Chapter 10: Introduction to Cell Culture Technology
In 1951, a woman named Henrietta Lacks came to the Johns Hopkins Hospital in Baltimore for treatment of cervical cancer. A small sample of her tumour was sent, without her knowledge or her consent, to the laboratory of a cell biologist named George Gey. Gey had been trying for years to keep human cells alive outside the body, and almost every sample he had ever received had died within days.
These cells did not die. They divided, and went on dividing, and filled every vessel they were given. Gey labelled them from the first two letters of the patient’s first and last names — HeLa — and began sending them free of charge to laboratories around the world, packed in tubes and kept cold in transit. Within a few years HeLa cells were being used to test the new polio vaccine, and they are still growing in laboratories today, decades after the woman they came from died.
What made the difference was not luck alone. It was the nutrient mixture the cells were fed, the sterile glassware they were grown in, the warm incubator that held them at body temperature, and the careful technique that kept a single stray bacterium from destroying the whole culture. Those tools are what this chapter is about.
Diagram
Fig. 10.1 – A cell culture laboratory scene: a laminar air flow cabinet with an open culture flask inside, an autoclave and hot air oven to one side, a CO₂ incubator holding T flasks, and a liquid nitrogen storage container; label each item.
This chapter will answer each of these questions as you work through it.
10.1 Instruments and Devices Used in Cell Culture Technology
Growing cells outside the body — or outside the plant — is really an exercise in taking over a job that nature normally does. A cell inside a living organism is supplied with nutrients, held at a steady temperature, given the right acidity, and protected from invading microbes. In a laboratory, every one of those services has to be provided by a machine.
That is why a cell culture laboratory looks the way it does. Each instrument on the bench replaces one condition the cell would otherwise have enjoyed inside its parent organism, and none of them is optional. For each device below, what matters most for your examination is what it is used for.
Table-Top Centrifuge
A centrifuge spins samples at high speed around a fixed axis. Spinning generates a force far greater than gravity, so denser particles travel outward and settle at the bottom of the tube while lighter material stays above. This is called sedimentation.
Laminar Air Flow Cabinet
A laminar air flow (LAF) cabinet, also called a tissue culture hood, is an enclosed bench on which sterile work is carried out. Room air is drawn in and pushed through a HEPA (High Efficiency Particulate Air) filter, which removes airborne dust particles, bacterial cells and fungal spores. The cleaned air then moves across the working surface in a smooth, unmixing stream — this is what laminar means — so that any contaminant released on the bench is swept away from the culture rather than swirled over it, as shown in Fig. 10.2.
Diagram
Fig. 10.2 – Sectional view of a laminar air flow cabinet; label prefilter, blower, HEPA filter, UV lamp, protective screen, working area and the direction of air flow.
The cabinet is made of stainless steel with no gaps or joints in which spores could lodge, and it usually carries a UV germicidal lamp that is switched on to pre-sterilise the chamber before work begins and switched off while anyone is working inside.
Uses: inoculating explants into culture medium, transferring cultures from one vessel to another, pouring sterile media into Petri plates, and any manipulation that would be ruined by contamination.
Biosafety Cabinets
A biosafety cabinet looks similar to a LAF cabinet but is designed to protect the worker as well as the work. In a plain laminar flow cabinet the filtered air blows outward towards the operator, which is fine for sterile media but not for anything infectious. A biosafety cabinet instead draws air inward at the front and exhausts it through a filter, so that whatever is inside the cabinet stays inside.
Autoclave
An autoclave sterilises by moist heat under pressure. Water is boiled in a closed chamber so that steam builds up to a pressure of 15 psi (pounds per square inch), at which point its temperature rises to \(121^\circ\text{C}\) — well above the boiling point of water at ordinary pressure. The load is held at this temperature for about 15–20 minutes, longer for bulky loads. In principle it is a laboratory pressure cooker, fitted with a safety valve, a pressure release point and a perforated inner cylinder.
Uses: sterilising culture media, glassware, distilled water, plugged flasks, instruments and any heat-stable material that will not be spoiled by wetting.
Hot Air Oven
A hot air oven sterilises by dry heat. It has double-walled insulation, a thermostat and a circulating fan that distributes heat evenly through the chamber. Ovens can generally be operated between \(50^\circ\text{C}\) and \(300^\circ\text{C}\); for sterilising glassware, a setting of \(160\text{--}180^\circ\text{C}\) for 2–4 hours is standard. Opening the door before the cycle finishes lets heat escape and leaves the load unsterile.
Uses: sterilising glassware such as Petri plates, pipettes and culture tubes, and also metallic instruments and powders — that is, articles that tolerate high temperature without burning or losing their edge.
pH Meter
A pH meter consists of a glass electrode, or probe, connected to a meter that displays the reading. The probe responds to the hydrogen ion activity of the solution around its thin glass bulb and generates a small voltage — about 0.06 volt per pH unit — which the meter converts into pH units.
Because the glass electrode drifts over time, the meter must be calibrated before use against at least two standard buffers that bracket the range being measured; buffers of pH 4 and pH 10 serve most general purposes. You will have used exactly this instrument in the Class XI practical on finding the pH of tap water and of water containing a little acid or base.
Uses: measuring and adjusting the pH of culture media and buffers before sterilisation.
Magnetic Stirrer with Hot Plate
A magnetic stirrer uses a rotating magnetic field, produced beneath the vessel, to spin a small magnetic stir bar dropped into the liquid. Because the bar is sealed and easily sterilised, nothing mechanical needs to be dipped into the solution. Most models combine the stirrer with a hot plate and a thermostat.
Uses: dissolving media components into a uniform solution and warming solutions gently while they mix — the routine first step in preparing any medium or buffer.
Electronic Weighing Balance
An electronic weighing balance measures mass to a very high precision — analytical balances read to 0.1 mg. The pan sits inside a transparent enclosure with doors, so that dust does not settle on it and stray air currents do not disturb the reading. Samples should be at room temperature before weighing, since a warm sample sets up convection currents inside the enclosure and the mass reading wanders.
Uses: weighing out salts, sugars, agar, vitamins and growth regulators in the exact quantities a medium recipe demands.
Vortex Mixer
A vortex mixer has a cupped rubber head mounted slightly off-centre on a motor shaft. When the head oscillates rapidly and a tube is pressed against it, the motion is transmitted to the liquid and a vortex forms, mixing the contents within seconds.
Uses: resuspending settled cells, mixing assay reagents, and mixing a sample with a diluent before counting or analysis.
Microfiltration Unit
Some of the most important ingredients of a culture medium — enzymes, vitamins, hormones, antibiotics and certain amino acids — are destroyed by heat. Autoclaving such a solution would sterilise it and ruin it at the same time. A microfiltration unit removes microbes mechanically instead, by passing the solution through a membrane whose pores are too small for bacterial cells to cross.
The membrane, made of cellulose acetate or cellulose nitrate, is available in pore sizes of 0.22, 0.45 and 0.5 micrometre; the \(0.22\ \mu\text{m}\) membrane is the one normally used for sterilisation. It is mounted in a holder of stainless steel, glass or autoclavable plastic, and the whole assembly is autoclaved before use. The solution is then drawn through by suction, and the sterile filtrate collects in a sterile container, as shown in Fig. 10.3.
Diagram
Fig. 10.3 – A microfiltration assembly; label the funnel holding the unsterile solution, the membrane filter, the receiving flask and the suction outlet.
Uses: cold sterilisation of heat-labile components of culture media.
Incubator
An incubator is an insulated chamber that holds cultures at a controlled temperature and humidity. Microbial cultures are typically incubated at \(37^\circ\text{C}\), plant tissue cultures nearer \(25^\circ\text{C}\). Keeping humidity up matters as much as temperature, because a small volume of medium in a warm chamber loses water rapidly and the salts left behind become concentrated enough to harm the cells.
Uses: growth and storage of bacterial, plant and animal cultures.
Carbon Dioxide Incubator
Animal cells in culture are held at a pH of about 7.4 by a bicarbonate buffer, and a bicarbonate buffer only works if the gas above the medium contains carbon dioxide. A \(\text{CO}_2\) incubator therefore controls carbon dioxide concentration along with temperature and humidity; oxygen and nitrogen content can be adjusted as well. Chamber volumes usually lie between 50 and 300 litres, and the working temperature is close to that of the human body, \(37^\circ\text{C}\).
Humidity is supplied either from water trays inside the chamber or from an external tank whose water is vaporised and fed in as steam, so that only sterile water reaches the cultures. The interiors are made of corrosion-free stainless steel, electrolytically polished so that germs cannot settle in surface irregularities and cleaning is easy.
Uses: growth and maintenance of animal and human cell cultures, and of any culture whose medium is bicarbonate-buffered.
Inverted Microscope
In an ordinary microscope the light source sits below the stage and the objective lenses above it. An inverted microscope reverses this: the light source and condenser are placed above the stage pointing down, and the objectives sit below the stage pointing up, as in Fig. 10.4.
Diagram
Fig. 10.4 – Side-by-side light paths of an upright microscope and an inverted microscope; label light source, condenser, stage, objective lenses and the culture vessel resting on the stage.
The reason is practical. Cells growing in a culture flask lie flat against the bottom of the vessel, under several millimetres of medium. Viewing from below means looking at the cells through the thin base of the flask rather than through the medium above them, and it leaves the space above the specimen free for handling large or heavy vessels.
Uses: examining living cells in culture flasks and dishes without opening them — checking whether cells have attached, how densely they have grown, and whether contamination has appeared.
T Flasks for Animal Cell Culture
Many animal cells will only grow while attached to a surface. A T flask is a flat-sided polystyrene culture flask, optically clear so that cells can be viewed through its base, whose inner surface has been treated so that cells attach and spread on it. The vent cap carries a filter that lets gases exchange with the incubator atmosphere while keeping microbes out, and opens with a third of a turn so that it can be worked one-handed inside a cabinet.
T flasks are named by their growth area in square centimetres, and are available in T25, T75, T175 and T225 sizes.
Uses: propagating animal cells in monolayer culture, and scaling up a culture in steps by moving it into progressively larger flasks.
Bioreactor
A bioreactor is a large closed vessel in which cells or genetically modified organisms are grown on a big scale under controlled conditions, so that a product can be harvested commercially. Because the syllabus asks for its components and their functions in some detail, the bioreactor is taken up separately in the next section.
Uses: large-scale cultivation of cells and large-scale production of fermentation products such as enzymes, antibiotics and single cell protein.
10.2 The Bioreactor: Types, Structure and Components
A flask holds a few millilitres of culture. An industry that must supply insulin, an antibiotic or an industrial enzyme needs thousands of litres. Scaling up is not simply a matter of using a bigger vessel, because as a culture grows larger it becomes harder to keep every part of it equally warm, equally aerated and equally well mixed. The vessel that solves this problem is the bioreactor.
The word fermentor is often used for the same vessel, particularly when the organisms inside are microbes. The two terms are used interchangeably in industry.
Why Control Matters
Inside a bioreactor the culture changes the very conditions it depends on. Growing cells consume sugars and oxygen, release acids and other metabolites, and give off heat. Left alone, a large culture would acidify itself, warm itself and starve itself of oxygen within hours. Every component described in this section exists to counteract one of these self-inflicted changes.
Types of Bioreactors
Two designs are prescribed for study. They differ in how the contents are mixed and aerated.
Continuous Stirred Tank Bioreactor
The continuous stirred tank bioreactor, also called the stirred tank reactor (STR), is the general-purpose design. A motor-driven shaft carries impellers through the culture broth, and this mechanical stirring both mixes the contents and breaks incoming air into fine bubbles so that oxygen dissolves readily.
Advantages of the stirred tank bioreactor
- Gas transfer to the growing cells is efficient.
- Mixing of the contents is thorough, and operating conditions are easy to work with.
- Vessels of this design are readily available commercially.
- It is comparatively inexpensive.
- It is easy to operate.
- Temperature is easy to control.
- Sterile conditions are easy to maintain.
Disadvantages of the stirred tank bioreactor
- Energy consumption is high, because the motor runs continuously.
- The culture medium must be agitated without a break.
- A large amount of foam is produced.
Bubble Column or Sparged Tank Bioreactor
In a sparged tank bioreactor there is no stirrer at all. Air or another gas is introduced at the base of the column through perforated pipes or plates, or through metal microporous spargers, and the rising stream of bubbles does the mixing. The flow rate of the gas therefore controls oxygen supply, temperature and mixing together. Perforated plates may be fitted along the column to break up the bubbles again and improve performance. The vessel is usually a tall cylinder, with a height-to-diameter ratio of about 4:1 to 6:1.
Advantages of the sparged tank bioreactor
- It is ideal for growing aerobic cultures, since gas is supplied continuously.
- Energy consumption is relatively low.
- It needs no agitator shaft.
- Heat is removed much faster than in a stirred tank bioreactor.
Disadvantages of the sparged tank bioreactor
- It requires a greater input of air.
- The air must itself be sterilised.
- High pressure is needed to deliver the air.
- There is no provision for a bubble breaker.
General Structure of the Bioreactor
The parts described below are those of a stirred tank vessel, which is the design shown in Fig. 10.5. Study the figure alongside the descriptions — in board papers this diagram is frequently set as a labelling or part-function question.
Diagram
Fig. 10.5 – Sectional view of a stirred tank bioreactor; label motor, sterile seal, viewing port, steam inlets, pH controller, exhaust, impellers, baffles, sparger, cooling jacket with cooling water in and out, culture broth, sterile air inlet and harvest outlet.
1. Body of the fermentor. The vessel body is made of stainless steel. It must resist corrosion, because a corroding surface would leak toxic metal ions into the broth and poison the culture, and it must tolerate the high temperature and pressure of steam sterilisation.
2. Baffles. These are small metallic strips fixed radially to the inner wall, each about one-tenth of the vessel’s dimension. Without them, a stirred liquid would simply rotate as a body around the shaft and form a whirlpool, with poor mixing between layers. The baffles interrupt this rotation and create turbulence, which distributes the oxygen supplied to the medium and improves aeration.
