Separating micro-organisms




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EXPERIMENTS

SEPARATING MICRO-ORGANISMS
Source: NQ Curriculum Support Intermediate 2 Biotechnology

(Unit 2 Student Materials)
When professional microbiologists isolate micro-organisms from the environment or an infected person, it is extremely rare to obtain a pure culture. It is therefore necessary to separate micro-organisms. Plating or streaking can be used to achieve this. Using isolated single colonies as inocula for further streak plates, pure cultures can be obtained.
Streaking out a mixed broth culture on an agar plate and incubating it to obtain single colonies of different types of bacteria or yeasts can simulate this.



Streak plate showing individual colonies

Instructions
These instructions are for right-handed people. If you are left handed, please reverse the instructions accordingly.


  1. Wear a lab coat.




  1. Prepare your work space on the bench, collect the materials and set them out correctly on the bench.




  1. Label the bases of the Petri dishes containing the appropriate sterile agar with initials, date and name of culture.




  1. Using the mixed broth culture as the inoculum, streak out applying the method you have used previously.




  1. Incubate the plates at room temperature for 48 hours.

If you have obtained well isolated single colonies, use these as inocula for further streak plates using the method you have used previously to obtain pure cultures.


CONJUGATION IN MUCOR (ZYGOSPORE PRODUCTION)
Source: James Watt College




  • Label an agar plate as shown here:

+ -



  • Sterilise a platinum stab wire or a scalpel blade.




  • Transfer a piece of mycelium from a Mucor (+) strain to one half of the agar plate.




  • Resterilise the wire/blade.




  • Transfer a piece of mycelium from a Mucor (-) strain to the other half of the agar plate







  • Carefully invert the plate and store at 25C for 3 – 7 days.




  • Note the growth which is found after this period of incubation.


EFFECTS OF TEMPERATURE ON MICROBIAL GROWTH
Source: James Watt College



  • Stock cultures (24 hours) of the following microbes:


Bacillus subtilus

Escherichia coli

Bacillus stearothermophilus

Saccharomyces cerevisiae


  • 5 nutrient agar plates.




  • Divide each plate into four quadrants and label appropriately with microbes name or initials.




  • Inoculate each quadrant with a different microbe by means of a single line of inoculation of each microbe in its appropriately labelled section.




  • Secure lids to plates.




  • Incubate in inverted position at appropriate temperature (10C, 20C, 37C, 50C and 60C) for 24 hours.




  • Record amount of growth:

(-) = Absence of growth

(1+) = Scant growth

(2+) = Moderate growth

(3+) = Abundant growth


  • Comment on the temperature ranges the 4 microbes grow best at.


ANTIBIOTIC SENSITIVITY: KIRBY-BAUER METHOD

Source: James Watt College
This technique is used to determine the sensitivity of various microbes to antibiotics present on discs.


  • Inoculate agar plates with 0.1 ml of test microbe:


E. coli

Klebsiella pneumoniae

Staphylococcus epidermis albus
i.e. set up spread plates


  • Allow culture to soak in for 10 minutes.




  • Add antibiotic disks using sterile forceps.


Do not move the disk once it is placed on the agar


  • Secure lids to plates.




  • Incubate at 30C for 24 hours. Do not invert.




  • Take a note of the specific antibiotics used here.




  • Determine whether the bacteria are resistant, intermediate or susceptible to each antibiotic by comparing ‘zones of inhibition’ ie areas of clearing around the antibiotic discs.




  • Deduce which antibiotic(s) would be best to treat each individual species of bacteria.



BIOCHEMICAL TESTS
Source: James Watt College

Oxidase Test
This identifies cytochrome c oxidase, an enzyme found in obligate aerobic bacteria. Soak a small piece of filter paper in a fresh solution of 1% (w/v) N-N-N1-N1-tetramethyl-p-phenylenediamine dihydrochloride on a microscope slide. Rub a small amount from the surface of a young, active colony onto the filter paper using a glass rod or plastic loop: a purple-blue colour within 10 s is a positive result
Note: Performing the oxidase test – never use a nichrome wire loop, as this will react with the reagent, giving a false positive result.
To avoid false negatives ensure you use sufficient material during oxidase and catalase testing, otherwise you may obtain a false negative result: a clearly visible ‘clump’ of bacteria should be used.
Catalase Test
This identifies catalase, an enzyme found in obligate aerobes and in most facultative anaerobes, which catalyses the breakdown of hydrogen peroxide into water and oxygen. Transfer a small sample of your unknown bacterium onto a coverslip using a disposable plastic loop or glass rod. Invert onto a drop of hydrogen peroxide on a slide: the appearance of bubbles within 30 s is a positive reaction. This method minimizes the dangers from aerosols formed when gas bubbles burst.

