Sterilization and Disinfection

Course : B.Sc. Biotechnology, Biochemistry

Sem II

Sub: Basic Microbiology

Unit 4.1

BASIC DEFINATION

• Cleaning is the physical removal of all foreign materials (dirt, organic matter, and microorganisms).

• Disinfection is the killing of many, but not all microorganisms. It is a process of reduction of number of contaminating organisms to a level that cannot cause infection, i.e. pathogens must be killed. Some organisms and bacterial spores may survive.

• Disinfectants are chemicals that are used for disinfection. Disinfectants should be used only on inanimate objects.

• Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill microorganisms, e.g. on the surface of skin and mucous membranes.

BASIC DEFINATION

• Sterilization is a process which achieves acomplete destruction and killing of allmicroorganisms, including bacterial spores byphysical procedures or chemical agents. It isprincipally

• Accomplished by:– Steam under pressure (Autoclaving).

– Dry heat (Hot Air Oven).

– Chemicals such as ethylene oxide.

ANTISEPTICS

• Antiseptics:

– A particular group of disinfectants – used to reduce the number of viable organisms in the skin. act differentially on organism and host tissue

• 1. Germicide

– chemical agent capable of killing microbes

• 2. Sporicide

– a germicide capable of killing bacterial spores

Relative Resistance of Microbial Forms

• Primary targets of microbial control: microorganisms that can cause infection or spoilage that are constantly present in the external environmentHighest resistance– Bacterial endospores; prionsModerate resistance– Protozoan cysts, fungal sexual spores, naked viruses,

resistant vegetative bacteriaLeast resistance– Most bacterial vegetative cells, fungal spores, enveloped

viruses, yeasts, protozoan trophozoites

Comparison of Resistance

Terminology and Methods of Microbial Control

• Sterilization

– Removes all viable microorganisms including viruses and bacterial endospores

– Material is said to be sterile

– Usually reserved for inanimate objects

– Mostly performed with heat

– Sometimes chemicals called sterilants are used

WHAT NEED TO BE STERLIZED?

1 2

3

5 6

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Disinfection

• The use of a physical process or chemical agent (disinfectant) to destroy vegetative pathogens

• Does not destroy bacterial endospores

• Usually used only on inanimate objects

• Also removes toxins

• 5% bleach solution, boiling water, iodine solutions

WHERE THE DISINFECTANT USED?

7 8 9

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Antisepsis

• Antiseptics: applied directly to exposed body surfaces to destroy or inhibit vegetative pathogens

• Sepsis: the growth of microorganisms in the blood and other tissues

• Asepsis: any practice that prevents the entry of infectious agents into sterile tissues

The Agents Versus the Processes• –cide: to kill

– Bactericide: chemical that destroys bacteria (not endospores)

– Fungicide: a chemical that can kill fungal spores, hyphae, and yeasts

– Virucide: a chemical that inactivates viruses– Sporicide: can destroy bacterial endospores– Germicide and microbicide: chemical agents that kill

microorganisms

• Stasis and static: to stand still, prevent multiplication– Bacteristatic, Fungistatic– Microbiostatic: materials used to control microorganisms in

the body, for example

Decontamination

• Used when actual sterilization isn’t needed but need to decrease the risk of infection or spoilage (ex. food industry)

• Sanitization: any cleansing technique that mechanically removes microorganisms to reduce contamination to safe levels

• Sanitizer: compound such as soap or detergent that sanitizes

• Sanitary: may not be free from microbes but are safe for normal use

• Degermation: reduces the numbers of microbes on the human skin (ex. alcohol wipes)

What is Microbial Death?• When various cell

structures become dysfunctional and the entire cell sustains irreversible damage

• A cell can no longer reproduce

• Death begins when a certain threshold of microbicidal agent is present

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PROCESS

• The rate of killing of microorganisms depends upon the concentration of the killing agent and time of exposure.

N = 1/CT

N – number of survivors

C – concentration of agent

T – time of exposure to the agent

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Process of sterilization

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Microbes in decreasing order of resistance to germicidal chemicals Pasteurization

Antiseptics on normal skin flora 14

MECHANISM OF STERILIZATION

• Damage cell membrane

• Denature proteins

• Modify functional groups of proteins and nucleic acids

• Activity of a particular disinfectant may result from one or combination of pathways

Cell Membrane

• All microorganisms have cell membrane

• Many viruses as well

• Disrupt cell membrane= cell loss of selective permeability

• Detergents (surfactants) disrupt membrane

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Protein and Nucleic Acid Synthesis

• Any level can be affected: Replication, Transcription, and/or Translation

• Some agents bind to ribosomes to stop translation

• Some agents bind irreversibly to DNA preventing transcription and translation

• Mutagenic agents

Protein Function

• Proteins must be in native state

• Disrupt the native state (denature)

