PHCT 401: Disinfectant Evaluation

Disinfectants, antiseptics and preservatives are

chemicals, which have the ability to destroy or

inhibit the growth of microorganisms.

Disinfection

• Destruction of harmful microorganisms (vegetative

pathogens)

• Inert surface or substance

• Chemicals, UV radiation, boiling water, and steam

BSI – not necessarily killing all microorganisms.

- reduction to acceptable level for defined purpose

- not harmful to health of individuals or quality of

perishable goods (pharmaceuticals, cosmetics)

Antisepsis

• Destruction or inhibition of harmful microorganisms (vegetative pathogens)

• Living tissue e.g. Skin

• Limiting / preventing harmful results of infection

• Not same as disinfection (BSI)

• Chemicals applied to skin, mucous membranes Not toxic or irritating to the skin

Decrease microbial population on the skin.

Preservatives

• Included in preparations

• Prevents microbial spoilage of the product

• Minimize risk of product-related infections to

consumers

• Limits proliferation of contaminants in non-

sterile preps e.g oral and topical preparations

• Kill microbial contaminants in sterile preps e.g

eye drops, multi-dose injs.

Sterilization

• Destruction of all forms of microbial life,

including endospores

• Are the microbes dead or not?

• -cide or cidal: Killing of microorganism

• -static or stasis: Inhibit growth or

multiplication of microorganisms

• Sepsis: Bacterial contamination

• Asepsis: Absence of bacterial contamination

Other terms

• Bactericide-substance that kills bacteria

• Sporocide- an agent which kills spores

• Bactriostat- an agent which prevents the reproduction and multiplication of bacteria

• Virucide (viricide)- is an agent which kill viruses

• Fungicide- an agent that kill fungi

• Fungistat- an agent that prevent fungi proliferation

• Sanitizer- an agent that reduce the number of bacterial contamination to a safe level.

Evaluation of Disinfectant Activity

• Tests prescribed varies in countries

• Regulatory bodies with laid out criteria for

comprehensive evaluation of antimicrobial

agents

• Standardised & internationally acceptable

guidelines exits.

Regulatory Bodies

BSI – British Standards Institutions

AOAC – Association of Official Analytical Chemists

AFNOR-Association Francaize de Normalisation

DGHM – German Society for Hygiene & Microbiology

NAFDAC ?

Evaluation of Disinfectant - How to determine

& compare effectiveness of disinfectants.

The tests can be either

• Bactericidal or

• Bacteriostatic

The bactericidal tests include

• Phenol coefficient tests

o The Rideal walker test

o The Chick Martin test

o The Crown agents test

o AOAC Phenol coefficient test

The Bacteriostatic tests include

• Serial dilution test

• Agar diffusion test

o Agar cup tests

o Surface contact tests

o Ditch plate tests

o Gradient plate method

o Test for combined action

• Viable counts

• Kelsey-Sykes Capacity test

• In-use test for hospital disinfectants

BACTERICIDAL TESTS

• Measure the ability of disinfectant to kill the test organism.

• The effects of the disinfectant must therefore be neutralized before incubation

• To prevent bacteriostatic effects.

The methods commonly used for testing the efficiency of disinfectants can be classified under

i. End point or extinction methods

ii. Counting methods

iii. Other methods

Extinction methods

Are of two types

• Methods in which the extinction time is

fixed and the concentration of disinfectant

that kills bacteria in the specified time is

calculated,

These methods are generally know as Phenol

Coefficient tests

• Methods in which the concentration of

bactericide is fixed and the extinction time is

estimated

Counting Method

• Viable Count Method

Quantitative Suspension tests

• Viable counts are taken after suitable contact

time

• Proportion of viable cells observed after

specific contact time used to characterise the

bactericidal activity.

Approaches to Disinfectant Evaluation

There are 2 broad approaches based on test

condition

i. In vitro test methods

ii. Practical test methods

In vitro test methods

• Phenol coefficient tests

• Suspension Tests of DGHM

Qualitative Suspension Test

Quantitative Suspension Test

• AOAC & DGHM Suspension Tests

• Kelsey-Sykes Capacity Test

•The Rideal Walker

•The Chick Martin

•The Crown agents test

•AOAC Phenol coefficient test

Practical Tests

1. Surface disinfectant test

Determines the reduction rate of S. aureus,

E. coli, by disinfectant on PVC plates and OP-tiles.

Other organisms included are Ps. aeruginosa,

C. albicans and Trichophyton mentagrophytes

on raw wood.

2. Hygienic hand disinfection test

3. Surgical hand disinfection test

4. Instrument disinfection test

5. Skin & hand disinfection test

6. Textile disinfection test

Substances Tested Bacteriostatic Tests Bacteriocidal Tests

Liquid disinfectants Serial dilution in fluid media

Serial dilution in solid media

Cup-plate, filter paper

methods

Gradient-plate method

Ditch-plate technique

All methods can be used

quantitatively; the final

method is usually used

qualitatively.

