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A Laboratory Manual for

Pharmacognosy

First Year Diploma in Pharmacy (PH)

Maharashtra State Board of Technical Education, Mumbai

CURRICULUM DEVELOPMENT CELL, MSBTE, MUMBAI.

LABORATORY MANUAL DEVELOPMENT PROJECT

Particulars

Team for design

EducationTechnology

Mr. D. M. Makone

Consultant

Project Institution

Padmashree Dr. D. Y. Patil Institute of Pharmacy, Akurdi, Pune - 411 044.

Project Period

November2005toMay 2006

Chief ProjectCoordinator

Prof. K. R. Deshmukh

Principal,D.Y.P.I.O.P.Akurdi, Pune - 411 044.

Project Coordinator

Mr. Yogesh. S. Bafana D.Y.P.I.O.P. Akurdi, Pune - 411 044.

Subject Experts

1.

Mr. Pravin V. Buge D.Y.P.I.O.P., Akurdi, Pune.

2.

Mr. Ravi B. Chintamani D.Y.P.I.O.P., Akurdi, Pune.

3.

Ms. R. S. Bende Shree FattechandJain College Of Pharmacy, Chinchwad, Pune.

O 2006, MaharashtraState Board of Technical Education,

49, Kherwadi,Aliyawar Jung Road, Bandra (East), Mumbai - 400 051 MaharashtraState, India. No part of this Laboratory Manual be reproduced, in any form or by any means, without permission in writing from MSBTE Mumbai.

MAHARASHTRA STATE BOARD OF TECHNICAL EDUCATION

Certificate

This is to certify that, Mr./Ms./Mrs. Roll No. of First Year Diploma in pharmacy of of First Year Diploma in Pharmacy has completed the term work satisfactorily in Pharmacognosy (0807) for the

academic year 200 -

to

200

as prescribed in the curriculum.

Place :

Enrolment No.:

Date :

Exam. Seat No.:

(

Subject Teacher

Seal of

Institution

(

Principal

Pharrnacognosy

LEARNING OVERVIEW

IMPORTANCE OF THE SUBJECT

Man knows disease since origin of human being, but causes of them were not known. They were assumed evil spirits. So people tried to cure them with plants, which are easily available. In that way ancestors accumulated knowledge of plants and preserved them in literature such as,

1.

Rigveda

2.

Ayurveda (ancient science of life)

3.

Materia rnedica

4.

Ebers papyrus

In all the old texts, preference has been given to description of plant as compared to other characteristics.

The word 'Pharmacognosy' was coined in 1815 by C. A. Seydler. Pharmacognosy initially known as materia medica may be defined as scientific study of those substances ( plant and animal origin )which are used or have been used in medicineand pharmacy. Pharmacognosycan be consideredas a valuable part of the cultural heritage of pharmacy. The name Pharmacognosy is formed from two Greek word 'Pharmakon' meaning drug and 'gnosis' meaning knowledge. The modern aspect of Pharmacognosy includes not only the crude drug but also covers their chemical scrutiny, leading to isolation of active principles.

This subject deals with biological, biochemical, therapeutic and economic features of natural drugs and their chemical constituents. Even we are movirlg with great pace in the 21st century, Herbal medicine, cosmetics, Ayurvedic dosage form and research in the field of herbal formulation are of great interest.Therefore it is need of time to explore into area of systematic knowledge about herbal drugs.

In recent year, popularity of natural drug is considered as an important contribution of Pharmacognosy in modern medicine.

Pharmacognosy

LINK 1 BLOCK DIAGRAM SHOWING INTER RELATIONSHIP OF SUBJECT AREAS, CURRICULUM 0B.IECTIVE AND JOB PROFILE.

1 CORE TECHNOLOGY

1)

2)

3)

HumanAnatomy and Physiology.

Bio-Chemistry and Clinical Pathology.

Health Education and Community Pharmacy.

4)

Pharmacognosy.

1

5)

Pharmaceutics - I

14) Pharmaceutical Chemistry - I

15)

PharmaceuticalChemistry - Il

JOB PROFILE

1)

Entrepreneur (Wholesaler, Distributor, Chemist and Druggist.

2)

Industry Skilled personnel (Bulk drug formulations, Cosmetics) Medical Representative.

3)

Hospital Pharmacist (Dispensing, Manufacturing) Supervisor

4)

Community Pharmacist.

5)

Academic Institution. (Laboratory Technician.)

6)

Repackqging of Drugs other than those specified in Schedule C and

C1 of

Drugs and Cosmetics Act

1940 and Rules 1945.

I

I

TECHNOLOGY SUBJECTS

I 1)

Pharmacologyand Toxicology.

1

2)

Hospital and Clinical Pharmacy.

/ 3)

Pharmaceutics - II

 

4)

Drug Store and Business Management

5)

Pharmaceutical Jurisprudence

6)

PracticalTraining.

 

CURRICULUM 0B.IECTIVES

 

1)

Develop attitude for personal development.

2)

Develop social skills for social development.

3)

Develop continued learning skills for life long learning.

4)

Gain basic knowledge of human body and various illness 1 disorders.

5)

Understand various drugs, their

Develop communication skills.

16)

formulations and counselingto patients for their appropriate use.

7)

Develop technical skills for industry and hospitalactivities.

I

Pharmacognosy

:

.

GRAPHICAL STRUCTURE OF SUBJECT AREA FIRST YEAR D. PHARMACY PHARMACOGNOSY (0807)

APPLICATIONS

PROCEDURE

PRINCIPLES

CONCEPT

FACTS

Understandingorganizedand unorganizeddrugs, their preparation and tests. Identifying the drugs by morphologicaland microscopic character, evaluation and extraction, etc.

Handlirlg Microscope, Preparationof sample for section cutting, Microchemical test and chemical test, etc.

Principleof magnification, Staining preparation, etc.

Staining view, signification and Clarity of field view.

Simple and Compound microscope, Dissection box, and DifferentMountings.

