What is a clinical trial?

What is a clinical trial?

Although there are many definitions of clinical trials, they are generally considered to be biomedical or health-related research studies in human beings that follow a pre-defined protocol. ClinicalTrials.gov includes both interventional and observational types of studies. Interventional studies are those in which the research subjects are assigned by the investigator to a treatment or other intervention, and their outcomes are measured. Observational studies are those in which individuals are observed and their outcomes are measured by the investigators.


Why participate in a clinical trial?

Participants in clinical trials can play a more active role in their own health care, gain access to new research treatments before they are widely available, and help others by contributing to medical research.


Who can participate in a clinical trial?

All clinical trials have guidelines about who can participate. Using inclusion/exclusion criteria is an important principle of medical research that helps to produce reliable results. The factors that allow someone to participate in a clinical trial are called "inclusion criteria" and those that disallow someone from participating are called "exclusion criteria". These criteria are based on such factors as age, gender, the type and stage of a disease, previous treatment history, and other medical conditions. Before joining a clinical trial, a participant must qualify for the study. Some research studies seek participants with illnesses or conditions to be studied in the clinical trial, while others need healthy participants. It is important to note that inclusion and exclusion criteria are not used to reject people personally. Instead, the criteria are used to identify appropriate participants and keep them safe. The criteria help ensure that researchers will be able to answer the questions they plan to study.


What happens during a clinical trial?

The clinical trial process depends on the kind of trial being conducted (See What are the different types of clinical trials?) The clinical trial team includes doctors and nurses as well as social workers and other health care professionals. They check the health of the participant at the beginning of the trial, give specific instructions for participating in the trial, monitor the participant carefully during the trial, and stay in touch after the trial is completed.

Some clinical trials involve more tests and doctor visits than the participant would normally have for an illness or condition. For all types of trials, the participant works with a research team. Clinical trial participation is most successful when the protocol is carefully followed and there is frequent contact with the research staff.


What is informed consent?

Informed consent is the process of learning the key facts about a clinical trial before deciding whether or not to participate. It is also a continuing process throughout the study to provide information for participants. To help someone decide whether or not to participate, the doctors and nurses involved in the trial explain the details of the study. If the participant's native language is not English, translation assistance can be provided. Then the research team provides an informed consent document that includes details about the study, such as its purpose, duration, required procedures, and key contacts. Risks and potential benefits are explained in the informed consent document. The participant then decides whether or not to sign the document. Informed consent is not a contract, and the participant may withdraw from the trial at any time.


What are the benefits and risks of participating in a clinical trial?

Benefits

Clinical trials that are well-designed and well-executed are the best approach for eligible participants to:

• Play an active role in their own health care.
• Gain access to new research treatments before they are widely available.
• Obtain expert medical care at leading health care facilities during the trial.
• Help others by contributing to medical research.

Risks

There are risks to clinical trials.

• There may be unpleasant, serious or even life-threatening side effects to experimental treatment.
• The experimental treatment may not be effective for the participant.
• The protocol may require more of their time and attention than would a non-protocol treatment, including trips to the study site, more treatments, hospital stays or complex dosage requirements.

What are side effects and adverse reactions?

Side effects are any undesired actions or effects of the experimental drug or treatment. Negative or adverse effects may include headache, nausea, hair loss, skin irritation, or other physical problems. Experimental treatments must be evaluated for both immediate and long-term side effects.


How is the safety of the participant protected?

The ethical and legal codes that govern medical practice also apply to clinical trials. In addition, most clinical research is federally regulated with built in safeguards to protect the participants. The trial follows a carefully controlled protocol, a study plan which details what researchers will do in the study. As a clinical trial progresses, researchers report the results of the trial at scientific meetings, to medical journals, and to various government agencies. Individual participants' names will remain secret and will not be mentioned in these reports (See Confidentiality Regarding Trial Participants).


What should people consider before participating in a trial?

