STERILIZATION AND

DISINFECTION-II

Dr Harender
MAMC, AGROHA
CHEMICAL
STERILIZATION
 A TRIBUTE TO IGNAZ SEMMELWEIS

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 1847- Hungarian physician, an
early pioneer of antiseptic
procedures. Semmelweis
proposed the practice of 
 Washing Hands
 with chlorinated lime
solutions in 1847 while working
in Vienna General Hospital's
First Obstetrical Clinic

3
A HYGIENIC AND SCIENTIFIC HAND
WASHING CONTINUES TO BE BEST

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PRAYER IN THE HOSPITAL

4
IDEAL ANTISEPTIC OR DISINFECTANT

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• An ideal antiseptic or disinfectant should have following
properties:

• Should have wide spectrum of activity

• Should be able to destroy microbes within practical period

of time

• Should be active in the presence of organic matter

• Should make effective contact and be wettable

• Should be active at any pH 5

•
 IDEAL ANTISEPTIC ……….

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• Should be stable, speedy, have long shelf life.

• Should have high penetrating power.

• Should be non-toxic, non-allergenic, non-irritative or

non-corrosive.

• Should not have bad odour.

•

6
 2/13/2020 10:21 AM
IDEAL ANTISEPTIC …….
• Should not leave non-volatile residue or stain.

• Efficacy should not be lost on reasonable dilution.

• Should not be expensive and must be available easily.

•

• Such an ideal
disinfectant is not yet
available. 7
CLASSIFICATION OF DISINFECTANTS

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• 1. Based on consistency
• a. Liquid (E.g., Alcohols, Phenols)
• b. Gaseous (Formaldehyde vapour, Ethylene oxide)

• 2. Based on spectrum of activity

• a. High level
• b. Intermediate level
• c. Low level

• 3. Based on mechanism of action

• a. Action on membrane (E.g., Alcohol, detergent)
• b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)
• c. Oxidation of essential sulphydryl groups of enzymes (E.g.,
H2O2, Halogens)
• d. Alkylation of amino-, carboxyl- and hydroxyl group (E.g.,
Ethylene Oxide, Formaldehyde)
• e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)
8
 2/13/2020 10:21 AM
9

 Photo: Biosafety in Microbiological and Biomedical Laboratories 5th Edition (2009); p330
 CLASSIFICATION BASED ON SPECTRUM OF ACTIVITY

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Vegetative M. Tb Spores Fungi Viruses
bacterial cells

Ethylene Oxide,
Gluteraldehyde,
High + + + + + Formaldehyde,
level Hydrogen
peroxide,
Peracetic acid

Interme Phenolics,
diate + + _ + + halogens
level

Alcohols,
Quaternary
Low + _ _ + +/- ammonium10
level compounds
 PHENOL

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 Mode of action: Act by disruption of membranes, precipitation
of proteins and inactivation of enzymes.

 Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol),

hexachlorophene, chlorhexidine, chloroxylenol (Dettol)

 Applications: Joseph Lister used it in 1865.

 Coal-tar derivatives
 Disinfectants at high conc. and as antiseptics at low concentrations.
 Bactericidal, fungicidal, mycobactericidal but are inactive against
spores and most viruses.
 Not readily inactivated by organic matter.
 Corrosive phenolics are used for disinfection of ward floors, in discarding
11
jars in laboratories and disinfection of bedpans ( E.g Lysol).
20% Chlorhexidine gluconate solution is used for pre-operative
hand and skin preparation and for general skin disinfection, mouth

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wash.
GPC > GNB, good fungicidal. very less active against viruses,
No activity against spores & tubercle bacilli,
Mixed with quaternary ammonium compounds e.g cetrimide to
get stronger and broader antimicrobial effects (eg. Savlon).

Chloroxylenols ( Dettol) are less irritant & less toxic. used for
12
topical purposes. Readily inactivated by organic matter.
 GPC> GNB, No activity against Pseudomonas.
 2/13/2020 10:21 AM
 Hexachlorophene is chlorinated diphenyl and
is much less irritant, potentially toxic & should
be used with care.
 GPC > GNB,very less active against viruses
,tubercle bacilli,fungi.