Pharmacognosy

DEVELOPMENT OF SKILLS

The objective of curriculum is to develop the desired skills in the students so that they can solve the field

problems.After undergoingthrough the laboratory experiments, it is expected that the following skills shall be developed in the students.

intellectual Skills : -

1. Logical thinking. ( I-,)

2. Identification of different stages I crude drug with the help of morphology. ( I-, )

3. Interpretation from transverse section I chemical and Microchemical test.( I-, )

4. Develop the creativity in section cutting and staining. ( I-, )

Motor Skills : -

1. To handle and observe instrument and crude drug correctly. ( M-, )

2. To follow step by step sequence of chemical test I operation. ( M-, )

3. Ability to

4. Labeling different component of T.S. ( M-, )

prepare thin transverse section. ( M-, )

GRID TABLE

Following table gives grid of the experiments and related intellectual and motor skills. Following table gives grid of the experiments and related intellectual and motor skills. Teacher shall ensure for development of generic skills during the practical. Students are expected to focus on acquiring specific skills mentioned therein.

No.

Experiment No. & Title

Intellectual skills

 

Motor skills

 

11

12

13

14

MI

.

M2

M3

M4

A.

TO KNOW YOUR PHARMACOGNOSY LABORATORY

1.

To study the compound microscope.

d

2.

To understand technique of Section Cutting, Staining and Mounting.

d

d

3.

To study the Microchemical reagent.

d

d

B.

GROSS ANATOMICAL STUDY OF CRUDE DRUGS.

4.

To study the Morphological and Microscopical characteristics of Cinchona Bark.

d

d

d

d

5.

To study the Morphological and Microscopical characteristics of Cinnamon Bark.

d

d

d

d

6.

To study the Morphological and Microscopical characteristics of Clove Buds.

d

d

d

d

7.

To study the lblorphological and Microscopical characteristics of Coriander Fruit

d

d

d

d

8.

To study the Morphological and Microscopical characteristics of Datura Leaf.

d

d

d

d

9.

To study the Morphological and Microscopical characteristics of Fennel Fruit.

d

d

d

d

10.

To study the Morphological and Microscopical characteristics of Ginger Rhizome.

d

d

d

d

11.

To study the Morphological and Microscopical characteristics of lpecacuanha Root.

d

d

d

d

iv

+

MAHARASHTRA STATE BOARD OF TECHNICAL

EDUCATION

Pharrnacognosy

No.

-

Experiment No. & Title

Intellectual skills

P

13

12

13

14

Mq

M2

M3

M4

12.

To study the Morphological and Microscopical characteristics of Nux-Vomica Seed.

d

d

d

d

13.

To study the Morphological and Microscopical characteristics of Senna Leaf.

d

d

---------

 

C.

MORPHOLOGICAL STUDY OF CRUDE DRUGS.

14.

To study Morphological characters of carminatives (Ajowan, Black pepper, cardamom and Nutmeg.)

and laxatives (Isapghula and Rhubarb ).

d

d

 

----

15.

To study Morphological characters of Drugs acting on Central nervous System (Aconite, Ashwagandha, Ephedra) and Antitussive (Tulsi and Vasaka).

d

d

16.

To study the Morphological characters of Anti tumor (Vinca),Antihypertensive (Rauwolfia) and Diuretic (Gokhru)

d

d

17.

To study the Morphological characters of Antiseptic (Curcuma and Neem ) , Vitamin (Amla) and

 

Antirheumatics (Colchicum).

4

d

-

-

-

-

-

18.

To study the Morphological characters of perfumes and Flavouring agents (Sandal wood) and F~bres(Cotton, Silk and Wool).

d

d

19.

To study the Morphological characters of Antidiabetics drugs (Gymnema) and unorganized crude drugs (Asafoetida and Kaolin)

d

d

20.

To study the Morphological characters of Miscellaneous drugs (Garlic, Liquorice, Shankpushpi and Shatawari)

d

d

D.

IDENTIFICATIONTEST FOR CRUDE DRUGS.

 

21.

To identify unknown unorganized powder drug with the help of physical and chemical tests.

a. Carbohydrates :Acacia, Agar, Algin, Honey, Tragacanth.

d

d

b. Protein : Gelatin.

 

22.

To identify unknown unorganized powder drug with the help of physical and chemical tests.

a. Tannins : Pale and Black Catechu.

d

d

b. Mineral : Kaolin.

 

23.

To identify unknown unorganized powder drug with the help of physical and chemical tests.

a. Lipids : Bees wax.

d

d

b. Resin : Benzoin myrrh.

 

24.

To identify unknown organized powder drug with the help of Physical and chemical tests.

a. Senna

d

b. Starch

c. Termeric

No.

Experiment No. & Title

J

Intellectual skills

11

12

13

14

Pharmacognosy

Motor skills

MI

M2

M3

M4

25 To identify unknown fibres with the help of Physical and chemical tests.

a.

b. Silk

C. wool

Cotton

NOTE : d- Identified Skills

d

d

STRATEGY FOR IMPLEMENTATION

It is expected that 45 to 50% experiment shall be completed in first term and remaining in te second term.

Pharrnacognosy

GUIDELINES FOR TEACHERS

Teachers shall discuss the following points with students before start of practical of the subject.

1.

Learning Overview : To develop better understanding of importance of the subject. To know related skills to be developed such as intellectual skills and motor skills.

2.

Link Diagram: context of the subject in the form of link diagram showing interrelationshipof various subject areas, curriculum, objectives and job profiles.

3.

Graphical structure: in this topics and sub topics are organized in systematic way so that ultimate purpose of learning the subject is achieved. This is changed in the form of fact, concept, principle, procedure, application and problem.

4.

Knowyour laboratory:to understandthe layout of laboratory,specificationof equipment, procedure, working in groups, planningtime etc. also to know total amount of work to be done in the laboratory.

5.

Teacher shall ensure that required equipment are in working condition before start of experiment, also keep operating instruction manual available.

6.

Explain prior concepts to the students before starting of each experiment.

7.

Involve the student's activity at the time of conduct of each experiment.

8.

While taking observation each student (from batch of 20 students) shall be given a chance to perform the experiment.

9.

List of questions given at the end of each experiment. Teacher shall instructthe studentsto attempt all questions given at the end of each experiment. Teacher shall ensure that each student writes the answers to the allotted questions in the laboratory manual after performance is over.

10.

If the experiment setup has variations in the specifications of the equipment, the teachers are advised to make necessary changes, wherever needed.

11.

Teacher shall assess the performanceof students continuously as per norms prescribed by MSBTE.

12.

Teacher is expected to share the skills and competencies to be developed in the students.

13.

Teacher may provide additional knowledge and skills to the students even though not covered in the manual but are expected from the students by the industries.

