School Science Lessons
UNBiology4a
2018-11-18
Please send comments to: J.Elfick@uq.edu.au
4.0.0 Microbiology

Table of contents
See: Microbiology, (Commercial)

4.3.0 Micro-organisms

2.1.0 Microbiology safety

3.3.0 Microbiology techniques

16.2.7 Yoghurts


4.3.0 Micro-organisms, [Based on "Practical Microbiology for Secondary Schools", Society for General Microbiology (SGM), UK]

4.3.14 Breakdown of pectin by micro-organisms

4.3.7 Breakdown of protein by micro-organisms

4.3.6 Breakdown of starch by micro-organisms

4.3.5 Estimate the number of bacteria in a water sample

4.3.4 Find and grow micro-organisms

4.3.16 Food preservation of peas

4.3.20 Micro-organisms and antiseptics

4.3.15 Micro-organisms and bread making

4.3.10 Micro-organisms and cellulose

4.3.18 Micro-organisms and food spoilage

4.3.19 Micro-organisms and milk quality

4.3.21 Micro-organisms and personal hygiene

4.3.11 Micro-organisms and water pollution

4.3.12 Nitrogen-fixing bacteria

9.2.20 Nutrient broth medium

4.3.8 Prepare alcohol using immobilized yeast cells

4.3.9 Prepare Euglena culture

4.3.17 Prepare yoghurt, test milk quality

4.3.13 Root nodules, Isolate micro-organisms from root nodules

4.3.22 Sensitivity of micro-organisms to antiseptics

9.2.30 Tensides, alkylphenol ethoxylates

2.1.0 Microbiology Safety
See: Safety, (Commercial)
See: Autoclaves, (Commercial)
See: Cleaning, (Commercial)
2.1.9 Animal tissue culture
2.1.6 Antibiotics, penicillin, Safety in school science
2.1.11 Bacteria and fungi NOT suitable for use in schools
2.1.4 Biogas
2.2.0 "Biosafety", Advances in Genetic Technology, BSCS, USA (edited)
2.1.5 Disposal, Safety in school science
2.1.3 Electrical safety, Safety in school science
2.1.8 Enzymes, Safety in school science
2.1.1 Fermentation, Safety in school science
2.1.10 Genetic engineering, Safety in school science
2.1.7 Plant growth substances
2.4.0 "Safety in the microbiology laboratory", by Eleanor Gough, Australian Science Teachers Journal Vol. 33, No. 3. (edited)
4.1.5 Safe microscopy of Penicillium camemberti and Mucor mucedo using the Petri slide technique
2.1.2 Spillage, Safety in school science
2.3.0 "Ten Rules for Safe Microbiology and Biotechnology in School", by Eckhard R. Lucius, IPN, Kiel, Germany, UNESCO / IUBS (edited)

3.3.0 Microbiology techniques
9.1.2.2 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 Heat-fixed stained bacterial smears
9.1.2.7 Streak dilution plate method for pure cultures from a mixed suspension
9.1.2.8.0 Lawn plate technique
9.1.2.8.1 Estimate aerobic mesophilic bacteria by the plate count method
9.1.2.9 Spread plate technique
9.1.2.10 Serial decimal dilution of a bacterial suspension
9.1.2.29 Microbiology chemicals
9.2.30 Tensides, alkylphenol ethoxylates

16.2.7 Yoghurts
9.3.0 "Yakult", Yoghurt and lactic acid bacteria
16.2.7 Yoghurts
4.2.1 Prepare yoghurt (Primary)
4.2.1a Prepare yoghurt (Turkey)
4.3.17 Prepare yoghurt, test milk quality
9.3.0 Yoghurt and lactic acid bacteria

