8-Lab Sterilization

Lab 9

Sterilization and Disinfection

Sterilization : removing or killing all the microorganisms and viruses on an object or any material.
Disinfection : The use of toxic chemical substance to destroy all bacteria and viruses ( it may destroy spores ) , this process can be used with glass wares, bench tops and similar laboratory articles.
 a. Physical Sterilization
Sterilization
b. Chemical Sterilization

a. Physical Sterilization :

1. Heat : Sterilization by heat depend on time, temperature, number of m.o., species of m.o. and nature of material containing bacteria.
 Flaming
Dry heat : Red heat
Hot air oven

Flaming : This method is used for sterilization, the mouth of culture tube, needles, spatula, glass slide and cover. This method in valuing passing of articles, through the flame of benzene burner with out allowing to become red.
Red heat : It mean holding the instrument in the flam of Bunsen burner until they become red. ex. Loops, needles and spatula.
Hot air oven : This is used to sterilize articles at a temp of 170 Co for 1hr. ex. Dry glass ware, pipettes.


Sterilization at a temp. Below 100 Co
Moist heat : Sterilization at 100 Co
Sterilization at temp. Above 100 Co

Sterilization at a temp. Below 100 Co :

This method is used to sterilize serum, body fluid containing coagulable proteins. Since high temp. cause coagulation of serum proteins.
Temperature of 56 Co for 1hr. used to sterilize vaccines in special water bath.
Pasteurization : This method is used in food industries, the temp. in employed is 63 Co for 30min or 71.6 Cofor 15 min. and then cooling it imediately.Its not a method of sterilization but its a process used for preservation of milk and other fruit juces-------------, 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.
Moist heat ( bellow 100 Co ) do not kill endospores and and the thermophilic bacteria.


Boiling at 100 Co : Vegetative bacteria or non spore bearing qre almost killed immediately at a temperature of 90 - 100 Co but speculated bacteria like B. anthracis ,Cl. tetani and Cl. welchii require longer periods within autoclave ( at temp. above 100 Co )
moist heat, above 100 Co [ autoclave ]
The auto clave is as team pressure sterilizer. Water boils when it's vapor pressure equals that of surrounding atmosphere & if the pressure inside a closed vessel increased so that temperature at which the water boils raised above 100 Co, within an autoclave, steam pressure can build to 15 30 pound per square inch pressure, bringing the temperature up with it to 121 Co - 123 Co .
Under autoclave conditions, pressurize steam kills bacterial endospore, vegetative cells and other microbial forms quickly and effectively at temp. much lower and less destructive to materials than are required in a dry heat oven (160 170 ) Co .
Autocleaving destroys the microorganism present by coagulation of their proteins .

Table 1 Pressure Temperature Time relation shipsin steam . pressure sterilization .
Steam pressure pound per square inch (Aboveatmospheric pressure )TempTime ( minutes required to kill exposed heat resistant endospore )0100 Co____10115.5 Co15 6015121.5 Co12 1520126.5 Co5 1230134 Co3 5Sterilization control :
chemical test : By using browns tube, with each load changing the color from green to brown as indicator for sterilization.
Bacteriological control : of proper autoclave function are essential to ensure that sterilization is being achieved with each run of steam pressure sterilizer .
 The spore of Bacillus sterothermophilus is used as the test organism, this organism is a thermopiles with an optimum growth ( 55 60 ) Co and their spores require an exposure of 12 minutes at 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 Co , a second strip that has not been autoclaved is incubated in broth at the same time. The endospore on the control strips have germinated and changed the color of the PH of indicator in the broth, but the broth of autoclaved endospores ((remains the original color)) .


Filtration : fluid likely to be damaged by heat are sterilized by filtration (plasma, serum, eg yolk medium, certain carbohydrate fluids, toxins
Type of filers : Berkefield filters, Mandlers filters, Asbestos filters, Cellulose membrane filters.

Radiation :
Articles may be sterilized by radiation includes :
Disposable rupper gloves, disposable syringes and other plastic equipments.
 Non ionizing type ex. Ultra violet rays
Types of Radiations
Ionizing type ex. Gama rays and X rays

Mode of action :

Protein coagulation.
Disruption of cell membrane.
Removal of sit groups ( oxidizing agent )
Damage of DNA.
Inhibit of DNA replication.



