sterile preparations, saline solution, glasses

1. Sterile Dosage
Forms
Akshata A. Jain
Asst. prof.
KYDSCT’s College of Pharmacy

2. Introduction:
• Parental preparation is a pharmaceutical product that is given
by other than oral routes.
• Transfusion fluids and injection are parenteral preparation.
• The injection is the sterile solution or suspension of drugs in
an aqueous or oily vehicle meant for introduction into the
body by means of an injectable needle under or through one
or more layers of the skin or mucous membrane.
• The injection should be sterile, isotonic, and free from foreign
particles, such as dust, fibers, etc.

3. Advantages Disadvantages
1)Onset of action is quick. 1. The injection causes pain at the site of
injection.
2) The drugs which cannot be administered by
the oral route, can be administered by this
route.
2. The trained persons are required to
administer the drugs.
3)Suitable for unconscious patients. 3. The administration of drugs through the
wrong route of injection may prove to be fatal.
4) The drug action can be prolonged by
modifying the formulation.
4. It is difficult to save a patient when an
overdose is given.
5) Transfusion fluids containing nutrients like
glucose and electrolytes such as sodium
chloride can be given by this route.
5. There are chances of sensitivity reaction of
allergic reaction of a drug by an individual.
These reactions are sometimes very fatal and
lead to death.

4. Routes of Administration of
Parenteral Products
Intradermal ( intracutaneous injection) :
These are given in between the dermis and epidermis. The skin of
the left forearms is usually selected for giving the injection.
Generally, 0.1 to 0.2 of the parenteral solution is injected by this
route. the route is used for diagnostic purposes and for testing the
sensitivity of the injectables.
Hypodermis ( subcutaneous ) injection :
-these are made under the skin, into the subcutaneous tissue.
-the volume of 1.0 ml or less, is usually injected into the upper arms.
-this is the most popular route because it is convenient for the
patient and the doctor.

5. Intramuscular injection
-These injections are given into the muscular tissues.
-The muscles of the shoulder, thigh, or buttock are usually selected.
-Generally, volume up to 2.0 ml is administered by this route and should not exceed 4.0 ml at
one site.
– Aqueous or oily suspensions and oily solution can be administered by this route.
Intravenous route
-These injections are made into a vein and therefore introduced directly into the bloodstream.
– The median basilic vein near the anterior surface of the elbow is usually selected because it is
easily located and connects with the major veins of the arm.
– Large volume of parenteral solution ranging from 1 ml to 500 ml or more than that can be
injected. The suspension and oily injections cannot be injected by this route.

6. Intraarterial injections-
• These are similar to intravenous injections and are
occasionally used for an immediate effect in a peripheral
area.
• These injections are given directly into the artery.
Intracardiac injections-
- These are given into the heart muscle or ventricles in an
emergency only
- For example; as a stimulant following cardiac arrest.
Intrathecal injections
-These are made into subarachnoid space that ventricle in an
emergency only
-This route is used for giving spinal anesthesia.

7. Intracisternal injection
-These are given in between the first and second cervical vertebrae.
-This route is used to withdraw C.S.F. ( Cerebrospinal fluid) for diagnostic purposes.
Peridural injections.
- These are given between the dura mater and the inner aspects of the vertebra.
- So it is that portion of the vertebral canal not occupied by the dura mater and its contents.
– This route is sometimes used for giving spinal anesthesia in special cases.
Intra- articular injections
– These injections are given into the liquid that lubricates the articulating ends of bones in a
joint.
Intracerebral injections
– These injections are given into the cerebrum.

9. Solution or emulsions of
medicaments suitable for
injections.
Sterile solids
Sterile suspensions
Transfusion fluids
1
2
3
4

10. 1. Solution or emulsions of medicaments suitable
for injections
These are commonly called as
injections. The parenteral
preparation in this form may be
supplied in single dose containers
or multiple dose containers. Its
volume varies from 0.5 ml to a litre
e.g., atropine sulphate injection,
dextrose injection.

11. Drugs which are not stable in
solution are prepared and
supplied as dry sterile solids
which are dissolved in a suitable
solvent immediately before its
administration.
e.g., benzyl penicillin G sodium
injection.
2. Sterile solids

12. 3.
STERI
LE
SUSP
ENSIO
NS:
3. Sterile Suspensions:
These are the sterile
suspension of drugs in a
suitable solvent which are
administered by intramuscular
route e.g sterile hydrocortisone
acetate suspension, and sterile
chloramphenicol suspension.

