

Osmotic pressure

• High concentration of salt causes water to leave the cell

• Used in preservation of food (high sugar concentration - fruit preserve)

Chemical methods of microbial control

• Effectiveness of the disinfectant depends on:

• Type of the chemical agent

• Type of microbes

• Concentration of a disinfectant

• Time of contact

• pH of the medium

• Temperature

Types of Disinfectants

Halogens

• Fluorine, bromine chlorine, and iodine

• Iodine is the oldest antiseptic

• Iodine tincture – skin disinfection

• Chlorine - gas (Ca-hypochlorite; Na-hypochlorite- bleach)

• Mode of action: oxidizing agent - alters cellular components

• Disinfection of drinking water, swimming pools, household (bleach)

Phenolics (derivatives of phenol)

• Used first time by Lister – carbolic acid

• Mechanism of action: damages the plasma membrane, enzyme inactivation

• Advantage: active even in the presence of organic compounds

• Hexachlorophene (bisphenol) used in antimicrobial soaps

Is antibacterial soap any better than regular soap?

• The antibacterial components of soaps need to be left on a surface for about two minutes in order to work.

Alcohols

• Ethanol or isopropanol 60% - 95%

• Kills vegetative cells of bacteria and fungi (not spores and nonenvelope viruses)

• Mechanism of action: protein denaturation

Is pure ethanol a better disinfectant than 70% ethanol? Why?

• 100% ethanol coagulates proteins in the cell wall

• 70% ethanol penetrates the cell wall and coagulates the proteins inside the cell

Hydrogen Peroxide

• 3% solution used as an antiseptic

• Skin and wound cleansing

• Mouthwash

• Contact lens

• Surgical implants

• Endoscopes

Chemicals with surface action: Detergents / Soaps

• Detergents are polar molecules - surfactants

• Decrease the surface tension among molecules and water

• Soaps and Detergents are not antiseptics – they break the oily film on the surface of skin

• They have microbicidal power when mixed with quaternery ammonium compounds

Heavy metals

• Silver, mercury, copper, gold, arsenic

• Only mercury and silver have germicidal significance

• Mechanism of action: ions combine with sulfhydril groups - protein denaturation

• 1% Silver nitrate - antiseptic

• Copper sulfate - controls algal growth

• Can be toxic to humans

Evaluation of a disinfectant

· Filter paper method

• Paper disks are soaked in a solution of disinfectant and placed on a agar previously inoculated with a test organism

• Observe the inhibition zone around the disk

Aldehydes (formaldehyde, glutaraldehyde)

• Most effective antimicrobials

• Formalin - used for preservation of biological specimens

– High level disinfectant

– Toxic - carcinogenic

• Glutaraldehyde –

– Used for disinfection of hospital instruments

– Mode of action: forms covalent cross-links with functional groups of proteins

– Kills bacterial spores, fungal spores and viruses

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