• Break the bonds of secondary or tertiary structures

• Coagulation: heat, alcohol, acids, phenolics 16

USE OF STERILIZATION/DISINFECTION/ANTICEPTICS

• Uses – Prevention of hospital infection depends on sterile

Equipments, instruments and dressings – Isolation facilities – Safe disposal of infected materials

• Microbiologists – production of sterile media and the laboratory

activities

• Central to almost all areas of medical practice like surgery(from hand washing to needles and prosthesis)

FACTORS AFFECTING EFFICIENCY

• Physical environment

• Presence of moisture

• Temperature and Ph

• Concentration of the agent

• Hardness of water

• Bioburden and the object

• Mature and state of microbes in bioburden

• Ability of microbes to inactivate the chemical agent

Methods of Sterilisation

Microbial Control

Methods

Physical Agents Chemical AgentsMechanical Removal

Methods

Microbial Control Methods

PHYSICAL METHODSPhysical Agents

Heat

Dry

Incineration

Dry Oven

Moist

Steam Under Pressure

Sterilization

Boiling Water/Hot Water

Pasteurization

Disinfection

Radiation

Ionizing

X Ray, Cathode,Gamma

Sterilization

Non Ionizing

UV

Disinfection

Methods of Physical Control• Heat as an Agent of Microbial Control

–Generally, elevated temperatures are microbicidal and lower temperatures are microbiostatic

–Can use moist heat or dry heat

–Moist heat is more effective

–Mode of Action

• Moist- coagulation and denaturation

• Dry –remove water from organisms, incineration

Moist vs. Dry Heat

Heat Resistance and Thermal Death of Spores and Vegetative Cells

• Bacterial endospores exhibit the greatest resistance

• Spore destruction requires temperatures above boiling

• Vegetative cells also vary in their heat sensitivity to both dry and moist heat

• Viruses fairly resistant to heat

WHY HEAT STERILIZATION?

• Heat – preferred choice

– Ease of use

– Controllability

– Cost

– Efficiency

HIGH TEMPRATURE

• High temperatures combined with high moisture is one of the most effective methods of killing microorganisms.

• Dry heat is used to sterilize surfaces, and materials which are not likely to break down in high heat and which do not contain any liquids, e.g., glass Petri dishes and culture vessels, and metal surgical instruments.

• Dry heat penetrates more slowly than moist heat which destroys microorganisms by coagulating their proteins and also destroys microorganisms by oxidizing their chemical constituents.

• Moist heat penetrates more quickly than dry heat, and is used to sterilize culture solutions and agar preparations, and to sterilize surgical instruments etc.

• Pressurized steam heat is needed to kill bacterial endospores, which can withstand boiling. Typically a pressure of 15 psi (pounds per square inch) is needed to create steam at a high enough temperature (121°C) to kill endospores. Spores of Clostridium botulinum are killed in within 20 minutes by moist heat at 120°C, whereas a 2-h exposure to dry heat at the same temperature is required.

THERMAL DEATH TIME AND DECIMAL REDUCTION TIME

• Thermal death time

• Decimal reduction time

The concept of decimal reduction timeThermal death time curve

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LOW TEMPRATURE

• Low temperature retards the growth of microorganisms by slowing their metabolism, but it does not always kill them and some bacteria (like Listeria) and fungi do grow at near freezing temperatures.

• Low temperatures are useful for preservation of cultures, since microorganisms have a unique capacity for surviving extreme cold.

• Refrigeration at 5° C retards the growth of many bacteria and fungi, freezing at – 10° to - 20° C (typical home freezer) is also an effective but not perfect means to retard microbial growth.

• Thus from a practical standpoint, high temperatures may be considered as microbicidal and low temperatures as microbistatic.

PHYSICAL METHOD: HEAT

• HEAT– STERILIZATION BY MOIST HEAT

• Moist heat is more efficient in contrast to dry heat; it causes coagulation and denaturation of proteins.

• At temperature below 100°C:– Pasteurisation: Food(dairy) Industry– Vaccine bath: (vaccine sterilisation)– Serum bath: (serum contaminants, does not kill spores survive)– Inspissation: (egg and serum containing media, can kill spores)

• At temperature 100°C:– Boiling: Boiling water (100°C)– Steam (100°C)

• At temperature above 100°C:– Autoclave

>100 OC

• Moist heat under pressure

– autoclave - most effective for sterilization (121oC, 15 mins.)

– pressure aids in penetration

– derived from thermal death curves for pathogens like Clostridium

– sterilization of surgical instruments, dressing and heat resistant pharmaceuticals

Autoclave

• Preparation of items for Autoclaving– containers should be unsealed

and articles should be wrapped in materials that allow steam penetration.

– Large packages of dressings and large flasks of media require extra time for heat to penetrate them.

