Laboratory Experimental Research

7/13/2019 Laboratory Experimental Research

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Laboratory experimental

research

Evy Sulistyoningrum

Histology department


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Outlines

Introduction

Animal experimental

Culture experimental


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Introduction

Laboratory experimental research : biomedical

research

In vivo : animal study

In vitro culture study

Confounding variables were strictly controlled

Research subject:

Certain Animals

Certain cell/tissue/organism culture

Pre-clinical trial


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Steps in biomedical research

1. Choose the appropriate subject

2. Choose research design models

3. Sample size

4. Animal/sample welfare guarantee

5. Treatments/intervention6. Data Collection

7. Analysis


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Subjects

Animals

Custom animal

Animal model for human condition Genetically modified animal

Human cells/ cell model for human cells

Pathogens Microbes

Paracytes

Fungus, etc


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Research designs

Post test-only with control group

Pre and post test with control group Post test-only without control

Pre and post test with control group

Complete Randomized Design

Control? No treatment at all

Disease model

Gold standard


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Post test-only with control group

scheme

T : (X) O1

C : (-) O2

T : treatment group

C : Control group

(X) : given treatment

(-) : no treatment or given placebo

O : observation in treated group


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Pre and post test with control group

scheme

T : O1 (X) O3

C : O2 (-) O4

T : treatment group

C : Control group

(X) : given treatment

(-) : no treatment or given placebo

O : observation


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Simple Complete randomized design

N animals

C

(-)

O

A

Xa

O

B

Xb

O

C

Xc

O

C : Control group (-) : no treatment or given placebo

(X) : given treatment O : observation


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Pre and post test CRD

N animals

C

O1

(-)

O2

A

O1

Xa

O2

B

O1

Xb

O2

C

O1

Xc

O2

C : Control group (-) : no treatment or given placebo

(X) : given treatment O 1 : observation before treatmentO2 : observation after treatment


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Sample size

Animal

Federer : (n-1)(t-1) > 15

WHO Minimal numberReduction

Cells

Literature based

Pathogens

Literature based


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Treatments

Variant depend on research intention

Routebased on research intention

Dosing :

Toxicology : LD50 , etc

Other dosing: literature-based

Human doseneed conversion


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Data collection

Based on research design

Based on data specification

Source: Animal behaviors

Parameters of animal specimen

Animal tissue

Specific products

Can be measuredresearch variables


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Types of Observations

Activity Level e.g., hypoactivity, hyperactivity, restlessness, lack of

inquisitiveness

Attitude e.g., arousal, depression, awareness of surroundings

Behavior, Spontaneous e.g., vocalization, self-trauma, isolation from cage mateswithout disturbing the animal

Behavior, Provoked e.g., vocalization, hiding, aggressiveness, minimal responsewith disturbing the animal


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Types of Observations Body Condition

e.g., missing anatomy

Food and Fluid Intake

e.g., elimination of feces and urine

Skin

e.g., cyanotic, pale, or congested mucousmembranes or skin (ears, feet, tail); skin lesions

Eyes

e.g., clarity/condition of lens, cornea; position ofglobe; condition of eyelids, encrustation

Posture


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Types of Observations

Locomotion e.g., gait, ataxia, action of each limb, position of tail when ambulating

Neurological e.g., tremor, convulsion, circling, paralysis, head tilt, coma

Vital Signs e.g., respiratory distress (open mouth breathing, pronounced chest

movement)

Other clinical parameters that are relevant e.g., presence and status of tumors, infection, or surgical wounds


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Examination

Behavior

Body weight

Surface lesions (wounds, masses)

Hydration status

Body temperature (telemetric methods)

Blood parameters

Other specimens parameters


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Variables & Analysis

Variables

Based on research designs and observation needed

Scalesbased on research preference

Specimen source must be stated

International unit or other common measurement units

Analysis

Based on research objective

Based on variables scales

Descriptive


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Examples

Nodul size

Blood level of AST, ALT, ALPreflect liverfunction

Blood level of ureum, creatininreflect renalfunction

Fasting glucose level, HbA1c

Inhibition zonereflect antibacterial activity

Balooning degeneration

Johnsons criteria for spermatogenic level


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Animal experiments


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Why perform animal experiments?

