Prac Classes Handbook 2016

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BMS1021 PRACTICAL UNIT GUIDE

IMPORTANT INFORMATION

You MUST bring your practical manual/guide to each practical class (or

alternatively a copy of the relevant prac printed from Moodle) as you will

work-off these during the prac, and may have to hand them in at the

conclusion of your class.

Please read the Practical Notes carefully before your scheduled class and

bring with you the relevant items. Some practicals have specific requirements

for clothing etc. please be aware of these. All information can be found in this

practical manual/guide and on Moodle.

Additionally, there may be some prior reading and interactive online-sites to

visit before coming to class. Make sure you complete these as your

assessment may depend on them.

Please bring your labcoat to practical classes in Week 1, 4-6 and 8-12.

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REQUIREMENTS FOR SAFE CONDUCT OF STUDENTS IN LABS Safety in the laboratory depends upon students achieving a recognized standard of behaviour .The following requirements shall apply to all students who use or enter the laboratory:

• Laboratory coats or surgical gowns shall be worn at all times while in the laboratory unless otherwise stated.

• Safety eyewear should be worn at all times while in the laboratory unless a risk

assessment has deemed it unnecessary. (You will be supplied with safety glasses when necessary) • Only non-slip closed in footwear is acceptable footwear for the laboratory. Do not

enter the laboratory wearing open-toed shoes. • Long hair should be tied back at all times. • Do not handle, store or consume food or drink in the laboratory. • Do not smoke within the laboratory or associated storage areas. • Do not apply makeup while in the laboratory. • Do not mouth-pipette. • Wash skin areas which come in contact with chemicals, irrespective of

concentration. Wash hands upon leaving the laboratory. • Always report hazards, faults, incidents and injuries to the demonstrators/practical

convener. • Do not indulge in reckless behaviour in the laboratory. • Always adopt an alert attitude and be conscious of potential hazards. • Regard all substances as hazardous unless there is definite information to the

contrary. • Dispose of specialized wastes (e.g. broken glassware, syringe needles, biological

and radioactive substances) in containers designated for the particular type of waste.

• Keep all fire-escape routes completely clear at all times.

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Footwear To Be Worn While Working In The Labs All students should wear proper protective footwear (closed all round and the

foot covered totally).

Slippers, sandals, straps & high heels not permitted

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PRACTICAL CLASSES

You can allocate yourself to a practical class through Allocate+.

Students only attend one session per topic, see below (note Week 4/5 and 8/9 where students attend once in each of the 2 weeks designated).

Additionally, some students may be allocated into a Microbiology practical in Wk9 or 10 (instead of Wk11 and 12). This means students may have 2 pracs for different topics in the same week. This is unavoidable due to location capacity and student numbers.

Clayton Map http://www.monash.edu.au/assets/pdf/about/who/clayton-campus-map.pdf

Week

Practical Class / Topics

Venue

Assessment%

Week 1

Practical 1 Microscopy

22 Rainforest Walk

3.5%

Week 2

Practical 2 Biochemistry Online

Online-only 3.5%

Week 3

Essay Workshop: Finding and referencing information for your essay

Online-only -

Week 4

Practical 3 Introduction to Developmental Biology

10 Chancellors Walk / CG63 or 22 Rainforest Walk

3.5%

Mid-semester Break

Week 5

Week 6 Practical 4 Histology


3.5%

Week 7

Mid-Semester Exam

10 Chancellors Walk / CG63

10%

Week 8 Practical 5

Metabolism

22 Rainforest Walk 3.5%

Week 9

Week 10

Practical 6 Immunology


3.5%

Week 11

Practical 7A Microbiology Part A

12 Innovation Walk Room G27/G38

4%

Week 12 Practical 7B Microbiology Part B

Practical Classes Total = 25% Mid-Semester Test = 10%

http://www.monash.edu.au/assets/pdf/about/who/clayton-campus-map.pdf

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TABLE OF PRAC DETAILS

Assessment type

Assessment due dates

Submission location

Assessment marking and return

Assessment feedback

PRAC 1: Microscopy Prac questions At the end of the practical

In prac Prac returned within 2 weeks

Feedback written on prac

PRAC 2: Biochemistry Online quiz Available 5pm 4th March until 5pm Mon 14th Mar

Online At quiz closing time

Online feedback

Week 3 Essay Workshop. No assessment PRAC 3: Introduction to Developmental Biology

Online assessment TBA

Available 5pm Mon 21st Mar until 5pm Mon 11th April

Online Prac mark/ comments returned within 2 weeks

Online feedback

PRAC 4: Histology Online activity Available 5pm Mon 11th April until 5pm Mon 18th April

Online Same as above

Online feedback

Week 7 - Mid-Semester Exam PRAC 5: Metabolism Prac questions At the end of

the practical In prac Prac

returned within 2 weeks


PRAC 6: Immunology Prac questions At the end of the practical

In prac Prac returned within 2 weeks


PRAC 7A: Microbiology

Tutor Assessment

During prac n/a See below See below

PRAC 7B: Microbiology

Tutor assessment & Quiz

During prac Toward the end of the practical

In prac Overall marks (Quiz and Tutor assessment)within 2 weeks via Moodle Grades

Grades-only

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This page should be left blank

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PRAC 1: Microscopy Location: 22 Rainforest Walk Assessment for this practical will be questions to be handed in at the end of the practical. Assessment pages to be handed in can be found at the end of this prac and should be filled in, torn-out and handed in at the end of the practical session.

USING THE COMPOUND LIGHT MICROSCOPE TO STUDY CELL STRUCTURE AND ORGANISATION IN A PROTIST Specific Learning Objectives:

1. To learn the mechanics and use of the compound light microscope

2. To become familiar with cells and tissues using the light microscope

3. To learn and apply some of the conventions associated with the drawing of biological specimens

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PRE-LABORATORY PREPARATION 1. Read “Campbell Biology” pp. 93-99 (Microscopy) (10th Edition) pp. 102-103 (Eukaryotic Cells) pp. 134-136 (Water balance in cells without walls) pp. 612-613 (Ciliates: Paramecium)

For some groovy images and information on Paramecium, go to: http://protist.i.hosei.ac.jp/PDB/Images/Ciliophora/Paramecium/index.html or http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/articles/param1.html

2. Before coming to the practical, using the textbook and these notes, make sure you understand and

are able to define the following terms

cell membrane cytoplasm oral groove vacuole osmoregulation protist micronucleus cilium (plural cilia) macronucleus 3. Bring 1-2 sharp HB pencils, a ruler and an eraser to this practical

In Part 1 of this practical, you will learn how to use the microscope and determine the relationship between

the magnification and field of view of different microscope objectives.

In Part 2 you will examine and draw a unicellular protist, applying standard conventions for biological drawings.

Please be aware that the answers to your questions and drawing of your specimen will be due before you

leave the session. SAFETY NOTES • Please do not wear mascara to this practical – it ends up on the microscope eyepieces and is difficult

to remove • Microscopes are expensive, precision instruments – please handle them with care

http://protist.i.hosei.ac.jp/PDB/Images/Ciliophora/Paramecium/index.html

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/articles/param1.html

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/articles/param1.html

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PART 1. INTRODUCTION TO THE COMPOUND LIGHT MICROSCOPE The compound light microscope is perhaps the single most important instrument used in cell biology.

It can magnify objects from 40 up to 1000 times life size, allowing observation of cell and tissue

details that are not visible to the naked eye. Light from a lamp is sent through the specimen and

passes two lenses, the eyepiece and objective (refer to the diagram illustrating the microscope).

Total magnification is the result of multiplying the magnification factor of each lens. The high power

objectives (total magnification of x400 or greater) on a compound microscope mean that most tissues

must be fixed, embedded in a resin, sliced into very thin sections and stained before they can be

observed. This obviously kills the cells. Only very small and very thin, transparent specimens can be

observed whole and alive. Adjustment of the iris diaphragm alters the properties of the light passing

through the specimen, affecting brightness, resolution and contract. It is possible to monitor the

activities of living cells and to observe changes in their subcellular organisation. The light microscope

may also be used to monitor certain operations such as cell fractionation and biochemical

characterisation of cellular components.