13. 4. Transfusion fluids
These are the parenteral
solutions which are
administered by intravenous
route. They are generally
used for nutrition and to
maintain the electrolyte
balance e.g., ringer solution.
dextrose injection, sodium
chloride injection.

14. Requirements For Parenteral Dosage Forms
The formulation of parenteral products involve careful
consideration of the following requirements:
1. Stability: The stability of parenteral preparation is very
important. The physical as well as chemical stability of parenteral
preparation must be maintained during storage.
2. Sterility: The parenteral preparations should be free from all
types of microorganisms. An aseptic conditions are required to be
maintained during the preparation of parenteral products and its
administration. The parenteral product must pass the test for
sterility.

15. 3. Free from pyrogens:
The parenteral preparations should be free from toxin and pyrogens.
It is necessary that the parenteral products must pass the test for
pyrogen, because contaminated parenteral product causes rise in
body temperature after its administration.
4. Free from foreign particles:
The parenteral products should be free from foreign particles such
as dust and fibres. To ensure this, the parenteral products must pass
the clarity test.

16. 5. Isotonicity:
The parenteral preparations should be isotonic with blood plasma
and body fluids. It is very important, in order to avoid any
complications on the administration of parenteral products.
6. Specific gravity:
The parenteral products meant for intra-spinal injections should
have the same specific gravity as that of spinal fluid into which the
same are to be injected.

17. The formulation of parenteral preparations need careful planning. thorough
knowledge of the medicaments and adjuvants to be used. The excess use of
adjuvants in parenteral products should be avoided as some of these may
interfere with the drug. In the preparation of parenteral products, the following
substances are added to make a stable preparation:
(1) Vehicles
(a) Solubilising agents
(b) Stabilizers
(c) Buffering agents
(d)Antibacterial agents
(e) Chelating agents
(f) Suspending, emulsifying and wetting agents
(g) Tonicity factors

18. 1.Vehicles: There are two types of vehicles which are commonly used
for the preparation of injections:
(a) Aqueous vehicles: Water is used as vehicle for majority of injections
because water is tolerated well by the body and is safest to administer.
The aqueous vehicle used are:
(i) Water for injection.
(ii) Water for injection free from CO₂.
(iii)Water for injection free from dissolved air.
Water for injection is a sterile water, which is free from volatile, non
volatile impurities and also from pyrogens.
Pyrogens are by-product of bacterial metabolism. Pyrogens are
polysaccharide, thermostable, soluble in water, unaffected by
bactericide and can pass through bacteria proof filters.

19. Pyrogens can be removed from water by simple
distillation process using an efficient trap which
prevents the pyrogen to enter into the
condenser. Immediately after the preparation of
water for injection, it is filled into the final
container, sealed and sterilised by autoclaving.
Water for injection, contaminated with pyrogens
may cause rise in body temperature if injected.
Hence, test for pyrogen is done to ensure that
water for injection is free from pyrogens.

20. (b) Non-aqueous vehicles:
The commonly used non-aqueous vehicles are oils and
alcohols.
Fixed oils, such as, arachis oil, cottonseed oil, almond
oil and sesame oil are used as vehicle. The oily
vehicles are generally used when a depot effect of
drug is required or the medicaments are insoluble or
slightly soluble in water or the drug is soluble in oil
e.g., dimercaprol injection by using arachis oil as
vehicle.
Ethyl alcohol is used in the preparation of
hydrocortisone injection. Hydrocortisone is insoluble
in water, hence the solution is made in 50% alcohol.
Propylene glycol is used as a vehicle in the preparation
of digoxin injection.

21. 2. Adjuvants:
- These substances are added to increase the stability or
quality of the product.
- They should be added in minimum possible quantity.
- The following adjuvants are commonly used in the
preparation of parenteral preparation:
a) Solubilising agents:
These are used to increase the solubility of drugs which
are slightly soluble in water.
The solubility of drug is increased by using surface active
agent like tweens & polysorbates.

