Practical 6 - A-Level Biology coursework PDF

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A-Level Biology coursework ...

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Polina Lobacheva December 21, 2018 Y12 AS Level Biology Ms. Tooley Lab partners: Nicholas Warren, Seonhu Jeon Required practical 6: Using aseptic techniques to investigate the bacterial sensitivity to antibiotics Aim The aim of this investigation is to test the sensitivity of two bacterial species to several antibiotics. Equipment 

two sterile Petri dishes

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sterile molten nutrient agar

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two sterile Pasteur pipettes

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Spirit burner

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Hydrogen peroxide solution

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Antibiotics

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Circular paper disks

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beaker of disinfectant

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forceps

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adhesive tape

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marker

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Antibacterial gel

Method 1. Dip a pair of forceps into ethanol to sterilize 1. Briefly put them above the flame to sterilise, make sure to leave it to cool down 2. Take a Q-tip and wipe it on a dirty surface then smear the Q-tip onto the agar 3. Divide the dish into 4 sections using a marker (antibiotic, control, hydrogen peroxide and antibacterial gel) 4. Take 4 separate paper disks to dip into each solution and place into the correct section on the dish. Repeat steps 1-2 before dipping the paper disk into another substance. 5. When inserting these paper disks make sure to open the lid only slightly and hold it near a spirit burner so that the microorganisms don’t get contaminated 6. When all 4 paper disks are in place hold the lid of the plate in place with four pieces of adhesive tape. 7. Place your plate upside down at 25 degrees Celsius for 1-3 days 8. Now wash your hands. Risk Assessment Hazard

Risk1

Control

Ethanol

Flammable

Wear eye protection

Microorganisms

Biohazard

Grow bacteria on solids rather than liquids to avoid spillage Work near a Bunsen burner to prevent contamination

Hydrogen peroxide

1 (CLEAPSS)

Irritant to the skin

Wear eye protection

Post-Laboratory Questions 1 When your plates are stored in the incubator, they are placed upside down. Why is this done? To keep the moisture condensing inside the plate which would condense onto the agar and destroy the cultured bacteria.

2 Agar plates are sealed by placing adhesive tape across the plate as shown in the diagram. What are the advantages of this method of sealing plates that have microorganisms growing on them? It’s not completely sealed so air can still enter and leave the dish; this was the microorganisms will breathe aerobically to aid with growth and respiration.

3 What is the control in these experiments? Temperature and species of bacteria.

4 Did the two bacteria respond differently to the antibiotics? Did all the groups obtain the same results? Discuss the class results. We failed to obtain class results perhaps because the agar didn’t have enough nutrients, the temperature was too high because it was placed near a heater or there weren’t enough bacteria to start a colony. Therefore, we weren’t able to observe the growth of bacteria and unable to say how the different solutions affected the growth of the bacteria.

5 Why were the bacteria resistant to some antibiotics?

Antibiotic resistance is when bacteria is able to survive and grow in the presence of an antibiotic, hence producing an infection. Because of the high mutation rates of bacteria, it allows them to resist several antibiotics as they won’t work on them anymore because the bacteria have changed its genetic code.

6 Find out more about the way in which antibiotics affect the growth of bacteria. There are different types of antibiotics that kill different types of bacteria. For example, BetaLactam (an antibiotic) kill bacteria that are encircled by a cell wall. It blocks the process of bacteria building a cell wall. A cell wall is built by linking the bacteria together and without it the pressure inside will exceed the standard and the membrane will burst, hence killing the bacteria. (Learn.genetics.utah.edu, 2019)

1 Explain what is meant by a zone of inhibition. It’s a circular spot around the antibiotic where bacteria don’t grow. The zone of inhibition is used to measure the sensibility of the bacteria to the antibiotic. (Ieeexplore.ieee.org, 2019)

2 There are strict regulations in place to ensure you do not become ill when working with microorganisms. Explain why you must follow these safety instructions: a

Clean benches with disinfectant = to prevent bacteria from spreading from the dish and harming us. To clean up any spillage (bleach irritate your skin if come in contact with)

b Incubate agar plates at a maximum of 25 degrees C = This reduces the risk of culturing microbes/pathogens. c

Do not seal plates completely = so that the microorganisms don’t start breathing anaerobically ad produce lactic acid

d Work aseptically throughout the procedure = to not get infected with the bacteria and contaminate the microorganisms

Dylan set up a culture plate of bacteria and placed three filter paper circles onto the plate. The discs were labelled 1, 2, and each disc contained a different disinfectant. Disinfectant chemicals kill bacteria, but are too strong to be used on huma tissue. Dylan then incubated the plate for 5 days. These are his resul

1a) Describe how Dylan inoculated the agar plate with bacteria. Dylan took a Q-Tip and wiped it on a dirty surface then smeared it along the entire agar plate b) State and explain two hazards that he controlled in his experiment. Disinfectant – can irritate the skin if come in contact with Bacteria – can cause an infection c) Describe the difference between antibiotics, antiseptics and disinfectants. Antiseptics – used in animals and animals to prevent infections or slow down the growth of those that are present. Heals and protects wounds. Disinfectants – a cleansing substance used on non-living things (ex. household products). Harmful to human body. https://www.quora.com/What-is-the-difference-between-an-antisepticand-a-disinfectant

Antibiotics – kills infection inside the body d) Explain why Dylan also included a piece of filter paper with nothing added to his agar. A control variable, to see if there is a relationship between the independent and dependent variable. 2a) Calculate the zone of inhibition for each disc. Disc 1 – 18mm Disc 2 – 14mm Disc 3 – 32mm b) State and explain which disinfectant was the most effective. Antiseptic 2 because it diffused the least meaning it slowed down the growth of bacteria hence reducing the spreading of the infection. c) Calculate the difference in effectiveness between the most and least effective chemical. Most effective – 14mm; least effective 32mm. The difference is 18mm. References Cleapss.org.uk. n.d. CLEAPSS. [online] Available at: [Accessed 10 April 2020]. Ieeexplore.ieee.org. (2019). Measurement of the Zone of Inhibition of an Antibiotic - IEEE Conference Publication. [online] Available at: https://ieeexplore.ieee.org/document/7544871 [Accessed 11 Jan. 2019]. Learn.genetics.utah.edu. (2019). What is an Antibiotic?. [online] Available at: https://learn.genetics.utah.edu/content/microbiome/antibiotics/ [Accessed 12 Jan. 2019]....