School Science Lessons
Biology Experiments - Biotechnology
Updated: 2008-03-01
Please send comments to: J.Elfick@uq.edu.au
See also: Interesting websites
Acknowledgements
WARNING!
The experiments in this document were devised by an international group of science educators and tested with students from schools in Germany.
However, some or all of the experiments may be illegal in your country. Before planning to teach any of the experiments below, you must get permission from the head of your school science department, the principal or head teacher of your school, and the Ministry of Education in your country. Also, you should check that you can follow the safety precautions in Appendix 1 to 3 below. Do not attempt any of the experiments below, apart from J1 or J2, if you have no experience of teaching biotechnology.
Comment: The biosafety advice given to schools in Germany, USA and the UK is significantly different in some aspects to the guidelines and legislation that apply in Australia for working with microbiological organisms (including bacteria, protozoa, fungi / yeast and mould) and genetically modified organisms. In Australia, see:
1. Australian / New Zealand Standard - Safety in laboratories, Part 3: Microbiological aspects and containment facilities (AS / NZS 2243.3:2002).
2. Gene Technology Act 2000, passed by the Federal Government In December 2000. The legislation came into force on 21 June 2001. The legislation is the Commonwealth's component of a new national scheme for the regulation of genetically modified organisms (GMOs), which will include legislation in every Australian jurisdiction. Copies of the Office of the Gene Technology Regulator's Handbook may be obtained from the OGTR.
Table of contents
4.1.0 Microbial systems
4.2.0 Fermentation processes in food production
4.3.0 Using in vitro culture techniques
4.4.0 Biotechnology genetics
4.5.0 Appendices
9.1.2.0 Techniques for studying bacteria
9.196 Fungi
9.209 Bacteria
9.207 Lichens
9.213 Viruses
4.1.0 Microbial systems
4.1.1 Observing colonies of different micro-organisms: fungi, yeasts, protists and Streptomyces bacteria
4.1.2 Enrichment of wild yeast strains
4.1.3 Making a fixed slide preparations
4.1.4 Making an India ink (China ink) preparation
4.1.5 Safe microscopy of Penicillium camemberti and Mucor mucedo using the Petri slide technique
4.1.6 Trace soil bacteria that decompose urea
4.1.7 Produce the antibiotic streptomycin with Streptomyces griseus, Bacillus mycoides, Candida utilis, Escherichia coli, Micrococcus luteus, Pseudomonas fluorescens
4.1.8 Effect of the antibiotic streptomycin on Bacillus subtilis and Saccharomyces cerevisiae using the small disc test
4.1.9 Show the presence of bactericidal substances with a coin and Bacillus mycoides
4.2.0 Fermentation processes in food production
4.2.1 Make yoghurt (activity for primary grade 4 students, about 9 years old)
4.2.2 Make sauerkraut (activity for primary grade 4 students, about 9 years old)
4.2.3 Make lactic acid in sourdough
4.2.4 Make wine from grape juice with Saccharomyces ellipsoideus and make vinegar from wine with Acetobacter sp. and Gluconobacter sp.
4.2.5 Make cider from apple juice
4.2.6 Make vinegar by continuous production with Acetobacter aceti
4.2.7 Microbial decomposition of thin paper, e.g. cigarette paper
4.2.7.1 Enzyme Technology
4.2.7.2 Experiments relating to pectinase in the industrial production of juice
4.2.8 Make apple juice gel when it is boiled
4.2.9 Make pectinase, an enzyme that decomposes pectin
4.2.10 Industrial uses of pectinase
4.2.11 Split lactose from milk or whey by using immobilized lactase
4.3.0 Using in vitro culture techniques
4.3.1 Grow the African violet; Usambara violet (Saintpaulia ionantha) with in vitro culture
4.3.2 Grow the African violet; Usambara violet (Saintpaulia ionantha), from pieces of leaf
4.3.3 Grow gerbera using in vitro culture
4.4.0 Biotechnology and genetics
9.107 Mitosis in onion root tip cells
9.4.2 DNA and RNA
9.6.1 Cultures of fruit fly (Drosophila melanogaster), for heredity experiments
9.6.2 Fruit fly strains
9.6.3 Mendel's 1st law, law of uniformity (inheritance of one pair of characteristics)
9.6.4 Mendel's 1st law, law of uniformity (inheritance of two pairs of characteristics)
9.6.5 Mendel's 2nd law, law of segregation
9.6.6 Mendel's 3rd law, law of independent assortment
9.6.7 Sex-linked inheritance, introduction of a recessive characteristic by female
9.6.9 Genetics, lethal factors
9.6.10 Giant chromosomes
4.4.1 Isolate DNA
4.4.2 Conjugation in bacteria, Escherichia coli
6.9.8a Meiosis in grasshopper testes
4.5.0 Appendices
Appendix 1 - How to produce sterile media or solutions
Appendix 2 - Information on safety issues in school-based biotechnology
Appendix 3A. List of bacteria suitable or unsuitable for use in schools
Appendix 3B. List of fungi suitable or unsuitable for use in schools
Appendix 4 - Chemicals
Appendix 5 - Abbreviations and addresses
1.0 Biology solutions
2.0 Microscopy adhesives
3.0 Microscopy stains
4.0 Biology fixatives
5.0 Standard buffer solutions
6.0 Culture media for routine cultivation and identification of fungi 
19.2.11 Yeast for fermentation and brewing
4.6.0 List of sterile media or solutions
9.1.2.0 Techniques for studying bacteria
9.1.2.1 Prepare stains
9.1.2.2 Make a simple staining rack
9.1.2.3 Aseptic transfer of bacterial cultures from a bottle or tube
9.1.2.4 Aseptic transfer of bacterial cultures from a culture plate
9.1.2.5 Prepare a heat-fixed stained bacterial smear
9.1.2.6 Gram stain
9.1.2.7 Streak dilution plate method for obtaining pure cultures from a mixed suspension
9.1.2.8 Lawn plate technique
9.1.2.9 Spread plate technique
9.1.2.10 Serial decimal dilution of a bacterial suspension
4.6.0 List of sterile media or solutions
20% "Domestos" solution
Agar media
BAP medium
Basal agar medium
Basal broth medium
Buffer reagent
Glucose nutrient agar
Liquid broth media
Malt agar medium
Minimal agar medium
MS agar medium
Nutrient agar medium
Nutrient broth medium
Ringer solution
Salt solution
Sterile solutions
Urea agar medium
Vinegar bacteria medium
4.1.1 Observing colonies of different micro-organisms: fungi, yeasts, protists and Streptomyces bacteria
See diagram 20.114: Required equipment - colonies
Moulds usually form a soft, stringy colony. Colourless mycelia may also grow below the surface of the agar medium. The aerial mycelium of Penicillium roqueforti usually has blue green spores. Spores of other fungi are also coloured brown / yellow or black. The diameter of a single colony is usually more than 10 mm.
Yeasts or Bacteria, other than Streptomyces, have shiny matt or slimy colonies often above the surface of the agar. The colonies are often grey / yellow. Bacillus subtilis bacteria colonies are usually grey. Yeasts may be grey, red, orange, yellow, brown. The diameter of a single colony is usually less than 10 mm.
Streptomyces bacteria have an earthy smell. In the Petri dish, a truncated, round, single colony less than 3 mm in diameter forms that grows in the shape of a lens. The aerial mycelium is often coloured after 6 days incubation. Streptomyces griseus produces a grey aerial mycelium. Other Streptomyces stain the surrounding agar brown.
Equipment: 1 Bunsen burner, 1 inoculation loop, 1 conical flask, 250 mL
Materials: 100 mL basal agar medium (see appendix), 3 Petri dishes, 1 felt tip pen, 50 mL basal broth medium (see appendix), cultures of the following micro-organisms, incubated overnight:
Penicillium roqueforti (DSM-No. 1079), Rhodotorula rubra (DSM-No. 70403), Streptomyces griseus (DSM-No. 40236, ATCC 23345), Bacillus subtilis (DSM-No. 1079, ATCC 6051)
Time needs:
1. Prepare overnight cultures in basal broth medium - 45 minutes.
2. Incubation of overnight cultures - 24 hours.
3. Inoculation -15 minutes, 4. Incubation - 6 days
Procedure:
1. On the day before the investigation, prepare cultures of the four species of micro-organisms and incubate overnight.
2. Prepare 100 mL of basal agar medium in 3 agar plates.
3. Divide the plates into 4 sectors on the underside, using a felt tip pen.
4. Inoculate each of the 4 sectors at one point with one of the 4 types of micro-organism.
5. Incubate the plates at 30oC for 6 days.
6. Identify the colonies.
4.1.2 Enrichment of wild yeast strains
You can produce yeast cultures derived from natural isolates within closed Petri dishes for demonstration purposes. However, for safety reasons they should not be used for further experiments. Some moulds spoil food but others can be used in the production of food, e.g. Camembert cheese, Indonesian bean cake tempeh, soya bean cheese. However, there are considerable safety risks in the open microscopy of mould. For example if a student who accidentally coughs into a spore culture can propel large numbers of spores into the air such as Aspergillus niger that can infect the respiratory tract and may be fatal for those whose immune system has been weakened. The Petri slide procedure for safe microscopy of mould avoids these risks.
Equipment: Petri dishes
Materials: Malt agar medium (see appendix), unwashed apple, or another piece of fruit with sugar content
Time needs: 45 minutes
Procedure:
1. Prepare malt agar plates according to the instructions in the appendix.
2. Roll a piece of unwashed fruit across the surface of the chilled agar, close the dishes immediately. Incubate the plates for a few days at 30oC.