The oxidase and catalase tests effectively allow us to sub-divide bacteria on the basis of their oxygen requirements.




  • Obligate aerobes will be oxidase and catalase positive;

  • Facultative anaerobes will be oxidase negative and catalase positive;

  • Microaerophilic bacteria, aerotolerant anaerobes and strict (obligate) anaerobes will be oxidase and catalase negative – the latter group will grow only under anaerobic conditions.

Once you have reached this stage (colony characteristics, shape, Gram reaction, oxidase and catalase status) it may be possible to make a tentative identification, at least for certain Gram-positive bacteria, at the generic level. To identify Gram-negative bacteria further tests are usually required.



SERIAL DILUTION
Source: HSDU Biology and Biotechnology Microbiological Techniques Intermediate 1-Advanced Higher Folder
Materials


Lab coat

Eye protection

Benchkote if necessary

Disinfectant and paper towels

Discard jar with disinfectant


Bunsen burner

Labels


7 sterile 1 cm3 syringes or pipette tips

7 sterile test tubes or bottles each containing 9 cm3 diluent

Broth culture of yeast or bacteria to be diluted



Instructions


  1. Wear a lab coat and use eye protection.

  2. Label the test tubes or bottles with the appropriate dilution, 10-1 – 10-7.

  3. Make sure that the lid of the culture tube is firmly attached then shake the culture vigorously to separate clumps of cells and to distribute the organisms evenly throughout the liquid.

  4. Remove a sterile syringe or pipette tip from its pack/container. Do not touch the parts which will come in contact with organism. If using a pipette tip, carefully attach to the dispenser.

  5. Using aseptic technique (i.e. flaming the neck of the tube or bottle after removal and before replacement of its lid), remove exactly 1 cm3 of the fluid and transfer to the dilution tube next in the series.

  6. Place syringe or pipette tips into discard jar.

  7. Mix the dilution well.

  8. Repeat steps 4 – 7 until the last dilution tube is reached.

This process dilutes the organisms in the original sample to a countable number.





COUNTING YEAST CELLS USING A HAEMOCYTOMETER
Source: HSDU Biology and Biotechnology Microbiological Techniques Intermediate 1-Advanced Higher Folder
Materials


Haemocytometer and coverslip

Tissue


Alcohol

Lens tissue

Water


Suspension of yeast cells

Capillary tube

Petri dish with moist tissue

Microscope




Instructions – setting up the slide


  1. Clean the haemocytometer with alcohol,

then wipe with lens tissue.
2 Using a damp tissue, moisten the slide as shown in diagram.




  1. Push the special coverslip on to the slide as shown in diagram, pressing down on the outside edges of the coverslip at the same time until you can see Newton’s rings (see diagram). If you push the centre of the coverslip, it is likely to break.




Instructions – loading the haemocytometer


  1. Shake the cell suspension gently.




  1. Insert the end of the capillary tube into the suspension. The liquid will rise into the tube.




  1. Run the end of the capillary tube along the edge of the coverslip between the arms of the ‘H’. The suspension should fill the area between the coverslip and the top half of the ‘H’ (shaded in diagram below). If the suspension flows into the troughs (the lines of the ‘H’), clean the slide and start again.






  1. Turn the slide through 180 and repeat for the opposite edge of the coverslip.





  1. Place the haemocytometer on a damp tissue in a Petri dish for at least two minutes to equilibrate.


Instructions – counting the cells
The haemocytometer has two grids situated as shown in the diagram:


1 Place the haemocytometer on the microscope stage
2 Using the instructions for use of the microscope, examine the

haemocytometer using the 4x objective lens. You should be

able to view one whole grid as shown in the diagram.




  1. Increase the magnification to the 10x objective lens.

You should be able to see the 25 central squares, each of

which is divided into 16 smaller squares (see diagram at

instruction 4).



  1. Increase the magnification to the 40x objective lens.

You will see one of the 25 central square made up of 16

small squares.






  1. Count the cells in each of the four corner squares and the central square; (see shaded squares in instruction 3). Note that you will count five groups of 16. Include in the count those cells touching the top or right side of the square; do not count those cells touching the bottom or left side. This

takes account of cells which are half in and half out the square.




