Cloning and biotechnology

Question 1

define agar

Answer 1

a polysaccharide of galactose obtained from seaweed, which is used to thicken the medium into a gel

Question 2

define aseptic technique

Answer 2

sterile techniques used in culturing and manipulating microorganisms

Question 3

what does typical nutrient agar contain

6

Answer 3

peptones
yeast extract
salts
water
may: glucose or blood

Question 4

what are peptones

Answer 4

what gelatine breaks down into using enzymes

Question 5

why were aseptic techniques developed

Answer 5

to reduce the risk of contaminating the medium with unwanted bacteria/ fungi

Question 6

what is a medium

Answer 6

refers to the substance that provides the necessary nutrients for the growth of microorganisms.

Agar plates are a common type of medium used for culturing bacteria and fungi.

Question 7

outline standard procedure of aseptic technique

Answer 7

1. Wash hands
2. Disinfect the working area
3. Have a bunsen burner operating nearby to heat the air
4. As you open the vessel, pass the neck of the bottle through a flame. The bottle should also be flamed as it is closed.
5. Do not lift the lid of the petri dish completely, open it enough to allow the introduction of the desired organism
6. Any glasswear/metal equip. should be passed through the flame before and after contact with the desired organism

Question 8

in the standard procedure of aseptic technique

why do we have a bunsen burner operating nearby to heat the air

Answer 8

Causes air to rise and prevents air-borne microorganisms settling.

Creates an area around it of sterile air.

Question 9

in the standard procedure of aseptic technique

why do we flame the neck of the bottle

Answer 9

prevent bacteria in the air from entering the bottle

Question 10

3 main steps to grow microorganisms on agar plates

Answer 10

1. sterilisation
2. inoculation
3. incubation

Question 11

sterilisation

hoow is the nutrient agar medium sterilised and what happens next

Answer 11

heating in an autoclave at **121 celsius **for 15 minutes

then, once the medium has cooled sufficiently to handle, it is** poured into sterile petri dishes and left to set.**

Question 12

sterilisation

when sterilising the nutrient agar medium, how is the high temperature achieved

Answer 12

boiling water under high pressure inside the autoclave

Question 13

sterilisation

why do we heat the nutrient agar medium?

Answer 13

kills all living organisms, including bacterial and fungal spores

Question 14

sterilisation

why is it important that the lid of the petri dish stays on after the nutrient agar medium has been sterilised

Answer 14

prevents infection

Question 15

what is inoculation

Answer 15

the introduction of microorganisms to a sterile medium

Question 16

inoculation

how is inoculation achieved

4

Answer 16

1. streaking
2. seeding
3. spreading
4. moisten a small cotton swab/bud with distilled water

Question 17

inoculation

outline streaking

Answer 17

a wire inoculating loop is used to transfer a drop of liquid medium onto the surface of the agar.

The drop is drawn out into a streak by dragging the loop across the agar.

the liquid medium is the microorganisms in** the broth**

Question 18

inoculation

outline seeding

Answer 18

a sterile pipette can be used to transfer a drop of liquid medium to the surface of the agar (or petri dish before the agar is poured in)

the liquid medium is the microorganisms in** the broth**

Question 19

inoculation

outline spreading

Answer 19

a sterile glass spreader used to spread the incoulated drop over the surface of the agar

another piece of equipment can be used instead of a glass spreader

Question 20

inoculation

outline how inoculation can be achieved through a small cotton swab/bud

Answer 20

a small cotton swab/ bud can be moistened w/ distilled water and used to collect microorganisms from a surface

AND THEN carefully wiped over the surface of the agar medium.

Question 21

inoculation

suggest a caution when streaking

Answer 21

take care not to break the surface of the agar

Question 22

outline how to inoculate an agar plate using a broth culture

Answer 22

1. heat inoculating loop in blue flame, allow to cool briefly
2. remove cap from broth culture and flame mouth of bottle.
3. Dip cool sterile inoculating loop in broth.
4. Flame and recap bottle.
5. Spread a streak of culture over surface of agar and cover with lid.
6. Reheat inoculating loop in blue flame.
7. Use adhesive tape to hold lid on petri dish and incubate at 25 celsius.

Question 23

incubation

the petri dish should be labelled and…

Answer 23

tape the top to the bottom using two strips of adhesive tape

Question 24

incubation

why should you NOT seal the petri dish completely

Answer 24

can lead to a selection of anaerobic bacteria which may be pathogenic

Question 25

# incubation why should you wash your hands thoroughly after handling a petri dish?

Answer 25

any moisture coming out of the dish could be a source of infection

Question 26

what will happen to the liquid broth when bacteria have grown?