3. Observe different yeast types. Some yeasts are brilliantly coloured. Some mould cultures can be recognized by their cotton-like texture.
4.1.3 Making a fixed-slide preparations
See Diagram 20.120: 1. Put a cover slide on a microscope slide
See Diagram 20.120: 2. Spreading a drop of liquid
Focussing sharply on living bacteria is difficult, so they are almost always observed in fixed preparations and stained.
Equipment: 1 microscope slide, 1 coverslip, 1 eye dropper, 1 Bunsen burner
Materials: Ethanol
Time need: 30 minutes
Procedure:
1. Remove grease carefully from a microscope slide with a lint free towel or a piece of tissue soaked in ethanol.
2. Place a drop of bacteria or yeast suspension in the middle of the microscope slide. The drop should flow out evenly and must not remain in globular form. The suspension eventually will flow back together, even when only traces of grease are present on the slide. This not only lengthens the drying time, but allows thick layers of bacteria to develop, as well. So it may no longer be possible to observe individual microorganisms. If attempts to remove grease from the slide are unsuccessful, use a drop of extremely diluted bacterial suspension and allow to air dry in place of the smear technique. This method is easier than executing a smear with the delicate coverslip.
3. Place a coverslip on the microscope slide at an angle of 45o so that the solution is collected in the space between the slide and slip and held by the properties of adhesion and cohesion.
It is important to pull and not push the suspension across the slide with the coverslip to ensure that the thickness of the coating decreases evenly.
4. Push the coverslip evenly across the entire surface of the microscope slide. This spreads the suspension across the slide, and the film of liquid becomes thinner.
This drying step must not be accelerated by heating. Bacterial structure changes when bacteria are heated in water.
5. Allow the smear to dry.
The first method is recommended. Because of the poor conducting qualities of glass, it is difficult to estimate the effect of heating on the bacteria with the second method. Organisms and the protein coagulated in the cells by heating adhere to the slide surface.
6. Fix the bacteria to the slide by briefly heating the slide in a flame. This can be done in one of two ways: with a low flame such as the pilot flame of a Bunsen burner and with the coated side of the slide oriented downwards, or with the high flame of a Bunsen burner and the coated side of the slide up. Pass the slide through the flame three times at a speed of roughly 30 cm per second.
4.1.4 Making an India ink preparation
If you stain the background uniformly black by the use of India ink, only the organisms are illuminated in the microscope, and they appear in bright contrast to the background.
Equipment: 2 microscope slides, 1 coverslip, 1 eye dropper,
Materials: India ink, ethanol
Time needs: 30 minutes
Procedure:
1 Thoroughly clean two microscope slides by wiping them with a lint free towel or a tissue soaked in ethanol.
2. Place a small drop of water on the microscope slide. The drop must spread out, otherwise further measures are necessary to remove grease from the slide.
3. Use a glass rod to mix a drop of India ink with the evenly spread drop of water.
4. Place a coverslip at a 45o angle on the microscope slide in such a manner that the solution is collected in the space between the slide and slip and held by the properties of adhesion and cohesion.
5. Push the coverslip evenly across the entire surface of the microscope slide. The suspension is thus spread across the slide. The thickness of the film of liquid decreases.
6. Allow the smear to air dry.
7. Prepare a second smear, using a drop of bacterial or yeast suspension instead of a drop of water.
8. Compare both smears are then compared under a microscope set at 400 X magnification. Open the condenser completely and use the brightest possible light source.
9. If observing bacteria with a microscope for the first time, prepare a control slide for comparison.
4.1.5 Safe microscopy of Penicillium camemberti and Mucor mucedo using the Petri slide technique
See diagram 20.120 (3): Inoculate a culture medium with fungal spores
See diagram 20.120 (4): Remove fungal spores from a pure culture
See diagram 9.202: Penicillium, Mucor
Petri slides are extremely flat, disposable Petri dishes that can be sealed tightly, height = 6 mm, inner diameter = 47 mm, area of the base plate = 52 x 75 mm. They are designed for counting microorganisms. Unknown microorganisms are placed on nutrient cardboard discs or membrane filters, and colonies are cultivated inside the chamber. The construction of the chambers also enables both the safe microscopy of sealed fungal cultures and the microscopy of sporangia from the side. A packet of Petri slides containing 100 slides can be ordered from laboratory suppliers. Petri slide cultures can be kept for several weeks. Basal agar medium is suitable for the cultivation of various moulds. Fungi do grow on other nutrients such as glucose nutrient agar, but in such cases they do grow more slowly.
Equipment: 1 autoclave or pressure cooker, 2 Petri slides, 1 Bunsen burner, 1 Pasteur pipette, sterile, 1 conical flask, 300 mL, wide necked, 1 inoculation needle, household aluminium foil
Materials: basal agar medium, 200 mL (see appendix), or glucose nutrient agar, 200 mL (see appendix), 1 M HCl, 1 pure culture of Penicillium camemberti (DSM-No. 1995), 1 pure culture of Mucor mucedo (DSM-No. 809, ATCC 38693)
Time needs: 45 minutes, inoculation of the Petri slides: 15 minutes
Procedure:
1. Place the culture medium in a conical flask, seal with aluminium foil, and autoclave in a pressure cooker. The time required for sterilization is 20 minutes after the sealing of the pressure valve. The Petri slides do not have to be sterilized, as they remain germ free inside during the production process. Sterilize Pasteur pipettes by wrapping them in aluminium foil and heating them to 180oC for 30 minutes in a drying cabinet.
2. Remove the aluminium foil from the conical flask. Fill a sterile Pasteur pipette with 5 mL of sterilized culture medium. Do not touch the tip of the pipette. Hold the Petri slide chamber upright between the thumb and index finger of the left hand. Lift the lid up far enough to reveal the side of the base of the filling hole. Introduce the tip of the pipette through the hole in the side into the middle of the chamber. The tip must not touch the outer parts of the chamber. Carefully pipette 3 mL of culture medium into the vertically held chamber without smearing the upper part of the chamber with culture medium.
3. Remove the Pasteur pipette from the chamber and replace the lid of the chamber. Ensure that the chamber remains vertical as the agar sets. Refill the pipette and prepare additional chambers in a similar manner. Pass the tip of the pipette through a Bunsen flame periodically.
4. After the agar has set inside the chambers for about 30 minutes, inoculate the chambers with different fungi. Use an inoculation needle that is sterilized by heating in the Bunsen burner flame and held to the sporangia of a pure culture of mould.
5. Open a Petri slide in the usual way. Insert the needle through the hole until the pointed end transfers the spores that adhere to it by contact with the surface of the agar.
6. Close the chamber again and incubate it for about a week in a vertical position at room temperature. Individual sporangia can be seen clearly even with microscopy using transmitted light. One can "take an optical walk" through the about 5 mm wide sporangia "wood" by using the fine focussing apparatus. Differences in the sporangia at the edge of the colony and in the middle can be seen clearly, and the mass of hypha in the nutrient agar can be examined up to its finest traces. The Petri slide cultures can be kept for several weeks in a dark cabinet at room temperature. It is inadvisable to keep them in a refrigerator, as the cold chambers become slightly steamed up if they are used again. The cultures hardly dry out at room temperature, and the fungi stops growing one or two weeks after they are inoculated because they lack oxygen and nutrients.
7. Place the Petri slides flat onto the stage of the microscope once the cultures have grown, examine with one of the two low power objectives (10 x or 40 x).
Notes:
1. Mouldy fruit or bread should not be examined using open microscopy because the types of mould that grow on them are often of the genera Penicillium and Aspergillus. The spores of Aspergillus may be harmful if students inhale them. Also, Aspergillus flavus (and Aspergillus parasiticus) produce aflatoxins, toxic and carcinogenic mycotoxins.
2. It is impossible to find the genus of a fungal colony and compare various species of mould by microscopic examination from above because the sporangia, which may be used to distinguish one genus from another, can only be seen from the side.
3. The tip of the pipette must not touch the outside of the Petri slide when the slide is being filled with agar because the pipette is contaminated with bacteria or fungal spores from the environment so the spores soon begin to sprout and grow in the sterilized agar. Any additional experiment with a nutrient base that has been contaminated in this way is invalid.
4.1.6 Trace soil bacteria that decompose urea
See diagram 20.120 (5): Proper use of Drigalski spatula
This experiment shows the importance of soil bacteria as decomposers of urea.
Equipment: Drigalski spatula, incubator, 10 basal agar medium plates (see appendix), 5 urea agar plates (see appendix), 1 beaker, 400 mL, 2 Bunsen burners, 1 graduated cylinder, 10 mL, 1 eye dropper, sterile, 1 glass plate, 6 test-tubes, sterile, 1 waterproof felt tip marking pen
Materials: 1 g soil, ringer solution, sterile, 500 mL (see appendix) or salt solution (see appendix) to dilute the micro-organism suspension
Time needs: steps 1 - 8: 45 minutes
Procedure:
1. Label six sterile test-tubes in series as follows: 10-1, 10-2, 10-3, 10-4, 10-5, 10-6.
2. Label the undersides of the agar dishes, not the lids, in series as follows: Nutrient plates: K, 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, urea agar plates: K, 10-1, 10-2, 10-3, 10-4.
3. Pipette 10 mL of the Ringer solution into the test-tube labelled 10-1. It is important to begin with the control sample because the sterile water is later needed for rinsing and so becomes contaminated
4. Sterilize the Drigalski spatula with ethanol. Apply a drop of sterile Ringer solution to the plate labelled "K". Spread the solution evenly over the surface of the dish with the sterilized spatula.
