Application for ethical approval of a research project involving humans

Application for ethical approval of a research project involving humans

APPLICANT INSTRUCTIONS

·         This form must be submitted to the IRC if you wish to undertake research activities involving human participants for which participant recruitment and/or data collection is to be conducted within any section of phect-NEPAL.

·         Please use the information provided in italics throughout this document as instructions of how to complete the relevant section, and delete these instructions before submitting the document (retain the bold subheadings). Follow the instructions carefully to ensure you include all necessary information as incomplete forms will not be accepted.

·         See section 7 for submission requirements and attach all relevant documents.

PRIVACY INFORMATION: The information you provide on this form is collected for the primary purpose of assessing your application. This information may also be entered on to a database to assist with administration, correspondence and statistical analyses. These records are only accessed by members of the IRC and affiliated staff at phect-NEPAL and are kept in a manner to ensure confidentiality. Although this information is not usually disclosed to other individuals, there may be some circumstances which require the information to be disclosed. If you choose not to complete all questions on this form it may not be possible for the IRC to assess your application. For privacy related concerns please contact the IRC; email: irc@phectnepal.org, ph: +977-1-422-2364.

PROJECT TITLE

Principal researcher OR responsible supervisor: (one individual only)

Note: This individual is responsible for effective oversight of the project in order to maintain proper ethical conduct and therefore must be appropriately qualified and experienced in carrying out the research process (ie. in the case of a student project, the research supervisor or guide should be listed here and details of the student undertaking the research should be listed in the section below).

·         Contact details: (including both phone and email)

·         Experience and qualifications relevant to the project: (attach curriculum vitae of maximum two pages, including passport-size photo)

Student researcher: (if applicable)

·         Contact details: (including both phone and email)

·         Experience and qualifications relevant to the project: (attach curriculum vitae of maximum two pages, including passport-size photo)

Other researcher(s) and/or supervisor(s): (if applicable)

·         Experience and qualifications relevant to the project: (please list here)

1.    PROJECT DETAILS

1.1    Summary in simple language: Provide a brief summary of the project outlining the broad aims, general background, research question(s), research design, research participants and what they will be asked to do and the importance of the research. This description MUST be in everyday language and free from any jargon, technical terms or discipline-specific phrases so that it may be understood by an individual without specialist training in the area of research. [No more than 300 words]

1.2    Background, literature review and justification: Provide a brief background of the study, literature review, including references, with justification as to why this research should proceed and an explanation of any expected benefits to the community. [No more than 500 words]

1.3    Aim(s) and objective(s): State the project aim(s) and objective(s) and, where relevant, the specific hypothesis/hypotheses to be tested. [No more than 100 words]

1.4    Method(s):

a)    Data collection method: Describe any data collection techniques which will be used. (If using a questionnaire or interview, attach copies.) [No more than 100 words]

b)    Participant tasks: Outline all tasks to be completed by participants, and the estimated time commitment involved. [No more than 100 words]

c)    Data collection period: State the proposed start and completion dates for data collection. (Note: The IRC will advise if the submission of progress report(s) will be required during this time period.) [No more than 50 words]

d)    Data collection personnel: List who will collect the data. (Note: If parts of the project will be carried out by independent contractors, the responsibility for effective oversight and proper ethical conduct of the project remains with the principal researcher OR responsible supervisor.) [No more than 100 words]

e)    Data analysis method: Describe how data will be analysed. [No more than 100 words]

1.5    Researchers involved: Specify which of the individuals listed on page 1 will actually carry out the research, and with which component of the methods they will be involved. Include whether they are; phect-NEPAL staff, student(s) (provide title of degree), personnel in conjunction with other centres (provide details of relevant ethics committees), or other external personnel. [No more than 50 words]

