Portfolio for infection and immunity PDF

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University of Sunderland

School of Pharmacy, Health and Well Being

Infection and Immunology I 2014-15

(HCS105)

MICROBIOLOGY PRACTICAL PORTFOILO

Name Programme

BSc Physiological Sciences

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Introduction.

This microbiology portfolio is a simplified replica of the practical schedule you have been using the laboratory classes. To complete the practical programme you should add your observations and results to this copy print off and submit as part of your assessment. Details of your investigations and results should be noted in the spaces provided. In addition you may wish to make additional comments or observations. Please use this document as a basis for broadening your microbiology experience. At the end of your studies it should be bound or secured in a file for assessment and for future reference.

Week 1. Culture of microorganisms. Aseptic techniques and sources of contamination. Week 2. Observation of microorganisms. Colony features and microscopy. Week 3. Enumeration of microorganisms. Counting cells. Week 4. Identification procedures I. Week 5. Identification procedures II. Week 6. Determination of bacterial growth. Measuring microbial growth.

Week 7. Antimicrobial action of lysozyme. Yeast mutation investigation. . Week 8. DNA isolation. Analysis of practical competence. Week 9. DNA isolation and analysis. Comparison of methods with agarose gels. Week 10. Lac operon investigation. Gene activity assessment. Week 11. Lac operon investigation. Gene control assessment. Week 12. No lab class.

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WEEK 1 CULTURE OF MICROORGANISMS Part 1. Aseptic techniques and transfers. The aim of this practical is to introduce you to the basic practical skills which are essential to work competently and safely with microorganisms. In addition, you will see how ubiquitous microorganisms are in the environment and how easily they can contaminate laboratory materials. The use of aseptic techniques is essential to prevent this contamination. Background Transferring microorganisms without contamination is a basic and essential microbiological skill. It is conducted by the use of aseptic techniques, which you must become competent with and use whenever handling microorganisms. If you don’t develop and use these skills you will contaminate your cultures and yourself and possibly other people. Your course work marks will suffer also. The following techniques will be illustrated for your use in this week’s practical: •

Use of the inoculation loop to obtain and transfer a sample.

•

Flaming of surfaces to kill microbes before opening bottles, flasks etc.

•

Use of Pasteur and micropipettes to remove and transfer samples.

•

Use of spreaders to spread a sample across an agar surface.

•

Use of the inoculation loop to streak a sample across an agar surface.

•

Use of an inoculation wire to transfer a sample into a tube of agar.

In addition you will be given instruction in the preparation of surfaces for microbiological work, in safe working procedures and in the cleaning of surfaces. You will also use a range of equipment from loops to pipettes. You may wish to make notes on some of these procedures for future reference. Space is available below for your comments. In addition you will be given instruction in the preparation of surfaces for microbiological work, in safe working procedures and in the cleaning of surfaces. Use the space below for notes on some of these procedures for future reference.

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Preparation and cleaning of surfaces before and after use. Preparation and cleaning of surfaces before use in order to disinfect the work bench area. This is because even though there isn’t any visual dirt, microbes and parasites may be on the benches. Preparation and cleaning of bench surfaces after use in order to disinfect any remaining bacteria or microorganism remaining after the practical. Preparation and cleaning of surfaces is done by pouring distel disinfectant onto a paper towel and carefully swab the working area bench.

Flaming of surfaces. In duration of a practical, the blue flame of the Bunsen burner is used in order to disinfect and sterilise the surrounding air that circumferences the Bunsen burner. Therefore, when doing the practical, it is better to do it near the blue flame in order to avoid any contamination from the air. The blue flame is also used to sterilise tubes as bacteria may be on the surroundings of the tube. The procedure of sterilising and preventing any contamination to the tube sample is by opening the tube and taking sample near the blue flame. After taking some sample, the top of the tube is then placed into the blue flame in order to sterilise and disinfect any remaining bacteria surrounding the tube and the lid is placed on the tube near the blue flame in order to avoid any contamination to the sample.

