1. 2 s.shatizadeh malekshahi m.sc student in virology

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S.Shatizadeh MalekshahiM.Sc Student in Virology

Historical Review (1):

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Semmelwise recognized a connection between autopsies and puerperal fever in

1847. After testing a few hypotheses , he found that the number of cases was

drastically reduced if the doctors washed their hands carefully before dealing with

pregnant woman.

Joseph lister pioneered antiseptic techniques in surgery. He used carbolic acid

sprays to decontaminate surgical wounds as he worked in 1865.

Louis pasteur : He developed the technique of pasteurization (heating wine to 60

°C in the absence of oxygen for 30 minutes ) to kill unwanted organisms.

Historical Review (2):

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Another giant on the scene in the development of microbiology and medicine was

Robert Koch . He ushered in the era of modern bacteriology with sterile technique,

pure cultures and solid media.

In the era of disinfection , he wrote in 1881 a comprehensive research paper titled

simply “on Disinfection”. It examined the ability of over 70 chemicals at different

concentrations , and at different temperatures to kill anthrax spores.

Definitions of terms (1):

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Many different terms are used for disinfection and sterilization. The following are

among the more common in biosafety :

sterilization : A process that kills and/or removes all classes of microorganisms

and spores.

Disinfection : A physical or chemical means of killing microorganisms ,but not necessarily spores. Antiseptic : A substance that inhibits the growth and development of microorganisms without necessarily killing them, and usually applied to body surfaces.

Definitions of terms (2):

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Decontamination : Any process for removing and/or killing microorganisms . The same term is also used for removing or neutralizing hazardous chemicals and radioactive materials.

Pasteurization : A process of heating milk, wine or other liquids to around 60 °C for

about 30 minutes to significantly reduce or kill the number of pathogenic organisms.

Sanitizer : A chemical agent typically used to reduce bacterial numbers. Sanitation

may simply refer to through washing with only soap or detergent.

Categorizing of sterilization procedures :

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Disinfectants:

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Disinfectant

chemical disinfectants-general consideration :

Must not be toxic . Must be chemically inert to an object . Must be stable . Must have rapid disinfection effect . Must have broad spectrum of effectiveness .

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only a small number of instances are simultaneousbactericidal, sporocidal, virucidal and fungicidal

Anti microbial power of chemical disinfectants depend on :

1) Concentration of antimicrobial agent

2) Time of exposure

3) pH

4) water hardness

5) Organic matter

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physical sterilization

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Autoclave (Steam sterilization)1:

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An autoclave is a pressurized device designed to heat aqueous solutions above

their boiling point to achieve sterilization.

This means that all bacteria, viruses, fungi, and spores are inactivated. However, prions , may not be destroyed by autoclaving at the typical 121 °C for 15 minutes or 134 °C for 3 minutes. But, 134 °C 18 minutes !

Under ordinary circumstances (at standard pressure), liquid water cannot be heated above approximately 100 °C in an open vessel.

Further heating results in boiling, which is the transition from liquid to gas, but does not raise the temperature of the liquid water.

Autoclave (Steam sterilization)2:

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Sterilization by autoclaving is invariably successful if properly done and if two rules are followed:

What can be autoclaved :

Surgical Instruments

Glassware

Plastic tubes and pipette tips

Solutions and water

Oven (dry heat sterilization) 1:

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The Dry-Heat sterilization process is accomplished by conduction; that is where

heat is absorbed by the exterior surface of an item and then passed inward to the

next layer the entire item reaches the proper temperature needed to achieve

sterilization.

Dry heat coagulates the proteins in any organism, causes oxidative free radical

damage, causes drying of cells and can even burn them to ashes, as in incineration.

Oven (dry heat sterilization) 2:

It is usually used to sterilize metal objects and glassware and is the only

suitable means of sterilizing oils and powders.

Objects are sterilized by dry heat when subjected to :

An open flame is a form of dry heat used to sterilize inoculating loops.

when flaming objects in the laboratory , you must avoid the formation of

floating ashes and aerosols ( droplets released into the air ).

These substances can be a means of spreading infections agents if the

organisms in them are not killed by incineration.

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Ionizing radiation : X-rays , which have wavelength of 1 to 40 nm, and gamma rays , which have even shorter wavelength , are forms of ionizing radiation, so named because it can dislodge electrons from atoms, creating ions.

Ionizing radiation damages DNA and produces peroxides , which acts as powerful oxidizing agents in cells.

It is used to sterilize plastic laboratory and medical equipment and pharmaceutical products.