Table 1

Some physical methods for control of micro organisms
AgentActionUse(Moist heat)CoagulationSterilization of bacteriological media, rubber gloves, syringes, tongue depressors, instruments.Autoclave or steam under pressurePasteurizationCoagulation changes in cellular proteinRemoving all pathogenic and some non-pathogenic organisms from milk.BoilingCoagulationGlass syringes and various equipment.(Dry heat)OxidationSterilization of empty glassware such as (test tubes, Petri dishes) , instrument, needles, syringes.Hot airRed heatBurning to ashesSterilization of inoculating loops, needle etc.(Radiation)Forms thymine dimmersReduces air borne infections in hospital, restaurants and school rooms.Ultra violetX raysIonization, peroxide formation. Used to induce mutationsDisposable gloves disposable syringes.FiltrationSeparation of bacteria from suspending fluidsSterilization of certain liquids injured by heat or chemical treatment separation of bacteria from toxins, enzymes.


Table 2
Some chemical agents for control of micro organisms

AgentActionPractical UsePhenol Destroy cell memb.Not generally effective against spores(Alcohols)Denaturation70% of alcohol is more effective that 100% because 70% was killing micro organisms by denaturation and dehydrationEthyl, Isopropyl(Halogens)HCIO (hydro chlorous acid)
NaCI0 (Sodium hypochlorite)Oxidation
Combines with protein to form protein halidesChlorine and compoundsIodineIodine active against spores, viruses and fungiMay be used to disinfect various equipment(Salts of hoary metals)Oxidation (toxic)Mercuric chloride skin antiseptic and preservative1. Mercuric chloride (Hgcl2)2. Silver nitrate (AgNo3)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 bacteria1. Crystal violet2. Acridine dyesAcridine appears to be enzymic inter-ferenceFor treatment of woundQuaternary ammonium compoundsDisrupts cell memb. inactivation of entzymes ,no effect of spores, denaturation of proteinsSkin anti sepsisFormaldehyde (HCHO)Alkylating agentMaybe 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)OxidationCleansing of woundsPotassium permanganate (KMno4)OxidationAnti microbial action on tissue surfacesEthylene oxideAlkylating agentSterilization of heat labile materials, effective against vegetative bacteria, spores and virusesGlutaraldehydeAlkylating agentUsed primarily on inanimate objects, effective against all forms of microbial life.
Disinfectants : A chemical agent that kill pathogenic and non pathogenic m.o. but not spores ( not necessary all microbial form ) generally applied to inanimate objects and not considered save for medical use disinfectants can be either 1. Germicides . 2. Micro static agents .
Germicides : Are chemical agents that kill all m.o. static activity : It's the antimicrobial agents merely inhibits the m.o. ( bacteria static, fungi static, virustatic ) .
Disinfectant that effectively vegetative bacteria may not destroy bacterial endo spores. Fungal conidia , tubercle bacilli or some viruses.
Tubercule bacilli are mor resistant than most other vegetative bacteria because of their waxy cell wall.
Other factor to consider when choosing a disinfectants include :
Temperature.
Time of exposure.
Concentration of m.o. present.
Concentration of antimicrobial agent.
PH for it's optimal activity.
Toxicity of the agent for skin or it's effect on material to be disinfected.
Disinfectant must kill m.o. while its in contact with them. So that they can not grow again when it is removed ( cidal, lethal ) or according to the type of m.o. it kills : bactericidal, fungicidal, virisidal and sporicidal.
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.
Various dyes used as antiseptic agents such as crystal violet.
Procedures :
The purpose of this method is to study the activity of some disinfectants and to learn the importance of time, germicidal, concentration and microbial species in disinfection.
Select one of the chemical agents provided. Add 5ml of the solution in to sterile test tube.
To 5ml of disinfectant, add 0.5ml of Esch . coli culture Gently shake the tube. Not the time.
Divide Nutrient agar plate in to 4 sections with a marking pen. (2, 5, 10, 15 ) minutes.
Transfer one loop ful of the disinfectant culture mixture to a section of the N.A. plate. Lable each plate with the name of m.o., the disinfectant, concentration ex. ( Esch.col : 1% phenol ) .
Incubate at 37o for 48 hours.


Result : Observe all plate. Section from growth ( + ) or absence of growth ( - ) .

Not : Gnoculate one half of anutrient ager plate directly from Esch. coli and the other half from the Staph . aureus culture, incubate at 37Co for 48 houre label each section of plate with the name of m.o. and control
DisinfectantCon.OrganismTime of exposurecontrolSodium hypochlorite5% E. coliStaph.
aureus2 min
5 minAlcoholAbsolute 70% E coliStaph.
aureus2 min
5 minHydrogen peroxide3%E. coliStaph.
aureus2 min
5 minMouth washE . coliStaph.
aureus2 min
5 min

Ecoli control

Staphi
control

Ecoli 2m Alcohol

Staphi15m
Alcohol