PRACTICAL FERMENTATION a guide for schools and colleges
Technical Guide
John Schollar and Benedikte Watmore Consultant Editor John Grainger National Centre for Biotechnology Education
Project sponsored by The Society for General Microbiology
Technical Guide: Practical Fermentation
Contents: Investigation 1 Sauerkraut – a natural traditional fermentation 3 Investigation 2 Two or three sugar substrate 4 Investigation 3 Balancing the loss of carbon dioxide 5 Investigation 4 Yeast cells and enzyme – together they can do it 6 Investigation 5 A sugary choice 7 Investigation 6 How do they like it? – alcohol levels and pH 8 Investigation 7 Deep purple! – a dark secret 9 Investigation 8 Nothing’s for free – you gain some, you lose some! 10 Investigation 9 Ester production – a fragrant or smelly fermentation? 10 Investigation 9 Demonstration of downstream processing 11 Investigation 10 Dextran production – a sticky fermentation 11 Investigation 11 Some sticky investigations – by gum! 12 Investigation 12 Probably the best yeast in the world 14 Investigation 13 Probably the best pigment in the world 14 Investigation 14 Vibrio natriegens – for a speedy growth curve 15 Technical Information 1 The bubble logger 16 Technical Information 2 Principles of a bioreactor 16 Technical Information 3 Basic microbiology – preparation of a streak plate 17 Technical Information 4 Basic microbiology – preparation of a dilution series 17 Good Laboratory Practice – GLP for all!
18
Information: This technical guide is intended for use by teachers and technicians in schools and colleges. It contains information to assist in the preparation of materials and the performance of the practical activities included in the student guide.
Acknowledgements: The authors are especially grateful to the following colleagues at the NCBE and SGM for their help and support in the production of these materials; Kate Porter (NCBE), Caroline Shearer (NCBE), Dean Madden (NCBE) Janet Hurst (SGM) and Jane Westwell (SGM). The authors are also grateful to the biology staff and students of Chislehurst and Sidcup Grammar School for trialing and testing of specific investigations, and participants of London Examinations in-service training courses for their constructive comments during the development of the materials. Finally the authors would like to thank Robin Sutton (Field Studies Council) for his help and advice in the construction of the suggestions for statistical analysis. JS/BW 1999
Copyright: Practical fermentation is copyright. The authors assert their moral rights to be identified as copyright holders under Section 77 of the Designs patents and Copyright Act, UK (1988). Educational Use. Electronic or paper copies of the resource, or individual pages from it, may be made for classroom and bona fide educational uses, provided that the copies are distributed free-ofcharge or at the cost of reproduction and that the authors and the NCBE/SGM are credited and identified as the copyright holders.
Technical Guide: Practical Fermentation
Investigation One Sauerkraut - a natural traditional fermentation
Equipment and materials
Reference and statistics
300 g finely shredded cabbage 300 cm3 3% w/v sodium chloride solution 1 dm3 glass beaker pH electrode and meter Temperature electrode (optional) 15 cm3 bent glass pipette with 3 cm rubber tubing Restriction clip (Hoffman clip) Large plastic bag (approx. 34 cm x 26 cm) Scissors Adhesive tape Elastic bands Small metal weights 3 x 99 cm3 sterile water for each population count Rogosa agar and GYLA plates (3 of each per count) (GYLA = Glucose Yeast Lemco Agar) Sterile 1 cm3, 2 cm3, 5 cm3 and 10 cm3 syringes Sterile spreader, and a capped beaker of IMS for flaming spreader Burette containing 0.1 M sodium hydroxide solution Flasks containing 10 cm3 deionised water Phenolphthalein indicator solution and dropping pipette
Points for consideration 1
2
3
4
5 6 7
The specially modified glass pipette is part of the NCBE bioreactor but can be ordered separately from the NCBE or made by a glass blower. Exclude as much air as possible from the fermentation to ensure anaerobic conditions predominate. If the fermentation becomes too aerobic then instead of a sweet pleasant smell a strong offensive odour will be noticed! The use of different media (glucose yeast Lemco agar and Rogosa agar) selects for different types of microorganisms. The GYLA predominately selects for aerobic microbes from the start of the fermentation. The Rogosa agar is fairly specific for lactic acid bacteria and colonies should become apparent by the second day. The viable count increases over the first seven days then levels off for the next two weeks. Normally a two week fermentation is sufficient. It is very likely that the third dilution plate will give sound viable counts for data interpretation (from days 2 - 7). Gram staining of the sample will reveal both Gram-negative and Gram-positive organisms. If the fermentation progresses as expected then the amount of acid present detected by the titration will relate approximately to the amount of lactic acid produced. If undesirable organisms are present or predominate early in the fermentation other acids such as acetic acid may be formed.
N.B. It is important to prepare the cabbage in as clean an environment as possible using clean utensils etc., however, the students should realise that if no naturally occurring bacteria were present the fermentation would not take place!
Amount of acid produced can be calculated by the formula: titre, cm3 x molarity of NaOH x mol. mass of lactic acid % lactic acid = cm3 sample x 10 3
Take 5 cm sample of liquid from the sauerkraut Titrate against 0.1 M NaOH using phenolphthalein indicator initial volume final volume titre
Blank (water) 13.05 cm3 13.10 cm3 ------------00.05 cm3 =======
Sample *1 14.00 cm3 26.60 cm3 ------------12.60 cm3 =======
Sample *2 08.00 cm3 20.65 cm3 -----------12.65 cm3 =======
Reference: This investigation was suggested in;
Sourcebook of Experiments for the Teaching of Microbiology Edited by S. B. Primrose and A. C. Wardlaw Academic Press, 1982, ISBN 0-12-565680-7 Out of print. Photocopies of the relevant chapter 86 can be obtained from Society for General Microbiology upon request. It has been adapted for school and college use. Statistics: To investigate the idea that older cabbages have more sugar and therefore produce better sauerkraut, compare results from at least six old and six young cabbages to give unmatched pairs and use the Mann-Whitney U Test. The Mann-Whitney U Test can also be applied to the other suggested extension activity.
Investigation data (information from fermentation trials) Sauerkraut Production (Beaker A) Date Time 06/December 15.20 16.20 17.00 07/December 09.30 08/December 09.30 16.30 09/December 09.30 16.30 10/December 18.30 12/December 08.48 12.30 16.30 13/December 09.30 16.45 14/December 09.25 16.30 16/December 09.40 16.30 19/December 09.20 Sauerkraut Production (Beaker B) Date Time 06/December 15.20 17.00 07/December 09.30 08/December 09.30 16.30 09/December 09.30 16.30 10/December 18.30 12/December 08.48 12.30 16.30 13/December 09.30 16.45 14/December 09.25 16.30 16/December 09.40 16.30 19/December 09.20
Temp0C 18.8 20.1 20.6 21.4 21.6 21.6 21.8 22.4 21.2 20.9 21.2 21.6 21.9 22.7 22.0 22.6 21.4 22.6 18.9
pH 6.80 6.40 6.25 5.90 5.80 5.60 5.50 5.30 5.20 5.16 5.20 5.13 5.14 5.14 5.10 5.10 4.82 4.77 4.45
Temp0C 19.7 20.3 21.3 21.4 21.3 21.7 22.2 21.2 20.7 21.1 21.5 21.8 22.5 21.8 22.3 21.0 22.1 18.9
pH 5.90 5.70 5.40 5.30 5.40 5.40 5.36 5.35 4.90 4.73 4.68 4.34 4.29 4.27 4.25 4.17 4.16 4.15
Sauerkraut Production (Beaker C) Date Time 18/January 16.10 16.20 17.10 20/January 09.46 23/January 09.25 16.55 24/January 09.15 16.55 25/January 09.30 16.30 31/January 09.30 01/February 09.30
Temp0C 17.2 17.5 19.1 20.0 17.8 20.3 19.6 20.5 20.1 21.0 20.7 20.8
pH 6.83 6.47 6.19 5.25 *1 3.95 3.90 3.77 3.68 3.62 3.62 3.58 *2 3.60
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
3
Technical Guide: Practical Fermentation
Investigation Two Two or three sugar substrate
Equipment and materials Culture of S. cerevisiae (e.g. Allinson’s dried active baking yeast) Culture of S. carlsbergensis
Investigation data (information from fermentation trials) Number of bubbles logged
2 x malt agar plate 40 cm3 GYEP broth (2% glucose, 1% yeast extract, 1% peptone) 400 cm3 RYEP broth (5% raffinose, 1% yeast extract, 1% peptone) 400 cm3 SYEP broth (5% sucrose, 1% yeast extract, 1% peptone) 4 x silicone rubber bung with a single hole 4 x glass fermentation lock Non-absorbent cotton wool and greaseproof paper or aluminium foil Sterile water 5 x Universal bottle 4 x sterile Pasteur pipette Inoculating loop 4 x wide-necked 250 cm3 flask Shaker (optional) 4 x magnetic stirrer and follower (optional) Universal indicator solution (full range) 4 x NCBE bubble logger 4 x sterile 10 cm3 syringe
Sucrose broth 29/March Time 12.34 13.45 14.00 14.30 15.00 15.30 16.00 16.30 16.45 30/March Time 09.00 13.00 16.15 31/March Time 09.00 13.00 16.30 01/April Time 21.05 22.15 02/April Time 08.30 03/April Time 09.20 12.00
Points for consideration 1
The media can be prepared and sterilised in advance and stored at 4°C until required. The neck and bung of each flask should be covered during autoclaving with greaseproof paper or aluminium foil. The cover should be removed just prior to use.
2
A total autoclave time of 15 minutes at 121°C should ensure sterilisation of the media without causing caramelisation of the sugars.
3
It is suggested that the small broth inoculum cultures are duplicated to allow for the selection of the best growing culture. This is to ensure a good inoculum for the start of the fermentation. The culture should look turbid when gently shaken with a good growth of yeast swirling around.
