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Before your first order: what plant tissue culture requires
Minimum equipment · Process · Safety
What has to be in place before your first culture
121 °CSterilisation in the autoclave at ≈ 1 bar overpressure
0.1 mgBalance readability for plant growth regulators
0.2 µmPore size for filter-sterilised additives
1 germis enough to lose a culture
Schematic of a horizontal laminar flow cabinet. The filtered airflow protects the culture, not the person in front of it.
Plant tissue culture rarely fails because of the plant. It fails because of microbes that grow faster than any explant.
Culture media contain sugar, vitamins and mineral salts. Bacteria and fungi multiply on them many times faster than plant tissue and overgrow it within a few days. Without an autoclave, a laminar flow cabinet and suitable balances, media, plant material and weeks of work are lost, usually without any way of finding out what went wrong.
This overview shows the minimum equipment required, how long a first run takes and which hazards are involved. Use the self-check at the end to see whether your setup is sufficient before you order media and active ingredients.
01
Minimum equipment
Eight devices without which sterile work cannot succeed reliably, and the materials needed for every batch. Where substitutes exist, their limits are stated.
Devices
01
Laminar flow cabinet
A fan forces room air through a HEPA filter and directs it across the work surface as an even, low-germ flow. Open vessels only stay sterile within this airflow.
Minimum
HEPA filter H14 to EN 1822, air velocity ≈ 0.45 m/s, regular filter testing
Substitute?
A glovebox or still-air box reduces the risk but does not replace the cabinet. Expect a considerably higher failure rate.
02
Autoclave
Sterilises media, water, vessels and instruments with pressurised steam and renders contaminated cultures safe before disposal.
Minimum
121 °C at ≈ 1 bar overpressure, at least 15 min holding time in the liquid, liquid programme with load sensor and door lock
Substitute?
A pressure cooker only with restrictions: many reach only ≈ 0.8 bar (≈ 117 °C), with no lock and no record. See hazards.
03
Analytical and precision balance
Plant growth regulators are weighed in milligrams, sugar and gelling agents in grams. This requires two balances.
Minimum
Analytical balance with d = 0.1 mg and draft shield, precision balance with at least d = 0.001 g, test weight for checks
Substitute?
Pocket, kitchen and jewellery scales are unsuitable: with d = 0.01 g or coarser, their display is too imprecise, see the worked example.
04
pH meter
The pH determines whether the gelling agent sets and whether nutrients remain available. It is adjusted before autoclaving, usually to 5.6–5.8.
Minimum
Glass electrode, two-point calibration with pH 4.01 and 7.00, 1 M KOH and 1 M HCl for adjustment
Substitute?
Test strips are too imprecise, their resolution is often 0.5 units. Calibration buffers have an expiry date.
05
Air-displacement pipettes
Stock solutions of plant growth regulators, vitamins and antibiotics are added in quantities from microlitres to millilitres.
Minimum
Two pipettes (100–1000 µL and 1–10 mL) with autoclavable tips, class A volumetric flasks and measuring cylinders
Substitute?
Disposable syringes and kitchen measuring jugs do not achieve the accuracy required.
06
Instrument steriliser
Forceps and scalpels are sterilised again between cuts so that microbes are not carried from one explant to the next.
Minimum
Glass bead steriliser (≈ 250 °C) or infrared steriliser, two sets of instruments to alternate
Substitute?
Flaming after dipping in ethanol disturbs the airflow and is a common cause of fires in the cabinet.
07
Culture vessels
Explants grow for weeks in closed vessels. These must let light through and allow gas exchange without letting microbes in.
Minimum
Tubes and jars with lids made of glass or PP, sterile Petri dishes, sealing film; stock for several subcultures
Substitute?
Preserving jars and baby food jars can be used if the lids are heat-resistant. Polystyrene (PS) deforms in the autoclave and is only usable as sterile-packed disposables.
08
Culture shelf
Cultures need even light and a constant temperature for months. Fluctuations slow growth and cause condensation in the vessel.
Minimum
LED lighting with timer (often 16/8 h light/dark), 22–26 °C, min/max thermometer, low-dust room
Substitute?