 Disadvantages:
 It is toxic, corrosive and skin irritant.
 Chlorhexidine is inactivated by anionic soaps.
 Chloroxylenol is inactivated by hard water.

13
 ALCOHOL

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• Mode of action: Alcohols dehydrate cells,
disrupt membranes and cause denaturation of
protein.

• Examples: Ethyl alcohol, isopropyl alcohol and

methyl alcohol

• Disadvantages: Skin irritant, volatile

(evaporates rapidly), inflammable. No action
on spores

14
• Application:
70% aqueous solution is more effective at

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•
killing microbes than absolute alcohols.
70% ethyl alcohol (spirit) is used as
antiseptic on skin.

• Isopropyl alcohol is preferred to ethanol.

Used to disinfect surfaces, disinfect
clinical thermo-meters.
•

• Methyl alcohol kills fungal spores,

hence is useful in disinfecting
inoculation hoods.
15
 ALDEHYDES

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Mode of action: Acts through alkylation of amino-,
carboxyl- or hydroxyl group, and probably damages
nucleic acids. It kills all microorganisms, including spores.

Examples: Formaldehyde, Gluteraldehyde, Ortho-

Phthaldehyde(OPA)

Disadvantages:
1) Vapours are irritating (must be neutralized by
ammonia),
2) has poor penetration,
3) leaves non-volatile residue,
4) activity is reduced in the presence of protein.
5) Gluteraldehyde requires alkaline pH and only those 16

articles that are wettable can be sterilized.

 FORMALDEHYDE

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 Application:
 40% Formaldehyde -surface disinfection and
fumigation of rooms, chambers, OT, biological
safety cabinets, wards, sick rooms, bedding,
furniture and books etc.

 10% formalin with 0.5% tetraborate sterilizes

clean metal instruments.

 2% formaldehyde at 400C for 20 minutes is used

to disinfect wool and
 0.25% at 600C for 6 hrs to disinfect animal hair
17
and bristles.
 2% GLUTERALDEHYDE

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 less toxic and irritant to eye & skin.

 used to sterilize corrugated rubber

anaesthetic tubes, thermometers,
cystoscopes, Endoscope, bronchoscopes,
centrifuges, etc.

 An exposure of at least 20 min at 200C at

alkaline pH is required for action by
gluteraldehyde.

 Available as cidex. 18

 Glutaraldehyde monitoring
 2/13/2020 10:21 AM
ORTHO –OPHTHALDEHYDE ( OPA)
 High level disinfectant.

 More stable during storage.

 OPA vapours may irritate resp. tract,

& eye.

 Can be used in place of

glutaraldehyde.

