ANALYSIS OF CRUDE DRUGS

Different techniques involved in the

analysis of crude drugs
• Macroscopic methods
• Microscopic methods
• Physical methods
• Chemical methods
• Biological methods
PHYSICAL ANALYSIS OF CRUDE DRUGS:
• Viscosity
• Melting point
• Solubility
• Moisture content and volatile matter
• Specific gravity
• Density
• Optical rotation
• Refractive index
• Bitterness value
• Hemolytic activity
• Swelling index
• Foaming index
• Ash value
• Astringency
VISCOSITY
 Viscosity of a liquid is constant at a given temperature and is an
index of its composition. Hence, it can be used as a means of
standardizing liquid drugs.

MELTING POINT
 In case of pure photochemical, melting points are very sharp and
constant.
 The crude drugs from plant or animal origin, containing the mixed
chemicals, are described with certain range of melting point.
 Their purity can be ascertained by determining their melting
points in that range
 for E.g. Colophony- 75-80˚c
 Cocoa butter- 30-33˚c
SOLUBILTY
 The presence of adulterant could be indicated by solubility studies
 E.g.pure Asafoetida is soluble in carbon disulphide

MOISTURE CONTENT AND VOLATILE MATTER

 The moisture content of the drug should be minimized in order to
prevent decomposition of crude drug either due to chemical
change or microbial contamination.
 The moisture content is determined by heating a drug at 105˚c in
an oven to a constant weight.
 For the drugs containing volatile constituents, toluene distillation
method is used
 E.g. – Aloe should have moisture content not more than 10% w/w
OPTICAL ROTATION
 Optically active compounds have the property of rotating the plane
of polarized light.This property is known as optical rotation.
 Normally, the optical rotation is determined at 25˚c using sodium
lamp as the source of light.
 E.g. castor oil has optical rotation from +3.5˚to +6˚

REFRACTIVE INDEX
 When a ray of light passes from one medium to another of
different density, then the ratio of velocity of light in vaccum
to its velocity in substance is termed as refractive index of
second medium.
 It is constant for a pure drug and varied with wavelength of
incident light, temperature and pressure
 E.g. Castor oil has refractive index 1.4758-1.527
ASH VALUES AND EXTRACTIVES
The residue remaining after incineration is the ash content of
drug
• Total ash method is used to measure the total amount of
material remaining after incineration
• Acid insoluble ash is the residue obtained after boiling the
total ash with dil. HCl and igniting the remaining insoluble
matter.
• Water soluble ash is the difference in weight between total
ash and residue after treatment of total ash with water.
DETERMINATION OF EXTRACTABLE MATTER:

HOT EXTRACTION:
1. Place 4 gms powdered material in a conical flask. Add water
and weigh to obtain total weight.
2. Shake and allowed to stand for 1hr. attach the reflux
condenser and boil for 1hr.
3. Readjust to the original weight with solvent. Shake and filter.
4. Transfer the filter to a flat bottomed disk and evaporate to
dryness on a water bath.
5. Dry at 105˚ c for 6hrs, cool and weigh immediately.
6. Calculate the content of extractable matter in mg per g of air
dried material.
COLD MACERATION
• Place the powdered material in a conical flask.
• Macerate with 100ml of solvent specified for 6hrs, shake
then allowed to stand for 18hrs.
• Filter and transfer the filtrate to flat bottomed disk and
evaporate to dryness on a water bath.
• Dry at 105̊ c for 6hrs, cool and weigh immediately.
• Calculated the content of extractable matter in mg per g of
air dried material.
BITTERNESS VALUE
 Medicinal plants having strong bitter taste are therapeutically used as
appetizing agents
 The bitterness is determined by comparing the threshold bitter
concentration of an extract material with that of quinine hydrochloride
 The bitterness value is expressed as units equivalent to the bitterness
of a solution containing 1gm of quinine hydrochloride in 2000ml.
 0.1gm of quinine hydrochloride is dissolved in 100ml drinking water
and the stock solution is prepared. Then it is diluted and tested and
compared with drug.
 Bitterness value in unit per gm = 2000*c
A*B
 Where, A = concentration of stock solution
B = volume of test solution in tube with threshold bitter
concentration
C = quantity of quinine hydrochloride in the tube with threshold
bitter concentration
HAEMOLYTIC ACTIVITY
• Haemolytic activity of plant material is determined by
comparison with that of reference material, Saponin R,
having haemolytic activity of 1000units/g.
  Method of preparation of standard:
• Fill a glass stopper flask to 1/10 of its volume with sodium citrate.
Ass sufficient volume of blood freshly collected from healthy ox
and shake, this can be stored for about 8 days at 2-4̊ c. place 1ml of
citrated blood in a volumetric flask with phosphate buffer pH 7.4.
Haemolytic activity = 1000* a/b
Where, 1000 = defined haemolytic activity of Saponin standard
a = quantityof saponin standard that produce total
haemolysis (g)
b = quantity of plant material that produce total haemolysis
(g)
SWELLING INDEX
• The swelling index is the volume in ml taken up by the swelling of 1gm of
plant material under specified conditions.
Its determination is based on addition of water or a swelling agent as
described in test procedure.
FOAMING INDEX:
• The foaming ability of an aqueous decoction of plant material and their
extracts is measured in terms of foaming index.
WATER AND VOLATILE MATTER:
• Azeotropic method is used to directly measure the water present in a
material.
• Loss on drying
• In order to measure volatile matter, plant is diluted with water and
distillate is collected in a graduated tube. The aqueous portion separates
and returns to distillation flask. A solvent of low mass density with a
suitable boiling point may be added to measuring tube to easily separate
the volatile oil.
 