3. Impellers or agitators. These are mounted on the central shaft and are nearly half the diameter of the vessel. They mix the nutrient medium with the cells of the organism, keeping cells suspended rather than settled and preventing pockets of exhausted medium from forming.
4. Sparger. The sparger sits at the bottom of the fermentor and admits air into the liquid medium. Introducing air at the base means the bubbles travel the full height of the broth before escaping, giving the maximum time for oxygen to dissolve.
5. Antifoam control system. Agitation and aeration whip up foam on the upper surface, and foaming is worsened by high cell densities and by media containing soya extract or yeast extract. A probe is held above the culture medium; when foam rises far enough to touch it, an electric circuit is completed through the foam. This switches on a pump that adds antifoaming agent to the medium, and the foaming stops.
6. pH control system. As nutrients are consumed and metabolites accumulate, the pH of the growth medium drifts. A changed pH alters the physiology of the growing organisms and lowers the efficiency of fermentation, so a pH probe is fitted inside the vessel in permanent contact with the medium. When the reading moves away from the set value, acid or base is supplied from a reservoir to bring it back.
7. Temperature control system. Heat is generated both by the continuous running of the motor and by the metabolism of the cells, which increases as cell density rises. To counter this metabolic heat, the fermentor is fitted with thermostatically controlled heating elements and a vessel jacket. When the temperature rises, cooling water is circulated through the jacket, and after it has taken up the excess heat it is discharged through the cooling outlet.
8. Additional ports. An inlet is provided for steam, so that the vessel can be sterilised in place before inoculation. If the vessel is made of glass the contents are directly visible; a steel vessel is fitted with a viewing port so that the internal operation can be observed.
Flowchart
FC 10.1 – Operating a bioreactor: sterilise vessel and medium with steam → cool to working temperature → inoculate with the culture → supply sterile air through the sparger and stir → hold pH, temperature and foam under automatic control → monitor growth → harvest product through the outlet at the base
Reading the Design as a Whole
Notice how each component answers a specific threat. Corrosion is answered by stainless steel, poor mixing by baffles and impellers, oxygen shortage by the sparger, foaming by the antifoam probe, acid build-up by the pH controller, and heat build-up by the cooling jacket. If any one of these fails, the culture does not simply grow a little more slowly; the conditions run away from the set point and the batch can be lost.
10.3 Sterilization Techniques and Aseptic Conditions
A culture medium is a rich broth of sugars, salts and vitamins held at a comfortable temperature. It is exactly as attractive to a bacterium or a mould spore as it is to the cells you intend to grow — and far more so, because microbes divide in minutes while a plant or animal cell may take a day. A single contaminant that lands in a flask will therefore outgrow the culture completely within a day or two, consuming the nutrients, acidifying the medium and leaving nothing behind.
This is why aseptic technique is not one precaution among many in cell culture; it is the condition on which everything else depends. Sterilisation means killing or removing every living organism and spore from a material, and in a culture laboratory it has to be applied to the room, the apparatus, the transfer area, the medium, the heat-sensitive additives and the living tissue itself — each by a method suited to what that material can survive.
Setting Up the Laboratory
Aseptic work begins with how the laboratory itself is laid out. Four areas are needed, arranged so that material moves from the dirtiest operations towards the cleanest.
Washing and storage facilities. A separate area for washing and storing glassware, with a large sink and hot and cold running water, a distillation apparatus, a washing machine, a pipette washer, a drier and cleaning brushes. Detergent residue left on glassware is toxic to cultured cells, so thorough rinsing here matters.
Media preparation room. A bench area for chemicals, labware, culture vessels and closures, equipped with a pH meter, a balance, hot plates, stirrers, burners, an oven, an autoclave, a water bath and a centrifuge.
Transfer area. The area where sterile material is handled in the open. Older laboratories used a plastic box fitted with a UV tube and swabbed with ethanol. Modern laboratories use laminar air flow hoods instead, which give a much larger sterile working area supplied with HEPA-filtered air.
Culture room. The room in which cultures are kept while they grow, with control over temperature, humidity, illumination and air circulation. The temperature is generally kept at \(25^\circ\text{C}\) for plant cultures, with relative humidity in the range of 20–98% and proper ventilation.
Diagram
Fig. 10.6 – Layout of a tissue culture laboratory showing the washing and storage area, media preparation room, transfer area with a laminar air flow cabinet, and culture room, with arrows showing the direction in which material moves.
Sterilisation by Heat
Heat kills micro-organisms by denaturing their proteins and nucleic acids. It comes in two forms, and choosing between them is the first decision in any sterilisation.
Moist heat — steam under pressure — penetrates well and works comparatively quickly, but it wets whatever it touches. Dry heat does not wet the load, but water conducts heat far better than air does, so dry heat needs a much higher temperature and a much longer time to achieve the same result.
Sterilisation of glassware. Petri plates, vials, culture tubes and pipettes are sterilised in a hot air oven at \(160\text{--}180^\circ\text{C}\) for 2–4 hours. Glass tolerates dry heat easily, and the load comes out dry and ready to use.
Sterilisation of instruments. Metallic instruments such as forceps, scalpels and needles are dipped in ethanol and passed through a flame, a process called incineration, then allowed to cool before they touch the tissue. A red-hot instrument would scorch an explant, so the cooling step is not optional. Instruments may also be autoclaved.
Sterilisation of nutrient media. The medium is dispensed into containers, plugged with cotton or sealed with special membranes, and autoclaved at 15 psi and \(121^\circ\text{C}\) for 30 minutes. Note that the time depends on the volume and the nature of the load: small instruments need only 15–20 minutes, while a bulky flask of medium takes longer because steam must penetrate to its centre before the clock effectively starts. This is the ISC Paper II experiment on wet physical sterilisation, in which Luria Bertani medium is autoclaved and then cooled to about \(60^\circ\text{C}\) before being poured into plates.
Sterilisation of the Culture Room and Transfer Area
Surfaces cannot be autoclaved, so they are disinfected chemically. The culture room and the transfer area are cleaned first with detergent, then with 2% sodium hypochlorite solution, and finally wiped with alcohol. Exposure to UV light is used as an additional measure — the UV lamp inside a laminar air flow cabinet is switched on before work begins and switched off while anyone is present, because UV damages skin and eyes.
Note that alcohol is used as a 70% solution rather than as absolute alcohol for surface disinfection, which is the concentration the ISC practical syllabus specifies. This seems wrong at first — surely stronger is better — but alcohol kills by denaturing proteins, and that reaction needs water. Absolute alcohol dries so fast that it fixes the outer proteins of a microbial cell and seals the cell rather than penetrating it. The diluted solution stays wet longer and reaches the interior.
Sterilisation of Media, Vitamins and Other Heat-Labile Components
Some components of a medium cannot go anywhere near an autoclave. Enzymes, vitamins, hormones, growth regulators, antibiotics and certain amino acids are denatured or chemically altered by heat, and a medium autoclaved with these ingredients in it emerges sterile but biologically useless. Such substances are sterilised by filtration instead, which removes microbes mechanically at room temperature.
The membranes are made of cellulose acetate or cellulose nitrate, with pore sizes of 0.22, 0.45 and 0.5 micrometre; the \(0.22\ \mu\text{m}\) membrane is standard for sterilisation. The filter holder is made of stainless steel, glass or autoclavable plastic so that the assembly can itself be sterilised before use.
Other Types of Filters
Filtration is also used more generally to separate cells and biomass from a culture medium. The efficiency of any filtration depends on the size of the organism, the presence of other organisms, the viscosity of the medium and the temperature.
- Depth filters have a filamentous matrix such as glass wool, asbestos or filter paper. Particles are trapped within the thickness of the matrix while the rest of the liquid passes through. Filamentous fungi are easily removed this way.
- Absolute filters have pores of a defined size, smaller than the particles to be removed, so that retention depends on pore size rather than on the depth of the matrix. Bacteria can be removed from a culture medium with these.
- Rotary drum vacuum filters are used for broths containing particles of \(0.5\text{--}10\ \mu\text{m}\), such as yeast cells and filamentous fungi. A drum partly immersed in the broth rotates, and biomass is deposited on its surface as a cake that can be scraped off easily.
- Membrane filters retain cells on a membrane of defined pore size, as described above.
Filtration through membranes is classified by the size of particle retained.
Sterilisation of Living Material
The last item to be sterilised is the tissue itself, and it presents an obvious difficulty: whatever kills the microbes on its surface must leave the tissue alive. Explants cannot be autoclaved and cannot be put in a hot air oven or flamed. They are therefore given a surface sterilisation — a brief chemical treatment strong enough to kill the microbes carried on the outside of the tissue, followed immediately by rinsing so that the chemical does not have time to penetrate and kill the cells.
The disinfectants used include sodium hypochlorite (\(\text{NaOCl}\)), mercuric chloride (\(\text{HgCl}_2\)) and ethanol. After treatment the explant is washed 6–7 times in distilled or double distilled water to remove every trace of the chemical.
Flowchart
FC 10.2 – Choosing a sterilisation method: material to be sterilised → is it living tissue? → yes: brief chemical surface sterilisation and rinsing → no: is it damaged by heat? → yes: membrane filtration at 0.22 μm → no: does it tolerate wetting? → yes: autoclave at 121°C, 15 psi → no: hot air oven at 160–180°C for 2–4 hours
10.4 Culture Media: Types and Composition
A cell taken out of a living organism loses its supply lines. A plant cell in a leaf receives sugars manufactured by photosynthesis elsewhere in the plant and minerals drawn up by the roots; an animal cell receives everything it needs from blood. Once that cell is placed in a glass vessel, all of it must be provided in dissolved form. The solution that does this is the culture medium.
Plant cells growing in vitro are, for practical purposes, heterotrophic — they are cut off from the whole-plant photosynthetic supply and must be fed a carbon source. It is the culture medium that allows a plant cell to express its totipotency and develop into a complete new plant, and it is the medium that governs in vitro growth and morphogenesis of plant tissues.
No single medium suits every kind of culture. Composition is altered according to the type of culture and the physiological requirements of the cells being grown, which is why a laboratory stocks several recipes rather than one.
Types of Culture Media
Media are classified by where their ingredients come from — whether every component is a known chemical, or whether some are natural materials of undefined composition.
1. Synthetic medium (defined medium). A medium whose components are chemicals synthesised in the laboratory, each present in a known amount. Because the exact composition is known, results obtained on such a medium can be reproduced anywhere. Czapek Dox medium, which contains agar and is commonly used for fungal culture, is a synthetic medium.
2. Semi-synthetic medium (differential medium). A medium containing synthetic chemicals together with natural complex compounds. It may be liquid or solid; a liquid culture medium is called a broth. Nutrient broth and brain heart infusion broth are examples, and such broths contain beef extract, yeast extract, peptone, potato or casein by-products.
3. Natural medium (complex medium). A medium obtained by using a natural complex compound whose precise composition is not known — soyabean seed extract broth, V8 juice extract, broth prepared from vegetables, or soil extract broth. Media of this kind supply carbon sources, nitrogen sources, growth factors, minerals, inducers, repressors, water and antifoams all together.
Composition of a Culture Medium
Whatever its type, a medium is assembled from the same categories of ingredient. Each category answers a specific need of the cell.
Inorganic Nutrients
Minerals are required either in large quantity or in very small quantity, and the two groups are separated by a concentration boundary. Nutrients needed at more than 0.5 mmole per litre are macronutrients; those needed at less than 0.5 mmole per litre are micronutrients.
They are supplied as salts — commonly \(\text{KNO}_3\), \(\text{KH}_2\text{PO}_4\), \(\text{NaH}_2\text{PO}_4\), \(\text{CaCl}_2 \cdot 2\text{H}_2\text{O}\), \((\text{NH}_4)_2\text{SO}_4\), \(\text{FeSO}_4\), \(\text{ZnSO}_4 \cdot 7\text{H}_2\text{O}\), \(\text{NaEDTA} \cdot 2\text{H}_2\text{O}\), \(\text{KI}\) and \(\text{MgSO}_4 \cdot 7\text{H}_2\text{O}\). Other elements such as manganese, molybdenum, boron, copper, cobalt and nickel are also provided.
Among the micronutrients, iron is the most critical and the most troublesome. Simple iron salts precipitate out of the medium as insoluble hydroxides at the pH at which cultures are grown, so the iron becomes unavailable just when the cells need it. This is why iron and copper are supplied in chelated form — bound to a carrier such as EDTA, which holds the metal in solution and releases it gradually to the cells.
Carbon and Energy Source
Since cultured plant cells cannot meet their own carbon requirement, an external carbon source must be supplied. Sucrose is the usual choice, and glucose is sometimes used. Fructose, lactose, maltose, galactose and raffinose are used rarely.
The sugar does two jobs at once: it is the respiratory substrate from which the cell draws energy, and it is the skeleton from which new cell material is built. It also contributes to the osmotic strength of the medium, which is why the sugar concentration cannot simply be raised at will.
Organic Supplements
Vitamins. Vitamins are required for good growth because they form the coenzymes that many metabolic reactions depend on. The vitamin B complex is essential; vitamin E, folic acid, ascorbic acid, myo-inositol and para-amino benzoic acid are also used.
Amino acids. Amino acids stimulate cell growth and help in establishing cell lines. They act as a readily usable source of nitrogen, saving the cell the energy of reducing nitrate and assembling amino groups itself.
Organic acids. Citric, malic, succinic, fumaric and pyruvic acids enhance the growth of cultured cells. These are intermediates of respiration, so supplying them gives the cell’s central metabolism a direct start.
Organic extracts. Casein protein, coconut milk, yeast extract, malt extract, orange juice, tomato juice, potato extract and ground banana are added to some media. Activated charcoal is also used: it stimulates growth and differentiation of certain plant cells by adsorbing toxic or inhibitory substances that the cultured tissue itself releases into the medium.
Antibiotics. Antibiotics such as streptomycin and kanamycin may be added to prevent the growth of microbes. They are an insurance policy, not a substitute for aseptic technique — a contaminated culture is still a failed culture, and heavy antibiotic use only selects for resistant contaminants.