Microscope Field of View (FOV)

Units of length

The basic unit of length used in all aspects of scientific measurement is the metre (m). In microscopy,

smaller units are used, which are:

millimetre: 1 mm = 10-3 m micrometre: 1 µm = 10-6 m nanometre: 1 nm = 10-9 m Determining the field of view diameters of a microscope To estimate the actual size of an object or structure being viewed, you first need to know the diameter of the field of view. The higher the magnification, the smaller the field of view. The diameter of the field of view for your microscope should be in the range 1.6-2.0 mm (1600-2000 µm) using the x10 objective (i.e. total magnification x100), and approximately 0.4-0.5 mm (400-500 µm) with the x40 objective (i.e. total magnification x400). You will use an object of known size (a micrometer) as the first specimen to calibrate the field of view.

Obtain a compound microscope by holding the microscope at the arm and under the base. Do not tilt the microscope or the eyepieces will fall out.

Collect a micrometer slide from the slide folder. Hold the slide up to the light – you should

just be able to see a small, straight, gray-black line (the micrometer), in approximately the middle of the coverslip. The micrometer is exactly 5 mm long, and the smallest divisions in the scale are 100 µm (or 0.10 mm) wide.

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Always start a microscope session with the lowest power objective (the x4 objective – it has a red band). With the x4 objective in position, use the coarse focus knob to position the microscope stage some distance below the tip of the objective, then place the micrometer slide on the microscope stage. Position the slide, using the stage adjustment knobs, so that the micrometer is in the middle of the beam of light shining through the hole in the stage.

Watching from the side, carefully raise the microscope stage to its upper limit.

Now, looking through the eyepieces, slowly lower the stage until the micrometer comes into

focus. Using the stage/slide adjustment knob, adjust the position of the slide until the micrometer is in the center of your field of view. When you move to a higher magnification (i.e. changing the objective by rotating the nosepiece), the micrometer should remain visible and you should only need to adjust the fine focus. Using the scale on the micrometer slide, estimate the field of view (FOV) diameter and write this value (in µm) into the ‘x40 mag.’ box on the first page of these notes.

Change to the x10 objective. Use the fine focus knob to focus on the micrometer.

Again, using the micrometer slide scale, estimate the FOV diameter and write this into the ‘x100 mag.’ box on the first page of these notes.

Answer Question 1. All questions and space for answers are provided at the end of these prac notes.

Now, without altering the focus, switch to the x40 objective focus, (please take care not to crush the objective into the micrometer slide), and measure the exact diameter of the field of view on your microscope. This diameter will vary between microscopes and should be measured individually for each microscope.

Answer Questions 2-5.

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PART 2. OBSERVING AND DRAWING A LIVING ORGANISM

The first living cells observed using the earliest microscopes were various unicellular microorganisms. Some unicellular eukaryotes are rather transparent, and thus need to be stained to give contrast to the cell and its contents. Others are fast swimmers, and need to be slowed down by immersion in methyl cellulose. In this part of the practical, you will observe and draw a ciliate protist and use different optical methods to enhance the observation of it and its subcellular components. Examination of Cells

View the organism at low (x100 mag.) and at high (x400 mag.) power. When examining cells, you should look for a number of different features. For example, cell size, shape and colour are important characteristics, along with the presence (or absence) of a cell wall, whether the nucleus is obvious, and the presence of particular organelles. A drawing will record much of this information. An accepted part of scientific drawings (and one that is useful for revision purposes) is to record any useful descriptive information on the drawing or in the caption. Paramecium, a freshwater ciliate The unicellular organism that you will examine is Paramecium, a freshwater ciliate. The surface of each cell is covered with hundreds of cilia arranged in longitudinal rows, which propel the organism through water by rhythmic beating.

1. Using the pasteur pipette provided, take a small sample of Paramecium culture (do not swirl the mixture and make sure your sample is taken from the bottom of the beaker) and place one drop of this culture onto a clean slide. Add a small drop of methyl cellulose.

2. Using a toothpick, mix the Paramecium culture and methyl cellulose solutions together, then carefully lower a coverslip over the mixture.

3. Touch a piece of filter paper to one side of the coverslip to remove any excess liquid.

4. Using the x4 and then the x10 objective, bring a Paramecium into focus, then switch to the x40 objective. Close the iris diaphragm (refer to the diagram of the microscope) to better examine the way Paramecium moves. HINT: if you are having trouble locating the Paramecium, focus along the edge of the cover slip and you should find some there.

Observe the cilia and the slow pulsation of the contractile vacuole.

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5. On the page provided, and using the x40 objective, draw a single Paramecium indicating the cilia (single = ‘cilium’), the cell membrane, the oral groove, and a contractile vacuole(s). Make sure that your drawing of the cell is at least 15 cm long (use half an A4 page) and that it conforms to scientific drawing standards. For examples of standard conventions such as title, labels, captions, scale bar, etc., please refer to the provided materials in the lab.

|--------------| 20 µm Figure 1. Photoimage of Paramecium.

6. Put an appropriate scale bar on your diagram (in the style of Fig. 1 above). To do this, focus on your specimen with the x40 objective (total magnification x400), and then use the following procedure:

Work out the ‘real’ length of the Paramecium you are observing. To do this, estimate how many times (lengthwise) it would fit across the field of view. It may be easier to work this out by placing it to one side of the field - e.g. at the right.

Note: Do not draw the edge of the field of view in your diagram.

Now divide the field of view diameter which you measured in order to answer Question 2, by the number of lengthwise Paramecium that would fit across your field of view. This gives you the actual size of your specimen in real life. Your answer should be in µm. For example, if the diameter of the field of view is 500 µm (0.5 mm) using the x40 objective (total magnification of x400), and the specimen fits across the field about 4 times, then this particular Paramecium is:

500/4 = 125 µm long in real life (i.e. its actual length is 125 µm)

Example of a scale bar

Edge of field of view Paramecium

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In Figure 1, the specimen is drawn about 10 cm long, so the ratio of ‘actual length’ to ‘drawn length’ is:

125 µm : 10 cm Now you need to draw a 1 cm scale bar next to your diagram. You need to indicate what this 1 cm represents in ‘real’ life (let this value = ‘y’). You can calculate the value of ‘y’ from the following ratio: 125 µm : 10 cm as y µm : 1 cm Thus, 125 µm / 10 cm = y µm / 1 cm So, y = (125 x 1) / 10 = 12.5 µm

Thus for this drawing, a 1 cm scale bar represents 12.5 µm in ‘real’ life. Therefore, a 2 cm scale bar would represent 25 µm in real life. If you generate a value like 24.2 µm, round up to the nearest whole number (i.e. 25 µm).

7. Observe and identify the different cellular components in Paramecium and label those that are asked for on your drawing.

Answer Questions 6 and 7.

Once Completed Make sure your microscope is clean. Remove the slide from the stage of the microscope. Wipe the x40 objective with lens tissue to remove any methyl cellulose which may be on it. Remove the power cord from the microscope and carefully return it and the microscope to their appropriate spots. Please ensure that the micrometer slide is returned to the slide folder, and place all used slides in the waste containers provided. Dispose of any rubbish and leave your workbench clean.

Assessment Make sure your name, authcate ID (not ID number) and prac session are written on each page that you are submitting. Staple your Paramecium drawing (Figure 1) and your answers to Questions 1-7 together and submit them BEFORE YOU LEAVE the lab. Late submissions will not be accepted.

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Prac 1: Microscopy prac Name: Authcate ID: Prac Session (Day & Time):

PART 1 Q1. What is the diameter of the field of view on your microscope using the x10 objective (i.e. at a magnification of x100)? [0.5 mark] Q2. What is the diameter of the field of view on your microscope using the x40 objective (i.e. at a magnification of x400)? [0.5 mark] Q3. What is the ratio of magnification between the x10 and x40 objectives? [0.5 mark] Q4. What is the ratio of the diameter of the field of view between the x10 and x40 objectives? [0.5 mark] Q5. What do these ratios tell us about the relationship between magnification and the diameter of the field of view? [1 mark]

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Prac 1: Microscopy Prac Name: Authcate ID: Prac Session (Day & Time):

PART 2 Using the x40 objective, draw a single Paramecium indicating the cilia (single = ‘cilium’), the cell membrane, the oral groove, and a contractile vacuole. Make sure that your drawing of the cell is at least 15 cm long (use half an A4 page) and that it conforms to scientific drawing standards. For examples of standard conventions such as title, labels, captions, scale bar, etc., please refer to the materials available in the lab. [5 marks] Note: Do not draw the edge of the field of view in your diagram.