5. Add one gram of soil to the test-tube labelled 10-1. Mix thoroughly by shaking and by rolling the test-tube back and forth between the palm of the hands. Apply one drop of the 10-1 suspension to each of the two plates labelled 10-1, spread the suspension evenly with the resterilized spatula (according to step 5). Finally, rinse the pipette with sterile water.
6. Transfer one drop from the test-tube labelled 10-1 to the test-tube labelled 10-2, mix carefully again. Distribute one drop of the 10-1 suspension evenly on each of the two Petri dishes labelled 10-2.
7. Proceed in the same manner until all other test-tubes are filled, each with one drop of suspension from the previous tube and nine drops of water, and until all Petri dishes are inoculated according to their respective labels.
8. Incubate the dishes for seven days at 30oC.
9. Count the colonies as soon as they can be easily recognized. Make complete counts of plates with 50 to 200 colonies.
10. To find the titre of the suspension, one must find the volume of one drop. For this purpose, fill a small graduated cylinder with drops (n) to a volume of 2 mL. The volume (V) of a drop is derived according to the following formula: V = 2 / m mL. For example if 32 drops are necessary to attain a volume of 2 mL, the volume of a drop, V = 2 / 32 mL = 0.06 mL.
11. Divide the number of counted colonies by the dilution factor. The results indicate the number of viable cells in one drop of the suspension. When this number is later divided by the drop volume (V), the number of viable cells in 1 mL of the suspension is obtained. For example: 186 colonies were counted on the plate labelled 10-1. The volume of a drop is 0.06 mL. The number of colonies is divided by the dilution factor: N = 186 cells / 10-4. Divide by the volume (V) of drops: N = 1.86 x 10-6 cells / 0.06 mL = 3.1 x 10-7 cells per mL. As with yeast, bacteria are transferred in conglomerates. They must be separated from each other because they will otherwise not disperse evenly in the suspension. The bacterial colonies on plates that have been diluted very little (10-3, 10-4) are smaller than colonies on plates that have been diluted a great deal (10-6, 10-7) because the bacterial colonies compete for the nutrients in the agar. On plates where the concentration is high, more colonies develop but they are more prone to "starve" than few colonies on plates that are very dilute. Fewer colonies grow on urea agar plates than on basal medium plates at a comparable concentration because only very few microorganisms possess urease and can use urea as a source of carbohydrates for biosynthesis. Most can, however, use glucose, which is present in large quantities in the basal medium. The decomposition of urea is visible by means of a red indicator zone. During the decomposition of urea, a basic ammonia is excreted from the bacterial cells and released to the environment.
4.1.7 Produce the antibiotic streptomycin with Streptomyces griseus
The following investigation employs Streptomyces griseus that produces the antibiotic streptomycin. Only those test organisms that are not sensitive to streptomycin can grow on the same culture medium plate as Streptomyces griseus. The use of Streptomyces or streptomycin is possible in school experiments because this antibiotic is no longer used in medicine, and the possible spread of resistant strains is no longer problematic from a medical point of view.
Equipment: 1 autoclave, 1 incubator, 1 Bunsen burner, 8 disposable Petri dishes, 2 sterile 300 mL conical flasks with sterile bungs, 1 inoculating loop, 6 culture tubes, 5 x 5 mL sterile pipettes, pipette aid
Materials: Pure culture of Streptomyces griseus (DSM-NO. 40236, ATCC 23345), a selection of the following pure culture strains as test organisms: Bacillus mycoides (DSM-No. 10, ATCC 6051), Candida utilis (DSM-No. 2361, ATCC 9950), Escherichia coli K-12 (DSM-No. 498, ATCC 23716), Micrococcus luteus (DSM-No. 20030, ATCC 4698), Pseudomonas fluorescens (DSM-No. 50090, ATCC 13525), distilled water, basal broth medium for overnight cultures, 100 mL (see appendix), basal agar medium for Petri dishes, 200 mL (see appendix)
Time needs: preparation and autoclaving of the solutions: 45 minutes, preparing the precultures: 15 minutes, waiting time: 24 hours, distribution of the test organism cultures: 16 minutes
Preparation: Prepare and autoclave the culture media. Place the basal agar medium into eight Petri dishes, 20 to 25 mL per plate. Suspend again the culture of Streptomyces griseus in 1 mL of sterile basal broth medium according to the manufacturer's instructions, pipette into a test-tube into which 5 mL sterile culture medium has been placed. Incubate for 24 hours in an incubator at 30oC, overnight culture.
Procedure:
1. Inoculate the basal agar media in the Petri dishes with the overnight culture of Streptomyces griseus. Sterilize an inoculation loop by passing it through a Bunsen burner flame, allow the loop to cool, then dip into the culture. Streak the germs that adhere to the loop onto the culture medium as a vertical line as far over to the right as possible so that the left part of the surface of the culture medium remains sterile. Sterilize the inoculation loop by passing it through a flame once more.
2. Incubate the plates for two days at 30oC.
3. In the interim, after incubating for 24 hours, prepare the overnight cultures of the selected organism, Bacillus mycoides, Candida utilis, Escherichia coli K-12, Micrococcus luteus, Pseudomonas fluorescens, using the same procedure as for the overnight culture of Streptomyces griseus.
4. Once the incubation time has elapsed, inoculate the culture media from four Petri dishes on which the Streptomyces sp. cultures are now visibly growing with the test organism, use an inoculation loop, as in step 1, to make horizontal streaks on the empty part of the medium, the left side of the dish. The streaks must always be vertical to the Streptomyces sp. culture and be drawn close up to the edge of it, but it must not be touched or contamination will occur. Keep cultures overnight.
5. Incubate the plates for two days at 30oC and keep them in a cool place for a day longer.
6. In the interim, after incubating for 24 hours, inoculate the culture media of the other four Petri dishes, on which the Streptomyces cultures are now even more vigorous, with the test organisms using an inoculation loop as in step 4.
7. Incubate the plates for two days at 30o3.
8. Compare and assess the first and second set of four plates. Some test organisms grow in the vicinity of the Streptomyces sp. culture and some do not. Streptomyces griseus produces an antibiotic, streptomycin, that diffuses into the culture medium. Some organisms, e.g. Bacillus mycoides, Escherichia coli, Micrococcus luteus, are sensitive, i.e. they are killed off by the antibiotic at a certain concentration. Other organisms are resistant, e.g. the yeast Candida utilis, are not susceptible to streptomycin because they are eucaryotes. The distance between sensitive test organisms and the Streptomyces culture is larger in older streptomyces cultures because production of antibiotic increases in the older cultures.
4.1.8 Effect of the antibiotuic streptomycin on Bacillus subtilis using the small disc test
The effectiveness of species of fungus to release antibiotics into the environment can be tested by using the small disc test.
Equipment: Petri dishes: 1 paper punch, or pair of scissors, 1 pair of tweezers, 1 Bunsen burner, 1 small glass beaker, 1 large glass beaker as a water bath, needles, corks and glass beaker or test-tubes with rubber bungs, filter paper, aluminium foil
Materials: Pure culture of Bacillus subtilis (DSM-No. 10, ATCC 6051), a yeast suspension that was incubated overnight, 1 g yeast to 100 mL water, nutrient agar medium, 200 mL (see appendix), malt agar medium, 200 mL (see appendix), streptomycin or another antibiotic, ethanol
Procedure:
1. Prepare nutrient agar as a bacterial culture medium and malt agar as a yeast culture medium
2. Sterilize the culture media and cool them in a water bath to 40oC, keep them at this temperature. These resistant microorganisms cannot damage our health because Streptomycin is no longer used in medicine.
3. Add 1 mL of a culture of Bacillus subtilis or yeast suspension (which was incubated overnight) per 200 mL to the still liquid culture medium, close the conical flask and mix the contents vigorously. Cooling is necessary because the hot agar would damage the organisms. If the agar is left to cool without putting it into a water bath, it sets too quickly.
4. Pour the culture medium that is inoculated in this way into Petri dishes and allow it to set.
5. Use a hole punch or a pair of scissors to produce small discs (0 = 5 mm) from a sheet of filter paper.
6. Sterilize two small discs by placing each one on a needle and then into 96% ethanol overnight, or by sticking them into a cork and placing them in a glass beaker sealed with aluminium foil and then into a drying cabinet at 135oC for three hours. Exposure to ultraviolet light for ten minutes will also work.
7. Produce a solution of streptomycin in sterile water. The concentration of the pure agent should be about 50 mg / mL.
8. Dip the sterile small discs into the antibiotic solution and dry them in a drying cabinet at 10o3. Dip at least one small disc into sterile water free of antibiotic, as a control. If the damp paper discs were placed onto the agar, the agents would begin to diffuse uncontrollably.
9. Use a sterilized pair of tweezers to place each of the dried antibiotic plates onto an inoculated labelled agar plate. As a control, use at least one petri dish without a small disc. The addition of small paper control discs shows that filter paper itself does not contain any substances that inhibit the growth of bacteria. The control plate without the small paper discs shows that a completely uniform bacterial lawn develops on an untreated plate.
10. Incubate the closed Petri dishes at 30oC for two days. A uniform bacterial or yeast lawn should be present on all of the control plates, and a circular zone free of bacteria should be visible around the small discs that contained antibiotics. The yeast is not affected by the antibiotics. Compare the streptomycin sensitivity of microbial strains (a) Bacillus subtilis (b) Saccharomyces cerevisiae. The antibiotic gradually diffuses into the agar. The diameter of the zone in which no bacteria grow is a measure of the concentration of the agent. If two different biocatalyst solutions of a similar concentration were used, the diameter of the bacteria free zone. The significance of colonies that grow within an antibiotic bacteria free zone is that each of them is formed from one mutant of the bacterium that is resistant to the antibiotic.
