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S.NO CONTENTS PG.NO

1. INTRODUCTION 3
1.1 General Introduction 3
1.2 Importance of medicinal plants 3
1.3 Benefits of herbal medicines 4
2. Preparation of Media 4
2.1 Bacterial count 5
2.2 Fungal count 5
3. Microbial test 6
3.1 Staphylococcus test 6
3.2 Ecoli test 7
3.3 Pseudomonas test 7
3.4 Serial Dilution 8
3.5 Pour Plate and Spread Plate Method 9
4. Instruments 12
4.1 B.O.D incubator 12
4.2 Auto clave 13
4.3 Hot air oven 15
4.4 Laminar air flow 16
4.5 Colony counter 18
4.6 Tablet compression machine 20
4.7 Hand operated capsule filling machine 22

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 Plants have been used for medicinal purposes long before prehistoric period. There medicinal
plants are also used as food, flavonoid, medicine or perfume, cosmetics and also in certain spiritual
activities. Some plants are considered as important source of nutrition and as a result of that they
are recommended for their therapeutic values. Some of these plants include ginger, green tea, also
pepper and turmeric etc. some plants and their derivatives are considered as important source for
active ingredients which are used in aspirin and toothpaste etc. medicinal plants such as aloe, tulsi,
neem, turmeric, and ginger cure several common ailments. These are considered as home remedies
in many parts of the country. Herbs that have medicinal quality provide rational means for the
treatment of many internal disease.

Herbal medicine is the fulcrum of complementary and alternative medicine, which in recent times
is increasingly gaining widespread popularity all over the world and gradually steaming toward
integration into the mainstream healthcare.

HM includes preparation of biologically active natural products that consist largely of herbs or
herbal materials such as minerals, ash, shells, insects, and animal part, and are used for the
maintenance of health and management of various diseases.

Medicinal plants are considered as a rich resource of ingredients which can be used in drug
development either pharmacopeial, non- pharmacopeial or synthetic drugs. A part from that, these
plants play a critical role in the development of human cultures around the whole world.

Importance of some herbs with their medicinal values:

❖ Herbs such as black pepper, cinnamon, myrrh, aloe, sandalwood, ginseng, red clover,
burdock, bayberry, and safflower are used to heal wounds, sores, and boils.
❖ Many herbs are used as blood purifiers to alter or change a long-standing condition by
eliminating the metabolic toxins. These are also known as 'blood cleansers'. Certain herbs
improve the immunity of the person, thereby reducing conditions such as fever.
❖ Some herbs are also having antibiotic properties. Turmeric is useful in inhibiting the growth
of germs, harmful microbes, and bacteria. Turmeric is widely used as a home remedy to heal
cut and wounds.
❖ Sandalwood and Cinnamon are great astringents apart from being aromatic. Sandalwood is
especially used in arresting the discharge of blood, mucus etc.
❖ Ginger and cloves are used in certain cough syrups. They are known for their expectorant
property, which promotes the thinning and ejection of mucus from the lungs, trachea, and
bronchi. Eucalyptus, Cardamom, Wild cherry, and cloves are also expectorants. Herbs such
as Chamomile, Calamus, Ajwain, Basil, Cardamom, Chrysanthemum, Coriander, Fennel,
Peppermint and Spearmint, Cinnamon, Ginger, and Turmeric are helpful in promoting good
blood circulation. Therefore, they are used as cardiac stimulants.

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Herbs can be…

• Adaptogenic – meaning they help the body adapt to stress by exerting a normalizing effect
upon bodily processes
• Nootropic – meaning they enhance memory or other cognitive functions
• Immunomodulatory – meaning they modify the body’s immune response
• Anti-microbial – meaning they fight bacteria or funguses
• Anti-viral
• Anti-inflammatory
• Antioxidants – meaning they stop the chemical reaction in the body that can produce free
radicals + damage cells

Some of the popular herbal medicines which many people were prefer by using the
Turmeric: Turmeric (Curcuma longa) is an herb that belongs to the ginger family. Used for
thousands of years in cooking and medicine alike, it has recently garnered attention for its potent
anti-inflammatory properties Curcumin is the major active compound in turmeric. It may treat a host
of conditions, including chronic inflammation, pain, metabolic syndrome, and anxiety.

