Sterilization

Sterilization and Disinfection

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Sterilization: Killing or removing all forms of microbial life (including spores) in a material or an object.
Disinfection: Reducing the number of bacteria and other m.o. as far as possible (but not bacterial spores). , this process can be used with glass wares, bench tops and similar laboratory articles. Thus, disinfection can never replace sterilization May use physical or chemical methods for these tech.

Why is Infection Control Important in Dentistry?

Both patients and dental health care personnel (DHCP) can be exposed to pathogens. Contact with blood, oral and respiratory secretions, mucosa of the eyes, nose, and contaminated equipment occurs. Proper procedures can prevent transmission of infections among patients and DHCP.


Disinfectants : A chemical agents that kill pathogenic and non pathogenic m.o. Not spores. generally applied to inanimate objects. Included: 1. Germicides . 2. Micro static agents . Germicides : Are chemical agents that kill all m.o. Micro static agents : It's the antimicrobial agents merely inhibits the m.o. ( bacteria static, fungi static, virus static). - Disinfectant that affect vegetative bacteria may not destroy bacterial endospores. Fungal conidia , tubercle bacilli or some viruses. Tubercule bacilli are more resistant than most other vegetative bacteria because of their waxy cell wall.


Antiseptics: Are similar to disinfectants but may be applied safely on biological tissue. Alcohol is more effective than soap and water to reduce m.o. on the skin surface Iodine is another antiseptic agent, hilling m.o. including spores.
Sterilization
a. Physical Sterilization
b. Chemical Sterilization


Several factors influence the effectiveness of Sterilization. 1. Number of Microbes: The more microbes present, the more time it takes to eliminate population. 2. Type of Microbes: Endospores are very difficult to destroy. Vegetative pathogens vary widely in susceptibility to different methods of microbial control. 3. Nature of material containing m.o : Presence of organic material tends to inhibit antimicrobials, pH etc. 4. Time of Exposure: Chemical antimicrobials and radiation treatments are more effective at longer times. In heat treatments, longer exposure compensates for lower temperatures.

a. Physical Sterilization :

Heat Filtration Radiation


a. Physical Sterilization : Heat : Kills microorganisms by denaturing their enzymes and other proteins. Thermal Death Point (TDP): Lowest temperature at which all of the microbes in a liquid suspension will be killed in ten minutes. Thermal Death Time (TDT): Minimal length of time in which all bacteria will be killed at a given temperature. heat can be divided to: Flaming Red heat Hot air oven

Flaming : This method is used for sterilization, the mouth of culture tube,, spatula, glass slide and covers by passing of articles, through the flame of benzene burner with out allowing to become red.


Red heat : It mean holding the instrument in the flame of Bunsen burner until they become red. ex. Loops, needles and spatula.

Sterilization at a temp. Below 100 Co

Sterilization at 100 Co Sterilization at temp. Above 100 Co
2- Moist heat :Kills microorganisms by coagulating their proteins. - In general, moist heat is much more effective than dry heat.
Moist heat :


Sterilization at a temp. Below 100 Co : This method is used to sterilize serum, body fluid containing coagulable proteins. Temperature of 56 Co for 1hr. used to sterilize vaccines in special water bath. Pasteurization :This method Developed by Louis Pasteur to used for preservation of milk and fruit juices.. Classic Method of Pasteurization: Milk was exposed to 65oC for 30 minutes. High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 seconds.


Ultra High Temperature Pasteurization (UHT): Milk is treated at 140oC for 3 seconds and then cooled very quickly in a vacum chamber. Advantage: Milk can be stored at room temperature for several months. this method will destroy non spore forming pathogenic bacteria . Such as : Brucella abortus Mycobacterium tuberculosis Salmonella typhi and other species of bacteria that may be found.

2- Sterilization at a temp 100 Co (boiling): Heat to 100oC. Kills vegetative forms of bacterial pathogens,almost viruses, and fungi within 10 minutes or less. but some viruses and sporulated bacteria like B. anthracis ,Cl. tetani and Cl. welchii require longer periods within autoclave to kill them.3- Sterilization above 100 Co [ autoclave ]: Autoclave: Closed chamber which is filled with hot steam under pressure. The pressure inside a increased, so that temperature at which the water boils raised above 100 Co . steam pressure can build to 15 – 30 pound per square inch pressure, bringing the temperature up with it to 121 Co - 123 Co . coagulation their proteins .


Under autoclave conditions, pressurize steam kills bacterial endospore, vegetative cells and other microbial forms quickly and effectively at temp. much lower than dry heat oven (160 – 170 ) Co for 1-2 hr.All organisms and endospores are killed within 15 minutes. Steam pressure pound per square inch (Above atmospheric pressure )
Temp
Time ( minutes required to kill exposed heat resistant endospore )
0
100 Co
____
10
115.5 Co
15 – 60 15
121.5 Co
12 – 15 20
126.5 Co
5 – 12 30
134 Co
3 – 5


Sterilization control : A. chemical test : By using browns tape, with each load changing the color from green to dark brown as indicator for sterilization
Autoclave tape - colour change in treated (bottom) indicator, right temperature has been reached.


b.Biological indicators : Use organism Bacillus sterothermophilus for this test, This organism is a thermophiles with an optimum growth ( 55 – 60 ) Co and their spores require 121 Co to be killed. Strips containing endospores are placed in the autoclave with materials to be sterilized.after the autoclave cycle is completed each strip is placed into broth medium and incubated at 56 CoThese spores are heat resistant. If the autoclave did not reach the right temperature, and second strip that has not been autoclaved is incubated in broth at the same time. the spores will germinate, and their metabolism will change the colour of a pH-sensitive chemical from rose to brown.

