This document provides an overview of bioassay procedures. It defines bioassay as the comparative assessment of the potency of a test compound to a standard compound using a living biological system. The basic bioassay procedure involves preparing tissues, attaching them to an organ bath, constructing dose-response curves for standard and test compounds, and calculating the potency of the test compound based on its curve's position relative to the standard. Sources of error include biological variation between tissues and methodological errors in experimental design or implementation.
Expt. 1 Introduction to in vitro pharmacology and physiological salt solutionsVISHALJADHAV100
Definitions of pharmacology & drug
Aims of experimental pharmacology
Pre-clinical pharmacology
Clinical pharmacology
Types of experiments in pharmacology
Assembly for isolated organ/ tissue related experiments
Recording (writing) levers
Physiological salt solution (PSS)
Introduction
Examples
Composition
Role of ingredients
Precautions in preparation of PSS
Selection of PSS
Common laboratory animals, Classification of Experimental Animals, Handling and application of different species and strains of animals,Different strains of laboratory animals, application and common diseases.
Expt. 1 Introduction to in vitro pharmacology and physiological salt solutionsVISHALJADHAV100
Definitions of pharmacology & drug
Aims of experimental pharmacology
Pre-clinical pharmacology
Clinical pharmacology
Types of experiments in pharmacology
Assembly for isolated organ/ tissue related experiments
Recording (writing) levers
Physiological salt solution (PSS)
Introduction
Examples
Composition
Role of ingredients
Precautions in preparation of PSS
Selection of PSS
Common laboratory animals, Classification of Experimental Animals, Handling and application of different species and strains of animals,Different strains of laboratory animals, application and common diseases.
Introduction to Screening Models of Anti-Atherosclerosis
Atherosclerosis, Screening models, In vitro models, In vivo models
Presented by
SHAIK FIRDOUS BANU
Department of Pharmacology
Expt. 7 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of frog ringer solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of histamine standard solution
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
Introduction to Screening Models of Anti-Atherosclerosis
Atherosclerosis, Screening models, In vitro models, In vivo models
Presented by
SHAIK FIRDOUS BANU
Department of Pharmacology
Expt. 7 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of frog ringer solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of histamine standard solution
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
An Over view on Bioassay, structure & principles, types & methods of bioassay. Also mention of other assay's like biotechnology, microbio assay, immunoassay etc.
Radha_Chafle_303_ALZHEIMER’S DISEASE AND PARKINSON DISEASE.pptxRadhaChafle1
The presentation is about the alzheimer's disease and Parkinson Disease. In this presentation, various screening models are given for both of the diseases.
Expt. 10 effect of spasmogens and spasmolytics using rabbit jejunumVISHALJADHAV100
Overview of Discussion
Objective
Principle
Requirements
Experimental specifications (conditions)
Drugs and solutions used in rabbit intestine experiment
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Result and interpretation
Induction of transformation by a deoxyribonucleic acid fraction isolated from...Babita Neupane
This is a highlight of the one of the groundbreaking paper of early 1940's in field of molecular biology.
Induction of transformation by a deoxyribonucleic acid fraction isolated from Pneumococcus Type III. Avery, O.T., Macleod, C. M., McCarty, M. (1944) The journal of experimental medicine 79(2):137-58.
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
2. Outline
• Types of assays
• Introduction
• Definition
• Indication and principles of bioassay
• Basic procedure
• Calculations
• Source of errors
• Summary
3. Types of Assays
• Biological assays
• Chemical assays:
– Spectrophotometer,
– Spectrofluorimetry,
– Chromatography,
• Radio Immunoassays
• Microbiological assays
4. Introduction
• Late 18th centaury- standardization of
Diphtheria antitoxin by Paul Ehrlich
• Bioassay literal meaning
o Bio – living tissue
o Assay- assessment / measurement
o Bioassay: Assessment of a biological substance
5. Definition
Comparative assessment of relative potency of
a test compound to a standard compound on a
living or biological tissue.
Quantitative measurement of the amount of
active principle or substance in a
pharmaceutical preparation or biological
material using a suitable biological system
6. Indications for Bioassay
• Active principle of drug is unknown
• Active principle cannot be isolated, e.g. insulin, posterior
pituitary extract etc.
• Chemical method is either
– not available
– if available, too complex,
– insensitive to low doses e.g. Histamine can be assayed in microgram
conc.