2.1.1 Fermentation
1. Fermentation experiments may be done out on a small or relatively large scale in a fermenter.
After initial inoculation, large numbers of micro-organisms may be produced.
Using a fermenter may require electrical connection, and gases may be produced, some of which may be flammable.
Risks from fermentation can be minimized by choosing suitable organisms and techniques, using safe ways of handling suitable
micro-organisms, and keeping the volume of the medium to a practical minimum.
2. Micro-organisms particularly suitable for studying fermentation technology at school level are yoghurt making bacteria, strains of
non-pathogenic yeast, and some unicellular algae, e.g. wine yeast, beer yeast, bread making yeast, dried yoghurt cultures.
3. For work at a more advanced level it is recommended that only micro-organisms with unusual growth needs are used,
e.g. micro-organisms requiring high salt, acid conditions, low or high temperatures, i.e. as far away from 37oC as possible, to
encourage growth of the "intended" organism at the expense of undesirable contaminants.
Bacteria with unusual growth needs include: Vibrio nutriegens (Beneckea nutriegens), Photobacterium phosphoreum,
Acetobacter aceti.
4. Organisms other than those listed above may be used, but only if the teacher responsible has had appropriate training in
microbiological techniques, i.e. giving a good knowledge of aseptic techniques, subculturing, recognition of contamination, and safe
disposal.
5. Cultures should be obtained only from recognized specialist UK schools suppliers.
Cultures should never be exchanged between schools, colleges, and other institutions.
Maintenance beyond two or three subcultures should be done only if those involved have had training in the necessary techniques.
The use of suitable techniques is particularly critical when working with fermentation.
Prepared and sterilized media and equipment must be used.
Aseptic technique must be used when inoculating the fermenter and when taking samples.
The volume of actively growing inoculum should be a significant fraction of the volume of the medium, e.g. 20% so that any chance of
contaminant getting in during the inoculation procedure has to compete with an established organism.

2.1.2 Spillage
When using fermenters, it is also important to remember that relatively large volumes of potentially hazardous substances, e.g. antibiotics,
enzymes, pesticides, or growth substances, can be produced, posing problems for dealing with spillages or safe disposal.
Spillages should be avoided, and if they happen, need to be carefully managed.
Materials and equipment must be safely sterilized and disposed of after use.
Any spillages must be cleared up immediately using a suitable disinfectant, e.g. clear phenolic, Clearsol, Hycolin, Putitol, Steticol, Sudol.
All disinfectants should be freshly prepared, and used at manufacturers' recommended dilutions.
Spills on clothing may be best disinfected using a surface active disinfectant, e.g. Harris "BAS clean" and "Griffin ASAB".
Any spillage may cause the formation of an aerosol that may remain for some time.
If a gross spillage occurs, the laboratory should be cleared immediately and that all accidental microbiological spillage incidents should
be recorded.
Aerosols are fine sprays or suspensions that, in this context, contain micro-organisms.
The scale of liquid culture involved may increase the chance of any contaminant being cultured in large numbers and then being released
back into the environment by the formation and escape of aerosols.
A fermenter must be adequately vented to prevent the build up of pressure in the vessel.
For work with yeast and similar organisms, a wine making trap or strong non-absorbent cotton wool plug attached to the air vent
should be sufficient to trap any fine spray.
The use of in-line microbiological filter cartridges should be considered.
In solid culture fermentation, e.g. cheese, tempeh, there is the possibility of fungal spores being produced in quantities sufficient to
produce sensitization and possible allergic reactions.

2.1.3 Electrical safety
Fermenters must heated and stirred, and much useful information can be gained by the use of monitoring equipment ranging from
stand-alone instruments to computer data logging packages.
However, most of these will be at less than 20 volts, which is relatively safe.
Care should be taken to keep mains and other leads tidy, and it is clearly wise to site electrical equipment as far as is possible from the
fermenter vessel and wet working areas.
Mains powered equipment, e.g. pH meter, used with a fermenter should be of a reputable design intended for school use.
Where apparatus other than purely proprietary devices intended for continuous operation, e.g. incubators, is left running out of school
time, then it should bear a warning notice.

