PHCT 401: Disinfectant Evaluation

Transcription

1 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)

2 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.

3 Preservatives Included in preparations Prevents microbial spoilage of the product Minimize risk of product-related infections to consumers Limits proliferation of contaminants in nonsterile preps e.g oral and topical preparations Kill microbial contaminants in sterile preps e.g eye drops, multi-dose injs.

4 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

5 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.

6 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?

7 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

8 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

9 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

10 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

11 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.

12 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

13 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

14 Substances Tested Bacteriostatic Tests Bacteriocidal Tests Liquid disinfectants Semi-solid antibacterial Formulations e.g. Creams, ointments & Gels. Tests for Disinfectant Activity 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. Cup-plate Ditch-plate End-point or extinction time methods Counting methods Turbidometric assessment In-use & other tests In-vivo tests additionally applied Modified end-point or extinction time methods In addition in-vivo tests are applied (e.g. skin tests) Solid disinfectants, Disinfectant powders Aerial disinfectants Inhibition on seeded agar Modified end-point or extinction Time methods Use of slit-sampler in test chamber.

15 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

16 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.

17 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.

18 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

19 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.

20 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.

21 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.

22 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 % of the germs are killed. Even though these tests are generally well standardized, their approach is less practical.

23 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.

24 Tube is inoculated to achieve a concentration of approximately 10 6 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.

25 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).

26 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.

27 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.

28 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.

29 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 o Protein factor o Protective factors o Spectrum of activity - Cleansing? Detergent agents - organic matter

30 In-vitro Tests Minimum Inhibitory Concentration Test (Test for determining bacteriostatic activity) Disinfectant bacterial cells Bacteriostatic activity acceptable activity on vegetative 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

31 Determination of MIC of disinfectant 10 8 cfu/ml Bacterial Suspension (overnight culture) 0.1ml 0.1ml 0.1ml 0.1ml 5ml NB (Double strength 50% 25% 12.5% 6.75% 5ml 5ml 5ml 5ml Dilution 1:2 1:2 1:2 Disinfectant solution 50% 25% 12.5% Serial dilution

32 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

33 Rideal-WalkerTest 0.2ml Bacterial Suspension 0.2ml 5ml 5ml 18 0 C 18 0 C Disinfectant Phenol solution solution 2½ 5 7 ½ 10 min Contact Time 2½ 5 7 ½ 10 min Loopful Loopful Nutrient Broth tubes Nutrient Broth tubes

34 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 C) From this, prepare five dilutions 1 in 95; 1 in 100; 1 in 105; 1 in 110 and 1 in 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 18 0 C 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

35 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.

36 Rideal-Walker Test Procedure contd 12.Incubate the inoculated NB tubes at 37 0 C 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. Dilution action of Time of Exposure (min) 2½ 5 7½ 10 Disinfectant 1 in in in in Phenol 1 in = Growth - = No growth

37 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. RW Coefficient RW Dilution of disinfectant killing in 7.5 min. but not 5 min. Dilution of Phenol killing in 7.5 min. but not in 5 min. coefficient 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 = Black fluid =

38 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

39 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.

40 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 20 0 C (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

41 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 37 0 C 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

42 Chick-Martin Test Sample Result Decreasing Concentration (%) A B C D E F G H I Phenol series Disinfectant series = Growth; - = No growth Average of Phenol series = C + D / 2 Average of Disinfectant series = E + F / 2 Chick - Martin Coeff. C E D 2 F 2 C E D F

43 Schematic Diagram of Chick-Martin Phenol Coefficient Test Bacterial Suspension 2ml 48ml Yeast Suspension (Sterile) 2.5ml 2.5ml 2.5ml Bacterial-Yeast Suspension Mixture 2.5ml 30mins loopful Disinfectant Solution Phenol Solution loopful 30mins 10ml 10ml Incubate NB tubes Incubate

44 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

45 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 20 0 C 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.

46 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: : : : Phenol 1: :

47 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

48 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

49 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. H 2 O 2, hypochlorite, chloramine T, chlorine, potassium permanganate etc. react vigorously with organic matter while acridines retain their activity.

50 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 (37 0 ), 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.