– Wrapping objects in aluminum foil is not recommended because it may interfere with steam penetration

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Principle of Autoclaving

• A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally.

• For example, when free flowing steam at a temperature of 100oC is placed under a pressure of 1 atmosphere above sea level pressure – that is, about 15 pounds of pressure per square inch (Psi) --- the temperature rises to 121oC. Increasing the pressure to 20 psi raises the temperature to 126oC.

Working of Autoclave

• Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket where steam is maintained.

• As steam flows from the steam jacket into the sterilizing chamber, cool air is forced out and a special valve increases the pressure to 15 pounds/square inch above normal atmospheric pressure.

• The temperature rises to 121.5oC, and the superheated water molecules rapidly conduct heat into microorganisms.

• The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes.

• For denser objects, up to 30 minutes of exposure may be required.

Uses of Autoclave

• Sterilize culture media, Instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items that can withstand high temperatures and pressures.

• Sterilize bacteriological media and destroy pathogenic cultures.

• The autoclave is equally valuable for glassware and metalware

• Large industrial autoclaves are called retorts, but the same principle applies for common household pressure cooker used in the home canning of foods.

INSTRUMENT USED

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Indicator of Sterilization Achievement

• The word "sterile" or "autoclaved" appears on wrappings or tapes. These tapes are not fully reliable because they do not indicate how long appropriate conditions were maintained. Tapes or other sterilization indicators should be placed inside and near the center of large packages of determine whether heat penetrated them

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=100 OC

• Fractional sterilization– Fractional sterilization or tyndallization is a method used to destroy

bacteria and endospores in preparation of grain spawn(rye, wheat, millet, birdseed...) and agar, which requires no pressure cooker.In this case, the jars fitted with a filter disc or a polyfill lid filter are boiled or steamed at 212°F (100°C) for 30 min in a pot with lid, three days in a row. Between the boiling steps the jars are kept warm, around 30°C(but room temperature will work too), to allow the remaining endospores to germinate.

– Hear method involves heating the material at 100 oC on three successive days with incubation period in between.

• Boiling Water – for a few minutes can be used as a rapid emergency measure to

disinfect instruments – kills vegetative bacteria but not all spores

Limitations and Disadvantages of Autoclave

• The autoclave also has certain limitations. For example, some plasticware melts in the high heat, and sharp instruments often become dull. Moreover, many chemicals breakdown during the sterilization process and oily substances cannot be treated because they do not mix with water.

• Heat requires extra time to reach the center of solid materials, such as caned meats, because such materials do not develop the efficient heat-distributing convection currents that occur in liquids. Heating large containers also requires extra time. Table 3 shows the different time requirements for sterilizing liquids in various container sizes. Unlike sterilizing aqueous solutions, sterilizing the surface of a solid requires that steam actually contact it.

<100 OC

• Pasteurization

– done at 62.8 – 65.6°C for 30 minutes

– used for fluids

– reduce the number of bacteria

– eliminate pathogen present in small numbers

– improve shelf-like of milk

Pasteurization

• Used to disinfect beverages

• Heat is applied to liquids to kill potential agents of infection and spoilage, while retaining the liquid’s flavor and food value

• Special heat exchangers

– Flash method: expose to 71.6°C for 15 seconds

– Batch method: expose to 63°C to 66°C for 30 minutes

• Does not kill endospores or thermoduric microbes

• Pasteurized milk is NOT sterile

PHYSICAL METHOD: HEAT

• HEAT

– STERILIZATION BY DRY HEAT

• Causes denaturation of proteins and oxidative damage.

• Techniques include:

– Red Heat (common uses: straight wires, bacterial loops and spatulas)

– Flaming (Common uses: bacterial loops, wires and spatula’s)

– Incernation (common uses: soil dressing, pathological bedding)

– Hot Air oven (discovered by Louis Pasteur, common uses: in dairy industry)

– Infra red rays (common uses: heat glassware and metallic instruments)

Flaming

• bacterial loops, wires and spatula’s

• Microbiological loop in abunsen burner flameSterilising a wire loop usedfor transferring bacteriafrom liquid culture to anagar plate. Heating it in abunsen burner flame until itglows red hot ensures thatthere is no contaminationbetween samples

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Dry Heat: Hot Air and Incineration

• Incineration– Ignites and reduces

microbes to ashes and gas

– Common practice in microbiology lab-incineration on inoculating loops and needles using a Bunsen burner

– Can also use tabletop infrared incinerators

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Dry Oven

• Usually an electric oven

• Coils radiate heat within an enclosed compartment

• Exposure to 150°C to 180°C for 2 to 4 hours

• Used for heat-resistant items that do not sterilize well with moist heat

• Glassware, metallic instruments

INSTRUMENT USED

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PHYSICAL METHOD: RADIATION

• Radiation– There are 2 types of Radiation:– Non-ionizing: wavelength longer then visible light.– UV Radiation has a wavelength of 200-280nm; it has a germicidal

effect on microorganisms.– Common uses: Surface disinfection, in hospitals, operating theatre and

laboratories.