4 5
It is suggested that the number of bubbles produced is recorded at hourly intervals to give a suitable amount of data.
X X
X
Number of bubbles
Reference: Further information relating to this topic can be found in
Microbial Biotechnology - Fundamentals of Applied Microbiology Alexander N. Glazer, Hiroshi Nikaido, W. H. Freeman and Company, 1995, ISBN 0-7167-2608-4 Chapter 11 Ethanol
An alternative investigation with two different yeasts and six different sugars can also be considered. The final idea of investigating effect of temperature should use both yeasts (S. carlsbergensis, S. cerevisiae) at 6 - 8°C and 15 20°C with a sugar that both can utilise, e.g. glucose or sucrose. 4
0 0 0 0 0 1 6 16 33
0 0 0 24 65 118 190 247 254
0 0 0 10 34 74 130 185 221
9317 10968 11144
10224 11194 11240
3567 6172 7279
2746 2763 2778
11257 11257 11259
11381 11386 11397
9050 9067 9095
2840 2840 2849
11282 11282
11423 11423
9128 9132
3389 3421
11286
11423
9154
3584
11286 11286
11423 11423
9180 9180
3707 3713
X X X X X X
XX XX
X X
X X X X
Sucrose broth S. cerevisiae Sucrose broth S. carlsbergensis
X
XX
Raffinose broth S. carlsbergensis
X
XX
Raffinose broth S. cerevisiae
X X X
10,000
Reference and statistics
The extension activities have been designed to help students think about trying to apply statistical tests. If students use at least six different species of yeast with two different sugars giving matched pairs they can use a Wilcoxon Matched Pairs Test.
0 6 23 79 137 206 295 366 414
Graph: Bubble number against time in days
The yeast S. carlsbergensis can use both sucrose and raffinose for anaerobic fermentation while the yeast S. cerevisiae can use only sucrose.
Statistics: The original activity would have to be repeated at least six times to obtain enough data to carry out valid statistical analysis.
Raffinose broth
S. carlsbergensis S. cerevisiae S. carlsbergensis S. cerevisiae
X X X
XX
8,000 X X
6,000
4,000 X
XX
X X X
X X X
2,000
X
XX X X X
29/3
30/3
31/3 1/4 Time in days
2/4
3/4
Comments on the trial data: • S. cerevisiae appears to be slow to take off in the sucrose • •
broth, this raises the issue of the importance of replicate samples. After 24 hours the difference between the two yeasts in sucrose broth is negligible. Is the final value for S. cerevisiae in the raffinose broth higher than expected? It should be noted that the yeasts were grown up overnight in a glucose broth and 5 cm3 of this was used as the inoculum. The sucrose and raffinose broths contain other ingredients at low levels which may include fermentable substances.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Investigation Three Balancing the loss of carbon dioxide
2 g dried baker’s or brewer's yeast 920 cm3 GYEP broth (2% glucose, 1% yeast extract, 1% peptone) 2 x Universal bottle 2 x sterile Universal bottle 2 x 500 cm3 wide necked flask 2 x silicone rubber bungs with a single hole Non-absorbent cotton wool Greaseproof paper Elastic bands 2 cm3 silicone antifoam and 1 cm3 syringe 2 x glass or plastic fermentation lock with lid or cotton wool plug in the exit vent Universal indicator solution (full range) and 1 cm3 syringe Balance suitable for weighing flasks up to 1000 g, sensitive to 0.1 g Boiling water bath
Points for consideration 1
It is recommended that the yeast used in this investigation is taken from an unopened packet. A one gram sample should be weighed into a sterile Universal bottle as aseptically as possible before transferring to the Universal bottle containing the broth.
2
A total autoclave time of 20 minutes at 121°C should be used to ensure maximum heat penetration of the media (450 cm3) and sterilisation.
3
If the fermentation locks are plugged with non-absorbent cotton wool evaporation from the indicator solution is greatly reduced and more accurate results will be obtained.
4
Plastic fermentation locks are suitable for this investigation and provide less of a hazard than the glass alternatives. Some types of plastic fermentation locks come with a cap to reduce evaporation but still allow escape of gas.
5
The addition of the extra sugar should be carried out using good microbiology practice..... as aseptically as possible.
Statistics and acknowledgment Statistics: Using the data collected from the investigation students should be able to use regression analysis to compare the different fermentations by comparing the gradients of the slopes obtained. It is important to remember that unless the points of the graph fall on an approximately straight line the comparison is not valid. Students should produce six flasks of glucose and six flasks of sucrose to be able to use the Mann-Whitney U Test.
Investigation data (information from fermentation trials) Date
Time
18/January 19/January 20/January 21/January 22/January 23/January 24/January 24/January
17.05 11.33 09.41 10.11 09.25 16.50 09.10 12.45
Mass of flasks on balance Flask A Flask B denatured (g) (g) 847.81 809.12 847.78 806.08 847.78 805.71 847.57 805.41 847.50 805.41 847.50 805.26 847.44 805.24 847.44 805.11 Add 10g glucose
24/January 25/January 25/January 26/January 29/January 31/January
16.55 09.50 16.30 14.17 09.09 13.27
847.42 847.36 847.36 847.32 847.00 845.77
813.92 810.59 810.49 810.41 809.38 807.43
Graph: Change in mass of flasks against time in days
Mass of flasks in grammes
Equipment and materials
Time in days Acknowledgment: This investigation was inspired by discussion with Jan Frings, from the Netherlands, a member of EIBE (The European Initiative for Biotechnology Education, Web site: www.reading.ac.uk/EIBE).
The Danish Biologists Association journal, BIOFAG in January 1995 contained a very interesting article by Eske Brunn. He describes six investigations in which the balance is used to monitor the loss of mass of carbon dioxide from the fermentations.
Investigation One: Sucrose concentration Six different sucrose concentrations: 32%, 24%, 16%, 8%, 4%, 2%, and 0% as a control. One gram of yeast is used for each fermentation and 24 hours later the change in mass is recorded.
Investigation Four: Inhibition of fermentation by copper ions A series of copper sulphate solutions are made from 500 µM, 250 µM, 125 µM, 62 µM, 31 µM, 15 µM, 0 µM as a control.
Investigation Two: Different sugars Sugars used in the investigation: sucrose, fructose, glucose, lactose, xylose, galactose and water as a control. One gram of yeast is used for each fermentation.
Investigation Five: pH and fermentation Eight different pH conditions are investigated: pH 1.8, pH 2.5, pH 3.0, pH3.6, pH 4.1, pH 5.0, pH 6.3, pH 7.5. A 10% sucrose solution with 1g of yeast is used for each pH. The fermentations are left for 24 hours and then the change in mass is recorded.
Investigation three: Concentration of yeast A 10% substrate of sucrose solution is used for each fermentation and six different yeast concentrations: 2.0 g, 1.5 g, 1.0 g, 0.5 g, 0.125 g, 0.062 g and no yeast as a control.
Investigation Six: Temperature and fermentation Five different temperatures are selected 5°C, 20°C, 30°C, 45°C, 65°C. A 10% sucrose solution is used as the substrate with 1 g of yeast for each.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
5
Technical Guide: Practical Fermentation
Investigation Four Yeast cells and enzyme - together they can do it
Equipment and materials: 4 x 5 g baker's or brewer's yeast 4 x 100 cm3 beakers 4 x 50 cm3 water (deionised or distilled) Glass rod 4 x 50 cm3 4% sodium alginate solution 2 x 10 cm3 lactase enzyme 4 x 200 cm3 2% calcium chloride solution 4 x 250 cm3 wide neck flask 4 x magnetic stirrers and follower (optional) 6 x 10 cm3 syringes Tea strainer 2 x 150 cm3 8% glucose solution in 0.5% calcium chloride 2 x 150 cm3 8% lactose in phosphate buffer (0.1 M pH 7.0) 4 x wide necked bung with glass fermentation lock Universal indicator solution (full range) and 1 cm3 syringe 4 x NCBE bubble logger
Points for consideration 1
It is important to use ordinary dried baker's or brewer's yeast, free of any additives that might react with the alginate solution. Some dried yeast preparations contain calcium compounds e.g. calcium sulphate and the calcium ions affect the sodium alginate solution causing premature gelling.
2
Distilled or deionised water should be used for similar reasons.
3
The 2% calcium chloride solution is prepared using the dihydrate (CaCl2.2H2O). If the anhydrous calcium chloride is used then a 1.8% solution can be made.
4
The 8.0% glucose solution is in a dilute calcium chloride solution (0.5%) to help stabilise the beads during fermentation.
5
The lactose solution is made up with 0.1 M phosphate buffer pH 7.0 to optimise the activity of the lactase enzyme. The enzyme recommended for this investigation is sensitive to acidic conditions and as the fermentation progresses the pH falls quickly.
6
The cloudiness that develops on the addition of the alginate beads to the lactose solution is due to excess calcium chloride solution reacting with the phosphate buffer. This can be reduced by washing the beads with distilled water but this may result in the destabilisation of some of the beads.
7
The glucose solution can be made in advance, autoclaved and stored. The lactose solution should not be autoclaved due to the presence of the phosphate buffer. However it can be made a few days in advance and stored at 4°C until required.
8
Instead of adding the enzyme to the yeast/alginate mix it can be added later. The yeast will produce just a few bubbles in the lactose solution but after the addition of the enzyme it will ferment quickly and can produce thousands by the end of the day. The bubbles produced before the addition of the enzyme are due to the fermentation of stored sugars in the dried yeast. The number of bubbles that are produced varies from 50 - 100 over a couple of hours in a warm classroom.
9
If the fermentation is very active, e.g. on warm days, it is advisable to add 1 cm3 of silicon antifoam. It is easier to add the antifoam to the flask before the fermentation lock is fitted 100 % relative but it can be added later if activity 80 necessary.
10 The data sheet sent out with the lactase enzyme gives further information about the influence of pH on the enzyme. 6
60
11 If the investigation is complete at the end of the day then the flasks and their contents can be disposed of by placing in a disinfectant solution. If the investigation continues for longer then the flasks should be sterilised by autoclaving.
