School Science Lessons
2024-10-28
(UNBiology4)
Microbiology
Microbiology websites
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Please send comments to: j.elfick@uq.edu.au
Contents
4.5.0 Enzymes
4.1.0 Microbiology cultures
4.2.0 Microbiology techniques
4.4.0 Prepare food. using microbiology
4.5.0 Enzymes
Enzymes are proteins that act upon substrate molecules and decrease the energy necessary for a chemical reaction to occur.
Enzymes speed up reaction rates and makes reactions appen at physiologically significant rates.
Enzymes bind substrates at key locations in their structure called active sites.
Enzymes are highly specific and only bind certain substrates for certain reactions.
Without enzymes, most metabolic reactions would not be fast enough to sustain life.
The active site is a crevice on an enzyme in which a substrate binds to facilitate the catalyzed chemical reaction.
A catalase is an antioxidant enzyme in all aerobic organismns that breaks down hydrogen peroxide into water and oxygen.
4.5.1 Amylase
4.5.2 Lactase
4.5.3 Protease
4.1.0 Microbiology cultures
4.1.1 Prepare colonies of different micro-organisms
4.1.2 Enrichment of wild yeast strains, Aspergillus niger
4.1.3 Grow African violet, with in vitro culture
4.1.4 Grow African violet, from pieces of leaf
4.1.5 Grow Gerbera using in vitro culture
4.1.6 Prepare pour plates
4.1.7 Prepare fixed slides
4.1.8 Prepare streak plates
4.1.9 Prepare a spread plate, lawn plate
4.1.10 Prepare India ink streak plates
4.1.11 Prepare streptomycin using Streptomyces griseus
4.1.12 Presence of bactericidal substances using a coin and Bacillus mycoides
4.2.0 Microbiology techniques
4.2.1 Aseptic transfer of bacterial cultures from a bottle or tube
4.2.2 Aseptic transfer of bacterial cultures from a culture plate
4.2.3 Colony counts using the calibrated drop method
4.2.4 Flaming and cotton wool plugs
4.2.5 Incubators
4.2.6 Inoculate with a Pasteur pipette
4.2.7 Lawn plate technique
4.3.1 Measure the amount of juice from apple mash with and without pectinase.
4.2.8 Microbial decomposition of cigarette paper
4.2.9 Microbiology chemicals
4.3.2 Pectins and pectinases
4.2.10 Pipettes (Safety)
4.2.11 Prepare heat-fixed stained bacterial smears
4.1.6 Prepare pour plates
4.2.12 Serial decimal dilution of a bacterial suspension
4.2.13 Spread plate technique
4.2.14 Spreaders
4.2.15 Staining rack
4.2.16 Streak dilution plate method for pure cultures from a mixed suspension
4.2.17 Wire loops
4.4.0 Prepare food, using microbiology
4.4.1 Prepare apple juice gel
4.4.2 Prepare cider from apple juice
4.4.3 Prepare lactic acid with sourdough
4.4.4 Prepare lactose from milk or whey using immobilized lactase
4.4.5 Prepare pectinase
4.4.6 Prepare sauerkraut
4.4.7 Prepare vinegar with Acetobacter aceti
4.4.8 Prepare wine from grape juice and vinegar from wine
4.4.9 Prepare yoghurt
4.1.1 Prepare colonies of different micro-organisms
See diagram 20.114: Equipment.
1. On the day before the investigation, prepare cultures of the four species of micro-organisms and incubate overnight.
Bacillus subtilis (DSM-No. 1079, ATCC 6051)
Penicillium roqueforti (DSM-No. 1079)
Rhodotorula rubra (DSM-No. 70403)
Streptomyces griseus (DSM-No. 40236, ATCC 23345)
2. Prepare 100 mL of basal agar medium in 3 agar plates.
1.1 Basal agar medium, 100 mL
3.4 Basal broth medium, 50 mL
3. Divide the Petri dishes 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 Petri dishes at 30oC for 6 days.
6. Identify the colonies:
Bacillus subtilis bacteria colonies are usually grey.
Streptomyces griseus have an earthy smell, produces a grey aerial mycelium and may stain the surrounding agar brown.
Penicillium roqueforti has an aerial mycelium and usually has blue-green spores.
Rhodotorula rubra is a unicellular pigmented yeast with orange/red colonies when grown on SDA (Sabouraud's Dextrose Agar).
4.1.2 Enrichment of wild yeast strains, Aspergillus niger
1. Prepare malt agar plates.
1.8 Malt extract agar medium
2. Roll a piece of unwashed fruit across the surface of the chilled agar, then immediately close the dishes.
3. Incubate the Petri dishes for a few days at 30oC.
4. Observe different yeast types.
This Petri slide procedure allows safe microscopy of moulds.
The incubated yeast cultures should not be used for further experiments.
4.1.3 Grow African violet with in vitro culture
See diagram 20.194: Construct a sterile tunnel from Plexiglas.
Saintpaulia ionantha, (now Streptocarpus ionanthus), African violet, Usambara violet, Gesneriaceae
Numerous shoots develop from pieces of shoot or leaf of the Usambara violet after 2 to 4 weeks if the pieces are placed onto a medium containing cytokinin.