• Ionising: 2 types:– Particulate (Electron beam)– Common uses: sterilisation of instruments such as syringes, gloves,

dressing packs, foods and pharmaceuticals.– Electromagnetic (Gamma rays)– Common uses: sterilisation of disposable petri dishes, plastic syringes,

antibiotics, vitamins, hormones and fabrics.

Modes of Action of Ionizing Versus Nonionizing Radiation

• Ionizing: ejects electron, causing ions to form

• Nonionizing: excites atoms but does not ionize them

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Ionizing Radiation: Gamma Rays, X Rays, and Cathode Rays

• Cold sterilization – no heat

• Dosage of radiation- measured in Grays

• Exposure ranges from 5 to 50 kiloGrays

• Gamma rays, most penetrating; X rays, intermediate; cathode rays, least penetrating

Applications of Ionizing Radiation

• Food products

• Medical products

• Main advantages:– Speed

– Penetrating power

– No heat31

Nonionizing Radiation: Ultraviolet Rays

• Wavelength approximately 100 nm to 400 nm

• Germicidal lamp: 254 nm

• Not as penetrating as ionizing radiation

• Powerful tool for destroying fungal cells and spores, bacterial vegetative cells, protozoa, and viruses

Ultraviolet Radiation• Wavelength:

– 200-300 nm

• Poor penetrating power

• Pyrimidine dimers

• Mutations induced

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Applications of Ultraviolet Radiation

• Usually disinfection rather than sterilization

• Hospital rooms, operating rooms, schools, food prep areas, dental offices

• Treat drinking water or purify liquids

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RECOMMENDED USE OF HEAT TO CONTROL BACTERIAL GROWTH

APPLICATION OF HIGH TEMP DESTRUCTION OF MICROORGANSM

• Study a time-temp relationship affecting to reduce the microbial population to desired level

LOW TEMPERATURE

• Temperature s below the optimum for growth depress the rate of metabolism, if the temperature is sufficiently low, growth and metabolism ceases.

• It is use full for preservation of cultures• Initial freezing kills a fraction of the population, but the survival may

remain viable for long periods.• Method used:

– Regenerator : temp 4 to 7– Deep freezer: temp -20 to -70– Liquid nitrogen: temp -196

• Application – Preservation of tissue cell, preserving viruses and microorganism– Used in application of animal virology research

DESICCATION

• Desiccation of the microbial cell causes a cessation of metabolic activity, followed by a decline in the viable population.

• Species of Gram negative cocci are very sensitive to desiccation : die in one hr

• Streptococci are much more resistant• TB dries in sputum remains viable for even longer period of time.• Dried spores remain viable for indefinably.• The time of survival of microorganism after desiccation varies,

depending on following factors:– The kind of microorganism– The material in or on which the organisms are dried– The completeness of the drying process– The physical conditions to which the dried organism are exposed

Lyophilization

• Lyophilization, or freeze-drying, preserves microbes and other cells for many years by freezing a culture in liquid nitrogen and removing residual water via a vacuum. Lyophilization prevents the formation of large damaging ice crystals, leaving enough viable cells to enable the culture to be reconstituted many years later. This is useful when storing a bacterial culture for future use in a laboratory.

Process of lyophilization

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OSMATIC PRESSURE

• If cells are exposed to solutions with higher solute concentration, water will be drawn out of the cell and the process is called plsmolysis and the reverse process, which is the passage of water from a low solute concentration into the cell, is known as plasmoptysis.

• The pressure built up within the cell as a result of this water intake is termed osmotic pressure.

• Plasmolysis results in dehydration of the cell and as a consequence metabolic processes are retarded partially or completely. Due to the great rigidity of the microbial cell, the cell wall doesn't exhibit distortions as a result of plasmolysis, but shrinkage of protoplast and changes in the cytoplasmic membrane can be observed during plasmolysis.

• High concentrations of salt or sugar inhibit microbial growth by osmotic pressure.

• Hyperosmotic conditions can preserve foods, because they cause water to be drawn out of bacteria and fungi so that they cannot thrive.

Osmatic pressure: Mode of Action

• The use of high concentrations of salts and sugars to preserve food is based on the effects of osmotic pressure. High concentrations of these substances create a hypertonic environment that causes water to leave the microbial cell, this effect is also called as plasmolysis.

• Loss of water severely interferes with cell function and eventually leads to cell death. This process resembles preservation by desiccation, in that both methods deny the cell the moisture it needs for growth.