People should know as much as possible about the clinical trial and feel comfortable asking the members of the health care team questions about it, the care expected while in a trial, and the cost of the trial. The following questions might be helpful for the participant to discuss with the health care team. Some of the answers to these questions are found in the informed consent document.

• What is the purpose of the study?
• Who is going to be in the study?
• Why do researchers believe the experimental treatment being tested may be effective? Has it been tested before?
• What kinds of tests and experimental treatments are involved?
• How do the possible risks, side effects, and benefits in the study compare with my current treatment?
• How might this trial affect my daily life?
• How long will the trial last?
• Will hospitalization be required?
• Who will pay for the experimental treatment?
• Will I be reimbursed for other expenses?
• What type of long-term follow up care is part of this study?
• How will I know that the experimental treatment is working? Will results of the trials be provided to me?
• Who will be in charge of my care?

What kind of preparation should a potential participant make for the meeting with the research coordinator or doctor?

• Plan ahead and write down possible questions to ask.
• Ask a friend or relative to come along for support and to hear the responses to the questions.
• Bring a tape recorder to record the discussion to replay later.

Every clinical trial in the U.S. must be approved and monitored by an Institutional Review Board (IRB) to make sure the risks are as low as possible and are worth any potential benefits. An IRB is an independent committee of physicians, statisticians, community advocates, and others that ensures that a clinical trial is ethical and the rights of study participants are protected. All institutions that conduct or support biomedical research involving people must, by federal regulation, have an IRB that initially approves and periodically reviews the research.


Does a participant continue to work with a primary health care provider while in a trial?

Yes. Most clinical trials provide short-term treatments related to a designated illness or condition, but do not provide extended or complete primary health care. In addition, by having the health care provider work with the research team, the participant can ensure that other medications or treatments will not conflict with the protocol.


Can a participant leave a clinical trial after it has begun?

Yes. A participant can leave a clinical trial, at any time. When withdrawing from the trial, the participant should let the research team know about it, and the reasons for leaving the study.


Where do the ideas for trials come from?

Ideas for clinical trials usually come from researchers. After researchers test new therapies or procedures in the laboratory and in animal studies, the experimental treatments with the most promising laboratory results are moved into clinical trials. During a trial, more and more information is gained about an experimental treatment, its risks and how well it may or may not work.


Who sponsors clinical trials?

Clinical trials are sponsored or funded by a variety of organizations or individuals such as physicians, medical institutions, foundations, voluntary groups, and pharmaceutical companies, in addition to federal agencies such as the National Institutes of Health (NIH), the Department of Defense (DOD), and the Department of Veteran's Affairs (VA). Trials can take place in a variety of locations, such as hospitals, universities, doctors' offices, or community clinics.


What is a protocol?

A protocol is a study plan on which all clinical trials are based. The plan is carefully designed to safeguard the health of the participants as well as answer specific research questions. A protocol describes what types of people may participate in the trial; the schedule of tests, procedures, medications, and dosages; and the length of the study. While in a clinical trial, participants following a protocol are seen regularly by the research staff to monitor their health and to determine the safety and effectiveness of their treatment.


What is a placebo?

A placebo is an inactive pill, liquid, or powder that has no treatment value. In clinical trials, experimental treatments are often compared with placebos to assess the experimental treatment's effectiveness. In some studies, the participants in the control group will receive a placebo instead of an active drug or experimental treatment.


What is a control or control group?

A control is the standard by which experimental observations are evaluated. In many clinical trials, one group of patients will be given an experimental drug or treatment, while the control group is given either a standard treatment for the illness or a placebo.


What are the different types of clinical trials?

Treatment trials test experimental treatments, new combinations of drugs, or new approaches to surgery or radiation therapy.

Prevention trials look for better ways to prevent disease in people who have never had the disease or to prevent a disease from returning. These approaches may include medicines, vaccines, vitamins, minerals, or lifestyle changes.

Diagnostic trials are conducted to find better tests or procedures for diagnosing a particular disease or condition.

