LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 1(a) Health and Safety (i)

(a) Health and Safety

(i) Identifying and controlling hazards and assessing risk.

(ii) The use of physical or chemical biological control measures including the use of personal protective equipment.

Standard laboratory rules and familiarity with risk assessment.

Chemicals or organisms can be intrinsically hazardous. Their use may involve risks to people, to other organisms or to the environment. The use of control measures, including personal protective equipment as a last resort, to reduce risk. Biological control includes using a more suitable strain of microorganism eg less virulent.

Health and Safety

Learning Outcomes

To know what a hazard and a risk are.

To be aware of how to reduce risk of injury in a laboratory.

Success Criteria

To understand the difference between the terms “Hazard” and “Risk.

To follow a Method of control to reduce risk of injury in a laboratory

What is a hazard?

‘A Hazard is a potential source of harm or adverse health effect on a person or persons’.

There are any hazards around us.

Take time to reflect on the hazards you may have encountered from getting up in the morning to arriving in this laboratory.

In Science, many hazards are given a symbol – can you identify them?

Answers to Hazard Symbols – how did we get on?

What is a risk?• The terms Hazard and Risk are often used

interchangeably but this simple example explains the difference between the two.

• If there was a spill of water in a room then that water would present a slipping hazard to persons passing through it. If access to that area was prevented by a physical barrier then the hazard would remain though the risk would be minimised.

Another example of reducing risk of personal injury...

Take a few moments to consider how we have reduced the risk of injury when using methane gas (a hazardous explosive)

It is everyone’s interest to minimise risk of injury

HASAW Act 1974

Google

“Workplace Accidents

- Prevent-It.ca”

Risk Assessments(Methods of Controls to minimise injury)

Risk Assessment• Risk Assessments (“Methods of Control”)must be carried

out when using hazards.

• A laboratory Risk Assessment form is usually used to guide the teacher in implementing the reduction of risk.

• A wealth of health and safety information is available in available to manage hazards correctly (e.g. HASAW).

• Many schools use the Scottish Schools Education Centre• (SSERC) for up to date health and safety information.

Risk Assessment continued• Using SSERC’s website

• Risk assess the use of concentrated hydrochloric acid in a science laboratory.

• Risk assess the use of the microbiological bacteria, Escherichia coli in the science laboratory.

PPE

This stands for personal protective equipment.

PPE is required when the hazard is not fully containable.

Why might this PPE be used?

What did this “PPE” try to protect you from?

Black Death / Bubonic Plague (Europe, circa 1347)

Health and Safety in the Laboratory

The Health and Safety at Work act (HASAW) 1974 ensures that hazards in laboratories are managed through clear risk assessments procedures / controls.

‘A Hazard is a potential source of harm or adverse health effect on a person or persons’. E.g. Wet floor-tiles are a slip hazard.

“A risk is the likelihood that a person may be harmed or suffers adverse health effects if exposed to a hazard. We seek to reduce risk of a hazard by using methods of control and when necessary the use of personal protective equipment (PPE).

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 2(b) Liquids and Solutions

Liquids and Solutions (1)

Learning Outcomes

1. That a range of apparatus is used to accurately measure volumes

2. That dilutions can be linear or serial

3. To know when log scales are useful

Success Criteria

To be able to produce a standard curve for the determination of an unknown

Measuring volumes

You may have used these apparatus for measuring volumes in Science.

Can you name them?

(Your teacher may give you an opportunity

to use some of these apparatus)

Measuring volumes

Measuring volumes

Measuring volumes

Measuring volumes

Measuring volumes

Measuring volumes

Measuring accurately using the meniscus

Measuring accurately using the meniscus

Measuring accurately using the meniscus

What apparatus would be used...

To titrate an acid against an alkali?

What apparatus would be used...

To measure an approximate small volume of liquid?

What apparatus would be used...

To measure an approximate large volume of liquid?

What apparatus would be used...

To measure an approximate large volume of liquid for medical purposes?

What apparatus would be used...

To measure an accurate volume of 25ml?

Note: Three pipette valves...

What apparatus would be used...

To measure a very accurate volume of 25ml?

What apparatus would be used...

What apparatus would be used...

Measure microlitres of volume (1000th of ml)

(b) Liquids and Solutions

(i) Dilution series are often linear or log.

(ii) Standard curves, measurement and determination of an unknown concentration.

(iii) The use of buffers to maintain and control pH.

(b) Liquids and Solutions (1)

Learning Outcomes

That dilutions are often serial or linear.

That Standard Curves, involve dilutions to determine an unknown concentration.

Success Criteria

To determine the concentration of an unknown glucose solutions using a prepared Standard Curve.

Dilutions

Come up with a definition for the term, “dilution”.

DilutionsDilution means to reduce the concentration of a chemical. Usually, dilutions involve using the solvent, water.

We can “dilute to taste” concentrated juice with water.

We can dilute a Stock Solution, for example, from 100% to 2% with water (how might this be done?).

Since biological systems are frequently in solution, we can dilute them too for analysis.

If a biological system or model is to be analysed, we may have to dilute them using one of two techniques:

1. Linear dilutions

2. Serial Dilutions

Please copy: Linear dilutionsA linear dilutions are often used when biological samples or models have a short range of dilutions. Linear dilutions use the “Stock solution” to prepare from.

Calculate the missing data to make linear dilutions.

Extension:

What apparatus would be used to accurately measure these volumes?

Stock volume (ml)

Water volume(ml)

Linear dilution(%)

100 0 100

95 5

10 90

25

70

Investigation using linear dilutionsTitle:

Estimating unknown glucose solutions (A,B,C) using a standard curve prepared from diluted glucose samples (2% to 12%).