Some experiments cannot be performed on humansor better performed on animals

Organs and body systems similar to humans andother animals

Susceptible to the same diseases that affect humans

Short life spanallows to be studied throughoutentire life

Environment easily controllable to keep

experimental variables to a minimum


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Benefits of Animal Research

Penicillin Mice

Blood Transfusions

Dogs

Tuberculosis Medicine Guinea pigs

Meningitis Vaccine Mice

Kidney Transplants Dogs and Pigs

Breast Cancer Treatments Mice, Rats and Dogs

Asthma Inhalers Guinea Pigs and Rabbits

Polio Vaccine Mice

Insulin for Diabetics Dogs

Deep Brain Stimulation forParkinson's Disease

Monkeys


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Benefits Continued Vaccine for Smallpox

Vaccine for Anthrax

Rabies Vaccine

Typhoid Vaccine Cholera Vaccine

Treatment for Beriberi

Treatment for Rickets Corneal Transplants Local Anaesthetics

Discovery of Vitamin C Canine Distemper Vaccine Coronary Bypass Operation German Measles Vaccine

MMR Vaccine Antidepressants and Antipsychotic CT Scanning for Improved Diagnosis Chemotherapy for Leukaemia

Medicines to Treat Ulcers Inhaled Asthma Medication Combined Therapy for HIV infection

Medicines for Type 2 Diabetes Cervical Caner Antibodies Avian Flu Vaccine Malaria Vaccine

Modern Anaesthetics

Tetanus Vaccine

Diphtheria Vaccine

Anticoagulants Streptomycin

Kidney Dialysis

Whooping cough Vaccine Heart Lung Machine Hip replacements

Cardiac Pacemakers High Blood Pressure Medicines Replacements of Heart Valves Chlorpromazine Psychiatric Medicine

MRI Scanning for improved Diagnosis Prenatal Corticosteroids for Premature Babies Treatment for River Blindness Life Support for premature Babies

Medicines to control Transplant Rejection Hepatitis B Vaccine Leprosy Treatment

Oral and Inhaled Insulin for Type 1 Diabetes Angiogenesis Inhibitors for Cancer and Blindness Gene Therapy for Muscular Dystrophy Alzheimers Vaccine


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Limitations of animal experimentation

Species differences

Some symptoms are hard to discover in animals

In some cases, genetically modified animals isbetter

discovery of gene functions

treatment and knowledge of genetic disease

minimisation of rejection followingxenotransplantation

development and production of therapeuticprotections


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Hazards of Working with animal

Injuries

Allergies Zoonoses


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Two Major Points of View

(1) Animal Rights- ending all animal use

no food, clothing, entertainment, medical research or hunting

(2) Animal Welfare - animals must be treated and

used humanely


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Rules for animal experiments

Animal Welfare Act, 1966 [USC Title 7, Sections 2131 to 2156] as amended in1970, 1976, 1985 and 1990.

Animal Welfare Regulations [Title 9 CFR, Subchapter A, Animal Welfare, Parts 1, 2and 3]

Health Research Extension Act, 1985 [Public Law 99-158, November 20, 1985,Section 495]

US Government Principles for the Utilization and Care of Vertebrate Animals Usedin Testing, Research, and Training, 1985

PHS Policy on Humane Care and Use of Laboratory Animals, 1986

2000 Report of the AVMA Panel on Euthanasia [JAVMA, Vol. 218, No. 5, March 1,2001]

Guide for the Care and Use of Laboratory Animals (Guide) [NRC, 5th Ed., 1996]

NIH Grants Policy Statement (03/01), Part II: Terms and Conditions of NIH GrantAwards Subpart A: General -- Part 2 of 7


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The 3 Rs

Russel & Burch (1959):

(a)Refinement: Improve the experiments forminimising animal pain and suffering

(b)Reduction: Use statistical methods so that a

smaller number of animals are required(c)Replacement: Use alternative, non-animal methods to achieve

the same scientific aim


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Refinements

Proper animal living conditions

Qualified and trained personnel

Medical care available and provided by a qualified

veterinarian Procedures avoid or minimize discomfort, distress, andpain

Procedures that may cause pain or distress : Analgesia unless well justified Unrelieved paineuthanize

Surgical procedures performed aseptically andappropriate

Animals not used in more than one major, survivaloperative procedure

Acceptable methods of euthanasia


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Examples of Refinement


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Reduction

Animal Numbers : minimum possible to achieve

statistically significant data and are well justified :