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Prac 1: Microscopy Prac Name: Authcate ID: Prac Session (Day & Time):

Q6. Which organelle in the Paramecium regulates its water content? What would you expect to happen to the cell if this organelle was inoperative? (Hint: Paramecium lives in freshwater) [2 marks]

Q7. Cilia allow the Paramecium to move through water. Do these cilia move randomly or in a coordinated manner? Describe how the cilia move in relation to each other to allow for movement. [2 marks]

ASSESSMENT SUMMARY Activity Marks

Questions 1-7 7

Drawing of Paramecium 5

Total 12

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PRAC 2: Biochemistry This is a self-directed online practical – go to Moodle for details Assessment for this practical will be the completion of a quiz on Moodle. Available 5pm Friday 4th March until 5pm Mon 14th March

Ionisation of aspartic acid. Protein function often relies on specific charged groups to

catalyse reactions and as a result each protein will have an optimal pH at which it can

function. The separation of amino acids and proteins often relies in differences in their

molecular charge. In order to achieve a separation of two amino acids of similar size,

conditions must be found under which they have different molecular charges.

These will be studied by looking at the ionisation of the amino acid aspartic acid at different

pH’s.

There will be a self-directed learning exercise available on Moodle.

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Essay Workshop:

Location: Online-only

Finding and referencing information for your essay:

The activities that are part of this workshop are an important part of your Biomedical Science degree. Once you understand and apply this information, you will find that starting, researching and writing scientific essays will come more easily.

This workshop will require completing two sets of benchmark activities: one in week 3 and one in week 6.

The activities are built around skills needed to complete the 15% Essay for BMS1021. They have been prepared by library staff including Tomas Zahora and Penelope Presta.

Topics covered will include:

• How do I source information online? (week 3) • What library resources can I use for the essay? (week 3) • How do I use and properly reference scientific articles? (weeks 3 and

6) • How do I organise my essay? (week 6) • How do I avoid common essay writing mistakes? (week 6)

All necessary information is included in the workshop activities. No additional reading or preparation is needed.

See Moodle for more details.

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PRAC 3: Introduction to Developmental Biology Location: 10 Chancellors Walk (CG63) or 22 Rainforest Walk (Friday only) Assessment for this practical will be the completion of an online Moodle assessment TBA. Available 5pm Mon 21st March until 5pm Mon 11th April EXPLORING THE BASIS OF MALE INFERTILITY

Infertility or subfertility is a common problem in Australia, with approximately 15% of couples

experiencing difficulties conceiving. This has been addressed by the introduction of artificial

reproductive technologies (ART) including in vitro fertilisation (IVF) and intracytoplasmic sperm

injection (ICSI). In Australia, more than one percent of births are now the result of ART. However,

these techniques cannot address all causes of infertility, particularly in cases where the underlying

defect is unknown. Further research is required to identify the underlying causes of infertility and

develop new individualised treatment options.

Male fertility is also an important consideration for industries where breeding of animals is

important either for agricultural purposes or for other purposes (for example selective breeding).

Artificial insemination can be used which requires the assessment of the quality of semen.

In this practical we will examine the process of spermatogenesis, which is responsible for the

continual production of sperm, and the characteristics of sperm that are often examined in fertility

testing.

PRACTICAL OBJECTIVES

1. To visualise the different cell types within the seminiferous tubules and the progressive steps in

the process of spermatogenesis.

2. To examine sperm and identify the structure and function of different regions of mature

spermatozoa.

3. To investigate the role of a secreted growth factor from Sertoli cells in male fertility.

4. To analyse a porcine semen sample and measure sperm concentration, motility, morphology

and viability.

A worksheet will be provided in class.

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PRAC 4: Histology Location: 10 Chancellors Walk (CG63) or 22 Rainforest Walk (Friday only) Assessment for this practical will be the completion of an online (Moodle) assessment. Available 5pm Mon 11th April until 5pm Mon 18th April The take home message from this practical is to recognise that organs are made up of the 4 primary tissue types. Activities will include microscope work, hands-on activity, group work and an opportunity to present student group-work. The prac will be led by experienced demonstrators from the Anatomy & Developmental Biology Department as has been designed to be a fun and engaging class, to help you apply information learned in lectures.

Please bring to class:

Your histology lecture notes, any histology textbooks, iPads/personal laptops/smart phones (there will be computers available in class on request).

Don’t forget your labcoat.

Background reading:

Chapters in Histology books such as ‘Kerr Functional Histology’ 1st or 2nd edition, or ‘Wheater’s Functional Histology’ or any other basic histology texts. Focus on the 4 primary tissue types.

Revision of lecture notes may also be useful.

Worksheets/other will be provided in class.

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PRAC 5: Metabolism

Location: 22 Rainforest Walk

You MUST check your week and lab allocations for this class.

If you need to change, please see

Yardenah Brickman or Christopher Wilson in the 1st Year Biology Teaching Laboratory,

23 Rainforest Walk, BEFORE missing your lab.

Space is limited so DO NOT assume that there will be space if you turn up to a session

which is different to the one allocated to you. Assessment for this practical will be questions to be handed in at the end of the practical. Assessment pages to be handed in can be found at the end of this prac and should be filled in, torn-out and handed in at the end of the practical session.

CELL METABOLISM: AMYLASE ACTIVITY IN GERMINATING BARLEY

Specific Learning Objectives:

• To extract an active enzyme from biological material and use it to catalyse a specific biochemical reaction

• To quantify the amount of enzyme activity present in the sample

• To correlate enzyme activity with metabolic function during development

• To identify the product of the enzyme-catalysed reaction

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PRE-LABORATORY PREPARATION 1. Read the following pages in Campbell et al. (2015), ‘Biology’ 10th Edn (Australian version):

pp. 871 Seed germination pp. 68-75 Carbohydrates pp. 151-154 Enzymes pp. 167 Overview of cellular respiration 2. Use the textbook and these notes to make sure that you understand and can define the following key words:

amylase enzyme reaction metabolism starch ATP glucose pH buffer substrate biosynthesis glycolysis polysaccharide control hydrolysis product dissacharide maltose reaction rate

INTRODUCTION Amylase is an enzyme that catalyses the chemical reaction that breaks down starch (a polysaccharide) into smaller disaccharide units which can be further broken down to glucose, the universal energy source. Different forms of this enzyme exist in plants and animals. In our own digestive system it is produced by salivary glands in the mouth and by the pancreas. In plants it plays a major role in making energy stored in seeds available for the development of seedlings. Germination of seeds starts when water is taken up (imbibition) and passes through the embryo (Figure 1). This results in gibberellic acid, a plant hormone, activating DNA that codes for the enzyme amylase. The amylase is shipped into interior of the endosperm (food store), where it hydrolyses the α (l - 4) glycosidic bonds between pairs of glucose units to release the disaccharide maltose, which is transported to the embryo. Maltose is then further hydrolysed to single glucose molecules by a second enzyme called glucosidase. Glucose can enter the glycolytic pathways of cellular respiration where it is utilized for producing ATP and carbon molecules for biosynthesis. As the embryo divides the seed expands, the seed coat ruptures and the radicle, the first part of the new seedling, emerges, completing the process of germination.

Figure 1. Activation of α-amylase production during germination in a barley seed.