Growth Regulators
The culture medium also contains plant hormones that control growth and differentiation. The two groups the syllabus requires are auxins and cytokinins.
Auxins mainly facilitate cell division and root differentiation, or root initiation. The commonly used auxins are IAA (indole acetic acid), IPA (indole pyruvic acid), IBA (indole butyric acid), NAA (naphthalene acetic acid), NOA (naphtho oxyacetic acid), p-CPA (para chlorophenoxy acetic acid), 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxyacetic acid). IBA and NAA are very widely used for root initiation and shoot proliferation, while 2,4-D and 2,4,5-T are effective for inducing the growth of callus.
Cytokinins are used for cell division and differentiation, and they direct the cell towards various developmental functions. Commonly used cytokinins are BAP (benzyl amino purine), 2iP (iso pentenyl adenine), kinetin (furfurylamino purine) and zeatin.
What matters most in practice is not either hormone alone but the balance between them. A medium with a relatively high auxin content pushes a culture towards root formation; a medium with a relatively high cytokinin content pushes it towards shoot formation; a balance between the two keeps the tissue growing as undifferentiated callus. This relationship is summarised in Fig. 10.7 and is a favourite subject for case-based questions, in which a culture forms roots when shoots were wanted and you are asked what to change.
Diagram
Fig. 10.7 – Effect of the auxin to cytokinin ratio on a callus culture: high auxin to cytokinin ratio leading to root formation, balanced ratio maintaining callus, high cytokinin to auxin ratio leading to shoot formation.
Solidifying Agents
A medium may be used as a liquid or set into a gel. Agar, a polysaccharide obtained from red algae such as Gelidium and Gerardiana, is the most commonly used solidifying or gelling agent. It suits the purpose because it does not react with the constituents of the medium, it is not digested by plant enzymes, and it remains stable at culture temperature. Gelatin, biogel (polyacrylamide pellets), phytagel, gelrite and purified agarose are used less frequently.
Solidification matters for a specific physiological reason. In a liquid medium that is not agitated, a piece of tissue sinks and becomes submerged, and the cells at its centre die for want of oxygen. On a gelled medium the explant rests at the surface, drawing water and nutrients from below while remaining in contact with air above. You will meet the same principle in the ISC Paper II practical on preparing culture media as plates, slants and suspensions.
Maintenance of pH
The optimum pH for most cultures lies in the range of 5.0 to 6.0. Different media are set to different values within and around this range: MS medium has a pH of 5.8, while White’s medium has a pH of 7.0.
Even a slight change in pH affects the growth of cells, chiefly because it changes the uptake of ions — the charge on a mineral and the charge on the transport proteins in the cell membrane both depend on the acidity of the surrounding solution. pH also governs the activity of every enzyme inside the cell, and, in a practical sense, whether the agar sets at all: agar gels poorly in a strongly acidic medium. The pH is therefore measured and adjusted with a pH meter before the medium is autoclaved, since autoclaving itself shifts the value slightly.
10.5 Media for Plant, Microbial and Animal Cell Culture
Having seen what goes into a medium in general, we can now look at the particular recipes a laboratory actually uses. Five are named by the syllabus: LB medium for microbial culture, MS and White’s media for plant culture, and RPMI-1640, DMEM and the supplement FBS for animal cell culture. What you need from each is a brief idea — what it is, what it contains in outline, and what it is used for.
Media for Microbial Culture
LB (Luria-Bertani) Broth and LB Agar
LB medium is the standard general-purpose medium for growing bacteria, and it is the medium in which Escherichia coli is routinely cultured in molecular biology laboratories. It is a semi-synthetic medium: the sodium chloride is a defined chemical, while tryptone and yeast extract are natural products of undefined exact composition.
Composition of LB liquid medium (broth)
| Component | Quantity |
|---|---|
| \(\text{H}_2\text{O}\) | 950 mL |
| Tryptone | 10 g |
| NaCl | 10 g |
| Yeast extract | 5 g |
The reagents are combined and shaken until the solutes have dissolved. The pH is adjusted to 7.0 with 5 N NaOH — about 0.2 mL is needed — and the final volume made up to 1 litre with \(\text{H}_2\text{O}\). The medium is then sterilised by autoclaving for 20 minutes at 15 psi on the liquid cycle.
Each ingredient has a defined job. Tryptone, a casein enzymic hydrolysate, supplies peptides and peptones as the source of amino acids and nitrogen. Yeast extract supplies the vitamin B complex. Sodium chloride provides sodium ions for membrane transport and maintains the osmotic equilibrium of the medium, so that bacterial cells neither swell nor shrink.
Luria Bertani agar medium is obtained simply by adding a suitable amount of agar — about 15 g per litre — to the liquid medium. The same nutrients are present; the agar only converts the broth into a solid surface on which colonies can develop as separate, countable spots.
You will prepare this medium yourself in the ISC Paper II practical on culture media, where LB medium is autoclaved, cooled to about \(60^\circ\text{C}\), and then poured into pre-sterilised Petri dishes under a laminar air flow cabinet, or into tubes held at a slant until they set.
Media for Plant Tissue Culture
MS Medium
MS medium was developed by Murashige and Skoog. It is used widely for many types of cultures and is meant for inducing organogenesis and the regeneration of plants in cultured tissues. Its distinguishing feature is a high concentration of inorganic salts — particularly nitrate and ammonium — which supports the vigorous growth needed when a callus is to be pushed into forming shoots and roots. Its pH is set at 5.8.
White’s Medium
White’s medium was developed by White and is one of the earliest plant tissue culture media, developed for root culture. Compared with MS it is a low-salt medium, and its pH is 7.0.
Read the nitrogen rows of that table together. White’s medium supplies 80 mg per litre of potassium nitrate and no ammonium nitrate at all; MS supplies 1900 mg per litre of the first and 1650 mg per litre of the second. That single difference is what makes MS a regeneration medium and White’s a maintenance medium for roots — organ formation is nitrogen-hungry, while an isolated root growing slowly is not.
Notice also that MS supplies iron as \(\text{FeSO}_4 \cdot 7\text{H}_2\text{O}\) together with \(\text{Na}_2\text{EDTA} \cdot 2\text{H}_2\text{O}\), in matched amounts. This is the chelated iron described in the previous section: the EDTA is present solely to keep the iron dissolved and available.
Media for Animal Cell Culture
Animal cells are more demanding than plant cells and less able to make their own metabolites, so their media are richer and more closely defined, and are usually supplemented with serum.
DMEM (Dulbecco’s Modified Eagle Medium)
DMEM is one of the most widely used culture media for growing animal cells in the laboratory. It is a modified form of Eagle’s medium containing higher concentrations of amino acids, vitamins and glucose, which support rapid cell growth and division. It is especially suitable for culturing cells such as fibroblasts and stem cells, and many continuous cell lines.
DMEM often contains phenol red as a pH indicator, which gives the medium its familiar pink colour and allows the acidity of a culture to be judged at a glance, as in Fig. 10.8. Depending on the requirement, DMEM is available in low-glucose and high-glucose forms, which makes it versatile for different cell types and experimental conditions. It is used as a medium for adherent cells such as fibroblasts and epithelial cells, and for stem cells and continuous cell lines.
Diagram
Fig. 10.8 – Three culture flasks of DMEM viewed side by side, showing the phenol red indicator as pink in fresh medium, orange in a healthily growing culture, and yellow in an exhausted or contaminated one; label the pH corresponding to each.
RPMI-1640 (Roswell Park Memorial Institute Medium)
RPMI-1640 is specially designed for the growth of suspension cells, particularly human lymphocytes and other blood cells. It contains a balanced mixture of amino acids, vitamins, inorganic salts and glucose, along with buffering systems that help maintain a stable pH.
Unlike DMEM, it is better suited to cells that grow freely in the medium rather than attaching to surfaces, and its composition supports cells that require specific nutrients and conditions, especially those derived from the immune system. It is commonly used in immunological studies, cancer research and cell signalling experiments, and for culturing suspension cells such as lymphocytes, T-cells, B-cells and hybridomas.
FBS (Foetal Bovine Serum)
FBS is not a complete medium by itself but an essential supplement added to culture media such as DMEM and RPMI-1640. It is derived from the blood of foetal calves and is rich in growth factors, hormones, proteins and the nutrients required for cell survival and proliferation.
FBS also provides attachment factors that help cells stick to the culture surface, and it protects them from mechanical damage during handling. It is typically added in concentrations of 5–20% of the culture medium. Because of its complex and rich composition, FBS plays a crucial role in keeping cells healthy and actively dividing in vitro, and it is used as a nutrient supplement for almost all cell types, supporting both growth and survival.
10.6 Preparation of Culture Medium
Knowing a recipe is not the same as being able to make the medium. The order in which components are added, the point at which the pH is set, the temperature at which the medium is poured — each of these affects whether the finished medium works, and each has a reason behind it. This section follows a medium from the weighing balance to the culture vessel.
The Sequence of Preparation
Preparation begins under conditions that will eventually have to be aseptic, and with the components measured out in their correct ratios. The different components are selected, weighed and transferred into a suitable container.
Agar, sugar and the other desired supplements are then added, and the final volume is made up using distilled or double distilled water. Ordinary tap water is never used, because its dissolved salts are neither known nor constant, and they would silently alter the mineral composition that the recipe took care to specify.
The pH is adjusted as the requirement demands, using dilute acid or alkali and a pH meter. Two points about the order matter here. The pH is set before autoclaving, because heating shifts the value slightly and a medium adjusted afterwards can no longer be sterilised. It is also set before the agar melts and gels, since a glass electrode cannot give a reliable reading in a setting gel.
The medium is then sterilised in an autoclave at 15 psi, at a temperature of \(121^\circ\text{C}\), for a period of 30 minutes to one hour depending on the volume of the load. A large flask needs the longer time simply because steam takes longer to raise its centre to the sterilising temperature.
If plastic culture vials are being used, they are placed inside glassware and then autoclaved, so that the softened plastic keeps its shape and remains supported through the cycle.
Any heat-labile component — vitamins, hormones, antibiotics, enzymes — is filter-sterilised separately through a \(0.22\ \mu\text{m}\) membrane, as described in Section 10.3, and added only after the autoclaved medium has cooled.
Flowchart
FC 10.3 – Preparing a culture medium: weigh out components in correct ratios → dissolve in distilled water → add sugar and supplements → make up to final volume → adjust pH with a pH meter → add agar if a solid medium is required → dispense into containers and plug → autoclave at 121°C and 15 psi → cool to about 60°C → add filter-sterilised heat-labile components → dispense as plates, slants or suspension
Forms in Which Medium Is Dispensed
The same sterile medium can be presented to cells in three different physical forms, and the choice depends on what the culture is meant to do. These are the three forms you prepare in the ISC Paper II practical on culture media, using Luria Bertani medium.
The medium is first autoclaved and then cooled to about \(60^\circ\text{C}\) before it is dispensed. This cooling step is deliberate. Agar stays molten well below its boiling point, so the medium is still pourable at \(60^\circ\text{C}\); but if it were poured hotter, steam would condense heavily on the lid of every dish and the drops falling back would spread contaminants across the surface. Cooling also protects any heat-sensitive additive that must go in at this stage.
Petri plate. Nutrient plates are prepared by pouring the cooled medium into pre-sterilised Petri dishes under a laminar air flow cabinet, where it is left undisturbed to set into a flat, level surface. A plate presents a large exposed surface area, which is what allows separated cells to grow into distinct, countable colonies.
Slant. To prepare slants, the medium is poured into several test tubes, which are plugged and kept in a tilted position at an angle of \(45^\circ\) until it sets. Setting the medium at a slope gives a much larger growth surface than a flat-bottomed tube would, while the tube itself remains easy to plug, handle and store — which is why slants are used for maintaining and storing stock cultures.
Suspension. A liquid medium with no gelling agent is dispensed into flasks or tubes and inoculated directly. The cells grow dispersed through the liquid, which is kept in motion so that oxygen and nutrients reach every cell, as discussed in Section 10.4.
Diagram
Fig. 10.9 – Three forms of culture medium: a Petri plate with a flat set surface, a plugged test tube containing medium set at a 45° slant, and a conical flask of liquid medium with cells dispersed through it; label each and mark the growth surface in the first two.
10.7 Cell Counting, Cell Viability and Cell Sorting
Once cells are growing in a suitable medium, they multiply, and three questions immediately follow. How many cells are there? How many of them are actually alive? And, if the culture contains more than one kind of cell, how can one particular kind be picked out from the rest? The syllabus prescribes one method for each — direct counting by haemocytometer, viability testing by Evan’s blue stain, and cell sorting by FACS.
These are not merely descriptive matters. Almost every quantitative statement made about a culture — its growth rate, the dose of a drug per cell, the number of cells seeded into a bioreactor — begins with a count.
Cell Counting by Haemocytometer
The principle behind the method is simple: if you can trap a known volume of cell suspension and count the cells inside it, you can calculate how many cells are present in any larger volume.
A haemocytometer, also called a counting chamber, is a thick glass microscope slide with a rectangular indentation that creates a shallow chamber. The floor of this chamber is engraved with a grid of precisely spaced perpendicular lines, so the area bounded by any set of lines is exactly known. Raised edges on either side hold a coverslip exactly 0.1 mm above the marked grid, which fixes the depth of liquid over the grid. Because both the area and the depth are known, every square encloses a defined volume, as shown in Fig. 10.10.
Diagram
Fig. 10.10 – A haemocytometer in two views: (A) from above, showing the moats, the two ruled grid areas and the coverslip in position; (B) in section, showing the coverslip supported above the grid with the chamber depth of 0.1 mm marked.
