Disinfectant classification of disinfectant

1. Prepared by
Mr. Sakhare V. G.
Assistant Professor
SBSPMs B. Pharmacy College Ambajogai
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2. Content
 Introduction
 Ideal properties of Disinfectant
 Classification of Disinfectant
 Factors affecting Disinfectant Action
 Evaluation of antimicrobial agent and Disinfectants
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3. Disinfection
 Disinfection is process of destruction or removal of microorganism and
reducing them to a level not harmful to health is known as disinfection.
 They generally kills the sensitive vegetative cells but not the heat resistant
Endospores.
Disinfectants
 The chemicals that kill vegetative bacteria, Fungi, Viruses, and bacterial spores
is known as disinfectants.
 Disinfectants do not necessarily kill all organisms but reduce them to a level,
which does not harm health or the quality of perishable goods.
Antiseptic
 Antiseptic are those agents which kill or prevent the growth of microorganism
and applied on living tissue is known as Antiseptic.
Antisepsis
 The destruction or inhibition of microorganism in tissue there by preventing the
harmful effect of infection caused by them is known as antisepsis.
“Disinfectant are usually Bactericidal but occasionally Bacteriostatic”
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4. ANTISEPTICS
 An antiseptic is a type of disinfectant, which destroys or inhibits growth of
micro-organisms on living tissues without causing injurious effects when
applied to surfaces of the body or to exposed tissues.
 Some antiseptics are applied to the unbroken skin or mucous membranes, to
burns and to open wounds to prevent sepsis by removing or excluding microbes
from these areas.
 Example :- Iodine has been modified for use as an antiseptic.
1. The iodophore, polyvidone-iodine , is effective against bacteria, fungi,
viruses, protozoa, cysts and spores and significantly reduces surgical wound
infections.
2. The solution of polyvidone-iodine releases iodine on contact with the skin.
3. Chlorhexidine has a wide spectrum of bactericidal and bacteriostatic activity
and is effective against both Gram positive and Gram-negative bacteria
although it is less effective against some species of Pseudomonas and Proteus
and relatively inactive against mycobacterium.
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5. An ideal Properties of Disinfectant
 It should have wide spectrum or Broad spectrum of activity.
 It Should be able to destroy microbes within practical period of time.
 It Should be active in the presence of organic matter.
 It Should make effective contact and be wettable.
 It Should be active in any PH.
 It Should be stable.
 It Should have long shelf life.
 It Should be speedy.
 It Should have high penetrating power.
 It Should be non-toxic, non-allergenic, non-irritative or non-corrosive
 It Should not have bad odour.
 It Should not leave non-volatile residue or stain.
 Efficacy should not be lost on reasonable dilution.
 It Should not be expensive and must be available easily.
 It should be economical.
 It should be dissolve easily.
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6. Classification of Disinfectants
1. Acid or Alkalies: Eg. HCl, H2SO4, HNO3
2. Halogens: Eg. Chlorine, Bromine, Fluorine and Iodine
3. Heavy metals: Eg. Silver, Copper, Mercury
4. Phenol and its derivative: Eg. Phenol, Cresol, Chlorocresol
5. Alcohol: Eg. Ethyl alcohol, Methyl alcohol, Isopropyl alcohol
6. Aldehyde and its derivative: Eg. Formaldehyde, Glutaraldehyde
7. Quaternary Ammonium Compounds: Eg. Cetrimide, Benzalkonium chloride
8. Dyes: Eg. Proflavine, Crystal Violet
9. Detergents and soaps: Eg. Sodium Lauryl Sulphate
10. Oxidizing agent: Eg. Potassium Permagnate, Hydrogen Peroxide
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7. 1. Acid or Alkalies
 Generally strong acid and Alkalies kill the bacteria but weak acids inhibit their
growth. The germicidal efficiency of acids is proportional to the hydrogen
ion(H+) Concentration of their solutions.
Strong acid: HCL, H2SO4, HNO3
Weak acid: Benzoic acid, Salicylic acid, Acetic acid
Strong acids and Alkalies or both not useful for disinfectant because they are
corrosive
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8. 2. Halogens
 Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds
(tincture iodine, iodophores), Bromine, Fluorine in the free state as well as
their compounds strongly acts as germicidal.
 The bromine and fluorine are irritants and difficult handle but chlorine and
bromine are commonly used as a sterilization.
 The iodine is good disinfectant in aqueous or alcoholic solutions.
 It having the sporicidal, fungicidal and active against many viruses.
 The iodine is also used as skin disinfectant and cold sterlisation of surgical
Sutures.
Mode of action: They are oxidizing agents and cause damage by oxidation of
essential sulphahydryl groups of enzymes. Chlorine reacts with water to form
hypochlorous acid, which is microbicidal. Protein inactivation
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9. 3. Heavy Metals
 Silver, Copper, Mercury are mostly used heavy metal for disinfectant and their
compounds acts as antimicrobial by combining cellular protein.
 High concentration of salts of heavy metal like mercury, copper and silver