Reference and statistics Reference: This investigation has been adapted for schools and colleges from a practical outlined in:
Immobilized Biocatalysts. An introduction Winifried Hartmeier Springer-Verlag, 1988,
Statistics: If students wish to compare fungal and bacterial lactase they will need to set up six fungal enzyme flasks and six bacterial enzyme flasks for each different pH e.g. pH 5 and pH 7.
Investigation data (information from fermentation trials) Flasks (single set of replicates) 1A
5.0
6.0
7.0 8.0 pH
9.0 10.0 11.0
5 May 10.30 10.40 11.15 11.30 12.00 12.30 13.00 13.30 14.00 14.30 15.00 15.30 16.00
0 103 874 1278 2106 3016 3834 4629 5444 6294 7176 8024 8802
0 71 876 1321 2230 3224 4060 4807 5542 6300 7257 8224 9188
6 May 9.00 10.00 11.00 12.00 13.00 15.00
10419 10420 10420 10420 10420 10420
13217 13218 13218 13218 13218 13218
7 May 9.00
10489 13282
2A
2B
Lactose
3A
3B
Glucose + lactase
4A
4B
Lactose + lactase
0 0 3 0 19 9 28 16 47 29 64 42 78 52 90 61 103 69 115 78 126 90 137 101 149 110
0 103 968 1462 2618 3950 5273 6226 7276 8271 9387 10395 11250
0 79 901 1362 2323 3365 4234 5043 5928 6808 7804 8765 9660
0 4 21 37 151 670 1138 1355 1475 1559 1636 1706 1775
0 8 26 196 810 1309 1784 1784 1883 1959 2025 2089 2156
276 280 289 297 302 309
12598 12604 12606 12607 12607 12610
11941 11942 11942 11942 11942 11943
3247 3247 3247 3247 3247 3248
3596 3597 3597 3597 3597 3589
347 347 347 347 347 347
341 341
12802 12019
3266 3681
Comments on the trial data: • • •
•
20
Effect of pH on the activity of LACTASE.
1B
Glucose
40
4.0
ISBN 0387-188088 ISBN 3540-188088
•
The investigation is normally complete in 24 hours. The importance of replicating samples can be shown. The beads in samples 3A and 3B were made using different size syringe nozzles resulting in beads of different sizes. This may be the cause of the apparent different rates of fermentation in the two samples that were otherwise the same. It should be noted that the final readings are very similar. There is a marked increase in the rate of fermentation of the lactose with the enzymes, however this increase may not be as great as expected. This is because the galactose produced probably acts as a competitive inhibitor on the enzyme lactase. In the presence of glucose and galactose S. cerevisiae preferentially ferments glucose.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Investigation Five A sugary choice
diammonium phosphate, (NH4)2 HPO4 , which is one of the ammonium compounds found in the yeast nutrient available from home brew shops.
Equipment and materials: 8 x 2 g dried baker's or brewer's yeast 200 cm3 0.2 M fructose solution 200 cm3 0.2 M galactose solution 200 cm3 0.2 M glucose solution 200 cm3 0.2 M lactose solution 200 cm3 0.2 M maltose solution 200 cm3 0.2 M raffinose solution 200 cm3 0.2 M sucrose solution 8 x 0.5 g ammonium dihydrogen orthophosphate, NH4 H2PO4 8 x 0.5 g ammonium sulphate, (NH4)2 SO4 (1 g "yeast nutrient" from home brew shops can be used as an alternative to the two ammonium salts) 8 x 250 cm3 wide necked conical flask 8 x silicone rubber bung with two holes 8 x glass fermentation lock Universal indicator solution (full range) and 1 cm3 syringe 8 x 15 cm3 glass pipette (specially modified) with 3 cm rubber tubing 8 x restriction clip (Hoffman clip) 8 x glass rod 50 cm3 burette 8 x 20 cm3 syringe (or equivalent) for sampling 8 x 100 cm3 flask for titration 0.1 M sodium hydroxide solution (about 400 cm3) Phenolphthalein indicator solution and dropping pipette
6
Used plastic syringes should be placed in a disinfectant solution and some of the solution drawn up into the syringe.
7
Flasks and titration samples should be sterilised after use by autoclaving.
8
The rate of fermentation can also be monitored by using the NCBE bubble logger.
9
The majority of modern brewing yeasts can normally ferment the same sugars but there is a difference in the rate at which the fermentations take place. To show yeast - carbohydrate specificity, different yeasts are required such as Kluyveromyces lactis which can ferment lactose and Saccharomyces diastaticus which can utilise starch.
Investigation data (information from fermentation trials) Sugar solution Volume of 0.1 M NaOH used 25 cm3 sample Titre set 1 Titre set 2 Mean
Glucose Lactose Galactose Sucrose Maltose Fructose Raffinose Water (control)
Points for consideration Good laboratory practice and sound microbial techniques should be encouraged. However, as a large inoculum and only a 24 hour incubation period are used, this practical is suitable for students with limited experience in aseptic techniques.
2
Any readily available sugars could be used as alternatives to the ones suggested.
3
To prepare 0.2 M sugar solution add the following amounts of sugar and make up to 200 cm3 fructose 7.2 g make up to 200 cm3 galactose 7.2 g make up to 200 cm3 glucose 7.2 g make up to 200 cm3 lactose 14.4 g make up to 200 cm3 maltose 14.4 g make up to 200 cm3 raffinose 23.8 g make up to 200 cm3 sucrose 13.6 g make up to 200 cm3
4
It can be easier and often more convenient to make up the total volume of ammonium compounds required in a single solution, (1.6 dm3) and use this solution to make up seven different sugar solutions and control. (4.0 g ammonium dihydrogen orthophosphate, NH4 H2PO4 and 4.0 g ammonium sulphate, (NH4)2 SO4 made up to 1.6 dm3).
5
Although the ammonium phosphate listed is ammonium dihydrogen orthophosphate, NH4 H2PO4 , it is also possible to use
Graph: Volume of alkali used Volume of alkali (0.1 M NaOH) cm3
20 Titre set 1 Titre set 2
10
cm3 17.3 8.8 8.95 19.85 19.55 18.3 18.1 8.6
Statistics: To investigate the extension activity that different types of yeasts ferment the same sugar equally well will require at least six replicates for each yeast used. The Mann-Whitney U Test can then be applied. (Different strains of brewing yeasts can normally be obtained from home brew shops.) By using two sugar solutions and the six yeasts in the investigation the Wilcoxon Matched Pairs Test can be applied to the data. Acknowledgment: This is an adaptation of a well known investigation frequently used in schools and colleges.
Graph: Mean volume of alkali used to neutralise seven different sugar fermentations 12
10
0
0
cm3 17.1 8.9 8.9 19.8 19.6 18.1 18.4 8.4
Statistics and acknowledgment
Volume of alkali (0.1 M NaOH) cm3
1
cm3 17.5 8.7 9.0 19.9 19.5 18.7 17.8 7.8
8
6
4
2
Glucose Lactose Galactose Sucrose Maltose Fructose Raffinose
Glucose Lactose Galactose Sucrose Maltose Fructose Raffinose Control Sugar solutions
Mean titres (control deducted)
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
7
Technical Guide: Practical Fermentation
Investigation Six How do they like it? - alcohol levels and pH
A. The effect of alcohol concentration on fermentation Equipment and materials
Investigation data (information from fermentation trials) Changes in the pH of each fermentation over the two days
Yeasts (ale, wine and champagne) 60 cm3 4% sucrose solution 60 cm3 water 12 cm3 ethanol Non-absorbent cotton wool 6 x 25 cm3 tube or 50 cm3 measuring cylinder Glass stirring rod and syringes (1 cm3, 5 cm3 and 10 cm3) 6 x malt agar plate and inoculating loop
Desired pH Actual pH Final pH
pH 4.0 4.4 4.2
pH 6.0 6.0 5.8
pH 7.0 7.2 6.3
pH 8.0 8.2 7.1
pH 9.0 8.9 6.5
Blank 7 .0 7.2 7.1
Number of bubbles produced by each fermentation Date 5 Nov
Points for consideration
Time 10.30 10.45 11.30 12.00 12.30 13.00 13.30 14.00 14.30 15.00 15.30 16.00 16.30 17.00 17.30 09.00
pH4.0 0 0 0 6 19 52 110 139 191 240 305 378 462 581 705 3180
pH5.0 0 1 81 211 532 923 1587 1890 2364 2638 2784 2819 2827 2832 2836 2850
pH7.0 0 0 21 54 127 228 558 750 1101 1398 1708 1941 2096 2182 2211 2215
pH8.0 0 0 0 0 7 22 69 99 161 222 300 371 431 486 515 609
pH9.0 0 0 0 3 3 3 3 3 3 3 4 8 13 22 30 1531
Blank 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 2
1
An example of a suitable tube to use is a plastic Universal graduated in 5 cm3 divisions to a maximum of 25 cm3.
2
Varieties of different yeasts can be obtained from either home brew shops or chemists stocking brewing materials.
3
Pure ethanol was used in all trial and evaluation work and is recommended. IMS can be used as an alternative if pure ethanol is not available.
4
If population counts are to be performed the students will have to decide whether they will use the agar plate method or microscopic examination of cells. The former will involve the students in making serial dilutions and then plating the samples on malt agar plates (or glucose nutrient agar). (See page 1 of the student guide). The latter will involve the students in either using a cell counting chamber such as a haemocytometer or the Breed smear method. (See page 12 of the student guide).
3
If plastic tubes are used then cultures must be disposed of by chemical disinfection, if glass then either chemical disinfection or autoclaving can be used.
Silicone rubber bungs have been suggested as they withstand repeated autoclaving and could be an investment in the long run. However, ordinary rubber bungs can be used. It is important to note that the rubber bungs required for this investigation are of a different size than others in this collection of investigations.
