Category: Essay Writing

microbiology portfolio

| May 19, 2015

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.

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 micro pipettes 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.

Preparation and cleaning of surfaces before and after use.

Flaming of surfaces.
Micro pipettes.
Spreaders.
Inoculating loops.

Safe working and personal protection.
indicate below two important safety requirements for working in the microbiology laboratory.

Objectives.

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 solid culture A or B onto a slope of solid agar and streak out. ?

4. A sample of solid 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 Fung: Moulds
B) Staphylococcus albus D) Alternaria, Fusarium and Trichoderma
C) A mixture of A) and B) E) Yeast (Saccharomyces cerevisiae)

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:
Can you rephrase or edit the highlighted sentences
Bacteria transfer. Features of the growth
Eg turbid broth, type of growth,
colonies of similar or different appearance etc.
1. Liquid to sterile broth.
Very cloudy, almost opaque
2. 0.5 ml to sterile broth.

Very cloudy, opaque
3. Streak on agar slope.
Clear surface growth
4. Plunge into agar deep.
Clear surface growth, no interior growth
5. 0.1 ml pour plate.
Large colonies break, the surface smaller. Numerous colonies.
6. 0.1 ml streak plate.
Large colonies on top half
7. 0.1 ml streak plate.
(mixed culture)

Clear colonies on first and second streak, none on third
8. 0.1 ml spread plate.
(mixed culture)

Large colonies numbers larger on edge of plate

Fungal transfer.
Diameter of the colony – mm Features of the growth
(colour, zonation)
1. Yeast streak.

Thick lines across the plate on surface of the agar.
2. Plug of mould.
Measure the diameter of the colony at different days of growth.
State the mould species here: – A brown background, there was a large colony at the plate centre with smaller colonies dotted around.
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
Escherichia coli
(Bacteria A) Staphylococcus albus
(Bacteria B)
Colony shape

Irregular, Circular
Circular, irregular
Edge features

Irregular
Circular
Elevation

Flat
Flat
Surface features

None
None

Colour
Pale cream
Pale cream

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.
b) Insert an image of your plate and one from the SunSpace 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 SunSpace streak plate
Can you insert a picture that it has been taken personally?

State an advantage of using spread plates and an advantage of using streak plates.

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?

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. Swab the back of a mobile phone and run the swab over a nutrient agar plate.

8. Take a nutrient agar plate, label and leave unopened. This will act as a control.

RESULTS.
In session two examine the plates you set up in session one.

Particularly 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? Account for your observations. Is the un-inoculated plate free of growth?
Plate.
Number of different colonies Observations.
Open settle plate.
(state location)

22
Orange and yellow colonies spread out, small cream and all circular.

Tap water

0

No change
Distilled water

20
No irregular or small colonies, large spread out colonies

Unwashed fingers

30
No colonies on bottom half
Yellow colonies on top half
Washed fingers
Water / soap
1 1 yellow colony on water side
No change on soap side
Hair / Coin

28
Small yellow + cream colonies their still visible
Mobile phone

4
Few colonies spread out are yellow

Everyday item

23 Yellow + cream colonies, large + small circular and irregular, large irregular mass near centre
Uninoculated control

0 N/A

Settle location Counts Fingers before wash Fingers after water wash Fingers after soap wash
Near bins 21 4 53 79
Stairs 21 4 17 81
Level 2 computers 6 31 22 30
Open se? 2 22 55 55
Hand rail 1 65 17 0
Computers 3 75 17 0
Under stars 11 19 18 0
Behind bins 14 4 0 2
Next to bin 8 113 227 329
Staff office door 4 55 23 0
Filling cabinet 4 100 N/A 18
Near bins 11 59 Too many 9

Average

View the overall findings of the settle plates for the different locations and comment on the counts found in the different areas surveyed.
View the overall findings of the finger counts for the different washing options and comment on the distribution before and after washing with water and with soap.

WEEK 2

OBSERVATION OF MICROORGANISMS.

Microscopical Examination of Microorganisms

Objectives.

This part of the practical asks you to observe the microscopic features of different microorganisms using different microscopical techniques. Tick each task when you have completed it.

1. Lactophenol cotton blue stain of Alternaria mould. ?

2. Wet mount of fresh Saccharomyces sorevisiae yeast. ?

3. Gram’s stain of Escherichia coli bacteria. ?

4. Gram’s stain of Bacillus subtilis bacteria. ?

5. Gram’s stain of Staphylococcus badius bacteria. ?

6. Gram’s stain of prepared mixed bacteria culture. ?

7. Capsule stain of Klebsiella aerogenes bacteria. (demonstration) ?

8. Hanging drop of Rhodospirillum rubrum bacteria to show motility. (demonstration) ?

To acquaint yourself with the basic procedures of microscopy ensure that you visit the virtual microscope at

http://www.udel.edu/Biology/ketcham/microscope/

Results: 1. Wet mounts.

Sample Cell shape Cell features
(size and grouping)
Alternaria mould.
Note the hyphal structure and shape of spores

Yeast

Note the cells shape and any budding and chaining.
2. Gram’s Stain.

This staining procedure is the most widely used in bacteriology. It not only reveals the shape and size of bacteria, but also enables them to be immediately classified into one of two categories, Gram positive (violet) and Gram negative (pink), depending on their staining reaction which is governed by the structure of their cell walls.

Examine heat-fixed smears (see below) of your cultures of bacteria and yeast using the Gram stain. Compare these with slides prepared from fresh cultures. Record the appearance (i.e. shape, clumping) and Gram reaction (colour) of each preparation.
Gram positive organisms resist decolourisation and stain deep violet.

Gram negative organisms are decolourised by treatment with alcohol and stain pink with the counterstain.

Prepare heat fixed slides of the samples from slope cultures of bacteria A – D and observe under the microscope using oil immersion procedure for viewing at 100x magnification. Record your results in the table below.

Sample Gram’s reaction (colour) Cell features
(shape and grouping) Comment on quality of staining

Comment here on the quality of the Gram’s stain shown above and how it could be improved, if at all.

3. Capsule stain – Modified Maneval’s technique

This employs Congo Red as the negative stain to colour the background. This background becomes blue/grey since it is in an acid environment. The cells are also stained, with Fuchsin Red in 5% acetic acid (Modified Maneval’s stain). An acid dye in an acid environment is attracted to the bacterial cell walls. You should observe pale red cells surrounded by a colourless capsule against a blue/grey background.

Capsule stain procedure. This will be demonstrated.

Record your observations in the summary table below.

Klebsiella aerogenes capsular reaction.

4. Motility tests. Demonstration.

Examine the cultures of Vibrio cholerae using the Phase Contrast microscope and record motility. Describe their manner and direction of movement.

Mobility tests.

WEEK 3
ENUMERATION OF MICROBIAL NUMBERS
Objectives.

This practical work asks you to estimate the number of microroganisms in some prepared bacteria cultures using two procedures – spread plating and pour plating. In both of these procedures small samples of the culture (0.1mL) are plated onto or into the agar and allowed to grow. The number of colonies developing is a close estimate of the number of cells initially present.

In order to obtain accurate counts it is necessary to dilute the sample so that a range of concentrations are available. These are plated and the most suitable growth is counted.

This experiment compares the growth of bacteria cells on spread plates and on pour plates. Spread plates leave the cells to grow on the surface of the agar where they will be exposed to oxygen but may dry out. Pour plates submerge the cells in agar but limit oxygen access. Different results may be expected depending on the features of the microorganism.
Results

Note the results of your spread and pour plate counts for E. coli in the tables on page 18. Record your haemocytometer and plate count results for the yeast culture into the table on page 18.

Your plates should show a range of growth for different dilutions. Some will be overgrown and others have few or no colonies. Record the counts in the table below and choose plates with suitable numbers to allow you to obtain an accurate estimate of the bacterial and yeast numbers present in the cultures.