Prepare 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 done in sterile conditions or other micro-organisms might be produced to overrun the pieces of plant tissue.
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
1. File down the sharp edges of the plexiglass plate.
Mark points A, B and C, D.
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.
4.1.4 Grow African violet from pieces of leaf.
See diagram 20.197: Cultivate a tissue culture in a sterile tunnel.
Saintpaulia ionantha, African violet, Usambara violet, Gesneriaceae
Materials:
See 9.2.23: MS agar medium
See 9.2.24: BAP medium
See 9.2.25: Buffer reagent, phosphate buffer reagent
See 9.2.26: 20% Domestos solution 10 mL
70% alcohol, 100 mL (or use denatured alcohol); 96 96% alcohol, 100 mL (or use denatured alcohol); sterile tap water 100 mL
Before tissue cultures are prepared, prepare Petri dishes, using MS agar medium as a culture medium.
Before this is done, add BAP medium 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 10 cm
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 to 2 minutes
Decant the solution as in 1.
3. Rinse the leaf in sterile tap water three times per 5 to 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.
Micro-organisms 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 to 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.
4.1.5 Grow Gerbera using in vitro culture.
Gerbera jamesonii, gerbera, Barberton daisy, Transvaal daisy, Asteraceae
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?
The gardener must buy both the young plants that have been produced in vitro and the seed.
The Gerbera seeds do not germinate very well, so the gardener must buy an average of 1 430 seeds 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 flower within 1-3 weeks, while 8 weeks elapse between the flowering of the first and last plants from seed.
So 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.
4.1.6 Prepare pour plates
1. Use a pipette to add inoculum from a broth culture to the centre of a Petri dish, then add previously molten, cooled agar medium.
Rotate the Petri dish to mix the culture and medium thoroughly and ensure that the medium covers the plate evenly.
Pour plates allow micro-organisms to grow both on the surface and within the medium.
Most of the colonies grow within the medium and are small and may be confluent.
The few colonies that grow on the surface of the medium are generally of the same size and appearance as colonies on a streak plate.
If the dilution and volume of the inoculum, usually 1 mL, are known, the viable count of the sample can be calculated, i.e. the number of bacteria or clumps of bacteria per mL.
The dilution chosen should produce 30 to 300 separate countable colonies.
2. Collect one bottle of sterile molten agar from the water bath.
Hold the bottle in the right hand then remove the cap with the little finger of the left hand.
Flame the neck of the bottle.
Lift the lid of the Petri dish slightly with the left hand and pour the sterile molten agar into the Petri dish and replace the lid.
Flame the neck of the bottle and replace the cap.
Rotate the Petri dish to mix the culture and the medium thoroughly and to ensure that the medium covers the plate evenly.
Leave the plate to solidify.
Seal and incubate the plate in an inverted position.
The whole base of the plate must be covered.
Do not let agar touch the lid of the plate.
The surface must of the inoculated medium must be smooth with no bubbles.
4.1.7 Prepare fixed slides
See diagram 20.120: Techniques.
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.
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.
5. Allow the smear to dry.
6. Fix the bacteria to the slide by briefly heating the slide in a low flame, e.g. the pilot flame of a Bunsen burner and with the coated side of the slide oriented downwards.
Pass the slide through the flame three times.
4.1.8 Prepare streak plates
See diagram 9.4.13: Streak plate.
Streaking causes a progressive dilution of an inoculum over the surface of solidified agar medium in a Petri dish, so that the colonies of bacteria or yeast grow separated from each other as single isolated pure colonies.
Professional microbiologists start with the Petri dish inverted on the desk.
Then they lift out the base, invert it, then inoculate the agar facing up.
1. Partially lift the lid of the Petri dish containing the solid medium.
Hold the charged wire loop parallel with the surface of the agar.
Smear the inoculum backwards and forwards across a small area of the agar medium on the left hand side of the plate.
Remove the wire loop and close the Petri dish.
Flame the wire loop and allow it to cool.
2. Turn the Petri dish through 90o anticlockwise.
Use the cooled wire loop to streak the agar plate across the surface in three parallel lines.
A small amount of culture must be carried over.
Remove the wire loop and close the Petri dish.
Flame the wire loop and allow it to cool.
3. Turn the Petri dish through 90o anticlockwise again and streak across the surface of the agar in three parallel lines.
Remove the wire loop and close the Petri dish.
Flame the wire loop and allow it to cool.
4. Turn the Petri dish through 90o anticlockwise, then streak the wire loop across the surface of the agar into the centre of the plate.
Remove the wire loop and close the Petri dish.
Use a marker pen to label the Petri dish at the edge off the plate.
Flame the wire loop.
Seal and incubate the plate in an inverted position so that condensation cannot occur on the lid and drip onto the culture, causing colonies to spread into each other.
4.1.9 Prepare a spread plate, lawn plate
The plate should have a growth of culture spread evenly over the surface of the growth medium.
Use it to test the sensitivity of bacteria to antimicrobial substances, e.g. disinfectants and antibiotics, and to determine the number of bacteria or clumps of bacteria per mL, colony count.