1.6    Monitoring: Outline how the conduct of this project will be monitored to ensure it complies with the protocols set out in this application, such as via regular meetings between all involved researchers/supervisors. (Note: This is especially relevant in cases of student research projects, or where several people are involved in recruiting, interviewing or administering procedures, or when research is carried out at some distance from the principal researcher OR responsible supervisor such as in rural areas.) [No more than 200 words]

1.7    Funding: If funding will be sought, provide details of any already granted and/or advise the IRC of the details as soon as any funding is granted. In the case of research projects independent from phect-NEPAL, please acknowledge that all necessary funds required for the research project to be carried out must be provided by the researcher. [No more than 50 words]

1.8    Risks to researchers: Outline any potential risks to the researchers and the strategies which will be in place to reduce these risks. [No more than 100 words]

2.    PARTICIPANT DETAILS

In the case of retrospective studies, the ‘participants’ are the data sets which will be collected.

2.1       Sample participant group: Describe the inclusion criteria which define the sample participant group (eg. age, gender, background, profession, location). If relevant, also describe the target group to which the results may be applied. [No more than 50 words]

2.2       Number and source of participants: Provide the proposed number and source of participants. Attach a signed letter of approval or no objection certificate (NOC) from all departments where participant recruitment and/or data collection will take place (electronic copies are preferred). [No more than 50 words]

2.3       Justification of participant numbers: Where applicable, provide a justification of sample size (including details of statistical power of the sample, where appropriate), explaining how this sample size will allow the aims of the study to be achieved. [No more than 50 words]

2.4       Exclusion criteria: Describe any exclusion criteria which will be applied to the participant group and justify why this criteria has been selected. (Note: These are the criteria which the researcher selects and not those factors which are out of their control, such as individuals declining to participate or failing to attend follow up sessions.) [No more than 50 words]

2.5       Potentially vulnerable participants: Identify if any potentially vulnerable participants are to be included in the study (eg. children, pregnant women, human foetuses, persons with intellectual disabilities, minorities, persons highly dependent on medical care, military personnel and inmates). Provide justification as to why the research must include these participants and cannot be performed with equivalent less vulnerable participants. [No more than 50 words]

2.6       Dependent relationships: Identify any participants in dependent or unequal relationships with any of the researchers (eg. doctor/patient, teacher/student, counsellor/client, warden/prisoner and employer/employee), particularly those individuals involved in recruiting for or conducting the project. Such a relationship may compromise a participant’s ability to give consent which is free from any form of pressure. Therefore if such relationships exist, explain both the relationship and the steps to be taken by the researchers to ensure that participation is purely voluntary and not influenced by the relationship in any way. Explain the special precautions which will preserve the rights of such people to decline to participate or to withdraw from participation once the research has begun. [No more than 200 words]

2.7       Participant recruitment:

a)    Method: Indicate all method(s) of recruitment:

(i)      Mail out (Describe who will be distributing it and attach a copy)

(ii)     Email (Describe who will be distributing it and attach a copy)

(iii)    Telephone

(iv)   Advertisement (Describe where it will be placed, eg. waiting room wall, newspaper, newsletter, and attach a copy)

(v)    Recruitment carried out by the researcher(s)

(vi)   Recruitment carried out by a third party, eg. employer, teacher, doctor (Attach an approval letter confirming the willingness of the third party to provide to potential participants)

(vii)  Contact details obtained from public documents, eg. phone book

(viii) Contact details obtained from private sources, eg. employee list, membership database (Attach an approval letter confirming the willingness of the proposed source to provide to potential participants)

(ix)   Personal contacts

(x)    Participants from a previous study (Provide details)

(xi)   Snowball, ie participants suggest other potential participants

(xii)  Other (Please explain) [No more than 50 words]

b)    Participant identification: Describe how, by whom and where potential participants are to be identified or selected for this research. [No more than 100 words]

c)    Participant invitation: Describe how, by whom and where potential participants are to be approached or invited to take part in this research. [No more than 100 words]

2.8       Requirement from participants: Provide details about what you will ask participants to do, or what is to be done to them, and the time commitment that will be required from them. Include a clear step-by-step description of what participants will experience if they choose to take part in your project, including medications, interventions, therapies, tasks, interviews etc. (Attach copies of any medication schedules, therapy descriptions, intervention methodologies, instructions, tasks, tests, questionnaires or interview guides to be used and also a flow chart if this will clarify the procedures.)