Screening trials test the best way to detect certain diseases or health conditions.

Quality of Life trials (or Supportive Care trials) explore ways to improve comfort and the quality of life for individuals with a chronic illness.


What are the phases of clinical trials?

Clinical trials are conducted in phases. The trials at each phase have a different purpose and help scientists answer different questions:

In Phase I trials, researchers test an experimental drug or treatment in a small group of people (20-80) for the first time to evaluate its safety, determine a safe dosage range, and identify side effects.

In Phase II trials, the experimental study drug or treatment is given to a larger group of people (100-300) to see if it is effective and to further evaluate its safety.

In Phase III trials, the experimental study drug or treatment is given to large groups of people (1,000-3,000) to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the experimental drug or treatment to be used safely.

In Phase IV trials, post marketing studies delineate additional information including the drug's risks, benefits, and optimal use.


What is "expanded access"?

Expanded access is a means by which manufacturers make investigational new drugs available, under certain circumstances, to treat a patient(s) with a serious disease or condition who cannot participate in a controlled clinical trial.

Most human use of investigational new drugs takes place in controlled clinical trials conducted to assess the safety and efficacy of new drugs. Data from these trials are used to determine whether a drug is safe and effective, and serve as the basis for the drug marketing application. Sometimes, patients do not qualify for these controlled trials because of other health problems, age, or other factors, or are otherwise unable to enroll in such trials (e.g., a patient may not live sufficiently close to a clinical trial site).

For patients who cannot participate in a clinical trial of an investigational drug, but have a serious disease or condition that may benefit from treatment with the drug,FDA regulations enable manufacturers of such drugs to provide those patients access to the drug under certain situations, known as "expanded access." For example, the drug cannot expose patients to unreasonable risks given the severity of the disease to be treated and the patient does not have any other satisfactory therapeutic options (e.g., an approved drug that could be used to treat the patient's disease or condition). The manufacturer must be willing to make the drug available for expanded access use. The primary intent of expanded access is to provide treatment for a patient's disease or condition, rather than to collect data about the study drug.

Some investigational drugs are available for treatment use from pharmaceutical manufacturers through expanded access programs listed in ClinicalTrials.gov. If you or a loved one is interested in treatment with an investigational drug under an expanded access protocol listed in ClinicalTrials.gov, review the protocol eligibility criteria and inquire at the Contact Information number. If there is not an expanded access protocol listed in ClinicalTrials.gov, you or your health care provider may contact a manufacturer of an investigational drug directly to ask about expanded access programs.

Clinical Trial Registry-India

Clinical Trials Registry-India (CTRI)

The Clinical Trials Registry- India (CTRI) has been set up by the ICMR's National Institute of Medical Statistics (NIMS) and is funded by the Department of Science and Technology (DST) through the Indian Council of Medical Research (ICMR). It also receives financial and technical support through the WHO, WHO-SEARO, and the WHO India Country office.

The CTRI is an online register of clinical trials being conducted in India. Any researcher who plans to conduct a trial involving human participants, of any intervention (drug, surgical procedure, preventive measures, lifestyle modifications, devices, educational or behavioral treatment, rehabilitation strategies and complementary therapies) are expected to register the trial in CTRI before enrollment of the first participant. Registration is voluntary but some fields marked* are mandatory for registration to proceed. Some fields marked WHO also need to be filled if the trial is to receive a registration number and fulfill WHO/ICMJE requirements. Incomplete entries will be given a provisional registration number that will not suffice for purposes of publication in journals that endorse the ICMJE recommendations for trial registration. Registration of trials in the CTRI is free. All registered trials will be made publicly available. The CTRI will be searchable by anyone free of charge. The Clinical Trials Registry- India (CTRI) has been set up by the ICMR's National Institute of Medical Statistics (NIMS) and is funded by the Department of Science and Technology (DST) through the Indian Council of Medical Research (ICMR). It also receives financial and technical support through the WHO, WHO-SEARO, and the WHO India Country office.