For an accurate count, the dilution and volume of the inoculum, usually 0.1 mL, must be known and the dilution chosen must produce 30 to 300 separate countable colonies.
Loosen the cap of the bottle or test-tube containing the broth culture.
Remove a sterile Pasteur pipette from its container and attach the bulb held in the right hand.
Hold a sterile pipette in the right hand and the bottle or test-tube containing the broth culture in the left.
Remove the cap or cotton wool plug of the bottle or test-tube with the little finger of the right hand and flame the neck.
With the pipette, remove a small amount of broth.
Flame the neck of the bottle or test-tube and replace the cap or plug.
With the left hand, partially lift the lid of the Petri dish containing the solid nutrient medium.
Place five drops of culture on the surface, an area of 0.1 cm3, or enough to cover a UK or AU 5 pence piece.
Replace the lid of the Petri dish.
Place the pipette in a discard jar of disinfectant.
Lift the lid of the Petri dish to allow entry of a sterile spreader.
Place the spreader on the surface of the inoculated agar and move the spreader in a top-to-bottom or a side-to-side motion to spread the inoculum over the entire surface of the agar.
Do this as fast as possible to reduce contamination.
Replace the lid of the Petri dish.
Put the spreader in a discard jar of disinfectant.
Leave the inoculum to dry.
Seal and incubate the plate in the inverted position.
To produce an agar plate inoculated with mould mycelium inoculated at the centre, invert the plate, lift the base of the Petri dish that contains the medium and inoculate onto the centre of the downwards-facing agar surface with a bent wire.
This method avoids the problem of spores falling off the piece of mycelium and producing unwanted inoculation sites.
4.1.10 Prepare India ink background stain
1. 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 so that the drop spreads out. 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.
5. Push the coverslip evenly across the entire surface of the microscope slide so that as the suspension is thus spread across the slide, the thickness of the film of liquid decreases.
6. Leave 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.
4.1.11 Prepare streptomycin using Streptomyces griseus
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.
4.1.12 Presence of bactericidal substances using a coin and Bacillus mycoides
If a coin in a culture medium 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 micro-organisms is often formed in the immediate vicinity of the coin.
These micro-organisms 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 micro-organisms.
This problem occurs quite frequently in hospitals, where certain micro-organisms suddenly occur in large numbers, e.g. the bacterium Serratia marcescens.
Inhibiting and encouraging growth of micro-organisms 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, stopper, 6 culture tubes, adhesive tape for sealing the Petri dishes, 5 X 5 mL sterile pipettes.
Material:
9.1.2.15 Basal broth medium, for cultures incubated overnight, 100 mL
9.1.2.14 Basal agar medium, 100 mL
Distilled water 100 mL,
Pure culture of Bacillus mycoides (DSM-No. 2048, ATCC 6462)
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.
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 micro-organisms 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 Aseptic transfer of bacterial cultures from a bottle or tube
1. Label the new bottle or plate with your name, the name and / or source of isolation of the organism, the date and the incubation temperature to be used.
2. Place the culture bottle, the loop, or pipette and filler, and the medium or slide to be inoculated near to the base of the flame.
3. Take up the loop and flame sterilize it.
4. Pick up the culture bottle with the other hand and hold onto the lid with the little finger of the loop hand.
Unscrew the bottle from the lid and continue to hold the lid with the little finger.
1. Flame the neck of the bottle quickly and place the loop into the suspension.
6. Reflame the bottle and screw on the lid.
7. Put the bottle aside and use the loop to inoculate the broth, plate or slide as required.
8. Flame the loop and place it down.
4.2.2 Aseptic transfer of bacterial cultures from a culture plate
1. Label the new bottle or plate with your name, the name and/ or source of isolation of the organism, the date and the incubation temperature to be used.
2. Place the inverted plate, the loop and the medium or slide to be inoculated near to the base of the flame.
3. Flame sterilize the loop.
4. When the loop is cool, lift up the plate by its base and expose the agar surface to the flame, but at some 10-15 cm from it.
5. Cut the colony with the loop and replace the plate on its lid.
6. Use the loop to inoculate the broth, plate or slide as required.
7. Flame the loop and place it down.
4.2.3 Colony counts using the calibrated drop method
Use this method only for pure cultures of bacteria and yeast.
The procedure is similar to the spread plate procedure, but the inoculum is added as drops from a dropping pipette calibrated to deliver drops of known volume, e.g. 0.02 mL.
About six drops from different cultures can be put on the same plate, thus saving the number of plates needed.
The method is not usually suitable for mixed cultures, e.g. soil samples.
4.2.4 Flaming and cotton wool plugs
Flame the neck of bottles and test-tubes.
Loosen the cap of the bottle.
Lift the bottle or test-tube with the left hand.
Remove the cap of the bottle or cotton wool plug with the little finger of the right hand.
Turn the bottle, not the cap.
Do not put the cap or cotton wool plug down on the desk.
Flame the neck of the bottle or test-tube by passing the neck forwards and back through a hot Bunsen burner flame.
After the procedure, replace the cap on the bottle or cotton wool plug using the little finger.
Label test-tubes and bottles with a marker pen where it will not rub off.