CHEMICAL METHODS OF ANALYSIS OF CRUDE DRUGS:

• It comprises of different chemical tests and assays. The

isolation, purification and identification of active
constituents are chemical methods of evaluation.
• Quantitative chemical tests such as acid value,
saponification value etc., are also covered under this
technique. Qualitative chemical tests are used in detection
of adulteration.
• The chemical evaluation also covers phytochemical
screening carried out for establishing chemical profile of a
drug. But before phyto screening, extraction of crude drug
should be done.
 EXTRACTION OF CRUDE DRUGS
Extraction may be defined as the treatment of the plant or animal
tissues with solvent, whereby the medicinally active constituents
are dissolved, and most of the inert matter remains undissolved.
The solvent used for extraction is known as Menstruum and the
inert insoluble material that remains after extraction is called
Marc

The various process used for extraction are:

1. Maceration
2. Infusion
3. Digestion
4. Decoction
5. Percolation
1. Maceration
In this process, the whole or coarsely powdered crude drug is placed in a
stoppered container with the solvent and allowed to stand at room
temperature for a period of at least 3 days with frequent agitation until
the soluble matter has dissolved. The mixture then is strained, the marc
(the damp solid material) is pressed, and the combined liquids are
clarified by filtration or decantation after standing.

2. Infusion
Fresh infusions are prepared by macerating the crude drug for a short
period of time with cold or boiling water. These are dilute solutions of
the readily soluble constituents of crude drugs.

3. Digestion
This is a form of maceration in which gentle heat is used during
the process of extraction. It is used when moderately elevated
temperature is not objectionable. The solvent efficiency of the
menstruum is thereby increased.
4. Decoction
In this process, the crude drug is boiled in a specified volume of water for
a defined time; it is then cooled and strained or filtered. This procedure is
suitable for extracting water-soluble, heat stable constituents. The
starting ratio of crude drug to water is fixed, e.g. 1:4 or 1:16; the volume
is then brought down to one-fourth its original volume by boiling during
the extraction procedure. Then, the concentrated extract is filtered and
used as such or processed further.

5. Percolation
Percolation is a continuous flow of the solvent through the bed of the
crude drug material to get the extract.
In this process, the powdered drug is moistened with an appropriate
amount of the specified menstruum and allowed to stand for
approximately 4 h in a well closed container, after which the mass is
packed and the top of the percolator is closed.
Additional menstruum is added to form a shallow layer above the mass,
and the mixture is allowed to macerate in the closed percolator for 24 h.
Hot Continuous Extraction (Soxhlet)
In this method, the finely ground crude drug is placed in a
porous bag or “thimble” made of strong filter paper, of the
Soxhlet apparatus.

The extracting solvent in flask is heated, and its vapors

condense in condenser . The condensed extractant drips into the
thimble containing the crude drug, and extracts it by contact.

When the level of liquid in chamber rises to the top of siphon

tube , the liquid contents of chamber siphon into flask. This
process is continuous and is carried out until a drop of solvent
from the siphon tube does not leave residue when evaporated.