14.

Teachers shall ensure that industrial visits recommended in the manual are covered.

15.

Teacher may suggest the students to refer additional related literature of the technical papers, reference books, seminar proceedirlgs,etc.

16.

During assessment teacher is expected to ask questions to the studentsto tap their achievements regarding related knowledgeand skills so that students can preparewhile submitting record of the practical. Focus should be given on development of enlisted skills rather than theoretical knowledge.

17.

Teacher should enlist the skills to be developed in the students that are expected by the industry.

18.

Teacher should organized group discussion, brain storming sessions, seminars to facilitate the exchange of knowledge amongst the students.

19.

Teacher should ensure that revised CIAAN-2005 norms are followed simultaneously and progressively.

20.

Teacher should give more focus on hands on skills and should actually share the same.

21.

Teacher shall also referto the circular No. MSBTEID-50lPharm. Lab. Manual/200613160 dated4th May 2006 for additional guidelines.

Pharmacognosy

INSTRUC'TIONS FOR STUDENTS

Students shall read the points given below for understanding theoretical concepts and practical applications.

Students shouldwear whiteApron, Cap, Mask, Gloves and Slipper beforeentering into the laboratory.

Students should keep their belongings in locker which are not required during practical like, Bag, Extra files etc.

Studentsshould always carry LaboratoryManual, rough notebook and practical requirementswithout fail.

Listen carefully to the lecture given by teacher about importance of subpct, curriculum philosophy, graphical structure, skills to be developed, information about equipment, instruments, procedure, methodof continuous assessment, tentative plan of working laboratoryand total amount of work to be done in a year.

Students should perform the practical only at the place which allocated to him 1her. (No change can be done without permission of subject teacher)

Students shall undergo study visit of laboratoryfor types of equipment, instruments, material to be used, before performing experiment.

Read write up of each experiment to be performed, a day in advance.

Organize the work in the group and make a record of

Understandthe purpose of experiment and its practical applications.

Write the answer of the questions allotted by teacher during practical hours if possibleor afterwards, but immediately.

Students should not hesitate to ask any difficulty faced during conduct of practical.

The students shall study all the questions given in the laboratory manual and practice to write the answers to these questions.

Students shall develop maintenance skill as expected by the industries.

Students should develop the habits of pocket discussion, group discussion relatedto the experiments so that exchanges of knowledge, skills could take place.

Students shall attempt to develop related hands-on -skills and gain confidence.

Students shall focus on development of skills rather than theoretical or codified knowledge.

Students shall visit nearby workshops, workstation, industries, technical exhibitions, trade fair etc. even not included in the lab manual. In short, students should have exposure to the area of work right in the student's hood.

Students shall insist for the completion of recommended laboratory work, industrial visits, answers to the given questions, etc.

Students shall develop habits of evolving more ideas, innovations skills etc. than included in the scope of the manual.

Students shall develop technical magazines, proceedingsof the seminars, refers websites related to the scope of the subjects and update their knowledge and skills.

Students should develop the habit of not to depend totally on the teachers but to develop self- learning techniques.

Students should develop the habit to react with the teacher without hesitation with respect to the academics involved.

Students should develop the habit to submit the practical exercise continuously and progressively on the scheduled dates and should get the assessment done.

Students should be well prepared while submitting the write up of the experiments.This will develop the continuity of the studies and he will not be over loaded at the end of the term.

Students should clean the platform before leaving the laboratory.

all observations.

Pharmacognosy

List of Experiments and Record of Progressive Assessment

Sr.

Page

Date of

Date of

Assess-

Teacher's

No.

Name of the Experiments

No.

Perfor-

submi -

ment

Signa-

 

mance

ssion

Max.

ture

 

Marks 10

A.

TO KNOW YOUR PHARMACOGNOSY LABORATORY

 

1.

To study the compound microscope.

01

2.

To

understand technique of Section

06

Cutting, Staining and Mounting.

3.

To study the Microchemical reagent.

14

B.

GROSS ANATOMICAL STUDY OF CRUDE DRUGS.

4.

To study the Morphological and lVlicroscopical characteristics of Cinchona Bark.

18

5.

To study the Morphological and Microscopical characteristics of Cinnamon Bark.

24

6.

To study the Morphological and Microscopical characteristics of Clove Buds.

30

7.

To study the Morphological and Microscopical characteristics of Coriander Fruit

36

8.

To study the Morphological and Microscopical characteristics of Datura Leaf.

42

9.

To study the Morphological and Microscopical characteristics of Fennel Fruit.

48

10.

To study the Morphological and Microscopicalcharacteristics of Ginger Rhizome.

54

11.

To study the Morphological and Microscopical characteristics of

-

12. To study the Morphological and Microscopical characteristics of Nux-Vomica Seed.

67

13. To study the Morphological and Microscopical characteristics of Senna Leaf.

73

Pharrnacognosy

Sr.

Page

Date of

Date of '

Assess-

Teacher's

No.

Name of the Experiments

No.

Perfor-

submi -

ment

Signa-

 

mance

ssion

Max.

ture

 

Marks 10

C.

MORPHOLOGICAL STUDY OF CRUDE DRUGS.

14. To study Morphological characters of carminatives (Ajowan, Black pepper, cardamom and Nutmeg.) and laxatives (Isapghula and Rhubarb ).

79

15. To study Morphological characters of Drugs acting on Central nervous System (Aconite,Ashwagandha, Ephedra) and Antitussive (Tulsi and Vasaka).

86

16. To study the Morpholog~calcharacters of Anti tumor (Vinca), Antihypertensive (Rauwolfia) and Diuretic (Gokhru)

93

17. To study the Morphological characters of Antiseptic (Curcuma and Neem), Vitamin (Amla) and Antirheumatics (Colchicum).

98

18. To study the Morphologicalcharacters of perfumes and Flavouring agents (Sandal wood) and Fibres (Cotton, Silk and Wool).

104

19. To study the

Morphological characters

110

of Antidiabetics drugs (Gymnema) and unorganized crude drugs (Asafoetida and Kaolin)

20. To study the Morphological characters of Miscellaneousdrugs (Garlic, Liquorice, Shankpushpiand Shatawari)

115

D.

IDEN'rIFICATIONTEST FOR CRUDE DRUGS.