19
 HOW TO USE CIDEX® OPA SOLUTION 4 Disinfection 6 Dry
Immerse clean, dry instru- ments completely in the CIDEX OPADry the instruments. Dis- infected equipment should be used
Solution. immediately or stored in a manner to mini- mize recontamination.
See package insert for complete instructions/information on drying
C IDEX® OPA Solution, containing 0.55% Following cleaning, rinse
instrument surfaces and
flexible endoscopes when using potable water
ortho-phthalaldehyde, is a fast and effective
lumens with large amounts of instrument manufacturer’s labeling for additional storage and/or
way to high level disinfect a wide range of
fresh water to remove residual handling instructions.
endoscopes and other semi-critical devices.
Follow these steps and refer to the specific detergent.
CIDEX OPA Solution label and package insert 7 Testing
for complete instructions/information. It is recommended that CIDEX OPA for Solution
rinsing. Refer to the
be tested before
Ensure all instruments are completely submersed in CIDEX OPAeach usage with CIDEX OPA Solution Test Strips to verify that
Solution, and if applicable, fill all lumens. the appropriate concentration of ortho-phthalaldehyde is present.
CIDEX OPA Solution must be discarded after 14 days even if
1 Don Personal Protective Equipment CIDEX OPA Solution Test Strips indi-
Personal protective equip- ment must always be worn when Remove excess moisture from
handling contami- nated instruments and equipment. Personal instruments prior to the Minimum Effective
protec- tive equipment includes gloves, eye protection and disinfection. This will help Concentration (MEC).
fluid-repellent gown. Contact with CIDEX OPA Solution may prevent excess water from 8 Disposal
discolor skin or stain clothing. If the solution diluting the CIDEX OPA
contacts skin, wash with soap and water for a few min- utes. Solution below its Minimum Cover the CIDEX Solution Tray with a secure lid. SoakCIDEX OPA Solution can be discarded down hospital and office
drains in accor- dance with local regulations.
The discoloration should disappear within 1 to 2 days. CIDEX Effective Concentration(MEC). instruments for 12 minutes at 20ºC to achieve high level
OPA Solution may also stain environmental surfaces such as disinfection.
Refer to instrument manufac-
countertops, walls and floors. Once personal protective turer’s labeling for additional Note: Cidex OPA Solution can Achieve high level disinfection in 5
equipment is donned, you are ready to begin the disinfection instructions on disassembly, minutes at a minimum of
process. decontamination, cleaning and 25oC in an automatic endoscope reprocessor.*
3 Using leak testing.
Clean Instruments cate a concentration above
3 Using CIDEX® OPA Solution
The first step in the high level disinfection process is 5 Rinsing Instruments
thorough cleaning.(a) Contaminated instruments must be Before using the solution, be
sure to read the directions Following disinfection, rinse instruments thoroughly, flushing the* When used or reused in a legallyomarketed automatic endoscope reprocessor
thoroughly cleaned prior to disinfection.
for use on the bottle label channels with potable or sterile water. Be sure to repeat thisthat can be set to a minimum of 25 C.
To remove debris, thoroughly clean all instrument surfaces proce- dure twice, for a total of 3 rinses. Each rinse should be aFor technical information on CIDEX® OPA Solution, contact your local
and package insert.
and the lumens of hollow instruments (e.g., endoscopes) with minimum of 1 minute in duration, and a large vol- ume of freshAdvanced Sterilization Products sales representative or call
a mild protein dissolving detergent such as ENZOL® The shelf life of an un- water (e.g., 2 gallons) must be used for each rinse.
2
Enzymatic Detergent.(b) opened bottle of CIDEX OPA
Solution is 2 years. The solu- Note: Please refer to the Instructions for Use for informa- tion on
CIDEX OPA Solution is compatible with enzymatic rinsing during processing in an automatic endoscope reprocessor.
tion requires NO activation.
detergents (e.g., ENZOL® Enzymatic Detergent), which are
mild in pH, low foaming, and easily rinsed from After opening the bottle,
or pour CIDEX OPA Solution
instruments. Detergents that are either highly acidic or alkaline
in the into a CIDEX Solution Tray
are contraindicated as cleaning agents.
or appropriate container. If there is still solution left in the
bottle, the bottle can be stored up to 75 days.

The use of CIDEX OPA

Solution in an automatic
endoscope reprocessor must be
part of a validated repro-
cessing procedure.
Note: CIDEX OPA Solution ASP Customer Support at 1-888-783-7723.
(b) can achieve high level
disinfection in 5 minutes at a
minimum of
25oC in an automatic
endoscope reprocessor.*
It is important to record the
date the solution was poured
from the original container
(a)
and the date its reuse life
ends (not to exceed 14 days).
Blank log book pages are
available at www.cidex.com or
through ASP Customer
Support.

Division of Ethicon,
Inc.

www.cidex.com

© ASP AD-09569-001
2003 Rev. C
 HALOGENS:

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 Mode of action: They are oxidizing agents and
cause damage by oxidation of essential
sulphydryl groups of enzymes. Chlorine reacts
with water to form hypochlorous acid, which is
microbicidal.