Read through instruction sheet and carry out the investigation.

Prepare a written Report to include:

1. Title

2. Aim

3. Materials

4. Method

5. Results table and Graph

6. Conclusion

7. Evaluation

Please copy: Serial DilutionsSerial dilutions are used when biological samples or models have an extensive range of dilutions e.g. : x10, x100, x1000 fold more dilute than the stock solution.

Serial dilutions are done in a stepwise method. If a ten-fold dilution is used (see below) then it can be referred to as a logarithmic dilution or log-dilution.

Serial Dilution table (please complete using “log” function on your calculator)

Sample volume (ml)

Water added(ml)

Serial dilution factor from stock

Logarithmic dilution factor

1 (from stock) 9 X 10 10-1

1 (from x10) 9 X 100 10-2

Serial Dilution table (please complete using “log” function on your calculator)

Sample volume (ml)

Water added (ml) Serial dilution factor from stock

Logarithmic dilution factor

1 (from stock) 9 X 10 10-1

1 (from x10) 9 X 100 10-2

1 (from x100) 9 X1000 10-3

Serial Dilution table (please complete using “log” function on your calculator)

Sample volume (ml)

Water added (ml) Serial dilution factor from stock

Logarithmic dilution factor

1 (from stock) 9 X 10 10-1

1 (from x10) 9 X 100 10-2

1 (from x100) 9 X1000 10-3

1 (from x1000) 9 X 10,000 10-4

Serial Dilution table (please complete using “log” function on your calculator)

Sample volume (ml)

Water added (ml) Serial dilution factor from stock

Logarithmic dilution factor

1 (from stock) 9 X 10 10-1

1 (from x10) 9 X 100 10-2

1 (from x100) 9 X1000 10-3

1 (from x1000) 9 X 10,000 10-4

1 (from x10,000) 9 X 100,000 10-5

Serial Dilution table (please complete using “log” function on your calculator)

Sample volume (ml)

Water added (ml) Serial dilution factor from stock

Logarithmic dilution factor

1 (from stock) 9 X 10 10-1

1 (from x10) 9 X 100 10-2

1 (from x100) 9 X1000 10-3

1 (from x1000) 9 X 10,000 10-4

1 (from x10,000) 9 X 100,000 10-5

1 (from x100,000) 9 X 1,000,000 10-6

Be careful when you interpret log scales

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 3(c) Separation Techniques (i)

Buffers

These are train buffers – what is their function?

Buffers in solutionBiological systems experience changes such as oxygen concentration, temperature variation and pH fluctuations.

pH is a measure of how “acid or alkali” a solution is. More correctly it is the measure of the concentration of hydrogen ions [ H+] .

To measure pH we take the inverse log of hydrogen ion concentration (pH = -log [H+]).

Task: Calculate the pH values of these numbers ([H+])

1)

2)

3)

Buffers in solution (continued)

As biological systems can experience changes in [H+ ] it may be useful to control this variation in pH using a buffer.

A buffer is a solution that resists pH change by removing the ions that cause change (namely [H+] and [OH- ] ). Buffers are useful as they can maintain a set pH value, for example, to ensure a biological system has ongoing optimal conditions e.g. pH7.

How buffers work

Buffers can react with both strong acids (top) and strong bases (side) to minimize large changes in pH.

(c) Separation Techniques

(i) Separation techniques using solubility, size, shape and charge: Centrifugation and Chromatography (paper, thin layer and affinity).

(ii) Separation techniques using solubility, size, shape and charge: protein electrophoresis and separation of proteins and iso-electric points.

Separation Techniques

Learning Outcome

That biological materials can be separated according to their:- Solubility- Size and density- Shape- Charge

To know examples of how to separating biological materials:

-Centrifugation- Partition techniques- Chromatography- Gel electrophoresis- Iso-electric point (IEP)

Success Criteria

To identify the stationary phase and mobile phase in three chromatography techniques.

To align an appropriate separation technique for a certain biological material.

Separation Techniques

This term refers to isolating different biological materials in a mixture. For example, separating cell organelles using centrifugation.

Centrifugation

Is a process which uses centrifugal forces to separate materials of different densities and sizes. (Note:g-force is a unit referring a gravitational force under acceleration)

A centrifuge is a piece of equipment that spins a sample at high speed.• Centrifugation allows substances to be separated

according to their density. • Remember that density is the heaviness of an object for a

given volume (d = Mass / Volume).

For example: Gold is 19.3 g/cm3 and silver is 10.49 g/cm3

What is the mass of 3500mm3 of pure lead?(d= 9.78 g/cm3 )

The largest and densest materials separate out first and form a pellet at the bottom of the tube. The liquid which remains above the pellet is called the supernatant.

How might you carefully remove the supernatant?

Which of these organelles have the greatest density (mass / volume)?

Health and Safety using a Centrifuge• A centrifuge can spin at extremely high speeds (up to 70,000

rpm for ultracentrifuges).• Note: car engine 2000rpm at 70mph

• What precautions should be taken?

1. Only open lid when centrifuging has completely stopped.2. Ensure samples are balanced (e.g. mass or volume)3. Balanced samples are placed on opposite sides from one another.4. Biological spillages are thoroughly cleaned using a manufacturer’s recommended cleaning agent (to avoid corrosion)

Chromatography

What can you remember about chromatography in school?

1. What is the solvent?

2. What does the solvent do?

3. What happens to the blue pigment?

4. What precautions are needed?

What is chromatography?Chromatography refers to a set of techniques which separates the components of a mixture e.g. separating amino acids found in a protein.

Chromatography allows scientists to identify and (in some types of chromatography) purify the components of a mixture.

This is achieved usually by the measure of a biological sample’s solubility.