• Chemical composition of drug is different but has same
pharmacological action e.g. cardiac glycosides isolated from
diff sources, catecholamines etc.
• To measure LD 50 and ED 50
• For biological standardization of drugs from natural sources
which cannot be obtained in a chemically pure form
e.g., vasopressin, oxytocin, insulin, heparin
7. Principles of bioassay
• Bioassay involves the comparison of the main
pharmacological response of the unknown preparation
with that of the standard.
• The reference standard and test sample should have same
pharmacological effect and mode of action, so that their
DRC curve run parallel and their potency ratio can be
calculated.
• The test solution and standard should be compared for
their established pharmacological effect using a specified
pharmacological technique.
• The method selected should be
reliable, sensitive, reproducible and should minimize errors
due to biological variation and methodology. ( Animals
should of same species, sex and weight and number of
animals should be large enough to permit statistical
analysis.)
8. Procedure
1. Prepare the physiological salt solution
2. Arrange the instrument and adjust the water bath.
3. Balance the lever
4. Tissue selection
5. Surgical process and collection of required tissue.
6. Tissue attachment to the water bath
7. Relaxation time given to the tissue
8. Prepare the standard drug( serial dilution)
9. Select the lowest possible measurable concentration
by trial and error method.
10. Prepare DRC for the standard drug.
11. Prepare DRC for the test drug.( serial dilution)
12. Select a assay method (3 point or 4 point assay)
13. Calculation
10. Various Physiological salt solutions
For 10 litres Frog- Kreb’s Tyrode Ringer- De Mc
pH- 7.3-7.4 Ringer Locke Jalon Ewen
NaCl 65 g 69 g 80 g 91.5 g 90 g 76 g
KCl 1.4 g 3.5 g 2.0 g 4.2 g 4.2 g 4.2 g
MgCl². 6H²O --- 1.1 g 1.0 g --- --- ---
NaH2PO4. H²O 0.1 g 1.4 g 0.5 g --- --- 1.4 g
NaHCO³ 2g 21 g 10 g 1.5 g 5g 21 g
CaCl² 1.2 g 2.8 g 2g 2.4 g 0.6 g 2.4 g
Glucose 20 g. 20 g. 10 g. 10 g. 5 g. 20 g
Aerating Gas air O² + O² or air Pure O² O² + O² + 5%
5%CO² 5% CO² CO²
•Calcium chloride to be added last.
•Calcium chloride and magnesium chloride are hygroscopic, so use stock solution.
11. Uses: Physiological salt Solutions
Physiological salt Uses
solutions
Frog-Ringer Amphibian tissue preparation
Kreb’s Mammalian/Avian skeletal muscle
preparation
Tyrode Intestine preparation
Ringer-Locke Heart muscle preparation
De Jalon Rat uterus preparation
13. Step 2: Arrange the instrument and adjust
the water bath.
Kymograph: Sherrington- starling
kymograph
To obtain a graphical amplified
measurable response of a muscle
or tissue
Two important parts: motor box
and drum
Speed lever: 1 revolution/ 96 min.
Paper:
glossy side outside – least
resistance
Rough side inside – stick to the
drum.
Fixing solution: shellac and
colophony saturated in alcohol
14. Student Organ bath
• Outer bath:-
First designed by rudolph
magnus
Perpex glass
Store water outside the
inner bath to maintain the
temperature
• Inner bath:-
– Glass
– To observe the tissue
during experiment
– 5-50ml (usually 10ml)
15.
16. • Tissue holder and oxygen supply:-
Tissue is attached inside the inner water bath to a
tissue holder.
Also supports the oxygen supply to the tissue.
17. Step:3 -Balance the lever
• Lever:
Three basic parts:
• Effort arm- where force in
applied
• Load arm- where effect of
force is observed
• Fulcrum
Classes of lever – 3
Types of lever
18. • Magnification :
= Distance from the fulcrum to the writing point
Distance form the fulcrum to the tied tissue
o For slow contracting muscles:- 10-15 times
o For fast contracting muscles:-5-10 times
19. Step:4-Tissue selection
S.No Compound Tissue used
1. Acetylcholine Guinea-pig ileum
Frog rectus abdominis muscle
Leech dorsal muscle
Rat uterus preparation
Isolated guinea-pig auricles
2. Serotonin Isolated oestrous uterus of rat
Isolated fundic strip of rat
Guinea pig ileum
Rabbit ear preparation
Isolated heart of the mollusc Venus mercenaria
20. S. No Chemical Tissue used
3. Histamine Guinea pig isolated ileum
Guinea pig tracheal chain.