Application of osmatic pressure

• Jam and pickles are classicexamples because of theirhigh solute loading – thismakes jams and pickles highlyhyperosmotic to thecytoplasm of bacteria andfungi which forces water toleave the cells by osmosis,but some microbes (someyeasts in brine pickles, orsurface molds in jam) do growin hyperosmotic conditions.

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Limitations of osmatic pressure

• As a general rule, molds and yeasts are much more capable than bacteria of growing in materials with low moisture or high osmotic pressures.

• This property of molds, sometimes combine with their ability to grow under acidic conditions, is the reason fruits and grains are spoiled by molds rather than by bacteria.

• It is also part of the reason molds are able to form mildew on a damp wall or a shower curtain

FILTRATION Filtration is used to sterilize heat labile liquids and

gases. Filtration is the passage of air or a liquid through a material that traps and removes microbes. These filters are made of different materials. The mean pore diameter in these biological filters are available in several grades, based on the average size of pores.

Apart from porosity, other factors such as the electric charge of the filter, the electric charge carried by the organism, and the nature of the fluid being filtered, can influence the efficiency of filtration.

The development of high-efficiency particulate air (HEPA) filters has made it possible to remove microbes and particles from air and to deliver clean air. This type of air filtration together with a system of laminar airflow is now used to produce dust and bacteria free air.

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Filtration

• Pass liquid or gas through a filter with sufficiently small pore size

• HEPA – high-efficiency particulate air

• Advantages: No thermal damage

• Disadvantages: viruses not eliminated and must be either liquid or gas

Filter

Sterilized

fluid

(a)

Vacuum

Pump suction

Filter

Liquid

Pore

(b)

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

b: © Fred Hossler/Visuals Unlimited

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CHEMICAL AGENT

• CHEMICAL AGENT– ALKYLATING AGENTS

– OXIDIZING AGENTS

– PHENOLIC COMPOUNDS

– QUARTERNARY AMMONIUM COMPOUNDS

– ALCOHOLS

– HEAVY METALS

– HALOGEN

– DYES

– DETERGENT

– ALDEHYDES

– GASEOUS AGENT

Chemical Agents in Microbial Control

• Approximately 10,000 different antimicrobial chemical agents are manufactured

• Approximately 1,000 used routinely in health care and the home

• Occur in liquid, gaseous, or solid state

• Aqueous: dissolved in ure water

• Tinctures: solutions dissolved in pure alcohol or water-alcohol mixtures

Factors that Affect the Germicidal Activity of Chemicals

• Nature of microorganisms being treated

• Nature of the material being treated

• Degree of contamination

• Time of exposure

• Strength and chemical action of the germicide

CHARACTERISTICS OF AN IDEAL ANTIMICROBIAL AGENT

Antimicrobial activity Solubility Stability Non toxicity to human and other animal Homogeneity Non combination with extraneous organic material Toxicity to microorganism at room or body temperature Capacity to penetrate Non corroding and nonstaining Deodorizing ability Detergent capacities Availablity

SELECTION OF CHEMICAL AGENT FOR PRACTICAL APPLICATION

• Nature of the material to be treated

• Types of microorganism

• Environmental conditions

o Disinfectants may be:

High level disinfectants.

Intermediate level disinfectants.

Low level disinfectants.

Disinfection by chemical disinfectants

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Sterilization and disinfection77

High level disinfectio

n

• large number of spores after prolonged exposure

• Vegetative bacteria

• Tubercle bacilli

• Fungi

• Viruses

Intermediate level

disinfection

• Few number of spores

• Vegetative bacteria

• Tubercle bacilli

• Fungi

• Enveloped viruses (HBV, HIV)

Low level disinfectio

n

• Mainly vegetative bacteria

• Some fungi

• Narrow range of viruses

1. Prions (the most resistant)

2. Spores

3. Tubercle bacilli

4. Non enveloped viruses

5. Fungi

6. vegetative bacteria

7. Enveloped viruses such as HBV, HIV

Most resistant

Least resistant

Organisms according to the innate resistance

Germicidal Categories According to Chemical Group

• Halogen Antimicrobial Chemicals

– Fluorine, bromine, chlorine, and iodine

– Microbicidal and sporicidal with longer exposure

• Chlorine compounds

• Kills bacteria and endospores

• Also kills fungi and viruses

• Example: Household bleach

• Iodine compounds

• Topical antiseptic

• Disinfectant

Phenol and its Derivatives

• Phenol coefficient: compares a chemical’s antimicrobic properties to those of phenol

• High concentrations: cellular poisons

• Lower concentrations: inactivate certain critical enzyme systems

• Bisphenols

• Triclosan38

Chlorhexidine

• Complex organic base containing chlorine and two phenolic rings

• Targets cell membranes and protein structure

• At moderate to high concentrations, it is bactericidal for both gram-positive and gram-negative bacteria but inactive against spores