End-point or extinction time

methods

Counting methods

Turbidometric assessment

‘In-use’ & other tests

‘In-vivo’ tests additionally

applied

Semi-solid antibacterial

Formulations e.g.

Creams, ointments &

Gels.

Cup-plate

Ditch-plate

Modified end-point or

extinction time methods

In addition ‘in-vivo’ tests are

applied (e.g. skin tests)

Solid disinfectants,

Disinfectant powders

Inhibition on seeded agar Modified end-point or

extinction

Time methods

Aerial disinfectants Use of slit-sampler in test chamber.

Tests for Disinfectant Activity

Disinfectant Evaluation Testing Schemes

The antimicrobial efficacy of a disinfectant is

examined at three stages

Phase 1

• laboratory tests

• verifies whether a chemical compound or a

preparation possesses antimicrobial activity

• preliminary screening tests essentially

quantitative suspension tests

• Absence of organic matter

• One strain of S. aureus & Ps. aeruginosa

• Determines basic bacteriostatic activity

Phase 2

• Carried out in the laboratory

• Simulating real-life conditions (in-use)

• Organic matter load

• Several test microorganisms

• Either as a suspension test (Step1)

• On surfaces (Step2)

• Determined in the Practical tests

• Conditions at which use-dilution after

a given contact time the preparation is active.

Phase 3

• “In-use” trials

• Field tests or pilot studies

• Tests verifies whether, after a normal period

of use, germs in the disinfectant solution are

still killed.

Carrier tests:

• Oldest tests. The test described by Robert Koch was a carrier test.

• The carrier such as a silk or catgut thread or a penicylinder (a little stick) is contaminated

• Submersion in a liquid culture of the test organism.

• Carrier is then dried and is brought in contact with the disinfectant for a given exposure time.

• Cultured in 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,

• Potentially active concentration-time relationships of the disinfectant is obtained.

Example carrier test

• Use-dilution test of the American Association of Official Analytical Chemists (AOAC, 1990). Limitation of the carrier tests

a) the number of bacteria dried on a carrier is hard to standardize and

b) the survival of the bacteria on the carrier during drying is not constant.

Suspension tests:

• Sample of the bacterial culture is suspended into the disinfectant solution

• After exposure it is subculture to verify whether this inoculum is killed or not.

• Suspension tests are preferred to carrier tests

the bacteria are uniformly exposed to the disinfectant.

Different kinds of suspension tests:

• the qualitative suspension tests,

• Phenol coefficient test(Rideal and Walker, 1903); (Chick and Martin, 1908)

• quantitative suspension tests.

Initially 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.

Disinfectant kill time test

• 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.

• Tube 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

• Selected dilutions plated onto agar.

• Colonies on agar surface are counted and

• Log reductions are calculated.

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.

• Ability to retain activity in the presence of an

increasing load is the capacity of the disinfectant.

• Disinfectant is challenged repeatedly by successive

additions of bacterial suspension until its capacity

to kill has been exhausted.

• Simulates the practical situations of housekeeping

and instrument disinfection.

• best known is Kelsey-Sykes test (Kelsey and Sykes,

1969).

Practical tests:

• Tests under real-life conditions

• Performed after measuring the time-concentration

relationship of the disinfectant in a quantitative

suspension test.

The objective of Practical tests

• to verify whether the proposed use dilution is still

adequate in the conditions under which it would be

used.

• Best known 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.)

• Contaminated with a standardized inoculum of the

test bacteria and dried

• 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.

Control series

• Determines the spontaneous dying rate of the

organisms caused by drying on the carrier,

• Disinfectant is substituted by distilled water;

• Comparison of the survivors in control series with

the test series

• The reduction is determined quantitatively.

Determination of Disinfectant activity

against vegetative bacteria

• Correct evaluation

• Standardise factors affecting effectiveness of agent

o Time

o Concentration

o Moisture

o pH

o Temperature

o Inactivating agent - Cleansing ? Detergent agents

- organic matter

o Protein factor

o Protective factors

o Spectrum of activity

In-vitro Tests

Minimum Inhibitory Concentration Test

(Test for determining bacteriostatic activity)

• Disinfectant acceptable activity on vegetative

bacterial cells

• Bacteriostatic activity determined by MIC

• Lowest concentration of disinfectant that will inhibit

the growth of a culture.

• Similar to test-tube serial dilution used in antibiotic

susceptibility test.

Graded doses of test substance (disinfectant)

Added to suitable liquid medium e.g NB

Tubes inoculated with standardised test organism

Tubes incubated at appropriate temperature

0.1ml

Serial dilution 50% 25% 12.5%

NB

(Double strength

Disinfectant

solution

5ml 5ml 5ml 5ml

Dilution

1:2 1:2 1:2

50% 25% 12.5% 6.75%

5ml

Bacterial Suspension

(overnight culture)

108cfu/ml

0.1ml 0.1ml 0.1ml

Determination of MIC of disinfectant

Phenol Coefficient Tests

• Devised by Lister

• First disinfectant was carbolic acid (coaltar derivative)

• Phenol Coefficient compares any disinfectant to

phenol.