Fall in BP of dog/cat
4. Adrenaline and Rat colon
noradrenalin Non pregnant rat uterus
Rat fundus
Rabbit aortic strip
Rabbit jejunum
Tracheal chain of guinea pig
21. Step 5: Surgical process and collection
of required tissue.
• Animal sacrificed by cervical dislocation.
• Tissue identified and isolated.
• Carefully dissect and separate unwanted tissue.
• Tissue kept in a physiological salt solution.
• Avoid excessive handling of tissue.
22. Step 6 : Tissue attachment to the
water bath
• Attach the ends of the tissue:-
– One end:- tissue holder
– Other end:- lever
• Method of attachment of tissue:
– Attach the thread at the end by a needle
– Intestine:- care should be taken not to block the lumen
23. Aeration
Pure oxygen (O2 ) For heart
Air For intestine
Carbogen ( 95% O2 & For uterus
5% CO2 )
Mixing of the test drug
Homogenisation of the solution
Keeping the tissue lumen patent
To maintain pH
( aeration by pure O2 causes losing of CO2 & solution
becomes alkaline )
24. Temperature
Rabbit intestine Physiological temp.(37°C ) is needed for
mammalian tissues
Guinea-pig ileum Temp. should be decreased in some
experiment to decrease spontaneous
contractions
Frog rectus muscle Amphibian tissue can survive in room
temperature
Temperature should be constant through out the experiment
25. Step 7:Relaxation time given to the
tissue
1. Intestine 30-45 min
2. Frog rectus 45-60 min
Measures to decrease spontaneous contraction:-
Hanging a weight of appropriate amount
Giving a antagonist
oE.g. Acetylcholine for blocking spontaneous
contraction of ileum.
26. Step 8: Prepare the standard drug
( serial dilution)
• Serial dilution: 10---10-9
27. Step 9: Prepare DRC for the standard
and test drug
•Select two std doses s1& s2 from linear part of DRC [ Let the
corresponding response be S1, S2]
•Also s2/s1 = t2/t1 = 3/2
28. Time cycle Start
Time ( Event kymogarp
h
min )
0 Start the kymograph Wait for
2 Add the Acetylcholine 11.5 Add Ach
min
2.5 Stop the kymograph & wash
the preparation
10 Wash the preparation
Wash Stop
15 Start the kymograph preparati kymograp
on h
Contact time
Time allowed for the drug (agonist) to remain in contact with the tissue
Frog rectus abdominis muscle Guinea-pig ileum
90 sec 30 sec
30. Types of Bioassays
• [1] Quantal Assays [ Direct endpoint ]
Elicits an ‘All or None’ response in different
animals
E.g.
Digitalis induced cardiac arrest in guinea pigs
Hypoglycaemic convulsions in mice.
Digitalis induced head drop in rabbits
• [2] Graded Response Assays
Graded responses to varying doses
Unknown dose response measured on same
tissue
31. [2] Graded Response Assays [ Direct comparison
on same tissues]
Interpolation:
Conc. of unknown is
read from a standard
plot of a log dose
response curve of at
least 4 sub maximal
concentrations
32. Matching & Bracketing:
Const dose bracketed with varying doses of standard till
exact match is obtained
• Used when test sample is too small
• Inaccurate & margin of error difficult to estimate
• Eg histamine on guinea pig ileum, Posterior pituitary on rat uterus
34. 4 point assay [2 +2 dose assay]
• Procedure [E.g. Ach bioassay]
Log dose response [LDR] curve plotted with varying conc of std Ach
solutions and given test solution
Select two std doses s1& s2 from linear part of DRC [ Let the
corresponding response be S1, S2]
Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ;
Also s2/s1 = t2/t1 = 2/3
Record 4 data sets [Latin square: Randomisation reduces error]
• s1 s2 t1 t2
• s2 t1 t2 s1
• t1 t2 s1 s2
• t2 s1 s2 t1
35. 3 point assay [2+1 dose assay]
• Fast & convenient
• Procedure [E.g. Ach bioassay]
Log dose response [LDR] curve plotted with varying conc of
std Ach solutions and given test solution
Select two std doses s1& s2 [ in 2:3 dose ratio] from linear
part of LDR [ Let the corresponding response be S1, S2]
Choose a test dose t with a response T between S1 & S2
Record 4 sets data [Latin square: Randomisation reduces
error] as follows
s1 s2 t
t s1 s2
s2 t s1
s1 s2 t
Log Potency ratio [ M ] = [ (T –S1) / (S2-S1) ] X log d
[d = dose ratio]
36. Step 11: Calculation
• Calculate the height of each response.