Citation of previous research: sufficient information toindicate that the previous research is similar enough in

concept and methodology

Power analysis: enough information to show ability in

analyzing data and using a power analysis

If need animal specimen: state clearly how many

amount of material is needed


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Replacement

In Vitro Testing

Computer Modelling

MRI Scanning

Micro dosing


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Animal models

Animal that biologically or habitually normative, canbe spontaneously or patogenetically investigated,and human phenomenon mimicking

Mice: most frequently used (mice + rodents :+ 90 %)

Other rodents: Rats

Guinea pig

Hamster etc

Dogs, cats, rabbits, farm animals, fish, frogs, birds,nonhuman primates, etc : 10%


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Animal models

Human condition mimicking: Diabetes

Dislipidemic

Hepatotoxic Ulcers

Cancers

Dosing Literature based

Conversion from human dose


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Animal handling


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Acclimation & sexingAcclimation

Period of time allows animals to adapt to a newenvironmentbiological stabilization

Effects of transportation stress : various blood parameters

food intake and animal behavior

Period of time necessary : depend on theparameters to be studied.


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Sexing


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Sexing

scrotum


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Mouse Pups

1 day old mice 2 days old pups day 3

4 days old pups 6 days old pups5 days old pups


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and more pups

Day 7 Day 8 Day 9

Days 12,13 and 14Day 11Day 10


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Blood Collection from Tail

TOOLS

Alcohol cotton ball forsurface disinfection

Small plastic bottlewith 1/2 cm diameterholes in both ends asmouse restrainer

Scissors Micropipette and tips

A vial for bloodcollection

For collection of small amount ofblood (Approximate 0.1 ml )


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Leave the tail of the mouseoutside the cover of the

restrainer

Amputate the tip of the

mouse tail by scissors


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Massage the tail and collect blood by pipette


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Blood Collection from Orbital Sinus in

Mouse

TOOLS Alcohol cotton ball for

surface disinfection

Kloroform for generalanesthetic

27 G needle with 1 mlsyringe for injection

Glass capillary tubeand vial for blood

collection

Anesthetic before blood withdraw

Collect amount up to 0.5 ml


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Anesthetize a mouse byintraperitoneal injection

Use a glass capillary tube to

penetrate the orbital conjunctiva

and rupture the orbital sinus


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Collect blood with a vial


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Cardiac puncture in Mouse

TOOLS

Alcohol cotton ball forsurface disinfection

Chloroform used asanesthetic

27G needle with 1 mlsyringe for injection

24G needle with 3 mlsyringe for bloodwithdraw

For collect up to 1 ml of blood within a shortperiod of time

Must be performed under general anesthetic


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Anesthetize a mouse by

intraperitoneal injection

Disinfect the thorax area

with 75% alcohol cotton

ball


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Search for the maximum

heart palpitation

Cardiac puncture in 90

angle


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Withdraw blood slowly by right hand


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Blood collection from Saphenous vein in mice

TOOLS Alcohol cotton ball

for surfacedisinfection

50 ml syringe tubewith small holes atthe end asrestrainer

a scalpel and shaverfor remove of hair

24 G 1 needle forrelease of blood

tips and pipette forblood collection

Multiple samples taken in the course of aday


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Place the mouse in the

restainer


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Pull out the leg and

removed the hair by a

assistant

Hair also be shaved by

using a small scalpel


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The saphenous veinis seen on the surface

of the thigh

Apply vaseline to the

surface area to reduceclotting and coagulation

during blood collection


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The saphenous veinis ruptured by scalpel

Blood is aspirated

through pipette


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Injection in Mouse

TOOLS

Alcoholcotton ballfor surfacedisinfection

25G 1/2needle with1 ml syringe

for injection


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Handle a mouseproperly


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The injection site : the lower left quadrant of the abdomen

because vital organs are absent from this area. Only the tip of

the needle should penetrate the abdominal wall to prevent

injection into the intestine.