For plates with more than 200 colonies record as “TNTC” – too numerous to count.

For each set of plates choose the dilution(s) which gives the most accurate countable number of colonies. These should have between 20 and 200 colonies. Count the colonies at this dilution and calculate from this the count in the original suspension of organisms by multiplying by the dilution factor. Insert this value into the shaded boxes in the fourth column.

In addition you will be able to compare the counts obtained by the two different viable count methods.

N.B. For details on the procedures to calculate cell counts per mL from haemocytometer data and from plate counts see the practical guide on pages 23 and 25 respectively – and support material on SunSpace.
Dilution plating results. Check the pictures to complete the following table:

Yeast culture Haemocytometer
Counts in 25 sq Counts per ml of original sample (**) Only one or two of the dilutions are likely to give you a valid result. Insert the best value below for each sample.
10-0 dilution Choose the result from counts of at least 200 colonies.
10-1
10-2

Spread plate
number per plate * Counts per ml of original sample Best estimate of counts per ml
10-3 dilution Choose the result from plates with between 20 and 200 colonies.
10-4
10-5
10-6
10-7

E. coli culture Pour plate
number per plate * Counts per ml of original sample Best estimate of counts per ml
10-3 dilution Choose the result from plates with between 20 and 200 colonies.
10-4
10-5
10-6
10-7

Spread plate
number per plate * Counts per ml of original sample Best estimate of counts per ml
10-3 dilution Choose the result from plates with between 20 and 200 colonies.
10-4
10-5
10-6
10-7

With reference to the values obtained answer the following questions:

The cfu numbers (*) on the agar plates are expected to decline by a factor of 10 between the plate count dilutions. Explain why this is so and comment on the extent to which your plate counts follow this pattern stating whether your dilutions were accurate or not.

The calculated (**) counts per ml for the original sample would be expected to be similar when calculated for different dilutions. Explain why this is so and comment on how similar your calculated counts are for the different dilutions you performed.
Suggest and explain which dilution of yeast would give the most accurate and reliable cfu/ml count of the original suspension when using spread plates.

Do the cfu/ml values of E. coil obtained by pour and spread plating differ greatly? If so state which is higher and suggest why this may be.

Do the cfu/ml values for the yeast count obtained by the haemocytometer and by the spread plating differ greatly? If so state which is higher and suggest why.

WEEK 4

THE USE OF SELECTIVE AND DIFFERENTIAL MEDIA TO IDENTIFY MIXED BACTERIAL POPULATIONS

Introduction

No single medium or set of conditions will support the growth of all the different types of organisms that occur in nature. Conversely, any medium suitable for the growth of a specific organism is, to some extent, selective for it.

In such a medium inoculated with a variety of organisms, only those that can grow on it will reproduce. All others will be suppressed. Thus a specially designed medium can be used to favour the development of certain types of organism and be used to select these from a mixed population in nature.

This can be done either by direct isolation on a solid medium or by enrichment in a liquid medium. In the first case, when a mixed inoculum is spread on the surface of the selective medium, all the bacteria that can grow will produce colonies. Dispersal of organisms across the surface eliminates, to a large extent, competition between species so that even slow growing organisms survive and produce colonies. In enrichment culture in liquid media, competition between species is encouraged such that the organisms most suited to the set of growth conditions will predominate.

Selective media can also be made differential for certain types. A differential medium is one in which a particular species exhibits differential colony characteristics that can be readily recognised. These media are used in the qualitative identification of organisms in samples, e.g. pathogens in clinical specimens, and in quantitative estimates of their prevalence.

Requirements

Two urine samples
9ml volumes of sterile saline in test tubes
Sterile 1ml pipettes
Glass spreaders and flaming alcohol
Plates of the following media:

• Nutrient agar (NA – Clear agar)
• MacConkey agar (MC – Red agar)
• Cystine lactose electrolyte deficient agar (CLED – Green agar)
• Mannitol salt agar (MS – Pink agar).

Method – work in pairs for this investigation.

1. Prepare a dilution series of the two urine samples provided as in previous weeks down to 10-7 cells per ml.

2. Take duplicate 0.1ml samples at dilutions 10-3 to 10-7 and carry out spread plates with all of the 4 different media.

3. Label the plates and leave for incubation at 37oC.
Before next week’s class you will need to be familiar with the properties and reactions of these media. Check these as background preparation to help you to identify the types of bacteria in the cultures provided.

Results.

As with previous weeks select the dilution which gives a countable number of colonies. Also examine the different types of colony visible on the selective media. With the assistance of the information given in class on the expected appearance of different bacteria types you should be able to deduce the identity of the organisms in the cultures.

Record your observations and conclusions in the following tables. Determine your calculated cell counts per ml from the most suitable plate count (between 20 and 200 colonies).

In addition make an estimate of the total counts in the cultures.

Check the pictures to complete the following table:

Urine sample 1 Colony counts on plate(s)
Calculated cell counts
per ml
Choose the result from plates with between 20 and 200 colonies Colony features
Nutrient agar 10-0
10-1
10-2 .

MacConkey agar 10-0
10-1
10-2
Cystine lactose electrolyte deficient agar (CLED) 10-0
10-1
10-2
Mannitol salt agar 10-0
10-1
10-2 .

Urine sample 2 Colony counts on plate(s)
Calculated cell counts
per ml Colony features
Nutrient agar 10-0
10-1
10-2 .

MacConkey agar 10-0
10-1
10-2
Cystine lactose electrolyte deficient agar (CLED) 10-0
10-1
10-2
Mannitol salt agar 10-0
10-1
10-2 .

From the results of the different media replicates summarise the calculated counts per ml in the table below.

Urine sample 1 Urine sample
2
Nutrient
agar

MacConkey agar

CLED
agar

Mannitol Salt agar
In addition make an estimate of the total counts in the two urine samples and suggest whether these indicate that treatment is required or not. N.B the total cell count is not the sum of counts on all the different media but the highest count per ml.

Total cell counts / ml in urine 1 =

Total cell counts / ml in urine 2 =

Comment here on how well the selective and differential media helped to distinguish the different bacteria in urine 1 and 2. Refer to examples from your own plates.
Explain why the counts of bacteria differ when plated on different media even though they come from the same sample.

2. Antibiotic testing.

Microorganisms can be affected by antibiotics with different responses according to the antibiotic and the species. Investigate the sensitivity of your urine microorganisms by preparing pour plates and adding antibiotic discs.

Procedure:

For each urine sample add 0.5 mL of the sample to a separate 20 mL bottle of molten agar. Mix by rotating carefully to avoid air bubbles developing. Flame the bottle and pour into an empty petri dish. Swirl the plate very gently to mix the culture with the agar. This is a pour plate and contains your microorganisms suspended in agar.

When the agar has set add an antibiotic disc to the surface of each plate. One student in each pair should use ‘Mastring 13’ discs and the other ‘Mastring 14’ so as to cover all the antibiotics available.

Note the results of your eyewash observations below by measuring the zone of inhibition around each antibiotic disc in mm.
Also list the names shown on the disks under the two disc headings.
Check the pictures to complete the following table:
Antibiotic Bacteria from Urine
1 Bacteria from
Urine
2
Mastring 13

List the different antibotics
from the mastrings
in these spaces.

Mastring 14

List the different antibotics
from the mastrings
in these spaces.

Comment on the extent of resistance shown by the two different bacteria to the various antibiotics, is one more resistant than the other and suggest which antibiotic would you use to combat each bacteria?
WEEK 5

IDENTIFICATION OF AN UNKNOWN BACTERIUM

Introduction

In order to successfully identify an unknown bacterium a logical sequence of steps must be followed. The source from which it was isolated and its microscopic appearance may suggest the initial identity of a bacterium. (That is the shape and whether it is Gram positive or negative) Microscopic observation is usually followed by noting the pattern of growth on selective, differential, enrichment or characteristic media. This in turn is followed by investigations of biochemical and metabolic capabilities.