21. To identify unknown unorganized powder drug with the help of physical and chemical tests

121

a. Carbohydrates : Acacia, Agar, Algin, Honey, Tragacanth.

b. Protein : Gelatin.

22. To identify unknown unorganized powder drug with the help of physical and chemical tests.

128

a. Tannins : Pale and Black Catechu.

b. Glycosides : Aloe.

c. Mineral : Kaolin.

Pharmacognosy

Sr.

Page

Date of

Date of

No.

Name of the Experiments

No.

Perfor-

submi -

 

mance

ssion

23. To identify unknown unorganized powder drug with the help of physical and chemical tests.

133

a. Lipids : Bees wax.

b. Resin : Benzoin myrrh.

24. To identify unknown organized powder drug with the help of Physical and chemical tests.

137

a. Senna

b. Starch

c. Termeric.

25. To identify unknown fibres with the help of Physical and chemical tests.

142

a. Cotton

b. S~lk

c. Wool.

Appendices Appendix-l

147

(Reagentsand solutions) Appendix-Il

149

(Botanical terms) Appendix-Ill

152

(Therapeuticalterms) Appendix-IV (Guidelines for Annual Practical Examination)

154

Sr.

No.

Appendix4

(Colour diagrams)

Examination

Experiment

No.

(From-To)

Total

WIarks

[No- of

Exp.

Assess-

ment

Max.

Marks 10

Average

Marks.

(Total marks

INo. of

X Exp.1

Max.

Marks

(lo)]

1. First sessional Practical Examination

2. Second sessional Practical Examination

3. Third sessional Practical Examination

Teacher's

Signa-

ture

Signature

of Subject

teacher

* To be transferred to Proforma of CIAAN - 2004 (Proforma I - 1)

Note :-

The guidelines for the conduct of Annual Practical Examination are enclosed at the end of page

no.177.

Pharmacognosy

Experiment No. 1

Experiment No. 1

.

.

1.0

TITLE :

 

To know the compound Microscope, the components and making it ready for working.

2.0

PRIOR CONCEPTS:

Simple microscope.

3.0

NEW CONCEPTS:

Proposition 1 : Illumination system

For distinctive and proper viewing of field object, it provides light, which may be either plane or concave mirror or electrically illuminated by tungsten filament.

Proposition 2

: Magnification system

Magnified real image can be viewed by objective and eyepiece.

4.0

LEARNING OBJECTIVES :

Intellectual Skill :

1.

Acquire skills of operating compound microscope.

2.

Develop skills to get magnified real image.

Motor Skill :

1.

To set the microscope for observations and operation.

5.0

REQUIREMENTS :

Apparatus :

Simple Microscope, Compound Microscope, Permanent slide etc.

Fig. 1.1Simple Microscope

Lens

Folder Arm

Stage

Adjust ment SCIrew

Mirror

Experiment No. i

Pharmacognosy

Objectiveturret

illumination System

.1-.,--Fine Focus

Fig. 1.2 Compound Microscope

6.0 STEPWISE PROCEDURE :

6.1 Microscope :

It is an optical instrument,comprising of a kns or a combination of lenses, which enables to view magnified images of a minute object.

The Englishman Robert Hooke developed an instrument that was the true compound microscope used today.

The compound microscope essentially consists of three major systems. Support system : It comprises of base, stage and body tube. Illumination system : It comprises of lightsource or mirror, iris diaphragm and condenser.

near the object,

forerunner of the

Magnificationsystem : This includesobjective (is a set of lenses placeda

it partially magnified the object) and eyepiece (a more magnified form of real image is observed)

6.2 Facts and figures about microscope

.

.

1. Magnifying power (M.P.) M.P. = magnificationof objective x magnification of eyepiece. e.g. M.P. = 15x X lox = 150x thus, the object viewed is magnified 150 times.

2. Resolving power of objective (R.P.) Resolving power of an objective is defined as the ability to separate distinctly two small elements of an object, which are, situated a short distance apart. R. P. can be measured by Numerical Aperture (N.A) of an objective. Greater the N.A. greater is the resolving power.

3.

Working distance :

The distance between the object and the objective is known as working distance. It decreaseswith increasingmagnification. This means higher the power of the objective, lesser is the working distance.

4.

Focusing :

Focusing an object while viewing through eyepiece means adjustment of working distance. This is done with the help of coarse and fine adjustment of knob.

*

Pharmacognosy

Experiment No. 1

5. Field of view :

The area of the object, which one can view through the eyepiece, is the field of view. The field of views narrows as magnification increases.

7.0

8.0

9.0

OBSERVATION :

(Subject teacher on the basis of permanent slide shall give one exercise to the students. In exercise he shall ask studentto focus the slide and observethe particulartissue component. Subjectteacher shall give idea about tissue components present in the permanent slide for confirmation of the learning. )

(Space for observations)

CONCLUSION :

From the observation it is concluded that the component of given permanent slide is found to be

QUESTIONS :

Group

C (Questions shall be allotted by the subject teacher. Subject teacher shall also add few

more relevant questions) GROUP :A

Write answers to Q

Q

from

Group A, Q

Q

from

Group B and Q

Q

from

1.

State the importance of graphical structure in understandingthe scope of the subject.

2.

List the parametersof the graphical structure in the hierarchy. (Refer the graphical structure)

3.

State two motor skills to be developed through this subject.

4.

Classify the curriculum in different groups of subject.

5.

State the importance of job description in designing the curriculum.

GROUP : B

1.

What is the contribution of Robert Hooke in Microscope?

2.

Mention mechanical and optical units of compound microscope.

3.

State the meaning of 'Field of View' in Microscope.

4.

State the meanirlg of

a. Magnifying power (M.P.)

b. Revolving power of objective (R.P.)

c. Working distance.

5. Why CEDAR WOOD OIL is used in case of oil immersion lenses? Give reason.

GROUP : C

1.

Which precautions should be taken while handling the microscope?

2.

State the condition where simple microscope is used in Pharmacognosy practical.

3.

When concave mirror is used in compound microscope?

4.

Name four types of microscope.

5.

What is the role of inclination joint in compound microscope?

Experiment No 1

Pharmacognosy

(Space for answers)

.

.

Pharmacognosy

Experiment No. 1

(Space for answers)

.

.

Experiment No. 2

Pharmacognosy

Experiment No. 2

1.0

TITLE :

To understand the method of Section Cutting Technique, Staining, Mounting and Observation of section under Microscope.