Ginger: Ginger is a commonplace ingredient and herbal medicine. You can eat it fresh or
dried, though its main medicinal forms are as a tea or capsule. Much like turmeric, ginger is a
rhizome, or stem that grows underground. It contains a variety of beneficial compounds and has
long been used in traditional and folk practices to treat colds, nausea, migraines, and high blood
pressure.

Bacteria and fungi are grown on or in microbial media of various types. Commercial culture media are
provided in dehydrated or powdered form in liquid concentrate or as working solutions. Dehydrated
media for broth cultured need to be prepared by dissolving in distilled water and adjusting the final
pH prior to sterilization. Powdered media for agar cultures must be dissolved in distilled water, stirred,
then boiled or autoclave prior to pouring into sterile petri dishes using aseptic techniques.

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Aim:
To prepare the media for bacterial count, soyabean casein digest agar must be used. It is a general-
purpose media for isolation and cultivation of a wide variety of fastidious and non- fastidious
microorganisms.

MATERIALS REQUIRED:
1) Measuring Cylinder
2) Pipette
3) Distilled Water
4) Autoclave
5) Cotton
6) Incubator

COMPOSITION:
1) Pancreatic digest of casein: 15.0 g/L
2) Papaic digest of soya bean: 5.0 g/L
3) Sodium chloride: 5.0 g/L
4) Agar: 15.0 g/L

PREPARATION:
1) Add 40-gram powder to 1.0 litre distilled water is added into conical flask and mix it
thoroughly.
2) Cover it with cotton plug and aluminium foil.
3) Autoclave at 121 c for 15 mins.

Aim:
To prepare the media for fungus count, savoured dextrose agar will be used. It is a agar which is
used for isolation, cultivation, and maintenance of non-pathogenic and pathogenic species of fungi
and yeast. The pH is adjusted to approximately 5.6 in order to enhance the growth of fungi,
especially dermatophytes and to slightly inhibit bacterial growth in clinical specimens.

MATERIALS REQUIRED:
1) Distilled Water
2) Autoclave
3) Agar
4) Measuring Cylinder
5) Pipette
6) Cotton
7) Petri Plates
8) Incubator

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COMPOSITION:
1) dextrose (glucose) :40 gm
2) peptone :10 gm
3) Agar :15 gm
4) Distilled water :100 ml

PREPARATION:
1) Combine all ingredients in 900 ml of distilled water.
2) Adjust pH 5.6 with hydrochloric acid and adjust final volume to 1 litre.
3) Heat by boiling the media to dissolve completely.
4) Autoclave at 121 c for 15 mins.

Aim:
To find out the presence of staphylococcus on the given sample.

Materials required:
1) Test sample,
2) Nutrient broth,
3) Mannitol salt agar,
4) Distilled water.

Procedure:
• Add 1 gram of sample in nutrient broth and incubate for 24 hours.
• Take 111.02 gram of mannitol salt agar in 1000ml of H20.
• Heat to boiling to dissolve the medium completely.
• Autoclave at 121c for 15 minutes.
• Add 5.0% egg yolk emulsion and mixed it well.
• After the mannitol agar plate is prepared to use, a loop full of culture is streaked on sterile
mannitol salt agar medium and incubate it for 24 hours.

Observation:
No yellow colour appears.

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Aim:
To find out the presence of Ecoli on the given sample

Materials required:
1) Test sample,
2) Distilled water,
3) Nutrient broth,
4) MacConkey broth,
5) Peptone broth,
6) Kovax reagent.