Rideal-Walker Test

• Qualitative suspension test

• Based on Phenol Coefficient

• Form the basis for other tests, with modifications

• Introduced in 1903 & became BSI test in 1934

• Three test organisms are recommended Salmonella typhosa ATCC 6539

Staphylococcus aureus ATCC 6538

Pseudomonas aeruginosa ATCC 15442

Rideal-WalkerTest

Loopful

Contact Time 2½ 5 7 ½ 10 min 2½ 5 7 ½ 10 min

Disinfectant

solution

Phenol

solution

5ml 5ml

Bacterial

Suspension

0.2ml

0.2ml

180C 180C

Nutrient Broth tubes Nutrient Broth tubes

Loopful

Test Procedure

1. Determine the MIC of the disinfectant

2. Make five dilutions of the disinfectant, differing by 1 in 100,

such that the second dilution is the MIC (e.g. If MIC is

1 in 400; then the dilution levels are 1in 300; 1in 400;

1 in 500; 1 in 600 and 1 in 700)

3. Prepare a 5% aqueous solution of Phenol (B.P. or USP, mp

40.50C) From this, prepare five dilutions 1 in 95; 1 in 100;

1 in 105; 1 in 110 and 1 in 115.

4. Set up 4 rows of five tubes containing 5ml of sterile NB

5. In front of these, set out 5ml amount of the five dilutions of

the test disinfectant. The tubes should be kept in a water

bath at 180C

6. At 30secs intervals add 0.2ml of the standardized overnight

culture (bacteria suspension)/ test organism to each of the

dilutions, starting with the lowest dilution. This will take

2mins

Rideal-Walker Test Procedure contd

7. After 30secs remove the first (lowest) dilution, shake and

transfer a loopful to the first tube in the front row of tubes

containing NB. Subsequently, at 30secs interval, remove a

loopful from the next dilution level into the next NB tube.

Thus, each broth tube in this row receives its loopful of

inoculum 2.5mins after the organisms were added to the

disinfectant dilution.

8. Repeat the operation with the second row of NB tubes at

30secs interval such that each tube receives its inoculum

5mins after the test organisms were added to the

disinfectant dilutions.

9. Repeat with the third row of NB tubes after 7.5mins

exposure to disinfectant.

10.Repeat with fourth row after 10mins exposure

11.Duplicate the whole procedure with phenol dilution.

Rideal-Walker Test Procedure contd

12. Incubate the inoculated NB tubes at 370C for 48hrs.

13.After incubation examine the tubes for presence or absence

of growth by comparison with a control tube containing NB

alone or Phenol alone.

A typical result and the method of calculating the coefficient is

shown below.

+ = Growth - = No growth

Dilution action of Time of Exposure (min)

2½ 5 7½ 10

Disinfectant 1 in 400 - - - -

1 in 500 + - - -

1 in 600 + + - -

1 in 700 + + + -

Phenol 1 in 100 + + - -

Calculation of the Coefficient

The Rideal-Walker coefficient is obtained by dividing the

dilution of test disinfectant which does not kill all the

organisms after 2.5 and 5minutes but after 7.5 and

10minutes, by the dilution of phenol that produces similar

effect.

A phenol coefficient greater than 1 means that the disinfectant

is better than phenol. Phenol Coefficient of commercial

cleaning agents is usually given on the label.

Phenol coefficient for some phenolics are:

Lysol = 3-4; Roxenol = 5 – 5.5;

White fluid = 10-11 Black fluid = 14-15

min. 5in not but min. 7.5in killing Phenol ofDilution

min. 5not but min. 7.5in killingnt disinfecta ofDilution t CoefficienRW

6100

600 t coefficienRW

Chick-Martin Test

• Qualitative suspension test

• Modification of Rideal-Walker test-disinfectant

dilution made in distilled water

• Disinfectants generally required to act in the

presence of organic or suspended matter.

• The 10mins exposure period considered inadequate

to test many disinfectant preparations.

• Chick and Martin (1908) proposed a test that takes

these into account.

• Originally, dried faeces were added to the

disinfectants, later modified by substituting a

standardized yeast suspension.

• BSI test with No. 808 in 1938

Chick-Martin Test Procedure

1. Determine the MIC

2. Make up a 1, 2 or 5% solution of the disinfectant

(whichever is nearest to 2 x MIC)

3. Prepare a 5% solution of Phenol (B.P or USP)

4. Make up a 5% suspension of commercial dried

yeast in distilled water & autoclave.

5. Set up a row of 9 test tubes. To each tube add 2.5ml

of diluted disinfectant, beginning with concentration

as in Step1, and decreasing in strength by 10% at

each tube.