2.9       Potential risks to participants: Define the risk of physical and/or psychological stress, inconvenience or discomfort beyond the normal experience of everyday life, in either the short or long term, from participation in this project. Confirm that these risks are all outlined on the information letter in simple language and, where relevant, on the consent form, or justify their omission. Outline the arrangements planned to minimise the risks involved in these procedures. [No more than 200 words]

2.10     Payments or incentives offered to participants: Describe and justify any proposed payments, reimbursements or rewards to be offered to participants in terms of amount, form and purpose. [No more than 50 words]

3.    INFORMED CONSENT

Before research is undertaken, the informed and voluntary consent of participants is required. Information needs to be provided to participants at their level of comprehension regarding the purpose, methods, demands, risks, inconveniences, discomforts and possible outcomes of the research. Such information should usually be provided in an information letter written in simple language, in the appropriate language for that participant or participant group. The participant must be provided with a copy of the letter which they may keep for their own records, should they wish to. Each participant’s consent needs to be clearly established and recorded, on a separate document to the information letter (eg. by using a signed consent form, returning a completed survey or recording an agreement for an interview).

3.1       Providing information for participants: Provide details of the method you will use to explain the research project to participants and invite their participation (eg. provision of information in a letter written in simple language). Describe arrangements which will be made to ensure that participants who have difficulty understanding certain written or spoken languages can comprehend the information provided about the research project (eg. provision of the information letter in their local language). [No more than 100 words]

3.2       Information letter in simple language (if applicable): Confirm that the information letter for participants will meet the following checklist of criteria (attach copies of the letter in all languages relevant to your study participants, ie. in English and/or Nepali, and any other relevant language):

(i)     Is printed on relevant letterhead (where possible) OR includes the full contact details of the institute with which the researchers are affiliated OR clearly states that the researcher is working independently and includes lists relevant qualifications

(ii)    Includes clear identification of all institutes and department(s) involved, the principal researcher OR responsible supervisor (including phone and email contact details), all other researchers and lists the study level if it is a student research project

(iii)   Advises that the project is for research purposes, states the title of the project and provides details of the purpose of the research project

(iv)   Provides details of the required participant involvement (eg. involvement in interviews, completion of questionnaire(s), provision of bodily fluid and/or tissue samples, audio/video taping of events) and estimated time commitment

(v)    Provides details of any risks involved and the procedures in place to minimise these

(vi)   Advises that the project has received ethical approval from the IRC

(vii)  If the sample size is small, identifies that this may have implications for protecting the identity of participants and explains the potential for participants to be (re)identified

(viii) Includes a clear statement that, if participants are in a dependent relationship with any of the researchers, their involvement in the project will not affect ongoing assessment/grades/management or treatment of health (if relevant)

(ix)   States that involvement in the project is voluntary and that participants are free to withdraw their consent at any time and to withdraw any unprocessed data previously supplied

(x)    Provides details as to whether or not data is to be destroyed after a minimum period (if relevant)

(xi)   Provides advice that if participants have any concerns about the ethical conduct of this research project they can contact the IRC; email: irc@phectnepal.org, ph: +977-1-422-2364

3.3       Obtaining consent: Explain how participant consent will be established (eg. by signing a consent form, by returning a completed survey or via a recorded verbal agreement). In the case of illiterate individuals, a consent form must be read out by the researcher in the presence of an independent witness, then the thumb print of the participant be obtained alongside the name and signature of the witness. In the case of participants who are unable to give informed consent themselves (eg. minors or intellectually disabled individuals) explain who will consent on their behalf and how such consent will be obtained. [No more than 50 words]