There are four types of chromatography:

1. Paper chromatography

2. Thin layer chromatography (TLC)

3. Affinity chrmoatography

4. High performance liquid chromatography (HPLC)

Although different from one another in technique, they each employ a stationary phase and a mobile phase (see later).

Optional Research Task

Prepare a short presentation on one of the four types chromatography and complete this Summary table to give to your class.

Name of technique

Outline of technique

Stationary phase

Mobilephase

Used to separateor purify

Paper ChromatographyThe stationary phase (not moving) is a strip of chromatography paper. This paper contains cellulose fibres, which are polar in nature, meaning that they have slightly charged molecular forces(+ and - ).

The mobile phase is the solvent being used. Solvents are often non-polar in nature (no charged molecular forces), such as acetone / propanone.

A sample of the mixture being separated is placed in a dot, or line, near the bottom of a strip of chromatography paper which is then placed into the tank of solvent (not above the dot). The end result is known as the chromatogram.

Can you label this diagram

A

B

D

C

Draw and label this diagram

Pencil application line.

X3 applications of the mixture (components) to be separated.

Solvent e.g. acetone.

Chromatography paper or chromatogram.

To think about:

1. Why might three applications of a pigment (coloured material) be used to create one dot?

2. Why must you not allow the “solvent front” to run-off the top of the paper?

3. Why is the lid helpful, during chromatography? (think about the solvent)

4. What might to the mixtures of pigments if the paper touches the side of the glass tank?

How does paper chromatography work?

This chromatography relies on the solubility of each of the mixtures being separated with the solvent (mobile phase) being used.

For example, we might say that the blue pigment has a higher solubility in the solvent, than the purple pigment.

How does paper chromatography work? (Continued…)However, it turns out to be not so simple as just about solubility (no surprise there then!)

We also need to take into account the stationary phase (the paper). This too will determine how quickly a component will travel.

For example, as the solvent moves up through the chromatography paper it will carry the dissolved components of the mixture with it. The most soluble travelling quickly upwards.

However, paper (stationary phase) contains cellulose fibres which are polar in nature and so components which are also polar will bond more readily with the cellulose fibres and will not travel as far up the paper.

Non-polar pigments of the mixture will not bind as readily to the paper and therefore travel further.

We might say then, that the component to be separated may have a stronger affinity either with the mobile phase or stationary phase.

Paper Chromatography Summary

1. Separation of mixtures is due to:

a) An affinity of the mixture to solubilise in the (non polar) solvent (mobile phase)

or

b) An affinity of the mixture to adhere to the cellulose (stationary phase).

2. Polar components of a mixture tend to be attracted to polar charges in the cellulose and readily “drop-out” of the (non polar) solvent.

Paper chromatography Summary

Align the correct statement to it’s correct label.

Statement 1. Component has more affinity to stationary phase than mobile phase

Statement 2. Component has more affinity to mobile phase than to stationary phase.

.

B

A

Thin Layer Chromatography (TLC)

Same principles of operation as paper chromatography except that the stationary phase (“thin layer”) can be altered. The thin-layer (commonly cellulose or silica gel) is thinly applied, usually onto a glass plate.

Affinity ChromatographyThe process of affinity chromatography differs from paper and thin layer chromatography as it relies the binding interactions between protein material and the chromatography material (stationary phase).

The chromatography material comprises of two substances:

1. An inert / unreactive packing material within a glass tube

2. A ligand* / binding material which is immobilised or trapped within the inert packing material.

This type of chromatography relies then on the shape of the biological material (see next slide).

* This could be a chemical bond (e.g. polar molecule) or an antibody (see later)

Note how the shape/ligand in the stationary phase is complementary to the shape of one of the proteins.

Is this a identification or purification technique?

Affinity Chromatography

1. A ligand (e.g. antibody) is immobilised on an inert support column (stationary phase).

2. A protein mixture is passed through the column using a liquid (mobile phase).

3. The protein, which is complementary to the ligand in the column, will bind to it and remain in the column.

4. When the other components are washed away, the target protein can then be stripped from the support, resulting in its separation and purification from the original sample.

Affinity Chromatography diagram

Stationary phase with bound ligand

Mobile phase

Google: YOUTUBE and “High Performance Liquid Chromatography HPLC Royal Society of Chemistry”

Title: High Performance Liquid Chromatography (HPLC)

Complete this table during the video

(Research opportunity: Find out HPLC is used by forensic scientists)

Chromatography technique

Outline of technique

Stationary phase

Mobilephase

Used to separateor purify

Homework: Extend your table and using Notes, complete the missing cells

Chromatography Technique

Outline how it works

Stationary phase Mobile phase Used to separate or purify

HPLC

Paper

Thin layer (TLC)

Affinity

Homework: Extend your table and using Notes, complete the missing cells

Chromatography Technique

Outline how it works

Stationary phase Mobile phase Used to separate or purify

HPLC Liquid sample passed though a tube a high pressure

Solid (inert) e.g. silica pushed into tube

Mixture of polar and non liquids with sample to be separated

e.g. steriod hormones

Paper Samples have an affinity for either stationary or mobile phase

Paper (cellulose – polar material)

Water (polar) or acetone (non-polar)

Separate pigments (e.g. chlorophylls)

Thin layer (TLC) Layer of silica gel or alumina placed over slide (e.g. glass)

Thin layer on slide. Liquid solvents or mixture solvents

See “paper”.Advantage is the polarity of the stationary phase can be adjusted.

Affinity Sample is passed through a column with a specific ligand (e.g. antibody) and trapped due to complimentary of 3d shapes

Inert gel with immobilised ligand.

Sample within a solvent (polar or non-polar).