22. b) Stabilizers:
• The drugs in the form of solutions are more liable to deteriorate due to oxidation &
hydrolysis the stabilizers are added in the formulation to prevent this.
• The oxidation can be prevented by adding a suitable antioxidant, such as thiourea &
ascorbic acid or the product is sealed in an atmosphere of nitrogen or carbon dioxide.
c) Buffering agents:
The degradation of preparation which is due to change in pH, can be prevented by adding
a suitable buffer to maintain the desired pH. For example,citric acid.
d) Antibacterial agents:
These substances are added in adequate quantity to prevent the growth of microorganism
during storage. So these substances acts as preservatives.

23. (e) Chelating agents :
Chelating agent such as EDTA (ethylene diamine tetra acetic acid) and its salts, sodium
or potassium salts of citric acid are added in the formulation, to chelate the metallic
ions present in the formulation. They form a complex which gets dissolved in the
solvent.
(f) Suspending, emulsifying and wetting agents :
The suspending agents are used to improve the viscosity and to suspend the particles
for a long time. Methyl cellulose, carboxymethyl cellulose, gelatin and acacia are
commonly used as suspending agents.
(g) Tonicity factors :
Parenteral preparation should be isotonic with blood plasma or other body fluids. The
isotonicity of the solution may be adjusted by adding sodium chloride, dextrose and
boric acid etc. In suitable quantities.

24. Units of Concentration
The concentration of ingredients in products may be expressed
in a number of different ways:
1. Weight per unit volume: e.g., Atropine sulphate injection
600 µg/ml, prednisolone injection 25 mg/ml.
2. Percentage weight / volume : e.g., Dextrose intravenous
infusion 5% w/v, and sodium chloride intravenous infusion
0.9% w/v.
3. Millimoles per unit volume : e.g., Sodium bicarbonate
intravenous infusion 150 m.Mol each of na+ and HCO3-/
litre, potassium chloride solution strong 2 m moles each of
K+ and cl/ml.

25. The containers used for parenteral products are either made from
glass or plastic material. The pharmacopoeia requires the following
conditions for a container and closure to be used for parenteral
preparations:
1. It should not yield foreign substances to the product.
2. It should be sufficient transparent to allow visual inspection of
the content in it.
3. It should not have any adverse effect on the product.
4. It should prevent diffusion in or across the walls.
The following three types of glasses are used for the preparation of
containers meant for storage of parenteral preparations.

26. 1. Type I glass :
It is also called borosilicate glass or neutral glass. It offers a high hydrolytic resistance
due to its chemical compo sition. It can withstand autoclaving and weathering.
2. Type II glass :
It is a soda-lime-silicate glass with high hydro lytic resistance as a result of treatment
with moist sulphur dioxide at high temperature. The containers made from this glass
are suitable for most acidic and neutral aqueous prepara tions.
3. Type III glass:
It is a soda-lime-silicate glass with only moder ate hydrolytic resistance. The
containers made from this glass are generally suitable for non-aqueous preparations
and powdersfor reconstitution immediately prior to administration.

27. Containers of type II and type III glass should be used once only. Containers for human
blood and blood components must not be reused.
Plastics are synthetic polymers such as polyethylene, polyvinyl chlo ride and
polypropylene which are used to make containers for injectables.
The standards for plastic containers for parenteral products are given in I.P.

28. Processing of Parenteral Preparations:
The following steps are involved in the processing of parenteral preparations:
(1) Cleaning of containers, closures and equipment
(2) Collection of materials
(3) Preparation of parenteral products
(4) Filtration
(5) Filling the preparation in final containers
(6) Sealing the containers
(7) Sterilisation
(8) Evaluation of parenteral preparations
(9) Labelling and packaging.

29. 1. Cleaning of Containers, Closures and Equipment :
All the containers, closures and equipment which are required during the prepa
ration of parenteral products are thoroughly cleaned with detergents and washing
is done with tap water, followed by clean distilled water and finally rinsed with
water for injection. Rubber closures are washed with hot solution of 0.5% sodium
pyrophosphate in water. The closures are then removed from the solution,
washed with water followed by rinsing with filtered water for injection. On a small
scale washing is done manually but on a large scale automatic washing machines
are used.