6. Repeat Step 4, using the 5% Phenol solution

7. Add 2ml of standardized overnight culture of the test

organism to 48ml of sterile yeast suspension.

Chick-Martin Test Procedure

8. Set up two rows of 10ml NB, one for each dilution of

disinfectant under test and of the Phenol (it is

common to use duplicate tubes, i.e two at each

disinfectant & phenol dilution)

9. At 30secs intervals, pippette 2.5ml of the yeast-test

organism mixture into each disinfectant and phenol

dilutions. Allow the reaction to proceed at 200C

(room temp) in a water bath.

10.Exactly 30min after the first dilution tube received

yeast-bacteria mixture (i.e. 30mins contact time),

transfer one loopful from the tube to the

corresponding tube(s) of NB

Chick-Martin Test Procedure

11.At 30secs intervals, transfer loopfuls from the other

dilution tubes in succession into corresponding NB

tubes.

12.Incubate the broth tubes at 370C for 24hrs

13.Note (record) the dilution level of the tube that

which fails to show growth and the first tube which

shows growth in both disinfectant and phenol

series.

The Phenol Coefficient is obtained by dividing the

mean of those of Phenol which do & do not show

growth with those concentrations of disinfectant

which do & do not show growth

Chick-Martin Test Sample Result

+= Growth; - = No growth

Average of Phenol series = C + D / 2

Average of Disinfectant series = E + F / 2

Decreasing Concentration (%)

A B C D E F G H I

Phenol series - - - + + + + + +

Disinfectant series - - - - - + + + +

F E

D C

2F E

2D C Coeff.Martin -Chick

NB tubes

10ml 10ml

Incubate Incubate

Schematic Diagram of Chick-Martin Phenol Coefficient Test

Bacterial

Suspension

Yeast

Suspension

(Sterile)

Bacterial-Yeast

Suspension Mixture

48ml 2ml

Disinfectant

Solution

Phenol

Solution

2.5ml 2.5ml

30mins 30mins

loopful loopful

2.5ml 2.5ml

Other Phenol coefficient-type tests

• The Crown agents test

Intended for testing white disinfectants

Finely dispersed emulsions of coal tar acids or similar

acids from petroleum

Similar to Chick-Martin test, but

Disinfectant diluted in sterile artificial seawater

Gelatin & rice starch included as organic matter

• FDA

• AOAC Phenol Coefficient Test

o Testing disinfectants miscible with water

o Neutralisation of bacteriostatic by using specific media

containing lecithin, tween 80, cysteine and thioglycolate

AOAC Phenol Coefficient Test

Procedure

• Make dilutions of disinfectant from 1% stock solution

• From 5% stock solution of phenol make further dilutions

1:80 and 1:90 for Pseudomonas aeruginosa

1:90 and 1:100 for Salmonella typhi

1:60 and 1:70 for Staphylococcus aureus

• Place tubes with 5ml of each final disinfectant dilution and

phenol in a water bath at 200C

• Place tubes with standardized suspension of test organisms

in the water bath

• Add 0.5ml of test culture to each dilution & phenol

• Mix gently & transfer a loopful to neutralizing media at 5, 10,

and 15minutes interval.

AOAC Phenol Coefficient Test

Phenol Coefficient is given by the highest dilution of

disinfectant killing test organism in 10 mins but not in 5 mins

divided by the highest dilution of phenol showing the same

result

Calculation of the Coefficient

Dilutions Disinfectant

5 min 10 min 15 min

1:300 - - -

1:325 + - -

1:350 + - -

1:375 + + -

Phenol

1:90 + - -

1:100 + + +

Applications of Phenol Coefficient Tests

Designed to test the ability of phenolic disinfectants to

destroy the causal organism of typhoid fever rapidly at room

temperature – hence the choice of the initial test organism.

Merits of phenol-Coefficient Tests

1. Tests are strictly standardized and this will make the results reproducible between different laboratories and from time to time.

2-.They are quick and cheap i.e. require little efforts and materials.

3. They provide a mean for sorting out disinfectants: either effective i.e. active or ineffective

Defects of phenol coefficient tests:

1. Apart from AOAC coefficient, tests use only a single strain of organisms: this is Salm. typhi, the use of other different strains will make the test more stringent but gives more information as the resistance of different organisms to different disinfectants varies.

2. Tests compare disinfectants at only one concentration. The tests are generally fix reaction time. Thus each test shows the efficacy of the disinfectant at such concentration. This would not matter if all disinfectants have the same concentration exponents, but this is not the case. Therefore the phenol coefficient of a disinfectant can be changed by altering the arbitrarily chosen time.

3. Tests compare activity of disinfectant at only one reaction temperature. In practice disinfectants are likely to be used at different temperatures. As the activity of the germicide changes with changes in the temperatures, then the information given by

phenol coefficient tests at the test temperature may be quite different if the disinfectant is used at a different temperature.