coagulate Cytoplasmic proteins resulting on the damage or death of cells.
Mode of action: Act by precipitation of proteins and oxidation of sulphahydryl
groups. They are bacteriostatic.
Examples: Mercuric chloride, Silver nitrate, Copper sulfate, organic mercury
salts (e.g., Mercurochrome, Merthiolate)
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10. 4. Phenol and its derivative
 These are obtained by distillation of coal tar and having the powerful
microbial action.
Cresol
 It is used as solution of cresol in soap(Lysol).
 It is used for disintegration of injected glassware in laboratory disintegration of
excreta, cleaning floors of words, operation theatres.
Chloroxylenol
It is an active ingredient of Dettol.
Mode of Action
 Phenolic group mainly destroy the micro-organisms by the process of
disruption of cells, precipitation of cell protein, inactivation of enzymes,
leakage of amino acid from the cells.
 Act by disruption of membranes, precipitation of proteins and inactivation of
enzymes.
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11. 5. Alcohol
The alcohol have bactericidal action against vegetative bacteria.
Ethyl alcohol
The concentration of ethyl alcohol 50-70% v/v is effective against the viruses.
Methyl alcohol
Methanol effective against fungal spores but it is toxic to eyes.
Isopropyl alcohol
50-70% better than ethyl alcohol in bactericidal properly used in disinfection of
clinical thermometers.
Ex:- Methanol, Ethanol, Isopropanol etc.
Mode of Action
Alcohol have protein denaturants activity. They may damage lipid complex in the
all membrane. They also have dehydrating activity.
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12. 6. Aldehyde and its derivative
Formaldehyde HCHO
 It is a bacteriocidal,sporicidal & Viricidal action.
 It in mucous solution containing 34 to 38% W/W of CH2O.
 Formaldehyde in solution is useful for sterilization of certain instruments.
Glutaraldehyde CHO-CH2-CH2-CH2-CHO
 It is more effective & less irritant then formaldehyde.
 It have bactericidal, sporicidal, fungicide.
 It is used in the medical field for sterilizing urological, lensed & other
instruments.
Mode of Action
 Denaturation of protein.
 Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably
damages nucleic acids. It kills all microorganisms, including spores.
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13. 7. Quaternary Ammonium Compounds
 They are highly active against Gram-positive bacteria & are also quite active
against Gram-negative bacteria.
 They are also active against fungi and certain pathogenic protozoa.
 It is widely used for control of micro-organisms on floors, walls, nursing
homes & other public places.
 They are also used for skin antiseptic, sanitizing agent in daily, egg & fishing
industries.
 Ex.: Cetrimide, Cetyl pyridinium chloride, Benzalkonium chloride.
Mode of Action
Quaternary ammonium compound which disruption of cell wall & membrane.
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14. 8. Dyes
 The number of Dyes have been found to inhibit the bacterial growth.
 Basic Dyes are more effective bactericidal than acidic dyes.
The Acridine Dyes
 Triphenyl methane Dyes are commonly used in 'Antimicrobial Agent'.
 Acridine Dyes:- Proflavin, Aminacrine, Enflavin, Acriflavin.
 Effective against Gram-positive bacteria.
 They kill or destroy the reproduction or capacity of the cell.
Aniline Dyes
 Ex. Brilliant green, Malchite green, crystal violet.
 The crystal violet has also been used a fungicidal.
Mode of Action
 Crystal violet inhibit the cellular oxidation process.
 Acridine dyes are bactericidal because of their interaction with bacterial
nucleic acids
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15. 9. Detergents and soaps
 They are widely used as surface active agents, wetting agents & emulsifiers.
 Soaps & sodium Lauryl sulphate are anionic compounds.
 Soap are prepared Gram saturated fatty acids & more active against Gram-
positive bacilli.
Mode of action
 They have the property of concentrating at interfaces between lipid containing
membrane of bacterial cell and surrounding aqueous medium.
 These compounds have long chain hydrocarbons that are fat soluble and
charged ions that are water-soluble. Since they contain both of these, they
concentrate on the surface of membranes. They disrupt membrane resulting in
leakage of cell constituents
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16. 10. Oxidizing agent
 Hydrogen peroxide H2O2 (Cleaning of wounds).
 Potassium Permagnate Kmno4.
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.
Application
It is used at 6% concentration to decontaminate the instruments, equipments such
as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and
de odorising wounds and ulcers. Strong solutions are sporicidal.
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17. Factors affecting Disinfectant
 Certain environmental factors can affect the rate and extent of antimicrobial
action of chemical disinfectants. The various factors affect the disinfectant
action there as follows:
1. Concentration of disinfectant
2. Temperature
3. Time of conduct
4. PH of the environment
5. Surface tension
6. Formulation of the disinfectant
7. Chemical structure of disinfectant
8. Type and number of microorganism
9. Interfering substances in the environment
10. Potentiation, Synergism and antagonism of disinfectants
