Core Practicals

Procedure

• Soak paper disc in an extract of antimicrobial substance.
• Mix a bacterial species in agar to make sure they are evenly spread throughout the agar, making sure to use sterile equipment in a sterile environment. 
• Sterilise a petri dish and pour the seeded agar in.
• Place the paper disc on the agar, along with one not soaked in the extract (as a control to make sure it is only the antimicrobial substance affecting bacterial growth - this increases the validity of the experiment) and seal the disc - however do not seal all the way round to maintain aerobic conditions.
• Incubate at 25 degrees celcius for a week to allow bacteria to grow.
• Look for a zone of inhibition around the antimicrobial extract
• A clear area means there is no bacterial growth - and so the antimicrobial substance is effective. The larger the zone of inhibition, the more the substance has diffused (eg, higher concentration of substance), so the more bacteria killed around it.

Safety and Validity

• Prevent contamination (aseptic techniques and use a lid) - may culture pathogenic bacteria. The other bacteria which may land on the dish may compete for food (agar), or may not be affected by the substance, which would not produce valid results.
• Make sure not to seal all the way around the lid, to allow oxygen to enter for aerobic respiration, as otherwise you would create an anaerobic environment and so encourage the growth of anaerobic bacteria which are harmful to humans.
• Make sure to incubate at 25 degrees - this is warm enough to allow growth of bacteria, but not so close to 37 degrees as to encourage the growth of bacteria harmful to humans.

Control Variables

• Incubate for the same amount of time
• Use the same strain of bacteria (unless this is the independent variable) as different strains of bacteria may react differently to the substance.

Method

• Extract the fibres from the plants. Retting the fibres may make this easier.
• Pick fibres with the same length and diameter, from the same parent plant to eliminate variation in genotype. (alter control variables according to the investigation)
• Clamp the fibre at one end, and attach the other to a pulley.
• Attach the masses to the fibre in small increments to increase the accuracy.
• Record the mass at which the fibre breaks
• Repeat with different variations of the independent variable.
• Repeat at each variation to increase the reliability of the experiment and to identify any anomalous results.

Control Variables

• All variables apart from IV: length and diameter of fibre; type of plant, area of plant; same parent plant to eliminate genetic variation (more valid results); temp and conditions of fibre

Safety

• Wear safety goggles to make sure the plant fibre does not hit an eye when it snaps.
• Make sure there is a foam cushion on the floor to absorb the impact of the masses when the fibre snaps. This also prevents the masses from hitting feet and causing pain.

Accuracy

• Add masses in small increments to determine the breaking mass in greater accuracy.
• The fibres can break at any point between two masses.
  For example, if you added masses is 100 grams, the actual breaking mass could range between 100g, whereas if you added them in 10 grams, the actual breaking mass will have a range of 10g so it is more accurate.

Method

• Obtain different concentrations or different mineral deficiencies in different test tubes (in which case make sure all other mineral ions are present, with the same concentration and volume). Make sure to have at least 5 different concentrations.
• Select cuttings/explants (same genotype, reduces genetic variation to increase validity) of a similar size. Measure their mass, then put one in each test tube.
• Cover the test tube with a clear lid/cling film to prevent pathogens entering.
• Leave for a week in amiable conditions of a nice, warm temperature, a good light intensity and high CO2 concentration (to allow for photosynthesis)
• Measure the final mass of each plant and calculate the percentage change in mass.
• Repeat three times at each concentration/mineral ion.

Plants need various mineral ions to survive:

Magnesium: needed to synthesize chlorophyll, which is needed as a for photosynthesis to take place, which then produces glucose and oxygen needed for the plant to respire and grow.

Calcium: component of the middle lamella (forms calcium pectate) between cell walls, which helps stick adjacent cells together to strengthen and support the plant. It is also involved in membrane permeability as well as helping uptake of other mineral ions. 

Nitrate: component of DNA and amino acids for proteins (growth), as well as being used in plant hormones.

• Sterilise the tissue using bleach/ethanol and sterile tweezers
• Place the plant tissue on a sterilised agar plate (and grow into a de-differentiated group of cells - a callus, which can then differentiate into other cells), with added growth regulators.
• Work in a sterile environment and use sterile equipment to prevent bacterial contamination
• Cover the top of the plate with clear film. This allows light to enter for photosynthesis, but prevents contamination from bacteria which may be pathogenic or compete for food.
• Leave the plant in an environment with plenty of light for a week. 
• Tissues which grow into other plant tissues/whole plants show totipotency.

blarh blarg blag blar blargggg

totipotent: an undifferentated cell which can keep dividing infintely to give rise to all other cell types, including other undifferentiated cells.

·         Cut root tip
Reason: Root tip (meristem) = site of active cell division (i.e. mitosis)
Safety: Cut away from oneself

·         Stain with orcein ethanoic (orc-ein = one orc)
Reason: To make the chromosomes visible
Safety: Wear safety goggles as it is an irritant, and wear gloves and a lab coat to prevent stains on skin or clothes

·         Add warm acid
Reason: Heat intensifies the stain whilst acid breaks the middle lamella (and so allows the cells to be spread out). Macerating also helps spread cells.
Safety: wear goggles to protect eyes from the hot acid - or generally just take care with the hot acid 

·         Squash between slide and coverslip
Reason: Spread all the cells into one layer for ease of viewing
Safety: Take care not to break the glass