Cotton wool plugs are used to plug test-tubes and pipettes to allow the passage of air, but prevent the passage of micro-organisms.
They must be made of non-absorbent cotton wool, be kept dry, and must keep its shape after being removed and returned to the test-tube.
4.2.5 Incubators
Incubators are not really necessary for microbiology in schools, because most of the cultures suitable for use in schools grow at room temperature so can be incubated in a cupboard.
Incubators can be set at a range of temperatures, but overlong incubation of a forgotten mould cultures may result in a massive formation of spores, which may cause contamination problems and be a health hazard.
The internal temperature of incubators may vary, so it is best to use a water baths for the accurately controlled temperatures needed for studying enzyme reactions and growth-temperature relationships.
4.2.6 Inoculate with a Pasteur pipette
Loosen the cap or cotton wool plug of the bottle containing the inoculum.
Remove the sterile Pasteur pipette from its container, attach the bulb and hold it in the right hand.
Lift the bottle or test-tube containing the inoculum with the left hand.
Remove the cap or cotton wool plug with the little finger of the right hand.
Flame the bottle or test-tube neck.
Squeeze the teat bulb of the pipette slightly, put the pipette into the bottle or test-tube and draw up some of the culture.
Always hold the pipette as still as possible.
Do not squeeze the teat bulb of the pipette after it is in the broth, because this could cause bubbles.
Remove the pipette and flame the neck of the bottle or test-tube again, before replacing the cap or cotton wool plug.
Place a bottle or test-tube on the bench.
When inoculating a Petri dish, lift the lid with the right hand just enough to insert the pipette and release the required volume of inoculum onto the centre.
Replace the lid.
Put the pipette into a discard pot of disinfectant.
Remove the teat while the pipette is pointing into the disinfectant.
4.2.7 Lawn plate technique
Use this technique when you wish to produce growth over the entire surface of the plate, like a lawn, i.e. not separate cultures.
1. Repeat steps 1 to 5 of: 4.2.12 Serial decimal dilution of a bacterial suspension
2. Spread the inoculum over the entire surface of the agar in one direction.
3. Rotate the plate and spread the inoculum over the surface across the direction of the previous streaks.
4. Replace the culture plate on its lid and sterilize the loop.
4.1.2.8.0a Estimate aerobic mesophilic bacteria by the plate count method.
An aliquot is a portion, a known fraction of the whole sample.
For counting the bacteria on cereals, bread and pasta the product to be investigated is mixed with a sterile physiological solution in and a decimal dilution series is prepared from this initial suspension.
Aliquots of the dilution stages are transferred to Petri dishes and mixed with a still liquid culture medium.
After the agar solidifies the individual bacterial cells are fixed in place and can multiply during incubation and to form colonies.
The number of colonies is counted as the number of bacteria per gram of the sample.
The accuracy of the method depends whether it is possible to separate completely the bacterial cells from the substrate, avoid damage to the cells during the necessary handling and get an even distribution of the cells in the culture medium.
4.2.8 Microbial decomposition of cigarette paper
Preparation: Sterilize 100 mL tap water in a closed conical flask for 30 minutes.
Sterilize half of a soil sample by using e a Bunsen burner 30 minutes.
1. Place the unsterilized part of the soil sample into a glass Petri dish.
Control: Dampen the sterilized soil sample with sterile tap water in a sterile Petri dish.
The contents of the two Petri dishes must be equally damp.
2. Place three strips of cigarette paper, 1 cm wide, on the dampened soil sample in each of the Petri dishes.
3. Cover both Petri dishes, seal the edges with adhesive tape, and place them into the larger covered dish.
4. Leave the experiment to stand in a safe place for about four weeks.
Only micro-organisms that decompose cellulose be enriched on cigarette paper, because cigarette paper does not contain lignin.
4.2.10 Pipettes, (Safety)
Use a sterile graduated pipette and filler or dropping (Pasteur) pipette to transfer cultures, sterile media and sterile solutions.
Remove the pipette from its container or wrapper by the end that contains a cotton wool plug.
Fit the teat.
Hold the pipette barrel as you would a pen, but do not grasp the teat.
Leave free the finger to take hold of the cotton wool plug or cap of a test-tube or bottle.
Leave the thumb to control the teat.
Depress the teat carefully to take up enough fluid, but not enough to wet the cotton wool plug.
Return any excess fluid if a measured volume is required.
Keep the pipette tip beneath the liquid surface while taking up liquid, to avoid taking up air bubbles.
Immediately after use, put the "contaminated pipette" into a discard pot of 0.25% v/v sodium chlorate I (sodium hypochlorite), then remove the teat.
Never use the mouth to "suck up" fluid into a pipette!.
4.2.11 Prepare heat-fixed stained bacterial smears.
1. Test a microscope slide for cleanliness by spreading a loop full of water across the glass.
If the water does not spread evenly, you must clean the slide with a powder cleanser, rinse in tap water and dry.
Repeat 1. Put the slide and culture near the base of the flame.
2. If the culture is a broth, aseptically remove a loop full of suspension from the bottle.
Spread the inoculum for 2 cm in the centre of the slide.