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As the seedling grows, by the time the endosperm is completely used up the seedling should have reached a stage where its energy and carbon requirements can be met by photosynthesis. SAFETY NOTES

Lab coats must be worn. Enclosed footwear must be worn in the lab. Steam from boiling water baths can burn – you must wear the safety gloves

provided. EXPERIMENTAL PROCEDURE

The specific aims of this practical session are to: 1. Determine and compare the mass-specific amylase activity among dormant barley seeds,

germinating barley seeds and barley seedlings by measuring their rates of starch hydrolysis;

2. Interpret these results in terms of the metabolic role of amylase during plant development;

3. Identify maltose as the product of starch hydrolysis by amylase.

Work in pairs throughout this practical. Refer to the flowchart on the last page of these prac notes for an overview of the experimental procedure.

Part 1 – Preparation of amylase extract from germinating barley 1.1 You have been provided with 10 germinating barley seeds (germinants, 3 days old). Using

paper towel, pat the germinants dry, then weigh and record the total weight of the 10 germinants.

1.2 Crush the 10 germinants to a fine paste with a mortar and pestle.

1.3 Slowly add 10 mL of buffer and continue crushing and mixing for 3 minutes to extract the amylase into solution.

1.4 Filter this solution through a tea strainer into the 100 mL beaker. Pour this amylase extract

into the measuring cylinder, record the volume, and then return the solution to the beaker.

Weight of germinants = ______ g

Volume of amylase extract = ______ mL

BEFORE YOU START YOUR EXPERIMENT: Make some predictions as to what level of amylase activity you expect to see in each life stage of the barley. Would all stages show presence of amylase activity? Would some stages have more or less amylase activity than others?

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1.5 Make a five-fold dilution of the amylase extract by auto-pipetting 20 mL of buffer into a measuring cylinder and then adding 5 mL of the amylase extract (use the 5 mL pipette) to make the total volume 25 mL. Do not be concerned if the volume on the measuring cylinder is not 25mL. The volume on the measuring cylinder is not as accurate as the auto pipette dispenser. Mix well. This is your diluted amylase extract.

Note: Save the excess amylase extract in case you make a mistake!

1.6 Prepare a control extract by adding 5 mL of the diluted amylase extract to a test tube and

place it in the boiling water bath for 10 minutes. Whilst waiting, start on Part 2. When the 10 minutes have elapsed, remove the test tube from the boiling water bath and leave it to cool at room temperature.

Part 2 – Determination of amylase activity in germinating barley by measuring the rate of starch hydrolysis The rate of starch hydrolysis can be determined by making up a reaction mixture containing starch in a buffer of appropriate pH. Amylase extract is added, and the samples are tested with starch indicator solution at constant time intervals. Starch indicator solution (iodine) gives a blue colour when bound to starch, but does not give a colour reaction with maltose. The colour intensity decreases as the reaction proceeds, until no colour develops when all the starch has been hydrolysed. This is called the achromic point, and the time taken to reach this point gives a measure of the rate of the reaction catalysed by amylase. If the amount of starch at the start of the reaction is known, then it is possible to calculate the activity of amylase as the amount of starch hydrolysed/unit time/g barley tissue. Carry out the following experiment to determine the activity of amylase per mass of germinating barley tissue. 2.1 Place one drop of iodine into each of 21 labelled wells on the ceramic test plates. Ensure the

volume of each drop is approximately equal for each well.

2.2 Add 5 mL of buffer and 1 mL of 0.5% starch solution to a test tube, and mix well. This is referred to as the reaction mixture.

2.3 Using a disposable pasteur pipette, add a single drop of this reaction mixture to a drop of

the iodine in the well labelled T (for test) on the test plate. It should turn blue/black, indicating the presence of starch in the reaction mixture.

Now that you are familiar with the colour change reaction that occurs when starch and iodine are mixed together, you are ready to start your experiment.

2.4 Thoroughly remix the diluted amylase extract and then add 1 mL diluted amylase extract

to the reaction mixture in the test tube (time = 0 min), mix well. This is the amylase reaction mixture.

You have combined amylase with a starch/buffer solution – think about what is NOW occurring in this tube and immediately do the step below!

2.5 Starting with well number 0, at 1 minute intervals add a drop of the amylase reaction

mixture (use the disposable Pasteur pipette) to sequential wells until the achromic point is reached.

2.6 Record the time taken to reach the achromic point. This should be between 5 and 20

minutes. If not, review your experimental procedure and ask a demonstrator for help.

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When the achromic point has been reached, save the amylase reaction mixture for the determination of maltose in Part 3.

2.7 Repeat the experiment using the identical volumes of buffer and starch that gave a satisfactory reaction rate for your germinants, but replace the diluted amylase extract with the same volume of boiled control extract.

Answer Question's 1 and 2. All questions and space for answers are provided at the end of these prac notes.

2.8 Calculate the activity of amylase in the germinating barley. You will notice that the formula for

this is not directly given to you. Part of the assessment is that you work it out yourself.

To make this calculation, you will need the following information:

• The concentration of the starch solution = 0.5%, i.e. 500 mg/100 mL • The time taken to reach the achromic point • The volume of diluted amylase extract added to the reaction tube • The total volume of the filtered, non-diluted amylase extract • The total weight of the barley seeds used to make the extract

Also note that the unit of amylase activity is mg starch hydrolysed/min/g barley tissue.

Suggested approach:

• Calculate the amount of starch added to the reaction tube. • Calculate the average amount of starch hydrolysed/min to reach the achromic point. • Assume that all the amylase in the barley grains was washed into the filtered

extract. Knowing the volume of diluted amylase extract added to the reaction tube, the total volume of filtrate, the filtrate dilution factor and the mass of barley tissue, it is possible to calculate the activity as mg starch hydrolysed/min/g barley tissue.

Answer Question 3.

Part 3 – Identifying maltose as the product of starch hydrolysis by amylase Benedict's reagent gives a red-orange precipitate of cuprous oxide when boiled with maltose. This reaction does not occur with starch. Carry out the following:

Please note the following: If the achromic point is reached in less than 5 minutes, repeat the experiment but add less amylase extract and additional buffer to maintain the total volume of the reaction mixture at 7 mL. Ensure to record the volume of amylase extract used. If the achromic point is more than 20 minutes, repeat the experiment but add more amylase extract and less buffer to maintain the total volume of the reaction mixture at 7 mL. Ensure to record the volume of amylase extract used.

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3.1 Using your saved reaction tube for maltose determination, add 2 mL of this amylase reaction mixture to a test tube containing 2 mL of Benedict's reagent.

3.2 Prepare a control by adding 5 mL of buffer to 1 mL of 0.5% starch solution in a clean test

tube. Mix well, and then transfer 2 mL of this into a new test tube containing 2 mL of Benedict's reagent. This is your control mixture. Note that for this experiment both the control and experimental tubes should contain equal volumes of solution (4 mL).

3.3 Place the two Benedict's reagent tubes in the boiling water bath for 10 minutes, then examine

them for the presence of cuprous oxide precipitate.

Answer Question 4.

Part 4 – Presence of amylase activity during barley development 4.1 Following the same procedure used with the germinating barley in Part 1 and Part 2, prepare

extracts of 10 dormant seeds (0 days old) and/or 10 barley seedlings (7 days old) as advised by your demonstrator. If necessary, include the use of a boiled control.

Between preparations, take care to clean the mortar, pestle, tea strainer, pipettes and measuring cylinder to avoid cross contamination of the extracts.

4.2 Determine the amylase activity of the extract(s) using the procedures outlined in Part 2.

Answer Question's 5-7. Once Completed

Before leaving the laboratory, empty the tubes containing Benedict’s solution into the toxic waste container. All other tubes can be emptied into the sink and placed in the appropriate tubs. All mortars and pestles, beakers, measuring cylinders, and ceramic plates are to be rinsed clean and left to drain on the trays provided. Clean up any rubbish, wipe away any spills on your bench and leave your work area clean. Assessment Make sure your name, authcate (not ID number) and prac session are written on each page that you are submitting. Staple your pages containing your answers to Questions 1-7 together and submit them BEFORE YOU LEAVE the lab. Late submissions will not be accepted. Additional Information to Consider

Malt (maltose produced from barley by the action of amylase), is used commercially as the starting material for producing beer. Dormant barley seeds are soaked in water to start germination, amylase is produced to convert starch to maltose, and the process is then stopped by heating ("kilning") to prevent seedling growth. The maltose is then extracted by heating in water and brewing yeast is added to provide a glycolytic pathway, which in the absence of oxygen converts maltose to ethanol and carbon dioxide (fermentation). The initial "malting" step is required because brewing yeasts cannot metabolise carbohydrates larger than trisaccharides.