Purification of a specific sample e.g. target protein (antigen)

Copy: Rf Values and Chromatography

The retardation factor (Rf) is defined as the ratio of the distance travelled by the centre of a spot to the distance travelled by the solvent front.

Calculate the following Rf values for the following coloured pigments (see next slide).

Calculating Rf values continued.

Pigment Distance from line to centre of spot (cm)

Distance of solvent front (cm)

Rf value

yellow 2 10 0.2

purple6 10 0.6

blue 8 10 0.8

Gel (or protein ) Electrophorsis

Gel electrophoresis is sometimes referred to as PAGEPOLACRYLAMIDE GEL ELECTROPHORESIS.

“Gel”

usually polyacrylamide (a toxic liquid that sets to provide “sieving” gel ).

Smaller biological components tend to move through these sieves/tunnels faster than bigger ones.

Where will the shortest nucleotide chains be?

Term “Electrophoresis” (PAGE)• Electrophoresis is a technique used in laboratories in

order to separate macromolecules based on size and charge. Treated proteins, and naturally occurring DNA and RNA are negatively charged. This means that these macromolecules will migrate down the gel towards the positive electrode.

As DNA and RNA(and some proteins) are negatively charged, they will be drawn down the gel towards the positive electrode.

Overall view of PAGE

Did you know?Not all proteins are negatively charged.

However, one form of protein electrophoresis is SDS-PAGE. During this procedure, the proteins are denatured and given a uniform negative charge; this means that the proteins can be separated based on their linear size as they migrate towards the positive electrode. Small proteins travel further through the gel than large proteins.

Sodium dodecyl sulfate (SDS) is used to denature the proteins and to impart an uniformed negative charge. The method is called:

“Sodium dodecyl sulfate polyacrylamide gel electrophoresis”– you won’t need to remember that!!

(SDS-PAGE for short!)

Before and after SDS

Health and Safety with SDS PAGE• 1. Acrylamide mix: Very toxic: danger of irreversible effects in contact

with skin and lungs (if inhaled). May cause cancer, heritable genetic damage, serious damage to health by prolonged exposure.

• 2. Sodium Dodecyl sulfate (SDS) risk of serious damage to eyes, may cause sensitisation by inhalation, harmful by inhalation &if swallowed

• 3. Electrophoresis equipment can pose significant electrical hazards in the laboratory typical electrophoresis units operating at 100 volts can provide a lethal shock of 25 milliamps.

• Worth risk assessing??!!• What PPE would YOU recommend?

Iso-electric point (IEP)Proteins are made up of amino acids which may carry a positive charge (e.g. -NH3+, amine group),

a negative charge (e.g. -COO- carboxyl group),

or neutral charge (e.g. same number of positive and negative charges).

The combined charges of the amino acids in a protein give the protein its overall charge.

What charge might this protein

(polypeptide) have?

ANSWER

No net charge / Neutral

What is the “iso-electric point”

The iso-electric point of a protein is the pH at which protein has no charge /neutral. The iso-electric point will be different from protein to protein.

Take for example this protein: its iso-electric point is actually pH7.

Did you know?When a protein reaches its iso-electric point (IEP) and becomes neutral, it actually becomes solid in suspension (precipitates) – a bit like albumin (egg-white protein) becoming solid when boiled.

When a suspension of protein precipitates into a solid, it indicates protein denaturation. This is reversible in most cases for a protein at its IEP but not for solid albumen in a boiled egg!

Changes in chargeAs you look closely at the iso-electric point of this particular protein you can see that a change in its charge is still possible.

Notice that as the pH drops (becomes more acidic / increases in [H+]) the protein becomes more positively charged.

And when the pH increases (becomes more alkaline / increases in [OH-]) the protein becomes more negatively charged.

How does IEP separation technique work?

Proteins can be separated using their IEP (iso-electric point). At their IEP they stop moving because…

At a specific pH, they have an overall neutral charge and then precipitate out of solution.

This technique both separates mixtures and purifies the components for further analysis.

Summary of Separation TechniquesTechnique Separates biological

components by…Purpose

Chromatography

Gel electrophoresis/PAGE

Centrifugation

Partition

Iso-electric point / IEP

Summary of Separation TechniquesTechnique Separates biological

components by…Purpose

Chromatography Affinity for a (non-polar) mobile or (polar) stationary phase

To identify components in a mixture e.g. in chlorophyll

Gel electrophoresis/PAGE

Centrifugation

Partition

Iso-electric point / IEP

Summary of Separation TechniquesTechnique Separates biological

components by…Purpose

Chromatography Affinity for a (non-polar) mobile or (polar) stationary phase

To identify components in a mixture e.g. in chlorophyll

Gel electrophoresis/PAGE Linear size/length of negatively charged proteins, DNA or RNA

Identify molecular size e.g. of a DNA digest

Centrifugation

Partition

Iso-electric point / IEP

Summary of Separation TechniquesTechnique Separates biological

components by…Purpose

Chromatography Affinity for a (non-polar) mobile or (polar) stationary phase

To identify components in a mixture e.g. in chlorophyll

Gel electrophoresis/PAGE Linear size/length of negatively charged proteins, DNA or RNA

Identify molecular size e.g. of a DNA digest

Centrifugation Size and density To isolate cell componentse.g. to pellet mitochondria

Partition

Iso-electric point / IEP

Summary of Separation TechniquesTechnique Separates biological

components by…Purpose

Chromatography Affinity for a (non-polar) mobile or (polar) stationary phase

To identify components in a mixture e.g. in chlorophyll

Gel electrophoresis/PAGE Linear size/length of negatively charged proteins, DNA or RNA

Identify molecular size e.g. of a DNA digest

Centrifugation Size and density To isolate cell componentse.g. to pellet mitochondria