Reading the Grid
The ruled area consists of several large squares of \(1 \times 1\) mm, that is \(1\ \text{mm}^2\), and these are subdivided in three ways: into squares of \(0.25 \times 0.25\) mm (\(0.0625\ \text{mm}^2\)), \(0.25 \times 0.20\) mm (\(0.05\ \text{mm}^2\)) and \(0.20 \times 0.20\) mm (\(0.04\ \text{mm}^2\)). The central \(0.20 \times 0.20\) mm marked, \(1 \times 1\) mm square is further subdivided into squares of \(0.05 \times 0.05\) mm (\(0.0025\ \text{mm}^2\)), which are small enough to count individual cells in a dense suspension without losing track.
The volume above one large square follows directly from its area and the chamber depth:
\[V = 1\ \text{mm} \times 1\ \text{mm} \times 0.1\ \text{mm} = 0.1\ \text{mm}^3 = 10^{-4}\ \text{mL}\]
From this, the concentration of cells in the original suspension is
\[\text{Cells per mL} = \frac{\text{total cells counted}}{\text{number of large squares counted}} \times 10^{4} \times D\]
where \(D\) is the dilution factor — the number of times the suspension was diluted before loading. The factor \(10^{4}\) is simply the reciprocal of the volume above one large square, in millilitres. Counting several squares and taking the mean matters, because cells never distribute themselves perfectly evenly and one square is not a fair sample.
Where cultured plant cells occur as clumps rather than as single cells, the aggregates are first broken up by treating them with pectinase or chromic acid, since a clump of twenty cells counted as one object gives a badly wrong answer.
This method gives an accurate total cell number, but it has two acknowledged drawbacks. It is tedious and time-consuming when large numbers of cells must be counted, and by itself it cannot distinguish between living and dead cells — every cell in the field is counted alike.
Assessment of Cell Viability
A count tells you how many cells are present; it does not tell you how many are alive. Viability of cells in the culture medium is assessed by direct microscopic observation or, more usefully, by staining.
Most viability stains work on a single idea called dye exclusion. A living cell has an intact plasma membrane that keeps the dye out, so it stays unstained. A dead or damaged cell has a leaky membrane, the dye enters freely, and the cell is coloured. Counting the coloured cells therefore counts the dead ones.
Evan’s Blue Staining
Evan’s blue is the viability stain prescribed by the syllabus, and it is the one used in the ISC Paper II practical on cell viability testing. Evan’s blue dye, used as a 0.025% w/v solution, stains the dead or damaged cells and does not stain the living or viable cells.
Viability is then expressed as a percentage:
\[\text{Percentage viability} = \frac{\text{number of unstained cells}}{\text{total number of cells}} \times 100\]
Other Methods of Assessing Viability
Trypan blue exclusion method. This is a simple and commonly used method to determine cell viability, based on the principle of cell membrane integrity. Living cells have an intact cell membrane that prevents the entry of the trypan blue dye, so they remain colourless or unstained. Dead cells have damaged membranes, allowing the dye to enter and stain them blue. By observing the staining pattern, viable and non-viable cells can be easily distinguished.
Phase contrast microscopy. For detecting the viability of cultured cells, the presence of healthy nuclei is observed under a phase contrast microscope, without any staining at all.
Fluorescein diacetate (FDA). The cell suspension is incubated with fluorescein diacetate, which is cleaved by the esterase enzyme of living cells. This results in the emission of green fluorescence under ultraviolet light, and because the product accumulates inside living cells, it helps in the detection of viable cells.
Cell Sorting
Animals and plants contain many different types of cells and tissues. To examine one specific type, the cells of that type must be isolated, or sorted, out of the mixture — and that begins by getting the tissue apart into single cells in the first place.
Cells of a uniform type are released from a tissue using proteolytic enzymes, or agents that bind or chelate the calcium ions on which cell-to-cell adhesion depends. Once these adhesive boundaries are broken, the tissue can be dissociated into single cells by gentle shaking.
Some separations then rely only on physical differences: smaller from larger, or denser from lighter cells, by simple centrifugation. Other techniques exploit the tendency of different cell types to adhere more strongly to plastic or to glass than other cells do. But the sophisticated methods involve labelling specific cells with antibodies or immunoglobulin proteins coupled to a fluorescent dye, and then sorting them by fluorescence activated cell sorter (FACS).
Diagram
Fig. 10.11 – A fluorescence activated cell sorter; label the cell suspension inlet, ultrasonic nozzle vibrator, sheath fluid, laser, detectors and analyser, drop charging signal, the deflecting plates at \(-2000\ \text{V}\) and \(+2000\ \text{V}\), the two cell collectors and the flask for undeflected droplets.
The specificity of the whole machine rests on one step — the antibody. An antibody binds only to its own antigen, so labelling with a fluorescent-tagged antibody marks only the cells carrying that surface antigen. Everything the sorter does afterwards is electronics and fluid mechanics; the biological decision has already been made at the moment of binding.
10.8 Cryopreservation
A culture that is growing is also a culture that is changing. Cells must be subcultured onto fresh medium again and again; every transfer costs labour, risks contamination, and gives mutations a chance to accumulate, so that after many passages the line is no longer quite what it was. For any material that must be kept unchanged for years — a valuable cell line, a rare plant variety, an engineered strain — growth itself is the problem.
Cryopreservation solves it by stopping growth altogether without killing the cells. The word comes from the Greek kryos, meaning frost.
The Need for Cryopreservation
The stock of genes present in a crop and its related species is its germplasm, and it is the raw material from which new commercial varieties are bred. Conventionally germplasm is conserved as seed, but this is not always possible: many crops produce recalcitrant or short-lived seeds, clonal crops cannot be conserved faithfully as seed because their seed is genetically heterogeneous, and roots and tubers lose viability while demanding a great deal of cold storage space. Material modified by genetic engineering may also be unstable and needs to be conserved intact.
For all such material, freeze preservation is one of the approaches used, alongside desiccated somatic embryos or artificial seeds, slow growth cultures, and the storage of DNA or gene clones. An international organisation, IBPGR (International Board of Plant Genetic Resources), was established to support the collection, conservation and utilisation of plant genetic resources throughout the world.
The Principle: Why Cold Alone Is Not Enough
The principle involved is to bring the plant cell and tissue cultures to a state of zero metabolism, or a non-dividing state, by reducing the temperature. But cooling a cell rapidly to a very low temperature usually kills it, and the reason is water.
As a cell freezes, water inside it forms ice crystals, and growing crystals pierce membranes and organelles from within. At the same time, as pure water separates out as ice, the solutes left behind in the remaining liquid become more and more concentrated, and this rising intracellular concentration of solutes damages the cell chemically even where the crystals do not tear it. Cells may also leak solutes outwards during freezing.
Cryoprotectants are added to counter this. They lower the freezing point and the supercooling point of water, which retards the formation of ice crystals during the process of cryopreservation. The cryoprotectants used include DMSO (dimethyl sulphoxide), glycerol, ethylene, propylene, sucrose, mannose, glucose, proline, acetamide and mannitol; of these, DMSO, sucrose and glycerol are the most widely used.
Precautions for Cryopreservation
- Formation of ice crystals inside the cell should be prevented, as they cause injury to the cell organelles.
- A high intracellular concentration of solutes may also damage the cell.
- Certain solutes leak out from the cell during freezing.
- Cryoprotectants themselves affect the viability of cells, since they are chemicals the cell would not normally tolerate at such concentrations — which is why they are washed out promptly after thawing.
- The physiological status of the plant material is also important; cells taken at the wrong stage of growth survive poorly.
Temperatures Used
Cryopreservation can be carried out at several temperatures:
- Over solid carbon dioxide at \(-79^\circ\text{C}\)
- In low temperature deep freezers at \(-80^\circ\text{C}\)
- In vapour phase nitrogen at \(-150^\circ\text{C}\)
- In liquid nitrogen at \(-196^\circ\text{C}\)
Among these, liquid nitrogen is the most commonly used. The reason is not simply that colder is better. Above about \(-130^\circ\text{C}\), ice crystals continue to form and grow inside the cells and reduce their viability, so storage must be below this threshold — around \(-150^\circ\text{C}\) — for the material to remain genuinely stable. Liquid nitrogen at \(-196^\circ\text{C}\) is comfortably below that limit, and is therefore most suitable for long-term storage.
Steps in Cryopreservation
(a) Development of sterile tissue cultures. Any tissue from a plant can be used for preservation — meristems, embryos, ovules, endosperms, seeds, protoplasts or suspension cell cultures. Among these, meristematic cells and suspension cell cultures selected in the late lag phase or the log phase are the most suited, because actively growing cells with small vacuoles and little free water survive freezing best.
(b) Addition of cryoprotectants and pretreatment. Cryoprotectants are added to prevent the damage caused to the cells by freezing or thawing, as described above. A typical pretreatment grows the culture for a few days on a medium containing mannitol before the cryoprotectant mixture — for instance DMSO with glycerol and sucrose — is applied.
(c) Freezing. Freezing is obtained in various ways.
- Slow freezing method: the tissue or plant part is subjected to freezing at a slow rate of \(0.5\text{--}5^\circ\text{C}\) per minute from \(0^\circ\text{C}\) to \(-100^\circ\text{C}\), and is then transferred to liquid nitrogen. It is used successfully for the cryopreservation of suspension cultures.
- Rapid freezing method: the temperature falls from \(-30^\circ\) to \(-100^\circ\text{C}\) per minute. Because the freezing process is very fast, only small ice crystals form within the cells. It is frequently used for the cryopreservation of shoot tips and somatic embryos.
- Stepwise freezing method: a combination of slow and rapid freezing procedures carried out in a stepwise manner. It is used for the cryopreservation of suspension cultures, shoot tips and buds.
- Dry freezing method: dehydrated cells are subjected to freezing, as they can withstand very low temperature — having less water, they have less ice to form.
(d) Storage. The frozen cells or tissues are kept for storage at temperatures in the range of \(-70^\circ\) to \(-196^\circ\text{C}\). Since ice crystals form inside the cells at temperatures above \(-130^\circ\text{C}\) and reduce viability, the cells are stored at a temperature around \(-150^\circ\text{C}\) in liquid nitrogen, which is most suitable for long-term storage.
(e) Thawing. Thawing is carried out by plunging the frozen ampoules into a warm bath at \(37^\circ\text{--}45^\circ\text{C}\) with vigorous swirling. As soon as thawing occurs and the ice melts, the ampoules are quickly transferred to a water bath at \(5^\circ\text{--}25^\circ\text{C}\) to prevent damage to the cells at high temperature. Thawing is deliberately rapid for the same reason that freezing is controlled: passing quickly through the intermediate temperature range gives ice crystals no opportunity to re-form and grow.
(f) Reculture. The thawed germplasm is washed several times to remove the cryoprotectants, and the material is then recultured in a fresh medium.
(g) Measurement of survival or viability. The viability of the frozen cells can be measured at any stage of cryopreservation, after thawing or after reculture, using the staining methods of the previous section. The best indicator, however, is not a stain but a behaviour: entry into cell division and regrowth in reculture. A cell that excludes a dye may still be too damaged to divide, and it is division that the whole exercise was meant to preserve.
(h) Plant regeneration. The plant parts are cryopreserved so as to regenerate the desired plant. The cells or tissues are carefully nursed and grown by adding suitable growth promoting substances and maintaining appropriate environmental conditions.
Diagram
Fig. 10.12 – A typical cryopreservation protocol shown as a cycle: (1) culture under standard conditions, (2) pregrow for 5 days in medium containing 6% mannitol, (3) cryoprotect with 0.5% DMSO, 0.5 M glycerol and 1 M sucrose in culture medium, (4) transfer cells and cryoprotectant to an ampoule and freeze in stages, (5) store in liquid nitrogen, (6) thaw in warm water, (7) pour onto semi-solid medium, (8) transfer recovered culture to liquid medium.
Flowchart
FC 10.4 – The cryopreservation cycle: sterile culture → pretreatment and cryoprotectant addition → controlled freezing → storage in liquid nitrogen at −196°C → rapid thawing at 37–45°C → washing and reculture → viability check by regrowth → plant regeneration
Applications of Cryopreservation
Cryopreservation is used to preserve germplasm in the form of seeds, cells, tissues, explants and callus. These preserved germplasms can be used to regenerate a new plant. At a very low temperature the germplasm ceases its metabolic activities, so it can remain inactive for a long duration of preservation, and once suitable conditions for metabolism are restored, it can be regenerated into new organisms.
10.9 Summary
Cell culture technology grows cells outside the parent organism, so every condition normally supplied by that organism — nutrients, temperature, pH, gas supply and protection from microbes — must be provided by laboratory instruments and media.
A centrifuge separates on the principle of sedimentation, denser material collecting as a pellet; it is used to separate cells from medium and organelles from one another.
A laminar air flow cabinet passes air through a HEPA filter and moves it smoothly across the working area, and is used for inoculation, transfer and pouring of sterile media. A biosafety cabinet draws air inward instead, so that it protects the worker as well as the culture.
An autoclave sterilises by moist heat, holding steam at \(121^\circ\text{C}\) under 15 psi; a hot air oven sterilises by dry heat at \(160\text{--}180^\circ\text{C}\) for 2–4 hours. Moist heat penetrates better, so it needs a lower temperature and a shorter time.
A pH meter measures the acidity of media and buffers through a glass electrode and must be calibrated against standard buffers before use.
A magnetic stirrer with hot plate dissolves and warms media; an electronic weighing balance weighs components to high precision; a vortex mixer resuspends cells and mixes reagents rapidly.
A microfiltration unit sterilises heat-labile solutions by passing them through a membrane of \(0.22\ \mu\text{m}\) pore size, which retains microbes without heating the solution.
An incubator controls temperature and humidity; a \(\text{CO}_2\) incubator additionally controls carbon dioxide, which is what holds the bicarbonate-buffered medium of animal cells at the correct pH near \(37^\circ\text{C}\).
An inverted microscope carries its objectives below the stage, so living cells can be viewed through the base of an unopened culture vessel.
T flasks are flat, surface-treated polystyrene flasks with filter vent caps, used to propagate animal cells as a monolayer; they are supplied as T25, T75, T175 and T225.
A bioreactor is a large closed vessel in which cells are grown at scale under controlled conditions so that products can be harvested commercially.