 Examples: Chlorine compounds (chlorine,

bleaching powder, sod. hypochlorite,
chloramine) and iodine compounds (tincture
iodine, iodophores)

21
 Applications:.

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 Chlorine gas is used to bleach water.
 Household bleach used in a stock dilution of 1:10
to disinfect floor.
 In higher conc. chlorine is used to disinfect
swimming pools.
 1% sodium hypochlorite is used in labs.

 10% in decontamination of spillage of infectious

material.
 Disadvantages: They are rapidly inactivated in
the presence of organic matter.
 Bleach solution is corrosive and will corrode
stainless steel surfaces
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 2/13/2020 10:21 AM
IODINE

 Bactericidal, virocidal, tuberculocidal, moderate

action against spores.
 Tincture of iodine (2% iodine in 70% alcohol) is
an antiseptic.
 Iodine can be combined with neutral carrier
polymers / surface –active agents to prepare
iodophores such as povidone-iodine.
 Iodophores permit slow release and reduce the
irritation of the antiseptic.
 For hand washing iodophores are diluted in 50%
alcohol. 10% Povidone Iodine is used undiluted in
23
pre and postoperative skin disinfection.
HEAVY METALS SALTS

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• Mode of action: Act by precipitation of
proteins and oxidation of sulphydryl groups.
They are bacteriostatic.

• Examples: Mercuric chloride, silver nitrate,

copper sulphate, organic mercury salts (e.g.,
mercurochrome, merthiolate)

• Disadvantages: Mercuric chloride is highly

toxic, are readily inactivated by organic
matter.

24
 HEAVY METALS SALTS
Applications:

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•

• 1% silver nitrate solution was applied on eyes as

treatment for ophthalmic neonatorum (Crede’s
method). No longer followed.

• Silver sulphadiazine is used topically to help to

prevent colonization and infection of burn
tissues.
•

• Mercurials are active against viruses at dilution

of 1:500 to 1:1000. Merthiolate at a
concentration of 1:10000 is used in preservation
of serum.
25
• Copper salts are used as a fungicide.
 DYES- ANILINE DYE

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• Mode of action: Aniline Dyes are bacteriostatic.
interact with bacterial nucleic acids.

• Examples: Crystal violet, malachite green and

brilliant green.
• GPC bacteria >GNB.
• No activity against tubercle bacilli,
• malachite green used in LJ Medium for M.
tuberculosis.
• Inhibited by organic material.
• Non-irritant, non toxic

26
• Applications:. The dyes are used as selective
agents in certain selective media.
ACRIDINE DYE

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• Mode of action : Impair DNA Complex of
organism , killing/destroying the reproductive
capacity of cell

• Examples: Acriflavin and Aminacrine.

Acriflavine is a mixture of proflavine and
euflavine.
• GPC > GNB and are more bacteriostatic in
action.
• Little affected by organic matter

• Applications: Impregnated in gauze, slow

27
release in moist condition.
 SURFACE ACTIVE AGENTS

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• Mode of actions: property of concentrating at
interfaces between lipid containing membrane of
bacterial cell and surrounding aqueous medium.
• Have long chain hydrocarbons that are fat soluble
and charged ions that are water-soluble. They
disrupt membrane resulting in leakage of cell
constituents.

• Examples: Soaps or detergents. Detergents can be

anionic or cationic. Anionic detergents include
soaps and bile salts. Cationic detergents are
known as quaternary ammonium compounds (or
quat) e.g. Cetrimide and benzalkonium chloride.
• . 28
SURFACE ACTIVE AGENTS ...

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• Application:
1. Active against vegetative cells,
Mycobacteria and enveloped viruses.
2. Widely used as disinfectants at dilution of
1-2% for domestic use and in hospitals.

• Disadvantages:
1. Their activity is reduced by hard water, and
organic matter.
2. Pseudomonas can metabolise cetrimide,
using them as a carbon, nitrogen and
energy source
29
 HYDROGEN PEROXIDE

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• Mode of action: It acts on the microorganisms
through its release of nascent oxygen.
Hydrogen peroxide produces hydroxyl-free
radical that damages proteins and DNA.