4.1.9 Show the presence of bactericidal substances with a coin and Bacillus mycoides
If a coin is placed onto a culture medium that is uniformly inoculated with Bacillus mycoides, a bacterial lawn grows with a bacterial free zone around the coin. The coin may consist of German silver, an alloy of copper, nickel, and zinc. Their metal anions kill cells of Bacillus mycoides by inhibiting growth and division. From the side, it is obvious that the colony is more dense at the edge of the zone than in the rest of the bacterial lawn. The metal anions encourage growth in small quantities. An area of resistant microorganisms is often formed in the immediate vicinity of the coin. These microorganisms can be traced to the coin and have become enriched in the course of time. They are resistant to ions of heavy metals. The demonstration is therefore also indicative of the pressure of selection that bactericides exert on a population of microorganisms. This problem occurs quite frequently in hospitals, where certain microorganisms suddenly occur in large numbers, e.g. the bacterium Serratia marcescens.
Inhibiting and encouraging growth of microorganisms by the use of bactericidal substances. the formation of resistance mutation selection
Equipment: 1 autoclave, 1 incubator, 1 Bunsen burner, 4 disposable Petri dishes, 1300 mL sterile conical flask, bung, 6 culture tubes, adhesive tape for sealing the Petri dishes 5 x 5 mL sterile pipettes
Material: pure culture of Bacillus mycoides (DSM-No. 2048, ATCC 6462), distilled water, 100 mL basal broth medium for cultures incubated overnight (see appendix), 100 mL basal agar medium (see appendix)
Time needs: preparation and autoclaving of the nutrient solution: 45 minutes, preparing the overnight culture: 15 minutes, waiting time: 48 hours
Preparation: Prepare and autoclave the basal broth medium. Suspend again the culture of Bacillus mycoides in 1 mL of sterile liquid basal medium according to the manufacturer's instruction, pipette this into a test-tube previously filled with 5 nil of the sterile liquid basal medium. Incubate for 24 hours at 30oC, culture incubated overnight.
Procedure:
1. Place the basal agar medium into conical flasks, autoclave and cool to 45oC under running water. The approximate temperature has been reached if you can hold the warm conical flasks to the back of your hand with no unpleasant sensation, back-of-hand test.
2. Add the culture that was incubated overnight and mix well with the culture medium by swirling the contents of the flask.
3. Pour the inoculated culture medium into four Petri dishes.
4. Once the agar is set, place a coin onto the surface of the culture medium in the middle of the Petri dish.
5. Close the Petri dishes and seal them with adhesive tape. The Petri dishes must be protected against accidental opening, and must be sealed because microorganisms that may grow on the coin, and possibly on the culture medium, are unknown, any risk that wild strains may pose are avoided in this way.
4.2.1 Make yoghurt (activity for for primary grade 4 students, about 9 years old)
Equipment: 1 balloon whisk or one wooden spoon, 20 cups or glasses, 1 oven ring, 1 saucepan, 20 teaspoons, 1 thermometer (100oC), incubator or insulated box made out of polystyrene foam (dimensions: 20 cm high x 35 cm long x 30 cm wide, thickness of the polystyrene: 6 cm) or "yoghurt machine"
Materials: 3 litres of milk, 3 beakers of yoghurt made from whole milk, cling film
Procedure:
1. Heat the milk to 72oC to kill any harmful bacteria in the milk.
2. Allow the milk to cool to 45oC.
3. Place a teaspoon of yoghurt and lactic acid bacteria into a beaker.
4. Add the cooled milk to the beaker.
5. Mix all of the ingredients.
6. Cover the beaker with cling film.
7. Place the yoghurt mixture into an insulated box.
8. After several hours the milk has thickened. The yoghurt is ready. It tastes acidic.
4.2.2 Make sauerkraut activity for (for primary grade 4 students, about 9 years old)
Equipment: 1 bowl, diameter 30 cm, chopping board, kilner jar, 2 mL, with rubber ring, lid, and clasp, 4 kitchen knives, 1 wooden cylinder, 43 cm, or 1 egg cup
Materials: 1 large cabbage, 20 - 40 g salt
Procedure:
1. Cut a white cabbage into strips on a chopping board.
2. Put the chopped cabbage into a bowl, together with the salt.
3. Mix together well the cabbage and the salt.
4. Put the salted cabbage into a kilner jar.
5. Press the cabbage together well with your fist.
6. Press a wooden cylinder or an egg cup onto the cabbage, attach the lid, and close the kilner jar with a clasp. Tell the students that the cabbage must remain like this for about two weeks, until sauerkraut has been formed. They can taste the sauerkraut at that time.
4.2.3 Make lactic acid in sourdough
See diagram 20.162: Making sourdough in glass beakers
Egyptians invented sour dough bread 3,500 years ago. They observed that dough made from rye flour can ferment and be used to bake light piquant bread. They could produce large quantities of sourdough from a small amount so they always saved a small amount of dough for next time. The souring of the rye flour is caused by consecutive fermenting of the dough by two groups of microorganisms: yeasts and lactic acid bacteria. Yeasts of the genera Saccharomyces and Kluyveromyces, together with lactic acid bacteria of the genera Lactobacillus and Lactococcus, stick to the grain and get into the flour in this way. Sourdough is made by mixing rye flour with water. The organisms take up their activity and enrich the "dough" in their substrate. Repeat inoculation of fresh dough with this culture encourages the yeasts to grow first and the lactic acid bacteria to grow later. During the growth of the yeast, the volume of the dough greatly increases and the dough smells of alcohol. After the third inoculation, you can measure the souring of the dough, pH 4.5. The sour dough contains mainly lactic acid bacteria.
Equipment: aluminium foil, 1 measuring cylinder, 100 mL, 1 felt tip pen, waterproof, 6 glass beakers, 400 mL, 1 shallow plastic bowl, 15 x 30 cm, 1 set of scales, 1 spatula, 1 thermometer, 50oC, 1 wooden spoon
Materials: rye flour, type 1250, warm tap water, 40oC, pH paper (3.5 pH - 5.5 pH
Time needs: mixing of the dough: 15 minutes, inoculating: 2 x 5 minutes waiting time: 3 x 24 hours
Procedure:
1. On the first day, mix dough made from 100 g of rye flour and 100 mL water, 40oC, with the spoon. Put the dough into the first glass beaker (dough 1), seal the beaker with aluminium foil and place it in a safe place at room temperature. Step 1. Spontaneous growth of the bacteria contained in the flour requires the addition of sufficient water of the right temperature, 40oC, and standing time. Constant humidity and temperature also are necessary. The lactic acid bacteria can develop their activity in the rye flour particularly well because rye contains very little gluten protein, in contrast to wheat. Dough made from wheat flour only "ferments" if bakers' yeast is added to it.
2. On the next day, prepare another dough as described in step 1, put it into a glass beaker (dough 2). Mix a quarter of dough 1 (from the day before) with 75 g of rye flour and 75 mL warm tap water, 40o3. Place this into a glass beaker (dough 3). Seal the glass beakers (dough 2 and dough 3) with aluminium foil and place them in a safe place. The rest of dough 1 is no longer required and can be put on the compost heap.
3. Three glass beakers are required on the third day. Fill the first with fresh dough prepared as described in step 1 (dough 4). Place a mixture of 75 g rye flour and 75 mL water, 40oC, into the plastic bowl which has been washed, add 50 g of dough 2. Place this mixture into the second glass beaker (dough 5). Finally, place a mixture of 75 g rye flour and 75 mL water, 40oC, into the plastic bowl which has been washed, add 50 g of dough 3. Place this mixture into the third glass beaker (dough 6). Seal the three (dough 4, dough 5, dough 6) glass beakers with aluminium foil and put them in a safe place. So dough 2 and dough 3 are no longer required and can be discarded. Steps 2 and 3. Fresh dough is prepared repeatedly because sourdough of various ages should be available for comparison on the third day. Water must be at the right temperature because the dough being prepared requires a specific temperature to promote the growth of yeasts and lactic acid bacteria. Dough 5 take up more space than dough 4 and dough 6 when the pH continuously increases from dough 4 to dough 6 because of the activity of the yeast cells that form carbon dioxide gas. The growth of the yeast is reduced as the dough becomes increasingly acidic. The lactic acid bacteria become increasingly more enriched after the dough has been inoculated several times.
4. After three hours, measure the volume of dough 4, 5, and 6, appraise the smell, and measure the pH with pH paper.
4.2.4 Make wine from grape juice and make vinegar from wine
The types of yeast that cause alcoholic fermentation belong to the genus Saccharomyces and can always be isolated from ripe fruit. Nowadays, the production of wine employs strains of Saccharomyces ellipsoideus, which is closely related to the brewers' yeast or bakers' yeast Saccharomyces cerevisiae. During this process, the fruit sugar is converted to ethanol and carbon dioxide. Certain bacteria, e.g. the genera Acetobacter and Gluconobacter, can oxidize ethanol to acetic acid. via intermediate stages. In the past, vinegar was produced at home. An industrial procedure for the production of vinegar was developed in the fourteenth century in the area of Orleans, France: one part of mash and one part of fresh wine vinegar were, put into wooden casks, which were lying on their sides, as a "starter." In later techniques, the vinegar bacteria were placed onto wooden lattices or beech shavings to encourage them to expand. These techniques, Fessel procedure, were eventually developed to such a degree that a solution containing alcohol was dripped onto the container that was filled with beech shavings from above, while a counter current of air was guided over the shavings from below.