In a stirred tank bioreactor, impellers on a motor-driven shaft mix and aerate the broth; it gives efficient gas transfer and easy control, but consumes much energy and foams heavily. In a sparged tank bioreactor, rising gas bubbles do the mixing; it needs no agitator shaft and removes heat faster, but demands a large supply of sterile, pressurised air.
The components of a bioreactor each counter one change the growing culture causes: a stainless steel body resists corrosion, baffles break the whirlpool and improve aeration, impellers mix, the sparger admits air at the base, the antifoam probe controls foaming, the pH probe corrects drifting acidity, the cooling jacket removes metabolic and mechanical heat, and additional ports admit steam and allow viewing.
Sterilisation kills or removes all micro-organisms and their spores, and is applied to the culture room, apparatus, transfer area, media, heat-labile additives and living material — each by a method the material can survive.
A tissue culture laboratory is arranged as washing and storage facilities, a media preparation room, a transfer area and a culture room, the culture room generally being held near \(25^\circ\text{C}\) for plant cultures.
Glassware is sterilised in a hot air oven, instruments by incineration in ethanol and flame or by autoclaving, media by autoclaving, and surfaces by detergent, 2% sodium hypochlorite, 70% alcohol and UV exposure.
Heat-labile components — vitamins, hormones, enzymes and antibiotics — are cold-sterilised by membrane filtration and added after the autoclaved medium has cooled.
Living material is surface sterilised briefly with sodium hypochlorite, mercuric chloride or ethanol, then washed 6–7 times in distilled water so that no residue remains to kill the tissue.
A culture medium supplies the nutrients cells need in vitro. Media are synthetic (defined), semi-synthetic (differential) or natural (complex), depending on whether their components are known chemicals or natural extracts.
Inorganic nutrients are macronutrients when required above 0.5 mmole per litre and micronutrients below it; iron and copper are supplied in chelated form so that they stay soluble and available.
Sucrose is the usual carbon and energy source for plant cultures. Organic supplements include vitamins as coenzyme precursors, amino acids as a nitrogen source, organic acids, organic extracts and antibiotics.
Auxins such as IAA, IBA, NAA and 2,4-D promote cell division and root initiation; cytokinins such as BAP, 2iP, kinetin and zeatin promote cell division and differentiation. It is the ratio between them that decides whether a culture forms roots, remains callus, or forms shoots.
Agar is the usual solidifying agent because it does not react with the medium, is not digested by plant enzymes and is stable at culture temperature; solidification keeps the explant at the surface instead of drowning it.
Most cultures are grown at a pH of 5.0–6.0, MS medium being set at 5.8 and White’s at 7.0; pH governs ion uptake, enzyme activity and the setting of agar, and is adjusted before autoclaving.
LB medium, containing tryptone, yeast extract and sodium chloride, is the standard microbial medium; adding agar to the broth converts it into LB agar for surface growth of colonies.
MS medium is a high-salt plant medium used for organogenesis and regeneration; White’s medium is a low-salt medium developed for root culture.
DMEM is an animal medium rich in amino acids, vitamins and glucose, used for adherent cells and often containing phenol red as a pH indicator; RPMI-1640 is designed for suspension cells such as lymphocytes and hybridomas; FBS is not a medium but a serum supplement, added at 5–20% to provide growth factors, hormones and attachment factors.
Media are prepared by dissolving weighed components in distilled water, making up the volume, adjusting the pH, adding agar if required, autoclaving, cooling to about \(60^\circ\text{C}\), and then dispensing as Petri plates, slants set at \(45^\circ\), or liquid suspensions.
A haemocytometer counts cells directly: its engraved grid and fixed chamber depth of 0.1 mm enclose a known volume, so that \[\text{Cells per mL} = \frac{\text{total cells counted}}{\text{number of large squares counted}} \times 10^{4} \times D\] where \(D\) is the dilution factor. The method gives the total count but cannot distinguish living cells from dead ones.
Viability is assessed by dye exclusion. Evan’s blue dye at 0.025% w/v stains dead and damaged cells but not viable ones, and \[\text{Percentage viability} = \frac{\text{number of unstained cells}}{\text{total number of cells}} \times 100\] Trypan blue works on the same principle of membrane integrity, while fluorescein diacetate instead detects esterase activity in living cells as green fluorescence.
Cell sorting begins by dissociating tissue into single cells using proteolytic enzymes or calcium-chelating agents. In FACS, cells labelled with fluorescent antibodies pass in single file through a laser beam, the stream breaks into droplets that are charged according to what is detected, and the droplets are deflected between charged plates into separate collectors.
Cryopreservation stores cells at very low temperature in a zero-metabolism state, so that valuable germplasm and cell lines can be kept unchanged without repeated subculture.
Cells are killed during freezing by intracellular ice crystals and by the rising concentration of solutes, not by cold itself; cryoprotectants such as DMSO, glycerol and sucrose lower the freezing point and retard crystal formation.
Freezing may be slow (\(0.5\text{--}5^\circ\text{C}\) per minute), rapid, stepwise or dry. Storage is in liquid nitrogen at \(-196^\circ\text{C}\), since ice crystals continue to form above about \(-130^\circ\text{C}\).
Thawing is rapid, in a bath at \(37^\circ\text{--}45^\circ\text{C}\), followed by transfer to a cooler bath; the cryoprotectants are then washed out, the material is recultured, and viability is judged by entry into cell division and regrowth rather than by staining alone.
Flowchart
FC 10.5 – The work of a cell culture laboratory: prepare medium (weigh → dissolve → adjust pH → autoclave or filter-sterilise) → sterilise apparatus, room, transfer area and living material → inoculate under laminar air flow → incubate in an incubator or CO₂ incubator, or scale up in a bioreactor → monitor by counting on a haemocytometer and testing viability with Evan’s blue → sort a required cell type by FACS → preserve the line by cryopreservation in liquid nitrogen
10.10 Key Terms
Sedimentation the settling of denser particles out of a suspension, which is the principle on which a centrifuge separates materials.
Laminar air flow (LAF) cabinet an enclosed work bench in which HEPA-filtered air flows smoothly across the working area to maintain aseptic conditions.
HEPA High Efficiency Particulate Air; the filter that removes dust particles, bacteria and fungal spores from the air entering a cabinet.
Biosafety cabinet a cabinet that draws air inward and exhausts it through a filter, so that it protects the worker as well as the culture.
Autoclave an instrument that sterilises by moist heat, holding steam at \(121^\circ\text{C}\) under a pressure of 15 psi.
Hot air oven an instrument that sterilises by dry heat, typically at \(160\text{--}180^\circ\text{C}\) for 2–4 hours.
Microfiltration unit an assembly that sterilises heat-labile solutions by passing them through a membrane of \(0.22\ \mu\text{m}\) pore size, which retains microbes.
CO₂ incubator an incubator that controls carbon dioxide concentration along with temperature and humidity, holding the bicarbonate-buffered medium of animal cells at the correct pH.
Inverted microscope a microscope with its light source above the stage and its objectives below, so that living cells can be viewed through the base of an unopened culture vessel.
T flask a flat, surface-treated polystyrene culture flask with a filter vent cap, used to propagate animal cells as a monolayer.
Bioreactor a large closed vessel in which cells or micro-organisms are grown on a large scale under controlled conditions so that their products can be harvested commercially.
Fermentor another name for a bioreactor, used particularly when the organisms grown inside are microbes.
Stirred tank bioreactor (STR) a bioreactor in which impellers mounted on a motor-driven shaft mix and aerate the culture broth.
Sparged tank bioreactor a bioreactor, also called a bubble column bioreactor, in which a rising stream of gas bubbles introduced at the base does the mixing, with no agitator shaft.
Baffles small metallic strips fixed radially to the inner wall of a bioreactor, which break up the rotating flow and create the turbulence needed for aeration.
Impeller the agitator mounted on the central shaft of a stirred tank bioreactor, which mixes the nutrient medium with the cells.
Sparger the device at the bottom of a fermentor through which air enters the liquid medium.
Sterilisation the process of killing or removing all living micro-organisms, including their spores, from a material or a surface.
Incineration the sterilisation of metal instruments by dipping them in ethanol and passing them through a flame.
Explant a piece of tissue taken from a plant and placed in culture.
Surface sterilisation a brief chemical treatment that kills the microbes on the outside of living tissue, followed by repeated rinsing so that the chemical does not penetrate and kill the tissue itself.
Depth filter a filter with a filamentous matrix such as glass wool or filter paper, in which particles are trapped within the thickness of the material.
Absolute filter a filter with pores of a defined size, smaller than the particles to be removed, so that retention depends on pore size alone.
Ultrafiltration filtration separating particles of \(0.001\text{--}0.1\ \mu\text{m}\), retaining compounds of molecular weight greater than 1000, such as enzymes.
Reverse osmosis filtration separating particles of \(0.0001\text{--}0.001\ \mu\text{m}\), retaining compounds of molecular weight less than 1000, such as lactose.
Culture medium a preparation containing the nutrients and other components required by cells for growth, multiplication and development under laboratory conditions.
Synthetic (defined) medium a medium whose components are chemicals synthesised in the laboratory, each present in a known amount.
Semi-synthetic (differential) medium a medium containing synthetic chemicals together with natural complex compounds.
Natural (complex) medium a medium obtained from a natural complex compound, such as soyabean seed extract broth, whose exact composition is not known.
Broth a culture medium in liquid form, containing no gelling agent.
Macronutrient a mineral nutrient required at a concentration greater than 0.5 mmole per litre.
Micronutrient a mineral nutrient required at a concentration less than 0.5 mmole per litre.
Chelated iron iron bound to a carrier such as EDTA, which keeps it dissolved and available to cells at culture pH instead of precipitating out.
Auxin a plant growth regulator that mainly promotes cell division and root initiation, for example IAA, IBA, NAA and 2,4-D.
Cytokinin a plant growth regulator used for cell division and differentiation, for example BAP, 2iP, kinetin and zeatin.
Solidifying agent a substance such as agar that sets a liquid medium into a gel, so that the explant rests at the surface instead of being submerged.
Agar a polysaccharide obtained from red algae, used as the standard gelling agent because it does not react with the medium, is not digested by plant enzymes and is stable at culture temperature.
LB medium Luria Bertani medium, a general-purpose microbial medium containing tryptone, yeast extract and sodium chloride, used as a broth or, with agar added, as a solid medium.
Tryptone a casein enzymic hydrolysate that supplies peptides and peptones as the nitrogen and amino acid source of LB medium.
MS medium Murashige and Skoog medium, a high-salt plant tissue culture medium used for inducing organogenesis and regeneration, set at pH 5.8.
White’s medium one of the earliest plant tissue culture media, low in salts and developed for root culture, set at pH 7.0.
DMEM Dulbecco’s Modified Eagle Medium, an animal cell medium rich in amino acids, vitamins and glucose, used chiefly for adherent cells.
Phenol red a pH indicator included in animal culture media, whose colour change reports the acidity of the culture at a glance.
RPMI-1640 Roswell Park Memorial Institute medium, designed for suspension cells such as lymphocytes, T-cells, B-cells and hybridomas.
FBS Foetal Bovine Serum, a supplement added to animal culture media at 5–20% to supply growth factors, hormones, proteins and attachment factors.
Slant a tube of solid medium set at an angle of \(45^\circ\) to give a larger growth surface, used mainly for maintaining stock cultures.
Haemocytometer a counting chamber consisting of a thick glass slide with an engraved grid, over which a coverslip is held 0.1 mm above the surface so that each square encloses a known volume.
Dilution factor the number of times a suspension has been diluted before counting, by which the counted concentration must be multiplied to give the original concentration.
Dye exclusion the principle that an intact plasma membrane keeps a stain out, so that only dead or damaged cells take up the dye.
Evan’s blue a viability stain used at 0.025% w/v, which stains dead and damaged cells but not living ones.
Trypan blue a viability stain that enters cells with damaged membranes and colours them blue, leaving living cells unstained.
Fluorescein diacetate (FDA) a viability reagent cleaved by the esterase enzyme of living cells, causing them to emit green fluorescence under ultraviolet light.
FACS Fluorescence Activated Cell Sorting; a technique in which cells labelled with fluorescent antibodies are identified by a laser in a stream of droplets, which are then charged and deflected into separate collectors.
Cryopreservation the storage of living cells and tissues at very low temperature in a frozen, zero-metabolism state from which they can later be revived.
Germplasm the sum of all the genes present in a crop and its related species, which serves as the raw material for developing new varieties.
Cryoprotectant a substance such as DMSO, glycerol or sucrose that lowers the freezing point and retards ice crystal formation during freezing.
Slow freezing freezing at \(0.5\text{--}5^\circ\text{C}\) per minute from \(0^\circ\text{C}\) to \(-100^\circ\text{C}\) before transfer to liquid nitrogen, used chiefly for suspension cultures.
Rapid freezing freezing at \(-30^\circ\) to \(-100^\circ\text{C}\) per minute, forming only small intracellular ice crystals, used for shoot tips and somatic embryos.
Thawing the rapid warming of frozen ampoules in a bath at \(37^\circ\text{--}45^\circ\text{C}\), carried out quickly so that ice crystals have no time to re-form.
IBPGR International Board of Plant Genetic Resources, the organisation established to support the collection, conservation and utilisation of plant genetic resources worldwide.
10.11 Points to Ponder
Every instrument in a cell culture laboratory replaces something a cell would have received free of charge inside a living organism. Choose any three instruments from this chapter and identify exactly which natural service each one is standing in for. Which service do you think is hardest to replace convincingly?
Sterilisation and cell culture are, in a sense, opposite activities — one kills every living thing, the other keeps living things alive. Think about how a laboratory manages to do both in the same room, and where the boundary between the two is drawn.
A defined synthetic medium makes results reproducible, yet animal cell culture still depends heavily on serum, a natural product whose exact composition nobody knows. What does this tension tell you about how far cell culture has actually progressed as a technology?
Cryopreservation halts mutation by halting division, which is presented in this chapter as a benefit. Consider whether there are situations in which preventing all genetic change in a stored line might not be desirable.