2.0

PRIOR CONCEPT

Preparation of sample for sectioning.

3.0

NEW CONCEPT

Proposition 1 : Transverse section

Transverse section is obtained by cutting along the -radialplane of a cylindrical portion of the stem, root, stolen and perpendicularto long axis.

Proposition 2 : Staining

To distinguish the arrangement of various tissues in the samples of crude drug, chemicals, dyes or colorants are used to impart colour to various tissue in section of drug sample.

Proposition 3 : Observation

For observation of section, selection of place in a laboratory where sufficient light is available is important. The low power observation helpsto draw a schematicdiagram. For distinctivetransverse section high power observation is used.

4.0

LEARNING OBJECTIVES :

Intellectual Skill :

1. Develop creativity in section cutting using different attributes and materials.

2. To discriminate different components of the section.

3. Identificationof different stages.

Motor Skill :

1. To observe the section under microscope.

5.0 REQUIREMENTS :

Apparatus :

Microscope,watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc

Chemicals :

Staining reagent, glycerin and water.

Crude Drug :

Subject teacher shall give any single drug to the students among the root, stem, seed, leaf, bark, fruit, rhizome, etc to see the understandingof the methods of section cutting, staining, mounting and its observation.

6.0 STEPWISE PROCEDURE :

6.1 Section of a stem, root, stolon :

Differentsection can be obtainedfrom a stem, root stolon, depending on the plane of cutting, each section revealing details from a different angle.

Pharmacognosy

Experiment No. 2

6.2 Transverse section (T.S.)

Transverse section is obtained by cutting along the radial plane of cylindrical portion of the stem, root, stolon and perpendicular to long axis.

Fig. 2.1

This section when prepared and observed under the microscope reveals the radial arrangement of tissues and shows concentric layers and vascular bundles.

Fig. 2.2

6.3 Section of leaf In case of leaf, the important aspect to study is a section through the midrib taken perpendicular to the midrib and Observation of a surface preparation.

Fig. 2.3

6.4 Section of bark In case of bark, transverse section is important as it reveals the horizontal section of cells and shows lenticels.

Experiment No. 2

Fharmacognosy

Fig. 2.4

6.5 Section of fruit and seed

in case of fruit and seeds, generallyT.S. of various parts are observed under the microscope. In case of fruit and seed drug, separate section technique is required for individualdrug.

Fig. 2.4

6.6 Section cutting technique

Following materials are required for Pharmacognosy laboratory work.

1. Napkin

7.

Test tube holder and stand

2. Adropper

8.

Needle

3. Filter paper

9.

Camel hair brushes

4. Stains

10.

Watch glass

5. Drug sample

11.

A sharp razor blades

6.

Forceps

12.

Micro-slide

 

13.

Cover slip.

Section cutting can be done by taking transverse as well longitudinalsection. Mostly transverse section is taken for study of crude drugs. Section cutting is a skill, which fac~litateeasy learningof the tissuecomponents. There are various techniques of section cutting depending on the part of crude drug used. Eg. Green unripe papaya or potato is usedfor easy sectioning of the leaves.

Section cutting includes the following steps :

Preparation of Sample for Sectioning

1. Boiling of the sample

Fig. 2.5

Pharmacognosy

Experiment No. 2

2. Section cuttiqg.

Fig. 2.6

3. Transfer the section in to watch glass containing water.

Fig. 2.7

Staning :

A stain is a chemical dye (colorant), which combines chemically or physically with a cell content to impart colour to it. e.g. Sudan red Ill dissolves in the fixed oil present in the oil seed to impart red color.

Staining Process

1.

Take a clean watch glass and add the staining solution to it.

 
 

Fig. 2.8

Fig. 2.9

3.

Transfer it to watch glass containing plane water, so that excess stain is washed away. This section is ready for mounting.

 

Fig. 2.10

Mounting Process

 

1.

Transfer the section to

be mounted on

the glass slide with the help of brush

 
 

Fig. 2.11

Experiment No. 2

Pharmacognosy

2. Add 1 - 2 drops of water on the section with the help of Dropper.

 

Fig. 2.12

3. Place a clean cover slip over the section with the help of a forceps and needle.

 

Fig. 2 13

4. With the help of blotting paper, wipe out excess of water present outside the cover slip. The slide is ready for observation

Fig. 2.14

Procedure described above is the routine laboratorytechnique, so there may be evaporation of water and slide preparedwill not last long. Glycerin is used to avoid evaporation of water and drying of section.

In order to prepare a permanent mount, a special process is adopted named as Double Staining Technique. A permanent preparation is useful for preservation of good sections for study and for preparationof standards,with which the sample can be compared.This process generally involves staining with two reagents, hence called as Double Staining Technique.

One of the stain Imparts colour to the lignified tissue and the other to the cellulose part.

Observation :

I

u i-4~~.

I

1. Select a place in the laboratory for microscope, where sufficient light isavailable. Set the microscope in a such a way that the C-Arm towards to you and the objective and mirror facing the light.

 

Fig. 2.15

2. Open the diaphragm completely withthe help of the substage mirror, Adjustthe position so that the field of view issufficiently illuminated.

 

Fig. 2.16

3. Place the slide prepared on the stage of the microscope at the centre, with the section placed exactly in line with the stage window lying above the condenser. Fix the slide between the clips. Now the slide Can be moved forward, backward or sideways above the stage with the helpof two screws provided on the mechanical stage. Take observations.

 

Fig. 2.17

Pharrnacognosy

Experiment No. 2

7.0

8.0

9.0

OBSERVATION :

(Subject teacher on the basis of permanent slide shall give two to three exercises to the student.) (Refer Exp. No.1)

(Space for observations)

CONCLUSION :

From the observation it is concluded that the given crude drug is found to be a ( stem, root, leaf, bark, seed, fruit, rhizome etc.) containing tissue components

QUESTIONS :

Q

teacher shall also add few more relevant questions)

Write answers to Q

Q

Q

(Questions to be allotted by the subject teacher. Subject

1.

How to focus the Transverse section in order to get fine image?

2.

What is role of condenser and iris diaphragm in critical illumination?

3.

State a role of a chemical dye during staining procedure.

4.

Give two examples of staining agent with their reactions.

5.

Write the reactions of cell wall and cell content with staining agent along with observation.