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Ultraviolet radiation :

UV light consists of light of wavelengths between 210 and 328 nm, but wavelength of

260 nm is most effective in killing microorganisms .

UV lamps are used to sterilize workspaces and tools used in biology laboratories and

medical facilities

UV light causes adjacent thymine molecules on DNA to dimerize . It is especially effective

in inactivating viruses . It kills far fewer bacteria than one might expect because of DNA

repair mechanisms.

UV light is of limited use because it does not penetrate glass, cloth, paper or most other

material , and it does not go around corners .

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Filtration (1):

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Filtration is the passage of a material through a filter, or straining device.

Filtration has been used since Pasteur’s time to separate bacteria from media

and to sterilize materials that would be destroyed by heat.

They are usually made of nitrocellulose and have the great advantage that

they can be manufactured with specific pore sizes from 25 µm to less than 0.025

µm.

Some filters can be attached to syringes so that materials can

be forced through them relatively quickly.

Filtration (2):

In the manufacture of some vaccines that require the presence of live viruses ,

it is important to select a filter pore size that will allow viruses to pass through

the filter but prevent bacteria from doing so.

Cellulose acetate filters with extremely tiny pores are now available and are

capable of removing many viruses from liquids.

High- efficiency particulate air (HEPA) filters are used in the ventilation

systems of areas where microbial control is especially important such as in

operating room and luminar flow transfer hoods in laboratories .

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Filtration (3):

Pore sizes of membrane Filters and Particles That Pass Through Them :

Pore Size (in µm)

Particles That Pass Through Them

10 RBC, Yeast, Bacteria, Viruses

5 Yeast, Bacteria, Viruses

3 Some yeast cell, bacteria, viruses

1.2 Most bacteria , viruses , molecule

0.45 A few bacteria, viruses

0.22 Viruses

0.10 Medium-sized to small viruses

0.05 Small viruses

o.o25 Only the very smallest viruses 20

Chemical sterilization

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Hydrogen Peroxide :

Oxidizing agents disrupt disulfide bonds in proteins and thus disrupt the

structure of membranes and proteins.

Hydrogen peroxide (H2O2) , which forms highly reactive superoxide ( O2 -), is

used to clean puncture wounds (6%) .

When Hydrogen peroxide breaks down into oxygen and water, the oxygen

kills obligate anaerobes present in the wounds.

Another Oxidizing agent , a potassium permanganate , is used to disinfect

instruments and ,in low concentrations ,to clean skin.

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Ethylene oxide (1) :

Gaseous ethylene oxide has extraordinary penetrating power.

Used at a concentration of 500 mg/l at 50 °C for 4 hours, it sterilizes rubber

goods, mattresses, plastics, and other materials destroyed by higher temperatures.

Also NASA has used ethylene oxide to sterilize space probes that might otherwise

carry earth microbes to other planets!

After exposure to ethylene oxide , Article such as catheters and rubber tubing

must be thoroughly flushed with sterile air to remove all traces of this toxic

gas

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Ethylene oxide (2) :

It is exceedingly important that workers be protected from ethylene

oxide vapors , which are toxic to skin, eyes and mucous membranes

and may also cause cancer.

Both the toxicity and flammability of ethylene oxide can be reduced

by using it in gas that contains 90% carbon dioxide.

Antimicrobiologic activity of ETO is due to alkylation of sulfhidryl, amino, carboxyl, phenolic and hydroxyl groups in the spore or vegetative cell.

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Formaldehyde :

Formaldehyde inactivates viruses and toxins without destroying their

antigenic properties .

The mechanisms of action of formaldehyde is assumed to be due to the

reaction with cell proteins and DNA or RNA.

Formaldehyde solution is too irritant to be used as a general disinfectant.

Althought formaldehyde is a high-level disinfectant, the hospital uses of

formaldehyde are limited by its irritating fumes and the pungent odor.

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Glutaraldehyde (Cidex) :

o Usually used as a 2% alkaline-buffered solution at room temperature .

o Has a Wide range of bactericidal, virucidal and fungicidal activity.

o Slow activity against bacterial spores.

o Active against mycobacteria, but slower against mycobacteria avium-intracellular.

o Irritant to skin, eyes and respiratory mucosa .

o Little inactivation by organic matter, but penetrate slowly

o Most preparations are non-damaging to metals and other materials.

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Disinfectants

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Alcohol (1) :

Overview : In the healthcare setting, “alcohol” refers to two water-soluble chemical compounds— ethyl alcohol and isopropyl alcohol.