Instructions – calculation


Length of side of grid = 1 mm
Area of grid = 1 mm2
Depth between coverslip and slide = 0.1 mm
Volume under squared area (25 = 1 mm2 x 0.1 mm = 0.1mm3

squares) of grid


Volume under 5 squares (the = 0.1/5 mm3 = 0.02mm3

number counted)

You have therefore counted the number of cells in 0.02 mm3 and can use the following calculation to estimate the cell concentration of your original suspension.
Number of cells (total in 5 squares) in 0.02 mm3 = n
Number of cells in 1 mm3 = n x 50
Number of cells in 1 cm3 = (n x 50) x 1000
If the cell suspension counted has been diluted, then the above result must be multiplied by the appropriate dilution factor to give the concentration of the original culture.
Viable count: pour plate method
Source: HSDU Biology and Biotechnology Microbiological Techniques Intermediate 1-Advanced Higher Folder
Materials

Lab coat

Eye protection

Benchkote if necessary

Disinfectant and paper towels

Discard jar with disinfectant

Bunsen burner



Dilution series of organism

Sterile pipettes or syringes (0.1 cm3)

Sterile Petri dishes

20 cm3 volumes of sterile molten nutrient agar at 45C

Glass spreader

Alcohol




Instructions

  1. Wear a lab coat and use eye protection.




  1. Label the underside of the plates with initials, date, sample and dilution. For greatest reliability/precision, each dilution should be plated in triplicate and the average of the three counts used.




  1. Remove a sterile 0.1 cm3 syringe or pipette tip from its pack/container. Do not touch the parts which will come in contact with organism. If using a pipette tip, carefully attach to the dispenser.




  1. Start with the highest dilution (i.e. 10-7).




  1. Using aseptic technique (i.e. flaming the neck of the tube or bottle after removal and before replacement of its lid), remove exactly 0.1 cm3 of the sample and transfer to the base of a sterile Petri dish.




  1. Using aseptic technique, pour 20 cm3 sterile nutrient agar over the sample and mix gently.




  1. Place syringe or pipette tip into discard jar.




  1. Repeat steps 5 – 7 for dilutions 10-6, 10-5 and 10-4.




  1. When the plates are solidified and dry, incubate upside down at the appropriate temperature for the appropriate time.




  1. After incubation, select plates for counting that contain 30 – 300 colonies (samples which contain <30 colonies/0.1 cm3 diluent are subject to large fluctuations in numbers or sampling errors, plates which contain >300 colonies are likely to have overlapping colonies).




  1. Count accurately and record the number of colonies on each plate.




  1. Calculate the concentration of viable cells or colony forming units (cfu) in the original suspension.

Viable count : spread plate method
Source: HSDU Biology and Biotechnology Microbiological Techniques Intermediate 1-Advanced Higher Folder
Materials

Lab coat

Eye protection

Benchkote if necessary

Disinfectant and paper towels

Discard jar with disinfectant

Bunsen burner



Labels

Dilution scrics of organism

Sterile pipettes or syringes (0.1 cm3)

Sterile nutrient agar plates

Glass spreader

Alcohol



Instructions

  1. Wear a lab coat and use eye protection.




  1. Label the underside of the plates with initials, date, sample and dilution. For greatest reliability/precision, each dilution should be plated in triplicate and the average of the three counts used.




  1. Remove a sterile 0.1 cm3 syringe or pipette tip from its pack/container. Do not touch the parts which will come in contact with organism. If using a pipette, carefully attach to the dispenser.




  1. Start with the highest dilution (i.e. 10-7).




  1. Using aseptic technique (i.e. flaming the neck of the tube or bottle after removal and before replacement of its lid), remove exactly 0.1 cm3 of the sample and transfer to the surface of an appropriately labelled sterile nutrient agar plate.

Note: Keep remainder of all samples


  1. Using aseptic technique, spread the sample evenly across the plate with the glass spreader.




  1. Place syringe or pipette tip into discard jar.




  1. Repeat steps 5 – 7 for dilutions 10-6, 10-5 and 10-4.




  1. When the plates are dry, incubate upside down at the appropriate temperature for the appropriate time.




  1. After incubation, select plates for counting that contain 30 – 300 colonies (samples which contain <30 colonies/0.1 cm3 diluent are subject to large fluctuations in numbers or sampling errors, plates which contain >300 colonies are likely to have overlapping colonies).