In the practical session you will carry out identification tests on the organisms you isolated from the urine samples. Traditional identification of microorganisms relies heavily on chemical tests to determine the metabolic abilities of the cells. These can be conducted in test tubes but this takes time and is expensive in preparation and materials.

A number of companies offer miniature systems for the rapid identification of certain types of bacteria and yeasts. Perhaps the best known of these is the API 20E system for the rapid identification of the Enterobacteriaceae and certain other Gram negative bacteria. Other systems are available for different groups such as API Staph for Gram positive bacteria.

The API 20E system consists of a plastic strip with 20 microtubes containing dehydrated substrates that can detect certain biochemical reactions. The test substrates in the 20 microtubes are inoculated with a pure culture of the bacteria to be identified suspended in sterile water. The strip is then incubated at 37oC overnight.

The tests are based on colour reactions and are scored positive or negative, in some cases after the addition of reagents. A test profile is built up which can be decoded by reference to a database. The database holds the profiles of a wide range of known bacteria and the closest match is found to identify the unknown organism.

You will be given strips to test the organisms isolated from your plates. Reagents will be added and the test profile obtained.

Requirements

Slope cultures of the microorganisms from the urine samples.
10% hydrogen peroxide – catalase reagent
Oxidase reagent
API strips set up before the class
API computer and identification charts

Method

Conduct the following tests on samples of the two bacteria and list the results in the table to obtain a presumptive identification.
Urine 1: aip 20e. Indicate positive or negative for each test and the resulting identification code in the table below.
Check the pictures to complete the following table:
Can you use the API computer programme to determine the identification.

ONPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR

RHA SAC MEL AMY ARA OX API identification.
Urine 2: aip Staph. Indicate positive or negative for each test and write the resulting code in the table below.

O GLU FRU MNE MAL LAC TRE MAN XLT MEL NIT PAL

VP RAF XYL SAC MDG NAG ADH URE LSTR API identification

Insert your code into the API computer programme to determine the identification.

Summary results.
Test Urine 1 Urine 2
Gram stain

Oxidase

Catalase

Agglutination for E.coli antibodies
Agglutination for Staphyloccus antibodies
Growth on NA media

Growth on MaConkey media
Growth on CLED media
Growth on MS media

API
Identification

Comment here on how your Grams stain, oxidase, cotalase and agglutination results provide supporting evidence for the API identifications – ie, do they agree with the features of the bacteria identified by the API test?

Comment here on how these results relate to the antibiotic sensitivity results you obtained from the disk samples you plated in week 4. You may need to research the effect of different antibotics on Gram + and Gram – bacteria to do this.

Comment on how the growth of the different bacteria on selective and differential media agree with your identification results.
WEEK 6

DETERMINING BACTERIAL GROWTH USING A SPECTROPHOTOMATER

Introduction

This practical illustrates the use of the spectrophotometer to estimate bacterial growth and from the data obtained to calculate the generation time of the growing culture. It also links this type of measurement with the methods of determining viable count that you carried out in last week’s practical.

Results.

Record your absorbance measurements in the table below.

Time (mins) Absorbance
at 600 nm Time (mins) Absorbance
at 600 nm
0
0.143 120 1.083
15
0.183
30
0.234
45
0.336
60
0.454
75
0.532
90
0.694
105
0.873

Record the colony counts from the plates in the table below.
Counts per plate 30 minutes Counts per ml
60 minutes Counts per ml
90 minutes
10-3 dilution 54 80 112
10-4 9 4 24
10-5 2 0 0
10-6 0 0 0
10-7 0 0 0
Counts per ml
30 minutes Counts per ml
60 minutes Counts per ml
90 minutes
Remember that this is based on the plates which have 20-200 colonies.

From the results above choose the best estimate for the numbers at 30 and at 60 minutes. Also insert into the table the average of the two figures (for 30 and 60 minutes) and the absorbance for each time.
Please solve the following calculations:

Time (mins) Count / ml Absorbance Count for 1.0 absorbance Average count for 1.0 absorbance
30

60

Also insert into the table the counts which would correspond to an absorbance of 1.0. To calculate this simply divide the cell count by the absorbance value. Finally average this value for 30 and 90 minutes to obtain an estimate of the relationship between absorbance and counts – ie to determine the cell counts for 1 absorbance unit.

Use the average count per ml you have calculated above to convert the absorbance values you measured into counts per ml.

To do this complete the duplicate table below by multiplying the absorbance value at each time by the average count you have calculated above.

For example if the average count for an absorbance of 1.0 was 5*106 and the absorbance at 10 minutes was 0.3 then the calculated count would be 0.3* 5×106 = 1.5×106.
Please solve the following calculations:

Time (mins) Absorbance at 600 nm Counts Time (mins) Absorbance at 600nm Counts
0
80
10
90
20
100
30
110
40
120
50
130
60
140
70
150
Finally plot the counts against time to obtain 2 growth curves – one plotted using the counts and one as the log of the counts. Insert your graphs in the boxes on page 20.
Counts
Log10 counts
Compare your graph against others of the group who added water or biocides to the culture at 60 minutes. Describe and explain below the differences found.
From the growth curve that you have plotted you should be able to calculate the generation time and specific growth rate of the bacterial culture. Note these values below and indicate how consistent they are to others of the group. Details of these aspects will be provided in the lecture sessions.
Please solve the following calculations:

Specific growth rate (?)
= maximum gradient of the log curve
= log increase / time

Generation time (h)
= time for population to double.

WEEK 7

1. MUTAGENESIS IN YEAST
(EFFECT OF RADIATION ON MICROORGANISMS).

In this practical you will apply techniques learnt in microbiology to a practical investigation in microbial genetics: specifically to practice accuracy of quantitative techniques using serial dilutions and spread plates and apply a method of mutagenesis in microorganisms.

Record your counts of the wild type and petite mutant colonies at different times of UV exposure in the table below.

Time (sec) 0 30 60 90 120 180
Total colonies
Petite mutants
Proportion of Petites
From the data plot a graph of number of colonies against time, log or number of colonies against time and proportion of petites against time. Draw the profile of the graph below.

What conclusions can you make about the effect of UV light on Saccharomyces sorevisiae?

What conclusions can you make about the experimental procedure you have used – was it rigorous and where could errors have been introduced?

Suggest how you might investigate in more detail the features and the genetic cause of the mutant colonies produced by the UV.
2. INVESTIGATIONS INTO THE EFFECTS OF LYSOZYME ON BACTERIA.

The following are short experiments that illustrate various aspects of the body’s antimicrobial defences. Lysozyme is present in human neutrophil granules and other secretions (eg. tears, saliva). It functions by hydrolyzing the glycosidic bond of peptidoglycans found in the cell walls of bacteria. This helps kill invading bacteria. Some bacteria have evolved virulence factors that render them resistant to lysozyme and therefore more effect pathogens.

Safety: In the following experiments you will be handling a variety of bacterial cultures. You should use careful aseptic techniques and must report any spillages immediately. If you have not done basic microbiology earlier in your course please inform a member of staff before beginning these experiments.

AIM: To illustrate the action of lysozyme on bacterial cultures. Specifically to estimate the lysozyme titre required to lyse cells of (a) Micrococcus lysodeikticus and (b) Staphylococcus epidermidis.

By the end of this practical you should be able to:
Visualise the effect of lysozyme on bacteria.
Calculate the weight of lysozyme required to destroy the bacterial culture.
Discover which of the two bacterial cultures is the more resistant to the action of lysozyme.

Method.

