Chemical Control of Microbial

Growth

• Physical and

Chemical Control of

Microbes

Physical and

chemical methods to

destroy or reduce

microbes in a given

area

History

• Humans vs. Microbes

– infections

– diseases

– plagues

– epidemics

– pandemics

Humans vs. Microbes

• 1. Most of History, microbes have been winning the battle

• 2. In the last 100 yrs or so the battle has swung in our favor

– Why?

– Because of our increasing knowledge of how to Control Microbial Growth..often by antibiotics, and a few other methods.

Smallpox

Variola virus

Eradicated in 1977 (Somalia)

• But now we have a very big problem!

• It is resistance of microbes to antibiotics

especially!

Relative resistance of microbes • Highest resistance

– Bacterial endospores

• Moderate resistance – Pseudomonas sp.

– Mycobacterium tuberculosis

– Staphylococcus aureus

– Protozoan cysts

• Least resistance – most vegetative cells

– Fungal spores

– enveloped viruses

– Yeast

– Protozoan trophozoites

• These are some of the techniques we

commonly used today to control microbes.

• Sterilization – a process that destroys all

viable microbes, including viruses &

endospores

• Disinfection – a process to destroy vegetative

pathogens, not endospores

• Sanitization – any cleansing technique that

mechanically removes microbes

• Degermation – reduces the number of

microbes

We need to distinguish between

• Sterilization and Disinfection

• Disinfectant and Antiseptic

• Bactericidal and Bacteristatic

• Sterilization refers to

• destroying all forms of life

• killing or removing ALL microorganisms,

including bacterial spores

• Disinfection refers to

• destroying pathogens or unwanted organisms

• killing of vegetative disease causing

microorganisms

• killing of many, but not all, microorganisms

• pathogens are killed but some organisms,

spores and viruses may survive.

• Disinfectants are

– chemicals (usually harsh) used to reduce or

kill microorganisms on the surface of

inanimate (non-living) objects

– usually toxic to living tissue.

• Disinfectant is an antimicrobial agent used on inanimate objects

• Antiseptic is an antimicrobial agent used on living tissue

• Antiseptic substances are used to reduce the number of organisms on living tissue.

• Antisepsis – prevention of infection of living tissues by microorganisms

• Aseptic technique – procedure designed to eliminate and exclude all infectious

microbes by sterilization of equipment, disinfection of the environment and cleansing of the body tissues with antiseptics.

• Sanitization – reduction of the microbial population to safe levels as

determined by public health standards. (kitchen disinfection)

Control of Microorganisms

• -cide

– suffix indicating that the agent will kill the kind of

organisms in question (i.e. virucide, fungicide)

• -static

– suffix indicating that the agent will prevent, inhibit the

growth of the type of organism is question.

• Bacteriostasis

– bacterial growth and multiplication are inhibited, but

bacteria are not killed.

Bactericidal vs. Bacteristatic

• Bactericidal agents - kills bacteria

• Bacteristatic agents - inhibits bacterial growth

• Fungicidal

• Fungistatic

• Algacidal

• Algastatic

Cellular targets of control by

Antimicrobial Agents

• 1. Cell wall

• 2. Cell membrane

• 3. Cellular synthetic processes

(DNA, RNA)

• 4. Cellular enzymes & proteins

Practical concerns for consideration

• Does the application require sterilization?

• Is the item to be reused?

• Can the item withstand heat, pressure, radiation,

or chemicals?

• Is the method suitable?

• Will the agent penetrate to the necessary

extent?

• Is the method cost- and labor-efficient & is it

safe?