30. 2. Collection of materials:
The various ingredients of the formu lation of parenteral preparations are
weighed and collected in the preparation room. The raw materials required in the
preparation of parenteral products should be pure. Water for injection free from
pyrogens and microorganisms are used in the preparation of parenteral products.
3. Preparation of parenteral product:
The pharmacist should decide the order of mixing and exact method of
preparation to be followed before preparing the parenteral products. The
parenteral prepa rations must be prepared under strict aseptic conditions. The
ingredients are accurately weighed separately and dissolved in the vehicle as per
method of preparation to be followed.

31. 4. Filtration :
The parenteral solution so formed is passed through bacteria proof filter, such as,
filter candle, seitz filter, membrane filter, and sintered glass filters. The primary
objective of filtration is to clarify the solution by removing foreign particles. If the
parenteral preparations are required to be sterilised by means of bacteria proof
filters, filtration should be done under strict aseptic condition to avoid
contamination of filtered solution, before it is finally transferred into final
container and sealed.

32. 5. Filling the preparation in final containers:
The filtered product is filled into final containers such as, ampoules, vials and
transfusion bottles, which are previously cleaned and dried. Ampoules are used for
filling single doses whereas, vials are used for filling multidoses. Bot tles are meant
for filling transfusion fluids. On small scale, filling is done manually by using
hypodermic syringe and needle. On large scale. Filling is done by automatic filling
machine.The sterile powders are filled into containers by individual weighing or by
using automatic or semi-automatic devices. The filling operation is carried out under
strict aseptic precautions.
During the filling of ampoules, the care should be taken that the solution should be
filled below the neck of ampoules and the solution should not touch the neck of the
ampoules. This will prevent the cracking and staining of the neck of ampoules at the
time of sealing.

34. 6. Sealing the containers :
Sealing should be done immediately after filling. Ampoules are sealed manually on a
small scale by rotating the neck of the ampoule in the flames of bunsen burner. But
on a large scale ampoule sealing machine is used in which tip of ampoule is fused to
seal it. The vials and transfusion bottles are sealed by closing its opening with a
rubber closures. The rubber closures are held in place by crimping the aluminium
caps which is done manually or by mechanical means.

35. 7. Sterilisation :
The parenteral preparations should be immediately sterilised after
sealing in its final containers. The sterilisation is done by any one of
the methods of sterilisation which depends on the nature of
medicament(s) present in parenteral preparations.
For thermostable medicament, the parenteral products are sterilised
either by autoclaving at a temperature of 115°C to 116°C for 30
minutes or 121°C for 20 minutes or in hot air oven at 160°C for two
hours. The thermolabile preparations are sterilised by filtration
through a suitable bacteria-proof filters. Parenteral preparations
which are sterilised by filtration method may contain a suitable
bacteriostatic agent to prevent the growth of microorganisms.

36. When the solutions are used for intravenous or intrathecal injection in
doses exceeding 15 ml, the bacteriostatic agent should not be used. The
sterilised product is filled into the final containers and sealed. The process
of filtration, filling and sealing are done under aseptic conditions.

37. 8. Evaluation Of Parenteral Preparations:
The finished parenteral products are subjected to the following tests, in
order to maintain quality control:
(A) Sterility Test
(B) Clarity Test
(C) Leakage Test
(D) Pyrogen Test
(E) Assay

38. a) Sterility Test:
The test for sterility is done by detecting the presence of viable forms of
bacteria, fungi and yeast in parenteral preparations. The sterility test must
be carried out under strict aseptic conditions in order to avoid accidental
contamination of the product during the test. All glasswares required for
the test must be sterile. Culture media required for the growth of aerobic,
anaerobic and fungi, are prepared as described in pharmacopoeia.
The test for sterility may be carried out either by:
(a) Membrane Filtration Method
(b) Direct Inoculation Method.

39. The test samples of parenteral preparation are transferred into test tubes
containing sterile culture media for aerobic, anaerobic bacteria and fungi. These
test tubes are incubated for stated period in the incubator. The presence of
turbidity in the culture media indicate the growth of microorganisms and the
sample fails to comply with test for sterility. This can be confirmed by repeating
the test.The detail of the test for sterility is discussed separately.

40. (B) Clarity Test:
Clarity test is performed to ensure that the parenteral products are free from
foreign particles. Each parenteral preparation in its final container is
subjected individually to a visual inspection to exclude the possibility of
foreign particles. The unlabelled containers are held by the neck against
strongly illuminated black and white screen. White screen is used for dark
coloured particles and black screen for the detection of light coloured
particles. The contents of the containers are slowly inverted and rotated. The
solution is examined for the presence of foreign particles. If any foreign
matter is visible, the same container is rejected. Nowadays, the various
instrumental methods such as coulter counter, has been developed which
are widely used in industry.