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18. 1. Concentration of disinfectant
 The rate of killing of microorganism varies directly with the concentration
Of the Disinfectant.
 The effectiveness is generally related to concentration exponentially not
linearly.
 E.X. The optimum concentration of phenol at about 1% beyond the
concentration the disinfectant becomes less effective.
The rate of killing α Concentration of disinfectant
 The rate of killing is directly proportional to Concentration of Disinfectant.
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19. 2. Temperature
 The rate of disinfection normally increase with temperature.
 The effect of temperature on bactericidal action may be expressed as
quantitatively by means temperature coefficient.
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20. 3. Time of conduct
 Sufficient time of contact must be allowed for disinfectant to exert its action
which shows that principle of first order kinetics.
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21. 4. PH of the Environment
 A change of PH during the disinfection process can affect the rate of
growth of inoculum, a potency of disinfectant and the ability of the
disinfectant to combine with a site on the cell surface.
 The PH OF 6-8 is optimal for growth of many bacteria and rate of growth
declines on either side range.
 Phenolic and acidic antimicrobial agents usually have greatest activity in
acidic conditions.
 Acridine dyes and quaternary ammonium compounds are usually more
active in alkaline than acidic solutions.
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22. 5. Surface tension
 The contact between aqueous solutions of disinfectant is facilitated if they
have surfactant properties.
 This helps in adsorption of surface active disinfectant on the surface of cells
as in wetting and spreading properties of the solutions
 Ex. A combination of soap with crude phenol ( Carbolic acid ) has excellent
disinfectant property.
 Soaps can be used to lower the surface tension and the extinction time until
the soap concentration is equivalent to critical concentration of micelle
formation.
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23. 6. Formulation of Disinfectant
 For effective use of disinfectant, its formulation is more important.
 Effectiveness of Chlorhexidine and quaternary ammonium compound may
be greater in 70% alcohol than in aqueous solutions.
 For convenience and economy it is essential to prepare disinfectants as
possible as concentrated and suitable for dilution with water immediately
before use.
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24. 7. Chemical structure of disinfectant
 The chemical structure of a compound affects the disinfection activity.
 The halogenations increases the antibacterial activity of phenol but nitration
increases antibacterial activity & systemic toxicity also.
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25. 8. Type and number of microorganism present
 The efficiency of disinfection greatly depends on the nature and number of
contaminating microorganisms and especially on the presence and absence
of bacterial spores.
 Ex. Aldehydes and halogens are effective against virus.
 Mycobacterium tuberculosis and other acid fast bacilli are fairly resistant to
many aqueous bactericide but are susceptible to iodine, formaldehyde,
alcohol and other Phenolic compounds .
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26. 9. Interfering substances in the environment
 Blood, body fluids, pus, milk, food residue or proteins may reduce the
effectiveness of disinfectants is present in small amount.
 It may due to chemical reaction or absorption from adequate contact with
germicide.
 The presence of oils and a fat markedly reduce the disinfectant ability of
Phenolics.
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27. 10. Potentiation, Synergism & Antagonism of disinfectant
Potentiation
 Potentiation of disinfectant leads to enhanced antimicrobial activity.
 Ex. Polysorbate 80, low concentration of non ionic surfactant.
Synergism
 Synergistic effects are often shown by two antimicrobial agents which is
giving an increased activity.
 Ex. P-hydroxy benzoate esters
Antagonism
 Antagonism leads to decrease antimicrobial effect.
 Ex. Sodium thiosulphate
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28. Evaluation of Antimicrobial agents & Disinfectants
The various methods or techniques used for the evaluation of disinfectant is
as follows:
1. Tube Dilution and agar plate Method
2. Filter paper and cup plate Method
3. Ditch-Plate Method
4. Phenol Coefficient Method
5. Kelsey Sykes Method
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29. 1. Tube Dilution and Agar plate Method
 The chemical agent is incorporated into nutrient agar medium or Nutrient
Broth medium inoculated with test microorganism.
 These tubes are incubated at 30-35◦c for 2-3 days.
 The result in the form of turbidity or colonies are observed.
 The results are recorded and the activity of the given disinfectant.
1. Tube dilution method
2. Agar Plate method
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30. A. Tube Dilution Method
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Nutrient Broth
Chemical agent
With Test Microorganism
Tubes are incubated at 30 to 35◦c
for 2 to 3 Days
Observe the growth