These alcohols are rapidly bactericidal rather than bacteriostatic against vegetative forms of bacteria.

They also are tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores.

The optimum bactericidal concentration is 60%–90% solutions in water.

Mode of Action. The most feasible explanation for the antimicrobial action of alcohol is denaturation of proteins

The bacteriostatic action was believed caused by inhibition of the production of metabolites essential for rapid cell division.

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Alcohol (2) :

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Microbicidal Activity : The bactericidal activity of various concentrations of ethanol was examined against a variety of microorganisms in exposure periods ranging from 10 seconds to 1 hour.

Pseudomonas, Serratia , Ecoli and Salmonella were killed in 10 seconds by all concentrations of ethanol from 40% to 100% .

virocidal activity : Ethyl alcohol, at concentrations of 60%–80%, is a potent virucidal agent inactivating all of the enveloped viruses (e.g., herpes, vaccinia, and influenza virus) and some non-enveloped viruses .

Alcohol (3) :

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Virocidal action of Ethyl and Isopropyl Alcohols

virus Ethyl Time of Exposure

Isopropyl

Time of Exposure

Poliovirus 70% 2 min 95% 10 min

Coxackie B 60% 10 min 95% 10 min

Adenovirus 50% 10 min 50% 10 min

Herpes Simplex 30% 10min 20% 10 min

Influenza 70% 2 min 30% 1o min

Vaccinia 70% 2 min 30% 10 min

Alcohol (4) :

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It now appears that high concentration alcohol mixtures(i.e.80% ethanol + 5%

isopropanol )are not only excellent antibacterials, but quickly inactivate HIV as

well as HBV and HCV.

Alcohols are commonly used topical antiseptics .

They are also used to disinfect the surface of medical equipment .

Alcohols require time to work and they may not penetrate organic material .

chlorine & chlorine compounds (1):

Ca(OCl) 2+ H2O Ca ++ + H2O+ 2OCl-

Ca(OCl) 2 + 2H2O Ca(OH)2 + 2HOCl

How HOCl destroy microorganisms?

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Mechanism of Chlorine Disinfection :

Early workers suggesting that hypochlorous acid was responsible for the

destruction of microorganisms. When elemental chlorine or hypochlorite are

added to water , they undergo the following reactions: Cl2 + H2O HOCl + H++Cl-


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Hypochlorites are the most widely used of the chlorine disinfectants and are

available in a liquid (e.g. sodium hypochlorite) or solid (e.g. calcium

hypochlorite) form.

The most common chlorine products are aqueous solutions of 4 to 6% sodium

hypochlorite, which are readily available as “household bleach”

Broad spectrum of antimicrobial activity

Unaffected by water hardness

Inexpensive and fast acting


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Some Disadvantages:

1- At the concentration used in household bleach (4-6%) may produce skin

and ocular irritation or oropharygeal, esophageal, and gastric burns .

2-Corrosiveness to metals in high concentrations (>500 ppm).

3-Inactivation by organic matter .

4-Release of toxic chlorine gas when mixed with

ammonia or acid .

Iodine And Iodophor (1):

Iodine , is able to penetrate the cell wall of microorganisms rapidly.

It can be assumed that iodine reacts

1- with basic N-H functions that are parts of some amino acids and the bases of nucleotides .

2- By oxidizing the S-H group of the amino acid cycteine, through which the ability of connecting protein chains by disulfide bridge , as an important factor in the synthesis of proteins, is lost.

The most important application of iodine in human medicine is the disinfection of skin.

The previously used aqueous iodine have been replaced by the iodophors , which cause less unwanted side reactions, such as staining and irritation of tissue.

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Among the investigated iodophors, povidone-iodine has been described as the

compound of choice.

An iodophor is a complex of elemental iodine with a carrier that has at least

three functions: 1- To increase the solubility of iodine 2- To provide a sustained

release reservoir of the halogen 3- To reduce the equilibrium concentration of

free molecular iodine.

The carriers are neutral polymers, such as polyvinyl alcohols, polyacrylic acid,

polyamids and poly saccharides.

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Practical applications of iodine as a disinfectant :

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Scope of application

concentration

Exposure time

Disinfective result

Drinking water

8ppm 10 min Kill all water born pathogens

General germicidal action

1:20000 1 min Most bacteria are killed

General germicidal action

1:200000 15 min Will destroy all vegetative form of bacteria

Disinfection of skin

1% 90 sec Will kill 90% of the bacteria

phenol (1) :

Phenol is commonly found in mouthwashes, scrub soaps and surface

disinfectants, and is the active ingredient found in household disinfectants.