3.4       Consent form (if applicable): Confirm that the consent form will meet the following checklist of criteria (attach copies of the form in all languages relevant to your study participants, ie. in English and/or Nepali, and any other relevant language) and will be retained by the researcher once signed:

(i)     Is printed on relevant letterhead (where possible) OR includes the full contact details of the institute with which the researchers are affiliated OR clearly states that the researcher is working independently and lists their relevant qualifications

(ii)    Includes the title of the project and names of all involved researchers

(iii)   States the project is for research purposes

(iv)   States that involvement in the project is voluntary and that participants are free to withdraw at any time, and free to withdraw any unprocessed identifiable data previously supplied

(xii)  Outlines particular requirements of participants (eg. involvement in interviews, completion of questionnaire(s), provision of bodily fluid and/or tissue samples, audio/video taping of events) and estimated time commitment

(v)    Includes details of the arrangements to protect the confidentiality of data

(vi)   Specifies that there are legal limitations to data confidentiality

(vii)  If the sample size is small, confirms that this may have implications for protecting the identity of the participants and explains the potential for participants to be (re)identified

The Bacterial Cell Wall Structure and Its Synthesis

 Figure 2: Cell wall structure of Gram negative bacteria

Figure 2: Cell wall structure of Gram positive bacteria

Sterilization

CHEMICAL METHODS OF DISINFECTION:

Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Some chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can sterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics. An ideal antiseptic or disinfectant should have following properties:

•      Should have wide spectrum of activity

•      Should be able to destroy microbes within practical period of time

•      Should be active in the presence of organic matter

•      Should make effective contact and be wettable

•      Should be active in any pH

•      Should be stable 

•      Should have long shelf life

•      Should be speedy

•      Should have high penetrating power

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

•      Should not have bad odor

•      Should not leave non-volatile residue or stain

•      Efficacy should not be lost on reasonable dilution

•      Should not be expensive and must be available easily Such an ideal disinfectant is not yet available. The level of disinfection achieved depends on contact time, temperature, type and concentration of the active ingredient, the presence of organic matter, the type and quantum of microbial load. The chemical disinfectants at working concentrations rapidly lose their strength on standing.

Classification of disinfectants:

1.   Based on consistency

a.      Liquid (E.g., Alcohols, Phenols)

b.      Gaseous (Formaldehyde vapor, Ethylene oxide)

2.   Based on spectrum of activity

a.      High level

b.      Intermediate level

c.       Low level

3.   Based on mechanism of action

a.      Action on membrane (E.g., Alcohol, detergent)

b.      Denaturation of cellular proteins (E.g., Alcohol, Phenol)

c.       Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2, Halogens)

d.      Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene Oxide, Formaldehyde)

e.      Damage to nucleic acids (Ethylene Oxide, Formaldehyde)

Spectrum of activity

	Vegetative cells	Mycobacteria	Spores	Fungi	Viruses	Examples
High level	+	+	+	+	+	Ethylene Oxide, Gluteraldehyde, Formaldehyde
Intermediate level	+	+	-	+	+	Phenolics, halogens
Low level	+	-	-	+	+/-	Alcohols, quaternary ammonium compounds

ALCOHOLS:

Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.  Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol

Application: A 70% aqueous solution is more effective at killing microbes than absolute alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to ethanol. It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers. Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods. Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

ALDEHYDES:

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

Examples: Formaldehyde, Gluteraldehyde

Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at 40^oC for 20 minutes is used to disinfect wool and 0.25% at 60^oC for six hours to disinfect animal hair and bristles.

Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration, leaves non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde requires alkaline pH and only those articles that are wettable can be sterilized.