Factors that influence action of

antimicrobial agents:

1. Number of microbes

2. Nature of microbes in the population

3. Temperature & pH of environment

4. Concentration or dosage of agent

5. Mode of action of the agent

6. Presence of solvents, organic matter, or

inhibitors

Pattern of Microbial Death

• Microbial death involves permanent

loss of reproductive capability, even

under optimum growth conditions

• Microorganisms are not killed instantly

when exposed to a lethal agent, but

rather the population is reduced by the

same fraction at constant intervals

Methods to Control Microbial

Growth

• 1. Physical

• 2. Chemical

Methods of Physical Control

1. Heat

2. Cold temperatures

3. Desiccation

4. Radiation

5. Filtration

Methods of Chemical control

1. Halogens

2. Phenolics

3. Chlorhexidine

4. Alcohols

5. Hydrogen peroxide

6. Detergents & soaps

7. Heavy metals

8. Aldehydes

•Physical Methods of Microbial Control

• Physical methods used to control Microbial Growth

• Physical factors that effect Antimicrobial Activity and influence the effectiveness of microbial Agents

• Methods of Physical Control

• 1. Heat or high temperatures

• 2. Time or Duration of exposure

– the longer a population is exposed, the greater the number of organisms killed

• 3. Concentration of Antimicrobial agent

• 4. Type of Microbe

• 5. Activity of Microbe

• 6. Presence of organic matter

• 7. Local environment

– environmental factors such as pH, viscosity, concentration of organic matter etc. can have a profound influence on the effectiveness of a particular antimicrobial agent.

Concentration of

Antimicrobial agent • Population size

– larger populations will take longer to kill than smaller

populations

• Population composition

– microorganisms differ markedly in their sensitivity to

various agents

• Concentration

– higher concentrations or intensities are generally

more efficient but results not linear

Mechanisms of Action of Microbial

Control Agents • Alteration of membrane permeability

– plasma membrane target of many control agents

– damage causes cellular contents to leak and interferes with growth of cell

• Damage to proteins and nucleic acids – disruption of hydrogen bonds

• heat and certain chemicals

• denaturation

– disruption of covalent bonds of DNA and RNA • damage by radiation and chemicals frequently lethal to cells

(prevents both replication and normal functioning)

Methods of Physical Control Heat or high temperatures: a higher temperature

will usually (but not always) increase the effectiveness of a particular antimicrobial agent.

• Heat works by denaturing enzymes and proteins and results in coagulation of proteins and breakage of H bonds

• A. Thermal Death Point (TDP) – lowest temp. at which all microorganism in a liquid culture

are killed in 10 minutes

• B. Thermal Death Time (TDT) – minimum length of time in which all microorganisms in a

liquid culture are killed at a given temperature

C Decimal reduction time (DRT) • time in minutes, in which 90% of the population of bacteria will be

killed at a given temperature

Heat or high temperatures continued

• Moist Heat • Boiling Water

• Steam Heat (Autoclave

• Dry Heat • Direct Flaming

• Incineration

• Hot Air Sterilization (Oven)

• Dessication

• Low or cold Temperatures

• Filtration

• Radiation – Ionizing Radiation

– Non-Ionizing Radiation

• Pasteurization (Heat)

MOIST HEAT

• Moist heat – use of hot (boiling) water or steam under pressure as in the autoclave

• Boiling at 100oC for 30 minutes to destroy non-spore-forming pathogens – kills all vegetative bacterial cells, most viruses and fungi within

30 minutes but may not kill bacterial endospores

– not effective for endospores and some viruses

– Hepititis (20 min)

– Some spores may survive boiling water for up to 20 hrs

– Boiling is effective for making food and water safe to eat or drink

MOIST HEAT

• Mode of action of moist heat – denaturation of proteins, destruction of membranes & DNA

• sterilization

• autoclave 15 psi/121oC/10-40min

• intermittent sterilization – unpressurized steam at 100oC 30-60 min for 3 days

• disinfection

• Pasteurization <100oC for seconds; kills Salmonella, Listeria & overall microbe count

• Boiling at 100oC for 30 minutes to destroy non-spore-forming pathogens

MOIST HEAT

• Autoclave (Steam under pressure)

– preferred method of sterilization

– Water boils at 100 C

– Increasing the pressure raises the Temp.