Foetal bovine serum, mercuric chloride and liquid nitrogen are all standard laboratory materials with real costs attached — to animals, to the environment and to safety. Should a school or college laboratory hold itself to a different standard from a research institute in deciding what to use, or should the standard be the same everywhere?
The FACS machine makes its biological decision at a single moment — when an antibody binds, or does not bind, to a cell surface antigen. What kinds of error could this introduce, and how much confidence should a researcher place in a “pure” sorted population?
Germplasm collected from farmers’ fields in one country is often conserved in gene banks located in another. Think through who should be regarded as the owner of such material, and what obligations, if any, follow for those who use it to breed commercial varieties.
A dye exclusion test scores a cell as alive if its membrane is intact, while an FDA test scores it as alive only if its enzymes still work. Both are called viability tests. What does this suggest about how carefully scientific terms need to be defined before results are compared?
Cell culture technology allows human tissue to be grown and studied indefinitely, sometimes long after the person it came from has died. Consider what a person should be told, and what they should be asked, before a sample of their tissue is taken for research.
Large-scale production in bioreactors has made medicines such as recombinant proteins widely available, but the equipment and expertise involved are concentrated in a few countries. What are the consequences of this for access to such medicines elsewhere, and what might reduce the gap?
Almost every failure described in this chapter — a contaminated plate, a dead explant, a poorly recovered ampoule — traces back to a single step performed carelessly rather than to a flaw in the method. What does that pattern suggest about what makes a good experimentalist, as distinct from a good student of experiments?
10.12 Objective Questions
- The volume of liquid held above one \(1\ \text{mm}^2\) square of a haemocytometer grid is:
- \(10^{-2}\ \text{mL}\)
- \(10^{-3}\ \text{mL}\)
- \(10^{-4}\ \text{mL}\)
- \(10^{-6}\ \text{mL}\)
- Evan’s blue dye is used for viability testing at a concentration of:
- 0.025% w/v
- 0.25% w/v
- 2.5% w/v
- 25% w/v
- In a cell suspension stained with Evan’s blue, the cells that remain unstained are:
- Dead cells
- Viable cells
- Cells with damaged membranes
- Cells in the process of division
- Which of the following is not a function of baffles in a stirred tank bioreactor?
- Increasing turbulence in the culture medium
- Improving aeration of the medium
- Preventing the contents from rotating as a single body
- Admitting sterile air into the liquid medium
- A bubble column bioreactor differs from a stirred tank bioreactor mainly in that it:
- Has no agitator shaft
- Requires no sterile air
- Cannot be used for aerobic cultures
- Consumes more energy
- Iron is supplied to plant culture media in chelated form because:
- Chelated iron is cheaper than iron salts
- Simple iron salts precipitate at culture pH and become unavailable
- Iron is required only as a micronutrient
- Chelation converts iron into an energy source
- A mineral nutrient required at a concentration greater than 0.5 mmole per litre is classified as a:
- Micronutrient
- Macronutrient
- Growth regulator
- Solidifying agent
- The pH of MS medium and of White’s medium are respectively:
- 5.8 and 7.0
- 7.0 and 5.8
- 5.8 and 5.5
- 7.4 and 7.0
- Which of the following is a supplement rather than a complete culture medium?
- DMEM
- RPMI-1640
- FBS
- LB broth
- RPMI-1640 is particularly suited to the culture of:
- Adherent fibroblasts
- Suspension cells such as lymphocytes
- Plant callus tissue
- Filamentous fungi
- Phenol red is included in animal culture media to:
- Supply an additional carbon source
- Prevent microbial contamination
- Indicate the pH of the medium by its colour
- Help cells attach to the culture surface
- In LB medium, the component that supplies the vitamin B complex is:
- Tryptone
- Sodium chloride
- Yeast extract
- Agar
- Cultures are stored in liquid nitrogen rather than in a deep freezer because ice crystals continue to form and grow at temperatures above about:
- \(-30^\circ\text{C}\)
- \(-79^\circ\text{C}\)
- \(-130^\circ\text{C}\)
- \(-196^\circ\text{C}\)
- Thawing of cryopreserved ampoules is carried out rapidly at \(37^\circ\text{--}45^\circ\text{C}\) in order to:
- Restart metabolism as quickly as possible
- Give ice crystals no time to re-form and grow
- Sterilise the outer surface of the ampoule
- Remove the cryoprotectant from the cells
- Which sequence correctly represents the steps of cryopreservation?
- Cryoprotectant addition → freezing → storage → thawing → reculture
- Freezing → cryoprotectant addition → thawing → storage → reculture
- Storage → freezing → cryoprotectant addition → reculture → thawing
- Thawing → freezing → storage → cryoprotectant addition → reculture
- In FACS, the stream of cells is broken into droplets so that:
- The cells can be counted more accurately
- Individual droplets can be given an electric charge and deflected
- The laser can pass through the stream
- The antibodies can bind to the cell surface
- The step in FACS at which the biological decision is actually made is:
- The charging of the droplet
- The deflection between charged plates
- The binding of the fluorescent antibody to a surface antigen
- The collection of cells in the flask for undeflected droplets
- Medium is cooled to about \(60^\circ\text{C}\) before pouring plates chiefly because:
- Agar will not dissolve above this temperature
- Hotter medium would produce heavy condensation on the lid
- The medium would otherwise remain liquid
- Cooling sterilises the medium a second time
- Culture medium is dispensed into tubes held at an angle of \(45^\circ\) in order to:
- Prevent the agar from setting
- Increase the growth surface available in the tube
- Allow the medium to be autoclaved a second time
- Keep the medium anaerobic
- The pH of a culture medium is adjusted before autoclaving rather than after because:
- The pH meter cannot read a hot solution
- Adjusting afterwards would introduce contamination into a sterile medium
- Autoclaving has no effect on pH
- Agar dissolves only at low pH
- Alcohol is used as a 70% solution rather than as absolute alcohol for surface disinfection because:
- Absolute alcohol is more expensive
- Protein denaturation by alcohol requires the presence of water
- Absolute alcohol evaporates too slowly
- 70% alcohol is flammable and absolute alcohol is not
- An explant is washed 6–7 times in sterile distilled water after chemical treatment mainly to:
- Remove residual disinfectant that would kill the tissue
- Supply water to the explant before inoculation
- Complete the sterilisation of the tissue
- Soften the cut surfaces of the explant
- Which of the following pairs is correctly matched?
- Ultrafiltration — retains compounds of molecular weight less than 1000
- Microfiltration — separates particles of \(0.1\text{--}10\ \mu\text{m}\)
- Reverse osmosis — used for cold sterilisation of vitamins
- Depth filter — retention depends only on a defined pore size
Assertion (A): A biosafety cabinet is preferred over a plain laminar air flow cabinet when working with pathogenic microbes. Reason (R): A biosafety cabinet draws air inward at the front and exhausts it through a filter, so that material inside stays inside.
Assertion (A): A hot air oven requires a much higher temperature and a longer time than an autoclave to achieve sterilisation. Reason (R): Dry air transfers heat less efficiently than steam, and moisture assists the denaturation of microbial proteins.
Assertion (A): A \(\text{CO}_2\) incubator is required for most animal cell cultures. Reason (R): Carbon dioxide is used by animal cells as their carbon source in place of sucrose.
Assertion (A): A callus supplied with a high proportion of cytokinin relative to auxin tends to form shoots. Reason (R): The ratio between auxin and cytokinin, rather than the amount of either alone, determines the direction of morphogenesis.
Assertion (A): Antibiotics are added to some culture media, but they cannot replace aseptic technique. Reason (R): Antibiotics are heat-labile and must be added by filter sterilisation.
Assertion (A): Solidifying a medium with agar prevents an explant from dying of oxygen shortage. Reason (R): On a gelled medium the explant rests at the surface, drawing nutrients from below while remaining in contact with air.
Assertion (A): A haemocytometer count alone is not sufficient to describe the state of a culture. Reason (R): Direct counting records every cell in the field and cannot distinguish living cells from dead ones.
Assertion (A): A cell scored as viable by a dye exclusion test may nevertheless be scored as non-viable by a fluorescein diacetate test. Reason (R): Dye exclusion reports membrane integrity, whereas fluorescein diacetate reports esterase activity.
Assertion (A): The best indicator of survival after cryopreservation is entry into cell division and regrowth in reculture. Reason (R): Cryoprotectants such as DMSO interfere with all staining methods used for viability testing.
Assertion (A): The sparger of a bioreactor is placed at the bottom of the vessel. Reason (R): Bubbles released at the base travel through the full height of the broth, giving the maximum time for oxygen to dissolve.
Assertion (A): Germplasm of clonal crops is not conserved satisfactorily as seed. Reason (R): Seed of clonal crops is genetically heterogeneous and does not reproduce the parent plant faithfully.
Assertion (A): Foetal bovine serum is added to animal culture media at concentrations of 5–20%. Reason (R): It supplies growth factors, hormones and attachment factors that the cells cannot make for themselves.
Ahmed uses an instrument to measure the pH of a medium. Which is it?
- Colorimeter
- pH meter
- Spectrophotometer
- Centrifuge
- Gurpreet sterilises media at \(121^\circ\text{C}\). Which method is used?
- Filtration
- Autoclaving
- Dry heat
- UV radiation
- Meena uses a device with grid lines to count cells. Identify it.
- Microscope
- Colony counter
- Haemocytometer
- Centrifuge
- Arjun uses agar in a medium. Its function is:
- Energy source
- Solidifying agent
- Growth regulator
- Antibiotic
- Fatima adds sucrose to a medium because:
- Cells are autotrophic
- Cells need a carbon source
- It sterilises the medium
- It acts as a hormone
- Rahul uses laminar air flow to:
- Increase growth
- Prevent contamination
- Measure pH
- Heat the medium
- Simran adds vitamins to a medium because they:
- Provide energy
- Act as coenzymes
- Kill microbes
- Solidify the medium
- David observes poor growth due to pH imbalance. This is because:
- pH affects enzymes
- pH affects colour
- pH affects size
- pH affects light
- Joseph studies a suspension culture. The cells grow:
- Attached to a surface
- In solid medium
- Freely in liquid
- Without nutrients
- Neha needs to sterilise heat-sensitive proteins. She should use:
- Autoclave
- Hot air oven
- Filtration
- Incineration
- Imran wants to separate cell organelles. He should use:
- Incubator
- Centrifuge
- pH meter
- Microscope
- Kavya transfers explants aseptically using:
- Incubator
- Laminar air flow
- Centrifuge
- Oven
- Rohit grows animal cells under \(\text{CO}_2\) control using:
- Incubator
- \(\text{CO}_2\) incubator
- Oven
- Desiccator
- Maria mixes reagents uniformly using:
- Centrifuge
- Vortex mixer
- Microscope
- Spectrophotometer
- Sanya observes contamination in a culture. The most likely cause is:
- Proper sterilisation
- Lack of aseptic conditions
- Correct pH
- Proper nutrients
- Tenzin finds low viability after freezing. The reason is:
- High temperature
- Ice crystal formation
- Low nutrients
- Excess vitamins
- Aditya fails to separate organelles properly. The reason is:
- Correct speed
- Incorrect centrifuge speed
- Proper setup
- Sterile medium
- Noor sees no differentiation in a culture. The reason is:
- Proper hormones
- Lack of growth regulators
- Adequate nutrients
- Correct pH
- Karan observes filtration failure. The reason is:
- Correct pore size
- Large pore size
- Proper pressure
- Sterile setup
- Ayesha chooses a sterilisation method for culture media. The best method is:
- Hot air oven
- Filtration
- Autoclaving
- UV light
- Vikram must sterilise antibiotics. The best method is:
- Autoclave
- Filtration
- Oven
- Incineration
- Harpreet selects an incubator for animal cells. The best choice is:
- Simple incubator
- \(\text{CO}_2\) incubator
- Oven
- Desiccator
- Zoya selects a solid medium. The advantage is:
- No oxygen
- Structural support
- Faster mixing
- No nutrients
- Rohan selects cryopreservation. The advantage is:
- High metabolism
- Long-term storage
- Rapid growth
- High temperature
- Pooja designs a medium but omits the carbon source. The result is:
- Rapid growth
- No growth
- Contamination
- Differentiation
- Aarav designs an aseptic setup. The key requirement is:
- Open air
- HEPA-filtered air
- High temperature
- Dry conditions
- Iqbal prepares a medium without hormones. The result is:
- Normal differentiation
- No growth
- Poor differentiation
- Contamination
- Lhamo designs a cryopreservation protocol. An essential step is:
- Heating
- Cryoprotectant addition
- Drying
- UV exposure
- Daniel designs a large-scale culture. The best system is:
- Flask
- Petri dish
- Bioreactor
- Slide
- For the following questions, choose from the options below:
- Both Assertion and Reason are true and Reason is the correct explanation of Assertion.
- Both Assertion and Reason are true but Reason is not the correct explanation of Assertion.
- Assertion is true, but Reason is false.
- Both Assertion and Reason are false.
- Assertion (A): Riya observes that autoclaving is used to sterilise culture media. Reason (R): Autoclaving uses high pressure and steam at \(121^\circ\text{C}\) to kill micro-organisms.
- Assertion (A): Ahmed finds that membrane filtration is used for sterilising enzymes. Reason (R): Enzymes get denatured at high temperature.
- Assertion (A): Gurpreet notices that agar is added to culture media. Reason (R): Agar provides nutrients to growing cells.
- Assertion (A): Meena observes that a laminar air flow cabinet prevents contamination. Reason (R): It provides sterile air through HEPA filters.
- Assertion (A): Arjun sees that centrifugation separates cell components. Reason (R): It works on the basis of density differences.
- Assertion (A): Fatima finds that vitamins are essential in culture medium. Reason (R): Vitamins act as coenzymes in metabolic reactions.
- Assertion (A): Rohit observes poor cell growth at improper pH. Reason (R): pH affects enzyme activity in cells.
- Assertion (A): Simran uses cryopreservation for long-term storage of cells. Reason (R): Very low temperature stops metabolic activities.
