Separating micro-organisms




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Part 1
Eye protection

Disinfectant and paper towels

10 mustard seeds

30 cm3 10% bleach in lidded container

forceps

clingfilm



new, clean, block shaped sponge

germination vessel, half filled with sterile water


Part 2 (after 2 – 3 days)

All equipment must be very clean
Disinfectant and paper towels

Bunsen burner

Eye protection

Forceps


Scissors

6 small glass containers:

2 half filled with 1% plain agar

2 half filled with 1% plain agar plus 2.2 g litre M&S salts

2 half filled with 1% plain agar plus 4.4 g litre M&S salts

clingfilm


Materials to be shared
Light bank

Hazard bags for disposal of plates


Instructions Part 1: Sterilising seeds for germination


  1. Prepare work space: clear area, swab bench with disinfectant.




  1. Collect materials and equipment for part 1.




  1. Place 10 mustard seeds in lidded container of bleach. Swirl seeds and leave for 10 minutes in the bleach.

Remember your seeds are now sterile. Do not touch with your hands.


  1. Pour off bleach.




  1. Rinse three times with fresh, sterile water, leaving them covered with a little water.




  1. Sprinkle the seeds onto the clean sponge, making sure the seeds are spaced out.




  1. Place sponge in germination vessel, with water level half way up sponge.






  1. Label vessel with your name date and seed type.




  1. Place under a light bank in a warm place (ideally 20 - 26C), for 2 to 3 days.




  1. Leave until germinated and cotyledons (young leaves) have just started to unfold.


Instructions Part 2 (after 2 – 3 days)


  1. Collect germination vessel and equipment for part 2 (see Student Guide).

  2. Select the 6 straightest and longest seedlings for your experiment.

  3. Label six glass containers with date and check there are 2 each of three medium types:

You should have 6 containers:

2 with agar and no M&S salts

2 with agar and 2.2 g/l M&S salts

2 with agar and 4.4 g/l M&S salts



  1. Cut off six cotyledons as shown below. These are your explants.






  1. Use cooled, sterile forceps to transfer one explant into each of the six containers. Push cut end of explant into the agar, making sure the cotyledons are not touching the agar’s surface.






  1. Cover containers with cling film and place in a warm area under a light bank for 1 to 2 weeks.

  2. Observe and note the number of explants with roots after 3, 6 and 9 days approximately.

  3. Collect class results and calculate % explants with roots for the three different media on the three different days.

  4. Present your results as three lines on a single graph (one line for each medium) with suitable scales and axes labelled with quantities and units.


CLONING CAULIFLOWER
Source: NCBE Practical Biotechnology at http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PRACBIOTECH/PDF/

cauli.pdf
A similar protocol, which includes colour photographs, can be found at http://www-saps.plantsci.cam.ac.uk/docs/tissue.pdf

Materials required for each student or group of students:
Fresh cauliflower curd (the white part)

Sterile distilled water (100 cm3)

70% ethanol (50 cm3)

20% Domestos solution (100 cm3)

Boiling tubes, each containing 2-3 cm3 of plant tissue growth medium

Sterile Petri dish

Metal forceps and scalpel

Non-absorbent cotton wool and aluminium foil

White tile or suitable cutting surface
Practical details


  1. Swab the working area with 70% ethanol. Keep the ethanol away from exposed flames!

  2. Cut out a small piece of cauliflower curd; roughly the size of a cherry. Working on a clean Petri dish, divide the curd into three.

  3. Place the pieces (explants) in bleach e.g. Domestos solution for 10 minutes to surface sterilize the tissue.

From this point on, quick, aseptic operations are important to prevent contamination.

  1. Rinse the explants in three successive beakers of sterile distilled water. Use flamed, cooled forceps to do this – the correct way to flame forceps and other instruments is to dip them in alcohol, then to pass them briefly through a flame to ignite the ethanol. As the ethanol burns off, it heats the surface of the instruments to 70C, killing any contaminating organisms. Do not heat forceps and scalpels until red hot, and remember to keep ethanol away from exposed flames.