The advantage of this method, compared to previously

described methods, is that large amounts of drug can be
extracted with a much smaller quantity of solvent.
SOXHLET APPARATUS
AQUEOUS ALCOHOLIC EXTRACTION BY FERMENTATION

It involves soaking the crude drug, in the form of either a powder or a decoction
for a specified period of time, during which it undergoes fermentation and
generates alcohol in situ; this facilitates the extraction of the active constituents
contained in the plant material.
The alcohol thus generated also serves as a preservative.
Some examples of such preparations are karpurasava, kanakasava, dasmularista.

COUNTER-CURRENT EXTRACTION
In counter-current extraction (CCE), wet raw material is pulverized and produce a fine
slurry.
Here the material to be extracted is moved in one direction within a cylindrical extractor
where it comes in contact with extraction solvent.
The further the starting material moves, the more concentrated the extract becomes.
Complete extraction is thus possible when the quantities of solvent and material and their
flow rates are optimized.
Finally, sufficiently concentrated extract comes out at one end of the extractor while the
marc falls out from the other end.
Ultrasound Extraction (Sonication)

The procedure involves the use of ultrasound with requencies ranging

from 20 kHz to 2000 kHz;this increases the permeability of cell walls and
produces cavitation.
The process is useful in some cases, like extraction of rauwolfia root, its
large-scale application is limited due to the higher costs.

Disadvantage
The deleterious effect of ultrasound energy (more than 20 kHz) on the
active constituents of medicinal plants through formation of free radicals
and consequently undesirable changes in the drug molecules.
Supercritical Fluid Extraction
The critical point represents the highest temperature and
pressure at which the substance can exist as a vapour and
liquid in equilibrium. The phenomenon can be easily explained
with reference to the phase diagram for pure carbon dioxide
Types of extract

Aqueous extracts
The medicinal preparations intended to be used immediately after
preparation or to be preserved for use, solvent used is water. The methods
used for their preparation are decoction, infusion, and digestion.

Hydro alcoholic or Alcoholic

These are prepared by the methods of maceration and percolation eg
tintures, here the solvent using is alcohol

Soft extracts
They are extracts with semisolid or syrup consistency Can be used in a
variety of dosage form like ointments and suppositories
Eg; glycerriza extracts

Dry extracts
They powdered extracts or dry powder Extract obtained from suitable
process is filtered and get concentrated under vacuum, dried completely
by spray or tray drying.
Eg; belladona used in dossage forms such as capsules, tablets etc.
PHYTOCHEMICAL SCREENING:
• Detection of alkaloids
• Detection of carbohydrates and glycosides
• Detection of phytosterols
• Detection of fixed oils and fats
• Detection of saponins
• Detection of phenolic compounds and tannins
• Detection of protein and free amino acids
• Detection of gums and mucilage
• Detection of volatile oils
Qualitative Reactions For The Detection Of Plant Constituents

Test for alkaloids 

1. Dragendorff’s test
1 ml of extract, add 1 ml of Dragendroff’s reagent (potassium bismuth iodide
solution). An orange-red precipitate indicates the presence of alkaloids.

2.Mayer’s test
1 ml of extract, add 1 ml of Mayer’s reagent (potassium mercuric iodide
solution). Whitish or cream colored precipitate indicates the presence of
alkaloids.

3.Hager’s test
1 ml of extract, add 3 ml of Hager’s reagent (saturated aqueous solution of
picric acid). Yellow colored precipitate indicates the presence of alkaloids

4. Wagner’s test
1 ml of extract, add 2 ml of Wagner’s reagent (iodine in potassium iodide).
Reddish brown colored precipitate indicates the presence of alkaloids
Test for glycosides

Bontrager's test
In this test boil test sample with 1ml of sluphuric acid in a test tube
for 5min,filter while hot. Cool the filterate and shake with equal
volume of dichloromethane or chloroform then seperate the lower
layer of chloroform and shake it with half volume of dilute ammonia.
A rose pink to red colour is produced in the ammonical layer.