1. A standard solution of lysozyme (0.02 mg/ml) is provided.

2. Prepare 2 series of tubes (A & B) with eleven tubes in each series.

3. Dispense 0.4ml sterile saline into each tube, and number them A1 – A11, and B1 – B11.

4. Add 0.4ml of the standard lysozyme solution to tube A1 and mix well on a whirlimixer, thus giving a 1/2 dilution.

5. Using a clean pipette transfer 0.4ml into tube A2, mix as before to give a 1/4 dilution. Continue until tube A10 is reached, when, after mixing its contents, discard 0.4ml. Tube A11 is a control, and contains no lysozyme.

6. Repeat the serial dilutions for the B series.

7. Add 0.4ml of a broth culture of M. lysodeikticus to each tube in series A and 0.4ml of a broth culture of S. epidermidis to each tube in Series B.

8. Mix the contents of all tubes and incubate them for 60min in a 37?C waterbath.

9. Read the titres as the last tube in each series to show clearing when compared with the control.

RESULTS:

Indicate for each tube whether it is clear or turbid (compared to the tube 11 control) in the table below.

TUBE 1 2 3 4 5 6 7 8 9 10 11
A
0.141 0.651 0.241 0.252 0.343 0.349 0.451 0.654 0.849 0.638 0.034
B
1.630 0.827 0.864 1.014 1.759 1.042 0.953 0.977 0.961 0.736 0.031

Calculate the weight of lysozyme present at this titre.
Please solve the following calculations:
Lysozyme titre required to lyse cells:

A M. lysodeikticus: ……………
B. S. epidermidis: …………….
Comment here on the levels of lysozyme required to lyse the cells and on any differences in levels between the two species.
Can you relate differences between the effect of lysozyme to differences in microbial physiology?
WEEK 8

Activity 1. Micro-pipetting skills.

1.1. Testing your accuracy and precision in micro-pipetting.

Indicate below the range of values for your pipetting along with the mean and CV.
Please solve the following calculations:

Sample Weight (g)
1 0.9913
2 0.9880
3 0.9874
4 0.9865
5 0.9878
6 0.9895
MEAN
CV
Analysis.

Use the data collected to calculate the mean and the coefficient of variation (CV) of the weight using the following formula:

CV = (Standard Deviation / ? mean)

The CV of a range of measurements indicates the degree of variability in comparison to the mean. Regard your accuracy as good if the % CV of your measurements is less than 5% and poor if greater than 5%.

Comment below on your interpretation of the data’s accuracy and precision.

1.2. Testing accuracy in complex procedures.

Replication of a simple pipetting is a basic requirement for laboratory work. Replicating in complex procedures is increasingly required. The second test requires you to conduct a dilution series across a microtitre plate starting with a concentrated solution of dye and finishing with a sample diluted by 1/128.

Analysis.

In Excel plot three lines to show the profile of absorbance from the three dilution series and of their mean values.

Note how linear the lines are by eye but also determine the R2 value (right click on the line and select trend analysis and “display R squared value on chart”). The R2 value indicates how close your line is to linear with 1.000 being perfect fit.

Note your results in the table below and insert a copy of your graph in the box.

Dilution Series A Series B Series C Mean
1.0 1.641 1.565 1.923
0.5 0.994 0.963 1.991
0.25 0.698 0.497 0.541
0.125 0.264 0.271 0.273
0.0625 0.053 0.065 0.051
0.03125 0.099 0.09 0.02
0.0156 0.067 0.065 0.067
0.0078 0.053 0.042 0.049
R2 value

Average class R2 value =

Comment on your accuracy.
Graph to show dilution of each series.

Activity 2. DNA extraction and purification.
This activity will conduct some basic DNA extraction procedures using bacteria and run the DNA obtained on a gel to estimate its size and compare with DNA extracted by different methods.

Insert the image of your gel in the box below.

State the size of the extracted DNA in the table below and comment on the quality – eg, clear bands, multiple bands, smeared bands etc.
Sample
Size Quality
E.coli
Heat-Freeze
Vibrio natriegens
Heat-Freeze
E. coli
commercial kit
Vibrio natriegens
commercial kit
Vibrio natriegens
purified extract
1. Analysis of DNA by spectrophotometry.

Protein and nucleic acids produce a bell shaped curve when measuring their absorbance in the UV light range. Nucleic acids show a peak absorbance at 260nm and proteins at 280nm. Measuring absorbance at these two wavelengths allows you to obtain a fairly accurate measure of the concentration of nucleic acids present as long as there is little protein contamination.

Readings taken at the two wavelengths and the value of the absorbance at 260nm is divided by the absorbance at 280nm. If the reading is greater than 1.8 it means that there is little protein contamination and that you can quantify the nucleic acids with reasonable accuracy. A 50µg/ml solution of pure DNA gives an absorbance of 1.0. Therefore to calculate your concentration of DNA you would use the following calculation:

50 x absorbance at 260nm x dilution factor from the original sample.

Eg, if you had a DNA sample and you diluted it by a factor of 100 and it gave a 260nm absorbance reading of 0.125 you would have a concentration of DNA in your original solution of

50 x 0.125 x 100 = 625µg/ml or 0.625µg/µl

With the help of a demonstrator and using a quartz curvette (which is needed for measuring in the uv), measure the absorption spectrum of your sample at A260nm and A289nm.

Insert your results and interpretations below:
I’m not sure about the below absorbance, If its wrong can you let me know please.
Sample
A260nm A280nm µg/µl DNA
Own
3.284 2.325
Class average

Comment on whether your DNA was pure or not and how you deduced this.
WEEKS 9 AND 10

REGULATION OF ENZYME SYNTHESIS IN BACTERIA.
In common with many other bacterial enzymes, ß-galactosidase, an enzyme hydrolysing lactose to glucose and galactose, is only produced when needed. A growth medium which lacks lactose (or a lactose analogue) fails to produce the enzyme. Lactose, when present, is said to induce the enzyme.

The natural substrate of ß-galactosidase is lactose, which it cleaves into glucose and galactose to be assimilated by the bacteria cell. ß-galactosidase will also cleave a number of sugars with similar structures as well as synthetically produced analogues such as o-nitrophenyl-ß-D-galactoside (ONPG).

In some experiments, the lactose analogue isopropylthio-ß-D-galactoside (IPTG) is used as a gratuitous inducer (ie, it induces the enzyme but does not act as a substrate. The enzyme is assayed by incubation with the artificial substrate o-nitrophenyl-ß-D-galactoside, (ONPG) since upon hydrolysis the yellow o-nitrophenyl-ß-D-galactoside released may be measured colorimetrically.

To do this o-nitrophenyl is measured quantitatively by determining its absorbance at 420nm and using a standard curve to convert the absorbance to ?M of 0-nitrophenol.
Insert your standard curve below and comment on its quality.
Standard curve
Comment on accuracy

As a result of your experiment you will have obtained data for the absorbance of the cells at 600nm and also the absorbance at 420nm developed by the enzyme assay of the control incubation and of incubations with the inducer IPTG.

Record your data in the tables below using the standard curve to covert the absorbance at 420 to ?M of 0-nitrophenol.

Growth comparison.

Sample Absorbance 600nm
1
0 min control 1.321
2
30 min control 1.319
3
60 min control 1.327
4
30 min + IPTG 1.239
5
60 min + IPTG 1.247
Enzyme comparison.
Sample Absorbance 420nm ?M
0-nitrophenol
1
0 min control 0.066
2
30 min control 0.080
3
60 min control 0.0105
4
30 min + IPTG 0.159
5
60 min + IPTG 0.228
Information for the above table:
Absorbance 600nm which is the absorbance of the bacteria indicating how much the culture has grown. This is important to know as we need to relate the enzyme activity to a comparative amount of cells. An increase in enzyme activity could be just a result of cell growth. We wish to know whether the enzyme activity per cell has increased and so need a comparative measure.
Absorbance 420nm which is the absorbance generated by the o-nitrophenol produced by the ß-D-galactosidase activity.
It would be possible to simply compare these two values but would not be very accurate as the 420nm measurement is not expressed in concentration of the o- nitrophenol. To do this we need to use the standard curve and convert the absorbance at 420nm to µM of o-nitrophenol. Use the formula you have determined from the standard curve graph and insert the values in the table above.