Partition Solubility between two different solvents

To extract a type of biological material e.g. DNA from a proteins (cell components)

Iso-electric point / IEP

Summary of Separation TechniquesTechnique Separates biological

components by…Purpose

Chromatography Affinity for a (non-polar) mobile or (polar) stationary phase

To identify components in a mixture e.g. in chlorophyll

Gel electrophoresis/PAGE

Linear size/length of negatively charged proteins, DNA or RNA

Identify molecular size e.g. of a DNA digest

Centrifugation Size and density To isolate cell componentse.g. to pellet mitochondria

Partition Solubility between two different solvents

To extract a type of biological material e.g. DNA from a proteins (cell components)

Iso-electric point / IEP By proteins becoming neutrally charged at a specific pH within a gradient of pH buffered solutions

To separate and purify proteins, or DNA or RNA

(d) Antibody techniques.

(i) Detection and identification of specific proteins using immunoassay techniques (using antibodies linked to reporter enzymes)

Antibody Techniques

Learning Outcomes

To understand what an antibody is and where it comes from.

To understand the term “monoclonal antibody” and how it is produced.

To know how monoclonal antibodies are used to separate proteins using ELISA; Immunohistochemistry and Protein blotting.

Success Criteria

To annotate how ELISA works.

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 9(d) Antibody techniques (ii)

What is an antibody and where does it come from? Take 1 min. and report back!

What is an antibody?

An antibody is a “molecular weapon” used by the immune system to identify and neutralize harmful pathogens (e.g. viruses and bacteria) cells (e.g. cancerous cells) as well as other materials that might enter the body.

Antibody (also known as immunoglobulin) is “Y” shaped.

Antibody made by the lymphocytes (type of WBC) in response to an alien antigen (e.g. on an invading pathogen)

Antibodies are “specific” what does this mean?

Antigen Antibody

Specificity means that one type of antibody is only complimentary (in shape) to one type of antigen – and there are thousands of antigens!

(d) Antibody techniques in Laboratories.

As thousands of specific antibody are available using the biological process of an immune response (i.e. creating antibody in response to an alien antigens)…

…we can then utilise an immune response to create specific antibody (see later monoclonal antibody) to help separate, identify and purify biological components (“antigens”).

Producing monoclonal antibodies (please copy)

This term refers to a mass production of one specific-type of antibody (hence, “monoclonal”)

1. Introduce antigen of interest into

mouse.

2. Remove mouse lymphoctyes

3. Fuse with cancerous (myeloma)

cells* (produce immortal hybridomas)

4. Select for hybridomas expressing

desired antibody.

5. Harvest the specific antibody

* Using PEG (polyethene glycol)

Using antibody: Immunoassays

An immunoassay uses specific antibody to detect the presence of a particular antigen e.g. human chorionic gonadotropin (hCG) which is in the blood or urine of expectant mothers (Pregnancy test)

How does the immunoassay work?

There are a number of “versions on a theme” when it comes to immunoassays, but basically it involves:

1. The specific antibody (grey)

2. The antigen (green) in question (e.g. hCG)

3. An attached label / marker (yellow) onto the

specific antibody.

Discussion..

How might a pregnancy test show a positive on an expectant mother?

ELISA – a more complex immunoassay

There are a number of types of ELISA (enzyme linked immunosorbent assay). They all involve specific antibody (orange) and an enzyme marker (green), which catalysis a reaction with its substrate (blue), e.g. a colour change.

Which region in this plate of ELISA tests has the most antigen in it?

ELISA - Summary

1. Usually, antigens from the sample are attached to a surface of a plate.

2. A specific antibody is applied over the surface so it can bind to the antigen in question.

3. The antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added.

4. The subsequent reaction produces a detectable signal, most commonly a colour change in the substrate.

5. This detectable signal is measurable (e.g. colour change; fluorescence; UV intensity and also gamma radiation) providing a powerful quantitative diagnostic tool or assay.

ELISA in the NHS

ELISA is a fast, efficient and quantitative method of detecting a dangerous antigen. It is often done in fully automated machines (costing millions of pounds) which may run a dozen or more different tests on the one patient sample provided (e.g. blood, faeces or urine samples).

ELISA tests include the following antigens:

1. detection of Mycobacterium antibodies in tuberculosis

2. detection of rotavirus in feces

3. detection of hepatitis B markers in serum

4. detection of enterotoxin of E. coli in faeces

5. detection of HIV antibodies in blood samples

(Notice that even “antibodies” can be used as “antigens” to be detected by enzyme linked specific antibody!).

Other Antibody techniques (1)Immunohistochemistry

Specific marker antibodies can be used to detect the presence of a particular antigen within a tissue sample; a process known as immunohistochemistry.

This technique is commonly used in the diagnosis of diseases such as cancer because it can identify abnormal gene expression protein antigens inside the tumour cells.

Technique

1. Remove sample of suspected tumour tissue (Biopsy)

2. Tissue treated and thinly sliced and mounted onto a slide.

3. Apply enzyme-linked antibody specific for the tumour antigen.

4. Apply ELISA staining technique to bring about a detectable colour change to indicate that the antibody has bound to the target protein.

The advantage of immunohistochemistry lies in the fact that it is capable of showing exactly where a certain protein is being expressed within a tissue sample.

Other Antibody techniques (2)Protein Blotting

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 10(d) Antibody techniques (iii)

(d) Antibody techniques.

(iii) Creation of monoclonal antibody techniques.

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 11(e) Microscopy (i)

(e) Microscopy

(i) Use of bright field and fluorescence microscopy

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 12(e) Microscopy (ii)

(e) Microscopy

(ii) Haemocytometers and flow-cytometry.

