CONTROL BY CHEMICAL AGENTS: DISINFECTANTS

DEFINITIONS:

1. Sterilization : The process of destroying all forms of microbial life.

2. Disinfectant : An agent, usually a chemical, that kills the growing forms but not
necessarily the resistant spore forms of disease-producing microorganisms. The term is
commonly applied to substances used on inanimate objects.

3. Antiseptic : A substance that opposes sepsis, i.e., prevents the growth or action of
microorganisms either by destroying microorganisms either by destroying
microorganisms or by inhibiting their growth and metabolism. Usually associated with
substances applied to the body.

4. Sanitizer : An agent that reduces the microbial population to safe levels as judged by
public health requirements. Usually it is a chemical agent that kills 99.9 percent of
growing bacteria.

5. Germicide (Microbicide) : An agent that kills the growing forms but not necessarily the
resistant spore forms of germs. In practice , a germicide is almost the same thing as a
disinfectant, but germicides are commonly used for all kind of germs (microbes) for any
application.

6. Bactericide : An agent that kills bacteria. Similarly, the terms fungicide, virucide and
sporicide refer to agents that kill fungi, viruses and spores, respectively.

7. Bacteriostasis : A condition in which the growth of bacteria is prevented. Agents that

have in common the ability to inhibit growth of microorganisms are collectively
designated as microbistatic agents.
8. Antimicrobial agents : One that refers with the growth and metabolism of microbes. In
common usage the term denotes inhibition of growth, and with reference to specific
groups of organisms, such terms as antibacterial or antifungal are frequently used.
CHARACTERISTICS OF AN IDEAL DISINFECTANT OR ANTIMICROBIAL
CHEMICAL AGENT:

1. Antimicrobial activity: The capacity of the substance to kill or inhibit microorganisms is

the first requirement. The chemical, at low concentration, should have a broad spectrum
of antimicrobial activity.
2. Solubility: The substance must be soluble in water or other solvents to the extent
necessary for effective use.
3. Stability: Changes in the substance upon standing should be minimal and should not
result in significant loss of germicidal action.
4. Non-toxicity to humans and other animals: Ideally, the compound should be lethal to
microorganisms and non-injurious to humans and other animals.
5. Homogeneity: The preparation must be uniform in composition so that active ingredients
are present in each application. Pure chemicals are uniform, but mixtures of materials
may lack homogeneity.
6. Non-combination with extraneous organic material: Many disinfectants have an
affinity for proteins or other organic material. When such disinfectants are used in
situations where there is considerable organic material besides that of the microbial cells,
little, if any, of the disinfectant will be available for action against the microorganisms.
7. Toxicity to microorganisms at room or body temperatures: In using the compound, it
should not be necessary to raise the temperature beyond that normally found in the
environment where it is to be used.
8. Capacity to penetrate: Unless the substance can penetrate through surfaces, its
germicidal action is limited solely to the site of application. Sometimes, surface action is
all that is required.
9. Non-corroding and non-staining: It should not rust or otherwise disfigure metals nor
stain or damage fabrics.
10. Deodorizing ability: Deodorizing while disinfecting is a desirable attribute. Ideally the
disinfectant itself should either be odorless or have a pleasant smell.
11. Detergent capacities: A disinfectant which is also a detergent (cleansing agent)
accomplishes two objectives, and the cleansing action improves the effectiveness of the
disinfectant.
12. Availability: The compound must be available in large quantities at a reasonable price.
SELECTION OF A CHEMICAL AGENT FOR PRACTICAL APPLICATIONS:
The major factors that need to be assessed in the process of selecting the most appropriate
chemical agent for a specific practical application are:
1. Nature of the material to be treated:
 To site an extreme example, a chemical agent used to disinfect contaminated utensils
might be quite unsatisfactory for application to the skin; i.e., it might do serious
injury to the tissue cells.
 Consequently, the substance selected must be compatible with the material to which
it is applied.
2. Type of microorganisms:
 Chemical agents are not all equally effective against bacteria, fungi, viruses and
other microorganisms.
 Spores are more resistant than vegetative cells.
 Differences exist between Gram-positive and Gram-negative bacteria; Escherichia
coli is much more resistant to cationic disinfectants than Staphylococcus aureus.
 Differences in action also exist between strains of the same species.
 Therefore, the agent selected must be known to be effective against the type of
organism to be destroyed.
3. Environmental conditions:
 The factors like temperature, pH, time, concentration, and presence of extraneous
organic material, may all have a bearing on the rate and efficiency of antimicrobial
action.
 The successful use of an antimicrobial agent requires an understanding of the
influence of these conditions on the particular agent, so it can be employed under the
most favorable circumstances.