Unprocessed

Positive For growth
Negative for growth


Filtration: Removal of microbes through a screen like material with small pores.Used to sterilize heat sensitive materials like vaccines, enzymes, antibiotics, and some culture media. Membrane Filters: Uniform pore size. Used in industry and research. Different sizes:0.22 and 0.45nm Pores: Used to filter most bacteria. Don’t retain spirochetes, mycoplasmas and viruses.0.01 nm Pores: Retain all viruses and some large proteins.High Efficiency Air Filters (HEPA):Used in operating rooms to remove bacteria from air.


Radiation: Three types of radiation kill microbes: 1. Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nanometer). Used to sterilize pharmaceuticals, disposable medical supplies. Disadvantages: Penetrates human tissues. May cause genetic mutations in humans.


2. Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. Used to disinfect operating rooms, nurseries, cafeterias. Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth.

Forms of Radiation

3. Microwave Radiation: Wavelength ranges from 1 millimeter to 1 meter. Heat is absorbed by water molecules. May kill vegetative cells in moist foods. Bacterial endospores, which do not contain water, are not damaged by microwave radiation.

Mode of action : Protein coagulation. Disruption of cell membrane.

Removal of sit groups ( oxidizing agent ) Damage of DNA. Inhibit of DNA replication.

Some physical methods for control of micro organisms

Agent
Action
Use
(Moist heat)
Coagulation
Sterilization of bacteriological media, rubber gloves, syringes, tongue depressors, instruments.
Autoclave or steam under pressure
Pasteurization
Coagulation – changes in cellular protein Removing all pathogenic and some non-pathogenic organisms from milk.
Boiling
Coagulation
Glass syringes and various equipment.
(Dry heat)
Oxidation
Sterilization of empty glassware such as (test tubes, Petri dishes) , instrument, needles, syringes.
Hot air
Red heat
Burning to ashes
Sterilization of inoculating loops, needle etc.
(Radiation)
Forms thymine dimmers
Reduces air borne infections in hospital, restaurants and school rooms.
Ultra violet
X rays
Ionization, peroxide formation. Used to induce mutations
Disposable gloves disposable syringes.
Filtration
Separation of bacteria from suspending fluids
Sterilization of certain liquids injured by heat or chemical treatment separation of bacteria from toxins, enzymes.

Some chemical agents for control of micro organisms

Agent
Action
Practical Use
Phenol
Destroy cell memb.
Not generally effective against spores
(Alcohols)
Denaturation
70% of alcohol is more effective than 100% because 70% was killing micro organisms by denaturation and dehydration
Ethyl, Isopropyl
(Halogens)
HCIO (hydro chlorous acid) NaCI0 (Sodium hypochlorite)
Oxidation Combines with protein to form protein halides
Chlorine and compounds
Iodine
Iodine – active against spores, viruses and fungi May be used to disinfect various equipment
(Salts of hoary metals)
Oxidation (toxic)
Mercuric chloride – skin antiseptic and preservative 1. Mercuric chloride (Hgcl2)


Silver nitrate – eye drops and lotion 1% solution is used to kill gonococcal infection in new born infants

(Dyes)

Combine with protein or interfere with reproductive mechanism
Inhibit G+ and isolation of G- pathogenic bacteria
1. Crystal violet
2. Acridine dyes
Acridine appears to be enzymic inter-ference
For treatment of wound
Quaternary ammonium compounds
Disrupts cell memb. inactivation of entzymes ,no effect of spores, denaturation of proteins
Skin anti sepsis
Formaldehyde (HCHO)
Alkylating agent
Maybe used to kill M. tuberculosis in sputum. Used in preparing vaccines Gas maybe used to disinfect rooms. Preservation of specimens. Alcoholic solution for instruments.
Hydrogen peroxide (H2o2)
Oxidation
Cleansing of wounds
Potassium permanganate (KMno4)
Oxidation
Anti microbial action on tissue surfaces
Ethylene oxide
Alkylating agent
Sterilization of heat labile materials, effective against vegetative bacteria, spores and viruses
Glutaraldehyde
Alkylating agent
Used primarily on inanimate objects, effective against all forms of microbial life.
Some chemical agents for control of micro organisms

Procedures :

The purpose of this method is to study the activity of some disinfectants and to learn the importance of Time and microbial species in disinfection.
1- Select one of the chemical agents provided. Add 0. 5ml of the solution in to sterile test tube. 2-To 0.5ml of disinfectant, add 0.0.5ml of Esch. coli or Staph. aureus culture Gently shake the tube. Note the time. 3- Divide Nutrient agar plate in to 4 sections with a marking pen. (2, 5, 10, 15 ) minutes.

4-Transfer one loopful from the mixture of disinfectant culture to a section of the N.A. plate. Lable each plate with the name of m.o., time and the disinfectant, concentration. ex. ( Esch.col : 1% phenol ) . 5- Incubate at 37o for 48 hours.
Result : Observe all plate. Section from growth ( + ) or absence of growth ( - ) .

Disinfectant

Con.
Organism
Time of exposure
control
Sodium hypochlorite
5%
E. coli
Staph. aureus
2 min 5 min 10min 15min
Staph aureus E. coli
Alcohol
Absolute 70%
E coli
Staph. aureus
2 min 5 min 10min 15min
Staph aureus E. coli
Hydrogen peroxide
3%
E. coli
Staph. aureus
2 min 5 min 10min 15min
Staph aureus E. coli
Mouth wash
E . coli
Staph. aureus
2 min 5 min 10min 15min
Staph aureus E. coli