• Mild, low toxicity, rapid action

• Preparing skin for surgery, obstetric antiseptic

Alcohols as Antimicrobial Agents

• Only ethyl and isopropyl alcohols are suitable for microbial control

• Mechanism of action depends in part upon its concentration

• Does not destroy bacterial spores at room temperature but can destroy resistant vegetative forms

• More effective in inactivating enveloped viruses than nonenveloped viruses

Hydrogen Peroxide and Related Germicides

• Germicidal effects are due to the direct and indirect actions of oxygen

• Oxygen forms hydroxyl free radicals which are highly toxic and reactive to cells

• Can be harmful to tissue

• Bactericidal, virucidal, and fungicidal

• In higher concentrations is sporicidal

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Chemicals with Surface Action: Detergents

• Act as surfactants

• Cationic detergents are more effective because the positively charged end binds well with the predominantly negatively charged bacterial surface proteins

• Soaps are weak microbicides but gain germicidal value when mixed with agents such as chlorhexidine or iodine

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Heavy Metal Compounds

• Hg, Ag, Au, Cu, As, and Zn have been used• Oligodynamic action: having antimicrobial effects in

exceedingly small amounts• Bind onto functional groups of proteins and inactivating

them• Drawbacks to using metals in microbial control:

– Can be very toxic to humans– Often cause allergic reactions– Large quantities of biological fluids and wastes

neutralize their actions– Microbes can develop resistance to them

Germicidal Effect of Heavy Metals

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Aldehydes as Germicides

• –CHO functional group on the terminal carbon

• Glutaraldehyde and formaldehyde (formalin- aqueous solution)- most often used in microbial control

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Gaseous Sterilants and Disinfectants

• Ethylene oxide (ETO)

• Propylene oxide

• Chlorine dioxide

• Block DNA replication, protein function

Dyes as Antimicrobial Agents

• Primary source of certain drugs used in chemotherapy

• Aniline dyes (crystal violet and malachite green) are very active against gram-positive species of bacteria and various fungi

• Yellow acridine dyes (acriflavine and proflavine) sometimes used for antisepsis and wound treatment

• Limited applications because they stain and have a narrow spectrum of activity

Acids and Alkalis

• Very low or high pH can destroy or inhibit microbial cells

• Limited in applications due to their corrosive, caustic, and hazardous nature

Glutaraldehyde

o Available in 2% concentration (cidex).

o High to intermediate level disinfectant.

o Disinfection of instruments that can not withstand heat such as endoscopes.

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Sterilization and disinfection91

Chlorine releasing compounds

o Example of chlorine releasing compounds is:

Hypochlorite solution (such as household bleach)

o Intermediate level disinfectant.

o Widely used in homes, hospitals and laboratories to disinfect table tops, incubators, spilled cultures.

o Disinfection of water supply.

Quaternary ammonium compounds

o Example:

Cetavlon, savlon

o Low level disinfectant.

o Used to clean floors, walls.

o Inactivated by organic matter.

o Not affect Gram negative bacilli.

3/18/2015Dr. Emad AbdElhameed Morad

Main Ingredients Antimicrobial Products

Concentration and Time Needed

Mode of Action of Disinfectant

• Damage to cell wall

• Alteration of membrane function

• Damage to proteins

• Damage to nucleic acids

Alcohol

• Ethanol (80%), propanol (60%), and isopropanol (70%)

• Cidal activity drops sharply when diluted below 50% concentration

• Optimum bactericidal concentration is 60%–90% solutions in water (v/v)

• effective against bacteria and fungi, less so against viruses. They do not kill bacterial spores.

• Most feasible antimicrobial action– Denaturation of proteins

– Dissolve membrane lipids

• Absolute ethyl alcohol, a dehydrating agent, isless bactericidal than mixtures of alcohol andwater because proteins are denatured morequickly in the presence of water

• Alcohol destroys the dehydrogenases ofEscherichia coli, and that ethyl alcohol increasesthe lag phase of Enterobacter aerogenes.

• Isopropyl alcohol (isopropanol) is slightly morebactericidal than ethyl alcohol for E. coli and S.aureus

halogens

• Chlorine, iodine, and derivatives of thesehalogens are suitable for use as disinfectants.Chlorine and iodine show a generalizedmicrobicidal effect and also kill spores.

• Chlorine denatures proteins by binding tofree amino groups;

• hypochlorous acid (HOCl), is produced inaqueous solutions, then disintegrates intoHCl and 1/2 O2 and thus acts as a powerfuloxidant.

Inactivation by chlorine can result from

• oxidation of sulfhydryl enzymes and amino acids• ring chlorination of amino acids• loss of intracellular contents• decreased uptake of nutrients• inhibition of protein synthesis• decreased oxygen uptake• oxidation of respiratory components• decreased adenosine triphosphate production• breaks in DNA• depressed DNA synthesis

• Chlorine is used to disinfect drinking water and swimming-pool water (up to 0.5mg/l).