MAJOR GROUPS OF CHEMICAL ANTIMICROBIAL AGENTS:

The major antimicrobial agents can be grouped into:
5. Dyes
1. Phenols and phenolic compounds
6. Detergents
2. Alcohols
7. Quaternary ammonium compounds
3. Halogens
8. Aldehydes
4. Heavy metals and their compounds
9. Gaseous agents
 MODE OF ACTION

Phenol and phenolic compounds Alcohols Halogens

 Disruption of cells  Protein denaturation  Iodine - irreversible oxidation and

 Precipitation of cell protein
 Inactivation of enzymes
 Leakage of amino acids from the
cells
 Physical damage to the membrane
structures in the cell surface, which
initiates further deterioration.
 Damage lipid complexes in cell
membrane
 Cell dehydration
 Cleansing and detergent action,
leading to mechanical removal of
microorganisms
inactivation of essential metabolic
compounds such as proteins with
sulfhydryl groups.
 Halogenation of Tyrosine units of
enzymes and other cellular proteins
requiring Tyrosine for activity.

Synthetic Detergents Heavy Metals and their compounds Dyes

 Mechanical removal of  Combining with cellular protein and Triphenyl methane dyes
microorganisms – Soap reduces inactivating them.  Interference with cellular oxidation
surface tension and thereby Example: processes.
increase the wetting power of the In case of mercuric chloride, the
water in which they are dissolved. inhibition is directed at enzymes which
Soapy water has the ability to contain the sulfhydryl group.
emulsify and disperse oils and dirt.  High concentrations of salts of HMs
The microorganisms become like Mercury, Copper and Silver
enmeshed in the soap lather and are coagulate cytoplasmic proteins resulting
removed by the rinse water. in damage or death to the cell.
 Salts also act as precipitants and in high
concentrations such salts could cause
the death of a cell.
 Chlorine and Chlorine compounds Quaternary Ammonium Compounds Aldehydes

 The antimicrobial action of chlorine  Denaturation of proteins  Formaldehyde which is an

and its compounds comes through the extremely reactive chemical,
 Interference with glycolysis
hypochlorous acid formed when free combines readily with vital organic
chlorine is added to water.  Membrane damage – especially nitrogen compounds such as
cytoplasmic membrane and alter the proteins and nucleic acids.
Cl2 + H2O HCl + HClO
(hypochlorous acid) vital permeability features

 Hypochlorites and chloramines

undergo hydrolysis with formation of
hypochlorous acid, which, formed Gaseous agent
under each instance, is further
decomposed:
Ethylene Oxide
HClO HCl + O
 The oxygen released in this reaction  Alkylation reactions with organic
(nascent oxygen) is a strong oxidizing compounds such as enzymes and other
agent, and through its action on cellular proteins.
constituents, microbes are destroyed.
β-Propiolactone
 Antimicrobial action of chlorine is also
due to the direct combination of  Sporicidal
chlorine with proteins of the cell  Fungicidal
membranes and enzymes.  Virucidal
 PRACTICAL APPLICATIONS