Micropipettes. When using micropipettes, green rubber bungs are used. The procedure of using micropipettes is firstly to allow a little bit of air into the micropipette then intake the amount of sample required into the micropipette and a little bit of air is then taken in order to prevent any sample droplets. After the usage of the micropipette, it is disposed in a pot of disinfectant.

Spreaders. The spreaders are used in order to evenly distribute bacterial sample across the petri dish. After use of the spreader, it is disposed in a pot of disinfectant.

Inoculating loops. Before using the inoculating loop, it is firstly placed into the blue flame until it glows orange. This is done in order to disinfect and kill any remaining bacteria on the inoculating loop. Before taking any bacterial sample, the loop must cool first and this can be done in several ways such as placing the tip of the inoculating hoop onto the agar, placing the inoculating loop on the surface of the petri dish lid and also by touching the side of the test tube. After using the inoculating hoop, it is placed back into the blue flame in order to disinfect and kill any remaining sample before storage or reusing.

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Safe working and personal protection. When working in a microbiology lab, long hair must always be tied back in order to avoid any contamination and burns; this is an example of a personal protection in a lab. Another way in which personal protection occurs in a lab is by wearing a white lab coat at all times to protect from any harmful microorganisms and chemicals. However, lab coats must only be worn inside the lab in order to prevent any contamination outside the lab. In order to work safely in a microbiology lab, Bunsen burners must be on a yellow safety flame when not in use. This will help identify Bunsen burners that are in use and prevent burning others, as it’s easier to be recognised in comparison to having a blue flame.

Please indicate below two important safety requirements for working in the microbiology laboratory.

One of the important safety requirements when working in a microbiology lab is to ensure lab coats are cleaned correctly in order to prevent bacterial contamination inside and outside of the lab. This is done by washing the lab coat at a temperature of 60°c. The reason for this is because at a temperature of 60°c, remaining bacteria on the lab coat would have been killed due to lack of bacterial survival at the extreme temperature of 60°c.

One of the important safety requirements when working in a microbiology lab is to ensure used equipment and cultures are disposed correctly in order to avoid any bacterial contamination in the water system. An example of this procedure is by disposing used micropipettes in the distel disinfectant pots in order to kill off any remaining bacteria on the equipment before disposing.

Objectives.

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This part of the practical asks you to perform the following transfers of microorganisms: Tick each task when you have completed it. 1. Bacteria colonies. Transfer the following: 1. A drop of liquid culture A or B to a test tube of liquid culture medium.

[√]

2. Exactly 0.5 ml of liquid culture A or B to a test tube of liquid culture medium. [√] 3. A sample of liquid culture A or B onto a slope of solid agar and streak out.

[√]

4. A sample of liquid culture A or B into a deep of solid agar and plunge in.

[√]

5. 0.1 ml of liquid culture A or B into a petri dish and cover with liquid agar.

[√]

6. A drop of liquid culture A or B to a solid agar plate and streak out.

[√]

7. A drop of mixed culture C to a solid agar plate and streak out.

[√]

8. 0.1 ml of mixed culture C (dilute) to a solid agar plate and spread out.

[√]

Make sure that you label your plates clearly on the base with your name, date and sample number. B. Fungi colonies. (Moulds and yeasts). Transfer the following: 1. A portion of a yeast colony onto a malt agar plate and streak out. [√] 2. A plug of one of the three mould colonies into the centre of a malt agar plate. [√] Requirements for this practical. Check you have the following before you start. • • • • • • • • •

Micropipettes and sterile disposable tips Sterile glass Pasteur pipettes Sterile disposable petri dishes Wire loops Straight and hooked wires Glass spreaders and flaming alcohol Universals of nutrient broth Nutrient agar (NA) slopes and deeps for bacteria transfers Nutrient agar (NA) plates for bacteria and malt agar (MA) plates for fungi

Cultures (Undiluted or diluted before streaking out to give discernible colonies): Bacteria: A) Escherichia coli B) Staphylococcus albus C) A mixture of A) and B)

Fung: Moulds D) Alternaria, Fusarium and Trichoderma E) Yeast (Saccharomyces cerevisiae)

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RESULTS. Observe your cultures in session 2. The samples will be available in trays. Be sure to identify your own plates accurately and take care removing plates to avoid displacing the tops. a) Examine broth and plate cultures from last week. In each case comment on the success and the purity of your cultures in the table below: Bacteria transfer.