  1. Count accurately and record the number of colonies on each plate.




  1. Calculate the concentration of viable cells or colony forming units (cfu) in the original suspension.

YEAST GROWTH CURVE
Source: Adapted from SAPS ‘Growth Curve: Determination of Doubling Time’

http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/double.htm
Technical guides are also available from the same source.
Read through the Student Activity Guide and consider the following questions.
Analysis of Activity
What is the aim of the activity?
What measurements are you going to make?
How will you record these measurements?
How will you determine the information you require to make the final calculation?
What constant will you calculate?
Getting organised for experimental work
In your group decide how the activity will be managed by allocating tasks to each member. It is very important that samples are removed at least three times per day: ideally early-morning, lunchtime and late afternoon. This will happen over 3 days. [A rota for removing samples may help].
Recording of data
Prepare tables and a graph to record your group results.
You should use a ruler, correct headings and appropriate units.
Evaluation
How effective were the methods which you used?
What were the limitations of the equipment?
What were the sources of error?
What possible improvements could be made to the experiment?
What ideas do you have for further work?
What is the economic importance of the process which you are studying and the calculations which you will make?
STUDENT ACTIVITY GUIDE
Introduction
Stages of Growth
Growth is the process during which living organisms synthesise new chemical components for the cell and as a result they usually increase in size. In unicellular organisms, such as bacteria and yeast, growth leads to cell division and consequently an increase in population size. The growth of a population of single-celled micro-organisms grown in a closed environment typically shows four stages: lag phase; exponential phase; stationary phase; death phase.


The lengths and characteristics of these phases will depend upon factors such as the nature of the growth medium and temperature of incubation.
In industry, it is important to understand the factors which affect the growth rate of a given micro-organism in order to generate maximum product by the most economic means. For example, if the desired product is a secondary metabolite such as an antibiotic which is produced when the organism has stopped growing, the manufacturer will want to provide optimum conditions for the culture to reach maximum numbers in stationary phase in the shortest time possible.
In some cases, the product is the organism itself e.g. the production of yeast biomass to be used as starter cultures for brewing or baking, or as the starting point for autolysis which produces a huge variety of food flavourings.
Growth of a population can be measured using the following methods:
Cell counts: total numbers of cells are counted directly using a microscope and a special slide called a haemocytometer.
Dilution plating: the culture is serially diluted and a known volume of each dilution plated out and incubated. Resulting colonies are counted giving a measure of viable numbers of cells in the original population.
Turbidometric methods: Cell density is measured using a colorimeter. This is a photometric method which measures the light scattered by the cells in suspension. Increase in cell density is an extremely accurate method of measuring cell growth rates.

In this practical, you will produce a growth curve of absorbance against time for a culture inoculated with a known dry mass of Saccharomyces cerevisiae (bakers’ yeast) then grown over several days. From this you will be able to calculate generation time and a growth rate constant.




Equipment and materials
Materials required
Materials required by each student/group:
1 x 5 cm3 sterile yeast glucose broth as blank

99 cm3 sterile yeast glucose broth in flask

dried yeast (not fast acting)

weighing boat

spatula

10 cm3 sterile water (if balance is accurate to 0.01g)



100 cm3 sterile water

sterile 1 cm3 pipette

discard jar containing 1% Virkon

semi-log graph paper


Materials to be shared:
water bath or incubator at 30C

balance (accurate to 0.001 g (preferably) or 0.01 g)

colorimeter (440 nm)
Instructions


  1. Start this experiment late afternoon at the start of a week.

  2. Draw a table showing date, time, hours of incubation and absorbance.

  3. Using aseptic technique, add 0.025 g dried yeast to 100 cm3 sterile distilled water at 30C. Shake gently to ensure that the cells are evenly distributed and suspended.

  4. Using aseptic technique, dilute 100 times by adding 1 cm3 to 99 cm3 sterile broth in a flask. This should give a starting concentration of 0.0025 g/l for your growth curve.

  5. Using sterile medium as the reference, calibrate the colorimeter (i.e. set it to zero).

Note: keep this reference medium (the blank) in the refrigerator throughout experiment.

  1. Shake the flask containing the yeast culture gently to distribute the cells evenly. Using aseptic technique, withdraw a 5 cm3 sample.

  2. Measure the absorbance of the sample you have just withdrawn. Record date, time and absorbance in the table.

  3. Incubate at 30C.

  4. Repeat instructions 5 – 8 three times per day for the next three days (early morning, lunch-time and late afternoon if possible). If it is not possible to measure the absorbance at the time of taking the sample, place it in a sterile container, label with initials, date and time and refrigerate until convenient to do so, preferably within 24 hours. Make sure that the yeast is fully suspended before reading the absorbance.