41. (C) Leakage Test:
This test is performed only for ampoules which
have been sealed by fusion to ensure that there
should not be any leakage in them. Leakage test is
performed in a vacuum chamber. The ampoules
are dipped in 1% solution of methylene blue in
vacuum chamber and vacuum is applied. When
vacuum is released, the coloured solution will
enter the ampoules with defective sealing. The
presence of dye in the ampoule, confirms the
leakage and hence rejected. Vials and bottles are
not subjected to this test because of flexibility of
rubber.

42. (D) Pyrogen Test:
The test is done as per I.P. 2007 to check the presence or absence of
pyrogen in all aqueous parenteral preparations. The test involves the
measurement of the rise in body temperature of rabbits following
intravenous injection of a sterile solution of a parenteral preparation being
examined. Rabbits are used to perform this test, because their body
temperature increases when pyrogens are introduced into their bodies by
parenteral route.
(E) Assay:
Assay is performed according to the method given in the monograph of
that parenteral preparation in the pharmacopoeia. Assay is done to check
the quantity of medicament present in the parenteral preparation.

43. 9. Labelling and Packaging:
After evaluation of the parenteral preparation, the ampoules, vials and transfusion
bottles are properly labelled and packed. The label should state:
(1) Name of The Preparation
(2) Quantity of The Preparation
(3) Mfg. Lic. No.
(4) Batch No. Preparation.
(5) Date of Manufacture
(6) Date of Expiry
(7) Storage Conditions
(8) Retail Price
(9) Manufacturer's Address.

44. Production Facilities:
The manufacture of parenteral preparation requires special precautions
and facilities in order to maintain sterility and freedom from particulate
matter. The production area where sterile parenteral preparations are
manufactured can be divided into five sections:
(1) Clean-up area
(2) Preparation area
(3) Aseptic area
(4) Quarantine area
(5) Finishing & packaging area

45. 1. Clean-up area:
It is not an aseptic area. All the parenteral products must be free from
foreign particles and microorganisms. The area and atmosphere of the room
where manufacturing of parenteral products are done must be free from
dust, fibres and microorganisms. Clean up area should be constructed to
withstand moisture, steam and detergent. Ceiling and walls can be coated
with materials which should prevent the accumulation of dust and
microorganisms. Exhaust fans should be fitted to remove heat and humidity
for the comforts of persons working in this area. This area should be kept
clean so that contami nants may not be carried into aseptic area. The
containers and closures are properly washed and dried in this area.

46. 2. Preparation area:
In preparation area, the ingredients of the parenteral products are mixed
and preparation is made for filling operation. Although it is not essential
that, this area should be aseptic, but strict precautions are required to
prevent any contamination from outside. Cabinets and counters should
be made of stainless steel and they are filled in such a way that no space
is left for the dirt to accumulate. Ceiling, walls and floor should be sealed
and suitably painted to keep them thoroughly clean.

48. 3. Aseptic area:
From preparation area the parenteral formulation will be transferred to aseptic
filling area. The parenteral preparations are filtered, filled into final containers
and subsequently sealed. The entry of personnel into aseptic area should be
through an air lock. To maintain sterility, specially trained persons should be
selected to work.
Ceiling, walls and floor of aseptic area should be sealed and painted, so that
they can be washed or treated with aseptic solution or sprayed before use.
Stainless steel counters should be fitted on the walls. Mechanical equipment
should be housed in stainless steel cabinet in order to prevent dirt to
accumulate on it. Mechanical parts that will come in contact with the
parenteral product should be separated so that they can be sterilised.

49. The air in the aseptic area should be free
from fibres, dust and microorganism. This can
be achieved by the use of high efficiency
particulate air filters (HEPA) which can
remove particles upto 0.3μ HEPA filters are
fitted in laminar air flow system, in which air
free from dust and microorganism flows with
uniform velocity. The air is supplied under
positive pressure which prevents particulate
contamination from sweeping from adjoining
areas. Ultra violet lamps are fitted in order to
maintain sterility.