The mission of the Clinical Trials Registry-India (CTRI) is to encourage all clinical trials conducted in India to be prospectively registered before the enrollment of the first participant and to disclose details of the 20 mandatory items of the WHO International Clinical Trials Registry Platform (ICTRP) dataset.

What is the primary purpose of the Registry?

The primary purpose is to make information regarding clinical trials being conducted in India freely available to anyone who desires the information. Through the Registry, information of all clinical trials taking place across the country in all areas of health - new drugs, treatments, therapies, surgical procedures and new medical devices will become publicly available for the first time

Why is it important to register clinical trials?

It is important to register clinical trials for both ethical and scientific reasons. Registration of trials ensures transparency, accountability and accessibility of clinical trials and their results. Further, it promotes greater trust and public confidence in clinical research. Trial registration also helps to prevent bias generated by selective reporting of only "positive" findings as well as reduce unnecessary duplication of research through greater awareness of existing trials and results.

Is it mandatory to register a clinical trial?

As of the present moment, registration is purely voluntary; however, registration is likely to have a lot of advantages both for the registrant as well as the public. Further, prior registration is now a condition of publishing clinical trial data. From 1st July 2005 the International Committee of Medical Journal Editors (ICMJE) have declared that their journals will not publish the results of any clinical trials not included on an authorised register at the trials inception.

Which clinical trials need to be registered?

All interventional clinical trials conducted in India and involving Indian participants need to be registered. An interventional clinical trial is any research study that prospectively assigns people to one or more health-related interventions (e.g., preventive care, drugs, surgical procedures, behavioral treatments, etc.) to evaluate their effects on health-related outcomes. Thus, early and late trials, trials of marketed or non-marketed products, randomized or non-randomized trials -- all should be registered.

Who is responsible for registering a trial?

The "Responsible Registrant" for a trial is either the principal investigator (PI) or the primary sponsor, to be decided by an agreement between the parties. The primary sponsor is ultimately accountable for ensuring that the trial is properly registered. For multi-center and multi-sponsor trials, it is the lead PI or lead sponsor who should take responsibility for registration. However, in case of multi-country trials, the Indian PI should also get the trial registered in CTRI quoting any other Registration number as its Secondary ID.

What is the procedure for registering a clinical trial?

Initially, a registrant will have to create a login name and password. This can be done by filling p the form that appears after clicking on the Register Trial button. Once all the mandatory information has been entered, the registrant should click the Submit button. 
Following submission of the personal data, a password confirmation note to the email ID of the registrant will be sent. Thereafter the registrant can use the chosen Username and password to upload the required information on a clinical trial proposed to be conducted. A registrant may fill in the data set items at his or her own convenience by clicking on the save button. A registrant may then edit the fields as required or take a print out of the entered information by clicking on the Print button. The information filled in the fields of the data set items will not be available for the System Administrator till the registrant clicks on the Submit button. Once the data is submitted and until the trial is registered, any trial information is not available for editing by the registrant. Once the data is submitted, the CTRI staff will check the submitted information for completeness and whether informative entries have been provided. If needed, the registrant will be contacted for any queries. Further, the staff will verify that the trial is being conducted through contact with Ethics Committee. Once queries, if any are clarified, the trial will be officially registered and allocated a unique registration number. Both the date of submission and date of registration will be recorded. Registrants are also required to regularly update information on each trial (including patient accrual, trial and publication status). Further, all the WHO fields need to be filled if the trial is to receive a registration number and fulfill WHO/ICMJE requirements. Incomplete entries will be given a provisional registration number that will not suffice for purposes of publication in journals that endorse the ICMJE recommendations for trial registration. If all the necessary fields are filled with valid and informative entries, the trial will be officially registered and allocated a unique registration number. Trials which are not verifiable from relevant sources despite attempts to do so by the CTRI staff, but appear complete with respect to trial information is provided, the trial will be fully registered but a note will mention that it is “Not verified”

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Some Important Definitions

Quality Assurance1

Quality assurance is a wide-ranging concept covering all matters that individually or collectively influence the quality of a product. It is the totality of the arrangements made with the object of ensuring that pharmaceutical products are of the quality required for their intended use.