Enzyme generated per unit of cells.

To determine the amount of ONPG cleaved per unit of cells for each sample use the following formula:
ONPG cleaved ?M
OD 600nm

Sample ONPG cleaved ?M
OD 600nm
1
0 min control
2
30 min control
3
60 min control
4
30 min + IPTG
5
60 min + IPTG

Effect of mutations and inhibitors.

Record your results from the second week experiment investigating the activity of ß-galactosidase in mutant strains and in the presence of antibiotics.
Control
E. coli G9 ONPG cleaved
OD 600nm Control
E. coli
G9 ONPG cleaved
OD 600nm

0 min control 2.72 0 min
IPTG 2.22

30 min control 2.84 30 min IPTG 4.09
45 min control 4.76 45 min IPTG 3.23

60 min control 3.52 60 min IPTG 4.17
Mutant
E. coli G6 ONPG cleaved
OD 600nm Mutant
E. coli
G7 ONPG cleaved
OD 600nm

0 min control 2.72 0 min control 2.72

30 min control 2.84 30 min control 2.84
45 min control 4.76 45 min control 4.76

0 min + IPTG 36.52 0 min +
IPTG 158.8

30 min + IPTG 48.2 30 min + IPTG 153.06

45 min + IPTG 39.16 45 min + IPTG 152.01

Rifampicin
E. coli
G6 ONPG cleaved
OD 600nm Chloramphenicol
E. coli
G7 ONPG cleaved
OD 600nm

0 min control 0.041 0 min
control 0.041

30 min control 0.043 30 min
control 0.043
45 min control 0.047 45 min
control 0.047

0 min + IPTG 2.529 0 min +
IPTG 0.046

30 min + IPTG 1.726 30 min +
IPTG 0.046

45 min + IPTG 3.911 45 min +
IPTG 0.05

In the box below explain your results in the light of your knowledge of the functioning of the lac operon.

Assessment.

Your practical work for HSC105 will be assessed both by observation of your skills in the practical class and in the portfolio you present. The profile of marks for the assessment of the practical work is as follows:

Practical assessment of streak and spread plating. 25%. 10% of module marks.

The assessment activity is for you to produce competent spread and streak plates from the mixed culture samples provided. You will have practiced these during the practical programme and will be observed in later weeks to show your competence.

The assessment mark gradings for this are as follows out of 10 maximum:

7-10. Competent microbiology skills to producing clear separation of colonies with no contamination using safe aseptic techniques.
4-6. Competent microbiology skills producing limited separation of colonies with no contamination but using safe aseptic techniques.
<4. Poorly competent aseptic skills producing poorly separated colonies with or without contamination and or damage to agar.

Three criteria will be used to assess the quality of your streak and spread plates as listed below. The competence score will comprise 25% of your practical assessment. The aseptic assessment will count towards your category 2 microbiology training which you will need for work in levels 2 and 3.

Streak plate Spread plate
Full and neat streak or spread using all available area. /4 /4 /4
Clear separation of colonies of both species. /4 /4 /4
Lack of contamination and no damage to agar. /2 /2 /2 Total score /20

The criteria for assessing the competence of your aseptic skills are listed below.
Preparation and clean-up of working area. /2 /2
Aseptic handling of materials and instruments. /2 /2
Use of suitable protective materials. /1 /1 Total score /5

Your portfolio. 75%. 30% of module marks.

A template for your practical results will be posted on the SunSpace site and should be completed in the relevant parts for submission. In some parts you will need to translate observations and comments you have made in class, in others you will need to import graphs you have drawn in another programme and in some areas you will need to import photographs of your slides and plates you have obtained in class. You should ensure that the version you submit has suitably spaced and separated sections with limited empty space but is not too crowded to make it difficult to follow. For example avoid splitting tables across two pages and ensure that each session starts on a separate page. Please note that the portfolio template contains some deliberate errors. You should note and correct these during your preparation.

The assessment mark gradings for the portfolio are as follows:

7-10. Full or extensive range of results, images and commentary reported. Accurate results and appropriate interpretations given. Excellent presentation with clear and fluent text and suitable spacing and positioning.

4-6. Some but limited range of results, images and commentary reported. Mostly accurate results and appropriate interpretations given. Good to adequate presentation – tables and content well positioned with limited wasted space.

<4. Incomplete results, images and commentary reported. <75% attendance recorded, inaccurate results and inappropriate interpretations given. Poor presentation eg pages jumbled with large spaces, split tables and mixed fonts.

Your portfolio marks.

The assessment criteria for the portfolio are listed below with more extensive details provided in the module guide.

Completeness of results and commentary in portfolio sections. /10
Accuracy of observations and interpretations. /10
Presentation. /5

Remember the following important guidelines for producing the portfolio.

• Complete all parts with relevant details. Some of this will be results of practical work, some comments on procedures and some interpretation.
• Do not worry if your results are unexpected or poor compared to your expectation. It is your interpretation of the results which is most important – ie, that you know why a result may be poor and can suggest how to improve.
• Ensure your calculations are accurate – particularly in the estimation of cell counts from dilution plates and the haemocytometer.
• Ensure that your final presented copy is well produced – avoid splitting sections, boxes, tables etc between pages, ensure each section (practical week etc) starts on a new page, ensure that you make appropriate comments and interpretations and refer to theory where possible. Use a consistent font.
• Add in relevant diagrams, photographs etc to illustrate your work.
• Bind the work securely. Staples are fine, plastic folders are fine but don’t put separate pages into individual plastic wallets – they may not be marked. Ensure your name is printed on the front of your portfolio and ideally in a footer on every page.

Do not e mail the portfolio – hand it into the library resources service.
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Nursing Research

| May 19, 2015

Introduction
Daytime sleepiness in aged care residents is a significant problem that needs to be addressed. To overcome the adverse health effects of daytime sleeping, there are three key recommendations that I wish to explore. These include treating sleep deprivation without medication, re-designing the current medical unit to be more accommodating for promoting night time sleep and adapting individual care-plans so specific schedules can be implemented for the needs of each resident. Through the analysis and collection of research literature various points of importance reflect these key findings. By engaging these recommendations and implementing each of the three key changes in practice, effective solutions will improve quality of life.

Part 1 Identifying Recommendations for Practice (approx. 1, 500)

Key findings:

Across the literature

The prevalence in sleep deprivation

Sleep is optimal cognitive function
Key findings and recommendations CHECK 500 WORDS ONLY

Poor sleep patterns, including excessive daytime sleeping affect the patient in a negative way, as cited by (Woodward, p. 131) “Long term deprivation can have severe adverse health consequences indicating the essential nature of this stage.” Woodward is referring to the compromise in attaining maximum rapid eye movement (REM) sleep, which is associated with dreaming and the consolidation of memory. REM sleep is essential for overall physiological balance and wellbeing. Quality REM sleep is critical for optimal cognitive function. Added to this evidence, Keage says, “Poor sleep can lead to various cognitive impairments in elderly people,” (Keage et al., p. 886; Blackwell et al).