Phenol disrupt cell membranes, denature proteins and inactivates enzymes.

When used on surfaces, it retains its antimicrobial action for several days.

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phenol (2) :

The addition of halogens to phenolic molecules usually increases their

effectiveness .

Hexachlorophene and dichlorophene , which are halogenated phenols, inhibit

staphylococci and fungi, respectively , on the skin and elsewhere.

Hexachlorophene is an excellent skin disinfectant. In a 3% solution, it kills

staphylococci and most other gram positive organisms.

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Quaternary Ammonium Compounds :

QAC or quats , have four organic groups, attached to a nitrogen atom.

A variety of quats are available as disinfecting agents, their chemical structures

vary according to their organic groups .

One problem with quats is that their effectiveness is decreased in the presence

of calcium or magnesium ions .

An even more serious problem with these agents is that they support the

growth of some bacteria of the genus Pseudomonas rather killing them.

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Dettol (parachlorometaxylenol) 1 :

• The key ingredient which defines its unique antiseptic property is an aromatic

chemical compound known as chloroxylenol

• This makes up 4.8% of Dettol's total mixture, with the rest composed of pine

oil, isopropanol, castor oil ,soap, caramel and water.

• Because several of the ingredients are insoluble in water, Dettol produces a

white emulsion of oil droplets when diluted during use.

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Dettol (parachlorometaxylenol) 2 :

Apart from its low toxicity and low metal corrosivity, it is also relatively cheap

compared to other disinfectants.

It is effective against gram positive/negative bacteria, viruses, fungi and yeast.

It is able to kill 98% of microbes in just 15 seconds.

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Soaps and detergents (1) :

Soaps and detergents remove microbes, oily substances and dirt.

In fact vigorous hand-washing is one of the easiest and cheapest means of preventing the spread of disease among patients in hospitals, in medical and dental offices.

Soaps contain alkali and sodium and will kill many species of Streptococcus , Micrococcus, Niesseria and will destroy influenza viruses.

Many pathogens that survive washing with soap can be killed by a disinfectant applied after washing .

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Soaps and detergents (2) :

A common practice after hand-washing and rinsing hands and inanimate

objects is to apply 70% alcohol solution.

Cationic detergent although not effective in killing endospores, they do

inactivate some viruses.

Anionic detergents are used for as household cleaning agents. They are less

effective sanitizing agents than cationic detergents, probably because the

negative charges on bacteria cell walls repel them.

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Spill clean up procedure (1) :

In the event of a spill of infectious or potentially infectious materials, the

following clean up procedures should be used.

1- Wear gloves and protective clothing , including face and eye protection .

2- Cover the spill with cloth or paper towels to contain it.

3- Pour an appropriate disinfectant over the paper towels and the immediately

surrounding area ( generally, 5% bleach solutions are appropriate ).

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Spill clean up procedure (2) :

4- Apply disinfectant concentrically beginning at the outer margin of the spill

area, working toward the center.

5- After the appropriate amount of time , clear away the materials.

6- Dispose of contaminated materials into a leakproof , puncture-resistant waste

disposal container.

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Ethidium Bromide (1) :

Ethidium bromide is a large, flat basic molecule that resembles a

DNA base pair .

Because of its chemical structure, it can intercalate (or insert)

into a DNA strand. The compound forms fluorescent complexes

with nucleic acids and these can be viewed under UV light.

Ethidium bromide (EtBr) is widely employed for the rapid visualization of

nucleic acids in electrophoretic gels.

It may be harmful by inhalation, ingestion, and skin absorption and should be

handled only when wearing gloves .

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Disposal of Ethidium Bromide :

Correct procedure for the disposal of EtBr , depend on the nature of the waste

materials and the concentration of EtBr that they contain.

Treatment of EtBr with hypochlorite (bleach) is not recommended as the

degradation products retain approximately 20% mutagenic activity .

Solid Waste :

Small amounts of solid waste, such as tissues, gloves or stained electrophoresis

gel , should be placed in appropriate packaging and sent for incineration .

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– Concentrated Liquid Waste ( Containing > 0.5mg/ml) :

1) Dilute solution with water to reduce the EtBr concentration to < 0.5 mg/ml

2) To the diluted solution , add 0.2 volume of fresh 5% hypophosphorous acid and

0.12 volume of fresh 0.5M sodium nitrite .