4. Most tests give no indication of activity of the disinfectant in the presence of organic matter. Apart from the Chick-Martin test, other tests are performed in absence of organic matter. Most disinfectants are affected to different extents by the presence of organic matter in the reaction medium. In the majority of cases the disinfectants are used in the presence of different quantities of organic matters and therefore the tests are of less value. Phenol is the least disinfectant not seriously affected by the presence of as much as 10% blood serum whereas the activity of emulsified disinfectants is seriously affected. Formaldehyde oxidizing agent’s e.g. H2O2, hypochlorite, chloramine T, chlorine, potassium permanganate etc. react vigorously with organic matter while acridines retain their activity.

5. Apart from A.O.A.C. coefficient test other tests give no indication to the effect of the recovery growth conditions for the organisms after treatment with the disinfectant. The tests rely on the fact that small volume of the disinfectant is transferred to the broth, thus no inhibitory effect if exerted, this is true for some disinfectants of high concentration exponents but not for disinfectants of low concentration exponents. The latter require antagonizing agents to be present in the broth. Further, the tests use one type of nutrient medium and one incubation temperature (370), however, it is now proved that the growth conditions of disinfectant treated bacteria are more stringent, and completely different for that required for untreated bacteria.

6. No indication of tissue toxicity. The tests are performed in reaction tubes, the results bear no relation or have any indication towards tissue toxicity, for example, Lysol is so toxic to tissue that its use on the body would never be considered. While chloroxylenol solution B.P. has the same phenol coefficient as Lysol but relatively less toxic.

R.W FDA CM AOAC Crown

agent

Medium pH 7.3-7.5 6.8 7.3 – 7.5 6.8 before

autoclaving

7.3 – 7.5

Medium vol 5.0ml 10 10 10 5

Vol of rxn

mixture

5 10 5 5 5

Diluent for

disinfectant

Water Water Yeast

suspension

Water Artificial

sea water

Rxn temp 17.5±0.5 20 30 20:37 19.0±1.0

Organism S. typhi S. typhi S. typhi S. typhi,

S. aureus

S. typhi

Sampling

time

2½, 5, 7½,

10 min

5, 10, 15 30min 5, 10, 15 10min

Calculation

of

Coefficient

Dilution test killing in

7½ but not in 5min

divided by same for

phenol

Dilution test

killing in 10 but

not in 5min

divided by same

for phenol

Mean highest

phenol concn

inhibiting & lowest

permitting growth

divided by same

for test

Greatest dilution

of test killing in

10min divided by

same for phenol

Greatest dilution

of test killing in

10min divided by

same for phenol

Essential differences between the Phenol Coefficient Tests

The DGHM Qualitative Suspension Test

• 5 test organisms recommended

Staphylococcus aureus ATCC 6538

Pseudomonas aeruginosa ATCC 15442

E. coli ATCC 11229

Proteus mirabilis ATCC 14153

Klebsiella pneumoniae ATCC 4352

There are several National and International Culture Collection

Centres. Some of them are given below:

§ ATCC (American Type Culture Collection Centre, Maryland,U.S.A.)

§ NCIB (National Collection of Industrial Bacteria, Britain)

§ DSM (Deutsche Sammlung von Mikroorganismen and Zelkulturen,

Germany)

§ NCTC (National Collection of Type Culture, London)

§ MTCC (Microbial Type Culture Collection, Osaka Japan)

§ MTCC (Microbial Type Culture Collection and Gene Bank Institute of

Microbial Technology, Chandigarh)

§ ICIM (Indian Culture of Industrial Microorganisms, National Chemical

Laboratory, Pune)

Bacterial Suspension Disinfectant Solution

0.1 ml 10 ml

108cfu/ml

Broth with

inactivator Taken for incubation and the pattern

of growth recorded

5 ml

Contact time 2 4 8 15 30 60 minutes

Schematic diagram of DGHM Qualitative Suspension Test

Quantitative Suspension Tests

• Viable count taken after known exposure time

• Comparing with initial count

Decimal reduction rate (DRR) or DRV = logNC-logND

where NC = # of colony forming units developed in

control series after specified time (i.e.

distilled water)

ND = # of cfu developed in disinfectant series

after contact time

DGHM Quantitative Suspension Test

• Contact time = 5minutes

• Good disinfectant should effect a 5log reduction

in the initial cfu.