Microscopy

Learning Outcomes

1. To understand Brightfield and Fluorescence microscopy.

Successful Criteria

1. To name several differences between Brightfield and Fluorescence microscopy.

Microscopy – a brief history

Bright-field microscopy

Is a relatively simple and straightforward microscopy technique for live organisms and tissue samples.

A sample is mounted on a slide and illuminated from below. Light is transmitted through the specimen to the objective lens (which magnifies the image) and then to the eyepiece at the top of the microscope where the image is observed. The image of the sample that is produced is usually darker than the background which appears bright, hence the name “bright field” microscopy. Samples are often stained before being viewed using a bright field microscope to increase contrast between structures.

Bright field micrograph image of a stem tissue (cross section). What magnification?

Electron microscopes

Why Brightfield (light) microscopes?

Light is a harmless form of wave energy (compared to an electron beam).

It therefore does not injure the specimen

It can look at specimens from “quite big (e.g. fly’s head)

To “quite small” (e.g. blood smear).

Tissues and cells

As many cells are transparent – light microscopy requires STAINS to view tissue structure.

Which tissue sample has not been stained?

Functions of a light microscope

Remember Total magnification = eye piece mag. x objective lens mag.

Light power magnification x 100

Light power magnification x 400

Light power magnification x1000

Note: Modern Bright field (light) microscopes can reach to x1500These magnifications use oil to refract scattered light into the objective lens.

Resolving power of a Bright field (light) microcsope

The resolving power of a microscope is a measure of how well we can distinguish two points from one-another.

Objects are “resolved” when you can distinguish between then – they are not “fudged together”.

Improving resolution

Which diagram shows a higher magnification? Which diagram a higher resolution? Which is more useful?

A B

Bright field microscopy looses resolution after x1500 specimen magnification

Resolution in a bright-field microscope is limited to about 0.2 micro-metres (10-6 m) due to the

SpecimenLight wavelength Image

< 0.2 m

Electron microscopes emit electron beams which have smaller wavelengths than light, and therefore provide better resolution.

Electron beam Specimen Image

Although Electron microscopy kills the specimen – the images can still be amazing to study!

SEM: 1 SEM: 2SEM: 1

TEM: 1 TEM: 2 TEM: 3

Although Electron microscopy kills the specimen – the images can still be amazing to study!

SEM: Hydrothermal worm

SEM: Butterfly proboscis SEM: Pollen grains

TEM: Hydrothermal worm

TEM: Dividing bacterium

TEM: Ciliated cells

Did you know?Although electron microscopes provide higher resolution and magnification (x400,000) there are some major drawbacks:

Electron beam operates only in a vacuum

Specimens therefore have to be dried-out (or they’ll vapourise!)

Electron beams produce high energy outputs

Specimens therefore need to be “stained” by fixing them in lead or uranium coating to this preserves the specimen (this fixing also deflects electrons too)

Fluorescence microscopy

This uses the fluorescence-light energy and not visible-light energy. Fluorescent-light energy is found in the UV range (“beyond the violet”) of the electromagnetic spectrum. UV was discovered 1801

Common uses of UV (“flourescent”)Light

In Laboratory Techniques…Fluorescent light wavelengths can be absorbed by particular protein structures (a “fluorescent marker molecule”).

A fluorescent marker molecule is one which absorbs a specific wavelength of light then emits a different (longer) wavelength.

This means that it absorbs light of one colour and emits light of a different colour and this type of protein can then be visualised.

Please copy: Fluorescence taggingSome proteins are naturally flourescent marker molecules. In some cases, antibodies are used to fluorescently tag protein structures (see also immunofluorescence).

A primary antibody*, which is specific to the protein (cancer ) being visualised, is introduced to a cell sample.

A secondary antibody*, attached to a fluorescent tag, is then added which binds to the primary antibody (* Monoclonal).

The fluorescent microscope1. Bright light passes through a filter (excitation filter)

2. This filter only lets through light at a specific wavelength (corresponding to the fluorescent marker being used).

3. When this light hits the fluorescent marker, it fluoresces and emits light of a different wavelength.

4. Viewed under fluorescence detector.

Flourescence

The following image shows endothelial cells under a microscope. Nuclei are tagged blue, microtubules are tagged green and actin filaments are tagged red, under fluorescent light

Brightfield and Fluorescence microscopy

Answer Brightfield of Fluorescence (or both) to questions

1. Uses objective lenses.

2. Bright or white light is initial source of wavelength energy.

3. Used specifically to view protein structures

4. Uses immunohistochemical techniques

5. No wavelength filters involved.

6. Specimens emit longer wavelengths compared to incoming wavelengths.

7. Useful for detecting cancerous cells.

Ethical issue for fluorescent techniques

Is it morally justifiable (“ethical”) to produce monoclonal antibodies for immunofluorescent techniques?

Identification of breast cancer cells.

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 13(f) Aseptic technique and cell culture

(f) Aseptic technique

Aseptic techniques are methodologies used to ensure that all equipment and solutions are free from contamination by biological organisms, such as bacteria or biological sources such as viruses and prion proteins (PrP).

Discuss the importance of being “free from contamination” and what “methodologies” would be needed.

Discuss the importance of being “free from contamination” and what “methodologies” would be needed.

Discuss the importance of being “free from contamination” and what “methodologies” would be needed.

Discuss the importance of being “free from contamination” and what “methodologies” would be needed.

Discuss the importance of being “free from contamination” and what “methodologies” would be needed.

Aseptic or Sterile Technique (“Methodologies”)

1. Radiation: UV Light

UV light has enough wave energy to significantly reduce bacterial load e.g. UV lamp in air flow cabinet overnight.