3. If the culture is a colony on a plate, select an isolated colony.
Place a loop full of tap water on the centre of the slide.
Aseptically, cut the colony with the edge of the loop and mix the inoculum into the drop of water to give an even suspension.
With the flat of the loop, spread the suspension 4 cm on the slide.
4. Allow the smear to air dry until barely visible.
1. Pass the smear quickly through the flame 3 times.
Check that the slide is not becoming too hot by holding it on the back of your hand between passes through the flame.
If you overheat the cells they will distort and burst.
Passing the slide through the flame is called heat-fixing of the cells, because the heat melts the sugars in the cell wall and causes them to stick to the glass.
For this type of smear, you do not need a coverslip.
6. Place the heat-fixed smear on a stain rack over a sink and flood with either crystal violet or safranin.
Leave to stand for 1-2 minutes.
7. Wash off the excess stain with water and tap the slide as dry as possible on the rack.
Fold the slide inside paper towelling and blot dry, but do not rub!
Allow the slide to air dry completely before placing on the microscope stage.
4.2.12 Serial decimal dilution of a bacterial suspension
See diagram 9.4.18: Series dilution.
1. Do this procedure in pairs with one person removing and replacing lids and flaming bottles while the other person uses the pipette and fillers.
2. Label the sterile dilution bottles by numbers according to the dilution to be made.
3. Use a sterile 10 mL pipette and filler to add aseptically 9 mL of dilutant to each of the dilution bottles.
4. Mix well the bacterial suspension, and use a sterile 1 mL pipette and filler with rubber bulb attached to transfer 1 nil, of the suspension to the first dilution bottle.
Be careful! Do not pipette and filler bacterial suspensions by mouth.
Discard the pipette and filler into disinfectant.
5. Mix well this first dilution and continue the routine from bottle to bottle, but use a new sterile pipette and filler for each transfer to prevent the carry over of large numbers of bacteria in residual drops or moisture left within or on the pipette and filler.
4.2.13 Spread plate technique
Use this technique when you wish to obtain an even growth over the entire culture plate and you are beginning with a broth (liquid) culture or you have made up a suspension of cells from a colony on a previous plate.
1. Aseptically transfer the required amount of inoculum into the centre of the plate (usually 0.5 to 1.0 mL).
2. Dip a bent glass rod into alcohol and flame.
3. Open the plate and spread the inoculum over the entire surface of the nutrient medium.
4. Close the plate and repeat step 2.
4.2.14 Spreaders
Use a sterile spreader to distribute inoculum over the surface of agar plates with a dry surface.
Dry the surface of agar plates by either incubating the plates for several hours, e.g. overnight, or put them in a hot air oven at 60oC for 60 minutes with the two halves of the Petri dish separated and the inner surfaces directed downwards.
Sterilize glass spreaders in a hot air oven.
Do not put the spreader down on the bench.
4.2.15 Staining rack
Cut a piece of chicken wire that will cover the top of a large jar.
Turn up one edge of this chicken wire to form a lip so that you can tilt the slide to wash the stain off by tilting the wire and slide together.
Do the staining in a sink that you clean with cleansing powder at the end of each practical session.
4.2.16 Streak dilution plate method for pure cultures from a mixed suspension.
See diagram 4.9.13: Streak plate.
1. When working with microbial cultures always work within the safety zone of a Bunsen flame.
2. Place the inoculum, the bacteriological loop and the labelled inverted NA plate near the base of the flame.
Check that the lid of the inoculation bottle is loose.
3. Take up the loop and sterilize by heating in the flame.
Remove the loop, but hold it beside the flame for 5-10 seconds to allow it to cool.
4. Take up the inoculation bottle in the other hand and hold the lid with the little finger on the loop hand.
Rotate the bottle to remove the lid and keep hold of the lid so that it does not touch the bench.
1. Pass the top of the bottle through the flame and insert the loop into the suspension.
6. Remove the loop, reflame the bottle and twist the bottle back onto its lid.
Place the bottle aside on the bench.
7. Pick up the agar plate by the base and expose the surface to the flame.
8. Spread the inoculum over 1/ 4 of the surface of the plate by a series of sideways strokes of the loop back and forth across the agar.
9. Place the plate back on its lid and reflame the loop.
Rotate the plate 75o clockwise, for a right-handed person.
10. When the loop has cooled, lift up the plate and angle it to the flame so that you can see where the initial inoculum was spread.
11. Prepare 4 strokes of the loop out of the initial inoculum across the surface of the agar.
Note that the inoculum being spread has been greatly diluted.
Again rotate the plate about 75 degrees clockwise.
12. Repeat the procedure a further 3 times, but at the end of each series of strokes remember to replace the plate on its lid, rotate the plate and reflame the loop.
13. If none of the final streaks recrosses the initial inoculum, the suspension will have been diluted to the point where single cells were deposited on the surface.
Each of these cells upon growth will give rise to a purified colony of the original.
4.2.17 Wire loops
Sterilize wire loops by heating in a Bunsen burner flame until red.
Hold the handle of the wire loop close to the top, like holding a pen, at an almost vertical angle, leaving the little finger free to take hold of the cotton wool plug or screw cap of a test-tube or bottle.