  5. Plot absorbance vs time.

  6. Identify on your graph the lag, log and exponential phases of the growth curve.

  7. From the exponential (log) phase of growth curve, work out the time in hours taken for the absorbance and hence the population size to double.

  8. Calculate growth rate constant.


SUPPLEMENTARY STUDENT INFORMATION
Calculation of growth rate constant
Growth rate constant, k, is a measure of the number of generations (the number of doublings) that occur per unit of time in an exponentially growing culture.
n 2

k = g
where n 2 is the natural log of 2 (determine this from your calculator) and g is the time in hours taken for the population to double during the exponential phase of growth.


DETERMINING DRY WEIGHT USING TURBIDITY: VERSION 1
Source: Adapted from SAPS ‘Growth Curve: Determination of Dry Weight from Standard Curve’.

http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/curve.htm
Technical guides are also available from the same source.

PREPARING FOR THE ACTIVITY
Read Through the Student Activity Guide and consider the following questions.
Analysis of activity
What is the aim of the activity?
What measurements are you going to make?
How will you record these measurements?
How will you determine concentration of yeast cells in a growing culture?
Getting organised for experimental work
In your group decide how the activity will be managed by allocating tasks to each member.
Recording of data
Prepare tables and graph paper to record your group results.
You should use a ruler, correct headings and appropriate units.
Evaluation
How effective were the methods which you used?
What is the significance of using dry mass as the measurement of concentration?
What were the limitations of the equipment?
What were the sources of error?
What possible improvements could be made to the experiment?
What ideas do you have for further work?
What is the economic importance of the process which you are studying and the calculations which you will make?
STUDENT ACTIVITY GUIDE
Introduction
In industry it is often important to determine the actual concentration of cells in a growing culture. This may involve counting total numbers of cells, numbers of viable cells or cell concentration in terms of dry mass.
In this experiment you will use commercially available dried yeast to produce a standard curve of absorbance at 440 nm against concentration (dry mass in grams/litre). You will then use your standard curve to determine the concentration of yeast in each of a series of diluted samples. [These can be from a previous experiment.
Equipment and materials
Materials required
Materials required by each student/group:
100 cm3 sterile yeast glucose broth in flask or bottle

11 large test tubes (or universals)

test tube rack

dried yeast (not fast acting)

weighing boat

spatula


4 x 10 cm3 syringes (or pipettors with tips)

1 cm3 pipette

cuvettes

discard jar containing 1% Virkon

graph paper

marker pen


Materials to be shared:
Balance (accurate to 0.01 g)

Colorimeter (440 nm)

Crushed ice (optional)
Instructions
In order to make up the appropriate concentrations of yeast you will weight out 0.2 g dried yeast and add it to 20 cm3 sterile broth to give a concentration of dry mass of 10 grams/litre. You will then use this suspension (the standard dilution) to make further dilutions giving you yeast concentrations in g/l of 5, 4, 3, 2.5, 2, 1.5, 1.0, 0.5, 0.25, 0.05. Look at the table carefully and make sure you understand it before you start.
You may need crushed ice to keep your tubes cool while you carry out the experiment.


  1. Withdraw 20 cm3 broth from a flask of sterile yeast glucose broth and add to a test tube (or universal).




  1. Add 0.2 g dried yeast to the broth in the test tube. This will give you a concentration of 10 g/litre. Shake gently occasionally until the cells are fully resuspended. This is your standard dilution. Use it to make further dilutions.




  1. Draw a table with test tube number, yeast concentration in g/l and absorbance at 440 nm.




  1. Label clean test tubes 1 – 10. Add the volume of sterile broth shown in Row A in the table below to each tube. Pay close attention as you are required to add different volumes to the tubes.




  1. From your standard dilution add the volume shown in Row B to tubes 1 – 5. Shake the standard dilution tube gently each time before removing the sample. Make sure that you understand how the yeast concentration in g/l is worked out.




  1. Add the volumes from the numbered tubes shown in Row B to tubes 6 – 10.



Note: Pay careful attention to tube numbers and volumes.

Remember to suspend cells by shaking gently before taking a sample and use a fresh syringe for each tube.

Dispose of used syringes and pipettes in the discard jar.

Make sure that you understand how the yeast concentration in g/l is worked out.