31. B. Agar Plate Method
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Nutrient Agar medium
Incubated at 30 to 35◦c for 2 to 3 days
Chemical agent
inoculated with test microorganism
Observe the growth

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Fig. Tube Dilution method and Agar plate method

33. 2. Filter paper, Cup Plate & Cylinder Method
A. Cup Plate Method
 The agar is melted and cooled at 45◦c and inoculated with test
microorganism and poured into the sterile plate.
 When the inoculated agar has solidify holes about 9mm in diameter are cut
in medium with sterile cork borer.
 The antimicrobial agent is directly placed into the holes.
 The zone of inhibition is observed after incubation at 30 to 35 ◦c for 02 to
03 days.
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Fig. Cup Plate method

34. Cup Plate Method
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The Nutrient ager medium
Melted & Cooled at 45◦c
Inoculated test Microorganism
Pour into sterile Petri plate
To make the holes about 09mm
Add the Antimicrobial agent
Incubate at 30 to 35◦c for 02 to 03 days
Observe the Growth

35. B. Filter paper and cylinder plate method
 The agar is melted and cooled at 45◦c and inoculated with test
microorganism and poured into the sterile plate.
 The antimicrobial agent is applied on surface of solidified and inoculated
agar.
 The filter disc or cylinder plate is placed into agar plate.
 The zone of inhibition is observed after incubation at 30 to 35 ◦c for 02 to
03 days.
 Observe the growth.
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36. Filter Paper and cylinder plate Method
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The Nutrient ager medium
Melted & Cooled at 45◦c
Inoculated test Microorganism
Pour into sterile Petri plate
Antimicrobial agent is applied on surface of solidified and inoculated agar
By using filter paper disc and cylinder
Incubate at 30 to 35◦c for 02 to 03 days
Observe the Growth

37. 37
Fig, Filter Paper and Cylinder Method

38. 3. Ditch-Plate Method
 Agar is melted and then solidified in a Petri plate.
 A ditch is made in the Petri plate by cutting the solidified agar.
 The disinfectant solution is made to run through the ditch carefully.
 The test organisms are streaked outwards from the ditch.
 The petriplate is incubated at desired temp. And time period.
 The microorganisms which are resistant to the disinfectant grow even near
the start at the ditch itself.
 The sensitive organisms show a zone of inhibition near the ditch or at center
of the Petri plate.
 The width of the zone of inhibition is an indication of activity against the
test organism.
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39. 39
Fig. Ditch Plate method