Phenol and phenolic compounds

Alcohols Halogens

 Bactericidal / bacteriostatic  Effective in reducing the microbial  Iodine is a highly effective

depending upon the concentration flora of skin and for the bactericidal agent and is unique in
used. identification of clinical oral that it is effective against all kinds of
 Bacterial spores and viruses are thermometers. bacteria.
more resistant  Alcohol concentrations above 60%  Iodine also possesses sporicidal
 Some are highly fungicidal are effective against viruses; activity; however, the rate at which
 Aqueous solutions of phenol from however, the effectiveness is the spores are killed is markedly
2 to 5 % can be employed to influenced considerably by the influenced by the conditions under
disinfect such materials as sputum, amount of extraneous protein which they are exposed, e.g., amount
urine, faeces, and contaminated material in the mixture. The of organic material and extend of
instruments or utensils. extraneous protein reacts with the dehydration.
 Derivatives of phenol diluted in alcohol and thus protects the virus.  Highly fungicidal
detergents or some other carrier  Virucidal to some extent.
find use in many commercial  Iodine: best for disinfection of skin,
antiseptic and disinfectant water and air, and sanitization of
preparations. food utensils.
 Synthetic Detergents
Dyes Quaternary Ammonium Compounds

 Reduction of the microbial flora Triphenyl methane dyes  Germicidal activity

from surfaces such as skin and  Certain media can be made selective by  Detergent action
clothing the incorporation of low concentrations  Skin disinfectants
of dyes crystal violet, brilliant green or  Preservative in Ophthalmic solutions and
malachite green. Gram-positive bacteria cosmetic preparations.
inhibited. Media of this kind is used  Control microorganisms on floors,
Gaseous agents
extensively in public health walls, and other surfaces in hospitals,
 Ethylene oxide has been microbiology. nursing homes and other public places.
established as an effective  Susceptibility to various dyes can also
 Sanitize food and beverage utensils in
sterilizing agent for heat and be used for identification of bacteria. restaurants as well as surfaces and
moisture-sensitive materials.
certain equipment in food processing
 The varieties of materials on which
plants.
it is used include spices, biological
 Other applications are to be found in the
preparations, soil, plastics, certain
diary, egg, and fishing industries to
medical preparations, and
control microbial growth on surfaces of
contaminated-laboratory
equipment and the environment in
equipment.
general.
Chlorine and Chlorine compounds Aldehydes

 Widely used to control

 Formaldehyde in solution is useful
microorganisms.
for sterilization of certain
 Water treatment in food industry,
instrumrnts.
for domestic uses and in medicine.
 In gaseous form (Formalin and
 Products containing calcium
paraformaldehyde) used for
hypochlorite are used for sanitizing
disinfection and sterilization of
dairy equipment and eating utensils
enclosed areas.
in restaurants.
 Gluteraldehyde (2% solution)
 Solutions of sodium hypochlorite
exhibits a wide spectrum of
of a 1% concentration are used for
antimicrobial activity. Effective
personal hygiene and as a
against vegetative bacteria, fungi,
household disinfectant.
bacterial and fungal spores.
 Higher concentrations of 5-12 %
 Used in medical field for sterilizing
used as household disinfectant and
urological instruments, lensed
for use as sanitizing agents in diary
instruments, respiratory therapy
and food processing
equipment and other special
establishments.
equipments.
 Compounds Used to disinfect open
wounds, to treat athlete’s foot, to
treat other infections and as a
general disinfectant.
TESTING OF DISINFECTANTS
 Disinfectants used in hospitals and laboratories must be tested periodically to ascertain its potency
and efficacy.
 As certain disinfectants lose potency on standing and addition of organic matter, their efficacy must
be tested.
 While certain methods help in selecting the right dilution of disinfectant for use others test the
efficacy of disinfectant already in use.
 Some methods compare the performance with that of phenol whereas other methods simply state if
the disinfectant is effective or not.
 There are several methods of testing disinfectants, with their own advantages and disadvantages.
 All these tests can be allocated to one of the following disinfectant tests: carrier test, suspension test,
capacity test, practical test, field test or in-use test.
 Disinfection process validation is defined as "establishing documented evidence that a disinfection
process will consistently remove or inactivate known or possible pathogens from inanimate objects."
 Robert Koch described a disinfectant test in the article Uber Desinfektion, in 1881.
 A silk thread was contaminated by submersion in a liquid culture of Bacillus anthracis.
 After drying, the contaminated thread was immersed in several disinfectant solutions for a given
exposure time.
 The thread was then cultured in a nutrient broth and no growth after incubation indicated activity of
the disinfectant.
 He concluded from the comparisons of disinfectant solutions that mercuric chloride was the most
active disinfectant.
 The results were erroneous as the disinfectants residues were also carried over to the subculture
medium.
 The problem was overcome by Geppert in 1890 by neutralising the disinfectant at the end of
exposure period.