Heat the end of the loop slowly, because after use it may hold culture that may splutter on rapid heating.
Hold the handle end of the wire in the light blue cone of the flame, the cool area of the flame.
Move the rest of the wire slowly upwards into the hottest region of the flame, above the light blue cone, and hold it there until it is red hot.
Heat the full length of the wire.
Use the wire loop as soon as it is cool.
Do not put the wire loop down on the desk and do not wave it around in the air.
Sterilize the wire loop again immediately after use.
4.3.1 Measure the amount of juice from apple mash with and without pectinase.
1. Grate two unpeeled apples over a plastic bowl, then divide the apple mash equally between the two glass beakers, A and B.
2. Pipette 10 mL of 5% pectinase solution into glass beaker A, and 10 mL water into glass beaker B, and leave them to stand for ten minutes.
3. Place a tea strainer into each funnel over two measuring cylinders A and B, then tip the apple mash A and B into the tea strainers.
4. Measure the quantity of juice in the measuring cylinders.
Commercial juice producers use pectinase to increase yield, save energy, because the juice is easier to press, more economic methods of transport.
However, some people do not like this use pectinase, because it interferes with the natural flavour and consistency of the juice, and less wild fruit is processed.
Australia New Zealand Food Standards Code - Standard 2.6.1 - Fruit Juice and Vegetable Juice.
Fruit juice means juice made from a fruit, juice means the liquid portion, with or without pulp, obtained from a fruit or a vegetable; or in the case of citrus fruit, other than lime, the endocarp only of the fruit; and includes a product that results from concentrating juice and then reconstituting it with water.
4.3.2 Pectins and pectinases
1. Pectins are vegetable polysaccharides, their main components are galacturonic acid and its methyl ester.
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, as a sort of "putty" in the middle lamella between the cells.
They occur in solution in the cell sap.
2. Pectin, beta-D-galacturonic acid, C6H10O7, molecular weight 194.14 g/mol, high molecular weight polysaccharides, in the cell walls of all plants, used as emulsifiers and stabilizers in the food industry, a conjugate acid of a beta-D-galacturonate.
3. 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.
4. Pectinases hydrolyses pectin, a component of 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 higher yield of juice.
Pectinases also are used to clarify fruit juice.
Pectin retains substances that make the juice cloudy, but once pectin has been destroyed, those substance can easily be precipitated out of the juice.
5. Commercial pectolytic enzyme preparation is produced from a selected strain of Aspergillus niger.
It contains mainly pectintranseliminase, polygalacturonase, and pectinesterase and small amounts of hemicellulases and cellulases.
Pectinase catalyzes the hydrolysis of (1-4)-Dgalactosiduronic linkages in pectin and other galacturonans.
6. Cellulases, amylases, proteases, and lipases are enzymes that are released by cells into the environment to help breakdown the large polymer food molecules that cannot be taken up into the cells whole.
Juice from oranges and lemons and from tropical fruits such as mango, papaya, and passion fruit is concentrated locally where the water that has been removed and 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.
4.4.1 Prepare apple juice gel
1. To prepare apple juice gel, cut an unpeeled apple into eight equal pieces, leaving the core intact.
2. Place the pieces into the larger glass beaker, and just cover them with tap water.
3. Boil the mixture for ten minutes, stirring all the time, until the pieces of apple must become mushy.
4. Cool the coarse puree and press it through a muslin cloth into the plastic bowl.
5. Weigh a glass beaker, put the juice into it, and weigh the beaker and juice.
6. Add an equal amount of sugar and heat the juice again, stirring all the time.
7. After the juice has simmered for about five minutes, do a gelling test by observing the drops that fall from the wooden spoon.
8. If the drops are thick and tend to remain on the wooden spoon, leave the gel mixture to stand and cool.
4.4.2 Prepare cider from apple juice
To prepare cider from apple juice, pure culture yeasts must be used for wine making, because the fermentation of wild wine yeasts is unpredictable.
The demijohn must not be filled to the top, because the carbon dioxide produced by the fermentation of alcohol can form 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.
4.4.2 Prepare cider from apple juice
Equipment: 1 rubber tube, internal diameter 5 mm, 1 rubber stopper 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
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 stopper 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, because 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 fermentation, the respiration processes of micro-organisms 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.
4.4.3 Prepare lactic acid with sourdough
See diagram 20.162: Making sourdough in glass beakers.
Prepare lactic acid with sourdough, remembering that 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 micro-organisms: yeasts and lactic acid bacteria.
Yeasts and 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.
Gluten
People with problems digesting gluten may benefit from eating sourdough bread, because the gluten becomes partially broken down to make wheat and rye more digestible and more easily assimilated.
Experiment
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 to 5.5 pH)
Time needs: mixing of the dough: 15 minutes, inoculating: 2 X 5 minutes waiting time: 3 X 24 hours
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, 40o.
3. 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.
4. 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 washed plastic bowl, 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 washed plastic bowl, 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.
5. After three hours, measure the volume of dough 4, 5, and 6, appraise the smell, and measure the pH with pH paper.
4.4.4 Prepare lactose from milk or whey using immobilized lactase.
See diagram 20.190: Split lactose.