6.

List the necessary material requiredfor section cutting.

7.

How to make a sample preparation for microscopic examination?

8.

What is the purpose of taking transverse section of crude drug?

9.

Mention special method for section cutting of leaf.

10.

Why young leaves are preferred to get fine section? Give reason.

Space for writing answers

.Experiment No. 2

Pharmacognosy

(Space for answers)

Pharmacognosy

Experiment No. 2

(Space for answers)

Experiment No. 3

Pharrnacognosy

Experiment No. 3

1.0

TITLE :

To study Microchemical reagent.

2.0

PRIOR CONCEPT :

Morphology or Sensory characters of crude drug.

3.0

NEW CONCEPT :

Preposition 1 : Microchemical Reagents Microchemicalreagents are used in Pharmacognosy practical for diagnostic identificationof crude drug, which stains the different components of T.S. of crude drug.

4.0

LEARNING OBJECTIVE :

Intellectual Skill :

1. To acquire skills of staining.

2. Ability to interpret from observation.

5.0

REQUIREMENT :

Apparatus :

Microscope, watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc

Chemicals :

Microchemical reagents and water.

Crude Drug :

Subject teacher shall give any single drug to the students among the root, stem, seed, leaf, bark, fruit, rhizome, etc to see the understanding of the procedure of staining.

6.0

STEPWISE PROCEDURE :

6.1.

Study Of Microchemical Reagents :

Pharmacognosy includes study of crude drugs obtain from natural origin. This drug can be studied by their morphological or sensory characters and microscopical characters. For microscopicalcharacters, they are treated with various reagentssuch as : -

1. Cleansing Reagents :

These reagents are used to make the tissue clear in appearance. e.g. Chloroform, Acetic acid, Chloral hydrate and water,

2. Dehydrating Reagents :

These reagents are used to remove water from the section or tissue and to make tissue clear in appearance. e.g. Absolute alcohol.

3. Bleaching Reagents :

Reagentsare used to bleach the tissues or section and to make tissue clear in appearance. e.g. Hydrogen peroxide.

4. Mounting Reagents :

These reagentsare used to mount the tissues or section and to prevent the drying of sections. e.g. Glycerin or mixture of glycerin and water.

6.2

Specific Microchemical Reagents. IndividualStaining Solutions

Pharmacognosy

Experiment No. 3

Name of Reagent

Eosin

Conc. SulphuricAcid + Iodine

Phloroglycenol+ Conc. Hcl

Safranin

RheutheniumRed

Alcoholic PicricAcid

Sudan Red - 111

Dilute Iodine

Conc. SulphuricAcid

Ferric Chloride

6.3 Procedure :

Component

Cellulose

Colour

Lignin

Lignin

Mucilage

Protein

Fixed Oils And Fats

Starch

Saponins I Stone Cells

Tannins

0bservation

Red Or Pink Colour

Pale Blue

Colour

Red Or Pink Colour

Red Or Pink Colour

Red Or Pink Colour

Yellow

Colour

Yellowish Brown

Colour

DeepI Pale

Blue

Green

Colour

BlueI Black

Colour

1.

Issue the glass watches and add microchemical reagents to it.

2.

Take section of crude drug and transfer section in to watch glass containing water.

3.

Place section in to watch glass containing microchemical reagents.

4.

Keep it for specified period.

5.

Mount the slide and observe it.

7.0 OBSERVATION :

Subject teacher on the basis of permanent slide shall give two to three exercises to the student.) (Refer Exp. No.I)

(Space for observations)

Experiment No. 3

Pharmacognosy

8.0

9.0

CONCLUSION :

From the above microscopicaltests, reagent) gives

QUESTIONS :

Write answers to Q

Q Q Q

(Name of the microchemical

colour indicating presence of components in given crude drug.

(Questions to be allotted by the subject teacher. Subject

teacher shall also add few more relevant questions)

1.

Define micorchemical reagent and list out different types of it.

2.

Why dehydrating agent is used in Microchemical test? Explain it with the help of example.

3.

Which Microchemical reagent is used for detection of tannin in cell?

4.

Which cell component is detected by ruthenium red?

5.

Define cleansing and bleaching reagent with help of example.

6.

Tabulate Microchemicaltests for fennel.

7.

Which lblicrochemicalreqgent is used for detection of Calcium oxalate crystal?

8. Write the stepwise procedure of staining and mounting?

9. Why glycerin is used as mounting reagent? Give reason.

10. Tabulate Microchemicaltest for Cinchona.

(Space for answers)

Pharrnacognosy

Experiment No. 3

(Space for answers)

Experiment No. 4

Pharmacognosy

Experiment No. 4

1.0

TITLE :

To study Morphological and Microscopical characteristics of Cinchona Bark.

2.0

PRIOR CONCEPTS :

Section cutting technique, staining, mounting and observation of transverse section of Cinchona Bark.

3.0 NEW CONCEPT :

Proposition 1 : Morphologicalcharacters It includes organoleptic characters and extra features. Proposition2 : Microscopicalcharacters It includes observation of important tissue components of transverse section of Cinnamon bark. Proposition3 :Adulterants It is debasement of genuine crude drugs, which proved harmful.

4.0

LEARNING 0B.IECTIVE :

Intellectual Skill :

1.

Ability to interpret the tissue components.

Motor Skill :

1.

Ability to prepare thin transverse section of cinnamon bark.

2.

To handle and observe instrument and crude drug correctly.

3.

Labeling different component of cell.

5.0

REQUIREMENT SSSS :

Apparatus :

Microscope, watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc.

Chemicals :

Phloroglucinol,Conc. HCI, Iodine solution, Glycerin, etc.

Crude Drug :

Cinchona bark.

6.0

DIAGRAM :

18 +

Fig. 4.1 Cinchona Bark and Plant

MAHARASKTRASTNE

BOARD OF TECHNICAL EDUCATION

Pharrhacognosy

Experiment No: 4

Fig. 4.2 T.S. of Cinchona Bark (Refer colour diagrams given in Appendix-V)

7.0 STEPWISE PROCEDURE :

7.1 Synonyms :

English : Jesuit's bark, Peruvian bark.

7.2 Biological source :

It consists of dried bark of the cultivated trees of Cinchona calisaya Wedd., C. ledgeriana.

hybrids of either of the last two

species with either of the first two. It contains not less than 6 percent of total alkaloids of

cinchona. Family : Rubiaceae.