1. Carrier tests:
 These tests are the oldest tests.
 The test described by Robert Koch was a carrier test.
 In these tests, the carrier such as a silk or catgut thread or a penicylinder (a little stick) is
contaminated by submersion in a liquid culture of the test organism.
 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.
 By multiplying the number of test concentrations of the disinfectant and the contact times, a
potentially active concentration-time relationships of the disinfectant is obtained.
 Example of a carrier test is the former use-dilution test of the American Association of Official
Analytical Chemists (AOAC, 1990).
 The AOAC Use-dilution test is a carrier-based test.
 The organisms used are Salmonella cholerasuis, S. aureus and P. aeruginosa.
 Carriers (stainless steel cylinders) are meticulously cleaned, sterilized by autoclaving in a solution of
aspargine, cooled and inoculated with a test organism by immersing in one of the culture
suspensions.
 The cylinders are drained on filter paper, dried at 37oC for 40 minutes, exposed to the use-dilution of
the disinfectant for 10 minutes, and cultured to assess the survival of the bacteria.
 A single test involves the evaluation of 60 inoculated carriers (one organism) against one product
sample.
 In addition to the 60 carriers, 6 carriers are required to estimate carrier bacterial load and 6 more
are included as extras.
 Thus, a total of 72 seeded carriers are required to perform a single test.
 A result showing no growth in all ten tubes confirms the result of phenol coefficient test.
 If any carrier produces growth, the test must be repeated using a lower dilution of the disinfectant.
Use:
 dilution test is performed to confirm the efficiency of disinfectant dilution derived from phenol
coefficient test.

Interpretation of result:

 Growth occur – less or ineffective disinfectants

 No Growth occur – more effective disinfectant.

Limitations of carrier test:

 Difficulty in standardizing number of bacteria dried on carrier

 Survival of bacteria on carrier during drying is not constant.

2. Suspension tests:
 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.
 There are different kinds of suspension tests: the qualitative suspension tests, the test for the
determination of the phenol coefficient (Rideal and Walker, 1903) and the quantitative suspension
tests.
 Initially this was done in a qualitative way.
 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’.
 In quantitative methods, the number of surviving organisms is counted and compared to the original
inoculum size.
 By subtracting the logarithm of the former from the logarithm of the latter, the decimal log reduction
or microbicidal effect (ME) is obtained.
 An ME of 1 equals to a killing of 90% of the initial number of bacteria, an ME of 2 means 99%
killed.
 A generally accepted requirement is an ME that equals or is greater than 5: at least 99.999% of the
germs are killed.
 Even though these tests are generally well standardized, their approach is less practical.
Interpretation of result:
 Growth occur – less effective disinfectant
 No growth occur – more effective disinfectant
Suspension tests are preferred to carrier test. This is because the bacteria are uniformly exposed to the
disinfectant.
Types of suspension test:
i. Qualitative suspension test
ii. Quantitative suspension test
iii. Phenol coefficient test

Phenol Coefficient Test

Determination of phenol coefficient:
 This method is used for evaluating the effectiveness of disinfectants.
 It is the best known disinfectant test.
 Staphylococcus aureus or Salmonella typhi are used for testing.
  Dilution of phenol and experimental disinfectant are inoculated with Staphylococcus aureus and
incubated at 20-37˚C for 2-3 days.
 Phenol coefficient of a disinfectant is calculated by dividing the dilution of test disinfectant by the
dilution of phenol that disinfects under predetermined conditions.
Two main methods under phenol coefficient test are:
i. Rideal Walker method
ii. Chick Martin test

i. Rideal Walker method:

 Phenol is diluted from 1:400 to 1:800 and the test disinfectant is diluted from 1:95 to 1:115.
 Their bactericidal activity is determined against Salmonella typhi suspension.
 Subcultures are performed from both the test and phenol at intervals of 2.5, 5, 7.5 and 10 minutes.
 The plates are incubated for 48-72 hours at 37°C.
 That dilution of disinfectant which disinfects the suspension in a given time is divided by that
dilution of phenol which disinfects the suspension in same time gives its phenol coefficient.