• Disadvantages:
• Decomposes in light,
• broken down by catalase,
• proteinaceous organic matter drastically reduces
its activity.

30
 2/13/2020 10:21 AM
H2O2 ….
• Application:
• Used at 6% concentration to decontaminate the
instruments, equipments such as ventilators.
•
• 3% Hydrogen Peroxide Solution is used for skin
disinfection and deodorising wounds and ulcers.
•
• Strong solutions are sporicidal.

31
 ETHYLENE OXIDE (ETO)

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• Mode of action: alkylating sulphydryl-, amino-, carboxyl-
and hydroxyl- groups of enzymes.
• Properties: a cyclic molecule, colourless liquid at room
temperature, boiling point of 107 OC.
• It has a sweet etheral odour, readily polymerizes and highly
flammable, explosive.
• Highly effective chemisterilant, capable of killing spores
rapidly.
• Since it is highly inflammable, 90% EtO is combined with
10%CO2 or dichlorodifluoromethane.
• It requires humidity.
• It has good penetration and is well absorbed by porous
material.

• Disadvantages: It is highly toxic, irritating to eyes, skin, 32

highly flammable, mutagenic and carcinogenic.

 2/13/2020 10:21 AM
ETHYLENE OXIDE (ETO)
• Application:
• Used to sterilize heat labile articles such
as
• Bedding,
• Textiles
• Rubber
• Sutures
• Plastics syringes
• Disposable petridishes,
• Complex apparatus like heart-lung
machine, respiratory and dental 33

equipments.
ETO STERILIZER

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DRAFT

34
 2/13/2020 10:21 AM
FORMALDEHYDE GAS
 Widely used for fumigation of operation
theatres and other rooms.
 Produced in sealed room by adding 150gms of
KMnO4 to 500 ml of 40% formalin for every
1000 cu. feet of room volume
 Heat producing reaction, so heat resistant
vessels must be used
 On generation of formaldehyde vapour , doors
should be sealed and left un-open for 48
hours.

35
BETA-PROPIOLACTONE (BPL)
Mode of action: It is an alkylating agent and

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•
acts through alkylation of carboxyl- and
hydroxyl- groups.

• Properties:
 colourless liquid with pungent to slightly sweetish
smell.
 More efficient for fumigation than formaldehyde

• Disadvantages:
 poor penetrating power and
 carcinogen.

36
GAS PLASMA

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 Plasma is a fourth state of matter which is
distinguishable from liquid, solid, or gas. In nature,
plasma is widespread in outer space.
 Gas plasma generated in an enclosed chamber under
deep vacuum using Radio frequency or Microwave
energy to excite gas molecules and produce charged
particles.

 Commercially available: Sterrad 100S steriliser, and

Plazlyte steriliser

37
 2/13/2020 10:21 AM
HOW GAS PLASMA WORKS.
 Many particles are in the form of free
radicals
 The mechanism of action of this device is
the production of free radicals within a
plasma field that are capable of
interacting with essential cell
components, i.e enzymes and nucleic
acids. And thereby disrupt the
metabolism of microorganisms.
38
 2/13/2020 10:21 AM
TESTING OF DISINFECTANTS

39
 2/13/2020 10:21 AM
TESTING OF DISINFECTANT
 Disinfectant must be tested periodically to
ascertain its potency and efficacy.

 Some methods help in selecting the right dilution

of disinfectant for use while others test the
efficacy of disinfectant already in use.