4.2.5 Make cider from apple juice
Pure culture yeasts must be used for wine making because the fermentation of wild wine yeasts is unpredictable.
Disinfecting effect. The demijohn must not be filled to the top because the carbon dioxide produced by the fermentation of alcohol can form several litres of foam together with the yeast cells. These can be. pushed through the air lock and out of the demijohn. As a safety precaution, the demijohn should never be kept on a surface that must remain clean. During fermentation, the formation of carbon dioxide creates excess pressure in the demijohn. The water contained in the air lock prevents large amounts of oxygen from entering the demijohn and encouraging the growth of vinegar bacteria.
Equipment: 1 rubber tube, internal diameter 5 mm, 1 rubber bung with air lock, 1 household funnel, 1 demijohn, 2 litres
Materials: 250 g granulated sugar, 2 x 0.7 litre bottles of unclarified apple juice, 1 package of wine yeast
Time needs: starting and inoculating the wine: 5 minutes, fermentation time: 6 months
Procedure:
1. Place 240 g granulated sugar into the demijohn, dry funnel, add a bottle of apple juice. Dissolve the sugar by carefully swirling the demijohn from side to side.
2. Add the wine yeast with the second bottle and swirl it round.
3. Seal the demijohn with a rubber bung and an air lock that is filled with water.
4. A cloudy development in the fermentation gases is visible after three days. Vigorous fermentation recedes after ten days.
5. After about six months the yeast has sunk to the base and the fresh wine appears clear. Use a piece of rubber tubing to siphon the wine off
from the yeast.
Step 1. The alcohol content determines the life of wine to a large degree. In Germany, table wines with an alcohol content of 8% by volume generally have to be preserved by the addition of sulfurous acid or potassium pyrosulfate but the wine called "port" with 15% alcohol by volume has a disinfecting effect so it preserves itself. Sugar must be added to achieve a high concentration of alcohol. Cider can be made from industrially produced apple juice in an extremely simple way, as it contains almost no pectin, but sufficient acid. Pectin might cause the fermenting wine to set or might prevent the deposition of particulate matter. Wines that contain very little acid, e.g. pear wine, often taste insipid and do not produce the esters necessary for good bouquet. So you do not need to clarify the wine to remove particulate matter that is linked to the pectin or to acidify the wine artificially. Also, you do not need to add yeast nutrient salt that contains nitrogen because apple "must" contains sufficient nitrogen compounds.
Step 2. At the beginning of the fermentation process, the respiration processes of microorganisms creates negative pressure in the demijohn. This must not be allowed to last for longer than three days. If the yeast culture does not grow, the juice must be inoculated again.
4.2.6 Make vinegar by continuous production
Continuous production of vinegar
Equipment: aluminium foil, 1 glass tube, 1 aquarium pump, 1 glass tube, right angled, 1 one way tap, right angled, 2 pipettes, 5 mL, sterile, 1 conical flask, 500 mL pipetting aids, 1 culture tube, 1 rubber bung, single bored, 2 glass bottles, 1.5 litres with stopper attachments at their bases, 1 rubber bung, double bored, rubber tubing, stand material
Materials: pure culture of Acetobacter aceti (DSM-No. 3508), beech tree shavings, cotton wool, distilled water, sterilized distilled water, 750 mL, wine (cider or unsulfured port), 250 mL, 1 M NaOH
Time needs: preparation and autoclaving of the solutions: 45 minutes, preparing the culture: 15 minutes, waiting time: 48 hours, constructing of fermenter and preparing main cultures: 45 minutes
Preparation: Suspend again the culture of Acetobacter aceti according to the manufacturer's instructions, inoculate the culture with 100 mL medium for the cultivation of vinegar bacteria in a 300 mL conical flask (see appendix). To ensure sufficient addition of oxygen, place the flask on to a magnetic stirrer for 48 hours. The stirring rods should be autoclaved with the culture medium before use.
Procedure:
1. Attach a 1.5 litre bottle that has a fixture for a bung at its base about 40 cm above the table, using the stand. A single bore rubber bung with an angled, one way tap seals the lower outlet of the bottle where the bung is attached.
2. Attach a second bottle of this kind directly beneath the outlet of the one way tap, or "fermenter". Seal its lower outlet with cotton wool inside, fill its interior with beech shavings. The beech shavings immobilize the vinegar bacteria to the fermenter. The cotton wool should retain coarser particles that can be separated from the wood shavings.
3. Close the lower outlet of the second bottle with a bung that has been bored through twice. In one of those openings, attach a glass tube as an attachment for the aquarium pump. In the other, insert a right-angled glass tube as a product outlet.
4. Use an aquarium pump to blow air constantly into the inside of the fermenter through the bung, the cotton wool filter keeps the system sterile.
5. As medium, use a mixture of unsulfured port and sterile distilled water in the ratio of 11. The pH value must be adjusted to 7.0 using 1 M NaOH. Pour 200 mL of the medium over the beech shavings in the fermenter. Allow the contents to stand for 48 hours. The wine must be unsulfured so that the vinegar bacteria do not die off. This is the case in home made wines and is usually true of ports, as well. The pH value of the medium must be adjusted to 7.0 so that the reduction of the pH value because of the formation of vinegar can be monitored.
6. Place the other 800 mL of the medium in the upper container. Adjust the tap so that it releases one drop per 5 minutes. 7. The product is continuously caught in a 500 mL conical flask at a rate of 1 drop per five minutes. Test the product once a day with indicator paper to monitor the development of acid. Air is blown into the bioreactor because without oxygen, the vinegar bacteria would die. The air must be filtered so that it is sterile because the air in the room contains fungal spores that develop in the fermenter and may cause the formation of mould on the beech shavings.
4.2.7 Microbial decomposition of thin paper, e.g. cigarette paper
Equipment: 1 autoclave or pressure cooker, 2 glass Petri dishes, 1 large dish that can be covered as a damp chamber, 1300 mL conical flask with cellulose bung, 1 drying cupboard or a Bunsen burner, tripod, pipe clay triangle, and crucible
Materials: 80 g soil, absorbent paper (approx. 90 cm2), sterile tap water, 6 strips of cigarette paper
Time needs: sterilization of the water: 30 minutes, sterilization of a part of the soil sample: 180 minutes, preparing the experiment in the damp chamber: 15 minutes, waiting time: about three to four weeks
Preparation: Sterilize 100 mL tap water in a closed conical flask for 30 minutes. Sterilize half of the soil sample in a glass Petri dish in a drying cupboard at 180oC for three hours, or use a Bunsen burner, tripod, pipe clay triangle, and crucible for 30 minutes.
Procedure:
1. Place the unsterilized part of the soil sample into a glass Petri dish. Dampen the sterilized soil sample with sterile tap water in the other sterile Petri dish. Ensure that both of the experiments in preparation are equally damp. The sterile soil serves as a control, no microorganisms should grow on the cigarette paper during the four weeks.
2. Place three strips of cigarette paper, 1 cm wide, on the dampened soil sample in each of the Petri dishes. For health reasons, cigarette paper does not contain lignin and is therefore more suitable for this investigation than is filter paper, which does contain lignin. Lignin prevents enrichment of organisms that decompose cellulose.
3. Cover both Petri dishes, seal the edges with adhesive tape, and place them into the larger dish, which has been covered with dampened absorbent paper. Cover the large dish. The Petri dishes must be sealed so that microorganisms do not accidentally escape and dangerous microorganisms do not develop. The damp chamber prevents the soil from drying out. 4. Allow the experiment to stand in a safe place for about four weeks. Only microorganisms that decompose cellulose be enriched on cigarette paper because cellulose is the only source of carbohydrate in the paper. Microorganism that grow on the paper also live off cellulose.
4.2.7.1 Enzyme Technology
See diagram 2.0: Cell walls and membranes
Cellulases, arnylases, proteases, and lipases are enzymes that are released by cells into the environment to help break down the large polymer food molecules that cannot be taken up into the cells whole.
1. Pectinase: Juice from oranges and lemons and from tropical fruits such as mango, papaya, and passion fruit is concentrated in the land of origin where the water that has been removed. The water is replaced in the country where it is to be consumed. However fruit juice contains pectin so jelly usually forms when fruit juice is concentrated. In the juice of fleshy fruit such as papaya when water is removed, pectin polymerizes and causes setting. To prevent this setting the fruit juice industry adds pectinase, an enzyme that splits pectin. A form of pectinase can be extracted from the mould Aspergillus niger.
2. Amylase: The enzyme amylase, which is also extracted from mould, is used in the textile industry to remove starch from cotton. Starch naturally adheres to cotton and inhibits the uptake of dye when textiles are being dyed. The baking industry mixes amylase with flour and supplements the naturally occurring amylase in flour. This enzyme is necessary to prepare the dough because it breaks down a small proportion of the starch in the flour to glucose, which serves the yeast as food. Manufacturers of liquid and powder detergents use amylase to break down the starch that forms as dirt on cutlery or in clothes. Protease is used in washing powder to decompose protein stains and lipases are used to break up fat stains. Cellulases are used in the processing of fruit and vegetables to destroy the cell walls.
3. Lactase: Lactase works inside the organism where it decomposes lactose molecules to alpha glucose and beta galactose. Whey contains relatively large quantities of lactose. Many adult humans cannot break down lactose in the digestive tract because they no longer produce the "infant's enzyme" lactase. Undigested lactose removes water from the intestinal wall, which results in diarrhoea. Bacteria in the intestinal flora that can split lactose decompose the products of splitting, developing gas in the process, the gas causes flatulence. Lactase is used to decompose lactic acid and to produce glucose.