Disinfectant Dilution Growth of test organism in subculture after exposure for:

2.5 mins 5 mins 7.5 mins 10 mins
1:400 NG NG NG NG
Test 1:500 G NG NG NG
disinfectant 1:600 G G NG NG
1:700 G G G NG
1:800 G G G G

1:95 G NG NG NG
1:100 G G NG NG
Phenol 1:105 G G G NG
1:110 G G G NG
1:115 G G G G

For example, after 7.5 minutes, the test organism was killed by the test disinfectant at a dilution of 1;600. In
the same period the test organism was killed by phenol at a dilution of 1:100.
  This result indicates that the test disinfectant can be diluted six times as much as phenol and still
possess equivalent killing power for the test organism.
 Disadvantages of the Rideal-Walker test are: No organic matter is included; the microorganism
Salmonella typhi may not be appropriate; the time allowed for disinfection is short; it should be used
to evaluate phenolic type disinfectants only.

ii. Chick Martin test:

 This test also determines the phenol coefficient of the test disinfectant.
 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 feces) to simulate the presence or
organic matter.
 Time for subculture is fixed at 30 minutes and the organism used to test efficacy is S.typhi as well as
S.aureus.
 The phenol coefficient is lower than that given by Rideal Walker method.

Rideal -Walker Chick-Martin

Volume medium 5.0 ml 10.0 ml
Diluent for test Water with yeast suspension or
Distilled water
disinfectant feces
Reaction temperature 17.5±0.5ºC 30ºC
Salmonella typhi,
Organism Salmonella typhi
Staphylococcus aureus
Sampling times 2.5, 5.0, 7.5, 10.0 min. 30.0 min.
Mean concentration of phenol
Calculation of Dilution test killing in 7.5 mins
showing no growth after 30 min.
coefficient divided by same for phenol
divided by same for test

 The phenol coefficient test recommended by AOAC included two test organisms (S. aureus and P.
aeruginosa) and included the disinfectant inactivators in the recovery medium.
 The recovery medium Letheen broth contains the inactivator Lecithin and Polysorbate 80.
 In separate tests, the bacterial suspensions are added to standard dilutions of pure phenol and several
 dilutions of the test disinfectant.
 After contact time of 5, 10 and 15 minutes, samples are transferred to the recovery medium by a
standard wire loop.
 When the positive and negative cultures have been recorded, the result of the test is expressed as
phenol coefficient.
 It is calculated by dividing the highest dilution of the disinfectant that kills the test inoculum in ten
minutes but not in five minutes by the dilution of phenol that gives the same result.

3. Disinfectant kill time test

 This test was designed to demonstrate log reduction values over time for a disinfectant against
selected bacteria, fungi, and/or mold.
 The most common organisms tested include: Bacillus subtilis, Bacillus atrophaeus, Bacillus
thuringiensis, Staphylococcus aureus, Salmonella cholerasuis, Pseudomonas aeruginosa,
Aspergillus niger, and Trichophyton mentagrophytes.
 A tube of disinfectant is placed into a waterbath for temperature control and allowed to equilibrate.
 Once the tube has reached temperature, it is inoculated to achieve a concentration of approximately
106 CFU/mL.
 At selected time points (generally five points are used including zero) aliquots are removed and
placed into a neutralizer blank.
 Dilutions of the neutralizer are made and selected dilutions plated onto agar.
 Colonies are enumerated and log reductions are calculated.
4. Capacity tests:
 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.
 Capacity tests simulate the practical situations of housekeeping and instrument disinfection.
 The best known capacity test is the Kelsey-Sykes test (Kelsey and Sykes, 1969).