– 15 lbs./ per sq. inch (psi) ------> 121 C

– 121 C for 15 min at 15 lbs. pressure.

– kills all forms of life including bacterial endospores

– Used to sterilize culture media, instruments,

dressings, IV equipment, solutions, syringes

Dry Heat

• 1. Direct Flaming

– Inoculating Loop and Needle 100% effective

• 2. Incineration

– disposable wastes (paper cups, bags,

dressings)

• 3. Hot Air Sterilization

– Oven ( 170 C for 2 hours)

– used on substances that would be damaged

by moist heat sterilization

• gauzes, dressings or powders

Dry Heat continued- Hot Air

Sterilization (Oven) • Dry heat using higher temperatures than moist heat, can also

sterilize

• Incineration at 600-1200oC combusts & dehydrates cells

• Incineration vaporizes organic material on nonflammable surfaces; used to get rid of paper cups, contaminated dressings, wipes, bags, carcasses..

• Dry hot air oven at kills by oxidizing (burning) organic compounds and coagulates proteins

• 160 C for 2 hours or 170 C for 1 hour

• used for materials that must remain dry and which are not destroyed at temperatures between 12O C to 170 C

• Good for glassware, metal, not plastic or rubber items

Dry

heat

Dry Heat continued- Dessication

• Dessication or drying is the gradual removal of water from cells. It leads to metabolic inhibition only, and is thus not an effective microbial control – because many cells retain ability to grow when water is reintroduced

• Most microorganisms cannot grow at reduced water activity but can remain viable

• Dessication is oten used to preserve foods

• Methods involve removal of water by heat, evaporation, freeze-drying, and addition of salt or sugar

• Ability to survive varies with species.

• Cold temperatures are a microbiostatic that slows

the growth of microbes

• Refrigeration (0-7 C) is bacteriostatic, since the

microbes’ metabolic rate is reduced, and they can't

reproduce;

• used to preserve and store food by slowing

spoilage,

• used to preserve and store media and cultures

» Lyophilization

• freeze-drying, preservation

• refrigeration 0-15oC & freezing <0oC

LOW OR COLD TEMPERATURE

Refrigeration (0-15oC) and freezing

(<0oC)

• Cold temperatures are a microbiostatic that slows the growth of microbes

• Refrigeration (0-7 C) is bacteriostatic, since the microbes’ metabolic rate is reduced, and they can't reproduce;

• used to preserve and store food by slowing spoilage,

• used to preserve and store media and cultures

• Lyophilization

• freeze-drying, preservation

• refrigeration 0-15oC & freezing <0oC

FILTRATION • Filtration is the physical removal of microbes from solutions

that might be damaged by heat by the passage of a liquid or gas through screenlike material ( a membrane filter composed of cellulose acetate or plastic polymers) with pores small enough to retain microorganism

• Filtration is thus an alternative for heat-labile substances that is used to sterilize heat sensitive liquids & air in hospital isolation units & industrial clean rooms.

• Filtration is used to sterilize heat-sensitive material, such as

• some culture media,

• enzymes,

• vaccines and

• antibiotic solutions

Filtration

IRRADIATION • 1. Ionizing Radiation -gamma rays cathode rays &

x-rays

• Gamma-irradiation has high energy and deep penetrating power and penetrates most substances effectively, , breaks DNA,

• Used on substances that could be damaged by heat and is therefore used to sterilize medical supplies & food products – used for sterilization of pharmaceuticals, disposable dental

and medical supplies such as

– plastic petri dishes

– plastic syringes

– catheters

– surgical gloves

– suturing materials

• 2. Non-Ionizing Radiation

• does not penetrate plastic, glass or proteinaceous matter

• has the disadvantage that uv light at 260 nm causes the formation of thymine pyrmidines in DNA, which interfere with replication

• this leads to genetic damage to cells and ultimate death

• Ultraviolet irradiation is an effective method of sterilizing work surfaces and air but has little penetrating power to sterilize water

• used to reduce microbial populations in hospital rooms, operating rooms, nurseries, and cafeterias