Geriatric sleep complaints include “insomnia, difficulty falling asleep, and difficulty maintaining sleep” (Missildine et al. p. 263; Hellstrom et al. p. 1). Issues in geriatric sleep patterns commonly stem from daytime sleepiness and napping, leading to nighttime sleep disturbances. Reyes states, “This may be directly related to poor health, increased risk of falls and mortality” (Reyes et al., p. 175). The importance of research is being increasingly recognized and is essential in addressing the problems in practice (Manian & Manian, p. 56). Keage et al., recognizes that sleep deprivation and daytime napping are all “modifiable behaviors open to intervention strategies,”.

Some intervention strategies to improve quality sleep could include increasing physical activities during the day such as walks around the facility grounds and participating in physical grous activities provided by the aged care facility . Spending time in sunlight is beneficial and also engaging in social intellectual activites like playing cards, chess, computer games benefits the resident sby balancing day time acitivity with exercises and prmotoing a restful sleep model. Social intell . acti… promote the memory and mental activity and also maintains social contacts and communication. Making these recommendations vital for normal sleep architecture. Sleep architure (the cycle of REM and non REM sleep) and achieving the optimum balance benefiting the resident.
. reflective by the various day time activity modifications, is crucial in the balance of day time activity and stimulation with nighttime sleep architecture improving quality of life for the residents
(p. 886) (maybe need to add in recommendations- other there any other reccomendations on the literature?)

have any recommendations been mentioned in more than one article?
Mannian et al., p 56 : Interventions at improving sleep quality in this patient population seem warranted.

What areas for nursing practice that could be changed or improved or strategies implemented
Medication

Another key recommendation to combat geriatric sleep deprivation explores treatment without medication (Woodward, pp. 137-9). Currently a method of combating sleep deprivation is to give patients sedatives. Woodward is critical of this approach. “It is relatively easy to immediately prescribe a hypno sedative such as benzodiazepine (BZD) yet the published literature is often critical of such an approach.”… Medication does not necessarily combat the problems of sleep deprivation and therefore an accurate diagnosis is needed to determine what strategies should be used in it’s place. As a result, alternate methods should be incorporated into nursing practice to address the use of sedatives. Woodward lists various alternatives to sedatives including psychological approaches, educational methods, light therapy and utilizing non – pharmacological approaches. Psychological treatment approaches have proved effective for the management of daytime sleepiness. These include approaches such as relaxation therapy and cognitive behavioral therapies (CBT) which can be administered by trained health professionals. Woodward states that the benefits of these methods have been established through two meta-analyses and systematic review, and therefore, would be worthwhile implementing in practice. (p. 138). This alongside other methods including education of sleeping patterns, light therapy and non- pharmacological approaches are strategies that could be used in place of sedatives. Hellstrom also identifies the benefits of using a non-pharmacological first stage response, finding that social intellectual activities were beneficial for sleep (Hellstrom et al, p. 10).
(There is a range of sleep assessment tools. A two week sleep diary designed by the American Academy of Sleep Medicine can be useful. The individual records use of stimulants such as coffee, taking medications, alcohol use and exercise. They record the time they go to bed and when they believe they fell asleep, and record whether they were asleep or awake throughout the 24-hour day. It is important to emphasize that they do not fill it in throughout the night. This diary, most importantly, can combat the mistaken perception that they are having very little sleep for instance it may reveal that they are having 14 hours in bed overnight and while awake for 7 hours they are still having 7 hours sleep. It can also reveal excessive use of stimulants late in the day and insufficient exercise, amongst other useful information.) (Woodward p. 136)
These new improvements to aged care facilities will help overcome daytime sleepiness in the first stage response by making this first hand approach individualized to the patients needs and focusing on alternative methods. Woodward states “treatment without medication should almost always be the first approach.” (p. 145). Health care in nursing practice can thus be improved by considering alternative to sedatives. Instead assess and individualize health care needs and then you can identify key problems in relation to an individuals sleeping patterns. By documenting these new improved suggestions which are non-pharmecautial and recording the results in the care plan, sedatives use may be reduced and other options considered.
Woodward states that sedatives have been associated with a greater risk of hip fractures and falls in older adults, and therefore by using alternate approaches the rate of falls in the aged care facility can be reduced. (Woodward, p. 145) and therefore by introducing these alternatives along with an individualized approach this issue may e reduced.
Ideally an aged care facility needs to reevaluate their system and put a process in place that will enable an individual-focused approach. One method of achieving this is to implement an interview process for when the patient first arrives to assess what is best suited for their immediate and ongoing care.

However, there may be challenges to implementing alternative practices, especially when sedatives are an easier alternative. The challenges to the implementation in the first stage response are how to ensure that treatment is sufficiently catered to the individual. It may be a difficult task to oversee that the nurse has sufficiently assessed the patient and isn’t over reliant on giving sedatives as an approach, but rather that they should be used a last resort. Woodward states that pharmacological management still has a place in the management of sleep disorders. (Woodward, p. 139) Therefore, despite it being a last resort it should not be disregarded altogether.

With the new changes to the aged care facility ethical considerations also have to be adapted and changed. Ethics, in nursing homes especially, are essential in ensuring that health professionals provide sufficient care to the patient. Therefore, any change in the procedures has to take into account ethical considerations and ensure they will be adhered to. One major ethical consideration in relation to these changes is the patient has a right to choose their treatment. By expanding first response methods the patient will be able to choose between a variety of methods that the nurse can recommend which is best suited for their care. As a result of increasing the choices the nurse should be aware of the patients right to either adhere to the new methods or to choose another alternative method that they may not necessarily be the best option.

• Sources for ethics?
• ‘Self Perceived Resources for Good Sleep’ has a lot in it about the patient being able to choose for themselves and technically they should not have their rights taken away

How recommendation could change the way nurses deliver care

Strength of the evidence that supports to recommendation

What outcomes of interests could be evaluated?

How to embed research into practice
p. 58

Re-designing of medical units 500 WORDS ONLY
Age old thing we do certain things at 6 or 7- changing – Missildine et al.
The next recommendation addresses the environmental physical surroundings impacting sleep cycles. Many aged care facilities are not designed to cater for low soft lighting and reduced noise from nursing staff activities. The setting is often rather hospital like in design. Looking at alternatives and redesigning units to promote quality sleep and a full sleep cycle of 90 minutes would allow patients to experience less fragmented sleep. Suggesting that staff should be encouraged to “reduce light and sound levels at night” (Missildine et al., p. 270) would be beneficial for the resident. It is argued that staff interruption making staff activities of central importance.

Pain and anxiety are the most commonly cited reasons for unsound sleep (Manian & Manian p. 59). By increasing staff numbers to care for these issues would promote better sleep management and a better outcome for the resident.

They don’t really recognise sleep patterns for patients they don’t get sleep because they have to take care of them they cannot just sleep residents rarely asked about at encounters with health professionals (Woodward p. 2) it was noted that few
It has been recommended that staff and GP’s take into account the impact of psychological barriers to sleep. These include Type 1 (non-psychological barriers, e.g. pain and noise), Type 2 (worry about actual situation and future) and Type 3 (traumatic memories) barriers (Hermann & Flick, pp. 484-6).
Feelings of being along, worrying at night, less staff to care for their phyciolgoical nees. Expand this.

What are the improvements?
How could this lead to improvements?
• Individualized
• Focused
• Fostering that type of care

What is eproblem 1?

How could it be implemented into the practice setting

Any challenges or barriers?

• They still have a place?

Any ethical considerations

How recommendation could change the way nurses deliver care

Strength of the evidence that supports to recommendation

What outcomes of interests could be evaluated
How to embed research into practice

Care plan 5OO WORDS ONLY

Care plan should be developed with the sleep quality part of the plan being a high priority. On an individual basis – puts everyone
Catered for the individuality- different meal times, sleep time- living in your home all your life want to go to bed early and then cant because you might have to eat when you want to go to bed- hard transitioning to an institution
Addressing Poor health and sociological barriers to sleep

Effectiveness of sleep management of agencies

Association between sleep deprivation and leisure activities

Cognitive leads to increased fall risk- this is a major health risk in elderly people and needs to be preventative

Preventative plan- it is no less important that we need to do activities to stay healthy- make sure it happens in those settings

Staffing issues

What are the improvements?
How could this lead to improvements?
• Individualized
• Focused
• Fostering that type of care

What is eproblem 1?
• Contributing to the increased rate of falls
• Physical activity

How could it be implemented into the practice setting

Any challenges or barriers?