Aseptic or Sterile Technique (“Methodologies”)

1. Radiation: Gamma rays

Very powerful wave-energy source to sterilse surgical equipment.

Aseptic or Sterile Technique (“Methodologies”)

2. Chemical : anti-bacterial disinfectant and alcohol

Useful to “swab” over bench surfaces to reduce risk of contamination (e.g. in using microbiology techniques)

Aseptic or Sterile Technique (“Methodologies”)

3. Heat: Dry heat (Oven)

Dry-Heat sterilization is 160 °C for 2 hours or 170 °C for 1 hour. Useful for glassware.

Aseptic or Sterile Technique (“Methodologies”)

3. Heat: Steam heat (autoclave).

Is a pressure chamber of saturated steam at high pressure at 121 °C for around 15–20 minutes. Ideal for sterilising solutions and agar.

Aseptic or Sterile Technique (“Methodologies”)

3. Heat: Flame

Bunsen flame (blue) used to sterilise (remove microorganisms) for metal inoculating hoop.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 1: Reduce bacterial load on hands by thoroughly

washing them in anti-bacterial soap or alcohol.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 2: Swab bench area with discinfectant to sterilise the

area where you will be working on.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 3: Using indelible pen, label the base of your agar

plate: Your name; Microbe’s name; Date; Class.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 3.5:Flame the metal inoculating hoop until it glows red

(lay over Bunsen’s inlet pipe to cool down).

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 4: Remove the lid of culture vessel using your pinky

finger, and then gently flame the lip of the culture vessel.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 5: In the vicinity of the Bunsen’s air canopy, pick up

the sterile loop and gently remove a small volume of yeast culture (either from liquid culture or agar slope).

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 6: While holding the loop, re-flame the bottle’s lip and

replace lid.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 7: Working within the protection of the air-canopy,

pick up the base of the sterile agar plate and with the inoculating loop and gently streak a pattern (similar to below) to dilute out your culture of yeast microbes.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 8: Place the base of the inoculated agar plate onto its

lid and then secure the lid onto the base using two small strips of cello tape.

Aseptic Technique: Inoculating a sterile agar dish with yeast microbe.Step 9: Place inoculated plate (lid down) into the incubator

LABORATORY TECHNIQUES FOR BIOLOGISTS: LESSON 15(f) Cell Culture (iii)

Counting and Detecting CellsOften a General Practitioner will request a blood sample from a patient to be analysed for certain proteins (e.g hormones, antibodies etc) or cell types.

For example, a patient with an elevated white blood cell count (e.g lymphocytes) may have a bacterial or viral infection or possibly leukaemia.

Counting and Detecting Cells

Therefore any technique that can help identify and count cells types is very useful in diagnosing disease.

Which blood smears show that a disease may be present?

Counting and Detecting Cells

Flow cytometry• Flow cytometry allows scientists to detect, count and

analyse cells as they flow past a detector in solution, one by one.

A Coulter Counter

Normal Blood Count

Write out the full number of red blood cells per litre (for male of female)

Counting and Detecting Cells (1)

Flow cytometry

1. Cells in solution flow through the chamber.

2. Cells interrupt and scatter a laser light source.

3. Interrupted/scattered light source collected by detector

4. Detector converts light to electrical signals.

5. Electrical signals provide information about each cell

(e.g. number/size/characteristics/name).

Note: Fluorescently tagged cells can also be detected.

Haemocytometer

A haemocytometer allows an estimation of the concentration of cells in a sample to be made.

HaemocytometerScientists often want to know how many cells are contained within a cell culture flask.

A haemocytometer can be used to provide an estimate of both the total and the viable (actively growing / dividing cells). A viable cell count is achieved using trypan blue dye, which is taken up by dead cells but not by living cells.

The percentage viability of a sample can be calculated using the following formula:

Please copy

Viable (Living) Cell Count Calculation.

Live Cell Count

Total Cell Count

Question. 460,000 cells were counted with 450,000 remaining clear in trypan blue dye. Calculate the % Cell Viability of the culture.

X100 = % Viability

Answer

Haemocytometer – getting ready

1. Pipette 100 µl (one tenth of 1 ml)

of cell suspension of cells from

culture to be counted.

2. Add 100 µl (one tenth of 1 ml) of stain e.g. Toluene blue to stain for viable (living).

Viable cells stain blue

3. Pipette the stained cells onto the haemocytometer.

.

Haemocytometer

3. Press down the special cover slip onto the haemocytometer.

The cell sample is now at a depth of 0.1mm

Haemocytometer

4. The etched counting grid has nine smaller squares each an area of 1 mm × 1 mm

HaemocytometerAnd so with depth of 0.1mm and an area 1 mm × 1 mm, each

grid has an overall volume under the cover slip of ...

1mm X 1mm X 0.1mm = 0.1 mm3 (or 0.1µl)

So the volume over one grid is 0.1 mm3

Question: How many times will 0.1 mm3 divide into 1cm3?

HaemocytometerQuestion: How many times will 0.1 mm3 divide into 1cm3?

a. 10 times?

b. 100 times?

c. 1000 times?

d. 10,000 times?

Remember… 1cm3 volume (lxbxh) is the same as 10mm x10mm x10mm, right?

Which is the same as saying 1cm3 = 1000mm3 , right?

Or, that 1mm3 thousandth of 1cm3

So, How many times will 0.1 mm3 divide into 1cm3?

Answer: D

Haemocytometer4. The cells are now counted on the five of the small grids.

1

4 3

2

5

Haemocytometer4. Here is an example from one of those grids.

Haemocytometer4. To avoid repeat counting a general rule of counting is applied e.g. If a

cell rests on a line, count only the top, right sides

Haemocytometer4. How many viable (stained) cells can you count?