In the following investigations, the students describe the effect of pectinase, explore the splitting of lactose, and decompose starch with Bacillus subtilis.
4.2.7.2 Experiments relating to pectinase in the industrial production of juice
Pectins are vegetable polysaccharides, their main components are galacturon acid and its methylester. The multiplicity of pectin is determined by the various degrees of polymerization and esterification. Together with cellulose, they are reticulum substances of vegetable cell walls, especially as a sort of "putty" in the middle lamella between the cells. They occur in solution in the cell sap.
Pectins have a great ability to combine with water, which accounts for the high gelling capacity of jams and jellies. For this reason, pectin are extracted from slices of sugar beet and from the remains of apples and lemons that have been used for making juice. They are then used as gelling agents in the food, cosmetic, and pharmaceutical industries, and in medicine.
Pectinases destroy the pectin in the cell wall and in the plasma so that it no longer retains juice in the chopped fruit. Fruit can therefore be pressed more effectively, resulting a high yield of juice.
Pectinases also are used to clarify fruit juice. Pectin retains substances that make the juice cloudy. Once pectin has been destroyed, those substance can easily be precipitated out.
4.2.8 Make apple juice gel when it is boiled
Equipment: 1 glass beaker, 800 mL, 1 chopping board, 2 glass beakers, 400 mL, 1 watch glass, 1 tripod, 1 plastic bowl, 1 ceramic net, 1 piece of muslin, 1 Bunsen burner, 1 gas lighter, 1 wooden spoon, 1 kitchen knife
Materials: 1 apple, sugar, tap water
Time needs: production of the juice: 20 minutes, production of the jelly: 25 minutes
Procedure:
1. Cut an unpeeled apple into eight equal pieces, leaving the core intact. Place the pieces into the larger glass beaker, and just cover them with tap water.
2. Boil the mixture for ten minutes, stirring all the time. The pieces of apple must become mushy. Cool the coarse puree and press it through a muslin cloth into the plastic bowl.
3. Weigh the empty glass beaker in advance. Place the juice into the small glass beaker and weigh it.
4. Add an equal amount of sugar and heat the juice again, stirring all the time.
5. After the juice has simmered for about five minutes, do a gelling test by observing the drops that fall from the wooden spoon. If the drops are thick and remain on the wooden spoon, you can allow the jelly to cool down. Divide the jelly and pour it into two glass beakers for this purpose. The gelling test can also be carried out by placing a little of the boiling juice onto a cold watch glass with a wooden spoon. If the juice gels on cooling, the boiling can be stopped. Do not boil the juice for longer than ten minutes, otherwise it will no longer gel.
4.2.9 Make pectinase, an enzyme that decomposes pectin
Freshly pressed apple juice is replaced with equal parts of pure alcohol. The pectin in the juice forms an insoluble gel with the alcohol. Juice to which pectinase has been added does not produce gel, however, and also deposits substances that make the juice cloudy
Equipment: 6 test-tubes with bungs, 3 pipettes, 5 mL, 1 test-tube stand, 2 pipetting aids, 1 measuring cylinder, 1 kitchen grater, 1 glass beaker, 50 mL, 2 bowls, plastic, 1 glass beaker, 100 mL, 1 cotton napkin, 1 glass rod
Materials: 1 apple, 5 mL 5% pectinase solution, 30 mL 96% alcohol or denatured alcohol
Time needs: 45 minutes
Procedure:
1. Grate the unpeeled apple into a plastic bowl. Squeeze the juice vigorously out of the puree over the second plastic bowl through a napkin folded double. Transfer the juice to a glass beaker. A medium sized apple such as a Granny Smith produces about 50 mL of juice.
2. Pour 10 mL of the juice and 2 mL of the pectinase solution into a glass beaker, shake the mixture. Position the glass beaker so that it will stand completely still so that substances that make the juice cloudy are deposited.
3. Put 5 mL of the juice and 5 mL of alcohol into a test-tube. Close the tube with a bung, shake it carefully twice, and let it stand.
4. Add 3 mL of pectinase solution to the remaining juice, 35 mL, stirring constantly. Start the stop watch. At three, six, nine and twelve minutes, pipette 5 mL of the juice out of the glass beaker into a test-tube, mix with 5 mL alcohol, seal, and rotate carefully twice. Place each of the test-tubes as still as possible in the test-tube racks.
5. After each test-tube has stood for at least five minutes, swirl it carefully to see whether the flocculation remains as a clump of gel on the surface or whether they collect as loose components at the bottom of the test-tube.
4.2.10 Industrial uses of pectinase
In this investigation, the students find the amount of juice produced from apple mash with and without the addition of pectinase. The fact that pectinase increases the juice yield indicates the significance of the use of pectinase for the fruit juice. Pectinase is used to increase yield, clarify juice and reduce transport costs. Trade terms include "naturally unclarified," "clear," concentrated," and "concentrate." However, some juice is still produced by the normal pressing technique. Reasons for the use of pectinase include increased yield, energy savings because the juice is easier to press, more economic methods of transport, and the ability to transport juice over longer distances. Reasons against the use of pectinase include interference with the natural flavour and consistency of the juice, and less wild fruit is processed, inability of small cider companies to compete with producers of cheap juices.
Equipment: 2 tea strainers, 2 funnels, 1 kitchen grater, 1 plastic bowl, 2 glass beakers, 100 mL, 2 glass rods, 2 spoons, 2 stands, sleeves, and stand clamps, 2 measuring cylinders, 50 mL, 1 set of scales Materials: 2 apples, 10 mL pectinase solution, freshly made, 5%, tap water
Time needs 25 minutes
Procedure:
1. Grate both unpeeled apples over a plastic bowl, using a household grater.
2. Divide the apple mash equally between the two glass beakers, A and B, with the help of the scales.
3. Pipette 10 mL pectinase solution into glass beaker A, and 10 mL water into glass beaker 2. Allow the glass beakers to stand as they are for ten minutes. Stir the mash at one minute intervals, using glass rods.
4. In the meantime, attach the funnels to the stands, using clamps. Place a tea strainer into each funnel and place the measuring cylinders under the funnels. Do not forget to stir the mash!
5. After the time has elapsed, tip out of the glass beakers the apple mash A and B into tea strainers. You may need spoons to do this. The mash may not be pressed into the tea strainer.
6. After five minutes, measure the quantity of juice in the cylinders.
4.2.11 Split lactose from milk or whey by using immobilized lactase
See diagram 20.190 Split lactose
Enzymes that are not released to the environment but that are active in the inside of cells are formed by microorganisms in relatively small quantities. The industrial production of such enzymes, of which lactase is one, is also quite tedious. The cells must first be broken open before enzymes of this kind can get into the culture medium from which they are produced. Dairies that use lactase for the treatment of whey therefore treat the expensive lactase with due care. It is immobilized before use, that is, it is bound to a vehicle. This allows several consecutive uses of the enzyme because it does not have to be thrown away with the waste products after it has been used the first time. The opposite is true of amylase, which is used in washing powder, this enzyme is naturally active outside the cell. Immobilized lactase can be used as often as desired for school experiments. It can be preserved with isopropanol and kept for six months in the refrigerator.
Equipment: 1 filter tube (Duran, pore size 40 - 100 mu, 20 mm), 1 suction flask with rubber bung attachment, 1 Woulfe bottle, 3 conical flasks, 500 mL, 1 water jet vacuum pump, 1 measuring cylinder, 100 mL, 1 glass beaker, 100 mL, 2 Pasteur pipettes, 2 glass beakers, 50 mL, 3 rubber caps for Pasteur pipettes, 1 conical flask, 100 mL, 1 pipette, 10 mL, with pipetting aid, 3 conical flasks, 300 mL, 1 stand with 2 clamps and 2 nuts
Materials: 1 piece of tubing, aluminium foil, 5 mL isopropanol lactase, sugar test strips, e.g. Diabur - Test 500, BOEHRINGER, Eupergit C, e.g. ROHM PHARMA Ltd whey from health food shop or skimmed UHT milk, 20 mL 1 molar phosphate buffer reagent (see appendix), 150 mL 0.1 molar phosphate buffer reagent (see appendix)
Time needs: Immobilization of the lactase: 10 minutes, waiting time: 2 days, splitting of lactose: 25 minutes
Procedure:
1. Several days before conducting the investigation, immobilize the enzyme lactase as follows: dissolve 0.1 g lactase in 20 mL 1 molar phosphate buffer reagent in a 100 mL glass beaker. Add 1 g eupergit C to this solution. Shake the suspension for a short while. Finally, seal the glass beaker with aluminium foil and allow it to stand for at least 2 days at 20oC, room temperature. Shake the beaker now and again about 2 to 3 times daily to facilitate the immobilization of lactase in eupergit.
2. After two days, place the suspension in the filter tube and place the tube onto the suction flask. Attach both to a stand and connect them to the Woulfe bottle with a piece of rubber tubing attached to a water jet vacuum pump.
3. Rinse the suspension in the filter tube with 40 mL 0.1 molar phosphate buffer reagent by rinsing it several times and removing the liquid by suction.
4. Finally, remove the suction flask. Place a 50 mL beaker under the glass beaker.
5. Add skimmed milk or whey drop by drop, using a Pasteur pipette. There should be a surplus 1 to 3 cm high above the eupergit. The milk products that drip out of the filter tube are caught in a glass beaker.
6. Test the milk products that have dripped through for glucose. The presence of glucose can be ascertained using glucose test strips that can be purchased from a supplier. The "untreated milk" or whey can be used as a control.
