Sterilization: complete removal, destruction,
inactivation of all forms of microbial life
viruses,
Fungi,
bacteria,
Spores.
• Disinfection: elimination of most pathogenic
organisms excluding sporicidal activity. There
are 3 levels;
• HLD, all vegetating bacteria, fungi, all
viruses
• ILD, all vegetating bacteria, majority of
fungi, majority of viruses
• LLD, vegetating bacteria, some fungi, some
viruses
•Antiseptics: chemical disinfectants applied to
skin, mm but not systemic.
•Cleaning: removal of dust, dirt, organics, FM.
•Decontamination: general term applied to any
procedure by which microorganisms are
reduced to a level where items are safe to
handle
Patient’s care items are divided into 3
categories:
Critical items
Semi-critical items
Non-critical items
Depending on the risk of infection due to their utilization
Critical
Semi critical
Items enter -sterile
tissues-cavitiesvascular system
Items come in
contact with
intact mm &
non intact skin
e.g. needles,
implants, catheters,
surgical instruments
e.g. endoscopes,
endotracheal tube,
thermometer
Sterilization: EO,
autoclave,
plasma.
High level disinfectant,
glutaraldehyde, chlorine
active, hydrogen
peroxide
Noncritical
Items come in
contact with intact
skin
e.g.
sphygmomanometers,
bed linens, floors
Intermediate and low
level disinfectants,
alcohols, phenols
Decreasing order of resistance of microorganisms
to disinfection and sterilization
Resistant
•
•
•
•
•
•
•
Susceptible
•
•
Prions
Bacterial spores
Mycobacteria
Small non-enveloped
viruses
Gram-negative bacteria
Fungi
Large non-enveloped
viruses
Gram-positive bacteria
Lipid enveloped viruses
Decontamination Steps
I. Heat
1- Moist heat or steam sterilization
2- Dry heat sterilization
II. Low temperature (cold):
1. chemical
a. ethylene oxide
b. liquid steriliants: GLD, PAA, H2O2
2- plasma sterilizer
III. Other sterilizers:
1- ionizing radiation
2- filtration
3- microwaves; in pharmaceutical industries
• Heat is the most practical, efficient, and
inexpensive method of sterilization and
disinfection of objects that can withstand high
temperatures.
• There are two forms:
– Moist heat is much more efficient than dry heat because:
• it kills microorganisms by coagulating and denaturing
their enzymes and structural proteins
• it is quicker in heating up the article to be sterilized.
– Dry heat is believed to kill microorganisms by causing
destructive oxidation of essential cell constituents.
Autoclave
principle>> saturated water steam under high pressure.
•heating water under controlled conditions in a closed vessel in order
that boiling point of water rises above 100°C.
•Saturated steam carries the same temperature of boiling water.
It is essential to make steam saturated and free of any residual air because
air acts as an insulator, reduces temperature and hinders penetration.
Sterilization temperature and exposure time :
at 121°C for 20-30 minutes at double atmospheric pressure (2 bar)
Or
at 134°C for 3-6 minutes (at 3 bar).
• Steam sterilization is
the most common and
preferred method for
sterilizing those items
that can withstand high
temperature and
moisture,
• e.g. culture media,
surgical instruments
and dressings.
Autoclave:
Advantages:
1- Non-toxic
2- liberation of latent heat due to condensation of steam
3- Inexpensive
4- Rapidly microbicidal
5- Least affected by organic/inorganic soils
6- Rapid cycle time
7- Penetrates medical packing, device lumens
Disadvantages:
1- Deleterious for heat labile instruments
2- not suitable for powders and oils
Sterilization monitored routinely by
combination of mechanical,
chemical, and biological parameters
• Physical - cycle time, temperature,
pressure
• Chemical - heat or chemical sensitive
inks that change color when
germicidal-related parameters
present
• Biological - Bacillus spores that
directly measure sterilization
(bacillus stearothermophilus)
Chemical Indicators
I.
Incineration:
II.
Red heat:
•
•
For dead animal bodies
Infectious hospital waste
for wires, loops, tips of forceps
they are flamed till reach
redness
III. Hot air oven:
Dry hot air used either
170c for 60 min
or 160c for 120 min
Micro-incinerator
• Hot air oven
- They are electrical devices used in
sterilization.
-There is a digitally controlled
thermostat controlling the
temperature.
- Their double walled insulation keeps
the heat in .
- There is also an air filled space in
between to aid insulation.