• Calcium hypochlorite (chlorinated lime) can be used in nonspecific disinfection of excretions. Chloramines are organic chlorine compounds that split off chlorine in aqueous solutions. They are used in cleaning and washing products and to disinfect excretions.

aldehyde• Formaldehyde is used as a disinfectant and

sterilant in both its liquid and gaseous states.

• Formalin - 37% formaldehyde by weight.

• The aqueous solution is a bactericide, tuberculocide, fungicide, virucide and sporicide .

• Mode of Action:– The mechanism of action of formaldehyde is based on

protein denaturation.

– Formaldehyde inactivates microorganisms by alkylating the amino and sulfhydral groups of proteins and ring nitrogen atoms of purine bases.

Use of formaldehyde

• Used to disinfect surfaces and objects in 0.5–5%solutions.

• In the past, it was commonly used in gaseousform to disinfect the air inside rooms (5 g/m3).

• Paraformaldehyde, a solid polymer offormaldehyde, can be vaporized by heat for thegaseous decontamination of laminar flowbiologic safety cabinets when maintenance workor filter changes require access to the sealedportion of the cabinet.

• Another aldehyde used for disinfection purposesis glutaraldehyde.

Ethylene oxide

• This highly reactive gas (C2H4O) is flammable,toxic, and a strong mucosal irritant.

• Ethylene oxide can be used for sterilization at lowtemperatures (20–60 8C). The gas has a highpenetration capacity and can even get throughsome plastic foils.

• This gas cannot kill dried microorganisms andrequires a relative humidity level of 40–90% in thesterilizing chamber.

• Ethylene oxide goes into solution in plastics,rubber, and similar materials, therefore sterilizeditems must be allowed to stand for a longerperiod to ensure complete desorption.

Phenol & Phenolic compounds• One common feature of phenolic substances is their weak

performance against spores and viruses.• Phenols denature proteins.• They bind to organic materials to a moderate degree only,

making them suitable for disinfection of excretedmaterials.

• Mode of Action• In high concentrations, phenol acts as a gross protoplasmic

poison, penetrating and disrupting the cell wall andprecipitating the cell proteins.

• Low concentrations of phenol and higher molecular-weightphenol derivatives cause bacterial death by inactivation ofessential enzyme systems and leakage of essentialmetabolites from the cell wall

Quaternary Ammonium Compounds • Some of the chemical names of quaternary ammonium

compounds used in healthcare are– alkyl dimethyl benzyl ammonium chloride,– alkyl dodecyl dimethyl ammonium chloride,– dialkyl dimethyl ammonium chloride.

• Mode of Action– inactivation of energy-producing enzymes,– denaturation of essential cell proteins,– disruption of the cell membrane

• Use - in ordinary environmental sanitation of noncriticalsurfaces, such as floors, furniture, and walls.

Heavy metals

• Hg, Ag, Cu

– Bind to -SH groups and cause protein denaturation.

• 1% silver nitrate for prevention of ophthalmia neonatorum (Neisseria gonorrhoeae)

Images:

1. http://finecrystoplast.com/pictures/Quality%20Control/Quality%20Control%203.jpg

2. https://www.naugraexport.com/science/Laboratory-Glassware.jpg

3. http://mikewilliamsonvalidation.files.wordpress.com/2014/07/vials.jpg?w=300&h=251

4. http://blog.globalknowledge.com/wp-content/uploads/2010/09/Photoxpress_1390512.jpg

5. http://www.bdl-cee.com/editor/image/eshop_products/eshop_products_types/4007860_m.jpg

6. http://i01.i.aliimg.com/photo/v0/434597507/falcon_tube.jpg_250x250.jpg

7. https://lh4.ggpht.com/U1kasMloWpROwg41zV_JY21hF_tTrbXYEn8yjzVSpsiOytRECF5y_PC3ZU-tRMOSlkXoRg=s149

8. https://figures.boundless.com/13234/full/disinfection-with-mop.jpe

9. http://kakbik.ru/wp-content/uploads/2014/03/vrach-250x166.jpg

10.http://www.contempclindent.org/articles/2011/2/4/images/ContempClinDent_2011_2_4_302_91793_f8.jpg

References

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12. https://lh5.ggpht.com/PDnjsA17MjULwBhdhZoXIf7l2gezcFE-ximjnlL4N_ZDDKbO1E6w5fXFGyH_HpnO534b=s85

13. https://lh3.ggpht.com/7PQew-hUPPIoMZW15igzrhVLP8e4zVY7PuYBTiRv3BffjpfrSAgjjJ-sXTbzASliSEgYY04=s170