4
An alternative to autoclaving the sample of yeast for the control flask is to place the Universal bottle in a boiling water bath for twenty minutes.
5
If a wider range of pH values are required for further investigations, e.g. ranging from 1 to 13, a table for the preparation of suitable buffer solutions is given in: CRC Handbook of chemistry and physics, 78th Edition (1997 - 1998), David R. Lide, Editor in Chief, It also contains a fascinating table of typical pH values of biological materials and foods.
5
6
Ethanol is toxic to yeast cells at certain concentrations depending on the strain of yeast and the metabolic state of the culture.
B. The effect of pH on fermentation Equipment and materials Yeasts (ale, wine and champagne) 0.5 M phosphate buffer solutions (pH 4, 6, 7, 8 & 9) Culture ingredients: sucrose, yeast extract, peptone Non-absorbent cotton wool 6 x 150 cm3 flask 6 x Universal bottle containing 5 cm3 sterile water 6 x NCBE bubble logger 6 x glass fermentation lock 6 x silicone rubber bung with single hole to fit flask Universal indicator solution and 1 cm3 syringe
References and statistics References:
Introductory Microbiology Gross, Faull, Ketteridge and Springham Chapman and Hall, 1995, ISBN 0-412-45300-2 Chapter 11 Microbial biotechnology
Points for consideration 1
Buffer tablets can be used for the preparation of the different buffer solutions. Alternatively stock phosphate buffer solutions can be diluted to give 100 cm3 0.5 M phosphate buffer pH 6.0, 7.0 and 8.0 as shown below: pH 6.0 7.0 8.0
volume of 1 M K2HPO4 6.6 cm3 30.8 cm3 47.0 cm3
volume of 1 M KH2PO4
volume of H2O
43.4 cm3 19.2 cm3 3.0 cm3
50 cm3 50 cm3 50 cm3
Usually buffer tablets come with instruction to make 100 cm3 of 0.1 M solution per tablet. To obtain a 0.5 M solution 5 tablets were used in 100 cm3. This relatively small volume was used so that the cost is not too prohibative for schools and colleges. 2
8
6 Nov
The buffer solutions can be made up the day before and stored in a fridge and brought to room temperature just before use.
Microbial Biotechnology: Fundamentals of applied microbiology Glazer and Nikaido, W.H. Freeman and Company, 1995, ISBN 0-7167-2608-4 Chapter 11 Ethanol Statistics: • Investigation A: If the students carry out a population count at the end of the fermentation for each alcohol concentration selected, a scattergram can be plotted (population count against alcohol concentration). Provided they have at least three replicates then a Spearman Rank Correlation can be applied. • Investigation B: It is possible to plot a scattergram of total number of bubbles produced at the end of the fermentation against each acid concentration selected. At least three replicate sets of data will be required to apply a Spearman Rank Correlation. It should be noted that this is population against acid concentration and not pH. If alkali values are included then a problem in interpretation is likely to be experienced.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Investigation Seven Deep Purple! - a dark secret
Equipment and materials Culture of Janthinobacterium lividum 2 x glucose nutrient agar plate (GNA) 500 cm3 glucose nutrient broth (GNB) (GNB: 6.5 g Oxoid dehydrated nutrient broth in 500 cm3 deionised or distilled water, 5 g glucose, pH 7.0) Bioreactor 2 x Universal bottle Sterile silicone antifoam Inoculating loop Sterile 1 cm3 syringe 2 x sterile 10 cm3 syringe Sterile 3-way tap Aquarium pump and tubing Magnetic stirrer and follower (optional)
Points for consideration 1
If a magnetic stirrer is to be used, a magnetic follower should be added to the bioreactor before autoclaving.
2
When selecting the colonies from the agar plate it is best to choose the darkest in colour. The overnight broth culture should be turbid but will not normally be coloured purple.
3
4
If two microbial cultures are grown together it may be necessary to add extra sterile antifoam to the bioreactor to prevent the broth foaming up into the air filter i.e. 2 cm3 rather than 1 cm3. This can be added at the start or as required during the fermentation. Further silicone antifoam can be added if necessary. Of the microorganisms suggested in the student guide both Erwinia carotovora and Rhizobium leguminosarum are Gramnegative bacteria and can be expected to produce the signalling molecule and therefore the purple pigment in
Janthinobacterium lividum. Initial cultures of Erwinia carotovora and Rhizobium leguminosarum can be grown up on GNA or NA plates. Even in the presence of Gram-negative bacteria it can take two days for the pigment to appear as the culture has to reach the correct age and population density to produce signalling molecules. 5
Extension activity 1: One might expect a positive correlation between the bacterial cell count and pigment production in Micrococcus roseus. However this is not the case with Janthinobacterium lividum unless the signalling molecule is present. Extension activity 2: One might expect a positive correlation between cell number and pigment production with Gramnegative bacteria that produce the signalling molecule and no correlation with Gram-positive bacteria that do not.
tubes are balanced and spin as before. Decant supernatant into disinfectant. f) Add 10 cm3 of IMS to each tube. Mix with the end of a pipette and transfer to a second 50 cm3 flask. Plug with cotton wool and incubate at room temperature for 48 hours to autolyse. Alternatively the pigment can be extracted using the following alkali and detergent method:
N.B. Care should be taken in the preparation and use of reagents for this method, including a full risk assessment. a) Follow steps a - e as above. b) Add 2 cm3 of alkali and detergent solution (1 cm3 40% NaOH and 1 cm3 10% SDS) to the pellet of bacterial cells. Mix well to resuspend the cells. Leave overnight at room temperature. c) Centrifuge the mixture to pellet cell debris and decant the supernatant into a clean centrifuge tube. Neutralise with 1 M HCl solution (pH 6 - 8). Centrifuge to obtain a clear supernatant which should be decanted into a clean tube or bottle. The collected pigment can be used for chromatography or trials for staining cloth e.g. S.D.C. Multifibre Test Fabric consisting of: secondary cellulose acetate (Dicel), bleached unmercerized cotton, nylon 6.6, polyester (Terylene), acrylic (Courtelle) and wool worsted.
References, statistics and acknowledgment References: a) Gram-negative bacterial communication by N-acyl homoserine lactone: a universal language? Trends in Microbiology, June 1994, Vol 2, No. 6 Simon Swift, Nigel J. Bainton & Michael K. Winson b) The bacterial 'enigma': cracking the code of cell-cell communication. Molecular Microbiology (1995) 16(4), pages 615 - 624 G.P.C. Salmond, B. W. Bycroft, G. S. A. B. Stewart & P. Williams Statistics: The Spearman Rank Correlation test would be suitable for investigating any correlation between pigment production and Gram-negative bacteria. Acknowledgment: We would like to thank Dr Emma Durant for her help and suggestions in the design of this investigation while working for her PhD at The University of Reading.
Molecular structure of deep purple pigment - violacein
Extension activity 3: The pigment can be extracted from the bacterial cells using the following method: a) Switch off the air pump from the actively growing culture and allow cells to settle to the bottom of the bioreactor. Place in fridge overnight to assist the settlement. b) Very carefully decant the majority of the supernatant into disinfectant solution. (Good Microbiology Laboratory Practice
should be followed). c) Swirl the remainder of the broth in the bioreactor and transfer to a 50 cm3 flask. Plug the flask with cotton wool. d) Pipette two 10 cm3 aliquots into each of two centrifuge tubes. Check tubes are balanced and spin at 3000 rpm (2.4 g) for 10 minutes. Decant the supernatant into disinfectant. e) Add 10 cm3 of sterile water to each tube. Mix well. check
More information about N-acyl homoserine lactone can be obtained by using the World Wide Web and searching it with a search engine such as ‘Excite’.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
9
Technical Guide: Practical Fermentation
Investigation Eight
Investigation Nine
Nothing’s for free -
Ester production -
you gain some, you lose some!
a fragrant or smelly fermentation?
Equipment and materials Fresh dried bakers' yeast, Saccharomyces cerevisiae
Equipment and materials Culture of Pichia anomala
500 cm3 of GYEP broth (10% glucose, 1% yeast extract, 1% peptone) Bioreactor 3 x Universal bottle 10 cm3 sterile water in a Universal bottle 2 x malt agar or glucose nutrient agar plate Sterile silicone antifoam Inoculating loop 3 x sterile 1 cm3 syringe Sterile 3-way tap Aquarium pump and tubing Magnetic stirrer and follower Containers and sterile 10 cm3 syringes for sampling
2 x malt agar plate 450 cm3 GYEP broth (10% glucose, 1% yeast extract, 1% peptone) 20 cm3 GYEP broth (2% glucose, 1% yeast extract, 1% peptone) Bioreactor 2 x Universal bottle Sterile silicone antifoam Inoculating loop Sterile 1 cm3 syringe 2 x sterile 10 cm3 syringe Sterile 3-way tap Aquarium pump and tubing
Points for consideration
Points for consideration 1 The yeast Pichia anomala is available from the NCBE, The
1
2
3
4
The method employed in this investigation ensures that the student uses a pure and viable culture for the inoculation i.e. dried yeast yeast slurry malt agar plate small broth inoculum. It has been suggested that both agar plate and small broth inoculum cultures are duplicated to allow for the selection of the best growing culture. If it is difficult to maintain 25OC the bioreactor can be incubated at room temperature but temperature fluctuations may affect the fermentation and this may have to be taken into account when considering the results. The Roche Diabur-Test® 5000 glucose test strips can normally be obtained from dispensing chemists as they are used by diabetics to test their urine.
5
Some of the extension activities suggested for this investigation in the student guide may require different equipment and materials such as the bubble logger to monitor carbon dioxide production and different strains of yeast.
6
Some of the questions posed by the suggested extension activities were designed to be thought provoking and students may need to carry out considerable research before embarking on any practical work.
7
The change in the glucose concentration can appear to remain constant and then suddenly drop very quickly. It is possible to make serial dilutions of the samples taken to obtain more sensitive readings with the test strips.