- An air circulating fan helps in uniform
distribution of the heat.
Advantage of dry heat
1.Hot air penetrates certain substances that cannot be steam sterilized (oil,
powder).
2. Dry heat can be used in laboratories to sterilize glassware
3. Dry heat is a protective method of sterilizing, no erosion or corrosion.
4. Instruments that cannot be disassembled may be sterilized in hot air.
5. Low cost
Disadvantages of dry heat
1. A long exposure period is required( because hot air penetrates slowly
possibly unevenly. )
2. Time and temperature vary for different substances.
3. Over exposure may ruin some substances.
4. It is destructive to fabrics and rubber goods
Monitored by B.subtilis spores.
• Ethylene oxide gas is commonly used to sterilize objects
sensitive to temperatures greater than 60 °C such as
plastics, optics and electrics.
• Ethylene oxide penetrates well, moving through paper,
cloth, and some plastic films and is highly effective.
• Ethylene oxide sterilizers are used to process sensitive
instruments which cannot be adequately sterilized by other
methods
• Advantages
– Very effective at killing microorganisms
– Penetrates medical packaging and many plastics
– Compatible with most medical materials not
withstanding heat
– Cycle easy to control and monitor
• Disadvantages
1. highly flammable
2. requires a longer time to sterilize than any heat treatment
3. The process also requires a period of post-sterilization
aeration to remove toxic residues.
Monitored by B. subtilis spores
1- Gamma rays (Co-60).
2-High energy electrons (electron accelerator).
This process does not result in rise in temp; but the
time required for sterilization is long (48hrs).
High penetration power so used in prepacked items, as gloves,
syringes, plastic IV lines,…
Monitored by B.pumilus spores
Filtration: Fluids
• Sterilization of fluids which would not
withstand heat such as antibiotic
solutions, blood products, hormones,
vitamins….etc.
• Fluids can be rendered free of bacteria by
passage through filters with a pore size of
less than 0.45 µm.
– However, most viruses and certain bacteria as
mycoplasma can pass through filters with a
pore size as small as 0.22 µm.
• Early filters made from glass and asbestos
– largely replaced by membrane filters made of
cellulose esters or other polymers.
• Membrane filters are preferred because:
– they filter more rapidly.
– they don't affect the filtrate in any way.
– they adsorb very little of the substance being filtered.
• The endopigment producing Serratia
marcescens can be used to test the efficiency of
bacterial filters.
• Filters can be used to remove microorganisms
from air supplied to critical areas such as
operating rooms, drug factories and laminar flow
ventilation system.
• A properly installed high efficiency particulate air
(HEPA) filter can achieve 99.997% arrest to
particles ≥ 0.5 µm, and can produce sterile air.
• Spores of the fungus Aspergillus can be used to
test the efficiency of HEPA filters.
Filtration: Air
1- Chemical disinfection
2- Boiling water
3- Pasteurization
4- Ultraviolet irradiation
Chemical disinfection
1- Alcohol 70% (ethyl and isopropyl).
2- Biguanides (chlorhexidine).
3- Chlorine- active compounds (hypochlorite, house hold
bleach).
4- Iodine based compounds
5- Phenol containing preparations.
6- Quternary ammonium compounds .
7- Hydrogen peroxide
8- Peracetic acid
9- Glutaraldehyde(cidex) and Formaldehyde
1- Alcohol 70% (ethyl and isopropyl):
Exposure time: 5 min at least
Uses: 1- disinfection of
a. external surfaces
b. non invasive instruments
2- skin antiseptic, alone or added to iodine or
chlorhexidine
2- Biguanides (chlorhexidine):
1- antiseptic for skin and mm, mouth wash (hexitol)
2- combined with
. Alcohol as hand rub
. Detergent as hand wash
3- Chlorine- active compounds (hypochlorite, house
hold bleach):
1- decontamination of blood splashes and lab.
Working surfaces (100-1000ppm)
2- linen bleaching
3- disinfection of water
4- Iodine based compounds (most effective antiseptics):
1.
2.
3.
4.
5.
include - tincture iodine
(iodine in alcohol), staining and irritant
- iodophores (iodine with neutral carrier):
slow release of free iodine.
more penetration.
no staining .
not irritant,
betadine (30-50ppm)used as skin antiseptic in surgical scrubbing
and before surgery.