Modified Borntrager’s Test:

To 1 gm of drug add 5 ml dilute HCl followed by 5 ml Ferric

Chloride (5% w/v). Boil for 10 minutes on water bath, cool and filter,
filtrate was extracted withcarbon tetrachloride or benzene and add
equal volume of ammonia solution, formation of pink tored colour
due to presence of anthraquinone moiety. This is used C-type of
anthraquinoneglycosides
Chemical tests for steroid and triterpenoid glycosides
Libermann Burchard test:
Alcoholic extract of drug was evaporated to dryness and
extracted with CHCl 3 , add few drops of acetic anhydride
followed by conc. H2SO4 from sidewall of test tube to the
CHCl3extract. Formation of violet to blue coloured ring at the
junction of two liquid, indicate the presence of steroid moiety.

Salkwovski test:
Alcoholic extract of drug was evaporated to dryness and extracted
withCHCl3, add conc. H2SO4 from sidewall of test tube to the
CHCl3 extract. Formation of yellow colored ring at the junction of
two liquid, which turns red after 2 minutes, indicate the presence
of steroid moiety
Chemical tests for cardiac glycosides
Keller Killiani test:
To the extract of drug equal volume of water and 0.5 ml of strong lead acetate
solution was added, shaked and filtered. Filtrate was extracted with equal
volume of chloroform. Chloroform extract was evaporated to dryness and
residue was dissolved in 3 ml of glacial acetic acid followed by addition of
few drops of FeCl3 solution. The resultant solution was transferred to a testube
containing 2 ml of conc. H2SO4. Reddish brown layer is formed, which turns
bluish green after standing due to presence of digitoxose.

Legal test:
Treat the test solution with 2ml of pyridine and sodium nitropruside 2 ml was
added followed by addition of NaOH solution to make alkaline. Formation of
pink colour in presence of glycosides or aglycon moiety.

Baljet test:
Treat the test solution with picric acid or sodium picrate solution, it forms
yellow to orange colour in presence of aglycones or glycosides
 Tests for tannins

Goldbeater’s skin test: Goldbeater’s skin is a membrane produced from the

intestine of Ox. It behaves just like untanned animal hide. A piece of
goldbeaters skin previously soaked in 2% hydrochloric acid and washed with
distilled water is placed in a solution of tannin for 5 minutes. It is then
washed with distilled water and transferred to 1 % ferrous sulphate solution.
A change of the color of the goldbeater’s skin to brown or black indicates the
presence of tannin. Hydrolysable and condensed tannins both give the
positive goldbeater’s test while pseudo tannins show very little color or
negative test.
Phenazone Test: To 5 ml of aqueous solution of tannin containing drug, add
0.5 g of sodium acid phosphate. Warm the solution, cool and filter. Add 2
% phenazone solution to the filtrate. All tannins are precipitated as bulky,
colored precipitate.

Gelatin Test: To a 1 % gelatin solution, add little 10 % sodium chloride. If a

1 % solution of tannin is added to the gelatin solution, tannins cause
precipitation of gelatin from solution.
Tests for flavonoids:

Shinoda Test: To the test solution, and few drops of conc.

HCl. To this solution 0.5 g of magnesium turnings were
added. Observance of pink coloration indicated the presence
of flavonoids.

With Lead Acetate: To the small quantity of test solution lead

acetate solution was added. Formation of yellow precipitate
showed the presence of flavonoid.

With Sodium Hydroxide: On addition of an increasing

amount of sodium hydroxide, the ethanolic extract showed
yellow coloration, this decolorized after addition of acid.
 Tests of protein
Biuret test 

On adding 1% copper sulphite to alkaline solution (4% NaOH solution) of

protein, a violet colour is developed. This test is due to the presence of peptide
linkage.
Xanthoproteic Test:
To 5ml test solution add 1ml of Con.HNO3 and boil, yellow ppt is formed.
On addition of NH4OH, yellow ppt. turned orange.

Ninhydrin test
  When protein is boiled with a dilute solution of ninhydrin, a violet colour is
produced.
SEPARATION AND ISOLATION OF CONSTITUENTS

The instrumentation for the structure for the structure

elucidation of organic compounds becomes effective and
allows the use of increasingly.

The most difficult operation in phytopharmacetical research

is the isolation and purification of plant constituents.

The physical methods used are chromatographic techniques

and methods such as fractional crystallisation, fractional
distillation, fractional liberation.

Chemical method is based on groups or moieties present in

the compound and chemical reactions.
FRACTIONAL CRYSTALLISATION

It is an important method for the purification of

compounds from mixture.