8
This investigation can be adapted to make use of available equipment. For example a two litre flask can be used with four times the volume of broth suggested. The broth can be inoculated in the late afternoon and tested the next morning. In trials 1% glucose was present at 10.00 a.m. and by midday no glucose was detected (30°C - 32°C). The benefit of performing the investigation on this scale is that larger samples (50 cm3 of broth) can be taken and the mass of yeast determined by centrifuging, drying and weighing.
Acknowledgments Acknowledgments: This investigation was inspired by discussion with Dr Bob Rastall, Department of Food Science and Technology, The University of Reading. We would like to thank Deborah Powell for her help in making constructive comments on this investigation while attending a London Examinations course on fermentation run by the NCBE. We would also like to thank all the other delegates who helped in the trials of this and other practicals. 10
University of Reading, Whiteknights, Reading, RG6 6AJ. 2
GYEP (glucose, yeast extract, peptone) agar plates can be used if malt agar plates are not available i.e. prepare extra GYEP broth and add 1.5% agar before autoclaving.
3
It has been suggested that both agar plate and small broth inoculum cultures are duplicated to allow for the selection of the best growing culture.
4
The investigation suggests that a 10% glucose solution is used. If a more concentrated solution is used, care must be taken when autoclaving so that the medium does not caramelise e.g. autoclave for 15 minutes at 69 kPa.
5
A suggested method for extracting the ester: A suitable volume of the broth is centrifuged to remove the cells from the spent broth. After centrifuging, decant the broth containing the ester into a flask for distillation. Attach a fractional distillation column and a condenser. Much of the ester can be driven off by heating the sample. The fragrant liquid, smelling of 'pear drops', can be collected. At the end of the investigation broth containing yeast cells and contaminated glassware should be autoclaved.
References and acknowledgment References: More information about the production of ‘esters in yeasts’ can be obtained by using the World Wide Web and searching it with a search engine such as ‘Excite’. The following website has proved interesting and informative; Research and Teaching Institute of Brewing, Berlin (VLB) www.vlb-berlin.org/english/kunze/ester.htm Excerpt of Chapter 4, Beer production (fermentation, maturation and filtration) Wolfgang Kunze: “Technology Brewing and malting” Textbook, International Version. 4.1.2.4. Esters
Acknowledgment: This investigation is based on discussions held with Frank and Dorothy Spencer while they were at Goldsmiths’ College, University of London.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Investigation Nine
Investigation Ten
Demonstration of downstream processing
Dextran production a sticky fermentation
Points for consideration
Dextran production : Equipment and materials Culture of Leuconostoc mesenteroides
1
Although the separation of food pigments by an isolute column is not directly related to the ester production investigation, it has been included as a demonstration of downstream processing. The green food dye suggested contains two pigments E104 (Quinoline yellow) and E131 (Patent blue V). The first colour to be obtained from the column is the Quinoline yellow; the second colour to be obtained is the Patent blue V.
E104 Quinoline yellow
2 x GNA (glucose nutrient agar) plate 20 cm3 starter broth (glucose nutrient broth + 4% sucrose) 400 cm3 fermentation broth (17% sucrose, 0.14% yeast extract, 4 x pH 7.0 tablets ) Bioreactor 200 cm3 medical flat or similar bottle 2 x Universal bottle Sterile silicone antifoam Inoculating loop 1 cm3 sterile syringe Sterile 3-way tap 2 x 10 cm3 sterile syringe Aquarium pump and tubing 2 dm3 plastic beaker or deep sided tray 250 cm3 flask, cotton wool, gauze, elastic band, greaseproof paper
Points for consideration 1 Leuconostoc mesenteroides can occasionally be difficult to grow. If it does not grow well on a GNA plate at first attempt, it can be grown in a small volume of GYLB, GNB or MRS starter broth.
E131 Patent blue V
GYLB: 5 g glucose, 2.5 g yeast extract, 8 g ‘Lab-Lemco’ powder, 10 g peptone make up to 1000 cm3 with demineralised water, check pH 7.
GNB:
10 g glucose, 13 g Oxoid dehydrated nutrient broth make up to 1000 cm3 with demineralised water, check pH 7.
MRS (de Man, Rogosa, Sharpe):
Ca
10 g peptone, 8 g ‘Lab-Lemco’ powder, 4 g yeast extract, 20 g glucose, 1 cm3 sorbitan mono-oleate, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate 3H2O, 2 g triammonium citrate, 0.2 g magnesium sulphate 7H2O, 0.05 g manganese sulphate 4H2O, make up to 1000 cm3 with deionised or distilled water, check pH 6.2. Alternatively MRS media can be purchased from biological suppliers.
R = C2H5 The different dyes can be collected on filter paper to provide a more permanent record, e.g. 7 cm diameter
Once a viable culture has been obtained in the small broth culture, a sample should be taken and grown up on a streak plate to ensure good, healthy colonies for the investigation. 2
MRS agar plates can be used, (recipe for MRS as above with the addition of 10 g of agar per 1000 cm3) instead of GNA plates.
3
It has been suggested that both agar plate and small broth inoculum cultures are duplicated to allow for the selection of the best growing culture.
4
It is advisable to place the bioreactor in a large plastic beaker or plastic tray in case not enough silicone antifoam has been added and the broth foams up through the air exit filter. If this looks likely to occur, more antifoam should be added via the 3-way tap. If a spillage does occur e.g. over night when the bioreactor is unattended, then the following procedures should be carried out.
Procedure: Run through the column 2 cm3 of a 95% ethanol solution Add 0.5 cm3 or 2 drops of green dye Wash with a little water (1 cm3) to remove first pigment Run through 2 cm3 of 20% ethanol solution to remove second pigment # If the column is still coloured wash again with a small volume of 95% ethanol solution
# # # #
References Isolute SPE columns (100mg C18 1ml) are obtainable from Jones Chromatography Ltd. Small numbers of the columns for use in school and college can be obtained from the NCBE. Jones Chromatography Ltd, New Road, Hengoed, Mid Glamorgan, CF82 8AU. There are two web sites that may be of interest when teaching about the use of resins for product separation and downstream processing. Jones Chromatography Ltd, www.jones-chrom.co.uk International Sorbent Technology Ltd. (manufactures of solid phase extraction products) www.ist-spe.com.
a) b) c) d)
The fermentation should be stopped. The aerator should be switched off. Contaminated surfaces should be disinfected. The bioreactor should be autoclaved.
The broth in the bioreactor can then be used as set out in the dextran investigations. N.B. The blocked sterile air filter cannot normally be re-used and should be discarded after autoclaving
Reference This investigation is based on: Unit 59, Dextran by Richard Badley, SATIS 16-19 The Association for Science Education, 1990, ISBN 0-86357-138-7
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
11
Technical Guide: Practical Fermentation
Investigation Eleven Some sticky investigations - by gum!
A. Alginate beads: Equipment and materials 10 cm3 1% sodium alginate soln. (from marine algae or bacteria) 100 cm3 0.5 M sodium chloride solution 150 cm3 0.5 M calcium chloride solution 100 cm3 0.5 M strontium chloride solution 4 x Pasteur pipette 1 cm3 syringe with short rubber tube 4 x beaker 250 cm3 1 cm3 0.1 M EDTA solution (sodium salt) Waterbath and 100 cm3 flask
Points for consideration 1
It is advisable to make the sodium alginate solution up with distilled or deionised water as any calcium ions affect the sodium alginate solution causing premature gelling.
2
Good laboratory practice must be observed when handling and making a 0.1 M EDTA solution. The powder is assessed as toxic by inhalation and contact with skin, it is also an irritant to the eyes, respiratory system and skin and therefore should be handled wearing protective clothing (gloves, mask and safety spectacles) and dispensed in a fume cupboard. There is less risk with the solution but it is harmful if swallowed. It is possible to obtain 0.1 M EDTA solution commercially (Philip Harris Education) and, due to the hazards associated with the solid, this is recommended.
3
The sodium alginate used in the trials of the investigation was obtained from BDH (product number 301054N) produced from Laminaria hyperborea.
B. Xanthan gum: Equipment and materials 3
This investigation does not appear in the student guide. It is included here as an additional investigation if students become particularly interested in gel work. Agar and gellan investigation: Equipment and materials 0.1 g gellan powder (Gelrite®) 0.1 g agar powder 2 x glass rod 2 x boiling tube 5 cm3 0.1 M magnesium chloride solution Waterbath
Procedure 1 Add 10 cm3 water to the gellan powder in a boiling tube. 2
Add 10 cm3 water to the agar powder in a second boiling tube.
3
Heat and thoroughly mix until the tubes and their contents are at 100°C. (Good Laboratory Practice must be observed when boiling liquids). Cool to room temperature. Observe the gels formed and compare with other gels that you have produced.
4
Add a few drops of 0.1 M magnesium chloride solution to both and repeat the heating and cooling cycle. Observe the gels and comment on both.
5
Add 0.1 cm3, 0.25 M Sodium hydroxide solution to both and repeat the heating and cooling cycle. Observe the gels and comment on both.
Points for consideration 1
It is advisable to make the solution up with distilled or deionised water. The Gelrite® used in the trials for this investigation was obtained from Sigma® : Gelrite Gellan Gum (product number G-1910) If three sets of solutions are prepared (3 gellan and 3 agar), one set can act as the control. Step 4 can be carried out on the two pairs and then one set aside. The final step 5 is performed on the remaining pair.
50 cm 0.25% xanthan gum solution 50 cm3 0.25% locust bean gum solution 50 cm3 0.25% guar gum solution 10 cm3 2 M calcium chloride solution 4 x 5 cm3 syringe 5 x test tube with bung or plastic Universal bottle Waterbath
2
Points for consideration
Reference and acknowledgment
1
The gums used in the trials of this investigation were obtained from Sigma® : Gum xanthan (product number G-1253) produced by fermentation of dextrose with Xanthomonas campestris. Gum, locust bean (product number G-0753) from seeds of Ceratonia siliqua.
