Posted: May 19th, 2015

This microbiology portfolio is a simplified replica of the practical schedule you have been using the laboratory classes.

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.