Quality Control

Quality control covers all measures taken, including the setting of specifications, sampling, testing and analytical clearance, to ensure that raw materials, intermediates, packaging materials and finished pharmaceutical products conform with established specifications for identity, strength, purity and other characteristics.

active pharmaceutical ingredient (API)

Any substance or mixture of substances intended to be used in the manufacture of a pharmaceutical dosage form and that, when so used, becomes an active ingredient of that pharmaceutical dosage form. Such substances are intended to furnish pharmacological activity or other direct effect in the diagnosis, cure,mitigation, treatment, or prevention of disease or to affect the structure and function of the body.

airlock

An enclosed space with two or more doors, which is interposed between two or more rooms, e.g. of differing classes of cleanliness, for the purpose of controlling the airflow between those rooms when they need to be entered. An airlock is designed for use either by people or for goods and/or equipment.

authorized person

The person recognized by the national regulatory authority as having the responsibility for ensuring that each batch of finished product has been manufactured, tested and approved for release in compliance with the laws and regulations in force in that country.

batch (or lot)

A defined quantity of starting material, packaging material, or product processed in a single process or series of processes so that it is expected to be homogeneous. It may sometimes be necessary to divide a batch into a number of sub-batches, which are later brought together to form a final homogeneous batch. In the case of terminal sterilization, the batch size is determined by the capacity of the autoclave. In continuous manufacture, the batch must correspond to a defined fraction of the production, characterized by its intended homogeneity. The batch size can be defined either as a fixed quantity or as the amount produced in a fixed time interval.

batch number (or lot number)

A distinctive combination of numbers and/or letters which uniquely identifies a batch on the labels, its batch records and corresponding certificates of analysis, etc.

batch records

All documents associated with the manufacture of a batch of bulk product or finished product. They provide a history of each batch of product and of all circumstances pertinent to the quality of the final product. bulk produc tAny product that has completed all processing stages up to, but not including, final packaging. Calibration The set of operations that establish, under specified conditions, the relationship between values indicated by an instrument or system for measuring (especially weighing), recording, and controlling, or the values represented by a material measure, and the corresponding known values of a reference standard. Limits for acceptance of the results of measuring should be established.

clean area

An area with defined environmental control of particulate and microbial contamination, constructed and used in such a way as to reduce the introduction, generation, and retention of contaminants within the area.

consignment (or delivery)

The quantity of a pharmaceutical(s), made by one manufacturer and supplied at one time in response to a particular request or order. A consignment may comprise one or more packages or containers and may include material belonging to more than one batch.

contamination

The undesired introduction of impurities of a chemical or microbiological nature, or of foreign matter, into or on to a starting material or intermediate during production, sampling, packaging or repackaging, storage or transport.

critical operation

An operation in the manufacturing process that may cause variation in the quality of the pharmaceutical product.

cross-contamination

Contamination of a starting material, intermediate product or finished product with another starting material or product during production.

finished product

A finished dosage form that has undergone all stages of manufacture, including packaging in its final container and labelling.

in-process control

Checks performed during production in order to monitor and, if necessary, to adjust the process to ensure that the product conforms to its specifications. The control of the environment or equipment may also be regarded as a part of inprocess control.

intermediate product

Partly processed product that must undergo further manufacturing steps before it becomes a bulk product.

large-volume parenterals

Sterile solutions intended for parenteral application with a volume of 100 ml or more in one container of the finished dosage form.

manufacture

All operations of purchase of materials and products, production, quality control, release, storage and distribution of pharmaceutical products, and the related controls.

manufacturer

A company that carries out operations such as production, packaging, repackaging, labelling and relabelling of pharmaceuticals.

marketing authorization (product licence, registration certificate)