7. After the experiment has been completed, purify the immobilized enzyme with about 100 mL 0.1 molar phosphate buffer reagent until the filtrate is clear. In the last rinse, add 2% isopropanol by volume (preservation buffer reagent) to the phosphate buffer reagent to preserve the enzyme.
8. Seal the lower end of the filter tube with a rubber cap that is pushed over the end.
9. Add a preservation buffer reagent that contains isopropanol to the eupergit lactase compound until there is a surplus of 1 to 2 cm 10. Close the filter tube with aluminium foil at the upper end and keep the tube in the refrigerator.
4.3.1 Grow the African violet; Usambara violet (Saintpaulia ionantha) with in vitro culture
See diagram 20.194 Construct a sterile tunnel from Plexiglas
Numerous shoots develop from pieces of shoot or leaf of the Usambara violet after 2 - 4 weeks if the pieces are placed onto a medium containing cytokinin. Make about 50 pieces from a piece of leaf 0.5 cm2. If the shoots are then transferred to a medium that does not contain hormones, it produces roots after about one week. The small plants can be cultivated further in plant pots. All of them produce flowers of the same colour and otherwise possess similar characteristics. Here the students experience the conspicuous production of clones. The work must be carried out in sterile conditions or other microorganisms might be produced that would overrun the pieces of plant tissue in a very short time.
Equipment: blowtorch, up to 600oC, with jet, paint stripper blow torch, 2 screw clamps, 1 wooden lath 1 cm x 1 cm x 50 cm
Materials: plexiglass 30 x 45 cm, 3 mm thick
Procedure:
1. File down the sharp edges of the plexiglass plate. Mark points A, B and C, 4. Put the piece of plexiglass on a wooden table so that line A-B is exactly on the edge of the table. Place the wooden lath onto the plexiglass exactly on the edge of the table, secure the lath on both sides with screw clamps so that the plexiglass is between them. Heat the A-B line with the blowtorch until the plexiglass softens. After 1 minute at about 600oC, bend the plexiglass that juts out beyond the A-B line upwards at the desired angle. Hold the plexiglass until it cools.
2. Bend the plexiglass along the C-D line. To achieve the desk form of the tunnel, the and angles should be 90o and 110o, respectively. Several sterile tunnels can be piled on top of one another.
4.3.2 Grow the African violet; Usambara violet (Saintpaulia ionantha), from pieces of leaf
See diagram 20.197: Cultivate a tissue culture in a sterile tunnel
Before tissue cultures are prepared, prepare Petri dishes, using MS medium as a culture medium (see appendix). Before this is done, add BAP strain solution in the ratio of 0.5 mL per litre of culture medium. (BAP: 6-benzylaminopurine, a cytokinin). Pour the medium into sterile, disposable Petri dishes (diameter 9 cm) while it is still hot (> 50oC). One litre is sufficient for 35 dishes. Stack the dishes to avoid the formation of condensation in the lids of the Petri dishes and to protect the surface of the table. Label five empty Petri dishes with the date and type of medium and pile the dishes on top of one another. Lift up the whole pile with the lid of the lowest dish, pour the medium into the lowest dish, cover it with the lid and the rest of the pile. Lift the lid of the next dish, together with the rest of the pile, place the medium into the next dish, and so on. After one week, place pieces of the leaf onto the sterile culture media. Plates that should be kept for longer periods of time are packed in cling film or plastic bags to prevent their drying out and to protect them from contamination.
Equipment: 1 bent pair of tweezers (sterile), 1 scalpel (sterile), 1 kitchen timer, 1 container for decanting liquids, 4 glass beakers, 200 mL, sealed by a glass Petri dish (sterile), 1 sterile tunnel, 1 Bunsen burner, adhesive tape, transparent freezer bags, wooden sticks ca. 10 cm
Materials, MS medium (see appendix), BAP strain solution (see appendix), 70% alcohol (denatured alcohol may be used), 100 mL, 96 96% alcohol (denatured alcohol may be used), 100 mL, dilute "Domestos" solution (disinfectant), 20%, 100 mL, sterile tap water, 100 mL.
Procedure:
1. Rinse a leaf of an Usambara violet in 70% alcohol in a sterile glass beaker for about 1 minute. The glass lid must only be opened for as short a time as possible and must be replaced immediately! Carefully decant the alcohol without removing the lid so that the objects do not slip out. The rinsing of the leaf increases the wetness of the surface.
2. Add dilute "Domestos" solution and shake the glass beaker. Sterilization time: 1 - 2 minutes. Decant the solution as in 1.
3. Rinse the leaf in sterile tap water three times per 5 - 10 minutes, shake slightly with the lid closed. Carefully decant the last water used for rinsing.
4. Sterilize tweezers in 96% alcohol then use them to take the leaf out and place it on an empty sterile Petri dish.
5. Cut away the tissue at the edge of the leaf that was damaged during the process of sterilization. Also, use the scalpel, sterilized in 96% alcohol to cut away the larger vascular tissue. Microorganisms that were not killed during the sterilization of the surface may be present in the vascular tissue.
6. The freezer bag is waterproof, but porous to air. The wooden stick prevents the plastic of the bag from pressing on the plants.
7. Cut the leaf into strips about 5 mm wide and 1 cm long.
8. Gently press the strips of leaf onto the culture medium that contains cytokinin. Close the Petri dish and seal it with adhesive tape. Place the Petri dish into a well lighted place for about 2 - 4 weeks at room temperature.
9. When shoots form, transfer them to culture medium without cytokinin so that they form roots.
10. After two weeks, as soon as small roots have been formed, transfer the plants to plant pots. These in turn are placed into freezer bags that are tied at the top. Place a small wooden stick into the soil.
4.3.3 Grow gerbera using in vitro culture
How much profit does a gardener make if she plants 1,000 plants that have been produced in vitro? How much profit does she make if she sows seedlings? She must buy both the young plants that have been produced in vitro, and the seed. Because the Gerbera seeds do not germinate very well, she must buy an average of 1,430 seeds if she wishes to grow 1,000 young plants. While they are being grown, there are losses, and a number of young plants do not blossom, so the gardener is only able to sell 700 of the 1,000 young plants grown from seed. The losses made from the young plants grown in vitro were less, 950 of 1,000 young plants could finally be sold as pot plants. "Overheads" refers to the financial cost of the required area in the greenhouse multiplied by the number of days during which this area is occupied by plants. The plants that have been produced in vitro can be compared to seedlings that are seven weeks old. The former come into flower within one to three weeks, while eight weeks elapse between the flowering of the first and last plants produced from seed. Therefore, the overhead for the plants produced in vitro is considerably less. Gerbera plants produced in vitro result in higher profit is higher than if the plants were sown from seed.
4.4.1 Isolate DNA
1. Isolate DNA from wheat germ. DNA occurs inside a nuclear membrane within a cell membrane. To see DNA, destroy the membranes and get a large enough quantity to be visible. Put wheat germ, or kiwi fruit liquidized in a food processor, in the test-tube to a depth of 1 cm. Add 3 mL of warm water and a drop of kitchen detergent. Plug the test-tube with cotton wool and roll it in the hands gently for three minutes. Float 2 mL of cold methylated spirit on top of the mixture and leave to stand. The cloudiness at the interface of the liquids is the DNA. Use an opened "slide-on" paper-clip to lift out the threads of DNA
2. Isolate DNA from sweetbread (calf thymus gland). Cells of the calf thymus gland possess large nuclei from which large amounts of DNA can be obtained. Buy some freshly-killed sweetbread from a butcher. Deep-frozen samples also produce comparable results. To produce a thymus suspension, cut up five sweetbreads in a mortar using a pair of scissors. Add clean washed sand to the tissue in the mortar and grind with the pestle. Add 20 mL of tap water and continue grinding until the water is completely clouded. Filter the suspension through gauze. Add five drops of kitchen detergent and shake until the suspension becomes clear and viscous. Cover the suspension with one and a half times the amount of cold methylated spirit solution in a narrow glass beaker. Use a glass rod to remove the white strands of DNA.
3. Isolate DNA from sweetbread (calf thymus gland)
Cells of the calf thymus gland posses relatively large nuclei from which large amounts of DNA can be obtained. Sweetbread can be bought from a butcher. Fresh and deep frozen samples produce comparable results
Equipment: 1 glass beaker, narrow, 100 mL, 1 mortar, 1 pestle, 1 pair of scissors, 1 scalpel, 1 glass rod, 1 pair of tweezers, surgical gauze
Materials: 5 -10 g sweetbread, abrasive sand, tap water, washing up liquid, containing tensides, ethanol 96%, or methylated spirit
(a) To produce a thymus suspension, cut up 5 - 10 sweetbreads in a mortar using a pair of scissors or a scalpel. (b) Add sand to the tissue in the mortar and grind with the pestle. Then add 20 mL tap water and continue grinding until the water is completely clouded. The water is clouded by cells and cell detritus that have been abraded and are suspended in the water. (c). Filter the suspension through 2 layers of surgical gauze. (d) To liberate DNA, add 5 - 10 drops of washing up liquid to the cell suspension and shake thoroughly. Each tensides molecule of the washing up liquid has a hydrophilic and a lipophilic end. When they are taken up, the field of charge of molecules of the lipoprotein membrane is shifted, and hydrolysis occurs. In this way, for example, the membranes of the cell and nucleus are destroyed and histone molecules split off from DNA strands. After the addition of washing up liquid, the suspension becomes clear and viscous. The viscosity is caused by liberated DNA molecules. (e) To obtain DNA, cover the clear, viscous suspension with one and a half times the amount of cold ethanol solution in a narrow glass beaker. (f) The DNA precipitating as whitish strands can be removed with a glass rod. Rotate the rod slowly and carefully dip it up and down occasionally to mix the suspension and the alcohol. This will precipitate a relatively large amount of nucleic acid. If the strands slip from the rod, they can be fished out of the beaker using a tweezers.