Both halogens exert their bactericidal action by irreversible
oxidation of the essential enzymes by the free radicals
5- Phenol containing preparations (phenol\lysol) ILD:
Used for floors, walls and furniture
Hexaphenol (detol) was used as antiseptic, was proven to
cause neurological damage
6- Quternary ammonium compounds LLD:
detergent either .anionic as soap
or . Cationic as cetylethyl-ammonium chloride.
Used for routine cleaning of walls, floors or furniture.
7- Hydrogen peroxide: (oxidizing agent) HLD& sterilants
1- antiseptic for open wounds( kill anaerobes)
2-disinfection of soft contact lenses, some endoscopes
8- Peracetic acid: (oxidizing agent) HLD& sterilants
HLD, strilizant of instruments as endoscopes
9- Glutaraldehyde(cidex) and Formaldehyde HLD& sterilants:
GLD: HLD, sterilizant of instruments as endoscopes,
respiratory and anaesthesia equipments
FLD: gas not used now, found to be carcinogenic, only used in
sterilization of HEPA filters.
Evaluation methods of disinfectants:
To evaluate an antiseptic or disinfectant, the phenol coefficient
test is used.
Phenol coefficient test (Rideal Walker
method)
Principle:
It is a measure of the bactericidal activity of a chemical
compound in relation to phenol.
Phenol Coefficient Test is done by measuring the concentration
at which a chemical is equal in effectiveness to phenol.
a. If a chemical is equal in effectiveness to phenol at the same
concentration, its phenol coefficient is 1.
b. If the concentration of the chemical to be tested must be
twice that of phenol, its phenol coefficient is 1/2.
c. If the solution is less concentrated than the phenol standard,
its phenol coefficient is greater than 1.
Phenol Coefficient = dilution of chemical
dilution of phenol
• Phenol Coefficient Test
– A series of dilutions of phenol and the experimental
disinfectant are inoculated with Salmonella typhi or
Staphylococcus aureus .
– Samples are removed at 2.5 min intervals and inoculated
into fresh broth.
– The cultures are incubated at 37°C for 2 days
– The highest dilution that kills the bacteria after a 7.5min
exposure, but not after 5 min, is used to calculate the
phenol coefficient
– The reciprocal of the maximum effective dilution for the
test disinfectant is divided by the reciprocal of the
maximum effective dilution for phenol to get the phenol
coefficient
– For example:
Suppose that, on the test with Salmonella typhi
The maximum effective dilution for phenol is 1/90
The maximum effective dilution for “Disinfectant X” is
1/450
The phenol coefficient for “Disinfectant X” with
S. typhi = 450/90 = 5
EVALUATION OF THE EFFICACY OF AN
ANTISEPTIC BY RIDEAL WALKER TEST
• Materials:
– Staph. aureus suspension
– 5ml of phenol (1:95)
– 5 ml of different dilutions of the
antiseptic under
test.(1:50,1:100,1:150……………….
– 4 sterile broth tubes for each dilution.
– Loop
– 1sterile pipette 1 ml
Methods:
• Add 0.2 ml of S.aureus suspension to each of the dilutions of the
phenol and antiseptic leaving 30 seconds intervals between each
tube.
• At 2.5 minutes intervals inoculate one loopful of each of the previous
dilution tubes into a sterile broth tubes.
• Shake all the tubes and incubate at 37oC for 2 days.
• Record the results as – if there is no growth and + if growth occurs.
• Calculate the phenol coefficient of the given antiseptic.
Results:
Time of
Sampling
(min)
phenol
1:95
1:50
Antiseptic
1:100
1:150
1:200
2.5
+
_
+
+
+
5
+
_
_
+
+
7.5
_
_
_
_
+
10
_
_
_
_
_
Boiling at 100c for 20 min = HLD ……Only in emergencies
Three methods of pasteurization
• Low temperature (62°C) for long time (30 minutes), rapid
cooling.
• High-temperature-short-time (HTST) method (72°C for 15
seconds) rapid cooling .
• Ultra-high-temperature (UHT) method (140-150°C for few
seconds) rapid cooling.
UV is a low energy, non ionizing irradiation
Its present in sun, artificially produced by mercury
lamps
Over exposure may cause eye damage and skin
irritation
Have extremely weak penetration power
Used only for:
Air and surface disinfection in
Operating rooms
Safety cabinet
Importance of bacterial
count
Various methods are used in
microbiology to measure the
numbers of microorganisms per unit
volume of a given sample
This measurement is needed
for:1. Standardization of inocula in
microbiological assay
• [e.g. evaluation of antimicrobial
agents, assay of vitamins]
1. Industrial fermentation.
2. Evaluation of sterilization
technique.