Windowsills are unsuitable: sunlight overheats closed vessels, and nights get too cold.
Materials and consumables
Dissecting instruments
Scalpel handle no. 3 with blades no. 10 and 11, long forceps (20–25 cm) so that your hand stays away from the vessel opening.
Sterile filtration
Syringe filters 0.2 µm for heat-sensitive additives such as gibberellic acid (GA₃) or antibiotics. Add to the cooled medium at ≈ 50 °C.
Glassware and stirrer
Beakers, bottles and measuring cylinders made of borosilicate glass 3.3, a magnetic stirrer with hotplate.
Laboratory-grade water
Demineralised or distilled. Tap water contains fluctuating amounts of calcium, magnesium, chlorine and trace metals.
Disinfection
Ethanol 70 % for surfaces and hands, sodium hypochlorite with a little Tween 20 for the explants. Concentration and duration are species-specific.
Personal protective equipment
Lab coat, safety goggles, nitrile gloves, plus heat-resistant gloves and a face shield for unloading the autoclave.
Storage
Stock solutions and media at 2–8 °C, some active ingredients at −20 °C. Labelled with contents, concentration and date, never stored with food.
Waste
Autoclave bags, a collecting tray and a puncture-resistant sharps container for blades.
Protocol and lab notebook
A protocol for your plant species and a notebook recording the date, batch and result of every run. Without records, contamination cannot be traced back.
Worked example
Weighing 10 mg of BAP
For 10 mL of a 1 mg/mL stock solution of 6-benzylaminopurine (BAP), you weigh out 10 mg. The balance display alone already determines how accurately this works.
Repeatability, static charge and the minimum weight add to this. The actual error is therefore larger than shown here.
Analytical balanced = 0.1 mg±0.5 %
Precision balanced = 0.001 g±5 %
Pocket scaled = 0.01 g±50 %
Kitchen scaled = 1 greads 0 g
Bars: rounding error of the display (± ½ d), relative to 10 mg. Dose small quantities via stock solutions instead.
02
Process and time required
From protocol to rooted plant takes months. The critical phase comes early: whether sterilisation has worked only becomes apparent after one to three weeks.
days to weeks
Find a protocol
Research medium, plant growth regulators and sterilisation times for your species in the literature. Not every species has an established protocol.
1 day
Prepare the medium
Make stock solutions, dissolve salts, sugar and additives, adjust the pH, add the gelling agent and dispense.
several hours
Sterilise
Autoclave media, water and instruments. Heat-up, holding time and cool-down together often take two to three hours.
1 day
Initiate explants
Surface-sterilise the plant material, rinse several times with sterile water, trim and place on the medium in the cabinet.
1–3 weeks
Contamination check
Inspect daily. Turbidity, slime or fungal growth indicate contamination. Remove affected vessels unopened and autoclave them.
months
Multiplication
Transfer growing shoots to fresh medium every 3–6 weeks. Every subculture is a new contamination risk.
2–6 weeks
Rooting
Transfer shoots to a rooting medium, often with auxin and without cytokinin.
2–4 weeks
Acclimatisation
Gradually accustom plants to substrate and room air at high humidity. Without this step, many plants dry out.
It usually takes several months to obtain the first rooted plant. Order active ingredients only once your equipment and protocol are in place.
03
Hazards at work
Tissue culture combines steam pressure, heat, flammable and corrosive liquids, and microorganisms. Two points are underestimated particularly often: opening a pressure cooker and handling contaminated cultures.
Pressure cooker instead of autoclave
Superheating: the medium boils once the lid is open
A laboratory autoclave with a liquid programme measures the temperature in a reference bottle and keeps the door locked until the load has reached a safe value, often 80 °C. A pressure cooker has no such lock (no thermolock). It can be opened as soon as the pressure has dropped.
The medium in the bottles cools much more slowly than the steam in the cooker and is often still above 100 °C at this point. A jolt while lifting a bottle out, or opening a cap, then triggers sudden boiling. Boiling medium shoots out and bottles can burst. The result is severe scalding of the hands and face.
Loosen caps by half a turn before autoclaving and fill vessels to no more than three quarters.