 Some methods compare the performance with

that of phenol whereas other simply state if the
disinfectant is effective or not
40
TYPES

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Carrier test
Suspension test
Capacity test
Practical test
Field test or in-use test
41
CARRIER TEST

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 oldest tests.
 Robert Koch’s Method- the carrier such as a silk
or catgut thread is contaminated by submersion
in a liquid culture containing spore of Bacillus
anthracis. The carrier is then dried and is
brought in contact with the disinfectant for a
given exposure time. After the exposure, it is
cultured in a nutrient broth; no growth indicates
activity of the disinfectant tested whereas
growth indicates a failing.
 Limitation : a) the number of bacteria dried on a
carrier is hard to standardize
 b) the survival of the bacteria on the
42
carrier during drying is not constant.
SUSPENSION TESTS

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 In these tests, a sample of the bacterial culture is
suspended into the disinfectant solution and after
exposure it is verified by subculture whether this
inoculum is killed or not.

 Suspension tests are preferred to carrier tests as

the bacteria are uniformly exposed to the
disinfectant.

43
 2/13/2020 10:21 AM
 Types: 1. Qualitative suspension tests, the
test for the determination of the phenol
coefficient (Rideal and Walker, 1903)
 2. Quantitative suspension tests.

 Qualitative Test: A loopful of bacterial

suspension was brought into contact with
the disinfectant and again a loopful of this
mixture was cultured for surviving
organisms. Results were expressed as
‘growth’ or ‘no growth’.
44
RIDEAL WALKER METHOD
 Based on Phenol Coefficient

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 The dilution of disinfectant which
disinfects the bacterial suspension in a 10
min time is divided by the dilution of
phenol which disinfects the suspension in
same time under predetermined
conditions gives its phenol coefficient.

45
 Phenol is diluted from 1:50 to 1:90 and

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 the test disinfectant is diluted from 1:150 to
1:250.

 Their bactericidal activity is determined

against Salmonella typhi suspension.

 Subcultures are performed from both the

test and phenol at intervals of 5, 7.5 and 10
minutes.

 The plates are incubated for 48-72 hours at

37°C.
46
• For example, after 10 minutes, the test organism

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was killed by the test disinfectant at a dilution
of 1:200. In the same period the test organism
was killed by phenol at a dilution of 1:80.
• Phenol coefficient= 200/80=2.5
• Test disinfectant can be diluted 2.5 times as
much as phenol and still possess equivalent
killing power for the test organism.
• Disadvantages :
 No organic matter is included
 Salmonella typhi may not be appropriate
microorganism;
 Time allowed for disinfection is short;
 It can be used to evaluate phenolic type
disinfectants only 47
CHICK MARTIN TEST

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• Also determines the phenol coefficient
• Unlike in Rideal Walker method where the test
is carried out in water, the disinfectants are
made to act in the presence of yeast suspension
(or 3% dried human faeces) to simulate the
presence of organic matter.

• Time for subculture is fixed at 30 minutes.

• Test efficacy against S.typhi and S.aureus.

• The phenol coefficient is lower than that given by

48
Rideal Walker method
 RIDEAL WALKER Vs CHICK -MARTIN

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Rideal-Walker Chick-Martin
Volume medium 5.0 ml 10.0 ml
Diluent for test Water Yeast suspension
disinfectant

Reaction temperature 37.5±0.5ºC 30ºC

Organism Salmonella typhi Salmonella typhi,

Staphylococcus aureus

Sampling times 2.5, 5.0, 7.5, 10.0 Min. 30.0 Min.

Calculation of Dilution test killing in Mean concentration of

coefficient 10 min divided by same phenol showing no
for phenol growth after 30 min.
49
divided by same for
test
CAPACITY TEST

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• Each time a soiled instrument is placed into a
container with disinfectant, a certain quantity of dirt
and bacteria is added to the solution. The ability to
retain activity in the presence of an increasing load is
the capacity of the disinfectant.
• In a capacity test, the disinfectant is challenged
repeatedly by successive additions of bacterial
suspension until its capacity to kill has been
exhausted.
• The best known capacity test is the Kelsey-Sykes test
(Kelsey and Sykes, 1969).