Gum guar (product number G-4129) 2
3
It is advisable to make the gum solutions up with distilled or deionised water. It is important to make sure the gums have dissolved thoroughly before carrying out the investigation. The use of a magnetic stirrer is advisable, otherwise leave overnight to soak and shake well before use. The synerginistic properties of gums are well illustrated with xanthan gum and locust bean gum. More information about xanthan, locust bean and guar gums can be obtained by searching the World Wide Web.
3
Reference: Further information about gums can be found in: Industrial Gums 3rd edition by Whistler, R. L. and BeMiller, J. N. Academic Press, 1993, ISBN 0-12-746353-8 This book is a valuable source of information with each chapter devoted to a different type of gum. Selected list of chapters: Chapter 1 Chapter 5 Chapter 7 Chapter 8 Chapter 10 Chapter 13 Chapter 20 Chapter 23
Introduction to Industrial Gums Agar Carrageenan Guar, Locust Bean, Tara, Fenugreek Gums Pectin Xanthan, Gellan, Welan, Rhamsan Sodium Carboxymethylcellulose Analysis of Gums in Foods
Acknowledgment: We would like to thank Professor Ian Sutherland, of The Institute of Cell and Molecular Biology, The University of Edinburgh for his help and suggestions in the design of these investigations . 12
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Investigation Twelve Probably the best yeast in the world
Production of yeast pigment Equipment and materials Culture of Saccharomyces cerevisiae (K5-5A) 2 x malt agar plate 1 dm3 GYEP broth (2% glucose, 1% yeast extract, 1% peptone) 2 x bioreactor 3 x Universal bottle Sterile silicone antifoam Inoculating loop 2 x sterile 1 cm3 syringe 2 x sterile 3-way tap 8 x sterile 10 cm3 syringe Aquarium pump and tubing
Points for consideration 1 The yeast Saccharomyces cerevisiae (K5-5A) is available from the NCBE, The University of Reading, Whiteknights, RG6 6AJ. 2
It has been suggested that the agar plate culture is duplicated to allow for the selection of the best growing culture. In this case choose the darkest red colonies. The small broth culture should be prepared in triplicate so the best two cultures can be used for the investigation.
3
It is suggested that one litre of GYEP broth is prepared for this investigation. Only 930 cm3 of GYEP broth is actually required. The remainder can be aliquoted into Universal bottles for future use. If correctly autoclaved the bottles can be stored at room temperature for at least a year and are suitable for growing up most yeast cultures.
4
Before samples are taken for population counts, students should be advised to carefully swirl the flasks to ensure an even distribution of cells in the broth.
5
If the samples are not used immediately they can be stored in the fridge until required.
Estimation of total cell population Breed Smear Method: Equipment and materials
4
There are a number of levels at which comparisons of cell populations can be made. The first is simply to examine at random the same number of fields of view for each sample. This gives a quick and simple comparison. The second is to calculate the number of adjacent fields of view and ultimately the total number of fields of view in the 20 mm x 10 mm rectangle. By counting the cells in a number of fields of view an estimation of the population in an approximate volume can be obtained. Finally, if a slide micrometer is available, the area of the field of view can be accurately calculated. From the calculations each field of view will represent a known volume. The number of fields of view that should be examined to give statistically reliable results depends on a number of factors, but the following procedure is often used. Average number of organisms per field 03 46 7 - 12 13 - 25 26 - 50 51 - 100 over 100
Number of fields to be counted 64 32 16 8 4 2 1
This allows for calculations such as the number of cells per cm 3.
Investigation data (information from fermentation trials)
The following calculations for the number of cells per cm3 were obtained using a two by one centimetre rectangle and 10 µl of yeast culture spread evenly over the rectangle.
Disinfectant Microscope 4 x slides Waterproof marker pen 4 x graduated micropipette tip or Pasteur pipette & inoculating loop
The number of fields of vision were estimated at 48 lengthwise and 24 widthwise making a total of 1152 using a x10 eye piece and a x40 objective.
Points for consideration
The original culture was diluted a hundred fold to help counting.
1
2
3
A 20 mm x 10 mm rectangle has been suggested with a 0.01 cm3 (10 µl) volume as this should give a thin spread of separate cells that can be seen clearly under the selected field of view. Microsyringes with graduated micropipette tips can be obtained from the NCBE to dispense 10 µl volume. Alternatively, to obtain a drop of known volume, a 50 dropper glass Pasteur pipette can be used. The volume of the drop will be 0.02 cm3 (20 µl) provided that the following conditions are satisfied: external diameter of the pipette is 1.07 mm; end of the pipette is cut square; pipette is clean; fluid being dispensed does not differ in viscosity from Ringer's solution; dropping rate is 40 - 45 drops per minute; pipette is held vertically Methylene blue solution or Gram staining can be used to stain yeast cells to make them easier to count. (Although Gram staining is usually associated with staining bacteria, yeast cells stain purple.)
Rectangle = 20 mm x 10 mm and contains 1152 fields of view Each field of view represents 10 µl divided by 1152 = 0.09 µl
Total number of yeast cells in 1 cm3 = ? Number of fields x average number of cells per field x the dilution value x (10µl/1000 =100). 73,728,000 cells per cm3 92,160,000 cells per cm3 88,704,000 cells per cm3 77,184,000 cells per cm3 81,792,000 cells per cm3 74,880,000 cells per cm3
student one student two student three student four student five student six
1152 x 64 x 100 x 100 = 1152 x 80 x 100 x 100 = 1152 x 77 x 100 x 100 = 1152 x 67 x 100 x 100 = 1152 x 71 x 100 x 100 = 1152 x 65 x 100 x 100 =
Mean value
= 74,218,000 cells per cm3
Acknowledgment The yeast culture used in this investigation was originally obtained from Frank and Dorothy Spencer when they were at Goldsmiths’ College, University of London.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
13
Technical Guide: Practical Fermentation
Investigation Thirteen Probably the best pigment in the world
A. Extraction of pigment from red yeast:
B. Chromatography of the pigment:
Equipment and materials
Equipment and materials
An actively growing culture of red yeast Saccharomyces cerevisiae (K5-5A) from Investigation Twelve Disinfectant solution 2 x 50 cm3 sterile conical flask Cotton wool (non-absorbent) 4 x sterile 10 cm3 graduated pipettes, plugged with cotton wool 2 x sterile centrifuge tube, plugged with cotton wool Centrifuge Sterile water, approx. 50 cm3 Balance Pipette and tubes for collecting pigment
Decanted pigment from yeast culture (K5-5A) Whatman No.1 filter paper, 2 cm x 15 cm Boiling tube with bung Micropipette (made by drawing out the end of a Pasteur pipette in a Bunsen burner flame) Drawing pin 20 cm3 solvent: glacial ethanoic acid : conc. HCl : water; 30 : 3 : 10
Points for consideration 1
If students wish to extract the pigment from the yeast cells it is necessary to continue immediately from Investigation Twelve or to store the bioreactor at 4°C until required.
2
If the centrifuge tubes are weighed before and after, some idea of the mass of cells produced can be obtained which can widen the scope of the investigation.
3
A Pasteur pipette can be used to transfer the supernatant from the centrifuge tubes into disinfectant instead of decanting. It is important not to disturb or dislodge the pellet of cells whichever method is used.
4
Alternatively the pigment can be extracted using the following alkali and detergent method:
(Good laboratory practice must be followed in the preparation and use of this solvent, including the use of a fume cupboard and eye protection).
Points for consideration 1
A single spot with an Rf value of approximately 0.7 (solvent front 93 mm, pigment 69 mm) is expected using the suggested solvent system.
2
The solvent recommended for the chromatography of the yeast pigment is also suitable for the separation of flower and fruit pigments.
N.B. Care should be taken in the preparation and use of reagents for this method, including a full risk assessment. a) Instead of adding 10 cm3 of water at step 5, add 2 cm3 of alkali and detergent solution (1 cm3 40% NaOH and 1 cm3 10% SDS) to the pellet of yeast cells in the centrifuge tube. Mix well to resuspend the cells. Leave overnight at room temperature. b) Centrifuge the mixture to pellet cell debris and decant the supernatant into a clean centrifuge tube. Neutralise with 1 M HCl solution to pH 6 - 8. Centrifuge to obtain a clear supernatant which should be decanted into a clean tube or bottle. 5
Enzyme preparations that lyse yeast cells are commercially available.
6
The collected pigment can be used for chromatography or trials for staining wool and cloth e.g. S.D.C. Multifibre Test Fabric: secondary cellulose acetate (Dicel), bleached unmercerized cotton, nylon 6.6, polyester (Terylene), acrylic (Courtelle) and wool worsted.
7
93 mm
69 mm
Some rewarding and interesting results have been obtained using mordanted wool samples. Students fascinated by this could find it a good subject for research.
Reference Reference: Further information about chromatography of plant pigments can be found in:
X Practical Biochemistry for Advanced Biology
X
Susan Aldridge, Cambridge University Press, 1994, ISBN 0-521-43782-2
14
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Investigation Fourteen Vibrio natriegens - for a speedy growth curve
Equipment and materials Culture of Vibrio natriegens on 2% saline nutrient agar (pH 7.5) 2 x 2% saline nutrient agar plate (a further 48 plates may be needed, see below) 3 x Universal bottle 156 cm3 of 2% saline nutrient broth (pH 7.5) 250 cm3 wide-necked flask Silicone rubber bung with two holes Glass fermentation lock Universal indicator solution (full range) and 1 cm3 syringe 15 cm3 bent glass pipette with 3 cm narrow rubber tubing Restriction clip (Hoffman clip) Non-absorbent cotton wool and aluminium foil Inoculation loop 12 x 10 cm3 sterile syringe Large beaker half filled with disinfectant solution for disposal Water bath and thermometer 10 x sterile Universal bottle Ordinary graph paper and 3-cycle semi-log paper Determination of cell population:
5
As so many samples are taken and dilutions made, the need for good labelling is paramount! (See page 15.)