• has less penetrating power to sterilize air, water & solid surfaces than ionizing radiation

PASTEURIZATION

• use of mild heat to reduce the number of microorganisms in a product or food – milk, certain drinks and alcoholic beverages

• heating 63 C for 30 minutes (batch method)

• kills most vegetative bacterial cells including pathogens

• heating 72 C for 15 seconds – flash pasteurization

– fewer undesirable effect on quality and taste

• 140oC for 3 seconds (ultra high temperature UTH) – allows milk to be stored without refrigeration

Methods to Control Microbial

Growth • 1. Physical

• 2. Chemical

Classification of Chemical Agents

• High level - effective against all life forms, including endospores; e.g. ethylene oxide

• Intermediate level - defined as tubercucidal (kills Mycobacterium tuberculosis), as well as more resistant viruses (hepatitis virus), Not effective against endospores

• Low level - not effective against tuberculosis or endospores, or viruses without envelopes (membranes) but do not kill vegetative bacteria and fungi; used extensively. Economical, not overly toxic to humans (Lysol, detergents)

Classification of Chemical Agents

• Qualities

– Effective killers of all microorganisms

including viruses

– Soluble in water for ease of preparation

– Low toxicity for humans

– Reasonably economic

CONTROL OF MICROBIAL

GROWTH BY CHEMICAL AGENTS • Halogens

• Phenolics

• Detergents & soaps

• Chlorhexidine

• Alcohols

• Aldehydes

• Heavy metals

• Oxidizing agents e.g Hydrogen peroxide

• Gaseous Chemosterilizers & aerosols

• Quaternary ammonium compounds (QUATS)

• Organic Acids

• Enzymes

Halogens

• Are powerful oxidizing agents

• chlorine and iodine are used widely for

disinfection

• become rapidly inactivated in the presence

of organic matter

• both highly irritating to humans

Chlorine

• Chlorine – Cl2, hypochlorites (chlorine bleach), chloramines – Denaturation of proteins by disrupting disulfide bonds

– Can be sporicidal

• used in low concentrations to prevent microbial growth in water supplies

• used in higher concentrations to disinfect swimming pools

• most commonly used chlorine preparation is sodium hypochlorite-household bleach - hypochlorite ion is active ingredient in Clorox

Iodine • Iodine - I2, iodophors (betadine)

– Denature proteins

– Can be sporicidal

– Milder medical & dental degerming agents, disinfectants, ointments

• effective antiseptic

• combines with tyrosine and oxidizes SH groups on other amino acids denaturing proteins

• Tincture: 1-2% iodine +2% NaI in 70% EtOH irritating…stains, local allergies, skin irritant

• Iodophor = iodine + organic molecule; permit slow release, reducing the irritation and prolonging the effectiveness; Wescodyne, Betadine, Isodine

Phenolics • low-intermediate level disinfectants

• phenol no longer used as a disinfectant because of its toxicity to tissues

• derivatives of phenol such as o-phenylphenol (Lysol), hexylresorcinol, and hexachlorophene (Phisohex) are used because they have been chemically altered to reduce irritating qualities

• Triclosan is an antibacterial additive to soaps

• remain active in presence of organic compounds.

• disrupt cell (plasma) membranes, ausing the denaturation & precipitation of proteins

• they are ; bactericidal, fungicidal, virucidal, BUT not sporicidal

Surfactants, Detergents & soaps

• Soaps and Detergents are usually quaternary ammonia compounds that act as surfactants to alter membrane permeability of some bacteria & fungi

• mechanically remove soil and grease containing microbes through scrubbing

• soap breaks oily film on skin (emulsification); little value as an antiseptic; they are NOT sporicidal