50
 KELSEY-SYKES TEST
Triple challenge test, designed to determine

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
concentrations of disinfectant that will be effective
in clean and dirty conditions.
 The disinfectant is challenged by 3 successive
additions of a bacterial suspension during the
course of the test.
 The conc. of the disinfectant is reduced by half by
the addition of organic matter (autoclaved yeast
cells), which builds up to a final concentration of
0.5%.
 A single test organism is selected from S. aureus, P.
aeruginosa, P. vulgaris and E. coli.
 The method can be carried out under 'clean' or
'dirty‘ conditions. The dilutions of the disinfectant
51

are made in hard water for clean conditions and in

yeast suspension for dirty conditions
KELSEY-SYKES TEST
• Test organism alone or with yeast is added at 0, 10 and 20
minutes interval.

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• The contact time of disinfectant and test organism is 8 min.
• The three sets of five replicate cultures corresponding to
each challenge are incubated at 32oC for 48 hours and growth
is assessed by turbidity.
• The disinfectant is evaluated on its ability to kill
microorganisms or lack of it and the result is reported as a
pass or a fail.
• Sets that contain ≥2 negative cultures are recorded as a
negative result.
• The disinfectant passes at the dilution tested if negative
results are obtained after the 1st and 2nd challenges.
• 3rd challenge is not included in the pass/fail criterion but
positive cultures serve as inbuilt controls. If there are no
positive cultures after the third challenge, a lower
concentration of the disinfectant may be tested.
• Kelsey and Sykes test gives a good guideline for the dilution
of the preparation to be used.
• Disadvantage -complicated
52
 KELSEY-SYKES TEST

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53

1 Test organism : S. aureus, P. aeruginosa, P. vulgaris and E. coli

 SPECIMEN RESULT OF A TEST

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Concentr Inoculum Challenge number Result
ation count

1 2 3

1.0 2X 109
+++++ +++++ +++++ Fail

1.5 2X 109
----+ --+++ +++++ Pass

2.0 2X 109
----- ----- ----+ Pass
54
 SUPPLEMENTARY TEST

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• Test for stability and long-term effectiveness:
• Recommended concentrations based on Kelsey Sykes
test apply only to freshly prepared solutions but if the
solutions are likely to be kept for more than 24 hours,
the effectiveness of these concentrations must be
confirmed by a supplementary test for stability of
unused solution and for the ability of freshly prepared
and stale solutions to prevent multiplication of a small
number of bacteria that may have survived the short
term exposure.
• P. aeruginosa is used a test organism.

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SUPPLEMENTARY TEST
Sufficient disinfectant solution is prepared for two

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
tests.
 One portion is inoculated immediately and tested for
growth after holding for seven days at room
temperature.
 The other portion is kept at room temperature for
seven days and then inoculated with a freshly
prepared suspension of test organism. It is also tested
for growth seven days after inoculation.
 If growth is detected, a higher concentration of
disinfectant must be tested in the same way.

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SUPPLEMENTARY TEST

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IN-USE TEST

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• A simple to use test was described by Maurer in
1985 that can be used in hospitals and
laboratories to detect contamination of
disinfectants..

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 In use testing of disinfectants already in use at hospital or lab

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1ml of disinfectant (to be tested) taken in a sterile syringe.
↓
Add 9 ml of Nutrient broth ( for alcohol, aldehydes and phenolic disinfectants)
or
Nutrient broth with 3% v/v Tween 80 for hypochlorite, quaternary ammonium
compounds, polyaminopropyl biguanide)
↓
Total 10 ml
↓
Place 10 drops (each of 0.02 ml) from the height of 2-3 cm on 2 Nutrient agar plates
↙↘
One plate incubated at 37 0C x 3 days 2nd plate incubated at room temp. x 7 days
↓ ↓
Count the colony after 3 days count the colony after 7 days
↓ ↓
Result: 1. ≥ 5 colonies in any plates means the disinfectant is ineffective. It has to
be changed.
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2. ≤ 5 colonies means the disinfectant is effective.
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•
 HAND HYGIENE

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THANK YOU VERY MUCH 61