PHENOL:

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

Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol)

Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar derivatives. They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. They are not readily inactivated by organic matter. The corrosive phenolics are used for disinfection of ward floors, in discarding jars in laboratories and disinfection of bedpans. Chlorhexidine can be used in an isopropanol solution for skin disinfection, or as an aqueous solution for wound irrigation. It is often used as an antiseptic hand wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand and skin preparation and for general skin disinfection. Chlorhexidine gluconate is also mixed with quaternary ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (eg. Savlon). Chloroxylenols are less irritant and can be used for topical purposes and are more effective against gram positive bacteria than gram negative bacteria. Hexachlorophene is chlorinated diphenyl and is much less irritant. It has marked effect over gram positive bacteria but poor effect over gram negative bacteria, mycobacteria, fungi and viruses. Triclosan is an organic phenyl ether with good activity against gram positive bacteria and effective to some extent against many gram negative bacteria including Pseudomonas. It also has fair activity on fungi and viruses.

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

HALOGENS:
Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups of
enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine,
iodophores)
Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such as povidone-iodine. Iodophores permit slow release and reduce the irritation of the antiseptic. For hand washing iodophores are diluted in 50% alcohol. 10% Povidone Iodine is used undiluted in pre and postoperative skin disinfection. Chlorine gas is used to bleach water. Household bleach can be used to disinfect floors. Household bleach used in a stock dilution of 1:10. In higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium hypochlorite is used in serology and virology. Used at a dilution of 1:10 in decontamination of spillage of infectious material. Mercuric chloride is used as a disinfectant.
Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is corrosive and staining. Bleach solution is corrosive and will corrode stainless steel surfaces.

HEAVY METALS:
Mode of action: Act by precipitation of proteins and oxidation of sulfydryl groups. They are bacteriostatic.
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (e.g., mercurochrome, merthiolate)
Applications: 1% silver nitrate solution can be applied on eyes as treatment for opthalmia neonatorum (Crede’s method). This procedure is no longer followed. Silver sulphadiazine is used topically to help to prevent colonization and infection of burn tissues. Mercurials are active against viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used in preservation of serum. Copper salts are used as a fungicide.
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

SURFACE ACTIVE AGENTS:
Mode of actions: They have the property of concentrating at interfaces between lipid containing membrane of bacterial cell and surrounding aqueous medium. These compounds have long chain hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they contain both of these, they concentrate on the surface of membranes. They disrupt membrane resulting in leakage of cell constituents.
Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents containing negatively charged long chain hydrocarbon are called anionic detergents. These include soaps and bile salts. If the fat-soluble part is made to have a positive charge by combining with a quaternary nitrogen atom, it is called cationic detergents. Cationic detergents are known as quaternary ammonium compounds (or quat). Cetrimide and benzalkonium chloride act as cationic detergents. 
Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals. 
Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter. Pseudomonas can metabolise cetrimide, using them as a carbon, nitrogen and energy source.

DYES:
Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial nucleic acids. 
Examples: Aniline dyes such as crystal violet, malachite green and brilliant green. Acridine dyes such as acriflavin and aminacrine. Acriflavine is a mixture of proflavine and euflavine. Only euflavine has effective antimicrobial properties. A related dye, ethidium bromide, is also germicidal. It intercalates between base pairs in DNA. They are more effective against gram positive bacteria than gram negative bacteria and are more bacteriostatic in action. 
Applications: They may be used topically as antiseptics to treat mild burns. They are used as paint on the skin to treat bacterial skin infections. The dyes are used as selective agents in certain selective media.

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

Application: It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal.

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

ETHYLENE OXIDE (EO):
Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and hydroxyl- groups. 
Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It has a sweet ethereal odor, readily polymerizes and is flammable. 
Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is highly flammable, it is usually combined with CO2 (10% CO2+ 90% EO) or dichlorodifluoromethane. It requires presence of humidity. It has good penetration and is well absorbed by porous material. It is used to sterilize heat labile articles such as bedding, textiles, rubber, plastics, syringes, disposable petri dishes, and complex apparatus like heart - lung machine, respiratory and dental equipments. Efficiency testing is done using Bacillus subtilis var niger.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and carcinogenic.