Mocns, C. officinalis Linn., C. succirubra Pav. ex. Klotzsch, or

7.3 Macroscopy :

Organoleptic characters :

Odour : Slight and Characteristic

7.4 Microscopy :

I. PERIDERM :

Taste : Intensely bitter and slightly astringent.

Cork : Several layers of thin walled, flat, polygonal cells with reddish brown content, impregnated with suberin. Phellogen : 2to 3 layers thin walled cells without any cellular content. Pheloderm : 6 to 8 layers of thin walled rectangular cells without any cellular content.

Experiment No. 4

Pharrnacognosy

2. CORTEX:

Severallayers of thin walled tangentiallyelongatedcells containing reddish brown matter. Calcium oxalate crystals : 2 to 6 m long, microsphenoidal crystals Starch grains : Rounded, 6 to 10 m in diameter. Sclerei&s are absent. Cavities (secretioncanals) are present.

3. SECONDARY PHLOEM :

Sieve Tubes : The compact cells being about 200 m long and 15 to 20 m wide and having narrowcompanioncells; most of the sieve tubes are compressedand collapsed. Fibres : Nemerous, large, fusiform, lignified phloem fibres, having striated walls and conspicuous tubular or funnel-shaped pits, mostly isolated, some times in groups of 2 to 3 fibres. Pholem parenchyma :Thin, dark-reddish brown walls, somewith micro-priims of calcium oxalate. Medullary rays : One to three seriate, extended up to cortex cells, redially elongated and contain starch grains.

7.5 Chemical constituents :

Alkaloids : Quinine, Quinidine,Cinchonine, Chichonidine

7.6 Uses :

1. Antimalarial

7.7 Allied drugs :

1. Cuprea bark,

7.8 Marketed preparations :

1. Cinchona extract,

7.9 Procedure

2. Antipyretic.

2. Colombian bark.

2.

Compound cinchona tincture.

1. Clean the platform and issue the apparatus.

2. Issue the sample of crude drug.

3. Preparation of sample for sectioning.

.

Boding ofthe sample.

.

Section cutting.

. Transfer the section in to Watch glass containing water. (If crude drug is too hard, or in any case where subject teacher may feel then the preparation of sample for sectioning is done before one hour or a day of the practical or may be varied in certain cases)

4. Staining Process.

.

Take a clean watch glass and add the staining solution to it.

.

With the help of brush, transfer the section taken from watch glass containing water to stain solution and keep it for 2 - 3 minutes.

.

Transfer it to watch glass containing plane water, so that excess stain is washed away. This section is ready for mounting.

5. Mounting Process.

. Transfer the section to be mounted on the glass slide with the help of brush.

.

Add 1 - 2 drops of water on the section with the dropper.

.

Place the clean cover slip over the section with the help of a forceps and needle.

. With the help of blotting paper, wipe out excess of water present outside the cover slip. The slide is ready for observation. 6. Observation.

.

Select a place in the laboratory for microscope, where sufficient light is available. Set the microscope in such a way that the C-Arm towards to you and the objective and mirror facing the light.

.

Open the diaphragm completely with the help of the sub stage mirror. Adjust the position so that the field of view is sufficiently illuminated.

.

Place the slide prepared on the stage of the microscope at the centre, with the section placed exactly in line with the stage window lying above the condenser.

20 +

MAHARASHTRA STATE BOARD

OF TECHNICAL EDUCATION

Pharmacognosy

Experiment No. 4

.

Fix the slide between the clips. Now the slide can be moved forward, backward or sideways above the stage with the help of two screws provided on the mechanical stage.

.

Take observations.

7.10

Staining :

1. T.S. + Phloroglucinol + Conc. HCI (1:1)

Pink colour. Lignified Phloem fibres

 

Fig. 4.3

 

2. T.S. + Iodine Blue colour. Starch.

 

Fig. 4.4

 

(Refer colour diagrams given in Appendix-V)

8.0

OBSERVATIONS :

 

8.1 Observation table for Macroscopy :

 
 

Sr. No.

Test

Observation

1.

Colour

2.

Odour

3.

Taste

8.2 Observation table for Staining :

 
 

Sr. No.

Test

Observation

Inferences

1.

2.

3.

9.0

CONCLUSION :

 

The given sample of

crude drug is found to

be

(Cinchona bark)

10.0 QUESTIONS :

Write answers to Q

Subject teacher shall also add few more relevantquestions)

Q

Q

Q

Q (Questions to be allotted by the subject teacher.

1.

Write two external characters of Cinchona.

2.

Differentiate between stem, root and bark of Cinchona with the help of external characters.

3.

The name 'Cinchona' is derived from which incident?

4.

Which staining test is used to detect starch?

5.

What will be the inferences after treatment of T.S. of Cinchona with Dil. Hydrochloric acid?

6.

Which microscopic component is present in periderm of Cinchona bark?

Experiment No. 4

Pharmacognosy

7.

Draw well labeled diagram of Cinchona bark.

8.

Which microscopical test is used for identification of Lignified phloem and fibres?

9.

Mention four species of Cinchona bark.

10.

Which parasites are responsible for infection of Malaria? Give two names.

(Space for answer)

Pharmacognosy

(Space for answers)

Experiment-No.4

Experiment No. 5

Pharmacognosy

Experiment No. 5

1.0

TITLE :

To study lblorphological and lMicroscopical characteristics of Cinnamon Bark.

2.0

PRIOR CONCEPTS :

Section cutting technique, staining, mounting and observation of transverse section of Cinnamon Bark seed.

3.0 NEW CONCEPTS :

Proposition 1 : Morphological characters It includes organolepticcharacters and extra features.

Proposition 2 : Microscopical characters It includes observation of important tissue components of transverse section of Cinnamon bark.

Proposition 3 :Adulterants It is debasement of genuine crude drugs, which proved harmful.

4.0

LEARNING 0B.IEC'I'IVES

:

Intellectual Skill :

1.

Ability to interpret the tissue components.

Motor Skill :

1. Ability to prepare thin transverse sedin of cinnamon bark.

2. To handle and observe instrument and crude drug correctly.

3.

Labeling different component ofcell.