Recommended test organisms

Ps. aeruginosa ATCC15442

Staph.aureus ATCC6538

E. coli ATCC11229

B. cereus

Saccharomyces cerevisiae

Disinfectant

Inactivator

100

1ml 1ml

Bacterial suspension

0.2ml 0.2ml

Disinfectant

series

Control

Series

Diluent

10ml 10ml

5mins Contact Time 5mins

10ml 10ml

1ml

9ml 9ml

Diluent

10-1 10-2 10-3 100 10-1 10-2 10-3

1ml 1ml 1ml 1ml

1ml 1ml 1ml 1ml

1ml 1ml

1ml

Test Procedure

• Standardize overnight broth culture of test organism to

contain 108cfu/ml

• Transfer 0.2ml of bacteria suspension into 10ml each of the

various disinfectant dilutions & 10ml of the diluent (NB) as

the control series

• After contact time of 5mins, take 1ml of the reaction mixture

(disinfectant solution + bact suspension) into recovery broth

containing about 10ml of specific inactivator for the

disinfectant.

• Make serial dilutions and plate out on NA plates

• Incubate at 370C for 24hrs

• Repeat the same procedure for the control series

• Determine the # of surviving organisms after 5min contact

time from the resulting colonies on the plates

• Calculate the log reduction factor for the disinfectant

Note

• Plates with more than 300 colonies or less than 50 colonies

are discarded

It is recommended that a good disinfectant should have a log

reduction factor (RF) of not less than 5.

RF = logNC-logND

= log107 – log103

Advantages

Disadvantages

CARRIER TEST

• Pre-treatment of the object (carrier) with bacterial suspension

Pieces of cloth (as in DGHM test)

Metal cylinder (as in the AOAC test)

• Then soaking carrier in disinfectant solution for a specific time

• Check to see if test organism is killed or not

• Carrier tests resemble Practical tests

• Classified as in-vitro tests

Carriers abstracted prior to pre-treatment

Carriers standardized (by sterilization) and in dimension

Best known examples of carrier test

• DGHM TestMethod

• AOAC Test Method

AOAC Carrier Test Method

Recommended test organisms

• Ps. aeruginosa ATCC 15442

• Staph. aureus ATCC 6538

• Salmonella choleraesuis ATCC10708

Carrier: Stainless steel penicillin cups (10 per dilution)

Contact time: 10 minutes

• Test confirms the phenol-coefficient results

• Determines the maximum dilution that is effective for

practical disinfection.

• Determines the in-use concentration of a test disinfectant

Standardized

Bacterial Suspension

Carrier

Stainless steel

108cfu/ml

0.1ml

15 mins Drying on

Filter paper

10 mins Contact time

Disinfectant

solution

+Inactivator

Subculture broth Incubate at 370C

for 24hrs

Schematic Diagram of AOAC Carrier Test Method

• Ten (10) subculture broth + inactivator tubes

incubated for each disinfectant concentration.

• The in-use dilution is that concentration at which

none of the 10 sub culture tubes show any

growth.

What is the in-use dilution for this disinfectant?

Disinfectant

concentration

Number of subculture broth

with growth out of 10

0.25 10

0.5 4

1.0 0

2.0 0

DGHM Carrier Test Method

Test organisms

• Staph. aureus ATCC 6538

• E. coli ATCC 11229

• Proteus mirabilis ATCC 14153

• Ps. aeruginosa ATCC 15442

• Candida albicans ATCC 10231

• M. tubeculosis ATCC 25618

Carrier: Standard cotton cloth, properly washed in

distilled water, cut in 1cm2 and sterilized by

autoclaving and dried.

Preparation of Bacterial Suspension

5ml of CSB is used to harvest surface colonies from

24hr agar culture of the test organism on a 90mm

diameter petri-dish. Standardize suspension to

contain ≈ 109 cfu/ml.

Test Procedure

• Soak 5 pieces of the sterilized, dried cotton cloth in

the standardized bacterial suspension for 15 mins

• Turn twice to remove air bubbles

• Transfer wet pieces of carrier onto a petri dish and

cover with 10ml of disinfectant solution

• After various contact times 15, 30, 60, & 120mins,

each piece is removed & immersed in a broth

containing inactivator & rinsed

• Transfer into another subculture broth & incubate at

370C for a max of 72hrs

• As control, similar cotton cloth pieces are treated

with standardized hard water instead of the

disinfectant solution, and similarly treated as

described

5

15

30

60

120

Rinsing broth

with inactivator

Incubate at 370C

for 72hrs

Subculture broth

Contact time

Petri dish Soak for

15mins

Carrier

(Cotton cloth, 1cm2)

Standardized

Bacterial suspension Disinfectant

solution

KELSEY-SYKES CAPACITY TEST

• In-vitro test formulated by Kelsey et al in 1965 and modified

by Kelsey & Sykes in 1969

• Current test protocol was a modification of Kelsey & Maurer in

1974

• Simulates as close as possible the practical situations of use

• Determines the ability of the disinfectant to withstand multiple

challenges of the inoculum as encountered in practical

situations

• Performed under two conditions of use

Clean and

Dirty – 5% yeast suspension as in CM or in some

cases horse serum is added

Three disinfectant concentrations used

I. Dilution as recommended by the manufacturer

II. 25% weaker than recommended in-use dilution

III. 25% stronger than recommended in-use dilution

Standard hard water used in preparing the

disinfectant solution

Contains: 10%w/v of MgSO4.7H2O

hardness is 300ppm.