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ASSESSMENT The following work must be submitted at the end of your session. Prac 6: Metabolic Prac Name: Authcate ID:

Prac Session (Day & Time):

Q1. Do you expect to reach an achromic point for the boiled control? What is the purpose for using the boiled control in this experiment? [1 mark] Q2. If an achromic point is reached using the boiled control, how would you use the boiled achromic point value to correct the amylase activity that is calculated for the germinating seeds? [1 mark] Q3. Show your calculations for determining the activity of amylase in germinating seeds. Include all the steps involved in your calculation. [3 marks]

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Prac 6: Metabolic Prac Name: Authcate ID:


Q4. What did you observe in both the control and the experimental tubes for Part 3? Was this expected? What does the Benedict's test confirm for us in this experiment? [1 mark] Q5. Draw a table showing the calculated amylase activity for each of the barley's life stages. Do not include raw data. Note that your table should have an appropriate number and caption and include the appropriate units of amylase activity. [2 marks]

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Prac 6: Metabolic Prac Name: Authcate ID:


Q6. Compare the observed amylase activity with the predicted amylase activity for each stage of the barley seeds. For each stage, answer the following:

• Do your observed results match your predicted results? • Why/why not? • What biological reasons explain why amylase activity would be present/absent or

higher/lower than other stages of the barley's germination process? [3 marks] Dormant Seeds (0 days old): Germinating Seeds (3 days old): Seedlings (7 days old): Prac 6: Metabolic Prac Name:

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Authcate ID:


Q7. Starch and cellulose are both polymers of glucose. Why is amylase able to break down starch into maltose, but unable to break down the cellulose in the cell wall into smaller molecules? [1 mark] ASSESSMENT SUMMARY Activity Marks

Questions 1-7 12

Total 12

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FLOWCHART OF STEPS FOR EXPERIMENTAL PROCEDURE

Prepare your amylase extract from the barley seeds

1. Weigh your 10 seeds (W = _____ g)

2. Grind up the seeds using 10 mL of buffer

3. Filter the solution (filtrate volume = _____ mL)

4. Make a five-fold dilution of 5 mL of the filtrate

8. Take 1 mL of diluted filtrate and add it to the reaction mixture – you are adding the enzyme that starts your experiment!!

Prepare your reaction mixture (RM = starch and buffer)

6. Mix together 5 mL of buffer and 1 mL of 0.5% starch solution. This is the reaction mixture.

5. Prepare a control extract by placing 5 mL of diluted extract in a boiling water bath for 10 minutes

7. Test for the presence of starch. Take a drop of the reaction mixture and add it to a well containing a drop of iodine – do you get a blue/black reaction? What does this mean?

9. At the start and then at 1 minute intervals, add 1 drop of the amylase reaction mixture to successive wells containing iodine – how long does it take to reach the achromic point? (Time = ___ min)

10. Calculate amylase activity = mg of starch hydrolysed min-1 g of barley tissue -1 (less any activity detected using the boiled control). Amount of tissue in 1 mL dilute extract is [mass/filtrate/5] g mL-1 Amount of starch hydrolysed was 5 mg in ‘X’ mins = 5/X mg min-1 Hence, reaction rate = 5/X/[mass/filtrate/5] mg starch hydrolysed

min-1 g barley tissue-1

Repeat steps 6-9 (in this flow chart) with boiled diluted amylase extract. Is an achromic point reached? What does this mean?

11. Keep the amylase reaction mixture and use Benedict’s reagent to test that maltose is the end product of the experiment.

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PRAC 6: Immunology Location: 10 Chancellors Walk (CG63) or 22 Rainforest Walk (Friday only) Assessment for this practical will be questions to be handed in at the end of the practical. Assessment pages can be found at the end of this prac-worksheet and should be filled in, torn-out and handed in at the end of the practical session. Ensure you include your name.

STUDY OF THE IMMUNE SYSTEM IN HEALTH AND DISEASE

Co-ordinator: A/Prof Robyn Slattery Department of Immunology and Pathology

PRACTICAL CLASS OVERVIEW: This class will demonstrate features of the normal immune system (blood cell types, primary and secondary humoral immune responses) and also disorders of the immune system (immunodeficiency and immunomalignancy). Students will work in groups to set up the Haemagglutination assay (exercise I). There will be time during the haemagglutination incubation period to commence the other exercises. At the end of the class, students will review their results with their demonstrator.

EXERCISE 1. THE ANTIBODY RESPONSE BY AN ANIMAL AFTER PRIMARY AND SECONDARY IMMUNISATION

NOTE: This experiment will be performed by the whole group and will be coordinated by your demonstrator.

Introduction:

The two fundamental features of the immune response are antigen specificity and memory. Immunological memory establishes a state of immunity and allows the body to be effectively prepared to resist a later invasion by the same organism. Immune memory is the basis of vaccination programs. In this experiment you will examine the antibody response by a rabbit after a first injection of an antigen and the antibody response after a second injection of the same antigen. The antigen used is sheep red blood cells, which is foreign to the rabbit. Antigens present on the surface of red blood cells will be cross-linked by antibodies produced following immunisation with the sheep red blood cells and this cross-linking can be detected by observing agglutination of cells (referred to as haemagglutination) in special trays called microtitre trays. The amount of haemagglutination that occurs is proportional to the concentration of antibodies in the serum – higher concentration of antibodies results in more haemagglutination.

Materials:

Sheep red blood cell suspension (2.5%) Microtitre trays Micropipette and tips Pre-immune rabbit serum (day 0) Rabbit serum taken on day 7, day 14, day 21, day 28 and day 100 after the first injection Phosphate-buffered saline (PBS)

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Procedure:

1. Examine the haemagglutination plate plan.

2. Prepare serial dilutions of the sera as follows:

• Place 50 μl of PBS in wells 1-12 of rows A, B, C, D and E. • Add 50 μl of the day 0 serum to well 1 of row A. This constitutes a 1:2 dilution of the serum in

well 1. o Serially dilute the serum by transferring 50 μl from well 1 to well 2, then mixing the

contents (which will give another 1:2 dilution of the serum in well 2 and therefore a 1:4 dilution of the original serum).

o Prepare a 1:8 dilution of the serum by transferring 50 μl from well 2 (1:4 dilution) and mixing it with the PBS in well 3.

o Continue serial dilutions to Well 11 of row A, and discard the final 50 μl of solution. o This procedure has produced serial dilutions of serum in the range 1:2 (well 1) to 1:2048

(well 11: refer to figure below).

3. Using a separate (i.e. clean) tip for each row, dilute the other sera in Rows B-E (refer to figure), as outlined above.

4. Add 50 μl of 2.5% (v/v) SRBC suspension to each well in rows A-E, mix by tapping the plate gently, cover with a lid, and incubate at 37°C for 1 hour. This step can be carried out at room temperature, but agglutination may take longer to occur.

5. Observe agglutination reactions. Tilt the tray slightly, view against a light background and observe ‘buttons” at the bottom of the wells. Beyond the titration endpoint, the buttons run on tilting.

6. Determine the endpoint (i.e. dilution of last agglutination reaction) for each serum sample.

• The antibody titre for the serum is the reciprocal of the endpoint. For example, if the endpoint of haemagglutination of a serum sample is 1:128, the antibody titre is 128.

• The higher the dilution of serum to give an endpoint, the higher is the antibody titre for the undiluted serum.

7. a) Plot the antibody titres of the sera against the time after immunisation on the graph provided.

b) What conclusions can you make about the strength and duration of the antibody response to this antigen?

c) What are the consequences of this result to immunisation procedures?

EXERCISE 2. CELLS OF THE IMMUNE SYSTEM

Cells of the immune system are produced in the bone marrow and then circulate in the blood. In this exercise you will identify the cell types in a human blood film and prepare a differential white cell count.