- Assertion (A): David observes that a hot air oven is used to sterilise culture media. Reason (R): Dry heat sterilisation is suitable for all types of materials.
- Assertion (A): Neha uses a haemocytometer to count cells. Reason (R): It has a grid of known dimensions to calculate cell number.
- Assertion (A): Imran observes contamination in culture media. Reason (R): Lack of aseptic conditions leads to microbial growth.
- Assertion (A): Kavya uses a \(\text{CO}_2\) incubator for animal cell culture. Reason (R): It maintains temperature, humidity and \(\text{CO}_2\) levels.
- Assertion (A): Tenzin observes cell damage during cryopreservation. Reason (R): Ice crystal formation can damage cell organelles.
- Assertion (A): Vikram selects a liquid medium for suspension culture. Reason (R): Suspension cells grow freely in liquid medium.
The pore size commonly used in membrane filtration is ______ micrometre.
The temperature used in autoclaving is ______ \(^\circ\text{C}\).
The instrument used to measure pH is called ______.
Laminar air flow works by passing air through a ______ filter.
A centrifuge works on the principle of ______.
Microfiltration is used to remove ______ from solutions.
Cryopreservation involves storage at very ______ temperatures.
To sterilise heat-sensitive substances, the ______ method is used instead of autoclaving.
For maintaining aseptic conditions during transfer, a ______ cabinet is used.
For measuring cell concentration, a ______ is used.
To grow cells at controlled \(\text{CO}_2\) levels, a ______ incubator is used.
For mixing small liquid samples in the lab, a ______ mixer is used.
The separation of organelles using a centrifuge depends on their ______ and weight.
The main reason for adding cryoprotectants is to prevent the formation of ______.
Agar is added to culture medium as a ______ agent.
The efficiency of filtration depends on the size of the organism and the ______ of the medium.
The most suitable temperature for animal cell culture in a \(\text{CO}_2\) incubator is around ______ \(^\circ\text{C}\).
Among sterilisation methods, ______ is preferred for culture media.
The most commonly used membrane filter size for sterilisation is ______ \(\mu\text{m}\).
The most commonly used cryopreservation medium involves ______ nitrogen.
For precise measurement of small masses, a ______ balance is used.
A culture medium must contain a ______ source for energy.
Vitamins in culture medium act as ______ in metabolic reactions.
Auxins are mainly responsible for ______ initiation.
Cytokinins promote ______ in plant tissue culture.
The combination of auxins and cytokinins controls the ______ of tissues.
10.13 Check Your Concepts
A cell culture laboratory is sometimes described as a set of machines that between them replace a living organism. Take any four instruments studied in this chapter and state, for each, the natural condition it is standing in for.
Why is it not enough to sterilise the culture medium alone? List the six categories of material that must be sterilised in a cell culture laboratory, and name the method used for each.
A technician sterilises a batch of culture medium in a hot air oven at \(180^\circ\text{C}\) for two hours. Explain what will be wrong with the medium afterwards, and state the method that should have been used.
Compare an autoclave and a hot air oven with respect to the type of heat used, the working conditions, penetration, and the materials each is suited to.
A laminar air flow cabinet and a biosafety cabinet look similar and both use HEPA filters. Explain how the direction of air flow differs between them, and why that difference decides which one may be used with pathogenic microbes.
What would happen to a batch of culture medium if the pH were adjusted after autoclaving instead of before it? Give two separate consequences.
Explain why alcohol is used as a 70% solution rather than as absolute alcohol for disinfecting a work surface.
An explant is treated with mercuric chloride for the correct time but is rinsed only once before being inoculated. The medium remains free of contamination, yet the explant does not grow. Account for both observations.
Arrange the following steps of medium preparation in the correct order and give a reason for the position of each of the three underlined steps: autoclave; dissolve salts and sugar; adjust the pH; weigh out components; add agar; make up the final volume; cool to about \(60^\circ\text{C}\); dispense as plates or slants.
Why is agar added after the salts and sugar have dissolved rather than at the beginning? Give two reasons.
Distinguish between a synthetic medium, a semi-synthetic medium and a natural medium, and state one advantage of a defined medium in experimental work.
A student prepares a plant tissue culture medium using tap water instead of distilled water. Explain why this is a serious error even though the water appears clean.
What is meant by chelated iron? Explain why iron cannot simply be added to a plant culture medium as a simple salt.
Sucrose is described as doing three jobs at once in a plant culture medium. State all three, and explain why its concentration cannot be raised freely to improve growth.
A callus culture on MS medium produces abundant roots but no shoots. State the change you would make to the medium and explain the principle on which your answer rests.
Explain why a culture that has been submerged in a still liquid medium dies at its centre, and state two different ways of preventing this.
Compare MS medium and White’s medium with respect to salt concentration, pH and the purpose each was developed for. Using the composition table in this chapter, identify the single largest compositional difference between them.
Name the three components of LB broth and state the function of each. What single change converts LB broth into LB agar, and what does that change make possible?
Compare DMEM and RPMI-1640 with respect to the type of cells each is designed for and the fields in which each is commonly used.
FBS is described as an essential supplement but also as a scientific problem. Explain both statements.
A flask of DMEM that was pink when prepared has turned yellow after three days of incubation. State two possible explanations, and describe how you would distinguish between them.
Explain why a \(\text{CO}_2\) incubator is needed for animal cell culture but not for the culture of plant tissues on MS medium.
Why does an inverted microscope place its objectives below the stage? Explain what this arrangement makes possible that an ordinary upright microscope does not.
Label the following parts of a stirred tank bioreactor on a diagram and state the function of each: impeller, baffle, sparger, cooling jacket, pH controller, antifoam probe.
Explain how each of the following components of a bioreactor answers a specific change that the growing culture itself causes: the pH control system, the temperature control system, and the antifoam control system.
Compare a stirred tank bioreactor and a sparged tank bioreactor with respect to the method of mixing, energy consumption, air requirement and heat removal.
Why is heat removal faster in a sparged tank bioreactor than in a stirred tank bioreactor? Give both reasons.
Arrange the following in the correct order to describe the operation of a bioreactor, and justify the position of the first and last steps: inoculate the culture; harvest the product; sterilise the vessel and medium with steam; hold pH, temperature and foam under automatic control; cool to working temperature; supply sterile air through the sparger.
Explain why the chamber depth of a haemocytometer must be exactly 0.1 mm, and what would happen to a calculated cell concentration if the coverslip were not seated properly.
Why is a mean count taken over several large squares of a haemocytometer grid rather than a count from a single square?
Cultured plant cells often occur as clumps. Explain why clumps must be dispersed before counting, and name the agents used for this.
State the principle of dye exclusion. Explain why a cell stained by Evan’s blue is scored as dead while an unstained cell is scored as viable.
Compare a dye exclusion test with the fluorescein diacetate test with respect to what is detected, which cells are marked, and the equipment required. Explain which of the two would detect a dying cell earlier.
Match each item in the first list with the correct item in the second list. | Column A | Column B | |—|—| | a. haemocytometer | i. removes airborne spores | | b. Evan’s blue | ii. reports medium pH by colour | | c. FACS | iii. determines total cell number | | d. phenol red | iv. separates a labelled cell population | | e. HEPA filter | v. distinguishes dead cells from living ones |
Describe how a tissue is dissociated into single cells before sorting, and explain why this step is necessary before FACS can be used.
In FACS, what would be the consequence of a droplet containing two cells instead of one? Relate your answer to the purity of the sorted population.
Explain why the specificity of FACS depends on the antibody rather than on the electronics of the machine.
Why is a growing culture unsuitable for the long-term preservation of a valuable line? Give two distinct reasons.
It is often said that cells are not killed by cold but by ice. Explain this statement with reference to the two kinds of damage that occur during freezing.
Explain how a cryoprotectant reduces freezing injury, and state why cryoprotectants must nevertheless be washed out promptly after thawing.
Compare the slow freezing and rapid freezing methods with respect to cooling rate, the ice crystals formed, and the material each is chiefly used for.
Cryopreserved material is stored at around \(-150^\circ\text{C}\) rather than in a deep freezer at \(-80^\circ\text{C}\). Justify this choice with reference to a specific temperature threshold.
Two ampoules from the same culture are thawed, one rapidly in a warm bath and one slowly on the bench. Predict which recovers better and explain the reason.
Why is regrowth in reculture regarded as a better indicator of survival after cryopreservation than a staining test?
Explain why germplasm of many crops cannot be conserved satisfactorily as seed, and name the approaches used for such material.
A student proposes to save time by inoculating explants on an open bench, arguing that the medium has already been autoclaved and the explant surface sterilised. Explain what is wrong with this reasoning.
A laboratory reports that its cultures grow well for two days and then stop, with no visible contamination. Suggest three possible causes drawn from different parts of this chapter, and state how you would test each.
What is the standard temperature used in autoclaving?
What is the function of a pH meter?
What is the pore size of commonly used membrane filters?
Why do plant cells require an external carbon source in culture medium?
Why is laminar air flow essential in cell culture?
Why is agar added to culture media?
Why are vitamins important in culture medium?
Why is sterilisation necessary in cell culture techniques?
Which instrument would you use to separate cell organelles?
Which method would you use to sterilise heat-sensitive solutions?
Which device is used to maintain aseptic conditions during transfer?
Which instrument is used to measure cell concentration?
Which equipment is used to grow cells at controlled temperature and \(\text{CO}_2\)?
What happens if contamination occurs in a culture medium?
Why is high temperature used in autoclaving?
Why are suspension cultures shaken continuously?
Why is pH control important in cell culture?
Which sterilisation method is best for culture media, and why?
Which is more suitable for heat-labile substances — autoclaving or filtration? Why?
Which type of incubator is preferred for animal cell culture, and why?
Why is membrane filtration preferred over heating in some cases?
Which culture method is best for large-scale production of cells?
Suggest one component necessary for preparing a culture medium.
Write one step to maintain aseptic conditions in a lab.
Suggest one way to improve cell growth in culture.
Write one method to store cells for a long time.
Write one modification to prevent contamination during transfer.
During sterilisation, a technician sets the autoclave at \(121^\circ\text{C}\) and 15 psi. Name the method and state its purpose.
A lab assistant uses a device to measure the acidity of culture medium before use. Identify the instrument and its function.
A researcher uses a glass chamber with a grid to count cells. Name the instrument and its use.
A scientist uses a filter of \(0.22\ \mu\text{m}\) pore size. What is this technique called and what is its purpose?
A culture medium without sugar fails to support plant cell growth. Give reasons.
A culture gets contaminated when transferred outside a laminar air flow cabinet. Explain the role of laminar air flow in preventing this.
A student notices that cells die when grown in liquid medium without support. Explain the role of agar in culture media.
A medium lacking vitamins shows poor cell growth. Explain the importance of vitamins in cell culture.
A culture room is not sterilised properly and shows fungal growth. Explain why sterilisation is essential.
You need to separate mitochondria from a cell extract. Which instrument will you use and how does it work?
A solution containing enzymes cannot be heated for sterilisation. Which method will you use and why?
During transfer of explants, contamination must be avoided. Which equipment will you use and how?
You need to grow mammalian cells under controlled \(\text{CO}_2\) conditions. Which equipment will you select?
A sample needs uniform mixing before analysis. Which laboratory device will you use?
A culture medium shows a sudden pH change during growth. Analyse the reason and its effect on cells.
A centrifuge is run at low speed but separation is poor. Analyse the reason behind the ineffective separation.
A filtration process fails to remove bacteria. Analyse what might be wrong with the setup.
Richard, a biologist, must sterilise culture media and glassware. Which methods will you recommend for each, and why?
Divya is choosing between autoclaving and membrane filtration for sterilising vitamins. Which method is better? Justify your answer.
A lab needs to grow animal cells efficiently. Which incubator would you suggest, and why?
Pawani wants long-term preservation of germplasm. Which method would you recommend, and why?
Gurmeet and Samrin are asked to design a basic culture medium for plant cells. Mention any two essential components and their role.
A new lab is being set up for tissue culture. Suggest two steps to maintain aseptic conditions.
Rehana needs to improve the growth of a slow-growing culture. Suggest two modifications in the medium.
Baldev wants to design a simple protocol to prevent contamination during transfer of explants. Mention any two steps.
Kastrov wants to preserve cells for future use. Design a basic approach using cryopreservation.
Gurpreet is preparing culture medium in the laboratory. List the major components of a culture medium and state their functions.
Joseph is shown a diagram of instruments used in a tissue culture lab. Identify any four instruments and explain their functions.
Arjun’s plant tissue culture experiment fails due to improper nutrient supply. Explain the role of inorganic nutrients, carbon source and vitamins in culture media.
Fatima observes that cells behave differently in solid and liquid media. Explain the differences and the significance of both types of media.
Simran notices frequent contamination during cell culture experiments. Explain the importance of aseptic conditions and the methods used to maintain them.
Rahul observes poor growth in culture despite proper nutrients. Explain how pH and growth regulators influence cell growth.
David observes demonstrations of laminar air flow and autoclave in class. Explain the working principles and importance of both in cell culture.
Neha is given a mixed sample of cell organelles. Explain how she would separate them using centrifugation, including principle and steps.
Imran needs to sterilise heat-sensitive substances like enzymes. Explain the method he should use and describe the procedure.
Kavya needs to transfer explants without contamination. Explain how she would use laminar air flow to achieve this.
Rohit is asked to measure and maintain the pH of a culture medium. Explain the procedure using a pH meter.
Maria wants to grow cells at a large scale. Explain how a bioreactor works and its advantages.
Sanya observes poor growth and contamination in her culture. Analyse the possible causes and suggest corrective measures.
Tenzin notices that many cells lose viability after cryopreservation. Analyse the reasons and explain how the process can be improved.
Aditya finds that centrifugation did not separate components properly. Analyse the factors affecting centrifugation efficiency.
Karan finds that filtration did not sterilise the medium completely. Analyse possible errors related to pore size, pressure and setup.