Never release the pressure quickly and never cool the cooker with water.
Only move bottles once a reference bottle of the same volume with a thermometer reads below 80 °C. This often takes considerably longer than the cooker itself needs to cool down.
Wear a face shield, heat-resistant gloves and a lab coat; move bottles upright and without jolting.
Check the water level, gasket and valves before every run. A cooker that boils dry overheats.
Indicator tape only shows that a temperature was reached, not that the medium is sterile. Verify the process with biological indicators (spores of Geobacillus stearothermophilus).
Schematic curve, not measured values. Large liquid volumes follow the temperature in the cooker with a considerable delay.
Microbial contamination
Autoclave contaminated cultures first, then dispose of them
Bacteria and moulds grow to high densities in a contaminated vessel, including species that can cause allergies or infections. Opening the vessel releases spores into the room air and onto other cultures.
Keep closed
Do not open, not even in the cabinet. Its airflow blows spores straight at the operator.
Collect
Collect in an autoclave bag inside a tray. Close the bag only loosely so that steam can enter.
Autoclave
121 °C, at least 30 min, longer for dense loads.
Dispose of
Only then dispose of according to local regulations, in a laboratory according to the institution's waste plan.
Ethanol and open flames
Ethanol 70 % is highly flammable (H225, flash point below 23 °C). Its vapours are heavier than air and collect on the work surface.
No flames in the cabinet, use a glass bead or infrared steriliser instead
Keep ethanol bottles closed and away from hot devices
Let disinfected gloves dry before reaching for the steriliser
Chlorine gas from hypochlorite
Sodium hypochlorite releases chlorine gas with acids and chloramines with ammonia. Both are severe respiratory irritants.
Never mix with acids, acidic cleaners or ammonia
Prepare in a well-ventilated room, make the solution fresh
Wear safety goggles, rinse splashes off with water immediately
Corrosive and toxic substances
1 M KOH for pH adjustment is corrosive (H314). Some additives are highly toxic, such as colchicine (H300, H340).
Read the safety data sheet before first use
Weigh powders at a draught-free place outside the cabinet, never in the airflow
Wear goggles and gloves, dispose of residues as per the safety data sheet
Hot surfaces
Glass bead sterilisers operate at ≈ 250 °C; the autoclave and hotplate stay hot long after being switched off. Hot glass looks exactly like cold glass.
Let instruments cool on a heat-resistant rest, blades that are too hot also damage the tissue
Never place hot glass on cold or wet surfaces
Keep heat-resistant gloves within reach
UV-C lamps
Many cabinets have a UV lamp. UV-C damages eyes and skin after a short time and can produce ozone.
Only switch on UV when nobody is in the room
Never look into the lamp, not even briefly
UV does not replace wipe disinfection, shaded areas remain untreated
Cuts
Scalpel blades are very sharp and are changed often. A cut at the cabinet is also a source of contamination.
Change blades with a blade remover or needle holder, never with your fingers
Put used blades straight into a puncture-resistant sharps container
Cut away from your body and hold the explant with forceps
Glass breakage and overpressure
Tightly closed bottles build up pressure in the autoclave and can burst. Scratched or thin glass cracks with temperature changes.
Loosen caps before autoclaving
Use only borosilicate glass 3.3, discard glassware with cracks or scratches
Pick up broken glass with a dustpan or tongs, never by hand
The cabinet only protects the culture
A horizontal laminar flow cabinet blows the filtered air towards the operator. Whatever produces dust, evaporates or grows on the work surface ends up in your face.
Do not handle pathogens, toxic powders or solvents in it
This requires a microbiological safety cabinet to EN 12469
No flames, they disturb the airflow and are a fire hazard
04
Self-check before ordering
Tick what you already have. If anything is missing, it is worth waiting before ordering media and active ingredients.
Not sure whether your equipment is sufficient? Write to us before you order. For method development, we offer consultancy services, which are subject to a fee.Contact us
This overview does not replace a risk assessment, operating instructions or safety training. The information provided by the device manufacturers, the safety data sheets and the regulations that apply to you are authoritative. All values are typical guide values and may differ depending on plant species and protocol.