Method

1. Examine the prepared human blood film stained with Leishman’s reagent under the microscope. Start by using the low power objective to select an area containing a suitable number of white cells (usually just back from the tail of the film), then move to the x40 objective for identification of cell types (see table below).

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2. Perform a differential white cell count by counting at least 100 white cells (ignore the red cells) and determining the percentage of lymphocytes, monocytes, neutrophils, eosinophils and basophils.

Morphology of human blood cell types

Cell Type

Morphology

Red cells (erythrocytes) The most common blood cell type; stained pink with paler centre due to biconcave disc shape; no nucleus

Neutrophil The most common white blood cell type (40-75%); 2-5 lobed nucleus; cytoplasm pale lilac with fine granules

Eosinophil 1-6% of white cells; bilobed nucleus; cytoplasm contains large red granules

Basophil <1% of white cells’ bilobed nucleus obscured by large purple cytoplasmic granules

Monocyte 2-10% of white cells; the largest white cell; indented nucleus (not always seen when settled on slide) grey-blue grainy cytoplasm

Lymphocyte 20-45% of white cells; dark blue round nucleus; thin clear blue rim of cytoplasm

EXERCISE 3: DISORDERS OF THE IMMUNE SYSTEM

Lymphocyte subsets such as T and B cells cannot be distinguished by morphology (i.e. in a blood film, T and B cells look the same). However, they do have different markers on their cell membranes which can be detected by using labelled antibodies against these markers. For example, the marker CD3 is found on T cells but not B cells, and CD19 is on B cells only. A common technique for examining a patient’s blood for abnormalities in the proportion of lymphocyte subsets is to stain blood cells with fluorescent-labelled antibodies against these cell membrane markers and then examine the stained cells using a flow cytometer.

In this instrument, a thin stream of the stained cell suspension is passed in front of a laser beam so that cells are in single file. On passing the laser, any cells which have bound the fluorescent-labelled antibody will emit fluorescence which is detected by a photomultiplier. The flow cytometer can analyse thousands of cells per second (quicker than counting cells down the microscope!) and generates data on the proportion of lymphocytes expressing a particular marker. Diagnosis of immunodeficiencies or immunomalignancies is routinely performed in this manner.

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1. In this exercise, you are provided with flow cytometry data for a normal blood sample and for three patient blood samples:

• one is from a patient with HIV infection,

• one is from a patient with a B cell leukemia,

• one is from a normal patient.

Which results fit which patient?

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PRAC 7: Immunology Prac STUDENT NAME: ………………………….STUDENT NO:……………………. STUDENT AUTHCATE USERNAME: ………………………….. SESSION TIME ……………………….

BMS1021 PRACTICAL 7: STUDY OF THE IMMUNE SYSTEM IN HEALTH AND DISEASE

This answer sheet is to be handed in to your demonstrator at the conclusion of the practical and will be used in your assessment.

EXERCISE 1:

1. Draw the observed pattern in the haemagglutination assay in the plate plan below: (+ indicates agglutination - indicates no agglutination)

[3 marks]

2. Answer to question 7 (a) of your worksheet. Plot the time course of the antibody response to primary secondary immunisation on the graph below (don’t forget to label the y-axis):

[5 marks]

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PRAC 7: Immunology Prac STUDENT NAME: ………………………….STUDENT NO:……………………. STUDENT AUTHCATE USERNAME: …………………………..

3. Answer to question 7(b) of your worksheet. What conclusions can you make about the strength and duration of the antibody response to this antigen?

[3 marks]

4. Answer to question 7(c) of your worksheet. What are the consequences of this result to immunisation procedures?

[3 marks]

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PRAC 7: Immunology Prac STUDENT NAME: ………………………….STUDENT NO:……………………. STUDENT AUTHCATE USERNAME: …………………………..

EXERCISE 2:

For your own reference, in your notes draw the morphology of the different leukocytes observed.

5. Indicate in the table below the absolute number and percentage of each cell type observed. This number will obviously be the same if you count 100 cells. Remember you do not need to count red blood cells.

[5 marks]

Neutrophils Lymphocytes Monocytes Basophils Eosinophils

EXERCISE 3:

6. From the flow cytometry data presented to you identify which patient has which disorder and indicate reasons for your choice

[2 marks each case, Total 6 marks]

Case A: Disorder

Reasons

Case B: Disorder

Reasons

Case C: Disorder

Reason

ASSESSMENT SUMMARY

Activity Marks

Questions 1-6

Total

/25

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PRAC 7 Part A and B: Microbiology ASSESSMENT SUMMARY

PRELIMINARY IDENTIFICATION OF UNKNOWN MICROORGANISMS The aim of this practical is to introduce students to the concept that bacterial pathogens differ from our normal

flora because they express virulence determinants, and that the production of certain virulence associated

determinants can be used to identify a suspected pathogen.

LEARNING OUTCOMES: At the end of this practical: 1. You should understand the biochemical basis of the differential Gram stain and the importance

of its role as a first stage identification test for unknown microorganisms.

2. You should be able to derive, transfer and grow pure cultures of microorganisms using aseptic techniques

3. You should be familiar with tests to identify bacterial structures, including flagella, spores and capsules and the production of enzymes such as haemolysin and catalase.

Activity Comments Week A marks

Week B marks

Demo Assessment

Punctuality (0.5 marks) Preparedness such as showing prior reading (1 marks) Active involvement in the lab and tutorials (0.5 marks) Ensuring work area is clean when you leave (0.5 marks)

2.5 2.5

Quiz Short answer questions which will be answered in 15 minutes at the end of the lab in Week 12 (or Wk10) - 15

Total overall mark /20 marks

Will contribute to 4% of final mark

Students must check in advance on the notice boards in 12 Innovation Walk (G27/28) to ensure you are in the correctly allocated practical session. Students will not be permitted to change their times. Assessment for this practical (see above) will be assessment from your demonstrator in both sessions AND a quiz completed during the practical classes in the second session. Marks will be put into your Gradebook on the BMS1021 Moodle site within 2 weeks of your practical session.

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PRE-LAB PREPARATION

Read these notes carefully and refer to “Biology” (pp. 528-529) for a description and discussion of the Gram

stain. Using the textbook and these notes, make sure that you understand and can define the following key

words:

coccus rod differential stain

Gram positive Gram negative colony

aseptic techniques pure culture sterile

spore virulence factor pathogen

capsule haemolysin motility

prion

INTRODUCTION

Microorganisms are described as organisms that cannot be clearly seen by the unaided eye. Viruses and

bacteria, together with many algae, fungi and protozoa are classified as microorganisms and are widely

distributed in nature. Many play an important and useful role in the production of various foods, alcohol,

enzymes, vaccines, antibiotics and other products. They are also important members of our ecosystems and

play an essential part in the recycling of nutrients in the environment. They are involved in the carbon, oxygen,

nitrogen and sulphur cycles that take place in aquatic and terrestrial systems. Thus a relationship usually exists

between the types of microorganisms present in a particular habitat and the physico-chemical characteristics

of the habitat itself. For example, very different types of microorganisms grow in the water of a boiling sulphur

spring compared with those growing in contaminated refrigerated food.

While most microorganisms present on and in our body are harmless, some are able to cause disease and

these are called pathogens. Microorganisms that are consistently found and multiply at a given site are known

as the resident flora. In normal, healthy individuals they are non-pathogenic at that site, but some can act as

opportunistic pathogens if they gain access to another site, e.g. Escherichia coli is considered part of the

normal resident flora of the bowel but is a common cause of urinary tract infection. Other organisms that may

be temporarily found at a given site but do not multiply there are called transient flora. These species are

acquired by contact with the surrounding environment and/or contact with people and are potential pathogens.

Infection with pathogenic microbes can lead to life-threatening illnesses such as AIDS, malaria, cholera and

typhoid. The difference between harmless and harmful microorganisms is that the pathogens possess

virulence factors that allow them to infect and damage the host.

Some of the microorganisms present on and in our body include Staphylococcus epidermidis which is present

in our skin and nasal region and Escherichia coli in the large bowel. Many of them also play an important role

in the gastro-intestinal tract (GI tract) and are also considered to be commercially useful, for example

Lactobacillus acidophilus and Bifidobacterium bifidum which are used in yoghurt manufacture (AB yoghurt).