Ayesha must choose between autoclaving and filtration for sterilisation. Evaluate both methods and justify which is more suitable in different situations.
Vikram is selecting between solid and liquid culture media. Evaluate the advantages and limitations of both types.
Rohan wants long-term preservation of germplasm. Evaluate cryopreservation as a method and discuss its advantages and limitations.
Pooja is asked to design a complete culture medium for plant tissue culture. List all the components and explain their role in supporting growth.
Aarav is setting up a tissue culture lab. Design a stepwise protocol to maintain aseptic conditions in the lab.
Lhamo is designing a protocol for cryopreservation of plant cells. Include steps such as pretreatment, freezing, storage and thawing.
Daniel is working in a biotech company on large-scale plant cell production. Design a strategy using bioreactors, including the key components and conditions.
10.14 Case-Based Questions
- A college laboratory prepares 2 litres of a plant tissue culture medium for a batch of student experiments. The technician weighs out the salts and sucrose, dissolves them in distilled water, adds agar, makes up the volume, and autoclaves the whole batch at \(121^\circ\text{C}\) and 15 psi for 30 minutes. Because the recipe also calls for a vitamin mixture and a growth regulator, these are weighed out and added to the flask along with everything else before autoclaving. The medium sets normally and remains free of contamination for three weeks, but the explants inoculated onto it fail to form any callus.
- Identify the mistake made during preparation.
- Explain why the medium remained sterile even though the experiment failed.
- Describe the correct procedure for the vitamin mixture and the growth regulator, naming the pore size involved.
- State the stage at which these components should be added to the rest of the medium.
- A research student is asked to determine the density and health of a cell suspension before setting up an experiment. She dilutes the suspension 1 in 20, mixes it with Evan’s blue solution, and loads it into a counting chamber. Counting four large squares of the grid, she records 96, 104, 88 and 112 cells in all, of which 50 cells across the four squares have taken up the dye.
- Name the instrument used and state the depth of liquid held above the grid.
- Calculate the total cell concentration of the original undiluted suspension, in cells per mL.
- Calculate the percentage viability of the culture.
- Explain why the stained cells are recorded as dead rather than as living.
- A biotechnology company cultures an aerobic bacterium to produce an industrial enzyme. The first batch is run in a stirred tank bioreactor. Growth is rapid, but the operators report heavy foaming, a steadily falling pH in the culture broth, and a rise in temperature well above the set point during the middle of the run. For the next batch they consider switching to a sparged tank vessel of the same working volume.
- Name the control system that should have prevented the foaming, and describe how it works.
- Explain why the pH of the growth medium falls as the culture grows, and how the vessel corrects it.
- Give two sources of the heat that accumulates in a stirred tank bioreactor.
- State one advantage and one disadvantage the company should expect if it switches to a sparged tank vessel.
- A field station holds a collection of a traditional banana variety that is propagated only vegetatively and produces seeds that lose viability within days. Maintaining the collection as living plants in the field is expensive and exposes it to disease. The station decides to conserve the material by freeze preservation instead. Suspension cultures are established, pretreated, mixed with a cryoprotectant solution, cooled at \(2^\circ\text{C}\) per minute from \(0^\circ\text{C}\) to \(-100^\circ\text{C}\) and transferred to liquid nitrogen.
- Explain why seed storage is unsuitable for this material, giving two separate reasons drawn from the passage.
- Name the freezing method used and calculate the duration of the controlled cooling stage.
- Name two cryoprotectants that could be used and state how a cryoprotectant reduces freezing injury.
- After a year in storage, a sample is thawed. Describe how the station should thaw it and how it should judge whether the material has survived.
- A hospital research unit needs to study one particular class of white blood cell present at low frequency in a blood sample. The cells are first treated to break the adhesion between them, then labelled with antibodies coupled to a fluorescent dye and passed through a sorting machine. The sorted population is afterwards cultured in RPMI-1640 supplemented with 10% FBS in a \(\text{CO}_2\) incubator.
- Name the sorting technique used and explain how a labelled cell is physically separated from an unlabelled one.
- Explain why the antibody, and not the machine, determines which cells are collected.
- Give one reason why RPMI-1640 rather than DMEM is the appropriate medium here.
- State two conditions the \(\text{CO}_2\) incubator maintains, and explain why carbon dioxide control is necessary for this medium.
- Two students set up identical cultures from the same stock on the same afternoon. Student A pours her plates inside a laminar air flow cabinet, allows the medium to cool to about \(60^\circ\text{C}\) before pouring, and inoculates within the cabinet. Student B pours his plates on an open bench directly after removing the flask from the autoclave, and inoculates beside an open window because the cabinet is occupied. Within two days, every one of Student B’s plates is overgrown with mould, and his plate lids are covered with water droplets. Student A’s plates are clear.
- Identify two separate errors made by Student B and state the consequence of each.
- Explain how a laminar air flow cabinet protects the exposed surface of a medium.
- Explain why the mould outgrew the intended culture rather than merely growing alongside it.
- Suggest one additional measure the laboratory could use to sterilise the transfer area before work begins.
- A newly established teaching laboratory is deciding on its purchasing and disposal policy. It must sterilise glassware, culture media, metal instruments and heat-sensitive vitamin solutions; it must surface sterilise field-collected plant material; and it plans to run animal cell cultures that will require serum. The head of department asks the staff to justify each choice on scientific grounds and to consider the ethical implications where these arise.
- Name the sterilisation method appropriate for each of the four categories of material listed, with a reason for each.
- The laboratory is offered a protocol using mercuric chloride for surface sterilisation. Set out the arguments for and against adopting it in a teaching laboratory.
- Explain why the composition of foetal bovine serum makes it both valuable and scientifically problematic.
- Suggest what the laboratory might do to reduce its reliance on serum, and state one limitation of your suggestion.
- Arjun is working in a plant tissue culture lab and transfers explants into culture media on an open bench. After a few days, contamination appears in all the culture tubes. His teacher explains that aseptic conditions were not properly maintained during transfer.
- Name the instrument that should be used for aseptic transfer.
- State its function.
- Mention one additional method used to sterilise the transfer area.
- A student prepares culture medium containing vitamins, enzymes and growth regulators. Instead of autoclaving, the teacher uses a membrane filtration method to sterilise certain components. The student is confused about why different sterilisation techniques are used.
- Name the sterilisation technique used for heat-sensitive substances.
- Why is autoclaving not suitable for these components?
- Mention the commonly used pore size in this method.
- During a lab practical, Meera uses a haemocytometer to count cells in a suspension culture. She also uses a stain to differentiate living and dead cells. The teacher asks her to interpret the results.
- What is the principle behind haemocytometer cell counting?
- What does the stain indicate about cell viability?
- Which cells, living or dead, take up the stain?
- Ravi is culturing mammalian cells but observes poor growth in a normal incubator. His mentor suggests using a specialised incubator that maintains \(\text{CO}_2\) levels along with temperature and humidity. After switching, cell growth improves significantly.
- Name the instrument used.
- State two conditions maintained by this instrument.
- Why are these conditions essential for animal cell culture?
- A researcher labels specific immune cells with fluorescent antibodies and passes them through a machine that separates cells based on fluorescence. The sorted cells are then used for further analysis. This technique helps isolate a pure population of cells.
- Identify the technique used.
- What is the role of fluorescent labelling?
- Mention one application of this technique.
- Priya prepares MS medium with auxins and cytokinins for plant tissue culture. After incubation, she observes excessive root formation instead of shoot development. Her teacher explains that hormone balance affects morphogenesis.
- Which hormone promotes root formation?
- What change should be made to promote shoot formation?
- Name one commonly used cytokinin.
- In a laboratory, a student prepares culture medium but forgets to adjust its pH before sterilisation. After incubation, the cells show poor growth despite proper nutrients being present. On checking, the pH is found to be outside the optimal range.
- What is the optimal pH range for most culture media?
- Explain how incorrect pH affects cell growth.
- Name the instrument used to measure pH.
- A researcher cryopreserves plant cells using liquid nitrogen and cryoprotectants. However, after thawing, many cells are found to be damaged and non-viable. It is suspected that ice crystal formation occurred during freezing.
- What is the temperature of liquid nitrogen used in cryopreservation?
- Why are cryoprotectants used?
- Suggest one reason for the reduced cell viability.
- Study Fig. 10.2, which shows a rectangular chamber with a working surface, a blower unit and a filter at the top, with arrows indicating uniform airflow across the workspace. This setup is essential for maintaining aseptic conditions during tissue culture work.
- Identify the instrument shown in the diagram.
- Write the complete name of the filter used in this instrument.
- State one function of this instrument in cell culture.
- Study Fig. 10.10, which shows a thick glass slide with a central grid divided into squares of known dimensions, with a coverslip placed above the chamber and cells visible within the grid.
- Identify the instrument shown.
- Explain how it is used to calculate cell concentration.
- Name the dye used to determine cell viability in this method.
- Study Fig. 10.5, which shows a large cylindrical vessel with components such as an impeller, sparger, inlet and outlet pipes and a temperature control system, used for large-scale cultivation of cells under controlled conditions.
- Identify the instrument shown.
- Explain the function of the impeller in the system.
- Analyse why aeration is essential in this setup.
- Label the parts marked “A”, “B” and “C”.
10.15 Practice With Data
A cell suspension is loaded undiluted into a haemocytometer. The counts recorded in five large squares of the grid are 42, 38, 45, 40 and 35 cells. Calculate the mean count per large square and the concentration of the suspension in cells per mL.
A dense culture is diluted 1 in 50 before counting. The mean count over four large squares is 62 cells per square. Calculate the number of cells per mL in the original suspension, and express your answer in standard form.
A student counts a suspension and obtains \(3.2 \times 10^{6}\) cells per mL. She needs to seed a flask with \(8.0 \times 10^{5}\) cells in a total volume of 10 mL of medium. Calculate the volume of the cell suspension she must transfer, and the volume of fresh medium she must add.
In a viability test, 320 cells are counted in all, of which 56 have taken up Evan’s blue stain. Calculate the percentage viability of the culture. If the total cell concentration is \(2.5 \times 10^{6}\) cells per mL, calculate the concentration of viable cells.
Two ampoules of the same culture are thawed and tested with Evan’s blue. Ampoule A gives 480 unstained cells out of 600 counted; ampoule B gives 315 unstained cells out of 700 counted. Calculate the percentage viability of each, state which recovered better, and suggest one difference in handling that could account for the result.
A suspension culture is frozen by the slow freezing method from \(0^\circ\text{C}\) to \(-100^\circ\text{C}\). Calculate the time taken for the controlled cooling stage at the slowest permitted rate of \(0.5^\circ\text{C}\) per minute and at the fastest permitted rate of \(5^\circ\text{C}\) per minute, and express both answers in hours and minutes.
A laboratory has a deep freezer operating at \(-80^\circ\text{C}\), a vapour phase nitrogen store at \(-150^\circ\text{C}\) and a liquid nitrogen store at \(-196^\circ\text{C}\). Using the temperature threshold above which ice crystals continue to form, state which of these are suitable for long-term storage and justify your answer numerically.
MS medium contains \(\text{KNO}_3\) at 1900 mg per litre and \(\text{NH}_4\text{NO}_3\) at 1650 mg per litre. Taking the molar masses as \(101\ \text{g mol}^{-1}\) and \(80\ \text{g mol}^{-1}\) respectively, calculate the concentration of each salt in mmol per litre and classify each as a macronutrient or a micronutrient.
Using the composition table of this chapter, calculate how many times greater the \(\text{KNO}_3\) content of MS medium is than that of White’s medium, and how many times greater the sucrose content of MS medium is. Comment on what these two ratios together suggest about the purpose of each medium.
A technician must prepare 250 mL of MS medium. Using the values in the composition table, calculate the mass of \(\text{MgSO}_4 \cdot 7\text{H}_2\text{O}\), \(\text{KNO}_3\) and sucrose required.
LB broth is made up as tryptone 10 g, NaCl 10 g and yeast extract 5 g per litre. Calculate the mass of each component needed to prepare 750 mL of the broth, and state the additional mass of agar required if the same volume is to be prepared as LB agar at 15 g per litre.
A laboratory prepares 1.5 litres of LB agar. Petri plates are poured using 20 mL each, and 150 mL is set aside for slants. Calculate the number of plates that can be poured and the volume of medium remaining unused.
FBS is to be added to DMEM at a final concentration of 10%. Calculate the volume of FBS and the volume of DMEM required to make up 500 mL of the supplemented medium. If the recommended range is 5–20%, calculate the smallest and largest volumes of FBS that would fall within it for the same total volume.
The table below records the pH of a bioreactor broth during a fermentation run in which the pH control system has failed.
| Time (hours) | 0 | 4 | 8 | 12 | 16 |
|---|---|---|---|---|---|
| pH of broth | 7.0 | 6.7 | 6.2 | 5.6 | 5.1 |
(a) Plot pH against time and describe the trend. (b) Explain, in terms of what the growing culture is doing, why the pH changes in this direction. (c) State what the pH control system would have done, and from where the correcting solution is supplied.
- The table below shows the percentage viability of a culture after freezing at four different cooling rates, all other conditions being identical.
| Cooling rate (\(^\circ\text{C}\) per minute) | 0.5 | 2 | 10 | 50 |
|---|---|---|---|---|
| Viability after thawing (%) | 71 | 84 | 52 | 30 |
(a) Identify the cooling rate giving the best recovery. (b) Explain why viability falls at rates faster than this. (c) Suggest why the slowest rate does not give the best result either. (d) State which of these rates fall within the range specified for the slow freezing method.
- A culture is sampled at intervals and counted on a haemocytometer, all samples being diluted 1 in 10.
| Time (hours) | 0 | 12 | 24 | 36 | 48 |
|---|---|---|---|---|---|
| Mean count per large square | 12 | 25 | 96 | 190 | 205 |
(a) Calculate the cell concentration in cells per mL at each time point. (b) Plot the results and describe the shape of the curve. (c) Identify the interval during which growth is most rapid. (d) Suggest two reasons why the increase between 36 and 48 hours is so much smaller than earlier.