DIFFERENT LABORATORY EQUIPMENT USED IN

TOXICOLOGY AND MOLECULAR BIOLOGY

Muhammad Kamran M.Sc(Hons) Agri.Entomology

University College of Agriculture,

University of Sargodha

Pakistan

Contents1. Personal Protective Equipment (PPE)

2. Volume Measuring Equipment

1. Non-volumetric containers

2. Volumetric labware

3. Equipment used for identification

1. Hand lens

2. Light Microscope

3. Inverted Microscope

4. Fluorescence Microscope

4. Analyzing Equipment (Analyzers)

1. Microplate Reader

2. PCR apparatus

3. Electrophoresis apparatus

4. Spectromer (UV/VIS )

5. Chromatography Equipment (GC,TLC, HPLC)

5. Other Equipment

1. Laboratory Refrigerators and Freezers

2. Instrument Sterilizers

3. Centrifuge

Chemical-resistant coveralls

Chemical-resistant gloves

Chemical-resistant footwear

Chemical-resistant hood or wide-brimmed hat

Chemical-resistant

apron

Chemical fume hoods

are used to protect

workers from

exposure to volatile

chemicals.

o For mixing and storing solutions and are generally not calibrated.

o Instead, the measurements, or graduations, on the side represent approximations of liquid capacity.

1.Non-volumetric

containers

Glass beaker

Erlenmeyer flasks

2. Volumetric labware

o Measure exact volumes of liquid

substances.

o Volumetric labware is denoted

with the capacity it is calibrated to

hold as well as the letters TC or

TD.

o Volumetric flasks prepare

solutions of a specific

concentration.

o Graduated cylinders are used to

measure volumes of liquid above

50 mL.

Volumetric flasks

Graduated cylinders

o Serological pipettes: Generally

used to measure and deliver

volumes in the range of 0.1 to 50

mL.

o Syringes: The use of syringes is

the most frequent method for

transferring liquid chemicals or

solvents in the laboratory.

o Micropipettors: For volumes of

0.2 microliters to 5 mL,

micropipettors should be used.

o Glass Hamilton syringes: When

plastic pipette tips are not

compatible with the liquid to be

measured, glass Hamilton syringes

are an alternative for accurate

measurement of volumes in the

microliter range.

o A magnifying glass is a

convex lens that is used to produce

a magnified image of an object.

o The lens is usually mounted in a

frame with a handle

o Hand lenses is used for identifying

gems, crystals, rocks, insects, leaves

or spiders from 5-30X magnification.

Hand lens

The light microscope

The light microscope is an

instrument used for

magnifying research

specimens.

Allow to view objects at

1000 times their original

size.

Inverted microscope

An inverted microscope is

a microscope with its light

source and condenser on the

top, above the stage pointing

down, while the objectives

and turret are below the stage

pointing up.

The main components of the light

microscope

Applications

• Light microscopy has the potential to visualize

a wide range of specimens.

• various configurations of the compound

microscope exists to suit many different

applications.

Fluorescence microscopes

A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances

Principle Components

• Main components of the fluorescent

microscope overlap greatly with the traditional

light microscope.

• The 2 main differences:

o The type of light source.

o The use of the specialized filter elements.

Applications

• Imaging of proteins

• To integrate the code for a fluorescent protein

such as green fluorescent protein, or GFP, into

the DNA of an organism.

• To investigate tumor cells

• Fluorescence Speckle Microscopy to study

movement and turnover kinetics of the protein.

Microplate reader

o The microplate reader allows for a variety of experiments to

be performed and measured simultaneously.

o Microplate readers can make absorbance, fluorescence and

luminescence measurements.

Multiplate readers are used

o To quantify protein

o Gene expression

o Various metabolic processes

such as reactive oxygen

species and calcium flux.

Components of a Microplate Reader

• Microplate readers come in different

shapes, sizes and set-ups.

• Multiwell plates: used to hold the

samples that are measured by the

machine.

• The loading tray: used to bring the

96-well plate into the machine.

• A computer interface: to operate the

plate reader and control its settings

and parameters.

Applications

• These are used in drug discovery, research, bioassay

validation, and biopharmaceutical manufacturing.

• ELISAs,

• Protein and nucleic acid quantification or enzyme activity

assays

• Bradford assay

• Fluorescent-based assays

• Luminescent assays

• High-throughput assays

PCR machine is used for an in-vitro technique

for amplification of a region of DNA whose

sequence is known or which lies between two

regions of known sequence

PCR Machine

Major Components

• Template DNA

• Oligonucleotide primers

• Thermostable DNA polymerase

• MgCl2

• dNTP’s

• Buffer • Working concentrations

• KCL

• Tris-HCl

• NaCl2 (sometimes)

• Thermocycler

• Detectors

Primers

DNA template

Buffer

+ +

A C T G

MgCl2

Principle

Typically, a PCR is a three-step reaction.

o Denaturation of DNAo In the first step of PCR, the sample is heated to 95–98°C, which

denatures the double-stranded DNA, splitting it into two single strands.

o Primer hybridization ( Annealing)o In the second step, the temperature is decreased to approximately

55–65°C, allowing the primers to bind, or anneal, to specific sequences of DNA at each end of the target sequence, also known as the template.

o DNA synthesis ( Primer extension)o In the third step, the temperature is typically increased to 72°C,

allowing the DNA polymerase to extend the primers by the addition of dNTPs to create a new strand of DNA, thus doubling the quantity of DNA in the reaction.