Microorganisms have also caused significant economic problems e.g.:

• the foot and mouth disease outbreaks;

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• contamination of food for local and export markets

They can also be the cause of great community concern, e. g.:

• Pollution of water by faecal contamination indicated by the presence of E. coli

• Legionella spp. In cooling towers

• Drug resistant bacteria in hospitals such as Methicilin Resistant Staphylococcus aureus (MRSA)

• the use of microorganisms by terrorist in biological warfare, egs., Bacillus anthracis spores causing

anthrax.

Although not microorganisms, Prions which are proteins present on the surface of brain tissue can also cause

the following diseases when they undergo changes.

• mad cow disease –Bovine Spongiform Encephalopathy (BSE) in animals.

• Chronic Wasting disease in deer.

• Creutzfeldt-Jakob disease (CJD) in older people.

• vCJD in young adults (attributed due to the consumption of meat from cattle with BSE)

Given that microorganisms live in all environments and proliferate in natural situations, they nearly always

occur as mixed populations in the environment. As such, in order to study microorganisms, they need to be

isolated under aseptic conditions as individual entities in pure culture so that their distinct characteristics can

be identified. A pure culture is one that has only a single type of microorganism present in the medium. There

are many methods of obtaining a pure culture. The three common methods used are the Streak plate, Pour

plate and Spread plate techniques. The spread plate and pour plate methods can also be used to count

the microorganisms present in a given sample so that effective treatment and level of contamination can be

studied.

In this practical you will perform a preliminary identification of unknown pure cultures of microorganisms

isolated from a clinical and an environmental sample. In the first week you will carry out the Gram stain

on the samples to detect any microorganisms present and identify their cellular morphological features

(Gram stain, shape, arrangement, etc.). In addition, you will subculture the microorganisms from the

samples provided for further identification. In the second week you will perform additional tests on the pure

cultures using specialised techniques to detect specific virulence associated characteristics that will aid

identification.

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PRAC 7 Part A: Microbiology

Scenario: The Unit Coordinator of BMS 1021 at Monash University recently developed a severe, acute respiratory tract infection after opening a letter containing a white powder.

Aim: To determine the likely cause of the Academic staff member’s unfortunate infection and identify the suspicious white powder

A. Isolation of a pure culture for the preliminary identification of unknown bacteria

Materials provided (per pair)

• sterile inoculating loops

• Photo of BMS 1021 coordinator opening envelope and scattering white powder

• one broth culture of bacteria grown from white powder (sample A) • one broth culture from a sputum sample (sample B)

• four agar plates, two each of horse blood agar (HBA) and nutrient agar (NA)

One student from each pair will plate out Sample A, the other will plate out sample B. 1. Label the base of the blood and nutrient agar plates with:

(i) Sample origin

(ii) your name, day, bay and group number

2. Using a flame-sterilised inoculating loop, transfer a loopful of Sample A or B broth culture and spread

over one section of a blood plate as shown below in step (a). Proceed to spread the bacteria across

the remaining agar surface by flaming the loop and allowing it to cool between each set of streaks (b-

d). Drag the loop over the agar plate with the loop at an acute angle to the plate to prevent the loop

cutting into the agar..

(a) area inoculated with loop

(b) first set of steaks

(c) second set of streaks

(d) third set of streaks

(e) final streak

3. Repeat the procedure for Sample B using the remaining blood and nutrient agar plates

4. Place all of your inverted plates into the labelled white boxes for incubation in air at 37°C for 24 hours.

You will examine the plates in the next practical session.

(a)

(c) (d)

(b)

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B. Motility test for the preliminary identification of unknown bacteria

Many bacteria have the ability to “swim” through liquid or semi-solid medium. This motion is termed motility

and is powered by bacterial flagella. Nevertheless not all bacteria are motile and thus the property of motility

can help to identify unknown bacteria.

There are three possible methods for determining the motility:

a) Hanging drop preparation – overnight culture has to be used and results could be obtained immediately

b) Craigie tube – needs to be incubated and incubated c) Mast method – need to be inoculated and incubated

In this experiment you will be using the MAST method to study motility.

Materials Provided (per pair)

• sterile inoculating wire

• samples A and B from part A.

• four motility agar bottles (Mast motility agar)

• control plate cultures of Escherichia coli (motile) and Staphylococcus epidermidis (non-motile)

1. Label the bottles with sample origin, your name, day, bay and group number.

2. Using aseptic technique and the inoculating wire, transfer the bacteria from the broth culture of

sample A and stab into the centre of the motility agar. Repeat with a new bottle each for sample B and the control strains provided.

3. Place the tubes into the 52abeled white boxes on the trolley for incubation in air at 37°C for 24

hours. You will examine the tubes in the next practical session.

C. Gram stain for the preliminary identification of unknown bacteria Bacterial cells are difficult to see under the bright field microscope even at high magnification because

there is very little contrast between the cell and the surrounding medium. To improve contrast, bacteria

can be stained to show their basic shape, size and arrangement. Differential stains are used to

distinguish between bacteria that differ from one another biochemically and physically, by their varied

reaction to a particular staining process.

The Gram stain is the most common differential staining technique and is used routinely for the preliminary

identification of unknown bacteria. Not only does it allow us to see the shape of bacterial cells easily using a

microscope, but it separates bacteria into two groups – Gram positive (purple) or Gram negative (pink) based

on their cell wall structure.

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Gram staining should be carried out on young cultures (less than 24 hours of incubation), otherwise variable

results would be achieved as some Gram positive bacterial groups take on increasingly Gram negative

character as the culture ages.

Here you will Gram stain the two test samples A and B to detect any bacteria present and determine if they

are Gram positive or Gram negative by comparing with the two controls provided.

Materials Provided (per student)

• 1 x microscope slide

• samples A and B from part A

• Gram stain reagents

• control plate cultures of E. coli (Gram negative) and S. epidermidis (Gram positive)

1. Make a heat fixed smear of sample A and B (as per appendix A) and the control strains provided setting

them out on a slide as below:

E. coli

S. epidermidis

Sample A or B

Mark underneath the slide and not in the middle with the grease pencil

2. Perform Gram stain (as per appendix B) and record your results below

Students should also observe the Gram stain of their partner’s specimen

Grease pencil marks*

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Table 1. Gram stain reaction and appearance of cultured bacteria.

Microorganism Gram reaction Cell shape Cell arrangement

Sample A

Sample B

E. coli

S. epidermidis

NOTE: When you have finished using your microscope, remove the immersion oil from the 100X

objective (and any other areas) using tissue paper.

A demonstrator must check your microscope before it is put away. This will ensure that the

microscope is clean and operational for the next user.

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PRAC 7 Part B: Microbiology This week you will continue with the preliminary identification of bacteria from samples A and B.

A. Motility agar test Materials provided (per pair of students) Motility test results of samples A and B from part A and controls.

1) Examine the motility agar tubes you set up in week 1. With reference to control tubes, record any

observed motility from samples A and B in the Table below.

Expected Results

Organism Sample A Sample B E. coli S. epidermidis

Motility

B. Growth of bacteria on blood agar Some pathogenic bacteria produce an enzyme called haemolysin that breaks down red blood cells.

Haemolysins can help bacterial survival in the host by destroying host immune cells and red blood cells (and

thus reducing the fitness of the host) but also by releasing the nutrient iron for bacterial growth.

Note the terminology for the type of haemolysis that occurs on blood plates

α-haemolytic: greenish brown zone surrounding colony

β-haemolytic: clear or yellowish zone surrounding colony (complete haemolysis).

Materials Provided (per pair of students) • Blood agar plates of samples A and B from Session 1.

• Gram reagents

• demonstration photographs of blood plate cultures of Streptococcus mitis (α-haemolytic),

Streptococcus pyogenes (β-haemolytic) and Enterococcus faecalis (non-haemolytic)

1. Appearance of bacterial colonies. Briefly observe the shape (round, irregular, wrinkled), colour (white,

yellow) and surface texture (smooth or rough) of the colonies from sample A and B grown on blood

plates.