5.0

REQUIREMENTS :

Apparatus :

Microscope, watch glass, camel hair brush, glass slides, cover slips, beaker, dropper, filter paper, forceps, test tubes, test tube holder, tripod stand, wire gauze, dissecting needle, sharp razor, etc.

Chemicals :

Phloroglucinol,Conc. HCI, Iodine solution, Glycerin, Ruthenium Red solution, etc.

Crude Drug :

Cinnamon Bark.

6.0

DIAGRAM :

Fig. 5.1 Cinnamon Bark and Plant

Pharmacognosy

Experiment No. 5

Fig. 5.2 Cinnamon Bark (Refer colour diagrams given in Appendix-V)

STEPWISE PROCEDURE :

7.1 Synonyms :

English : Cinnamon Bark

Hindi

: Kalmi - Dalchini

7.2 Biologicalsource :

It consist of dried inner bark of the shoots of coppiced trees of Cinnamomum zeylanicum Nees. It contains not less than 1.O% vlw of volatile oil, belonging to family Lauraceae.

7.3 Macroscopy :

Organoleptic characters :

Colour : Outer surface, dull yellowish-brown; Odour : Fragrant,

Extra features : Bark is free of cork, siqgle or double, closely packed compound quills.

Fracture : Splintery.

Inner surface darker in colour Taste : Warm, sweet and aromatic

7.4 Microscopy :

1. PERICYCLE (stone cell layers) :

Produce the light coloured wavy, longitudinal lines on the outside of the bark.

Experiment No. 5

Pharmacognosy

.

.

Pericyclic fibres : Small groups of about 6 to 15 pericyclic fibres (lignified) occur at intervals. Sclerides : 3 to 4 layers of pitted sclerides,thickened lignifiedwalls, isodiametric,slightly elongated tangentially (U-shapedthicknening ), with starch grains.

2. SECONDARY PHLOEM :

Parenchymatous:few cells contains acicular calcium oxalate crystals and starch grains (diameter upto 10 p). Medullary rays : Biseriate, narrow at innersight, wider in thescleride band side, contains starch, acicular raphides. Pholem fibres : single, isolated,circular, lignifiedwith stratification, being above 12 to 22 to 35 p wide and 200 to 500 to 600 p long Mucilage cells : can be identified after staining with Ruthenium red (shows pink / red colour). Oil cells : big, isolated. Cork and cortex are absent.

7.5

Chemical constituents :

 

Volatile oil (0.5 to 1%), cinnamic aldehyde (55 to 65%),eugenol(4 to lo%), terpenes, mucilage, starch, calcium oxalate, tannins.

7.6

Uses :

 

1.

Carminative,

2.

Flavouring agent,

3. Mild astringent,

4. Powerful germicide.

7.7

Allied drugs :

1.

Cassia bark or Chinese Cinnamon,

3.

Java Cinnamon,

2. Wild or Jungle Cinnamon, 4,Saigon Cinnamon,

5.

Oliver bark.

7.8

Marketed preparation :

 

1.

Cinnamon is an ingredient of

Compound-cardamomtincture I.P.

7.9

Procedure

1. Clean the platform and issue the apparatus.

2. Issue the sample of crude drug.

3. Preparation of sample for sectioning.

.

Boiling of the sample.

.

Section cutting.

. Transfer the section in to Watch glass in to Watch glass containing water. (If crude drug is too hard, or in any case where subject teacher may feel then the preparation of sample for sectioning is done before one hour or a day of the practical or may be varied in certain cases)

4. Staining Process.

.

Take a clean watch glass and add the staining solution to it.

.

With the help of brush, transfer the section taken from watch glass containingwater to stain solution and keep for 2 - 3 minutes.

.

Transfer it to watch glass containing plane water, so that excess stain is washed away. This section is ready for mounting.

5. Mounting Process.

.

Transfer the section to be mounted on the glass slide with the help of brush.

.

Add 1 - 2 drops of water on the section with the dropper.

.

Place the clean cover slip over the section with the help of a forceps and needle.

.

With the help of blotting paper, wipe out excess of water presentoutside the cover slip. The slide is ready for observation.

6. Observation.

.

Select a place in the laboratory for microscope, where sufficient light is available. Set the microscope in such a way that the C-Arm towards to you and the objective and mirror facing the light.

.

Open the diaphragm completely with the help of the sub stage mirror. Adjust the position so that the field of view is sufficiently illuminated.

Pharrnacognosy

Experiment No. 5

Place the slide prepared on the stage of the microscope at the centre, with the section placed exactly in line with the stage window lying above the condenser. Fix the slide between the clips. Now the slide can be moved forward, backward or sideways above the stage with the help of two screws provided on the mechanical stage. Take observations.

7.10

Staining:

Subject teacher shall ask student to draw diagrams of staining in the space provided below.

1.

T.S. + Phoroglucinol+ Conc. HCI (1:l) Pink colour Lignified cells: Pericyclic fibres, stone cells, cork cells.

2.

T.S. + Iodine Blue colour Starch

3.

T.S. +Acetic acid Insoluble Calcium oxalate crystals

4.

T.S. + Dil. HCI Soluble Calcium oxalate crystals

8.0

OBSERVATIONS :

 

8.1 Observation Table for Macroscopy

 
 

Sr. No.

Test

Observation

1. Colour

2. Odour

3.

Taste

4.

Fracture

5.

Extra features

 

8.2

Observation Table for Staining :

 
 

Sr. No.

Test

Observation

Inferences

1.

2.

3.

9.0

CONCLUSION :

 

The given crude drug is found to be

(Cinnamon bark)

Experiment No. 5

Pharmacognosy

10.0 QUESTIONS :

Write answers to Q

teacher shall also add few more relevant questions)

Q

Q

Q (Questions to be allotted by the subject teacher. Subject

Give biological source of Cinnamon bark. Draw neat labeled macroscopical diagram of Cinnamon bark. Which microscopic character is detected by Ruthenium Red in case of Cinnamon bark? Write two crude drugs, which contain eugenol as main active chemicalconstituent. Mention four allied drugs of Cinnamon bark. Give the process of chemical test by which tannins are detected from Cinnamon bark. Write three regional names of Cinnamon bark other than title. Which volatile oil constituents are present in Cinnamon bark? Write four Therapeutic uses of Cinnamon bark. Why is Cinnamon presented in compound quill form? Give reason.

(Space for answers)

Pharmacognosy

Experiment No. 5

(Space for answers)