How to determine bacterial
growth?
1. Direct microscopic count:
Used to determine the number of
both dead and living bacterial cells
(haemocytometer).
2.Turbidimetricdetermination
(viable & dead)
• Increased turbidity in a culture is
another index of bacterial growth
and cell numbers.
The increase in the number of cells
during growth increases the
turbidity.
Turbidimetric determination is done
using a spectrophotometer.
SPECTROPHOTOMETER
 Measure growth rates with a
spectrophotometer
– Measure living and dead cells
• Gives immediate assessment of the
number of cells in a population.
• Direct relationship between cell number and
absorbance, Otherwise known as Optical Density
 More bacteria= higher absorbance.
 less light reaches sensor
 Cells scatter light, not absorb light
How to USE THE SPECTROPHOTOMETER
 Bacterial suspensions are measured at wave
length equals 600 nm .
 A clear solution will allow almost all of the
light through (BLANK).
 Light entering a cloudy solution will be
absorbed.
 The amount of absorbance obtained
measures what fraction of light passes
through a given solution and compared to
that absorbed by a clear solutio
The amount of
 cells in the solution is directly
proportional to the
absorbance reading
(linear relationship)
 A graph of absorbance vs.
concentration will give a straight line.
Dry weight and nitrogen content
determinations:
. In this method, the bacterial cells are
collected by centrifugation, then
dried in an oven overnight at 85℃.
• The dry weight of bacterial mass will
be proportional to their number.
• Also the nitrogen content of the dry
sample can be determined by micro
kjeldahl method
MICRO KJELDAHL METHOD
• Bacterial cells are heated with conc H2SO4
• Ammonia is liberated.
• Ammonia is distilled & captured in boric
acid solution.
• Titrate with 0.01N HCL
• Using methyl red- bromo cresol green as
indicator.
4. Measurement of microbial
activity
• Many microbial activity measured
quantitatively and used as a measure
of microbial growth.
e.g; the growth of acid forming
bacteria may be measured by simple
titration of the culture using
standard alkali.
5. Viable count of bacteria:
• in this method only viable cells which
are capable of reproduction are
counted.
• Principle:
Based on the fact that if the viable
cells are allowed to grow apart from
each other on a solid medium, each
cell develops into one visible colony.
The number of colonies obtained is
equal to the number of viable cells.
Viable count of bacteria
• There are different procedures used
to determine viable bacterial
count:a. Pour plate method.
b. Spread plate method.
c. Surface viable count
A. pour plate method
• In this method different dilutions are done from
•
•
•
•
the bacterial suspension.
1 ml of each dilution is then poured on a sterile
empty Petri dish.
15 ml of melted nutrient agar whose
temperature is about 45oC is poured in each
plate with swirling.
The plates are left to dry and then incubated at
37oC for 48 hours.
The plates containing number of colonies
between 30-300 only are counted to eliminate
error,Calculate CFU/ml by multiplying number of
colonies by the dilution factor.
B. Spread plate technique
In this method different dilutions are done from
the bacterial suspension.
Sterile nutrient agar plates are prepared for
each dilution.
1 ml of each dilution is then poured on the agar
plates.
Using a glass or metal spreader- previously
sterilized by dipping it in alcohol and flaming-the
bacterial suspension is then uniformly spread on
the plate
The plates are left to dry and then
incubated at 37oC for 48 hours.
The plates containing number of colonies
between 30-300 only are counted to
eliminate error.
 Calculate CFU/ml by multiplying number
of colonies by the dilution factor.
C. Surface viable technique
Materials:
Culture of S. aureus.
 Nutrient agar plate.
Ringer solution.
3 Test tubes.
Sterile 3ml pipettes.
Sterile 10ml pipette.
Sterile Pasteur pipette
9 ml ringer solution
1ml
1ml
1ml
bact.
Susp.
R.S
S
1
1:10
2
1:100
3
1:1000 dilution
1 drop
in each
sector
Don’t Invert and incubate
After incubation
Each sector
from 10-30
colonies
Count the colonies in each sector
and record the results
Result
• Count the number of colonies on
each sector in the plate which are in
the range of 10-30.
• Over 30 reported as TNTC.
• Under 10 reported as TFTC.
Result
Sector number
No. of colonies /sector
1
a
2
b
3
c
4
d
Result
No of cell / 1ml of original culture
(cfu/ml)=( ( a+b+c+d )/4 )X20X
dilution factor