  2. The explants can be left in the final beaker of sterile water (covered with a Petri dish lid) until required.

  3. Take the first tube of growth medium withdraw the cotton wool plug, then briefly flame the neck. Use flamed, cooled forceps to pick up an explant and quickly drop it into the tube. Return the forceps to the ethanol beaker. Flame the neck of the tube once more, then replace the plug.

  4. Repeat Step 6 with the two remaining explants and two fresh tubes of growth medium.

  5. The tubes should be kept in a warm, light place. Growth should be visible within 10 days. Contamination, if it has occurred, should also be visible by this time. Failure of anything to grow usually indicates that the bleach has not been rinsed from the plant tissue.


Safety
Students should wear safety goggles when using bleach solution.

Ethanol used for sterilizing working surfaces should be kept away from naked flames.


Results
Observe periodically throughout the 10 day incubation process and describe growth.




ENZYMES & FRUIT JUICE PRODUCTION
Source: Adapted from NCBE Practical Biotechnology

http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PRACBIOTECH/PDF/

juice.pdf

Extracting Fruit Juice
The enzyme pectinase can break down the pectin found in cell walls. Fruit juice companies use pectinase to improve the juice extraction and to produce a clearer juice.
Read the following procedure and think carefully about what apparatus you will need and what information you will need to record.
Remember to label your beakers and measuring cylinders


Experiment 1
Practical details


  • Collect a piece of apple and grate it.

Place an equal weight of grated apple into two beakers


  • To one beaker add 2 ml of pectinase and to the other add 2 ml of distilled water.




  • Stir each beaker and leave for 5 minutes.




  • Filter the juice from the apples.




  • Record the volume of juice every 2 minutes.


Apparatus
Results
Use the following grid and design your results table







































































































Conclusion
ENZYMES
Extracting Fruit Juice
Questions


  1. What effect did the enzyme pectinase have on the volume of juice produced?

___________________________________________________________




  1. What effect did the enzyme have on the clarity of the juice?

___________________________________________________________




  1. Why did you set up a beaker which had the apples and distilled water in it?

___________________________________________________________




  1. What other factors might affect the volume of juice produced?

___________________________________________________________


___________________________________________________________


You will investigate some of these factors when you know a little more about juice extraction.
ENZYMES
Clarifying Fruit Juice
You have already discovered some effects of the enzyme pectinase. Pectinase was first used by industry to clarify juice. In this experiment you are going to look at the effects of pectinase and amylase on the clarity of juice.
What does the enzyme amylase do?
______________________________________________________________

Experiment 2
Practical Details
Set up the following test tubes


ENZYMES
Clarifying Fruit Juice
Add 10 ml of cloudy apple juice to each tube and stir
Incubate for 30 minutes at 40C
Observe the tubes every 5 minutes and record their appearance
Results: Make a results table to record your results


Questions


  1. What effect did each enzyme have on the clarity of the juice?

AMYLASE
___________________________________________________________


PECTINASE
___________________________________________________________


  1. What was the effect of using both enzymes?

___________________________________________________________


___________________________________________________________
DEHYDROGENASE ACTIVITY IN YEAST
Source: NCBE Practical Fermentation

http://www.ncbe.reading.ac.uk/ncbe/protocols/fermentation.html

Yeasts are living organisms. They belong to the group of microbes known as FUNGI.




Yeasts are single celled organisms which reproduce by splitting into two. This process in yeast is known as budding.
Yeast has been used for thousands of years to make beer, bread and wine. These technologies have developed because yeast cells can produce carbon-dioxide and alcohol when they grow. The process is known as fermentation.
Nowadays yeast is still used in the brewing and baking industries but it is also used to produce new products such as flavourings for foods, fizzy drinks and the enzymes needed to make cheese.
Dehydrogenase Enzymes in Yeast
During aerobic respiration glucose is gradually broken down and energy is released.
glucose + oxygen  energy + carbon dioxide + water
In a metabolic pathway such as this, it is usually the removal of hydrogen from the glucose which allows the energy to be released. The removal of hydrogen is called oxidation. As the glucose is oxidised, hydrogen is released at various stages along the pathway. This hydrogen binds to a chemical called a co-enzyme and each reaction is catalysed by an enzyme known as a dehydrogenase.
Therefore dehydrogenase enzymes catalyse the oxidation of substrates by transferring hydrogen to co-enzymes such as NAD. These co-enzymes carry the hydrogen as they become reduced.