3) Incubate 24 hours at room temperature. ( A check for loss of fluorescence can

be used to monitor completion of the inactivation process.) Add a large excess

of 1M sodium bicarbonate before discarding.

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– Dilute Liquid Waste ( Containing < 0.5mg/ml, e.g. electrophoresis buffer)

1) Add 100mg powdered active charcoal to each 100ml solution.

2) Keep at room temperature for 1 hour, shaking intermittently.

3) Filter through a whatman No.1 filter

4) Wrap the filter and charcoal in a plastic bag. Place inside a bag and send for

incineration.

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Cleaning of Equipment and Laboratory Surfaces Contaminated With EtBr:

1) Unplug all electrical equipment before decontamination and wear appropriate

protective equipment , including rubber gloves, lab coat and goggles.

2) Make up decontamination solution just prior to use. This consist of 4.2 g of sodium

nitrite and 20 ml hypophosphorous acid (50%) in 300 ml H2O.

3) Wash the contaminated surface once with a paper towel soaked in the

decontamination solution , taking care to avoid wetting electrical components. Then

wash five times with water-soaked paper towels using a fresh towel each time.

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Cleaning of Equipment and Laboratory Surfaces Contaminated With EtBr:

4) Soak all the towels in decontamination solution for 1 hour before disposal by

incineration.

5) Use a portable UV lamp to check that decontamination is complete. EtBr

absorbs a broad range of UV light, so short (245nm), medium(300-315nm) or

long(365-6nm) wavelength lamps can be used. Appropriate eye protection

must be worn to guard the user against UV light while the lamp is switched on.

6) Neutralize the used decontamination solution with sodium bicarbonate and

discard as aqueous waste. 52

10 Ways to Work Rnase Free (1) : Working with RNA? What fun! Those little, nearly indestructible RNases are

everywhere - on your skin and mucous membranes, in the water and on lab

surfaces. Here are 10 ways to keep the RNases at bay, and keep your precious

samples safe:

1. Clean everything; bench surfaces, pipettes, electrophoresis equipment and

anything else you can think of with an RNase cleaning product, such as

RNaseZap from Ambion (or 0.5% SDS followed by 3%H2O2). Establish a

regular cleaning routine; a quick daily clean and a deeper weekly or monthly

clean… and stick to it.

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10 Ways to Work Rnase Free (2) :

2. Treat your solutions. Good old DEPC is a fine way to keep your solutions

RNase free. Use 0.5 mL DEPC/L, incubate for 2 hr, autoclave for 45 minutes

minimum.

3. Designate a workspace, and a set of pipettes, if possible, that are dedicated to

RNase-free work.

4. Use barrier tips. Barrier tips stop cross-contamination of your reagents and

samples by preventing aerosols reaching the barrel of your pipette. They are a

must-have for RNA work.

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5. Wear gloves and a lab coat. The obvious ones are the best. Gloves and a lab

coat will stop you from contaminating your samples with your own RNases.

6. Bake your glasswear. No enzyme can withstand baking for 300°C for 2 hours,

but your glasswear can.

7. Use RNase-free enzymes. Enzymes isolated from bacteria (e.g. DNase) can be

full of RNase. Make sure you use certified RNase-free enzymes on your RNA

samples where possible.

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8. Use an RNase inhibitor when it’s not possible to keep things completely RNase-free.

9. Store RNA in ethanol at -80°C. Make aliquots if the sample is to be used a number of

times to avoid freeze/thaw cycles. Before use, centrifuge to pellet the RNA, air dry

then resuspend in an RNase-free buffer.

10. Be completely paranoid, work as far away from your colleagues as possible,

and shower in RNaseZAP five times per day. Just kidding !

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Microbiology , principles and explorations, Jacquelyn G. Black.

Disinfection in Healthcare, Petter hoffman.

Antiseptic and Disinfectants: Activity, Action, and resistance. Gerald Mcdonnel

and A.denever russel.

Mechanisms of action of disinfectants, S.P.Denyer, B. Stewart.

Antiviral Activity of alcohol for surface disinfection, WR Moorer.

Disinfection Sterilization and Preservation , Lea & Febiger Philadelphia

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References (1) :

گندزداها و ضدعفونی کننده ها ، دکتر کرامت الله ایماندل

راهکارهای مقابله با میکروارگانیسم با استفاده از ضدعفونی کردن و استریلیزاسیون ، فاطمه فالح- گیتا اسالمی

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References (2) :

59Thanks for your attention

1. 2 s.shatizadeh malekshahi m.sc student in virology

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