4. Isolate DNA from anything living, e.g. strawberries, broccoli, split peas, spinach leaf, lettuce, onions or chicken liver. Put in a kitchen blender one half cup (100 mL) of extraction material, one eighth teaspoon (less than 1 mL) of table salt and 1 cup (about 200 mL) of cold water. Run the kitchen blender on high for 30 seconds or until there is a runny pea soup consistency. Pour this blended material through a strainer into another container. Add 2 tablespoons (about 30 ml) of liquid detergent to the strained mixture and swirl to mix. Let the mixture stand for 5-10 minutes. Transfer the mixture into test-tubes one third full. Tilt the test-tube and slowly pour cold ethanol into the test-tube down the side so it forms a layer on top of the blended mixture. Observe the DNA rising into the alcohol layer from the blended mixture. Use an opened "slide-on" paper-clip to draw the DNA into the alcohol.
Cells of the calf thymus gland posses relatively large nuclei from which large amounts of DNA can be obtained. Sweetbread can be bought from a butcher. Fresh and deep frozen samples produce comparable results
Equipment: 1 glass beaker, narrow, 100 mL, 1 mortar, 1 pestle, 1 pair of scissors, 1 scalpel, 1 glass rod, 1 pair of tweezers, surgical gauze
Materials: 5 -10 g sweetbread, abrasive sand, tap water, washing up liquid, containing tensides, ethanol 96%, or methylated spirit
Procedure:
1. To produce a thymus suspension, cut up 5 - 10 sweetbreads in a mortar using a pair of scissors or a scalpel.
2. Add sand to the tissue in the mortar and grind with the pestle. Then add 20 mL tap water and continue grinding until the water is completely clouded. The water is clouded by cells and cell detritus that have been abraded and are suspended in the water.
3. Filter the suspension through 2 layers of surgical gauze.
3. To liberate DNA, add 5 - 10 drops of washing up liquid to the cell suspension and shake thoroughly. Each tensides molecule of the washing up liquid has a hydrophilic and a lipophilic end. When they are taken up, the field of charge of molecules of the lipoprotein membrane is shifted, and hydrolysis occurs. In this way, for example, the membranes of the cell and nucleus are destroyed and histone molecules split off from DNA strands. After the addition of washing up liquid, the suspension becomes clear and viscous. The viscosity is caused by liberated DNA molecules.
4. To obtain DNA, cover the clear, viscous suspension with one and a half times the amount of cold ethanol solution in a narrow glass beaker.
5. The DNA precipitating as whitish strands can be removed with a glass rod. Rotate the rod slowly and carefully dip it up and down occasionally to mix the suspension and the alcohol. This will precipitate a relatively large amount of nucleic acid. If the strands slip from the rod, they can be fished out of the beaker using a tweezers.
4.4.2 Conjugation in bacteria, Escherichia coli
The strain Escherichia coli GY767 (DSM-No. 1562) is streptomycin sensitive. The F plasmid is integrated into its chromosomal DNA, and it is able to transfer chromosomal genetic information to strains of E. coli that have no F plasmid. In this experiment, the recipient is E. coli AB1157 (DSM-No. 1563). This streptomycin resistant strain is characterized by a large number of mutations and can no longer produce for itself the amino acids proline, leucine, arginine, threonine, or histidine, or the vitamin thiamine, which it requires to survive. These substances must therefore be present in the culture medium if growth is to occur. In the following experiment, donor cells (E. coli GY767, DSM-No. 1562) and recipient cells (E. coli AB1157, DSM-No. 1563) are mixed in a ratio of 1:9. Within two hours the chromosomal genes for the biosynthesis of proline, threonine, and leucine are transferred from the donor to the recipient. Recipient cells are distinguished from the donor cells by their resistance to streptomycin. After transfer of the genes for biosynthesis of the amino acids proline, leucine, and threonine, recombinant recipient cells, now also called transconjugants, grow on minimal agar that contains arginine, histidine, thiamine, and streptomycin, but not proline, leucine, or threonine. Neither the donor, with its streptomycin sensitivity, nor the unchanged recipient, which would need the missing nutrients proline, leucine, and threonine, can grow on this minimal agar. Conjugation between E. coli GY767 (DSM-No. 1562), a wild strain with chromosomal integrated F-plasmid, and E. coli AB1157 (DSM-No. 1563), a multiple auxotrophic mutant. Neither strain can grow on streptomycin containing minimal agar. Transconjugants of E. coli AB1157 (DSM-No. 1563) develop on this medium only after conjugative transfer of genetic information
Equipment: incubator, 1 glass beaker, 250 mL, 1 conical flask, 250 mL, 3 Petri dishes, 3 test-tubes with aluminium caps or cotton wool bungs, 1 pipette, 5 mL, sterile, 4 pipettes, 1 mL, sterile, pipette aids, 1 Drigalski spatula, 1 Bunsen burner
Materials: overnight culture of E. coli GY767 (DSM-No. 1562), overnight culture of E. coli AB1157 (DSM-No. 1563), nutrient broth medium for the overnight culture (see appendix), minimal agar medium (see appendix), streptomycin, 0.02 g, distilled water, alcohol, 70%.
Procedure:
1. Using the Erlenmeyer retort, prepare minimal agar with 100 mL distilled water and adjust to a pH value of 7.2 using 1 M NaOH. Seal the retort with aluminium foil and sterilize, then allow to cool to 55o3. Add 0.02 g streptomycin and firmly shake the retort to dissolve. Pour into three agar dishes.
2. Prepare 5 mL each of overnight culture of E. coli GY767 (DSM-No. 1562) and E. coli AB1157 (DSM-No. 1563).
3. Mix 0.3 mL E. coli GY767 (DSM-No. 1562) overnight culture and 2.7 mL E. coli AB1157 (DSM-No. 1563) overnight culture in a sterilized test-tube and incubate at 208 37oC for two hours.
4. Spread 0.1 mL of this mixture onto a minimal agar dish using the Drigalski spatula, sterilized with 70% alcohol and flamed in the Bunsen burner.
5. Prepare two control dishes with 0.1 mL of E. coli GY767 (DSM-No. 1562) and E. coli AB1157 (DSM-No. 1563) overnight culture respectively. Incubate all three dishes at 37oC for 72 hours. E. coli GY767 (DSM-No. 1562) shows no signs of growth on the nutritive medium, as it is streptomycin sensitive. Nor can E. coli GY767 (DSM-No. 1562) grow on this medium, because the amino acids required by this strain, proline, leucine, and threonine, are not present. Cells of E. coli AB1157 (DSM-No. 1563) can grow on this medium only when they have received from the donor the genes for biosynthesis of these amino acids. During the experiment, genes for arginine, histidine, and thiamine biosynthesis are also transferred, but not detected.
Acknowledgements
The above experiments have been edited from Chapter 4 "Suggestions for teaching biotechnology" and "Appendix 2" of UNESCO Science and Technology Education Document Series 39 "Teaching Biotechnology in Schools", Section of Science and Technology Education, ED-90 / WS / 33, Paris, 1990, edited by Joseph D. McInerney, Commission for Biological Education, International Union of Biological Sciences (IUDs). In that document the individual experiments were attributed as follows:
Contributors: Horst Bayrhuber, Christian Gliesche, Christine Labahn-Lucius, Eckhard R. Lucius, Uta Nellen, Ronald Westphal
Institute for Science Education (IPN), University of Kiel, Germany
1. Division of microorganisms into large groups by observing colonies (3. Gliesche and E.R. Lucius)
2. Enrich wild yeast strains (R. Wistful, 1989)
3. Make fixed slide preparations (after R. Westphal, 1989)
4. Make an India Ink preparation (after R. Westphal, 1989)
5. The Safe Microscopy of Mould Using the Petri Slide Technique (after E.R. Lucius and M. Fries, 1990)
6. Trace soil bacteria that decompose urea (E.R. Lucius after R. Westphal, 1989)
7. Demonstrating Production of an Antibiotic (E.R. Lucius and M. Fries)
8. Show the effect of Streptomycin in the Small Disc Test (E.R. Lucius and M. Fries, after R. Westphal, 1989)
9. Show the presence of bactericidal substances (E.R. Lucius and M. Fries)
10. Make yoghurt and sauerkraut (Primary grade four students) (after H. Bayrhuber, M. Fries, and Th. Heineken, 1990)
11. Make lactic acid in sourdough (E.R. Lucius and L. Rohweder)
L. Make wine from grape juice and make vinegar from wine (E.R. Lucius)
M. Microbial decomposition of cigarette paper (E.R. Lucius)
N. Why apple juice gels when it is boiled? (after Ch. Labahn-Lucius, 1990)
O. Pectinase enzyme decomposes pectin (after Ch. Labahn-Lucius, 1990)
Q. Split lactose from milk or whey by using immobilized lactase (after Ch. Labahn-Lucius and 8. Plainer)
R1. In vitro culture in the ornamental Usambara violet (IPN)
R2. Production of Usambara violets from pieces of leaf (from Nellen, 1988)
S. Cultivation of Gerbera by using in vitro culture (after H. Bayrhuber, 1990)
T. Isolating DNA from sweetbread (calf thymus gland) (from H. Bayrhuber, Ch. Gliesche, 5. R. Lucius, 1990)
U. Observe conjugation in bacteria (from Ch. Gliesche, 1990)