It depends upon the compound which form crystals at

the point of super saturation in the solvent in which it is
soluble

Many natural products are crystaline nature even in

mixture, process such as concentration, slow
evaporation, refrigeration are using for crystalisation
FRACTIONAL DISTILLATION

This method is used for the separation of

the components from volatile mixtures
Largely using in the separation of
hydrocarbons from oxygenated volatile
oil eg citral, eucalyptol

FRACTIONAL LIBERATION

In this proces the groups of compounds

having the tendency of precipitation
from the solution.
Incertain cases the compounds may
modified by converting to its salt form.
This proces is often used in separation of
cinchona alkaloids, morphine etc.
 SUBLIMATION

Here the compound is

heated the solid state
changes to gaseous state
without passing via liquid
state. Such compounds get
deposited in form of
crystals or cake.

This method is traditionally

used for the separation of
camphor from chips of
cinnamomum camphora.
 CHROMATOGRAPHY

Chromatography is widely used for the separation &

identification of components of a mixture.

Separation of chemical compounds is carried out by mobile

phase and stationary phase.

Chromatography can be classified according to mechanism of

separation as:
adsorption chromatography,
 partition chromatography,
 ion exchange chromatography,
 size exclusion chromatography and
 affinity chromatography.
 PAPER CHROMATOGRAPHY

The principle is partition

Mainly the stationary phase is
moisture present in the cellulose
fibers and mobile vary as we using.
The components separated based
on their solubility

The ratio between the distance travelled on the paper by a

component of the test solution & the distance travelled by the
solvent is termed the RF value. Under standard conditions, this
is a constant for the particular compound.

In practise, however, variations of the RF value often occur & it

is best to run a reference compound alongside the unknown
mixtures.
ADVANTAGES
i. Simple & inexpensive
ii. Sensitive – gives good separation of very
small amounts, of especially water-soluble
compounds, e.g. sugars.
DISADVANTAGES
iii. Fragile – chromatogram may be destroyed
by chemicals used for visualization
iv. May be time-consuming.
THIN LAYER CHROMATOGRAPHY (TLC)

TLC is an e.g. of adsorption chromatography, the stationary

phase being a thin layer adsorbent held on a suitable backing.
Separation of the compounds present in the plant extract
depends on the differences in their adsorptive/desorptive
behaviour in respect of the stationary phase.

TLC involves a thin layer of adsorbent, mixed with a binder

such as CaSo4, which is spread on a glass plate & allowed to
dry.
The plant mixture to be separated is applied as a spot near the
base of the plate, which is then placed in a closed glass tank
containing a layer of developing solvent.
ADVANTAGES OF TLC OVER PAPER CHROMATOGRAPHY

Separation of compounds
can be achieved more
rapidly & with less plant
material.
-The separated spots are
more compact & clearly
demarcated from one
another
-Reagents such as
concentrated H2SO4 would
destroy a paper
chromatogram, but ma be
used to locate the separated
substances on a TLC plate.
COLUMN CHROMATOGRAPHY

It  is a method used to purify

individual chemical
compounds from mixtures of
compounds the principle of
separation is adsorption.

The classical preparative

chromatography column, is a
glass tube with a diameter from
5 mm to 50 mm and a height of
5 cm to 1 m with a tap and some
kind of a filter (a glass frit or
glass wool plug – to prevent the
loss of the stationary phase) at
the bottom.
GAS CHROMATOGRAPHY (GC)
It  is an analytical technique for separating compounds based primarily on
their volatilities.
GC provides both qualitative and quantitative information for individual
compounds present in a sample.
Compounds move through a GC column as gases, either because the
compounds are normally gases or they can be heated and vaporized into a
gaseous state.
The differential partitioning into the stationary phase allows the compounds
to be separated in time and space.
 APPLICATIONS
Quality control
contamination of plant and plant based products with pesticides, herbicides and many
other materials that are considered a health risk, all such products on sale today must
be carefully assayed

Identification of Source /Origin

The source of many plants (herbs and spices) can often be identified from
the peak pattern of the chromatograms obtained.
Technique of fingerprint could really identify the false herbal products.
The fundamental reason of quality control of herbal medicines is based on
the concept of phytoequivalence of herbs, and then to use this conception to
identify the real herbal medicine and the false one, and further to do
quality control.