Media: Nutrient broth

Yeast suspension – 5% as in Chick-Martin or

Horse serum – at initial concentration of 20%.

Subcultured tubes incubated at 370C for 48hrs

Contact time 8 18 28 minutes

Disinfectant

solution

3ml 1ml 1ml at

10mins

1ml at 20mins

(+8) (+8) (+8)

108cfu/ml

Bacterial suspension

I drop

Subculture

broth Plated out on NA plates

At Time 0

Schematic diagram of Kelsey-Sykes Capacity Test

Interpretation of Results

Growth in broth no growth in 2 or more tubes of the

5 subculture tubes after the second incremental

addition of the bacterial suspension.

Growth in agar shows a total of 5 colonies or less

from all the 5 broth cultured after the second

incremental addition of bacterial suspension.

Concentration No. of tubes out of 5 with no growth

8 min 18 min 28 min

A 0 0 3

B (A + 25%) 0 0 1

C (A – 25%) 2 5 5

MIC of various test organisms are determined

Ps. aeruginosa NCTC 6749

Proteus vulgaris NCTC 4635

E. coli NCTC 8196 (ATCC 11229)

Staph. aureus NCTC 4163 (ATCC 6538)

• The most resistant organism (determined from the

MIC results) is used in the test.

• The Kelsey-Sykes test is more severe than

suspension test

• It is a valuable evaluation of the efficacy of

agents used for floor disinfection.

Practical Tests

1. Surface Disinfection Test

2. Hand disinfection test

i. Hygienic Hand Disinfection

ii. Surgical Hand Disinfection

3. Test for Textile Disinfection

i. Chemical Textile Disinfection

ii. Chemothermic Laundry Disinfection

4. Instrument Disinfection Test

Test for Surface Disinfection

• Methods of evaluation varies in countries

• Kelsey-Sykes used in UK

• DGHM recommends a more comprehensive practical

test

• Design of any surface disinfection test must take into

consideration

i. The differential contamination potentials of the area

e.g. food industry vs an operating theatre

ii. The contamination level likely to be encountered

• In heavily contaminated surfaces(food industry),

disinfection proceeded by cleansing with detergent

• Possibility of detergent attached to the surfaces

inactivating disinfectant when applied

• Contact time chosen to take into account the fact

that the disinfectant when applied must

First spread on the surface

Be transported across the surface especially to crevices &

pores in the material (covering the surface)

Have enough time to irreversibly damage the bacterial

cells

• Humidity of the environment important in determining

effectiveness of the agent

Affects rate of evaporation of the agent from surface being

disinfected

Affects contact time of agent

DGHM Surface Disinfection Test

Test Procedure

Test Organisms

• Staph. aureus ATCC 6538

• E. coli ATCC 11229

• Pr. mirabilis ATCC 14153

• Ps. aeruginosa ATCC 10231

• Candida albicans ATCC 10231

• Klebsiella pneumoniae ATCC 4352

Test surfaces

• PVC floor tiles

• OP tiles (Operating)

• Sheets of synthetic material 50 x 50mm

• Thoroughly washed with water, treated with 70% alcohol

• Stored away from dust

Contamination Process

• Test organism cultured CSA or (NA), incubated for 24hr or

48hr at 370C

• Harvest with CSB (or NB)

• Homogenize for 5min using a vortex mixer in a container with

glass beads and diluted Standardized) to give ~108-109cfu/ml

• 0.1ml of suspension spread carefully & evenly on each test

surface

• Contaminated test tiles held under a relative humidity of

40%±5% at 20-250C and allowed to dry for 90 minutes.

• After drying, contaminated tiles are sprayed with the

disinfectant solution from a distance of 30-40cm ensuring that

the disinfectant completely covers the surface of the tiles.

• After contact times of 1, 2, 3, and 4hrs (30, 45, 60 and

90mins) for rapidly acting disinfectants, the number of

survivors is determined by impression or using RODAC plates

• When the number of are too many to be accurately

counted using the impression method, a rinsing

technique is used

• Test tiles are rinsed in a diluent and the number of

bacteria in the rinsing fluid determined.

• It may be necessary to make appropriate dilution of

the rinsing fluid before plating to count the colonies.

Drying time 90mins

1 2 3 4 hrs

Tile(PVC or OP)

(5x5cm)

Bacterial

suspension

108-109

cfu/ml

Rodac

plate

Disinfectant

solution

Diluent

Hard water

For one disinfectant

series

Control Series

4

Schematic diagram of the DGHM Surface Disinfection Test

Germicidal effect = logNc - logNo

Nc = Average # of bact in the control

No = Average # of bact in the test disinfectant

(after specific contact time)

• Disinfectants used on surfaces mostly

Aldehyde compounds

Phenols

Less frequently, chlorine compounds

• Frequently combined with detergents (mostly QACs)

• Advantages of such combinations

o Time saving; cleansing is done with disinfection

o Provides optimal disinfection where there is no inactivation

since some detergents have antimicrobial activity.