Answer : 15

Haemocytometer4. Usually an average from all five grids is taken.

What is the average of this cell count: 15 + 13 + 18 + 7 + 11.

Answer: 13 viable cells per grid.

1

4 3

2

5

Haemocytometer5. The next step is to calculate the overall cell count in 1cm3 (or 1ml).

Let’s recap!• We added 100 µl stain to 100 µl our of cell suspension

(That means we have diluted the cells by half)• We counted an average of 13 cells in 0.1 mm3 or 0.1µl

So our volume of viable cells in 1cm3 (or 1ml) will be:

a) 13 x 2 (our dilution factor)

b) 13 x 2 x 10 (to bring to 1.0mm3 or 1.0µl)

c) 13 x 2 x 10 x 1000 (to bring to 1cm3 or 1ml – still with us?).

d) Answer per 1cm3 will be..

Haemocytometer5. Answer: 260,000 viable cells per 1cm3 or 2.6 X 105

A culture flask contained 250cm3 of the cell suspension.

6. Question: Estimate how many viable cells will be in the culture flask?

260,000 x 250 = 65,000,000 viable cells

Haemocytometer6. Answer: 65,000,000 viable cells in 250cm3

7. Express this with 10n notation.

Answer: 6.5 X 107 cm-3 or 6.5 X 107 per cm3 or 6.5 X 107 / cm3

Please remember “cm3 “ and “ml” are interchangeable.

Answer: 6.5 X 107ml -1

Haemocytometer

Steps1. Calculate the average number of cells in five small squares (1mm2 ).

2. Calculate the volume within one small square (using the grid’s information)

e.g. 1mm X 1mm X 0.1mm = 0.1 mm3 (or 0.1µl =1.0 x 10-4 cm3 )

3. Work out how much of the volume (step 2) divides into 1cm3

(Remember: 1cm3 = 10mm x10mm x10mm = 1000mm3 )

e.g. 1cm3 / 0.1mm3 = 1.0 x 104 mm3 (x10,000)

4. Times the average number of cells by 1.0 x 104 to bring the cell count from 0.1mm3 to 1cm3

Note: Viable cells mean “living cells”. Tolulene Blue is a stain that differentiates living cells from dead cells (living cells take up this stain and become blue).

(f) Cell Culture

(ii) Use of explants or cells in inoculum. Estimation of vialble and total estimation cell counts

Cell Culturing Technique• Cell culture is the ability to grow cells in an artificial laboratory

environment. Cell culture is necessary for growing bacterial cells , culturing mammalian cells for cancer studies and many other processes.

• Cell culture is often performed in flasks such as those shown in the following image. Cell culture requires environmental factors, for example temperature and pH, to be controlled and the growing cells must be given an opportunity for gas exchange. Both plant and animal cell cultures require complex culture media which contain all the requirements of the cells. Typical culture media contains water, salts (Murashige and Skoog salts for plants), amino acids, vitamins and glucose. Animal cell culture also requires media containing growth factors from serum, for example fetal bovine serum (FBS) which contains growth factors that promote cell proliferation.

Cell Culturing Technique

Cell culture is the ability to grow cells in an artificial laboratory environment.

Cell Culturing TechniquesAlthough seen as a “laboratory procedure” cell cultures have been with us for a long time, for example:

1. Growing yeast cells for the baking and brewing food industries.

2. Growing microbes in sewage works to digest sewage.

3. Using microbes in agriculture for the production of silage (animal fodder).

Cell Culturing TechniqueIn laboratories, cell culturing can be for:

1. Growing mammalian cells to study growth cycles e.g. cancer cell lines.

Why is the cell culturing done within an air-flow cabinet?

Cell Culturing TechniqueIn pharaceutical laboratories, cell culturing can be for:

2. Growing genetically-engineered bacteria to produce human hormones (e.g. Human growth factor and Insulin) in a fermentor.

What aseptic techniques out to be considered when culturing bacteria within this fermenter?

Cell Culturing TechniqueIn laboratories, cell culturing can be for:

3. Producing plants from plant cell cultures for conservation, genetic modification (e.g. disease resistance) and economical purposes.

How could opportunities for fungal contamination be reduced?

Practical: Cauliflower Cloning (ww.saps.org.uk)

Demo videos: Procedure and Growth Medium: wwwyoutube.com/watch?v=cEbGnJnVuLi

Cell Culturing Techniques: How it is Done

1. Employ Aseptic Techniques throughout (e.g. sterilised medium).

2. Select appropriate growth medium for cell type (e.g. Murashige and Skoog Medium).

Note: There is two types of medium:

a) Complex Medium

Contains

b) Defined Medium

1. Select inoculum (the cell line or explant or type of microbe) to culture.

Note: There are two types of cultures:

a) Primary cultures: These are taken from fresh tissue. Mammalian tissue is first treated with a proteolytic enzyme (e.g. trypsin) to release them from the source material, such as tissue biopsy. Plant cells are released from source (e.g. from explant tissue) using cellulase and pectinase with mannitol sugar (helps with plasmolysis).

Cell Culturing Techniques: How it is Done

3b) Continuous cell line.

Are pre-existing cell cultures that have acquired the capacity for “infinite” growth, either as cells derived from tumours (see HeLa) or cells that have been transformed by producing hybridomas (see monoclonal antibody production and myelomas).

HeLa continuous cell line originated from a cervical tumour

biopsy shortly before Henrietta died. Her legacy is that her cells have

been grown to investigate research into cancer, AIDS, the

effects of radiation and toxic substances, gene mapping,

and many other scientific pursuits. Some estimate that there

have been 50 million metric tonnes of HeLa cells produced

since the 1950’s

Henrietta Lacks Born 1930, Died 1951, aged 31