Qualification and Quantification of Phytoconstituents

Alkaloids
 
Capillary gas chromatography (GC), often coupled with a mass spectrometer
as a detector (GC-MS), is a well established technique for analyzing
complex mixtures of alkaloids.
Terpenes

A qualitative comparative study was performed for terpenes

from volatile oils by GC and GC-MS technique
Flavanoids and Flavones

Flavonoids receive considerable attention in the literature,

specifically because of their biological and physiological
importance. Gas Chromatography Coupled to Mass
Spectrometry GC-MS is established as a routine technique
for the analysis of flavonoid aglycones.
Essential Oils /Volatile oils

Many pharmacologically active components in herbal

medicines are volatile chemical compounds. Thus, the
analysis of volatile compounds by gas chromatography is
very important in the analysis of herbal medicines
 High-performance liquid chromatography (HPLC)

High performance liquid chromatography is a powerful tool in

analysis. This page looks at how it is carried out and shows
how it uses the same principles as in thin layer chromatography
and column chromatography.
BIOLOGIAL/ TOXICOLOGIAL ANALYSIS OF CRUDE DRUGS
• Drugs which cannot be assayed by chemical, or
physical means are evaluated by biological methods

Indication for biological evaluation:

This is true for the substances having an
• Interfering obstacles
• When quantity is too small.
• No specific chemical test is available
• When the action of drug is due to a mixture of
substance
• Purification of drug is not possible
BIOASSAY
• When the estimation of crude drug or its preparation is done by means
of its effect on living organism like bacteria, fungi, or animal tissue or
entire animal it is known as BIOASSAY.

TYPES OF BIOASSAY
• QUANTAL:-It is all or none phenomenon
• GRADED:-Based on observation that there is a proportionate increase
in the observed response with increase in concentration or dose.

Graded bioassay can be performed by using any of the following

techniques
• Matching bioassay
• Interpolation Method
• Bracketing Method
• Multiple point bioassay
TOXICOLOGICAL ANALYSIS OF CRUDE DRUGS
• Determination of pesticides.
• Determination of arsenic and heavy metals
• Determination radioactive contamination
• Determination of aflatoxins.

PESTICIDES
• Fungicides
• Herbicides
• Insecticides
• Acarcicides
• Nematocides
• Rodenticides
• Bactericides
 ACCEPTABLE RESIDUE LEVEL(ARL)
ARL = ADI*E*60
MUI*100
• ADI=maximum acceptable daily intake of pesticides
(mg/kg of body weight)
• E= extraction factor, which determines the transition rate
of the pesticides from the plant material into the dosage
form
• MDI=Mean Daily Intake of medicinal palnt products
• 60 in numerator=adult body weight
• 100 in denominator=consumption factor
Determination of arsenic and heavy metals
• Arsenic and heavy metals are even in trace
amounts but they are dangerous removed from
herbal drugs.
• Amount is estimated by matching the depth of
colour with of standard stain
Determination of arsenic and heavy Limit test for cadmium
metals and lead
1. Coarsely ground material in kjeldahl
flask. Add water nitric acid and then • Material weighed
sulphuric acid. Material is destroyed. • Add digestion mixture
No further darkening with heating
• Heat
2. Clear solution with sulphur trioxide
vapors, cool and add ammonium • Dissolve in nitric acid
oxalate • Determination of the
3. Heat with sulphur trioxide vapors
metal concentration
4. Cool
5. Add potassium iodide and • Maximum amount
granulated zinc; keep for 40 minutes should not exceed
6. Compare the stains with standard than 10 mg/kg;
solution on mercuric bromide paper cadmium 0.3 mg/kg
Standard: Standard chloride + dilute
arsenic + water = Gives stain on
mercuric bromide paper
Radioactive contamination
• The exposure cannot be avoided because of
many naturally occurring sources including
radionucleotides occurring in ground and
atmosphere

Health risk depend on

• Specific radionucleotides
• Level of contamination
• Quantity of food
 Aflatoxins
• Aflatoxins are the poisonous substance in the
spores of the fungus Aspergillus flavus
• The toxin is known to produce cancer in human
beings living in warm and humid region of the
world
• Stored nuts and cereals are contaminated by
the fungus
• They should therefore be determined after
using a suitable clean up procedure.
 TEST FOR SPECIFIC MICROORGANISMS

• Tests are designed to minimize the accidental

contamination of micro organisms.

• Steam sterilization is conducted in an

autoclave and employs steam under
pressure. It destroys microorganism by
cellular protein coagulation.