Kelsey-Sykes test
 It is a triple challenge test, designed to determine concentrations of disinfectant that will be effective
in clean and dirty conditions.
 The disinfectant is challenged by three successive additions of a bacterial suspension during the
course of the test. The duration of test takes over 30 minutes to perform.
 The concentration 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%.
 Depending on the type of disinfectant, 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 are made in hard water for clean conditions and in yeast suspension
for dirty conditions.
 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 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 and not as a coefficient.
 Sets that contain two or more negative cultures are recorded as a negative result.
 The disinfectant passes at the dilution tested if negative results are obtained after the first and second
challenges.
 The third 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.
 Specimen result of a test
Inoculum Challenge number
Concentration Result
count 1 2 3

1.0 2 x 109 +++++ +++++ +++++ Fail

1.5 2 x 109 - - - -+ - -+++ +++++ Pass

2.0 2 x 109 ----- ----- ----+ Pass

Advantage
It gives a good guideline for the dilution of the preparation to be used
Disadvantage
It is a complicated test
5. 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. Sufficient disinfectant solution is prepared for two 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.

6. Practical tests:
 The practical tests under real-life conditions are performed after measuring the time-concentration
relationship of the disinfectant in a quantitative suspension test.
 The objective is to verify whether the proposed use dilution is still adequate in the conditions under
which it would be used. The best known practical tests are the surface disinfection tests.
 Surface tests assess the effectiveness of the selected sanitizer against surface-adhered
microorganisms.
 The test surface (a small tile, a microscopic slide, a piece of PVC, a stainless steel disc, etc.) is
 contaminated with a standardized inoculum of the test bacteria and dried: then a definite volume of
the disinfectant solution is distributed over the carrier; after the given exposure time the number of
survivors is determined by impression on a contact plate or by a rinsing technique, in which the
carrier is rinsed in a diluent, and the number of bacteria is determined in the rinsing fluid.
 In order to determine the spontaneous dying rate of the organisms caused by drying on the carrier, a
control series is included in which the disinfectant is substituted by distilled water; from the
comparison of the survivors in this control series with the test series, the reduction is
determined quantitatively.

 There is an essential difference between a carrier test and a surface disinfectant test: in the former
case the carrier is submerged in the disinfectant solution during the whole exposure time, whereas in
the latter case the disinfectant is applied on the carrier for the application time and thereafter the
carrier continues to dry during the exposure.

 Surface tests can reflect in-use conditions like contact times, temperatures, use-dilutions, and surface
properties.

7. Surface Time kill Test

 A 24 hour culture in nutrient broth culture is prepared.
 A volume of microbial culture (usually 0.010 mL to 0.020 mL) is placed onto the center of each of a
number of sterile test surfaces.
 This inoculum can be spread over the sterile test surface in a circular pattern to achieve a thin,
uniform coverage with the test microorganism if desired.
 To measure initial microbial concentrations, one or more untreated, inoculated test surfaces are
harvested and microorganisms are enumerated.
 The remaining inoculated test surfaces are treated with the disinfectant, each for a different length of
time.
 Immediately after the treatment times have elapsed, the test surfaces are placed into a solution that
 neutralizes the disinfecting action of the product, and microorganisms surviving treatment with the
disinfectant or sanitizer are cultured and enumerated. Results of the timekill study are tabulated and
reported, usually by charting microbial concentrations on the test surfaces as a function of treatment
time with the disinfectant or sanitizer.

8. In-use test:
 A simple to use test was described by Maurer in 1985 that can be used in hospitals and laboratories
to detect contamination of disinfectants.
 A 1 ml sample of the disinfectant is added to 9 ml diluent which also contains an inactivator.
 Ten drops, each of 0.02 ml volume of the diluted sample are placed on each of two nutrient agar
plates.
 One is incubated at 37oC for three days and the other at room temperature for seven days. Five or
more colonies on either plate indicate contamination.