Tube no

1

2

3

4

5

6

7

8

9

10

A

Volume sterile broth (cm3)

5

6

7

7.5

8

5

5

5

5

9

B

Volume yeast suspension (cm3)

5

4

3

2.5

2

5 cm3 from tube 3

5 cm3 from tube 5

5 cm3 from tube 7

5 cm3 from tube 8

1 cm3 from tube 8




yeast conc. (g/l)

5

4

3

2.5

2

1.5

1

0.5

0.25

0.05



  1. Using sterile broth as the reference, calibrate the colorimeter (i.e. set it to zero) at 440 nm.

  2. Starting with tube 10 shake the test tube to distribute the cells evenly and use a pipette to transfer about 3 cm3 into a cuvette.




  1. Measure the absorbance at 440 nm of the sample you have just withdrawn. Record it in the table.




  1. Return the sample to its original tube and repeat steps 7 and 8, using the same pipette and cuvette, to obtain readings for tubes 9 – 1.




  1. Draw a graph of absorbance at 440 nm vs. yeast concentration (grams/litre). This is the standard curve which you will use to determine yeast concentration from absorbances of serially diluted samples in earlier experiments e.g. viable count experiment.


Using the standard curve to determine concentration in grams/litre from known absorbance



  1. Use the absorbances measured in the previous experiment.




  1. Draw a table showing time of sample, absorbance and concentration.




  1. Fill in the time and absorbance rows.




  1. Carry out the following for each absorbance:




    • Mark the value on the absorbance (vertical) axis of your standard curve

    • Draw a horizontal line till it meets the standard curve (a). Mark the point

    • Draw a vertical line from there to meet the yeast concentration axis (b)

    • Read the value and complete the concentration row on the table




      1. Determine the dry weight of yeast (in g/litre) present in the original sample.




      1. Write a report on your practical placing particular emphasis on evaluation of the equipment and methods used with respect to the resulting accuracy and reliability.


DETERMINING DRY WEIGHT USING TURBIDITY: VERSION 2
Source: James Watt College


  • Take stock culture of micro-organism & serial dilute to 10-7.




e.g.



1.0 ml


stock

1 ml


9 ml diluent
= 10-1

1 ml


9 ml diluent
= 10-2

1 ml


9 ml diluent
= 10-3





10-7


This will give 8 samples in total (including original stock).




  • Measure the turbidity of each of these samples using a spectrophotometer set to 600 nm.




  • Plot graph of absorbance against dilution factor: is there a direct relationship between dilution and turbidity?




  • Given the following piece of reference information, convert your absorbance readings to quantities of actual micro-organism (a sample calculation is provided to help).


Absorbance at 600nm value of 0.5 is equivalent to a total cell mass of 1 mg dry cells per ml.
Example of calculation
A600 = 0.298 from machine for 10-1 dilution
0.298 x 1.0 mg/ml = 0.596 mg/ml for 10-1 dilution

0.500
Correcting for dilution factor, this becomes


0.596 x 10  5.96 mg/ml.
CARBOHYDRATE FERMENTATION
Source: James Watt College
Carbohydrate utilization tests
Some bacteria can use a particular carbohydrate as a carbon and energy source. Acid end-products can be identified using a pH indicator dye while CO2 is detected in liquid culture using a Durham tube (inverted small test tube). Aerobic breakdown is termed oxidation while anaerobic breakdown is known as fermentation. Identification tables usually incorporate tests for several different carbohydrates.

Durham tube in carbohydrate utilization broth. Air within the Durham tube is replaced by broth during the autoclaving procedure.



  • Collect the following materials:

    • 24 hour broth cultures of E. coli, Staphylococcus epidermis albus, Erwinia carotovora and Micrococcus luteus.

    • Phenol red dextrose, lactose and sucrose peptone broths in

test tubes also containing Durham tubes.

  • Label one tube of each type of medium and inoculate with E. coli.

Label one tube of each type of medium and inoculate with Staphylococcus epidermis albus.

Label one tube of each type of medium and inoculate with Erwinia carotovora.

Label one tube of each type of medium and inoculate with Micrococcus luteus.

Label one tube of each type of medium and do not incubate i.e. control tubes.



  • During incubation do not tip the fermentation tube as this may accidentally force a bubble of air into the Durham tube to give a false positive result.

  • Mix tubes by rolling them back and forth between the palms of the hands.

  • Incubate tubes at 30C for 24 – 48 hours.