Step 1:

Denaturation

dsDNA to ssDNA

Step 2:

Annealing

Primers onto template

Step 3:

Extension

dNTPs extend 2nd strand

Vierstraete 1999

extension products in one cycle serve as template in the next 28

Applications

• Genome mapping and gene function determination

• Biodiversity studies ( e.g. evolution studies)

• Diagnostics ( prenatal testing of genetic diseases, early detection of cancer, viral infections, mutation detection...)

• Detection of drug resistance genes

• Forensic (DNA fingerprinting)

• Microarrays, molecular cloning, recombinant DNA research.

Detection of amplification products

• Gel electrophoresis

• Sequencing of amplified fragment

• Southern blot

• etc...

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Gel Electrophoresis Equipment

Electrophoresis

apparatus is used for the

separation of charged

molecules in an applied

electric field.

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Major components

o Box to hold the gel

oComb to create small wells in the agarose gel to

put the DNA sample into at the beginning of the

gel

o Positive and negative electrodes to create the

electrical current

o Power supply

oGel photo imaging system

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Principle

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Applications of Gel Electrophoresis

DNA can be separated by electrophoresis to:

• Visualize bands of a molecular marker to genotype individual

• Verify amplification by PCR or sequencing reactions

• Check the quality and quantity of genomic DNA after DNA

extraction

• Used for investigating the DNA

• To investigate various binding modes of small molecules to

supercoiled DNA

• Complex mixtures can be separated to very high resolution by

this process (Sheehan, D.; 2000).

Chromatography Equipment

(TLC)

The solvent travels up

plate via capillary action

and the compounds

separate depending on

compound solubility.

Detection: Color changes

or by using UV light to

observe bands.

(HPLC)

A highly improved form of

column chromatography.

Solvent is forced through

under high pressures of up

to 400 atmospheres. That

makes it much faster.

(GC)

Gas chromatography is a

technique used for

separation of volatile

substances, or substances

that can be made volatile,

from one another in a

gaseous mixture at high

temperatures.

Detection: By comparing retention times of peaks

in a sample to retention times for standards.

HPLC Detectors

oUV/Vis

oRefractive index

oFluorescence

oEvaporative light scattering (ELSD)

oMS

oDiode Array Detector (DAD)

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Application of HPLC

1. Pharmaceuticals industry

• To control the drug stability

• Quantity of drug determination from pharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet

• Quantity of drug determination from biological fluids, ex: blood glucose level

2. Analysis of natural contamination

3. Forensic test

- Determination of steroid in blood, urine & sweat.

- Detection of psychotropic drug in plasma

4. Clinical test

5. Food and essence manufacture

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Spectrometers

Spectroscopy is based, principally, on the study of the interaction

between radiation and matter. This interaction causes in the atom

an electronic transition from a lower energetic level, m, to a

higher level, l, occurring energy absorption from the atom equal

to the energy difference between both levels, El - Em.

UV and Visible Spectroscopy

o UV-VIS spectroscopy is used to detect the presence of chromophores

like dienes, aromatics, polyenes, and conjugated ketones, etc

o It uses light in the visible and adjacent ranges.

o The absorption or reflectance in the visible range directly affects the

perceived color of the chemicals involved.

o This technique is complementary to fluorescence spectroscopy.

Application

o Quantitative Analysis

o Rate Measurements

o Analysis of Mixtures

o Chemical Reaction

o Biochemistry

o Molecular biology.

o Used as a detector for HPLC.

• Measuring tapes

• Insect-proof containers

• Scissors

• Fine forceps

• Fine pointed brush

o Instrument sterilizers

o Analytical balance can measure

down to ten thousandths or even

hundred thousandths of a gram.

Laboratory refrigerators and freezers

Centrifuge

A centrifuge is used to separate components of a

complex mixture.

Principle component

o The rotor, which is the moving part that spins

at high speeds.

o Many centrifuges will have a refrigeration unit

that allows the internal temperature to be

controlled during the spin.

Applications

The centrifuges can be used:

• To purify biological specimens like animal cells.

• Useful for quickly isolating one cell type from another, or for isolating individual organelles.

• The ultracentrifuge can spin in excess of 70,000 rpm, which makes it well suited for the isolation of small particles, like DNA or viruses.