See 9.1.2.25: Buffer reagent, phosphate buffer reagent.
Enzymes not released to the environment, but are active inside cells are formed by micro-organisms in 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 to 100 mu, 20 mm) 1 suction flask with rubber stopper 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 pipette aids, 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 to 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, 150 mL 0.1 molar phosphate buffer reagent
Time needs: Immobilization of the lactase: 10 minutes, waiting time: 2 days, splitting of lactose: 25 minutes.
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.
4.4.5 Prepare pectinase
1. Grate unpeeled apples, e.g. "Granny Smith", into a plastic bowl to produce about 50 mL of juice.
Then squeeze the juice out of the puree into a second plastic bowl by using a folded napkin.
2. Pour 10 mL of the juice and 2 mL of 5% pectinase solution into a glass beaker, shake the mixture, then leave to stand.
3. Pour 5 mL of the juice and 5 mL of 96% alcohol into a test-tube, shake the mixture, then leave to stand.
Observation:
The pectin in fruit juice formed an insoluble gel with the alcohol.
Fruit Juice + pectinase does not produce gel, but deposits substances that make the juice cloudy.
4. Add 3 mL of pectinase solution to the remaining juice and stir the mixture constantly.
After 3 minutes, 9 minutes and 12 minutes, pipette 5 mL of the juice out of the glass beaker into a test-tube, mix with 5 mL alcohol, and rotate carefully twice.
Leaves each test-tube to stand for 5 minutes in a test-tube rack, then swirl to see if flocculation remains as a clump of gel on the surface or collect as loose components at the bottom of the test-tube.
4.4.6 Prepare sauerkraut
Prepare sauerkraut 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
To prepare sauerkraut you need 1 large cabbage, and 20 to 40 g salt
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.
4.4.7 Prepare vinegar with Acetobacter aceti
Equipment: | aluminium foil | 1 glass tube | 1 aquarium pump | 1 glass tube right-angled | 1 one-way tap right angled | 2 X 5 mL sterile pipettes | 1 conical flask 500 mL || pipette aids | 1 culture tube | 1 rubber stopper single bored | 2 glass bottles 1.5 litres with stopper attachments at their bases | 1 rubber stopper double bored | rubber tubing | stand material |
Materials: | pure culture of Acetobacter aceti (DSM-No. 3508) | beech tree shavings | cotton wool | 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 vinegar bacteria medium in a 300 mL conical flask.
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.
1. Attach a 1.5 litre bottle that has a fixture for a stopper at its base about 40 cm above the table, using the stand.
A single bore rubber stopper with an angled, one way tap seals the lower outlet of the bottle where the stopper 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 stopper 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-angle glass tube as a product outlet.
4. Use an aquarium pump to blow air constantly into the inside of the fermenter through the stopper, 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 1 to 1.
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.
4.4.8 Prepare wine from grape juice and prepare 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, 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.
This technique, Fessel procedure, was 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.
A common method to produce vinegar in dilute alcohol solutions from fermenting wine with Mycoderma aceti.
4.4.9 Prepare yoghurt
(activity for primary grade 4 students, about 9 years old)
Yoghurt is made from milk inoculated with a mixed culture of Lactobacillus bulgaricus and Streptococcus thermophilus, then inoculated for hours then left to cool so that the milk proteins coagulate at about pH 4.3.
Equipment: 1 one 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 containers of yoghurt made from whole milk, cling film.
1. Heat the milk to 72oC to kill any harmful bacteria in the milk.
2. Allow the milk to cool to 45oC or body temperature, 37oC.
3. Place a teaspoon (5 mL), of unpasteurized yoghurt and lactic acid bacteria starter into a plastic container.
4. Add the cooled milk to the container.
5. Mix all of the ingredients.
6. Cover the container with cling film.
7. Place the yoghurt mixture into an insulated box to let the contents incubate at 45oC for 3-4 hours or overnight at room temperature.
8. After the milk has thickened, the yoghurt is ready.
It tastes acidic.
9. If yoghurt is made from whole milk, the product is smoother but, unless homogenized, a fat layer may separate.
To avoid this separation, add 3% of skim milk powder to the fresh milk before adding the yoghurt starter.
4.5.1 Amylase
Amylase, n-amylene, C5H10, amylene, n-pentene, 2-methyl-2-butene, 1-pentene, pent-1-ene, Highly flammable
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 breakdown the starch that forms as dirt on cutlery or in clothes.
9.9.3.9 Tests for diastase activity
9.3.6 Tests for breakdown of starch to sugars
4.5.2 Lactase
Lactase works inside the organism where it decomposes lactose molecules to α-glucose and β-galactose.
Whey contains relatively large quantities of lactose.
Many adult humans cannot breakdown lactose in the digestive tract, because they no longer produce "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 gases causing flatulence.
Lactase is used to decompose lactic acid and to produce glucose.
The following experiments, describe the effect of pectinase, splitting of lactose, and decompose starch with Bacillus subtilis.
4.5.3 Protease
A protease (peptidase, proteinase), is a proteolysis enzyme that hydrolyzes peptide bonds in proteins and peptides.