Intraperitoneal injection


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Pick up a nude mouse and spin its tail


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Subcutaneous injection


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Oral Feeding in Mouse

TOOLS

A 18 G

stainlesssteel, balltipped

needle a glove

Gastric intubation ensures that all the material wasadministered

Feeding amount limited to 1% of body weight


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Gastric intubation


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Dissection


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Genetically modified animal


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Transgenic mice

Transgenic: an organism that has had DNA

introduced into one or more of its cells artificially

transgenic: DNA is integrated in a randomfashion by injecting it into the pronucleus of a

fertilized ovum

Random (+ 10% disrupt an endogenous gene important

for normal development)

multiple copies


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Knock-out mice

knockout: DNA is introduced first into embryonic

stem (ES) cells

ES cells that have undergone homologous

recombination are identified and injected into a 4 dayold mouse embryo - a blastocyst

targeted insertion


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Pronuclear injection in transgenic


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Pronuclear injection in transgenic

technology

Implanting 1(or 2) cell embryos


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Implanting 1(or 2) cell embryos

(cont )


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(cont.)


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Transgenic mice


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Trangenic mouse embryo in which the promoter for a gene

expressed in neuronal progenitors (neurogenin 1)

drives expression of a beta-galactosidase reporter gene.

Neural structures expressing the reporter transgene are dark

blue-green. (Dr. Anne Calof)


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Tail tip9.5 day embryos -

GFP and wt

GFP transgenic mouse (Nagy)


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GFP transgenic mouse (Nagy)

ES cells growing in culture in knock out


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ES cells growing in culture in knock out

technology

Successfully transformed ES cells are


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Successfully transformed ES cells are

injected into blastocysts

l bl


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Implanting blastocysts

1 2

l bl ( )


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Implanting blastocysts (cont.)

3 4

i


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Littermates

Black mouse -

no apparent ES cell

contribution

Chimeric founder -

strong ES cell

contribution

Chimeric founder -

weaker ES cell

contribution


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Cell culture

I d i


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Introduction

Cell culture is the process by which prokaryotic,

eukaryotic or plant cells are grown under controlled

conditions In practice it refers to the culturing of cells derived

from animal cells.

Cell culture was first successfully undertaken by Ross

Harrison in 1907

Genetic engineering can be done to cellsproduce

desired substance (recombinant)

E l f bi t


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Examples of recombinant

products

Viral vaccine

Hybridoma capable of secreting a monoclonal

antibody.

Human insulin recombinant protein

Human growth hormone produced fromrecombinant bacteria

Lymphoblastoid IFN

Tissue-type plasminogen activator (tPA) from

Wh i ll lt d f ?


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Why is cell culture used for?

Model systems for:

Studying basic cell biology

Interactions between agents and cells

Effects of drugs on cells

Toxicity testing

Study the effects of new drugs on cells

Cancer research

Study the function of various chemicals, virus & radiation

to convert normal cultured cells to cancerous cells

Study of the effects of drugs in cancer cells

Contd


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Contd.

Virology

Cultivation of virus for vaccine production

Genetic Engineering

Production of commercial proteins

Gene therapy

Cells having a functional gene can be replacedto cells which are having non-functional gene

P i lt


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Primary culture

Cells when surgically or enzymatically removed

from an organism and placed in suitable culture

environment will attach and grow

Primary culture contains heterogeneous

population of cellssub culturing of primary

cells leads to generation of cell lines

Cell lines have limited life span, they passage

several times before they become senescent

S C ll li


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Some Common cell lines

Human cell lines

MCF-7 breast cancer

HL 60 Leukemia

HEK-293 Human embryonic kidney

HeLa Henrietta lackscervical cells infected by HPV 16

HUVEC human Umbilical Vein Endothelial cells

Primate cell lines Vero African green monkey kidney epithelial cells

Cos-7 African green monkey kidney cells

C t i t f ll lt


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Contaminants of cell culture

Chemical:

difficult to detectinvisible

caused by endotoxins, plasticizers, metal ions or traces of

disinfectants

Biological:

cause visible effects Mycoplasma, yeast, bacteria or fungus or cross-

contamination of cells from other cell lines

Basic equipments used in cell culture


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q p

Laminar cabinet

Incubation

Refrigerators

Inverted microscope

Tissue culture ware- Culture plastic waretreated by polystyrene

P th lt


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Pathogen cultures

Similar with cell culture

Larger organism

Proper & specific media

McConkey

Sabouroud

etc

Can be treated with various substance

produce specific effect that can be

measurable


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Laboratory Experimental Research

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