A legal document issued by the competent drug regulatory authority that establishes the detailed composition and formulation of the product and the pharmacopoeial or other recognized specifications of its ingredients and of the final product itself, and includes details of packaging, labelling and shelf-life

master formula

A document or set of documents specifying the starting materials with their quantities and the packaging materials, together with a description of the procedures and precautions required to produce a specified quantity of a finished product as well as the processing instructions, including the in-process controls.

master record

A document or set of documents that serve as a basis for the batch documentation (blank batch record).

packaging

All operations, including filling and labelling, that a bulk product has to undergo in order to become a finished product. Filling of a sterile product under aseptic conditions or a product intended to be terminally sterilized, would not normally be regarded as part of packaging.

packaging material

Any material, including printed material, employed in the packaging of a pharmaceutical, but excluding any outer packaging used for transportation or shipment. Packaging materials are referred to as primary or secondary according to whether or not they are intended to be in direct contact with the product.

pharmaceutical product

Any material or product intended for human or veterinary use presented in its finished dosage form or as a starting material for use in such a dosage form, that is subject to control by pharmaceutical legislation in the exporting state and/or the importing state.

production

All operations involved in the preparation of a pharmaceutical product, from receipt of materials, through processing, packaging and repackaging, labeling and relabelling, to completion of the finished product.

qualification

Action of proving that any premises, systems and items of equipment work correctly and actually lead to the expected results. The meaning of the word “validation” is sometimes extended to incorporate the concept of qualification.

quarantine

The status of starting or packaging materials, intermediates, or bulk or finished products isolated physically or by other effective means while a decision is awaited on their release, rejection or reprocessing.

reconciliation

A comparison between the theoretical quantity and the actual quantity.

recovery

The introduction of all or part of previous batches (or of redistilled solvents and similar products) of the required quality into another batch at a defined stage of manufacture. It includes the removal of impurities from waste to obtain a pure substance or the recovery of used materials for a separate use.

reprocessing

Subjecting all or part of a batch or lot of an in-process drug, bulk process intermediate (final biological bulk intermediate) or bulk product of a single batch/ lot to a previous step in the validated manufacturing process due to failure to meet predetermined specifications. Reprocessing procedures are foreseen as occasionally necessary for biological drugs and, in such cases, are validated and pre-approved as part of the marketing authorization.

reworking

Subjecting an in-process or bulk process intermediate (final biological bulk intermediate) or final product of a single batch to an alternate manufacturing process due to a failure to meet predetermined specifications. Reworking is an unexpected occurrence and is not pre-approved as part of the marketing authorization.

VALIDATION OF INCUBATORS

 1.0      OBJECTIVE

           To lay down a procedure for validation of incubator in microbiology laboratory.

2.0        RESPONSIBILTY

            Microbiologist / Executive.

3.0        ACCOUNTABILITY

            Quality  Assurance Control

4.0               PROCEDURE

4.1       DETAILS OF the Standard thermometer used for validation of  Lab thermometer.

Ø      Type    : Liquid - Glass lab Thermometer.

Ø      Place the standard thermometer dipped in glycerin in incubator at various locations as mentioned in annexure-II.

Ø      The incubator is set for desired temperature with the knob on control panel of incubator e.g 32.5 ⁰C,  35 ⁰C & 22.5 ⁰C.

Ø      Wait till the temperature reaches at set point & note down the temp. displayed on the small screen of incubator and compare this temp. with standard thermometer kept near the RTD probe.Likewise check the temperature after every 15 minutes up to one hour and note down the readings on the format.

Ø      Record any difference between the displayed temp. & temp. showed by standard thermometer and set the incubator accordingly

Ø      Frequency of validation :  once in a year.

Ø      The observations are noted in the format of annexure-I

             

4.2       PROCEDURE FOR VALIDATION OF D.H.S (HOT AIR OVEN):

Ø      Keep all the apparatus wrapped thrice with aluminum foil inside DHS as locations shown in the diagram.                