Procedure:
• Take 1gram of sample to add in 50ml of nutrient broth.
• Incubate for 37c for 24 hours per day.
• Take 1ml of nutrient broth sample to add 5ml of MacConkey broth.
• Incubate at 37c for 24 hours
• Take 1ml of nutrient broth with MacConkey broth sample to add peptone broth.
• Incubate in 37c for 24 hours.
• Take 1ml of nutrient, MacConkey, and peptone broth sample to add 0.5ml of kovax reagent

Observation:
Pink layer not present.

Aim:
To find out the presence of pseudomonas on the given sample.

Materials required:
1) Test sample,
2) nutrient broth,
3) distilled water,
4) glycerol,
5) Petri plates,
6) pseudomonas agar.
Procedure:
• Add 1ml of sample in nutrient broth and incubate for 24 hours.
• Take 38 gram of pseudomonas agar in 1000ml of H2O and add 10ml of glycerol.
• Heat to boiling to dissolve the medium completely. 6
• Autoclave at 121c for 15 minutes. • Cool it for 45-50c and Pour the medium into Petri plates.
• After the pseudomonas agar plate is prepared to use, a loop full of culture is streaked on the
plate and incubate for 24 hours.

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Observation:
No green colonies present

Serial dilution, as the name suggests, is a series of sequential dilutions that are performed to
convert a dense solution into a more usable concentration. It is a process of stepwise dilution of a
solution with an associated dilution factor. It is often associated with reducing the concentration of
cells in a culture to simplify the operation.

Calculation:
The dilution factor in a serial dilution can be determined either for an individual test tube or can
be calculated as a total dilution factor in the entire series. Then a small measured volume of each
dilution is used to make a series of pour or spread plates.

The dilution factor of each tube in a set:

𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆
𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 + 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒅𝒊𝒍𝒖𝒆𝒏𝒕

For a ten – fold dilution, 1ml of sample is added to 9ml of diluent. In this case, the dilution factor for
that test tube will be:

Dilution factor: 1ml \ 1ml + 9ml = 1\10 = 10-1

After the first tube, each tube is the dilution of the previous dilution tube.

Now, for the total dilution factor

𝐓𝐨𝐭𝐚𝐥 𝐝𝐢𝐥𝐮𝐭𝐢𝐨𝐧 𝐟𝐚𝐜𝐭𝐨𝐫 𝐟𝐨𝐫 𝐭𝐡𝐞 𝐬𝐞𝐜𝐨𝐧𝐝 𝐭𝐮𝐛𝐞

= 𝐃𝐢𝐥𝐮𝐭𝐢𝐨𝐧 𝐟𝐚𝐜𝐭𝐨𝐞 𝐨𝐟 𝐭𝐡𝐞 𝐟𝐢𝐫𝐬𝐭 𝐭𝐮𝐛𝐞 × 𝐃𝐢𝐥𝐮𝐭𝐢𝐨𝐧 𝐟𝐚𝐜𝐭𝐨𝐫 𝐨𝐟 𝐭𝐡𝐞 𝐬𝐞𝐜𝐨𝐧𝐝 𝐭𝐮𝐛𝐞

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Procedure:
• The sample is taken in a test tube and six tubes, each with 9ml of sterile diluents, which can
either be distilled water or 0.9% saline are taken
• A sterile pipette is taken.
• 1ml of properly mixed sample is drawn into the pipette,
• The sample is then added to the first tube to make the total volume of 10ml. this provides
an initial dilution of 10.
• The dilution is thoroughly mixed by emptying and filled the pipette several times.
• The pipette tip is discarded and a new pipette tip is attached to the pipette.
• Now, 1ml of mixture is taken from the 10 dilution and is emptied into the second tube. The
second tube now has a total dilution factor 10^2
• The same process is then repeated for the remaining tube, taking 1ml from the previous
tube and adding it to the next 9ml diluents.
• As six tubes are used, the final dilution for the bacteria/cells will be 10^6 ( 1 in 1,000,000)

Observation:
White dots of colonies will be present.

