AGRI10051 2024 Prac

AGRI10051 2024 Prac 1

PRACTICAL INFORMATION AND APPENDICES

Welcome to the Biology Laboratory! This first page contains important information that will help you during your practical classes, so you may wish to read through it carefully so that you are familiar with how the practicals run.

LOCATION, ATTENDANCE etc.

Both practical sessions across all 4 weeks are held in Level 2 (Rm 210) of the Redmond Barry Building. Please ensure that you attend your registered class as per your timetable. Attendance will be taken at each class.

Please be punctual (or even early) and come with the appropriate safety equipment/PPE.

If you are more than 15 minutes late you would have missed the safety information and start of the practical activities so will NOT be allowed to enter the practical. If this happens and you have a valid reason for being late or absent, you should email subject staff for further information.

ASSESSMENT

In this subject, there are 3 assessed practical classes (Week 1 practical is not assessed but serves as great practice). Each practical assessment is worth 7.5% of the overall subject mark. Passing the practicals is the first hurdle requirement for this subject: a mark of at least 50% in the combined in-semester assessments (Weeks 4, 5, 7 pracs run by BioSciences, and a workshop assignment run by SAFES).

Each practical assessment consists of two parts: a pre-prac quiz based on the preparation material and your prior knowledge (worth 1.5%), and a post-prac test which draws on all the completed practical activities (worth 6%). You may use your notes during the assessment (i.e., these are open-book assessments), but you must complete this on your own.

BEFORE EACH PRAC

•    Download and read the prac notes thoroughly. Pracs can be quite busy, and you will struggle if you are not prepared.

•   Watch the  TechTip videos as instructed on the preparation page for each Practical.  Information covered in these videos will not be repeated in class, as it is assumed that you have completed all preparation and therefore have knowledge of these techniques already.

•   Complete any other activities listed on the Practical Preparation page.

•   Complete the pre-prac test before your scheduled prac day (except for Week 3).

PRACTICAL 1: Chromosomes, Cell Replication and DNA

In this prac, we will:

•    Become familiar with basic microscope techniques

•   Visualise chromosomes in plant cells to understand mitosis

•    Extract DNA from strawberries

•   Set-up bacterial plates for the auxotroph experiments which you will characterise next prac

BEFORE THE PRAC

•    Download and read these prac notes, including the appendices

•    If you haven’t completed AGRI10050 last semester, watch the following videos on Canvas:

o Using a compound microscope

o Using a dissecting microscope

•   Watch the following videos on Canvas:

o TechTip: Spreading an agar plate

o TechTip: Setting up the Auxotroph experiment

ASSESSMENT

•    None for the first prac – a practice post-prac quiz will become available at the end of prac to get you used to the process, and to help you consolidate your knowledge.

IN THE PRAC

Time (approx.)	Activity
25 mins	Welcome & introduction to Practical 1
10 mins	Activity 1: Using a microscope Practical Task 1.1: Familiarise yourself with the use of a microscope
30 mins	Activity 2: Visualising chromosomes, part 1 Practical Task 2.1: Garlic root-tip staining Practical Task 2.2: Mitotic Index
20 mins	Activity 3: DNA extraction Practical Task 3.1: Extracting DNA from strawberries
20 mins	Activity 4: Characterising auxotrophs Practical Task 4.1: Experimental set-up

Your safety in the laboratory is very important:

•   At all times wear a lab coat, suitable shoes with enclosed heel and toe and safety glasses. •   Always work to ensure your safety and the safety of those around you. •    Immediately report any injuries or spills to a demonstrator. •    Microorganisms will be used in this class, avoid touching things to your mouth and wash hands carefully after the class. A risk assessment has been carried out for the practical classes and identified risks minimised.

ACTIVITY 1: USING A MICROSCOPE                                                                                      10 MINS

Practical Task 1.1: Familiarise yourself with the use of a microscope

The light microscopes in this lab can magnify images up to 400x. This is large enough to view most cells in good detail. During your practicals, you will use 2 different microscopes – a compound microscope and a dissecting microscope.

(A)     Read Appendix 1 (pp. 11-12) and familiarise yourself with the compound microscope. 

(B)     Read Appendix 2 (pp. 13) and familiarise yourself with the dissecting microscope.

(C)     Read Appendix 3 (pp. 14) and learn how to estimate size with both microscope types.

Prior to attending Practical 1

If you did not complete AGRI10050 last semester (or would like to revise), you will need to watch the two videos below BEFORE attending the first practical class. It will be assumed that you have completed this preparation or are already familiar with the basic features of microscopes and how to use them.

(A)     On Canvas, watch the short video about how to set up the compound microscope.

(B)     On Canvas, watch the short video about how to set up the dissecting microscope.

During Practical 1

We will only be using the compound microscope for today’s practical activities. Spend a few minutes looking at the compound microscope on your bench. Practice adjusting the magnification and focus using the provided newsprint.