  • Examine all cultures for evidence of acid (pH change), and/or acid and gas production for each sugar.

  • Determine type of fermentation occurring.


Digestion of Cellulose
Source: “Microbial Friends & Allies” BBSRC publication (originally developed by NCBE http://www.ncbe.reading.ac.uk/ )


Cellumonas bacteria secrete cellulase enzyme so could potentially be used to utilise cellulose waste (eg paper) as feedstock for their fermentation process, converting low cost start materials into products of greater value.



  1. Use sterile forceps to place a disc of sterile filter paper on the surface of a nutrient agar plate. Use the tips of the forceps to smooth the paper onto the agar, to ensure that there is a good contact.




  1. Take the lid off the Cellulomonas culture bottle. Keep the bottle top in your hand – do not place it on the bench. Briefly pass the neck of the bottle through a Bunsen burner flame.




  1. Dip a sterile cotton wool bud into the culture. Do not allow the culture to drip on the bench. Flame the neck of the bottle again and replace the lid.




  1. Quickly ‘paint’ a message or picture on the filter paper with the culture (see diagram below). Dispose of the cotton wool bud into a beaker of disinfectant.




  1. Seal the Petri dish diagonally with a small amount of tape. Label the base of the Petri dish with your initials, the date, and the name ‘Cellulomonas’.




  1. Incubate the Petri dish at 25 - 30C in an inverted position: the filter paper should stick to the surface of the nutrient agar.


2 or 3 weeks later
After incubation, the sealed plates may be examined for digestion of the filter paper under a binocular microscope or using a hand lens. You should be able to read the message/see the picture as the paper should have been digested where the Cellulomonas bacteria were painted.


Testing viability of yeast at different stages of the autolysis process
Source: SAPS

http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/yeast.htm
Teacher and technical guides can also be found at this site.

Preparing for the Activity
Read through the Student Activity Guide and consider the following questions.
Analysis of activity
What is the aim of the activity?
What is being varied in the activity?
What measurements are you going to make?
Getting organised for experimental work
What precautions you must be taken to prevent contamination of the agar plates?
Can you successfully examine material under a microscope at x400 magnification?
In your group decide how the activity will be managed by allocating tasks to each member. It is important that you play an active part in setting up the experiment and in collecting results.
Recording of data
Prepare summary tables to record your group results.
You should use a ruler, correct headings and appropriate units.
Evaluation
How effective were the tests which you used?
What were the limitations of the equipment?
Were there any possible sources of error?
What possible improvements could be made to the experiment?
What is the biological importance of the process which you are investigating?


Student Activity Guide
Introduction
Yeasts are versatile micro-organisms which have been used for centuries by man to produce bread and alcoholic drinks.


In more recent years they have been used to produce flavourings for the food industry. The yeast is grown in huge fermenters to produce biomass – Upstream Processing and then it is treated in different ways to produce different flavourings. These flavourings are found in most of the savoury snack foods which we eat – crisps, soups, snacks etc.


The yeast goes through a series of different treatments to develop the huge variety of different end products. All of these treatments involve the process of autolysis and are examples of what is known as Downstream Processing. The yeast products may be powders, granules or pastes and they are then incorporated into processed foods to provide natural flavourings.
This process of AUTOLYSIS (auto-self; lysis-splitting) involves killing the yeast and encouraging the breakdown of the cells by enzymes. These may be the cells own endogenous enzymes or enzymes may be added. It is these products of enzyme degradation which produce the specific flavour molecules. Autolysis usually begins with the addition of salt to the cells, causing water to leave the cells by osmosis and beginning the process of cell breakdown. The cells are then heated encouraging further breakdown of the cells.
In this practical you are going to carry out the process of autolysis and try to find at what point in the process the cells actually die. You will salt and heat yeast and then test the viability by plating out the treated yeast to see if it will grow. You will also test the autolysed product to see if the dehydrogenase enzymes are active and to see if the cells take up methylene blue dye.
Yeast Autolysis
Equipment and materials
Materials required by each student/group:
10 g fresh yeast
salt
5x 200 cm3 beakers
stirring rod
Materials to be shared:
Ovens at different temperatures
Instructions


  1. Autolyse the yeast in 4 different ways. You could alter the temperature at which the yeast is autolysed or the amount of salt which is added to the yeast. One possible regime is suggested below:




    • 2 g yeast 1 g salt at room temp overnight

    • 2 g yeast 1 g salt at 40C overnight

    • 2 g yeast 1 g salt at 60C overnight

    • 2 g yeast 1 g salt at 80C overnight




      1. Collect dried yeast samples and rehydrate by slowly adding water to the dried sample, stirring constantly. Continue to add water to the samples to make them up to 100 cm3.