• They still have a place?

Any ethical considerations

How recommendation could change the way nurses deliver care

Strength of the evidence that supports to recommendation

What outcomes of interests could be evaluated?
How to embed research into practice

Ethical considerations
Across the research literature it has been identified that poor sleep patterns in the population lead to a number of detrimental health issues. Stone et al. asserts that “as many as 50% of older adults report sleep problems” (p. 299). Koch et al. argue that “older adults are prone to increased night awakening and sleep fragmentation” (p. 1268), it has been found that the prevalence of sleep deprivation in the population can range between 64%-69% (Herrmann & Flick, p. 482).
Woodward maintains the first recourse to action in combating geriatric sleep deprivation should be treatment without medication (pp. 137-9). Nonetheless, the risk of hypnotic agents may be acceptable when compared with the dangers of “undertreated sleep disturbance” (p. 145). In accordance with a non-pharmacological first stage response, it was found that social intellectual activities are beneficial for sleep (Hellstrom et al, p. 10).
Stone et al. found that “actigraphic short sleep duration and lower sleep efficiency” were positively related to recurrent falls in the population (p. 303). It was concluded that there was an association between poor sleep and the risk of falls, factors leading to this may include, “impaired cognitive function, depression, balance problems, and use of medications” (p. 304). Woodward reaffirms the position that a greater risk of falls is associated with insomnia/sleep deprivation (p. 135). Cumbler et al. (p. 580) highlighted the importance of a growing literature that emphasises the increasing occurrence of falls in patients/community dwellers receiving sedative medication. Based on the analysis of the chosen articles it is possible to argue that cognitive and functional impairments, developed through sleep deprivation, will have an increasing probability in the occurrence of falls in the target population.
Missildine et al. recommended the redesign of unit activities to revolve around a full sleep cycle—90 minutes—to allow patients to experience sufficient sleep. Suggesting that staff should be encouraged to “reduce light and sound levels at night” (Missildine et al., p. 270), it is argued that staff interruptions—making staff activities of central importance—pain and anxiety are the most commonly cited reasons for unsound sleep (Manian & Manian p. 59). It has been recommended that staff and GP’s take into account the impact of psychological barriers to sleep. These include Type 1 (non-psychological barriers, e.g. pain and noise), Type 2 (worry about actual situation and future) and Type 3 (traumatic memories) barriers (Hermann & Flick, pp. 484-6).
Part 2: Strategies to Facilitate Research Utilisation (500)

Part 3: Identifying and Evaluating Outcomes(300)

Part 4: Disseminating Evidence-Based Practice Outco
Conclusion (100)

Given the prevalence of daytime sleep disturbance problems in aged care importance of good quality sleep to overall physical and psychological well-being, it is imperative that the most effective sleep assessment, diagnosis and management strategies be implemented in aged care facilities.

Sleep disorders are a major clinical issue in aged care although advances have been made in diagnosis and management, there is still a major need for effective approaches to safely treat the spectrum of sleep problems. The dangers of under- treated sleep disturbance may outweigh the risk of adverse effects of the hypnotic agent, but treatment without medication should almost always be the first approach.
Socio intellectual activities are beneficial for sleep. Physical activities, such as strolling in the country or gardening,
Excessive daytime sleepiness was related to fall risk, Future studies using comprehensive objective measures of sleep should confirm the interrelationships between sleep characteristics to determine whether they contribute independently to risk of falls. Further research into daytime sleepiness is clearly warranted
FOOTNOTE MORE INFO FOR THE WRITER
• Assignment 2 is considering what key nursing recommendations come from the evidence, patient and nursing outcomes that would be the focus of any interventions, strategies on how to embed research into practice and finally how to evaluate potential evidence-based practice change and then how findings could be disseminated
Page 1 of2
USQStudyDesk: My home > My courses > Fac of Hith, Eng & Sciences > 2015 Semester 1
NUR2300_2015_,1 > ePBL (Problem-Based Learning) >Assessment ebook
USQ TIME 4:15 pm Tue, 28 Apr 2015
Research Methods for Nursing
Assessment ebook

11 Assignment 2 Rationale
Rationale:
The purpose of this assignment is for you to demonstrate your knowledge and skills in
relation to:
1) analytical skills in the critical appraisal of the research evidence and applicability of
research evidence in the provision of nursing care
2) written communication skills, inciuding analysis and synthesis of information, and
appropriate use of language and literacy
3) an ability to synthesise research evidence and consider applications within clinical
practice using an EBP process
Assignment Links to Evidence-Based Practice Steps:
This course has been structured around the 7 steps of evidence-based practice. This
assignment is focused on the following key stages:
1. Critically Appraising the Evidence
2. Integrating Evidence into Practice
3. Evaluating the Outcomes of Practice Decisions based on Evidence
http://usqstudydesk.usq.edu.au/m2/mod/book/view.php?id=305744&chapterid=16684 28/ 04/201 5
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Project Management

| May 19, 2015

Your role in this case study for the assignment is that of the new Senior Project Officer overseeing all project management activities at Engineering Consultancy Ltd (ECL). You are reporting directly to the Managing Director.

ECL is a leading company delivering projects and consultancy in the field of engineering and infrastructure. Customers are both from the public and private sector. It is located in the capital and has a proven history of successful projects for international customers. It was founded in 1980 and today, it employs 250 people.

Two years ago, representatives of investors on the board of directors of ECL had required that the company demonstrates how it is managing and improving its project processes. As a response, several ideas were explored and a budget made available to implement project process management at ECL. However, these activities have not led to any improvements or successes that could be presented to investors.

Therefore, ECL has decided to advertise the position of Senior Project Officer for which you were headhunted. In preparation for the next meeting with key shareholders, the Managing Director found in the literature the following quote: “Many organisations, particularly from sectors that run large-scale projects, use project offices to assist in all aspects of the management of project work” (Maylor, 2010:63). Accordingly, the Managing Director wants you to write a study focusing on the introduction of a Project Management Office (PMO) at ECL.
This report is expected to serve as an input for the discussions at the board of directors. In the meantime, this has become a critical issue as several customers of the company have also required documentation about sustainable process and resource management at ECL. The Managing Director has made it clear that your report is of strategic importance both for the company’s market position and also for some of its customers, and which should not fall into the hands of competitors.
For ECL, the stated objectives of your report are:

• Identify all relationships between project process management and a project management office (PMO);
• Contact all project managers of ECL about how the PMO can help to improve projects;

• Provide evidence (e.g. through secondary data collection from the literature on Project management) about the effectiveness of PMOs;

• Explain how the interaction between project management, project portfolio management and strategic programme management can lead to better and more sustainable process and resource management at ECL; and

• Organise meetings with customers of ECL to understand how a PMO can support their projects.
A significant IT solution was discussed to be an essential component as part of the PMO in case you can demonstrate that it supports the objectives listed above.
Timescale

Although your report is time critical, a 4 month period is given to prepare, before presenting in front of the board of directors of ECL. Please consider progress meetings or other milestones as part of your time planning.

Cost

A total project budget of £147,000 is considered appropriate and sufficient by the Finance Director. This sum is intended to cover all costs to finish your report including all consultant and travel costs, and new hardware and software for the project planning software solution.
The budget includes all costs for contacting and meeting customers of ECL at your discretion.
The only costs this budget would not cover would be the salaries of any ECL project managers seconded full or part-time to support you. They would draw their salaries exactly as usual.