BETA-PROPIOLACTONE (BPL):
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl- and hydroxyl- groups.
Properties: It is a colorless liquid with pungent to slightly sweetish smell. It is a condensation product of ketane with formaldehyde.
Application: It is an effective sporicidal agent, and has broad-spectrum activity. 0.2% is used to sterilize biological products. It is more efficient in fumigation that formaldehyde. It is used to sterilize vaccines, tissue grafts, surgical instruments and enzymes
Disadvantages: It has poor penetrating power and is a carcinogen.

PHYSIO-CHEMICAL METHOD:
Mode of action: A physio-chemical method adopts both physical and chemical method. Use of steam-formaldehyde is a physio-chemical method of sterilization, which takes into account action of steam as well as that of formaldehyde. Saturated steam at a pressure of 263 mm has a temperature of 70^oC. The air is removed from the autoclave chamber and saturated steam at sub-atmospheric pressure is flushed in. Formaldehyde is then injected with steam in a series of pulses, each of 5-10 minutes. The articles are held at this holding temperature for one hour. Formaldehyde is then flushed by inflow of steam.
Disadvantages: Condensation of formaldehyde occurs and induction of large volume of formaldehyde wets the steam resulting in loss of latent heat.
Sterilization control: using paper strips containing 10⁶ spores of G. stearothermophilus.

TESTING OF DISINFECTANTS:

A disinfectant must be tested to know the required effective dilution, the time taken to effect disinfection and to periodically monitor its activity. As disinfectants are known to lose their activity on standing as well as in the presence of organic matter, their activity must be periodically tested.

Different methods are:

1.      Koch’s method

2.      Rideal Walker Method

3.      Chick Martin test

4.      Capacity use dilution test (Kelsey-Sykes test)

5.      In-use test

Koch’s method: Spores of Bacillus anthracis were dried on silk thread and were subjected to action of disinfectants. Later, it was washed and transferred to solid medium.

Rideal Walker method: This method relies on the estimation of phenol coefficient. Phenol coefficient of a disinfectant is calculated by dividing the dilution of test disinfectant by the dilution of phenol that disinfects under predetermined conditions. Both the phenol and the test disinfectant are diluted from 1/95 to 1/115 and 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. 

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.

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). 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 disinfectant	Water	Yeast suspension
Reaction temperature	17.5±0.5ºC	30ºC
Organism	Salmonella typhi	Salmonella typhi, Staphylococcus aureus
Sampling times	2.5, 5.0, 7.5, 10.0 min.	30.0 min.
Calculation of coefficient	Dilution test killing in 7.5 min divided by same for phenol	Mean concentration of phenol showing no growth after 30 min. divided by same for test

The classical tests such as Rideal - Walker or Chick - Martin are not practicable.

Capacity use dilution test (Kelsey-Sykes test):

Inoculum of four different test organisms, namely Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris are added to the disinfectant in three successive. Dried yeast is included to simulate presence of organic matter. 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 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. The capacity test of Kelsey and Sykes gives a good guideline for the dilution of the preparation to be used. Disadvantage of this test is the fact that it is rather complicated.

In-use test:

The routine monitoring of disinfectant in use can be done by the ‘in use’ test of Maurer. This test is intended to estimate the number of living organism in a vessel of disinfectant in actual use. The disinfectant that is already in use is diluted 1 in 10 by mixing 1 ml of the disinfectant with 9 ml of sterile nutrient broth. Ten drops of the diluted disinfectant (each 0.02 ml) is placed on two nutrient agar plates. One plate is incubated at 37^oC for 3 days while the other is held at room temperature for 7 days. The number of drops that yielded growth is counted after incubation. If there growth in more than five drops on either plate, it represents failure of disinfectant.

Source: Sridhar Rao PN, Assistant Professor, Department of Microbiology, JJMMC, Davangere (www.microrao.com), Thank you very much to DR. Rao.