• detergents-more effective against gram positive than gram negative

• nonionic detergents have no germicidal activity

• anionic (acid) sanitizes, food and dairy industry

• cationic (positive)-antiseptic for skin, instruments, utensils

Chlorhexidine

• Hibiclens, Hibitane

• A surfactant & protein denaturant with

broad microbicidal properties

• Not sporicidal

• Used as skin degerming agents for

preoperative scrubs, skin cleaning & burns

Alcohols

• Alcohols such as methanol, ethanol, and isopropanol in solutions of 50-90%

• act as surfactants by dehydrating cells, dissolving and disrupt membrane lipids and coagulating proteins of vegetative bacterial cells and fungi

• they kill bacteria and some viruses, but do not kill spores- they are NOT sporicidal

• 70% aqueous solution is more effective than absolute alcohol

• used as skin antiseptic (isopropanol most common)

Aldehydes • high level disinfectants (formaldehyde and glutaraldehyde)

• formaldehyde and glutaraldehyde

• formalin - 37% solution of formaldehyde in water…. used for preserving dead tissues, making toxoids from toxins and creating whole killed viral vaccines.

• Glutaraldehyde & formaldehyde kill by alkylating protein & DNA; i.e they form covalent crosslinks with organic functional groups on proteins and nucleic acids denaturing them

• kill most microbes including spores

• 2% glutaraldehyde: 3- 10 hours sporocidal - can actually sterilize

• glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments

• formaldehyde - disinfectant, preservative, toxicity limits use

Heavy metals • Solutions of silver & mercury kill vegetative cells in low

concentrations by inactivating proteins

• These metals ions combine with -SH groups on proteins (oligodynamic action) but they are NOT sporicidal

• 1% silver nitrate -weak solution, eyedrops (gonorrhea)

• mercuric chloride (inorganic) preservative for wood paper, leather-toxic

• mercurochrome, merthiolate (organic) skin antiseptic

• copper sulfate-algicide

• zinc chloride-mouthwashes

• zinc oxide-antifungal in paints

• silver sulphadiazine - topically used to prevent colonization and infection of burn tissues

Oxidizing Agents

• Hydrogen peroxide Weak (3%) to strong (25%)

• Produce highly reactive hydroxyl-free radicals that damage protein & DNA while also decomposing to O2 gas – toxic to anaerobes

• Hydrogen peroxide is a high level disinfectant BUT NOT a good open wound antiseptic but a good disinfectant Strong solutions are sporicidal

• Ozone is used in some water purification systems

Gaseous Chemosterilizers & aerosols

• Ethylene oxide, propylene oxide, betapropiolactone & chlorine dioxide are all strong alkylating agents and are sporicidal

• excellent sterilizing agent for those material destroyed by heat

• denatures proteins

• highly penetrating

• used in hospitals for plasticware, sutures, textiles, anesthesia equipment, catheters, implantable devices (pacemakers, artificial hips)

• explosive, carcinogenic

• irritating effect on tissues-blisters on contact

• toxic fumes need to be eliminated before objects used

Quaternary ammonium compounds

• cationic, colorless, odorless, stable (bezalkonium chloride-Zephiran; cetyl pyridinium chloride-Cepacol)

• more germicidal than other detergents

• widely used as disinfectants for domestic use and in hospitals

• kills about everything except: endospores, M. tuberculosis

• not very effective in the presence of organic compounds

• neutralized by soaps, various anionic detergents and fibers

• Pseudomonas can metabolize these compounds

Organic Acids

• various organic acids and their salts are common antimicrobials in foods

• preservatives to control mold growth

• sorbic acid ( Ca, Na, K) used in cheeses, baked goods, soft drinks, fruit juices, jams, jellies

• benzoates (sodium benzoate, methy-p-hydroxybenzoate [methylyparaben]) fruit juices, jam, jellies, soft drinks, salad dressings, margarine, and many pharmaceutical products

• boric acid used in eye washes

• calcium propionate-prevents mold growth in bread

Enzymes

– Biological catalysts that lower the energy of

activation required for a chemical reaction to

occur inside a living cell.

• General characteristics

– enzymes acts on substrates and produce

products

– active site of an enzyme is the site where the

substrate binds