• Take mean of all S1, S2, T1 and T2 values.
• Plot a graph
39. Calculation of the strength of the solution from
graph :
• We know that D1=D2
• EG..
• 0.675 ml of 1 µg/ml= 0.425 of D2 conc.
• D2 = 0.675/ 0.425
= 1.59 of 1 µg/ml
• Strength of D2 = 1.59 µg/ml
40. Log potency ratio :
• The horizontal separation M of the two curves
represents the log potency ratio of the
concentration of test solution and of standard
41. Direct calculations
• M={(T1-S1) +(T2 –S2)}/{(S2-S1) +(T2-T1)}×log d
• Log d = log[s1/s2]
Where,
• M = Potency of the drug
• S1 & S2 = Length of the standard dose
response selected between 25-75 %
• T1 & T2 = Length of the test drug response
• s1 & s2 = Standard drug dose which came in
contact with tissue and had given the
response S1 & S2 respectively
• Dilution of the inner water bath has to be taken in to
account
42. • Strength of test solution = s1/t1 × antilog of M
• Dilution of the inner water bath has to be taken
in to account
43. Calculation of the percentage error:-
• Percentage error = ACT-OCT × 100
ACT
Where,
• ACT = Actual concentration of test
• OCT = Observed concentration of test
• The permissible limit of percentage error is <10%
44. Errors in bioassays
• Margin of error of bioassay should be < 10%
• Two types:-
1. Biological variation:
2. Methodological variation
45. • Biological variation:-
1. Variation in response to a drug.
2. Down regulation of receptor (repeated washing
of tissue)
3. Loss of tissue sensitivity (change the tissue)
4. Laboratory condition may be variable.
46. • Methodological variation:-
1. Human error: done by the experimenter
2. Experimental error: faulty procedure selection or
calibration error.(proper balancing the lever, and
by maintaining the ph and temperature at a
physiological level.)
47. • Reasons for methodological error:
1. Lack of standardization of procedure
2. Over handling of tissue
3. Preparation of physiological salt solution.
4. Drug preparation or in dilution
50. Summary
1. Prepare the physiological salt solution
2. Arrange the instrument and adjust the water bath.
3. Balance the lever
4. Tissue selection
5. Surgical process and collection of required tissue.
6. Tissue attachment to the water bath
7. Relaxation time given to the tissue
8. Prepare the standard drug( serial dilution)
9. After relaxation test any concentration of the drug
10. Then standardize the tissue response with same drug. ( take subsequent
two response)
11. Select the lowest possible measurable concentration by trial and error
method.
12. Prepare DRC for the standard drug.
13. Prepare DRC for the test drug.( serial dilution)
14. Select a assay method (3 point or 4 point assay)
15. Measure the height of each response
16. Calculation
51. Time cycle
Time ( mins) Event
0 Raise the 1 gm weight & start the kymograph
2 Add acetylcholine
3.5 Stop the kymograph, wash rectus & lower the 1 gm weight
6 Raise the weight & start the kymograph
Contact time
Time allowed for the drug (agonist) to remain in contact with the tissue
Frog rectus abdominis muscle Guinea-pig ileum
90 sec 30 sec
52.
53. Principles of Bioassay
• Active principle to be assayed should show the same
measured response in all animal species
• The degree of pharmacological response produced should
be reproducible under identical conditions [Eg Adrenaline
shows same rise in BP in the same species under identical
conditions: wt, age, sex, strain / breed etc]
• The reference standard must owe its activity to the
principle for which the sample is being bioassayed
• Activity assayed should be the activity of interest
• Individual variations must be minimised / accounted for
• Bioassay might measure a diff aspect of the same
substance compared to chemical assay [Eg testosterone &
metabolites
54. Biological objects
Whole Isolated organ Isolated Isolated
animal tissue cells
Assay of Assay of Assay of Assay of
insulin in gonadotropins oxytocin on antibiotics
rabbits on ovary isolated on bacterial
uterine cells
tissue