40. 4. Phenol Coefficient Method
 Bacteria potency is assessed by measuring the rate of killing a selected
range of bacteria under specified conditions.
 In this method the efficacy of the disinfectant in use is rated by comparing it
with the activity of Phenol taking as a standard.
 The test is carried out by adding an increasing amount of phenol and
disinfectant in test tubes containing microorganisms.
 In UK the test organism used is Salmonella typhi while the USA uses
Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa.
 The official phenol coefficient tests include,
 Rideal-Walker Test (RW Test).
 Chick-Martin Test.
 United States FDA Test for Phenol Coefficient. (FDA Test)
 The US Association of Official Agricultural Chemists Test (FDA Test)
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41. Rideal-Walker Test (RW Test).
 Introduced by Rideal and Walker the British chemists in 1903 are still in
use.
 RW Coefficient is a measure of effectiveness of an antiseptic or disinfectant
as compared to phenol.
 Higher than 1 RW Coefficient values indicates that the chemical is more
effective than phenol. While lower than 1 RW coefficient values indicates
that poor disinfectant power of the test disinfectant than phenol.
 The method is prescribed under drug and cosmetics rule (Rule 126,
Schedule o) is briefly explained below
 The test uses Rideal, Walker Broth and Salmonella typhi as a test organism.
Broth prepared by dissolving
20gm- Meat extract
20gm – Peptone
10gm- Sodium Chloride
1000ml – Water
PH is adjusted to 7.6 and it is sterilized by Autoclaving.
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42. Procedure
 Different dilutions of the phenol and test disinfectants are made and 5 ml of
each is inoculated with the 0.5 ml of the 24 hr broth culture of the test
microorganism.
 All the test tubes are placed in a water bath at 17.5 ℃.
 Subcultures from each test tube are taken and transferred to 5 ml sterile
broth after 2.5, 5, 7.5 and 10 minutes.
 The broth tubes are incubated at 37 ℃ for 2 - 3 days and are examined for
the presence or absence of the growth and the Phenol coefficient is
calculated using the formula,
 The Phenol Coefficient for phenol is considered as “1” any value for
disinfectant coming below one is considered as less while above it is more.
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43. 43

44. Advantages
 Quick.
 Inexpensive.
 Reproducible results.
 Useful to eliminate useless products.
 Sets standard for the preparations.
Disadvantages
 Most tests use only one test organism i.e. Salmonella typhi which represents
inadequate information.
 Compares the activity of disinfectants at only one concentration at fixed
conditions.
 Presence of organic matter at action time is not considered.
 Does not give information about tissue toxicity of the disinfectant.
 Sampling errors are large.
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45. 5. Kelsey Sykes Method
 This method is developed in 1969, this test overcomes many drawbacks of
the RW Test.
 The test uses several test organisms viz. Staphylococcus aureus, Proteus
vulgaris, Escherichia coli and Pseudomonas aeruginosa.
 Tests can be carried out in a clean as well as dirty environment.
 In both clean and dirty cases the final bacterial concentration should be
about 109/ml.
 Clean conditions are simulated by using a clean broth while dirty conditions
are simulated by using a yeast suspension or inactivated horse serum as a
the suspending fluid.
 The dilutions of the disinfectant are made in hard water.
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46. Principle
Kelsey-Sykes test is a triple challenge test, designed to determine
concentrations of disinfectant that will be effective in clean and dirty
conditions.
Procedure
 Test organism alone or with yeast is added at 0, 10 and 20 minutes interval.
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 32◦C 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 and not as a coefficient.
 Sets that contain two or more negative cultures are recorded as a negative
result.
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47. 47
Incubate all the test tubes at 32◦c for 48 Hours

48. 6. Koch’s test Method
 These test is performed for detergents and soaps disinfectant.
 In this test performed for to check cleaning of cloth.
 Spores of Bacillus Anthracis were dried on silk thread.
 Wash and transferred to solid medium
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Silk thread
Spores
Dried the Silk
Thread
Disinfect the Silk Thread
Transfer the thread on agar slant
Solid Media
No Evidence Growth
Action of Disinfectant
Evidence Growth
Fail the test
Disinfectant

49. ANY QUESTIONS
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