Download Lab 9 Sterilization and Disinfection Sterilization : removing or killing

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 C o
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 it’s 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
Time ( minutes required to kill
Temp
pressure )
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 tube, with each load changing the
color from green to brown as indicator for sterilization.
b. 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 C o , 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
Agent
Action
(Moist heat)
Autoclave or steam under
Sterilization
Coagulation
Coagulation –
changes in
cellular protein
Boiling
Coagulation
(Dry heat)
bacteriological
media,
rubber gloves, syringes, tongue depressors,
Removing all pathogenic and some nonpathogenic organisms from milk.
Glass syringes and various equipment.
Sterilization of empty glassware such as
Oxidation
Hot air
of
instruments.
pressure
Pasteurization
Use
(test tubes, Petri dishes) , instrument,
needles, syringes.
Red heat
(Radiation)
Ultra violet
Burning to
ashes
Sterilization of inoculating loops, needle
etc.
Forms thymine Reduces air borne infections in hospital,
dimmers
restaurants and school rooms.
Ionization,
peroxide
X rays
formation. Used Disposable gloves disposable syringes.
to induce
mutations
Separation of
Filtration
bacteria from
suspending
fluids
Sterilization 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
Agent
Action
Destroy cell
Phenol
memb.
Practical Use
Not generally effective against spores
70% of alcohol is more effective that
(Alcohols)
Denaturation
Ethyl, Isopropyl
100% because 70% was killing micro
organisms
by
denaturation
and
dehydration
(Halogens)
HCIO (hydro
chlorous acid)
Chlorine and compounds
NaCI0 (Sodium
hypochlorite)
Iodine – active
Iodine
against spores,
viruses and fungi
Oxidation
Combines with protein to form protein
halides
May be used
to
disinfect
various
equipment
(Salts of hoary metals)
1.
Mercuric
chloride
Oxidation (toxic)
(Hgcl2)
Mercuric chloride – skin antiseptic and
preservative
Silver nitrate – eye drops and lotion 1%
2. Silver nitrate (AgNo3)
solution is used to kill gonococcal
infection in new born infants
(Dyes)
Combine with
Inhibit G+ and isolation of G- pathogenic
protein or interfere
1. Crystal violet
bacteria
with reproductive
mechanism
Acridine appears to
2. Acridine dyes
be enzymic inter-
For treatment of wound
ference
Disrupts cell memb.
inactivation of
Quaternary
ammonium entzymes ,no effect
compounds
of spores,
Skin anti sepsis
denaturation of
proteins
 Maybe used to kill M. tuberculosis in
sputum.
Formaldehyde (HCHO)
Alkylating agent
 Used in preparing vaccines Gas maybe
used to disinfect rooms.
 Preservation of specimens.
 Alcoholic solution for instruments.
Hydrogen peroxide (H2o2)
Oxidation
Potassium
Oxidation
permanganate
(KMno4)
Alkylating agent
Ethylene oxide
Cleansing of wounds
Anti microbial action on tissue surfaces
Sterilization of heat labile materials,
effective against vegetative bacteria,
spores and viruses
Alkylating agent
Glutaraldehyde
Used 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.
1. Select one of the chemical agents provided. Add 5ml of the
solution in to sterile test tube.
2. To 5ml of disinfectant, add 0.5ml of Esch . coli culture Gently
shake the tube. Not the time.
3. Divide Nutrient agar plate in to 4 sections with a marking pen. (2,
5, 10, 15 ) minutes.
4. 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 ) .
5. 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
Disinfectant
Sodium
hypochlorite
Alcohol
Hydrogen
peroxide
Mouth wash
Con.
5%
Organism
E. coli
Absolute
70%
3%
E coli
E. coli
E . coli
Time of
exposure
Staph.
2 min
aureus
5 min
Staph.
2 min
aureus
5 min
Staph.
2 min
aureus
5 min
Staph.
2 min
aureus
5 min
control
Ecoli
control
Staphi
control
Ecoli 2m
Alcohol
Staphi15m
Alcohol