9. British standard tests for quaternary ammonium compounds:

 This test was initially described in 1960 to distinguish bactericidal action from the high level of
bacteriostatic activity, which is characteristic of QACs.
 This test is also applicable to other bactericides such as chlorhexidine and synthetic phenols.
 The inactivator used contains 2% lecithin and 3% non-ionic detergent (polysorbate 80).
 The test is performed using suspensions of gram negative and gram positive bacteria with or
without the inclusion of organic matter.
 If a series of samples are taken from a dilution of the disinfectant containing 5 x108 to 5 x 109
bacteria per ml at the start of the test, a death curve may be prepared from the colony counts on the
agar containing recovery medium and reduction factors up to 106 (99.99% kill) can be verified.
 In order to determine the antimicrobial value of QAC, it was revised as the highest dilution of the
disinfectant that, under the test conditions, will reduce the microbial population to a colony count
not greater than 0.01% of that in the control.
 In this revision, E. coli was used and the contact time was 10 minutes.
 The challenge medium is E. coli culture suspension with equal amount of horse serum.
 One ml of the challenge is added to nine ml of each dilution of the disinfectant along with two
control tubes containing diluent alone.
 At the end of exposure period, one ml each of the mixture is added to 9 ml of inactivator and the
surviving bacteria are counted as colony forming units on agar plates.
 Testing schemes:
 The antimicrobial efficiency of a disinfectant is examined at three stages of testing.
 The first phase concerns laboratory tests in which it is verified whether a chemical compound or a
preparation possesses antimicrobial activity: for these preliminary screening tests essentially
quantitative suspension tests are considered.
 The second stage is still carried out in the laboratory but in conditions simulating real-life
conditions.
 Not disinfectants, but disinfection procedures are examined.
 It is determined in the practical tests in which conditions and at which use-dilution after a given
contact time the preparation is active.
 The third phase comprises the field tests or pilot studies, and the variant of in-use tests. In these tests
it is verified whether, after a normal period of use, germs in the disinfectant solution are still killed.
 Most studied are the bactericidal tests in which the activity towards vegetative bacteria is examined.
AOAC has schedules that are applicable for fungi and yeasts too (fungicidal tests), for mycobacteria
(tuberculocidal tests), for viruses (virucidal tests) and for spores of bacteria (sporicidal tests).

EVALUATION OF OTHER DISINFECTANTS

The evaluation methods described above basically apply to liquid disinfectants.
Solid disinfectants
 Solid disinfectants include metals such as,
1. Gold
2. Copper
3. Aluminum
4. Silver
 Exposure of disinfectant onto particular organism
 Inoculated onto a Solid Agar.
 Disc may be cut out from agar and sub cultured for enumeration of survivors.
 The inhibition of solid disinfectant activity is also evaluated by dusting the powders onto the
surface of seeded agar plates.
 The extent of growth is then observed following incubation

Air disinfectants
 Disinfection of air is very important for infection and contamination control.
 Air is the commonest route of transmission of infection in hospitals and other places
 Disinfection of air can be done via filtration (HEPA filters).
 Via chemical agents (H2O2, formaldehyde).
 Also by means of ultra violet light.
 The number of viable bacteria present in air can be assessed by exposing Petri dishes (of solid
nutrient medium) to the air (settle plate).
 Filtration sampling, where the air is passed through a porous membrane which is then cultured
can also be used.
INVITRO ANTIBACTIRIAL ACTIVITY SCREENING

1. BACTERIOSTATIC AGENTS

These are the agents which prevent the growth of bacteria, but it does not kills the organism.