1. Liquid to sterile broth.

2. 0.5 ml to sterile broth.

3. Streak on agar slope.

4. Plunge into agar deep.

5. 0.1 ml pour plate.

6. 0.1 ml streak plate.

7. 0.1 ml streak plate. (mixed culture)

8. 0.1 ml spread plate. (mixed culture)

Features of the growth E.g. turbid broth, type of growth, colonies of similar or different appearance etc. Turbidity broth cluster of cells with a colour of clear pale yellow with a cream coloured ring of bacterial deposit at the bottom of the test tube with the similar appearance throughout. When the test tube is mixed, the bacterial deposit remains at the bottom of the test tube. Turbidity broth colour is clear pale yellow with a cream coloured bacterial ring deposit with the similar appearance at the bottom of the test tube. The bacterial deposit is smaller in comparison to the bacterial deposit for bacteria transfer 1. When the test tube is mixed, the bacterial deposit remains at the bottom of the test tube. There is a colony of similar white bacterial growth in the middle of the agar slope shaped as a solid triangular shape. There is a build up of similar white bacterial growth in the centre of the agar. It is shaped in a straight line where the thickness decreases from the top to the bottom of the agar. There is a thin white layer of bacterial growth covering the surface of the agar plate with similar appearances. There are different colony appearance growths on top of the agar plate. There is a white thick bacterial streak line on the top half of the agar plate, which then varies into small rounded colonies. However there was only a selected few of the white small rounded shaped colonies on the 3rd section of the streak plate. There is a thick colony of bacterial growth on the first section of the streak. Then as the streak lowers down the bottom of the streak plate, the thickness of the bacterial growth streak lines decreases. The streaks show different colony types, which is a small white rounded colony and a cream sculptured irregular shaped colony. There are different colonies of bacterial growth found on the spread agar plate. There was a mixture of cream irregular sculptured colony and while round small colony. It all covers the top surface later of the agar where there is a variation of both thick and thin growth layers.

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Fungal transfer.

Diameter of the colony mm

1. Yeast streak.

2. Plug of mould. Measure the diameter of the colony at different days of growth. State the mould species here: • Fusarium

Fusarium: Day 1 – 10mm Day 2 – 51mm Day 3 – 63mm Day 4 – 71mm Day 5 – 85mm

Features of the growth (colour, zonation) Cream coloured thick lines of bacterial growth colony until the centre of the petri dish, which then varied to small round colony shapes reaching the other end of the petri dish. Fusarium: Red coloured base with white filamentous growth consistency across the top layer of the petri dish, which is mostly situated in the middle of the agar plate.

b) Describe the colony morphology of the two bacteria from the mixed culture plates using the following terms. Details of how to assess each are shown below. Bacteria Colony shape

Escherichia coli (Bacteria A) Irregular

Staphylococcus albus (Bacteria B) Round

Edge features

Undulating

Smooth

Elevation

Domed

Flat

Surface features

Sculptured

Smooth

Colour

Cream

White

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Colony features. The illustrations below show some of the common features seen on microbial colonies. You may need to use a magnifier to view details of small colonies.

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b) Insert an image of your plate and one from the Sun Space image gallery in the spaces below. Comment on the quality of both streak plates in the space below. In particular state how well isolated colonies were achieved? How could these streaks be improved? Own streak plate

Sun Space streak plate

Comments The technique looks to have been properly demonstrated. This is because the colonies were successfully isolated on the second and third sections. On the second section of the agar plate, there were a large amount of big colonies isolated. Following onto the third section, there were only a few bacterial colonies showing a good quality streak test. However, in order to improve the streaks, a larger amount of sample should be taken onto the inoculating loops as this will enable to isolate more bacterial colonies onto the third section.