50. 4. Quarantine area:
A batch of parenteral products after its filling, sealing and sterilisation are held
up in this area. The random samples of parenteral product of different batches
are in the analytical laboratory for its evaluation. Those parenteral products
which passes all the quality control tests are transferred into finishing or
packaging area.
5. Finishing and packaging area:
The parenteral products are properly labelled and packed. Proper packaging is
essential to provide protection against physical damage from transportation,
handling and storage. The labelled containers should be packed in cardboard or
plastic containers. Ampoules should be packed in partitioned boxes.The packed
parenteral products are temporarily stored till it is finally disposed off.

52. Preparation of intravenous fluids:
Large volume of parenteral solutions intended to be administered by intravenous route
are commonly called intravenous fluids. The intravenous fluids are administered to
supply body fluids, electrolytes or pro vide nutrition to the body. Intravenous fluids are
available in packs of 100 ml to 1000 ml.
Examples were study from book

53. Preparation of Intravenous Admixtures
When one or more sterile products are added to an intravenous fluid for administration,
the resulting combination is known as intravenous admixture. The drugs are incorporated
into a bottle of large volume transfusion fluids by injected it through a rubber closure. This
practice is very dangerous and it may lead to:
1. Microbial contamination:
The intravenous fluids contains no bactericide but contains sugar and amino acid,
which are richest source of multiplication of bacteria. So there are more chances of
bacterial contamination if the mixing is not done under aseptic condition.
2. Incompatibility :
Physical, chemical and therapeutic incompatibilities may occur due to interaction between
additives and in travenous fluids.

54. Methods for safe and effective use of intravenous admixtures
Following are the few methods for safe and effective use of intravenous admixtures:
(1) Proper training should be given to nurses and pharmacists for preparation of
admixtures.
(2) Nurses should be instructed that they should attach a label bearing date and time
of addition of a drug. The quantity of drug mixed should also be given in the
label.
(3) The pharmacy department of the hospital, should provide latest information
regarding drug stability and compatibility to the paramedical staff.
(4) The pharmacist should always try to avoid addition of medicaments to
transfusion bottles by using some other suitable formulations.

55. Total Parenteral Nutrition (TPN)
Intravenous administration of calories, nitrogen and other nutrients in
sufficient quantity to produce tissue synthesis and anabolism is called
parenteral nutrition or parenteral hyperlimentation. The source of
nitrogen in TPN fluid is either protein hydrolysates or crystalline amino
acids.
The normal calories requirement for an adult is approximately 2500 per
day which can be supplied by injecting dextrose 25%.

56. Dialysis Fluids
Dialysis is a process by which the substances are separated from one another due to their
difference in diffusibility through membranes. The fluids used in dialysis are known as
dialysis fluids. In cases of renal failure, transplantation of kidney or certain cases of
poisoning, dialysis is needed to save the patient. In the case of renal failure, it becomes
necessary to remove the waste products and to maintain electrolyte balance. This can be
done by haemodialysis or intraperitoneal dialysis.
Haemodialysis
haemodialysis is done to remove toxins from the blood. In haemodialysis, the blood from
an artery is passed through an artificial dialysing membrane, bathed in dialysis fluid. The
dialysing membrane is permeable to urea, electrolytes and dextrose and not to plasma
proteins and lipids. So urea which is excess in the blood, pass out in dialysis fluid.

57. After the dialysis, the blood is returned back to the body circulation through a vein. A
kidney unit may require more than 1200 litres of solution in a week. So
haemodialysis fluid is prepared in concentrated form which can be diluted with
deionised water or distilled water before its use.
Intraperitoneal dialysis
In this method, the peritoneal cavity is irrigated with the dialysis solution and the
peritoneum acts as the semi permeable membrane thereby the toxic substances
normally excreted by kidney are removed. Intraperitoneal dialysis fluid should be
sterile and free from pyrogens.

58. Sterility Test
• Test for sterility is intended for detecting the presence of viable forms
of bacteria, fungi & yeasts in substances, preparations or articles
which are required to be sterile as per I.P
Principle:
• The test is based on the principle that if bacteria or fungi are placed in
a medium which provides nutritive material & water, & kept at a
favourable temp. The organism will grow & their presence can be
indicated by a turbidity in clear medium.

59. Steps involved in sterility testing:
1. Selection of the sample size.
2. Selection of the quantity of product to be used.
3. Method of testing.
4. Observation & results.