6
The investigation suggests preparing a full set of serial dilutions from 10-1 to 10-8 by adding 1 cm3 to 9 cm3 of saline solution for the spread plate method. However if 0.1 cm3 is added to 9.9 cm3 or 1 cm3 to 99 cm3 dilutions of 10-2, 10-4 and 10-6 can be obtained, reducing the number of bottles required. Dilutions of 10-7 and 10-8 will still be required. (See page 15.)
7
Recommended dilutions for the spread plate method are 10-6 to 10-7 for the first few samples and 10-7 to 10-8 for the remainder. For further investigative work other dilutions may be found to be more suitable.
8
Dilutions should be stored in a fridge at 4°C if spread plates cannot be prepared immediately.
9
This investigation lends itself to team work as one student can be responsible for sampling, one for preparing the dilutions and one for producing the spread plates.
30 x sterile plugged Pasteur pipette 1 cm3 syringe with 3 cm wide rubber tubing (to fit over syringe barrel and pipette) 80 x 9 cm3 of sterile saline (0.85%) in Universal bottle Sterile spreader, and a capped beaker of IMS for flaming spreader 48 x 2% saline nutrient agar plate
10 The rate at which microbial cells increase in number is called the growth rate. The time taken for a population of cells to double is called the population doubling time or the mean generation time. The population doubling time is related to the growth rate. Generation time or doubling time is the unit of measurement of microbial growth. A bacterium with a doubling time of 15 minutes has a growth rate of 4 doublings an hour.
Points for consideration
For a growing microbial culture the generation time is:
Colorimeter, spectrophotometer or turbidity meter with cuvettes
or
1
2
3
4
This investigation is quite time consuming in the preparation of materials if the spread plate method is used. It is hoped that this will not deter anyone from trying it as it produces valuable data for interpretation and has many advantages due to the nature of the organism. It has been suggested that the agar plate culture is duplicated to allow for the selection of the best growing culture. The small broth culture should be prepared in duplicate so the best culture can be used for the investigation. A 2% saline nutrient broth (20 g sodium chloride, 13 g nutrient broth powder made up to 1 litre with distilled water, pH 7.5) has been recommended for this investigation. References in text books may suggest the use of brain-heart infusion. We have intentionally not used this medium and have obtained satisfactory results with the saline nutrient broth. For this investigation it is important to use the correct sampling technique as numerous and regular samples are required. With this in mind it is recommended that students carry out some sampling practice using spare equipment and the following method, using coloured water instead of broth.
Sampling Check that the syringe plunger is withdrawn to the middle position. The piston is slowly and very carefully withdrawn so that culture from the vessel is drawn up the tube and falls into the expanded region of the bent pipette. Then slowly and gently push back the piston so that a little air bubbles through the broth retained in the expanded region. The piston is gently withdrawn again and the broth enters the syringe. Careful repetition of this process should ensure that all the sample ends up in the syringe - none in the expanded region. The remaining broth in the sampling tube should be returned to the same level as the broth in the flask. With practice and patience exact volumes can be withdrawn.
g = generation time, t = time and n is the number of generations The number of cells in a growing population is expressed as: g =
t n
Nt = No x 2n No= number of cells at zero time, Nt= number of cells at any time (t) after start, n = number of generations. Since the number of microbial cells in a culture can range from a few to many millions, a mathematical transformation is performed and logarithmic values are used. It is easier to plot the logarithm of microbial cell number rather than the arithmetic which can get into thousands of millions of cells. The equations are rewritten as; t g=
n=
log10 Nt - log 10 No
3.3 (log10 Nt 2 log 10 No)
log10 2
Reference and acknowledgment Reference:
Microorganisms in our world, R. M. Atlas, Mosby, 1995, ISBN 08-8016-7804-8 Acknowledgment: This investigation was suggested in;
Sourcebook of Experiments for the Teaching of Microbiology Edited by S. B. Primrose and A. C. Wardlaw Academic Press, 1982, ISBN 0-12-565680-7 Out of print. Photocopies of the relevant chapter 22 can be obtained from Society for General Microbiology upon request. It has been adapted for schools and college use.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
15
Technical Guide: Practical Fermentation
Technical information 1
Technical information 2
The bubble logger
Principles of a bioreactor
NCBE Bubble logger
Construction of NCBE bioreactor and simulation exercise.
Constructed bubble loggers, ready for use, can be purchased from the NCBE but for those wishing to construct their own the following information is included.
Before a bioreactor is used by students for growing microorganisms it is advisable that they become familiar with its construction and function. This activity involves the assembly of a simple bioreactor (fermenter) by the student and its use in a simple titration exercise.
Circuit diagram of NCBE bubble logger R6 680Ω
R2 100Ω R1 360Ω
VR1 10k
R3 100kΩ
2-
7
Counter I/P
OP-90 6 3
+
INDICATOR LED
+
BATTERIES 3 volts
Basic information on construction:
-
+V
COUNTER
INFRARED LED
0V
PHOTO TRANSISTOR
R5 16kΩ R4 100kΩ
1
Check glassware is smooth and will fit the holes in the silicone bung.
2
Place a small amount of silicone grease on the glassware and moisten the bung with water. Carefully twist each piece into the correct position in the bung. (Observe Good Laboratory Practice when doing this).
3
Place the silicone bung into the neck of the bioreactor. Items such as the silicone tubing, filters and syringes can then be added. Fit ties to hold all the silicone tubing in place on the glass tubes. N.B. This is very important if autoclaving the bioreactor.
4
RESET SWITCH
TO COMPUTER OR DATA LOGGER
SLOTTED OPTO SWITCH
PCB Foil pattern (actual size)
Suggested solutions: Alkali solution: 4 cm3 1 M NaOH in 450 cm3 of water Inoculum: 10 cm3 of Universal indicator solution Acid solution: 10 cm3 0.5 M HCl
68mm
•
Components Item
74mm
Source
LCD counter MC33171P OP-AMP
or
2 x AA battery box Tactile switch 10K finger adjust Low current red LED Slotted Opto switch 6 x resistors as shown below (R1 = 360Ω R2 = 100Ω, R5 = 16kΩ, R6 = 680Ω) Printed circuit board material 4 x stick on feet Glass fermentation lock
Part No.
RS 343-442 Maplin AD99H RS 641-796 Maplin CL17T Maplin KR88V RS 186-968 RS 588-386 RS 219-2511 RS R3 and R4 = 100kΩ, Maplin RS
223-859
Acknowledgment Acknowledgment: The authors would like to thank Roy Palmer in the School of Animal and Microbial Science, University of Reading. He took an idea and a crude electronic device and redesigned it into the present bubble logger which ensured the development of many of the investigations found in this project. 16
•
•
Once constructed, the system is trialled and tested using an alkali solution to represent the growth medium. Universal indicator solution in used as the inoculum and is added using a syringe via the addition port - just as one would the microbial inoculum. The system is aerated by using an aquarium pump. The action of the bubbles in the alkali solution should mix the contents slowly and the solution should turn an even purple colour. The action of aeration on the alkali and indicator solution often causes some foaming. If a syringe of silicone antifoam is prepared in advance it can be added just before the foam fills the flask and affects the exit filter. By adding the antifoam just at the last minute it shows very spectacularly the role it plays in preventing foaming. A sample of the coloured alkali solution should be taken using the side arm sampling device and kept for later. The correct procedure and a little practice is required to sample correctly, so no liquid remains in the side arm. (See student guide page 16.) Acid is slowly added via the addition port and allowed to mix with the alkali until the solution has turned blue in the bioreactor. A sample is taken and stored with the previous one. A difference in the colour of the two samples should now be seen. The process is repeated until the student has a full collection of samples that represent different pH solutions. Just like a conventional titration it is easy to over shoot with the acid, time is needed to ensure good mixing in the flask.
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Technical information 3
Technical information 4
Basic microbiology preparation of a streak plate
Basic microbiology preparation of a dilution series
Streak plate
Dilution series
2 1 1 2 3 4 5
Illustration of the dilution series suggested for the Vibrio growth investigation
Sterilise inoculating loop until red hot. Allow loop to cool. Remove bottle top and flame neck of bottle. Stroke culture from agar surface with loop. Flame neck of bottle and replace bottle top.
Undiluted sample
4 3
5
6
6 Stroke loop back and forth on agar surface. 7 Sterilise loop in flame until red hot.
7
9
8
10
8 When loop is cool stroke loop over agar surface. Streaking should cross previously streaked area.
11 12
11 Sterilise loop in flame until red hot. 12 When loop is cool stroke loop over agar surface. Streaking should cross previously streaked area.
13
13 Sterilise loop in flame until red hot. 14 Incubate plate until colonies appear.
Plate after incubation
10-5
10-6
10-7
10-8
Time minutes 0 30 60 90 120 150 180 210 Suggestion for running as a class practical with eight groups. Each group is responsible for two different time samples which also ensures that the class has a replicate set. Group
1
Sample time minutes
0
9 Sterilise loop in flame until red hot. 10 When loop is cool stroke loop over agar surface. Streaking should cross previously streaked area.
(dilute 1 in 9 each time) 10-1 10-2 10-3 10-4
120
Set one 2 3 30
150
4
5 0
60
180
90
120
Set two 6 7 30
150
210
8
60
90
180
210
Illustration of an alternative abbreviated dilution series for the Vibrio growth investigation Undiluted sample
(dilute 1 in 99 each time) 10-2 10-4 10-6
10-8
Time minutes 0 30 60 90 120 150 180 210 Suggestion for an abbreviated version. It should be noted that this method may be more time consuming with regard to the counting of colonies
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
17
Technical Guide: Practical Fermentation
Good Laboratory Practice GLP for all!
Safety All investigations should be carried out using good laboratory practice. Chemicals and procedures requiring special care are marked with a warning symbol in the text of both the student and technical guide. The following information on safety has been provided in the student booklet and is considered a sound minimum but teaching staff may wish to supplement it with additional information in light of local situations and circumstances that prevail in their own teaching institutions.