Staffing

Personnel allocated for your study are: two secretaries with a salary at £ 3,250 p.c.m. (per current month), and two consultants at £ 300 per hour or 2,400 per day.

The implementation of the Project Management Office (PMO)

Work for your PMO report should start immediately. Please elaborate on the following issues as preparation for the future implementation of the PMO at ECL:

Requirements Analysis which needs to be discussed with project managers and should take no more than 20 working days. This should include information about current projects, staff project experience, and current project process management.

This should be followed by Detailed Analysis of the Effectiveness of the PMO (5 days) and Improvement of Project Processes (15 days) – these activities can run at the same time and can run roughly in parallel. As soon as Detailed Description of the PMO is complete you could start Selection of Project Planning Software solution (30 days).

Computing Centre Review should run for 10 days and can be done in parallel.

Depending on your requirements for the PMO, you can create up to 10 more activities and/or milestones supporting the creation of a PMO.

Presentation of final PMO report is a final milestone.

Please check for public holidays during the project duration.

When asked about who works on what tasks the following information was handed over to you (the letters in brackets stand for: S1 and S2 = Secretaries, C1 and C2 = Consultants). Please note that these are only suggestions. As Senior Project Officer you can adapt this as necessary:

Requirements Analysis (no more than 20 working days) (SPO, C1, C2, S1) Detailed Analysis of the Effectiveness of the PMO (5 days) (SPO, C1, C2) Improvement of Project Processes (15 days) (SPO, C1, C2)

Detailed Description of the PMO (SPO, C1, C2, S1, S2)

Selection of Project Planning Software solution (30 days) (SPO, C1, C2) Computing Centre Review (SPO, C1, C2)

Draft of PMO report (SPO, C1, C2, S1, S2)

Discussion of report with Managing Director (SPO, C1, C2) Writing of final PMO report (SPO, C1, C2, S1, S2) Presentation of final PMO report (SPO, C1, C2)
Assignment Questions

In the role of the Senior Project Officer you are required to develop a report (including academic references), structured along the following lines:
Part A – General overview (60%) – approx. 2,500 words

Critically evaluate the relationships between a project management office (PMO) and project process management. What steps can be taken to improve the overall performance of project process management at ECL? What are the key weaknesses roles of a PMO? What are the key measures and tactics to implement a PMO?
Part B – Project Planning (20%) – Gantt Chart report (added explanations approx. 250 words)
Given the importance of your report, develop a one-page project plan in Gantt chart form (as if the project had not commenced) for the 4 month duration. The chart should clearly indicate the critical tasks and the planned end date but ignore progress and resources at this point. Include the Gantt chart as appendix Part B.1.

Suggest any tactical options to reduce the project duration to 3 months.

Update your planning and include a second updated Gantt chart as Appendix Part B.2.
Part C – Budget creation (20%) – Excel report and approx. 250 words of added explanation

According to below part C is progress management and budget creation part D
Assuming that the project will run perfectly to the schedule outlined by you in Part B.1 with all contract personnel working as defined on the tasks indicated, generate an overall budget planning for your report. Include the 2 secretaries and 2 consultants, accommodation costs at £650 per week inclusive, your own salary at £ 5,250 p.c.m. (per current month), all new PC hardware (total fixed cost: £35,000) and project planning software (total fixed cost: £27,000), and total miscellaneous fixed costs of £25,000. Create budget positions for meetings, travel costs and other activities at your discretion within the overall budget limit. Show all calculations and totals via suitable report formats. Include as Appendix C.1.
The agreed total project budget for your report is £147,000. When you discuss this budget on your first day with the Finance Director, he asks that you prepare an alternative where total costs are only £117,000. Show all calculations and totals via suitable report formats, and include as Appendix C.2.
Part A – General overview (60%) – approx. 1,800 words
Part B – Project Planning (10%)
Part C – Progress Management (10%)
Part D – Budget creation (10%)
Part E – Budget management (10%)
Word limit

The written element of the report relating to part A should be approximately 2,500 words in length.

Explanatory notes supporting the creation of information using MS Project (Parts B and C) should be approximately 500 words in length.
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Literature Review

| May 19, 2015

Does clinical debriefing in Emergency Departments help reduce work related stress for Nurses Working in the Emergency Department?

critique of the 5 peer-reviewed articles. Please See Attched and Include
• a brief introduction reiterating your research question and the purpose of the literature review
• a synopsis of the methodologies used in your five articles
• a presentation of the common themes that emerged from your critique of the articles
• agreements and disagreements within the articles
• an evaluation of the conclusions and recommendations presented in the articles
• commentary on the relevance of this review to your context of paediatric nursing, i.e. the answer to your research question
• a conclusion that addresses potential areas of practice review/development
• state what your literature review adds to the body of nursing knowledge.

Articles to be used for review:

Ross-Adjie, G.M., Occupational stress in the ED: what matters to nurses? Australasian emergency nursing journal, 2007. 10(3): p. 117-123.
Theophilos, T., et al., Debriefing critical incidents in the paediatric emergency department: current practice and perceived needs in Australia and New Zealand. Emergency Medicine Australasia, 2009. 21(6): p. 479-83.
Magyar, J. and T. Theophilos, Review article: Debriefing critical incidents in the emergency department. Emergency Medicine Australasia, 2010. 22(6): p. 499-506.
Adeb-Saeedi, J. Stress amongst Emergency Nurses. Australian Emergency Nursing Journal, 2002. 5 (2): p:19-24.
Tuckey, M.R., Issues in the debriefing debate for the emergency services: Moving research outcomes forward. Clinical Psychology: Science and Practice, 2007. 14(2): p. 106-116.

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Religious studies

| May 18, 2015

. REQUIRED RESOURCE
Oswalt, John. N. The Bible Among the Myths: Unique Revelation of Just Ancient Literature. Grand Rapids, MI: Zondervan, 2009. ISBN: 9780310285090.

The first Book Summary will be a summary of the Oswalt text, The Bible Among the Myths, and must be at least 11 pages, not including the title page. Please be sure to summarize each chapter, as well as the introduction and conclusion.

General Instructions:
Write a one-page summary for each introduction, chapter, conclusion, and postscript found in the books. The summaries can be written as the reading is being done then collected into one coherent paper for submission. Each section should have its own heading in the papers. The major topics discussed in each section of the books should be summarized. These are not intended to be book reviews, but summary re-statements of the authors’ works in your own words.
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business

| May 18, 2015

What are the patterns of business failure in the UK? What are the main explanations for why businesses do not survive? Which explanations are the most and least convincing?

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Sydney tourism branding(a tourism location,place branding)

| May 18, 2015

Introduction to the case study brand you’re researching(Sydney tourism branding)
a. Overview of the case study brand
b. Detailed description of two branding tactics animated by your topic
(select two actual micro-level events, examples, or instances of your case study brand’s tactics. E.g., describe the elements of George Clooney as a brand ambassador in his work for Nespresso in the 1st tactic, and maybe describe aspects of his “brand identity” that enables him to star in certain film genres etc. for the 2nd tactic. Focus on only one example for each brand tactic to keep your analysis focused and tight. You may include visuals of print ads / YouTube screen shots or other evidence etc. to enhance the descriptive elements of “the what,” “the when,” and “the how” of your brand case study’s tactics)
i. Branding tactic 1
ii. Branding tactic 2
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BUSINESS

| May 18, 2015

Briefly describe Porter’s “generic strategies”
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MANAGEMENT

| May 18, 2015

Briefly describe the basic idea behind Porter’s five forces model.
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MANAGEMENT

| May 18, 2015

If someone asks you what the term “vertical integration” means, how would you describe it? Also, what would you consider as a key reason for why firms vertically integrate into different stages of the value chain?
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