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Walk-in chamber

The growth chamber (Benatska 2, basement, nr. 13) with 12 banks for separate plant growing.

Rules for the growth-chamber 13

General
The growth chamber (Benatska 2, basement, nr. 13) is a plant growing facility meant primarily for employees and students of the Department of botany. The chamber consists of 12 growing banks that can be used and programmed separately (chamber 10 - 35°C, each bank 2 - 12°C above the chamber) using the panel outside the room. One bank is approx. 1,5m2 and 55 cm high with maximum 300 μmol of LED visible light (30 cm from source) and far-red lighting.

Training
Everybody who uses the chamber has to be trained to use the growth chamber and sign to acknowledge that he agrees to these rules at the end.

Scheduling and payments
The schedule of the growth chamber is publicly available at (http://bit.ly/walkinchamber), it is only visible for Prf UK accounts and can only be changed by the manager. Larger projects and reservations are discussed in a short meeting every half a year. To schedule, write an e-mail to the manager.
All users (or groups) should participate in covering the basic running costs. These can be material and equipment like detergents, sterilisation agents, biocontrol, shoe covers, cans etc. A table with paid and possible future expenses will be kept by manager.

Cleanliness and pest protection
This is to avoid any contamination by pests such as thrips, aphids and sciarids (dark-winged fungus gnats) as well as plant diseases. Please try to be as cautious as possible.

Measures:
1) clean material
a) plant material – strictly no living material can be brought to the chamber without treatment; seeds have to be treated by heat/freeze treatment (find it in the protocol section); in case adult plant material has to be used, it should be surface sterilised by alcohol and bleach (protocol section)
b) soil – either naturally sterile substrate (perlite) or commercial soil treated with biocontrol agents is used (we use bacterial larvicide and/or parasitic nematodes, see protocols); if there’s no risk of side effects, the soil can be heat or γ-sterilised
c) pots – all pots should be either fresh or well washed with detergent and surface sterilised (e.g. by 70% ethanol or bleach solution)
d) utensils, labels, watering cans – the same as with pots; only supplied cans can be used for the watering
e) strictly no storage of material (especially soil and pots) outside the banks, small objects like pens can be stored on the shelf to the left
f) potting should be preferentially done in a clean lab within the building, in case of small experiments or small changes, the potting can be done at the table outside the chamber or very cleanly within the chamber; when transferring potted plants inside, avoid accidental contamination e.g. by a lid
g) if your experiment becomes source of pest, be prepared that it might have to be moved to greenhouse or other growth chamber to avoid spread of the pest

2) clothing and behaviour
a) clothing – use a supplied or your own dedicated lab coat when working in the chamber; avoid bright (especially yellow) shirt when you plan to enter the chamber that day, no access to the chamber if you visited greenhouse, garden or field the same day (the other direction is of course possible, shower and change of clothes can help as well)
b) shoes – if you work in the chamber often, bring you own clean shoes and store them in cabinet outside (then they should not be used even within the building), for short visits, use supplied shoe-covers
c) no food in the chamber
d) room door should be closed before opening the chamber and generally the chamber door should be closed all the time except when entering or leaving the chamber e) water your plants reasonably, sciarids love overwatered plants

3) everybody has to thoroughly clean his banks after finishing the experiment (using supplied vacuum cleaner, detergent and sterilisation if needed

4) if your plants are about to have seeds and you are sharing a bank, be sure to bag them to avoid “weeds” in others experiments

General safety
1) no spraying of pesticides or solvents within the chamber – the air in the chamber is circulating through all the banks
2) no one except the manager manipulates with banks other than his own; if you are not 100% sure how to program the banks, ask the manager to help you – be totally sure you are not changing someone’s parameters
3) everybody has to label his banks with name, duration and what is inside and a warning sign in case of potential infestation
4) all pest outbreaks, other serious problems or equipment failures should be reported to the manager immediately
5) UV light and sterilisation should be avoided because it damages the paint in the chamber and banks

Supplied utensils and pest protection measures
1) these are supplied for free use (paid collectively)
• vacuum cleaner, watering cans, stairs
• basic cleaning utensils, detergents and disinfectants, rubber gloves
• biocontrol agent (bacterial larvicide e.g. Vectobac) – not available right now
• shoe covers, some lab coats
• tags, sticky traps for pests
• sterilisation sacks
2) fresh start once a year with thorough cleaning (and sterilisation by ethanol if needed)
3) yellow and blue sticky pads in the banks as a permanent check for pests

Protocols:
heat/freeze for seeds
• 37°C for 2-3 days (e.g. ovens in geobotany lab)
• -18°C for 2-3 days (any freezer)

Thrips lay eggs into the seeds. The 37°C is for the eggs of thrips to hatch and -18°C is to kill the larvae (as eggs, they would survive the freezing)

surface sterilisation for plants
• dip the plant material (seeds, seedlings) by immersion in 90 % (v/v) ethanol in a sterile Petri plate, tube, beaker, etc., depending on the size of your material for 1 min.
• dip the plant material by immersion in a sterilizing solution (0.25-0.5 % sodium hypochlorite = 5-10% SAVO) + 0.01% Triton-X or Tween20 surfactant) for 10 min with gently mixing.
• remove the sodium hypochlorite and rinse the material five times with sterile deionized water.
bacterial larvicide and other measures against sciarids
• the substrate has to be watered/soaked with Bacillus thuringiensis var. israelensis that kills larvae of dark-winged fungus gnats, e.g. Vectobac, then the plants can be immediately planted – not available right now
• all biocontrol measures can be inefficient sometimes so in case this doesn’t work, we will also use Nemaplus or similar product (containing nematodes Steinernema feltiae) which has to be used fresh

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