Introduction Just like any other practical activity in a laboratory all of these investigations require the user to adopt good laboratory practice. Given here are a few brief notes and hints to help those involved in the various activities to carry them out safely. Remember that before any practical activity is undertaken a risk assessment should be performed to ensure there is minimal hazard to all concerned. If there is any doubt about the assessment of the risk, reference must be made to safety texts or expert advice taken.
Safe microbiology The practical activities selected in this package and the microorganisms suggested present minimum risk given good practice. It is therefore essential that good microbiology laboratory practice is observed at all times when working with any microbes. There are five areas for consideration when embarking on practical microbiology investigations which make planning ahead essential. 1 Preparation and sterilisation of equipment and culture media. 2 Preparation of microbial cultures as stock culture for future investigations and inoculum for current investigation. 3 Inoculation of the medium with the prepared culture. 4 Incubation of cultures and sampling during growth. 5 Sterilisation and safe disposal of all cultures and decontamination of all contaminated equipment. Good organisational skills and a disciplined approach ensure that every activity is performed both safely and successfully.
Protection Food or drink should not be stored or consumed in a laboratory that is used for microbiology. One should not lick labels, apply cosmetics, chew gum, suck pens or pencils or smoke in the laboratory. Hands should be washed with disinfectant soap after handling microbial cultures and whenever leaving the laboratory. If hand contamination is suspected then the hands should be washed immediately with disinfectant soap. To ensure that any wounds, cuts or abrasions do not get infected or infection is passed on, protect them by the use of waterproof dressings or wear disposable surgical gloves.
General personal safety Each individual embarking on these or any other microbial investigation is responsible for their own safety and also for the safety of others affected by their work (other students, technicians, teachers). The individual must include in the planning and performance of the investigation a risk assessment to assess any hazard that the investigation may pose and ways of
18
minimising it. Points to consider include safe storage and culturing of microorganisms, emergency procedures such as dealing with spillages and safe disposal of all contaminated material. No one should perform any microbiological procedures without receiving appropriate training from a competent person. To minimise the chance of contamination of the user, any other individual, the environment or the microbial culture good laboratory practice is required. GLP requires us to consider all cultures as potentially pathogenic.
Aseptic technique Sterile equipment and media should be used to transfer and culture microorganisms. Aseptic technique should be observed whenever microorganisms are transferred from one container to another. Contaminated equipment should preferably be heat sterilised by either incineration or autoclaving. A suitable chemical disinfectant can be used but this may not ensure complete sterilisation.
Electrical safety Many of the investigations use bioreactors that require aeration and this is usually supplied by the use of an aquarium air pump. Care should be taken to ensure that no liquid comes into contact with electrical mains power. The same care should apply if a magnetic stirrer is to be used to mix the growth medium in a bioreactor.
Glassware Great care must be taken when assembling the glassware for the bioreactor. The insertion of the glass fermentation lock used in some of the investigations requires particular care as it is not laboratory grade glass. Hands should be protected during the insertion of the glass into the bung. Both the bung and the glass should be lubricated with water and either a small amount of silicone grease or washing up liquid. Very gentle twisting should be used to assist fitting but not too much so that it breaks! (See Information 1. The bubble logger)
Organisations able to provide information and advice on aspects of safety in schools and colleges CLEAPSS School Science Services Brunel University, Uxbridge, UB8 3PU Telephone number: 0189 525 1496 Website: http://www.cleapss.org.uk Microbiology in Schools Advisory Committee (MISAC) c/o Society for General Microbiology Marlborough House, Basingstoke Road, Spencers Wood. Reading, RG7 1AE. Website: http://www.sgm.org.uk/PA/edu_car/res_list.htm National Centre for Biotechnology Education, The University of Reading Whiteknights, Reading, RG6 6AJ Telephone number: 0118 987 3743 Website: http://www.reading.ac.uk/NCBE Scottish Schools Equipment Research Centre (SSERC) St. Mary’s Land, 23, Holyrood Road, Edinburgh, EH8 8AE Telephone number: 0131 558 8180 Website: http://www.svtc.org.uk/resources/sserc/
© National Centre for Biotechnology Education / Society for General Microbiology Practical Fermentation 1999
Technical Guide: Practical Fermentation
Resources: Books on Microbiology:
Books on Safety:
Microbial Biotechnology Fundamentals of Applied Microbiology Alexander N. Glazer, Hiroshi Nikaido W. H. Freeman and Company 1994 ISBN 0-7167-2608-4
Safety in Science Education Department for Education and Employment HMSO Publication Centre 1996 ISBN 0-11-270915-X
Principles of Fermentation Technology, 2nd ed. P. F. Stanbury, A. Whitaker and Stephen J. Hall Butterworth Heinemann 1999 ISBN 0-7506-45016 Biotechnology, 2nd ed. A Textbook of Industrial Microbiology Wulf Crueger and Anneliese Crueger Sunderland, M A: Sinauer Associates 1990 ISBN 0-87893-131-7 Biotechnology from A-Z, 2nd ed. William Bains Oxford University Press 1998 ISBN 0-19-963693-1
Living Biology in Schools Institute of Biology Chapter 2 Microbiology Edited by Michael Reiss ISBN 0-900490-32-2 Information and Advice: Microbiology in Schools Advisory Committee (MISAC) c/o Society for General Microbiology Marlborough House, Basingstoke Road, Spencers Wood. Reading, RG7 1AE Books on Statistics:
Power Unseen: How Microbes Rule the World Bernard Dixon W. H. Freeman and Company 1996 ISBN 0-7167-4550-X
Maths for Advanced Biology Alan Cadogan and Robin Sutton Nelson 1994 ISBN 0-17-44 8214-0
Instant Notes in Microbiology J. Nicklin, K. Graeme-Cook, T. Paget and R. Killington βios Scientific Publications 1998 ISBN 1-85996-156-8
Statistics for Science Projects Sallie Powell Hodder and Stoughton 1996 ISBN 0-340-66409-6
Biotechnology of Malting and Brewing J .S. Hough Cambridge University Press 1991 ISBN 0-521-39553-4
Multimedia Resources:
Biotechnology, 3rd edition Studies in Biology John E. Smith Cambridge University Press 1996 ISBN 0-5214-4911-1
Bt media P.O. Box 1017, Kingshurst, Birmingham, B37 6NZ.
The Science of Brewing The Essential Multimedia Guide to the Science of Brewing
Data logging: The NCBE bubble logger was designed to be a very simple and cheap device for monitoring carbon dioxide bubble production from anaerobic fermentations. The bubble logger is very pupil interactive, in that students need to manually record the number of accumulative bubbles produced. With the addition of a suitable data logger or computer it is possible to record the number of bubbles against time. To do this two leads need to be attached to the underside of the circuit board. One lead has to be attached to the base region (OV) of the circuit board and the other lead between the OP-AMP and the LCD counter. The diagram shows how the two leads can be connected. The board can be drilled and leads attached through the front to make a more pleasing and permanent logging device.
PRACTICAL FERMENTATION a guide for school and colleges The National Centre for Biotechnology Education
NCBE
Microbiology In Schools Advisory Committee
The Society for MISAC
General Microbiology
SGM
The National Centre for Biotechnology Education was established as part of the Department of Microbiology at the University of Reading and is now part of the School of Animal and Microbial Sciences. The Centre's principal aim is to encourage and support biotechnology education in schools and colleges. To achieve this, the NCBE produces material and runs training courses for teachers and students. Novel practical work designed especially for the school laboratory is developed at the Centre. Where specialised equipment is required, the Centre endeavours to supply this to schools at a reasonable cost. As biotechnology has become increasingly drawn to the attention of the public, demand for courses and workshops for the adult population has widened the Centre's activities.
The purpose of the Microbiology in Schools Advisory Committee (MISAC) is to promote the teaching of microbiology in schools. Founded in 1969, MISAC consists of representatives of a wide range of educational and scientific bodies who work to help teachers recognise the potential of micro-organisms as educational resources in a variety of ways. In particular, MISAC is a recognised source of authoritative advice on the safe use of microorganisms, prepares practical protocols, produces fact sheets on a range of resources related to the teaching of microbiology, provides a list of speakers on microbiological topics, responds to Government reports and maintains contact with examination boards. The annual MISAC competition for schools is a major activity supported by special sponsorship from a range of professional organisations and industry.
The UK-based Society for General Microbiology (SGM) has over 5000 members throughout the world. Its members are expert scientists who work in all branches of microbiology. An important function of the Society is the promotion of microbiology to the public. To meet this objective the Society produces and distributes resources to support microbiology teaching in schools and colleges, attends events to promote microbiology as a career to school leavers and organises activities to promote the public understanding of microbiology. A wide range of free material is available to schools and the Society also offers an information service to teachers.
Contact: National Centre for Biotechnology Education The University of Reading Whiteknights Reading, RG6 6AP
Contact: MISAC General Secretary, c/o SGM, Marlborough House, Basingstoke Road, Spencers Wood, Reading RG7 1AE
Telephone: 0118 987 3743 Fax: 0118 970 5901 Website http://www.ncbe.reading.ac.uk
Telephone: 0118 988 1800 Fax: 0118 988 5656 E-mail:
[email protected]
Contact: External Relations Office, SGM Marlborough House, Basingstoke Road, Spencers Wood. Reading, RG7 1AE Telephone: 0118 988 1800 Fax: 0118 988 5656 Email:
[email protected]. Website http:/www.socgenmicrobiol.org.uk
NATIONAL CENTRE FOR BIOTECHNOLOGY EDUCATION School of Food Biosciences The University of Reading Whiteknights, Reading, RG6 6AP Tel: 0118 9873743 Fax: 0118 9750140
http://www.ncbe.reading.ac.uk
Published by The Society for General Microbiology © John Schollar and Benedikte Watmore
ISBN 0 9536838 0 X