Q1.1a. Take note of the information in the appendices about microscopes – what are some of the main components/features of these microscopes?

Q1.1b. Recognise that the two different types of microscope are used to examine different samples in different contexts – why might this be important?

ACTIVITY 2: VISUALISING CHROMOSOMES, Part 1                                                           30 MINS

Practical Task 2.1: Garlic Root Tip Staining

DNA is so long that when it is condensed, the resulting chromosomes are large enough to be viewed quite  easily under a light microscope. To see this, you need to view cells that are actively dividing, since it is only during cell replication that chromatin (DNA and its associated proteins) will condense into chromosomes. Most of our mature, specialised cells do not actively divide; those that do divide are often unspecialised cells called stem cells. These are found in certain locations in the body. For example, although our skin constantly replenishes itself as the outer layers die, the adult skin cells do not replicate; it is a layer of stem cells under the adult skin cells that constantly produce new skin cells for the body.

In plants, there are also regions where cell replication facilitates plant growth. These regions are called meristems. For this activity, we use garlic root tips which have a meristematic region near the tip. We have stained these cells with an appropriate stain that binds to chromatin and squashed them onto a glass slide  in order to view a single layer of cells. You may want to watch the short TechTip video (Staining a Garlic Root Tip) to see how this was performed.

Place one of the prepared slides onto the stage of the compound microscope. Look around the preparation at scanning power (x4) until you see small squarish cells. These small, cuboidal shaped cells form chains because they were recently dividing. As cells leave the meristematic region, they start to grow in size rather than divide. Hence, any regions with larger or longer cells are unlikely to be actively dividing – ignore these regions and only zoom in on areas with cuboidal cells. Using low power (x10), look for a cell in which the chromosomes are visible.

When you do, you should see some cells arrested during mitosis (Fig. 1). Ensure that you can identify the stages of mitosis seen. The four main stages are prophase, metaphase, anaphase, and telophase.

Fig. 1 Cells of the meristematic region – cuboidal and in chains, with some arrested during mitosis

Q2.1  In Figure 1, label a cell at each of the stages of mitosis.

Practical Task 2.2: Mitotic Index

One common variable we measure in dividing cells is the mitotic index of the tissue. The mitotic index refers to the proportion of dividing cells within the tissue. We can estimate this for the meristematic region of the root tip.

You will need to observe three separate fields of view of the meristem under high power (x40). Be careful that there is sufficient clearance between the objective and the slide. Count the total number of cells in the field of view (only whole cells). Count the number of cells in mitosis (any stage where chromosomes are condensed and visible). Then divide the cells in mitosis by the total number of cells you see. This is an estimate of the mitotic index. Repeat this for each field of view to get a more accurate estimate.

Q2.2a. Record your data in the table below and calculate an estimate of the mitotic index for this tissue.

	Number of cells in mitosis	Total number of cells	Proportion of cells in mitosis
Field of view 1
Field of view 2
Field of view 3
Totals
Mitotic index

In the lab you would repeat this 10 times using different fields under high power to get a more accurate estimate.

Q2.2b. Using a pie chart, show the relationship between the time spent at interphase compared to mitosis.

Q2.2c. Suppose the cell cycle for garlic meristematic tissue is 24 hours. How long would a cell spend in mitosis each day?

High mitotic rates are often indicative of cancer, which is simply uncontrolled cell growth. Therefore, mitotic indices are useful when observing cancer pathology. For some cancers, mitotic indices are the most reliable predictor of whether a cancer will reoccur.

Consider the data below on meningiomas, which are tumours arising from the meninges – membranes surrounding the brain and spinal cord. 

Fig. 2 Relationship between mitotic index and survival with no recurrence of cancer in 99 meningioma patients. Solid line denotes a mitotic index

<4, dashed line denotes a mitotic index of 4 or more. Adapted from Kim et al. 2007

Q2.2d. Describe the relationship between mitotic index and the probability of survival as shown in Fig. 2.

Q2.2e. In the study above, an average number of mitoses in 10 high-power fields of view was calculated, and reported as a single number, rather than a proportion. Why do you think this repetition is important in cancer studies?

ACTIVITY 3: DNA EXTRACTION                                                                                               20 MINS

DeoxyriboNucleic Acid (DNA) is found in the cells of living organisms, and encodes the blueprint instructions for life. Extracting DNA  allows us to determine the genotype of individuals and conduct other genetic analyses. We now know enough about  how to extract  DNA that we can even use common household reagents to do this, even from your own cheek cells. Some organisms contain more DNA in their cells than others, which makes them a good choice for some bucket biochemistry DNA extraction. Fruits in particular contain lots of DNA, and today we will extract DNA from strawberries.