The image above shows that the technique has not been applied properly due to various reasons. One of which is that the inoculating loop has not been sterilised for each section. The streaks are continuous throughout each section therefore the colonies are not isolated. As the streaks are constant on each section, it suggests that the inoculating loop has been re-dipped into the sample, which will then hinder the isolation of the colonies. Another reason of which why the quality of the streak plate is poor is because the streaks on the plate are thick. The streaks being drawn on thickly results the streaks to overlap. Another reason is that the streak lines are not drawn from one end of the streak plate to the other; therefore the bacterial colonies are only situated in the middle of the agar plate. The isolated colonies were unachieved as the streaks in each section are constant therefore no colonies were isolated.

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State an advantage of using spread plates and an advantage of using streak plates. The advantage in using the spread plate method is that it is easier to deal with large liquid volumes in comparison to other methods. This is because the sample is evenly distributed across the agar plate. Spread plates will also enable you to count the bacterial colonies. However, the advantage of using streak plate method is that it is used to isolate and separate colonies in order to identify them, as only a small amount of sample is required in order to perform a streak plate method. On the other hand, the separated/isolated colonies are only able to be used for identification and not to count the colonies.

Both streak and spread plates can be used to produce separate colonies on agar plates. What is the disadvantage of using spread plates if you wished to obtain separate colonies? When trying to obtain a separation of colonies in a sample, it is a disadvantage to use the spread plate method. The reason for this is because it is difficult to fully control where the colonies will form using a spreader. Another reason for the spread plate method being a disadvantage to produce separate colonies on agar plates is that you will have to dilute the sample first before spreading the sample onto the agar plate. This will allow a wider range over a large surface area.

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Part 2. Sources of contamination The aim of these experiments is to show how easily external contamination of cultures can occur. As a result of the contamination produced you should appreciate the need for good aseptic technique. Methods. Work in pairs for this practical. 1. Leave one of the nutrient agar plates open in an area of your choice until the end of the practical session – settle plate. 2. Spread 0.1ml of tap water onto a nutrient agar plate using a sterile pipette. 3. Spread 0.1 ml of sterile deionised water onto a nutrient agar plate using a non sterile pipette. 4. Draw a line across the base of a nutrient plate to divide the area in half and label A and B. Apply your fingerprints to the A side of the plate. Wash your hands in just water or with soap and allow them to drip dry. Then apply your fingerprints again to the B side of the plate. 5. Pluck a hair from any part of your body. It is important that the hair root is obtained. Carefully lay the hair on the surface of a nutrient plate, ensuring that your fingertips do not touch the agar. Also place a coin onto the surface of the plate, leave for a moment and remove. 6. Swab the surface of an everyday object and run the swab over a nutrient agar plate. 7. Take a nutrient agar plate, label and leave unopened. This will act as a control.

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RESULTS. In session two examine the plates you set up in session one. In particular note the microbial status of the plates. Describe in your own words the kind of growth you see. Which samples show the greatest amount of growth? Which show the greatest variety ? inoculated plate free of growth?

Account for your observations.

Is the un-

In the table below for settle and finger plates insert the data from your group to discuss. Plate.

Number of different colonies 12

Observations.

Tap water

100

There were different types of colony bacterial growth shapes on the agar plate such as white and cream domed shape colonies. This agar plate showed the greatest amount and types of bacterial growth in comparison to the other plates.

Distilled water

0

There was no bacterial growth found

Unwashed fingers

21

There was a small amount of white sooth rounded bacterial colonies found that were scattered across the agar plate

Washed fingers Water / soap

1

There was only one white small rounded colony found on the centre of the agar plate.

Hair / Coin

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