14. https://lh6.ggpht.com/MW412SwFUwKtGACS6lF5N7iMyjBYYvUSzCaHwdLm_8-NKFTJ6dDRF_8crIg41sU_TSuHaQ=s85

15. http://www.austincc.edu/rlewis3/docs/lec08a_notes_control.pdf

16. http://4.bp.blogspot.com/-86EThnaVL-A/T3zpMW0OPPI/AAAAAAAAAFc/RQ7pe5aylRA/s200/cow95289_11_04.jpg

17. http://www.discoverbiotech.com/image/image_gallery?uuid=95160db9-9b70-49ca-a928-94306a01c793&groupId=11406&t=1353908326618

18. http://cdn.instructables.com/F89/801K/HF2444F2/F89801KHF2444F2.MEDIUM.jpg

19. http://www.puneetind.com/images/STEAM%20STERILIZER.jpg

20. http://2.imimg.com/data2/AA/XU/MY-3840018413/autoclave-cooker-type-stainless-steel-500x500.jpg

References

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22. http://www.steritecproducts.com/mobile/ci122.gif

23. http://wellcomeimages.org/indexplus/obf_images/c6/a8/60f8670ef1124b7b67a0fa7f0578.jpg

24. http://www.argos-tech.com/images/products/reusableloops.jpg

25. http://upload.wikimedia.org/wikipedia/commons/a/a7/Spatulas-lab.jpg

26. http://o.quizlet.com/xsLsa4QFciyAyllSGPLZ.Q.jpg

27. https://lh6.ggpht.com/dUZL2Rw2ndKQ8m-qArAsUqCViACGjHITm68Sz6OeeKE6f5JVVeHa5meY4VpWPx0hlmZV=s121

28. http://3.imimg.com/data3/OA/DA/MY-1498410/hot-air-ovens-250x250.jpg

29. http://www.jeffreythompson.org/blog/wp-content/uploads/2012/01/SterilizationForBacterialCulturesWithMomTatoo.gif

30. https://lh5.ggpht.com/L6kgEpRfrwqgx9CVnb9w832NlTMEFlgKQtr_aluda1jWFwIOgEhEGFwrxzxOnkzkY1cZ=s85

References

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31. https://albeiroumi.files.wordpress.com/2012/04/4411.jpg

32. https://lh6.ggpht.com/DJHXf5TpoFbWjLRdAYWGePoVufbRjXbD4dcmHItSz4OHGNpcxGIw2mUu1LBXe7sgWhuFwQ=s85

33. https://lh5.ggpht.com/a--e6XUI9OEIfxfmOwGw1kwqp_5b1zWzDAfpxL3FCr7mwqib2MfRhKLeQ4Oj-Z1A4r7FUKc=s109

34. http://3.imimg.com/data3/WK/IU/MY-5936247/embryos-cryopreservation-250x250.jpg

35. http://extension.usu.edu/duchesne/images/uploads/Website%20Pics/Canning%20Pic2.jpg

36. http://www.discoverbiotech.com/image/image_gallery?uuid=7063fb77-1e31-419e-9b73-639521361845&groupId=11406&t=1353909618265

37. https://lh6.ggpht.com/gXyjWU01i_gSNkd5H233yXqFylpeJjqBNcU5sC2h63smo8u4AveeinH_Xok1pDa4M9m0=s85

38. https://lh5.ggpht.com/5LZ02AzBKtQJPGBPxDxCEz0np0kA-VsLVY8ZTmIDt1k8yyXcWbZWEAnQ1431RBFWAURG7gw=s85

39. http://www.somatechnology.com/images/medium/steris_system_1_endoscope_sterilizers.jpg

References

Images:

40. https://lh4.ggpht.com/XNQPNpc4ya2boer7dZfAQ3ygZU2EO7pePrhYbyK0Z-QhgfitdckDqQRjCf6QfHnV0MzQAJQ=s107

41. https://lh5.ggpht.com/eZ2Gjv0jviJl9FIkv5CY5T9MF4Nrqsd2RRXmtem2t4H-AYYJgdebmqQGi_DJ8eXkWhmRvRg=s110

42. https://lh5.ggpht.com/LOkbHxjeeSQ-WVxsmTM3WAUnyVzn2LY2Ptbu4i7KA0FXirOF_ri14KQ8JNQ_nFWSUWJGA2s=s85

Websites:• http://generalbacteriology.weebly.com/sterilization-and-

disinfection.html• http://apps.who.int/phint/en/p/docf/• http://www.microrao.com/ugslides.htm• http://www.discoverbiotech.com/wiki/-

/wiki/Main/Physical+Methods+of+Microbial+Control;jsessionid=20E17C7B9AA81D34A4B37371FA2F2ECE

Book:• Microbiology by pelczar

References