To extract DNA, we need a reagent to perform. each of the following steps:

1.       Rupture the cell membrane with a detergent.

2.      Separate the DNA from its associated proteins.

3.       Precipitate the DNA out of solution using alcohol.

Materials

•   Strawberry

•   Ziplock bag

•    Lysis buffer

•    Plastic cup

•   Coffee filter

•    Ethanol (cold)

Method

1.    Remove leaves from the top of a strawberry.

2.    Place the strawberry in a Ziplock bag and pulverise the fruit by pressing down with your hand or rolling a pen back and forth over it (don’t break the bag!)

3.    Add 10 ml of lysis buffer. (To make 100 ml of this solution, we

mixed 90 ml of water with 10 ml of shampoo and ¼ teaspoon of salt.)

4.    Mix lysis buffer with the fruit by pressing down on the Ziplock bag or rolling a pen back and forth over it for 2 minutes.

5.    Place a coffee filter into a cup. Pour the contents of the Ziplock bag into the coffee filter and set aside for 10 minutes.

6.    Discard the coffee filter and its contents. Keep the liquid in the cup (the filtrate).

7.    Pour 30 ml of cold ethanol into a tube, then pour in the filtrate. The DNA should become visible as a white precipitate at this

stage!

8.    Take a glass rod and give it an electrostatic charge it by rubbing it on your shirt a few times. Spool the DNA onto the rod.

Compare your extraction with the others on your bench. Who precipitated the most DNA?

In a research lab, the DNA extracted would be cleaned and dried, and then resuspended in an appropriate solution for subsequent use and/or storage.

Q3.1a. How do you think a detergent helps to rupture the cell membrane? Explain.

Q3.1b. Deduce the function of the salt in the extraction process. (i.e. Why not use plain or distilled water?)

Q3.1c. Consider the steps required to extract DNA listed above. How would you modify this protocol if you were trying to extract DNA from bacterial cells instead?

ACTIVITY 4: CHARACTERISING AUXOTROPHS                                                                   20 MINS

Practical Task 4.1: Setting up the experiment

Your average wildtype bacterium can produce all the organic compounds it requires, such as amino acids and vitamins, from inorganic sources in the environment. This type of bacterium is known as a prototroph. Prototrophs can grow on minimal media agar, which only contains inorganic ingredients.

However, if there is a block in a pathway to manufacture one of these compounds, the bacteria must be provided with the compound in the environment and will not grow on minimal media without it. This strain of bacterium is known as an auxotroph. By adding different compounds to minimal media and seeing which compound allows bacteria to grow, we can establish in which pathway this block occurs.

In this activity, you will determine the supplement requirements of 4 strains of E. coli auxotrophs. Because the bacteria need to time to grow, you will perform the set-up for this experiment this week and analyse the results in the next practical in Week 3.

Work in groups of 4 – you will need to share your results so that all 4 strains can be analysed between you.

Equipment per group

•   4 agar plate with minimal media

•   4 Eppendorf tube containing bacterial suspension – each student in group to choose 1 auxotroph strain (A, B, C or D)

•    Petri dishes with paper tabs labelled: Arg (arginine), Leu (leucine), Meth (methionine), Tyr

(tyrosine), Phe (phenylalanine). These tabs have been soaked in the corresponding compound.

•    Micropipette and tips

•   Sterile spreader

•    Forceps

•   Alcohol

Procedure

When working with the agar plates you want to minimise the exposure of the agar to air and other contaminants like your hands. Therefore, keep the lid on the plate whenever possible.

1.   Collect the Eppendorf tube containing the strain of bacteria that you will work with (A, B, C, or D).

2.   Clearly label around the edge of the base of your petri dish with your seat number, prac day/time, and your bacterial strain (A, B, C or D).

3.   Using a micropipette and tip, transfer 100 µL of the bacterial strain onto the surface of the agar.

4.   Using the sterile spreader, spread the bacteria over the surface of the agar as demonstrated in the TechTip video. Replace the lid on the petri dish.

5.   Sit the agar petri dish on top of the laminated template provided on your bench (see Figure 3 below).

6.   Dip the forceps into the alcohol and carefully shake off excess alcohol.

7.   Using the now dry and sterile forceps, pick up the first paper tab e.g., Arg, and without touching  anything else, lift the lid of the petri dish and place the tab on the surface of the agar, above the corresponding position of the template. Lower the lid of the dish.

8.   Dip the forceps into the alcohol and carefully shake off excess alcohol. Repeat step 7 until each paper tab is in the correct position as per Figure 3 below.

Do NOT invert the plates. Leave them in the tray for incubating, with the tabs facing upwards. These plates will be returned to you in the next practical (Week 3).

Each strain has been spread on minimal media. The paper tabs that you are placing on the agar plate are each infused with a different amino acid. The amino acids in the paper tabs will diffuse out into the agar.

Fig. 3 Positions of paper tabs impregnated with amino acids on each petri dish

Q4.1a. Without using the word ‘control’, explain why one paper tab is infused with water only.

Q4.1b. If, when you check the plates next practical, you were to observe growth of E. coli around the disc impregnated with water, how would this influence the conclusions you could draw from your experiment?