51 Essential differences between the Phenol Coefficient Tests R.W FDA CM AOAC Crown agent Medium ph before autoclaving Medium vol 5.0ml Vol of rxn mixture Diluent for disinfectant Water Water Yeast suspension Water Artificial sea water Rxn temp 17.5± : ±1.0 Organism S. typhi S. typhi S. typhi S. typhi, S. aureus Sampling time 2½, 5, 7½, 10 min S. typhi 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

52 The DGHM Qualitative Suspension Test 5 test organisms recommended Staphylococcus aureus ATCC 6538 Pseudomonas aeruginosa ATCC E. coli ATCC Proteus mirabilis ATCC Klebsiella pneumoniae ATCC 4352

53 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)

54 Bacterial Suspension 10 8 cfu/ml Disinfectant Solution 0.1 ml 10 ml Contact time minutes 5 ml Broth with inactivator Taken for incubation and the pattern of growth recorded Schematic diagram of DGHM Qualitative Suspension Test

55 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

56 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

57 Disinfectant series 0.2ml 10ml 0.2ml Bacterial suspension 10ml Control Series Disinfectant Diluent 5mins Contact Time 5mins 10ml 10ml 1ml 1ml Inactivator 1ml 1ml 1ml 1ml 9ml 9ml 1ml 1ml 1ml 1ml 1ml Diluent 1ml 1ml 1ml

58 Test Procedure Standardize overnight broth culture of test organism to contain 10 8 cfu/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 37 0 C 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

59 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 = log10 7 log10 3 Advantages Disadvantages

60 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

61 AOAC Carrier Test Method Recommended test organisms Ps. aeruginosa ATCC 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

62 Standardized Bacterial Suspension Disinfectant solution 0.1ml 10 8 cfu/ml Carrier Stainless steel 15 mins Drying on Filter paper 10 mins Contact time +Inactivator Incubate at 37 0 C for 24hrs Schematic Diagram of AOAC Carrier Test Method Subculture broth

63 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. Disinfectant concentration Number of subculture broth with growth out of What is the in-use dilution for this disinfectant?

64 DGHM Carrier Test Method Test organisms Staph. aureus ATCC 6538 E. coli ATCC Proteus mirabilis ATCC Ps. aeruginosa ATCC Candida albicans ATCC M. tubeculosis ATCC Carrier: Standard cotton cloth, properly washed in distilled water, cut in 1cm 2 and sterilized by autoclaving and dried.

65 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 10 9 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

66 Transfer into another subculture broth & incubate at 37 0 C 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

67 Standardized Bacterial suspension Disinfectant solution Soak for 15mins Petri dish Rinsing broth with inactivator Carrier (Cotton cloth, 1cm 2 ) Contact time Subculture broth Incubate at 37 0 C for 72hrs

68 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

69 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 MgSO 4.7H 2 O Media: Nutrient broth hardness is 300ppm. Yeast suspension 5% as in Chick-Martin or Horse serum at initial concentration of 20%. Subcultured tubes incubated at 37 0 C for 48hrs

70 Disinfectant solution 3ml 1ml At Time cfu/ml Bacterial suspension 1ml at 10mins 1ml at 20mins (+8) (+8) (+8) I drop Contact time minutes Subculture broth Plated out on NA plates Schematic diagram of Kelsey-Sykes Capacity Test

71 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 B (A + 25%) C (A 25%) 2 5 5

72 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.

73 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

74 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 ii. e.g. food industry vs an operating theatre 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

75 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

76 DGHM Surface Disinfection Test Test Procedure Test Organisms Staph. aureus ATCC 6538 E. coli ATCC Pr. mirabilis ATCC Ps. aeruginosa ATCC Candida albicans ATCC 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

77 Contamination Process Test organism cultured CSA or (NA), incubated for 24hr or 48hr at 37 0 C Harvest with CSB (or NB) Homogenize for 5min using a vortex mixer in a container with glass beads and diluted Standardized) to give ~ cfu/ml 0.1ml of suspension spread carefully & evenly on each test surface Contaminated test tiles held under a relative humidity of 40%±5% at C 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

78 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.

79 Bacterial suspension cfu/ml Tile(PVC or OP) (5x5cm) For one disinfectant series Drying time isinfectant solution 90mins Control Series hrs 4 Diluent Hard water Rodac plate Schematic diagram of the DGHM Surface Disinfection Test

80 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.

81 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

82 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

83 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

84 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

85 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

86 Test Organism E. coli ATCC 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 (10 8 cfu/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.

87 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% Nadeoxycholate. Disinfection Treat the other fingers with 60% isopropanol (3ml) for 1min. Wash under flowing water for 1s Determine the viable count

88 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

89 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 RF L - log (cfuprel cfupostl disinfection) FRR - log(cfuprer cfupostr disinfection)

90 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) R L Pre-disinfection X 0 Y 0 Post-disinfection X 1 Y 1 RF R = log (X 0 X 1 ) RFL = log (Y 0 Y 1 ) 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