      1. Autolyse a fresh sample of yeast. Mix 2 g yeast with 1 g salt and add water to make this sample up to 100 cm3.

You can now test the viability of these yeast samples by 3 different methods.


Method 1: Plating Out Autolysed Yeast Samples
Streaking an agar plate
Equipment and materials
Materials required by each student/group:
Disinfectant and cloth
3 Yeast agar plates
Metal inoculating loop
Rehydrated yeast samples
Marker pen
Sellotape
Materials to be shared
Incubator at 30C
Instructions


  1. Make sure that you are working in an area which has been swabbed with disinfectant.




  1. Turn your petri dish upside down and use a pen to mark the base, as shown, and label it with the yeast samples to be used, the date and your initial.




1 2


  1. Open your petri dish and using a sterile inoculating loop, place your sample of yeast onto one side of the petri dish. Make a shape like this.














  1. Repeat with other yeast samples and plates.




  1. Seal your plates with tabs of sellotape and place it in an incubator at 30C.




  1. Record any growth of yeast over the next 5 days.

Method 2: Comparing the activity of the dehydrogenase enzymes present in autolysed yeast samples.
Background Information
During a metabolic pathway such as aerobic respiration glucose is gradually broken down and energy is released. Hydrogen is released from the glucose in a process knows as oxidation. This hydrogen binds to a co-enzyme and each reaction is catalysed by an enzyme known as a dehydrogenase.
Although it would not be possible to detect this reaction in a test-tube some chemicals such as resazurin dye change colour when they gain hydrogen.
It changes colour in the following ways:

BLUE LILAC MAUVE PINK COLOURLESS


Unreduced Partially Reduced

reduced

You can use this reaction to compare the activity of the dehydrogenase enzymes present in each of your autolysed yeast samples.


The time it takes for the dye to change colour will indicate the activity – the faster the colour change takes place the greater the activity of the dehydrogenase enzymes.
Activity of enzymes such as dehydrogenases would indicate that the yeast is likely to be viable.
Equipment and materials
Materials required by each student/group:
Rehydrated yeast samples
1 test-tube rack with 5 test tubes
5 labels
1 stop clock
1 pair safety spectacles
1 syringe/measuring cylinder


  1. cm3 Resazurin dye

20 cm3 5% glucose solution


colour chart
Materials to be shared:
Waterbath at 35C
Instructions


  1. Collect the materials indicated above.




  1. Label 5 test-tubes and add 3 cm3 of resazurin dye to each tube.




  1. Add 3 cm3 of the appropiate yeast suspension to the labelled tubes.




  1. Shake each tube and place in a water bath at 35C.




  1. Using the colour chart, record the colour of each tube every 2 mins for 20 mins.

If you do not get a reaction in any of the tubes after 10 minutes add 3 cm3 of 5% glucose solution to each test-tube and shake.




  1. Record the results in a table with suitable headings.

Method 3: Testing the yeast samples with methylene blue
Methylene blue dye will diffuse into the yeast cells. If the cells are living they will pump the blue dye out but if they are dead they will remain blue.
Equipment and materials
Materials required by each student/group:
rehydrated yeast samples
microscope
microscope slides and coverslips
dropper
0.1% methylene blue solution
50 cm3 beaker
stirring rod
distilled water
Instructions


  1. Place a drop of one of the diluted yeasts onto the microscope slide and add a drop of methylene blue dye and wait 5 minutes.




  1. Place the slide onto the stage of your microscope and focus.




  1. Count all the blue cells and clear cells in your field of view.




  1. Repeat steps 1-3 for each of the yeast samples.

You may find that there are too many cells to count, if this is the case then you can dilute your samples and start again.




  1. Record your results in a suitable table.




  1. Calculate the percentage of viable ie unstained cells.


TISSUE CULTURE: EFFECT OF M&S SALTS ON EXPLANT GROWTH
Source: LTScotland National Curriculum Support Materials

Intermediate 2 Biotechnology, Unit 3 Biotechnological Processes, Student Materials

Background information
This tissue culture technique uses (cotyledon) explants taken from the top of a seedling. Micro-organisms grow faster than plant cells. It is important to keep seeds, medium and equipment sterile. Follow aseptic technique throughout.
Equipment and materials required by each student/group:
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