Proteases breaks down proteins into amino acids.
Proteases are used to degrade proteins, to study protease inhibitors and to study thermal inactivation kinetics.
Protease is used in washing powder to decompose protein stains and lipases are used to break up fat stains.
Proteases may be isolated from bacteria.
For example: Alcalase is a protease from Bacillus licheniformis.
Savinase is a protease from Bacillus species.
Subtilisin is a protease from Bacillus subtilis.
Proteinase K is a protease from Tritirachium album.
Proteases from Aspergillus oryzae contains both endoprotease and exopeptidase activities.
A protease usually begins the hydrolytic breakdown of proteins by splitting them into polypeptide chains.
An endopeptdase catalyzes the hydrolysis of a peptide chain within the chain, not near either terminus, e.g. pepsin, trypsin.
An exopeptidase catalyzes the hydrolysis of the terminal amino acid of a peptide chain, e.g. carboxypeptidase.
An endopeptidase is an enzyme which breaks peptide bonds other than terminal peptide bonds in a peptide chain, and includes ficin from fig tree latex, papain derived from papaya latex, bromelain (bromelase), extracted from pineapple stem.
Food additive E1101 Proteases: papain, bromelain, ficin, enzymes (stabilizer, flour treatment agent, tenderizer, flavour enhancer), avoid skin contact.
Proteases are used in bread making as a bread improver / flour treatment agent and raising agent.
Proteases acts on the yeast and gluten to improve the extensibility of the dough and strengthen the structure of the bread to retain the carbon dioxide produced, which causes the bread to rise.
4.2.9 Microbiology chemicals
agar (MERCK No. 1614, SIGMA No. A 7002) 96% alcohol (ethanol)
agar (MERCK No. 818760, SERVE No. 11094) 70% alcohol (ethanol)
agar (MERCK No. 818760, SERVE No. 11094)
D, L-arginine (SIGMA No. A 4881)
BAP (6-benzylaminopurine) (SIGMA No. B 6894)
casein peptone (MERCK No. 2239, SIGMA No. C 9386)
"Domestos" household cleaning fluid (containing sodium hypochlorite)
Eupergit C (from ROHM gumbo, D-6700 Darmstadt, FR Germany)
Fe2(SO4)3 NHL (MERCK No. 3965, SERVE No. 20917)
glucose (MERCK No. 8342, SIGMA No. G 8270)
granulated sugar or sucrose (MERCK No. 7651E, SIGMA No. S 8501)
HCl (MERCK No. 9057, SERVE No. 34616)
D, L-histidine (SIGMA No. S H 7875)
Indian ink isopropanol (MERCK No. 9634, SIGMA No. 405 -7)
KH2PO4 (MERCK No. 4873, SIGMA No. P 0662)
K2HPO4 (MERCK No. 5099, SIGMA No. P 3786)
KOH (MERCK No. 9918, SIGMA No. P 1767)
lactase (SERVE No. 22075)
liver extract (MERCK No. 5347, SIGMA No. 201 -1)
malt extract (MERCK No. 5391E, SIGMA No. M 0383)
malt extract agar (MERCK No. 5398)
mantel (MERCK No. 5982, SIGMA No. M 9647)
meat extract (MERCK No. 3979, SERVE No. 48020)
MgSO4.7H2O (MERCK No. 5886, SIGMA No. M 9397)
Mn(SO4)3.4H2O (MERCK No. 5963, SERVE No. 28405)
MS-powder (after Murashige and Stooge) (SIGMA No. M 68.99)
Nalco (MERCK No. 6404, SIGMA No. S 8888)
NaOH (MERCK No. 9137, SIGMA No. 930 -65)
(NH4)2SO4 (MERCK No. 1211, SIGMA No. A 5132)
nutrient agar (MERCK No. 5450)
nutrient broth (MERCK No. 5443)
pectinase (SIGMA No. P 5146)
peptone from meat (MERCK No. 7214, SIGMA No. P 7750)
phenol red (MERCK No. 7241, SIGMA No. P 2167)
Ringer tablet (MERCK No. 15525)
sodium citrate (MERCK No. 6448, SIGMA No. C 7254)
sugar test strips
streptomycin (MERCK No. 10117, SIGMA No. S 6501)
thiamine HCl (SIGMA No. T 4625)
Tween 80 (polyoxyethylenesorbitan) (SIGMA No. P 1754)
urea agar (Christens) (MERCK No. 8492)
urea (MERCK No. 818710, SIGMA No. U 1250)
washing liquid (containing tensides)
yeast extract (MERCK No. 3753, SIGMA No. Y 4000)
Warning!
1. The experiments in this document from an international group of science educators were 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, check that you can follow the safety precautions below.
Do not attempt any of the experiments if you have no experience of teaching biotechnology.
2. 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 or yeast and mould) and genetically modified organisms.
In Australia, see the following:
* Australian or New Zealand Standard, Safety in laboratories, Part 3: Microbiological aspects and containment facilities (AS or NZS 2243.3:2002)
* Gene Technology Act 2000, passed by the Federal Government In December 2000 and came into force on 21 June 2001
This 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.