AH2

substrate

e.g. glucose



+

2 NAD

co-enzyme




A

oxidised substrate

+

2 NADH

reduced co-enzyme

Although it would not be possible to detect this reaction in a test-tube some chemicals such as resazurin dye change colour when they gain hydrogen. We say the chemical has become reduced.


It changes colour in the following ways:


blue

(unreduced)



lilac

mauve

(partially reduced)



pink

colourless

(reduced)


This reaction enables you to test to see if respiration is taking place. You can also use it to investigate respiration rates as you alter variables. Yeast is a suitable organism to use for these investigations.


Experiment – Testing respiration rate in yeast
Apparatus
3 test tubes

3 labels


5 ml or 10 ml syringe

resazurin dye

fresh yeast solution

boiled and cooled yeast solution

glucose solution

water bath



timer
Method


  1. Label 3 test-tubes A, B & C




  1. Add 3 ml of resazurin dye to each tube




  1. Add 3 ml of glucose solution to A & C

Add 3 ml of water to B


  1. Add 3 ml of fresh yeast suspension to A & B

Add 3 ml of boiled and cooled yeast suspension to C


  1. Shake each tube and place in a water bath at 35C


Result
Record the colour in each tube in the table below


Time in minutes

A

B

C







0













Blue

3













Lilac

6













Mauve

9













Pink

12













Colourless

15
















18
















21
















24

















QUESTIONS
1 Give an example of a metabolic pathway __________________________
___________________________________________________________


  1. What happens to the hydrogen when it is first released from the glucose?

___________________________________________________________


___________________________________________________________


  1. What do dehydrogenase enzymes do?

___________________________________________________________


___________________________________________________________


  1. What is a reduced coenzyme?

___________________________________________________________




  1. What happens to resazurin dye when hydrogen is added to it?

___________________________________________________________




  1. In which test-tube did the yeast respire most rapidly?

___________________________________________________________




  1. What was the purpose of test-tube C?

___________________________________________________________


DNA FROM KIWI FRUIT
Source: Adapted from NCBE Illuminating DNA

http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PRACBIOTECH/PDF/

onion.pdf
In this practical you are going to isolate DNA from plant cells (i.e. not from micro-organisms).
Detergent is used to degrade the cell and nuclear membranes. Cell fragments are separated by filtration leaving the DNA and the soluble proteins in the filtrate. A protease enzyme called neutrase breaks down the protein and then the DNA is precipitated using ice cold ethanol.
QUESTIONS

  1. Which part of the cell and nuclear membrane is the detergent degrading?

___________________________________________________________




  1. What will the neutrase enzyme break its substrate into?

___________________________________________________________


APPARATUS
A balance

Weigh boat

Sodium chloride

Washing up liquid

5 ml syringe

2 x 250 ml beaker

50 g of kiwi fruit

stirring rod

coffee filter paper

measuring cylinder

boiling tube

ice cold ethanol


A liquidizer, an ice bath and a water bath at 60C must be available in the lab.




METHOD
Part 1 Preparing the Fruit extract


  1. Add 1.5 g of sodium chloride and 5 ml of

washing up liquid to a 250 ml beaker.


  1. Make up to 50 ml with distilled water.



  1. Now, collect 50 g of fruit and chop it up, then add it to the salt/detergent solution.

  2. Stir the mixture and incubate for 13 minutes at 60C




  1. Cool the mixture in an ice bath for 4 minutes.




  1. Pour the mixture into a liquidizer and blend for 5 seconds

at high speed.



  1. Filter this mixture into a second beaker.


Separating the DNA




  1. Add 4 ml of fruit extract to a boiling tube




  1. Add 2 drops of neutrase enzyme to the extract




  1. Slowly trickle 6 ml of ice-cold ethanol very slowly

down the side of the test-tube.
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