Ø      Keep the Spore loaded strips (having spore of B subilis) & Endotoxin indicator indicators having 10,000EU/vial in DHS(hot air oven) kept in 30ml vial wrapped thrice with aluminum foil at locations shown in the diagram keep one vial unbaked as PPC.   

Ø      Set the hot air oven at 250°C ,wait till the temperature reaches upto the set temperature.

Ø      After reaching the set temperature, note the time & temperature, hold for one hour

Ø      After completion of depyrogenation cycle ,switch off the power supply and take out Indicators for testing.

4.3       Procedure for testing reduction of endotoxin as under.

Ø      Take out the baked endotoxin loaded vial from the DHS.

Ø      Reconstitute 1ml of LRW in the baked vial and transfer 100ml of sample to the depyrogenated reaction tube kept in heating block at 37⁰ C and add 100ml  of LAL in the same tube in duplicate.

Ø      Prepare the dilutions for PPC for confirming 10,000EU/vial .

Ø      Note the gel clot after incubation of one hour

4.4.       Procedure for testing of reduction of prepared spore loaded strip(B subtilis):

Transfer (spore loaded) strip from backed vials and are inoculated in100ml sterile SCD media and incubate at 30 – 35⁰C for 7 days to observe for any turbidity, if any, report to Manager QC

5. 0          REFERENCES:         

USP 25 page no.-1890 & 2251

 Annexure 1. Formats for Instrument validation

DILUTION FOR PPC (Use depyrogenated glass-wares for testing as per SOP no.:K/QC/052)

 CSE contains 1,00000 EU Reconstituted  with  1000  ml of LRW distributed 100ml each in 10ml vials.

Keep one vial as positive.

Dilution for PPC
10,000 EU / vial--> 0.1 ml of CSE + 4.9 ml of LRW---> 0.1 ml of above + 3.9 ml of LRW---> 
0.1 ml of above + 4.9 ml of LRW (0.5 EU / ml) --->  500ml of above  +  500ml of LRW (25 EU / ml)-->
500ml of above  +  500ml of LRW (0.125 EU / ml) --> 500ml of above  +  500ml of LRW (0.06 EU / ml)
--> 500ml of above  +  500ml of LRW    (0.03 EU / ml)

FOR SAMPLE : Reconstitute 1 ml in each backed vial vortex for at least ten minutes and take 100ml of sample  + 100ml of Limulus amoebocyte lysate in the reaction tube and incubate for one hour at 37 °C  ± 1°C and record the results.

HEATING BLOCK TEMPERATURE: 37°C ± 1°C    TIME FROM:           TO:           HRS

S. no.	DETAILS	GEL	S. NO.	DETAILS	GEL
1.	Blank	-ve     -ve	10.	Sample (NPC) in duplicate	-ve     -ve
2.	2 l dilution in duplicate (PPC)	+ve    +ve	11.	Sample (NPC) in duplicate	-ve     -ve
3.	2 l dilution in duplicate (PPC)	+ve     +ve	12.	Sample (NPC) in duplicate	-ve      -ve
4.	l dilution in duplicate (PPC)	+ve    +ve	13.	Sample (NPC) in duplicate	-ve      -ve
5.	l dilution in duplicate (PPC)	+ve    +ve	14.	Sample (NPC) in duplicate	-ve       -ve
6.	l/2  dilution in duplicate (PPC)	-ve     -ve	15.	Sample (NPC) in duplicate	-ve       -ve
7.	l/2  dilution in duplicate (PPC)	-ve     -ve
8.	l/4  dilution in duplicate (PPC)	-ve     -ve
9.	l/4  dilution in duplicate (PPC)	-ve     -ve

.

OBSERVATION OF SPORE LOADED STRIP OF B.subtilis

NAME OF MEDIA	SCD media              INCUBATION TEMP. 30-35°C
NO. OF DAYS  ®	1	2	3	4	5	6	7
GROWTH OBSERVED Location:- B.subtilis