2. Compare zones of haemolysis around colonies from sample A and B with control plates and record the

result below.

3. Using an isolated colony from each plate, prepare a heat fixed film and perform a Gram stain. Examine

the slides by microscopy and record results below, in particular noting the presence of any spores which

appear as refractory (non-staining) bodies.

C. Capsule stain

Many bacteria produce an extracellular capsule that provides protection from desiccation and allows some

pathogens to evade detection by the host immune system. Capsules are usually made from polysaccharide

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and can make the surface of the colony appear mucoid (sticky). Again not all bacteria produce a capsule and

thus the presence of a capsule can help to identify unknown bacteria. Materials Provided (per pair of students)

• Nutrient agar plates of samples A and B from Session 1.

• Maneval’s Stain, Congo red

• Control plate cultures of Klebsiella pneumoniae (encapsulated) and E. coli (non-encapsulated).

For each of samples A and B and the controls, perform a Maneval’s stain as outlined in appendix D. Examine

the fields by microscopy and record results in the table below.

E. coli

K. pneumoniae

Sample A or B

D. Catalase test

Many bacteria also produce the enzyme catalase, which catalyses the breakdown of hydrogen peroxide to

oxygen and water. Catalases can be detected easily by adding Hydrogen peroxide to bacterial cells. Again

not all bacteria produce the catalase enzyme and detection of catalase activity can help further identification,

particularly of Gram positive organisms.

Materials Provided (per pair)

• Nutrient agar plates of samples A and B from Session 1

• Catalase reagents

• Control plate cultures of Enterococcus sp. (catalase negative) and E. coli (catalase positive)

1. For each of sample A and B and the controls, perform a catalase test as outlined in appendix E. Record

your results in the table below.

Table 2. Characteristics of organisms present in Samples A and B (Results)

Microorganism Motility Haemolysis Gram stain Spores Capsule Catalase

Sample A

beta

Sample B

beta

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…………………………………………………………………………………………………………

CONCLUSIONS

You have now completed the preliminary identification of two unknown samples of microorganisms using the

flow chart provided.

1) Based on the information below, is it possible that the BMS 1021 Coordinator was sent a virulent

organism in the envelope?

2) What is the likely cause of the BMS 1021 Coordinator’s respiratory infection?

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FLOW CHART TO IDENTIFY THE ORGANISMS

S. pneumoniae S. pyogenes S. mutans B. anthracis B. cereus B. subtilis

Gram stain + cocci + cocci + cocci + rods + rods + rods

Growth in air Yes Yes Yes Yes Yes Yes

Catalase - - - + + +

Capsule Yes Yes/No Yes Yes Yes/No Yes

Spores No No No Yes Yes Yes

Motility - - - - + +

Haemolysis α β - - β β

Egg Yolk

Lecithinase

NA NA NA + + -

Gram positive bacteria

Cocci Rods

Growth in air Spores

Catalase Anaerobic Cocci

Growth in air Listeria, Corynebacteria

+ _ + _

+ _

Bacillus Clostridium

+ _

Staphylococcus, Micrococcus

Streptococcus

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Appendix A Preparation of fixed films

It is essential that slides for the preparation of films of bacteria are clean and free from grease. Prior

to use, the surface of the slide should be degreased by passing through a Bunsen flame several times.

To prepare the film:

(1) Label the slide at one end with a code for the particular colony to be sampled.

(2) Sterilize an inoculating loop by flaming and allow to cool.

(3) If the sample is a liquid culture, with the sterile loop transfer a loopful of the culture to the slide

and spread over about a quarter of the surface of the slide. Sterilize the loop by flaming and

proceed to step (7).

(4) If the sample is a plate culture (solid medium), place a loopful of sterile water in the middle of

the slide.

(5) Sterilize the inoculating loop by flaming and allow to cool.

(6) With the sterile loop transfer a very small quantity of cells from the selected colony to the

drop of water on the slide, emulsify and spread thinly over about a quarter of the surface of

the slide. Sterilize the loop by flaming.

(7) Allow the film to dry in air

(8) With the film uppermost, fix the film by slowly passing the slide three or four times through the

hottest part of the Bunsen flame. (When the slide is just too hot to be borne on the back of the

hand, fixation is complete).

(9) Allow the slide to cool before staining the film.

(10) Remove the carbol fuchsin by washing with water, drain off the excess water.

Gram positive bacteria appear purple and Gram negative bacteria

Appendix B Gram stain (modified)

(1) Circle the area beneath the bacteria film with a grease pencil mark. This will be the area

within which the solutions will be applied.

(2) With the film uppermost, pour crystal violet solution onto the slide within the grease pencil

area and allow to remain for 30 seconds.

(3) Remove the crystal violet by washing briefly with water.

(4) Add iodine solution and allow to remain for 30 to 60 seconds.

(5) Remove the iodine solution by washing briefly with water.

(6) Decolourise with 95% ethanol by holding the slide at an angle of 45° over the sink and adding

the ethanol to the top of the slide, allowing it to run down the slide across the film.

NOTE: (a) Make sure all of the film is treated with the ethanol.

(b) Ideally, the ethanol treatment should be continued until no more colour comes out

of the film. In practice, with thin films, decolourization is usually complete within 5

seconds.

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(7) Wash immediately with water.

(8) Counterstain by adding dilute carbol fuchsin for 30 seconds.

(9) Remove the carbol fuchsin by washing with water, drain off the excess water.

(10) Air dry or blot dry the slide.

(11) Typical Gram positive bacteria appear purple and Typical Gram negative bacteria appear pink.

Appendix C Microscopic examination of stained films

(1) Place a small grease pencil mark across the centre of the film or you can focus on the grease

pencil around the smear. (2) With the stained film in position on the stage (place the grease pencil mark across the centre

of the substage condenser), set up the microscope for use with the x10 objective lens.

(3) Focus on to the grease pencil mark on the stained film. (Do not attempt to describe the

stained bacteria at this stage.)

(4) Swing the x40 objective lens into position and refocus on to the grease pencil mark smear using the fine focus knob.

(5) Swing the x40 objective lens aside and place a drop of immersion oil on to the film directly

above the substage condenser.

(6) Swing the x100 objective lens into position.

(7) With the substage condenser in its highest position and the condenser iris diaphragm fully

open, focus on to the grease pencil mark and then move the slide so as to view the stained

bacteria. Appendix D Maneval’s Stain

This is a negative staining technique. The stain does not bind the bacteria but stains the background.

Therefore the capsule appears as a clear halo surrounding the cell.

(1) Place a loopful of Congo red onto a slide and mix into it a small amount of growth from the agar

plate.

(2) Make a well spread smear and allow to dry. DO NOT HEAT FIX

(3) Flood the slide with Maneval’s stain and leave for one minute.

(4) Rinse with water and blot dry.

(5) Observe using oil immersion lens.

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Results

Organisms: Red

Background: Grey

Capsule: Clear halo around organism (capsule is negatively stained)

Appendix E Catalase test

(1) With a wire loop remove cells from the agar surface and make a thick suspension in a drop of

hydrogen peroxide on a slide. Catalase activity is indicated by the appearance of bubbles of gas

(oxygen) within a minute.

NB: Media containing blood will give a false positive reaction Appendix F Approved Tests for the Detection of Bacillus anthracis in the U.S. Laboratory Response Network (LRN)1

1Protocols for Level A tests are publicly available at www.bt.cdc.gov/agent/anthrax. Protocols for Level B are available only to laboratories in the LRN. These laboratories include state public health laboratories and many federal laboratories (below).

Test Procedure

Laboratory level

Preliminary testing A B C D

Gram stain (micromorphology) X X X X

Capsule (microscopic observation) X X X X

Colonial morphology X X X X

Haemolysis X X X X

Motility X X X X

Sporulation X X X X

Confirmatory tests

Lysis by gamma-phage X X X

Direct fluorescence assay (capsule specific) X X X

Antimicrobial susceptibility testing X X

Advanced technology (PCR-testing) X X

Molecular typing and characterization X

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