Assessment of Bacteriostatic Activity

1. Serial dilution in fluid media
2. Serial dilution in solid media
3. Cup plate methods
4. The gradient-plate method
5. The ditch-plate technique

1. Serial Dilution in fluid media:

 In this method, graded concentrations of the test substance in a nutrient medium are
inoculated with the test organism and incubated.
 The minimum concentration preventing detectable growth (MIC) is taken as a measure of
bacteriostatic activity.
2. Serial Dilution in solid media
 A suitable volume of double strength nutrient agar is diluted with an equal volume of
bacteriostatic solution and poured into a sterile petridish.
 When solidified the surface is dried by incubating at 370 C. Drops of 24hrs broth culture
of the test organisms are placed on the dried surface and incubated for 2 to 3 days.
 Upto 27 cultures can be tested on each plate if a multi-point inoculator is used.
 This method is mainly used for solutions which give turbidity with fluid nutrient media.

3. Cup – Plate method

 In these methods the agar is melted, cooled suitably, inoculated with the test
organism and poured into a sterile petri dish.
 In the cup-plate method, when the inoculated agar is solidified, holes about 8mm
in diameter are cut in the medium with a sterile cork borer.
 In all cases zones of inhibition may be observed, the diameter of the zones giving
a rough indication of the relative activities of different anti-microbial substance.
4. The Gradient – Plate method
 Two layers of agar is poured.
 The plates are then incubated over night to allow diffusion of anti-microbial
substance.
 The agar is streaked in the same line as the slope of the agar and re-incubated.
 An approximate MIC can be obtained from the following equation:

MIC = C (x/y) mg/ml

Where,
C = concentration in mg/ml, in total volume
X = length of growth, in cm,
Y = total length of possible growth, in cm

5. The Ditch – Plate technique

 An agar is poured in a Petri-plate, allow to solidify, and ditch cut out is made of
the agar.
 A solution of the antimicrobial substance or a mixture of this with agar is
carefully run into the ditch so as to about three-quarters fill it.
 A loop-full of each test organism is then streaked outwards from the ditch on the
agar surface.
 Organisms resistant to the antimicrobial grow right up to the ditch whereas
susceptible organisms show a zone of inhibition adjacent to the ditch.
  The width of the inhibition zone gives an indication of the relative activity of the
antimicrobial substance against the various test organisms.

i. BACTERIOSTATIC AGENTS

These are the agents which kills the bacteria including its spores is called as the bactericidal
agents.

Assessment of bactericidal activity

End-point or extinction time methods
There are two types of extinction time method;
Method 1: Phenol coefficient type tests
In which the extinction time is fixed and the concentration of disinfectant needed to kill in the
specified time is estimated.
Method 2:
In which the concentration of bactericide is fixed and the extinction time is estimated.

Method 1
Phenol coefficient test
i. Rideal Walker Test
ii. Chick Martin Test
 i. Rideal Walker Test
Principle: dilutions of the test disinfectant is compared with the standard dilutions of the
phenol (usually 1 in 95 to 1 in 115) further activity against salmonella typhi.
Procedure:
 Take 24 hrs culture of the S. typhi
 Test disinfectant solution or phenol is added about 0.2 ml to the S. typhi culture of 24
hrs
 At intervals of 2.5, 5,7.5, 10 min sub cultures are taken and transferred to the fresh
broth media
 The broth tubes are incubated at 370c for 48-72 hours and growth is measured.

Rideal walker Dilution of test disinfectant killing in 7.5min not in 5 min

=
co-efficient Dilution of the standard phenol killing in7.5 min not in 5 min

ii. Chick Martin Test

Principle:
This test is carried in the presence of organic matter like 3% human faecas or dried yeast.
Procedure:
 Serial dilutions of test solution and phenol is prepared in distilled water
 To this 3% yeast suspension is also added.
 To this solution the s typhi organsim is added
 After contact time of 30 minutes the above mixture is transferred to the freshly
prepared 10 ml of broth.
 The test tubes are incubated at the 370c for 48 hours.
 Presence or absence of the growth is calculated.

Mean of highest phenol concentration inhibiting and lowest

Chick martin permitting growth
=
Co-efficient
Mean of highest disinfectant concentration inhibiting and
lowest permitting growth
Method 2
 In this method the concentration of the test disinfectant is maintained constant.
 The extinction time is altered i.e., the contact between the disinfectant and the
organism is increased or decreased.
 By this the extinction time is estimated.