Hand Disinfectant Test

• Main principle in hand disinfection

Kill or inactivate as much as possible transient or resident

infectious organisms on the hand

Prevent transmission of infection

Utmost importance during surgery or in the production of

sterile products

Direct contact with hands

Wearing of gloves designed to minimise such effect

Not able to solve problem

Gloved surgeon’s hands must intermittently be disinfected

anytime it comes in contact with bare skin.

• Hand disinfection requires

Rapid, quick killing or inactivation of pathogenic organisms

Contact Time in test designs ranges from

30s to 1min in hygienic disinfection

Minimum of 5mins in surgical hand disinfection

Hand disinfection desired in

• Production of cosmetics & foods

• Manufacture of sterile products

• In hospitals – especially in routine examination

• Fight against infection outbreaks

Hygienic Hand Disinfection

• Indicated when hand has prior contact with

infectious materials

After toilet visit

Coming from patients’ bedside

General method of using hand disinfectants

• Disinfectant solution dispensed into the hand/palm

• Spread around for required contact time

• Hands then washed with water

• Effective time is ½ - 1 min

• In massive infectious contact, extended to 5mins

Surgical Hand Disinfection

• Procedure relatively different

Hands first cleaned by washing with soap & brush

Rinsed with water

Dried with hand (paper) towel

Hand rinsed & spread with 2x50ml of the disinfectant for

1min

• Usually necessary when surgeon is going for

operation

Prevents shedding of transient or resident flora

Prevents infecting the patient

• Contact time is usually 5mins

Procedure for Hand Disinfection Evaluation

(DGHM Method)

Principle

• Effect of disinfectant compared with reference

• On the same day

• On the same group of volunteers

• Basis of activity

Degree of reduction of artificially contaminated finger tips

Difference between contamination level before and after disinfection

Volunteers

• 15 recommended but results of only 12 used

• Categories of people excluded as volunteers

With hyperkeratosis

Skin injuries (open wounds)

Long fingernails

Test Organism

E. coli ATCC 11229

Contamination Procedure

• Hands washed with soap (proved to have no antimicrobial

effect) under flowing slightly warm water for 2mins

• Dried with tissue paper

• Finger tips immersed for 5secs in petri dish containing

standardised suspension of test organism (108cfu/ml)

• Fingers held vertically in hanging position to dryin air for

3mins

• Care taken to prevent drops of bacterial suspension on finger

tips falling off.

Determination of pre-disinfection contamination level

• Finger tips of one hand immersed in broth containing

appropriate inactivator for reference disinfectant in petri dish

• By scratching & rubbing of the finger tips against each other

for 1min

• The artificially contaminated test organisms released into

liquid medium

• Perform viable count on agar containing 0.05% Na-

deoxycholate.

Disinfection

• Treat the other fingers with 60% isopropanol (3ml) for 1min.

• Wash under flowing water for 1s

• Determine the viable count

Disinfection Process for the Reference

Determination of Pre-infection Count

• Dip all the finger tips of each hand a solution containing broth

+ appropriate inactivator for disinfectant in a 9mm diameter

petri dish

• By scratching & rubbing the finger tips together for 1min

reduce some organism into solution

• Plate out and perform viable count to determine cfu/ml in

solution

• Where counts are likely to be too high, make dilutions and

plate out and incubate

Disinfection

• Disinfect the hand immediately after, without recontamination

• 2 x 3ml 60% Isopropanol

• On cupped palm, distributing on rest of fingers & tips for 1min

• Wash hands by placing under running tap water for 1sec

• The count is obtained as previously described

• After this, wash the hand with soap, dry & re-contaminate for

the evaluation of the test disinfectant

• Pre- and post-disinfection counts of test organism is done as

described for reference disinfectant (alcohol)

• The disinfectant will be applied as described by manufacturer

• Disinfectants with instructions to add some water before use

are not appropriate for the test.

L – Left hand

R – Right hand

RF – Reduction factor

RFL - log (cfuPreL – cfuPostL disinfection)

FRR - log(cfuPreR – cfuPostR disinfection)

• For each volunteer, there are 4 Pre-values and 4 Post-values

• (2 for Reference & 2 for test disinfectant)

• Find the log of the values.

• Where the value is zero, take the log (i.e value changed to 1)

RFR = log (X0 –X1)

RFL = log (Y0 –Y1)

• Find the mean of all Pre-values(L) for Ref

• Find the mean of all Pre-values(R) for Ref

• Find the mean of all Pre-values(L) for Test

• Find the mean of all Pre-values(R) for Test

R L

Pre-disinfection X0 Y0

Post-disinfection X1 Y1