Answer 26

clear to cloudy

Question 27

what is a liquid broth useful for?

Answer 27

**increase** **numbers** of **microorganisms** before transferring to agar plates for counting/identification

Question 28

what can the liquid broth be used to investigate

Answer 28

population growth

Question 29

# exam qu explain how flaming the tube (contaning the liquid broth) helps avoid contamination

Answer 29

causes air to expand and push bacteria away so less likely to settle into tube

Question 30

# exam qu List two precautions that should be taken when preparing a bacterial culture in order to ensure that the procedure is aseptic

Answer 30

Use sterile equipment eg broth Use stopper flask to prevent contamination Disinfect surfaces Use bunsen burner to create upward air flow

Question 31

1. what can be used to measure the growth rate of a microorganism population? 2. Outline how it can be used.

Answer 31

1. a liquid broth 2. A sterile broth is inoculated and the population size is measured at regular intervals during incubation

Question 32

how can the population size of a species of microorganism be measured?

Answer 32

transfer a small sample of microorganisms to an agar plate and incubate = each individual microorganism will produce a colony

Question 33

closed culture def

Answer 33

a culture which has no exchange of nutrients or gases with the external environment

Question 34

serial dilution def

Answer 34

a sequence of dilutions used to **reduce** the concentration of a solution or suspension

Question 35

# method Investigating population growth using aseptic techniques

Answer 35

1. Thoroughly shake the broth and incubate the broth at 25 degrees celsius. 2. At time zero take a small sample of the broth using a sterile pipette that gives a standard size drop. 4. Inoculate the agar plate and use a sterile glass spreader to spread the drop over the agar surface. 5. At intervals of 2 hours, shake the broth and take another sample to inoculate another nutrient agar plate. 6. When the broth starts to look cloudy, carry out a serial dilution and use each dilution to inoculate a sterile agar plate. 7. Incubate the plates at 25 °C. 8. After 24 hours, record the number of bacterial colonies visible on each plate and multiply by any dilution factor used. 9. Use the volume of the drop added to the plate to calculate the density of cells per cm?. 10. Plot the population density calculated (in cfu/cm*) against time. Typically the y-axis should be as a log10 scale.

Question 36

Will the population growth of a closed culture be a predictable or unpredicatble pattern?

Answer 36

predictable

Question 37

tissue culture def

Answer 37

a series of techniques used to grow cells, tissues or organs from a small sample of cells or tissue

Question 38

conditions needed for tissue culture

Answer 38

carried out on a nutrient medium under sterile conditions

Question 39

micropropagation def

Answer 39

taking a small piece of plant tissue (explant) and using plant growth substances to encourage it to fully grow into a new clone of the original plant

Question 40

conditions needed for micropropagation

Answer 40

ensure to use aseptic techniques to avoid fungi from colonising the growth medium

Question 41

why is micropropagation used by scientists

Answer 41

to reproduce endangered species of plants

Question 42

micropropagation method cauliflower

Answer 42

1. wear eye protection at all times 2. ensure that the environment is sterile by wiping surfaces with disinfectant and soaking all apparatus in sterilant 3. Break off a small section of the healthy cauliflower 4. using a scalpel, cut a thin section of the cauliflower. This is the explant 5. Sterilise the explant by soaking and swirling it in sterilising solution for 15 minutes 6. Using sterilised forceps take out the explant and place it in a sterilised container of agar growth medium 7. Leave on a sunny windowsill for a week

Question 43

why should the environment be sterile in micropropagation

Answer 43

so that no fungi contaminate the experiment, which would result in seeing a fungal growth rather than an explant growth

Question 44

adv of plant cloning (9)

Answer 44

* plants are same genotype and phenotype * plants produced are free of disease * plants can be genetically modified to give immunity to certain diseases * process is rapid * process yields large numbers of new plants * small plants produced can be transported easily to other sites * plants that can be difficult to grow from seeds can be produced by plant cloning * Plants can be grown in any country, in any season * Rare and endangered species can be propagated to save them from extinction

Question 45

disadv of plant cloning

Answer 45

* expensive * labour intensive process * process is susceptible to microbial contamination * no genetic variation, so all of the offspring are susceptible to the same diseases = risks large-scale loss * new plants have to be carefully screened for abnormalities that could lead to the new plants being infected * risk of an unexpected secondary metabolic chemical reaction that could cause stunted growth or even death in the new explants

Question 46

how to take a plant cutting

Answer 46

1. Ensure to cut a shoot from a healthy plant 2. Cut the stem at a slant between nodes 3. Dip cut in rooting powder 4. Place cutting in soil and add water (OR place cutting in moist soil) 5. Cover cutting with plastic bag or remove some leaves to reduce the rate of transpiration