Uses:
• It is used in microbiology to estimate the concentration or number of cells/organisms in a
sample to obtain an incubated plate with an easily countable number of colonies.

• In homeopathy, homeopathic dilutions are used where a substance is diluted in distilled water
or alcohol. It believed that dilution increase the potency of the diluted substance by activating its
vital energy.

Pour Plate Method:

Pour plate method is usually the method of choice for counting the number of colony-forming
bacteria present in a liquid specimen. Because the sample is mixed with the molten agar medium, a
larger volume can be used than the spread plate.

The number of microorganisms present in the test sample is determined using the formula:

CFU/Ml = CFU*dilution factor * 1/aliquot

Materials required:
1) Test sample
2) Plate count agar (PCA) or nutrient agar
3) Hot water bath 45c
4) Sterile Petri plates
5) Flame
6) Colony counter with a magnifying glass
7) Sterile capped 16*150 mm test tubes
8) Pipettes of various sizes (e.g., 01,1.0 and 2,0 ml)

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 Procedure:

• Prepare the dilution of the test sample expected to contain between 30-300 CFU/mL (follow
the serial dilution technique)
• Inoculate labelled empty petri dish with specified mL (0.1 or 1.0 mL) of diluted specimen

Pour the molten agar and incubation:

1) Collect one bottle of sterile molten agar (containing 15ml of melted plate count agar) from the
water bath
2) Hold the bottle in the right hand; remove the cap with the little finger of the left hand.
3) Flame the neck of the bottle.
4) Lift the lid of the petri dish slightly with the left hand and pour the sterile molten agar into the
petri dish and replace the lid.
5) Flame the neck of the bottle and replace the cap.
6) Gently swirl the plate on the benchtop to thoroughly mix the culture and medium. Ensure that
the medium covers the plate evenly and do not slip the agar over the edge of the petri dish
7) Allow the agar to gel completely without disturbing it. It will take approximately 10 minutes.
8) Seal and incubate the plate in an inverted position at 37c for 24-48 hours.

Results:
After 24-48 hours, count all the colonies (note that the embedded colonies will be much smaller
than those on the surface) A magnifying colony counter can aid in counting small embedded
colonies.

Calculate CFU/mL using the formula = (number of colonies X dilution factor / volume of culture
plated*)

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Spread Plate Technique:
Spread plate technique is the method of isolation and enumeration of microorganisms in a
mixed culture and distributing it evenly. The technique makes it easier to quantify bacteria in a
solution. In this method, the substance to be tested if not in liquid form is ground and dissolved in a
suitable liquid medium. The sample is then diluted in 10- fold serial dilutions and plated in an
appropriate medium.

Materials required:
1) Glass wares
2) Screw-capped test tubes
3) Sterile pipettes
4) Sterile bent glass roads (bent in the shape of a hockey stick) or commercially available
sterile spreaders
5) Medium: plate count agar or nutrient agar. The surface of the plate must not be too
moist because the added liquid must soak in, so the cells remain stationary.
6) Alcohol

Procedure:
• a dilution series from a sample
• Pipette out 0.1ml from the appropriate desired dilution series onto the centre of the surface
of an agar plate.
• Dip the L- shaped glass spreader into alcohol.
• Flame the glass spreader (hockey stick) over a Bunsen burner.
• Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully
rotating the Petri dish underneath at the same time.
• Incubate the plate at 37c for 24 hours.
• Calculate the CFU value of the sample. Once you count the colonies, multiply by the
appropriate dilution factor to determine the number of CFU/mL in the original sample.

Result:
After incubation, isolated countable colonies are present in the evenly spread across the surface
of the agar. Count the number of colonies and multiply it by the appropriate dilution factor to
determine the colony-forming units (CFU) present per ml in the original sample.

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