2.1.4 Biogas
Some schools are interested in investigating "biogas" generation by the fermentation of silage or grass clippings.
Methane is produced slowly when the mixture is inoculated by the addition of well rotted garden compost or rich pond mud and the
presence of broad bean husks is helpful.
Such procedures are acceptable, provided the methane is handled with the care because of a flammable gas.
However, this document does not recommend the use of animal manure as an inoculum, although of course it is widely used for fuel gas
production.
While many people handle horse manure in stables or when gardening, deliberate culturing in a fermenter may introduce unacceptable
risks of infection, e.g. from Salmonella

2.1.5 Disposal
See: 2.1.6 Antibiotics, penicillin
Antibiotics can cause allergic reactions, so penicillin-producing cultures should not be used in schools.
Culturing of large quantities of any micro-organism that produces an antibiotic could be hazardous.
There is growing concern over the promiscuous use of antibiotics and the increasing prevalence of antibiotic resistant strains of
micro-organisms.

2.1.7 Plant growth substances
Plant growth substances, known wrongly as plant hormones, are extensively used both in growth investigations and in plant tissue culture.
Many are toxic.
A few may also be carcinogenic.
They are normally used in very low concentrations in experiments and media, but technicians and teachers handling solids or more
concentrated solutions should be aware of the hazards, and take appropriate precautions, e.g. by wearing disposable gloves.

2.1.8 Enzymes
Hazards from enzymes are associated with their increased use both in quantity and variety.
Teachers and technicians should be aware of the hazards involved in handling enzymes in solid and liquid form, to avoid spillage and the
formation of aerosols.
Enzymes are biologically active proteins, which can irritate the skin or eyes and cause allergic reactions.

2.1.9 Animal tissue culture
Practical, work with animal tissue cultures is not recommended for use in schools because of the risk of serious infection.
If such work is to be done, ensure that the culture is obtained from a recognized specialist UK schools supplier.

2.1.10 Genetic Engineering
Experiments called "genetic engineering" should not be done in schools.
However, "genetic engineering" is a loose and somewhat general term.
The Health and Safety Executive (HSE) regulations and guidance adopt a more precise definition of the term "genetic manipulation."
"Genetic manipulation" is defined by HSE as: (the) formation of new combinations of heritable material by the insertion of nucleic acid
molecules, produced by whatever means outside the cell, into any virus, bacterial plasmid, or other vector system so as to allow their
incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation.

2.1.11 Bacteria and fungi NOT suitable for use in schools
Fungus
Rhizomucor (Mucor) pusillus
Bacteria
Bacillus cereus, Chromobacterium violaceum, Clostridium perfringens (C. welchii), Clostridium tetani,
Photobacterium phosporeum, Proteus vulgaris, Pseudomonas acruginosa, Pseudomonas aeruginosa (It is said to be
responsible for one-in-ten hospital-acquired infections), Pseudomonas solanacearum, Pseudomonas tabaci, Serratia marcescens,
Staphylococcus aureus, Vibrio fischeri, Xanthomonas phaseoli

2.2.0 "Biosafety"
See: Safety, (Commercial)
See: Autoclaves, (Commercial)
See: Cleaning, (Commercial)
See: Containers, sharps, (Commercial)
See: Packaging, (Commercial)
See: Syringes, (Commercial)
Following containment procedures:
1. The laboratory door must be kept closed, except as needed for access.
No special laboratory design or special containment equipment is required to work with Biosafety Level 1 organisms.
2. There is to be no eating, drinking, smoking, or storage of food in the laboratory.
3. Workers must wash their hands thoroughly after handling organisms, and upon leaving the laboratory.
4. Do not use a pipette and filler by mouth.
Only use a mechanical pipette and filler device.
5. Take special care to avoid skin contamination with potentially biohazardous organisms.
Wear gloves when skin contact with the agent is unavoidable.
6. Avoid use of hypodermic needles and syringes when alternative methods are available.
Take care to minimize the creation of aerosols or splatters that occur when a hot loop or needle is inserted into a culture, an
inoculating loop is flamed so that it splatters, or fluids are forcefully ejected from pipette and fillers and syringes.
7. Separate biological waste from office trash and general laboratory trash.
8. Collect solid and liquid biological waste in specially marked containers, e.g. autoclave plastic bags or biosafety disposal bags, and
disinfect by with autoclave or pressure cooker or by treatment with a solution of household bleach.
The containers should be airtight and must be secured tightly and marked with tape that reads "AUTOCLAVED after with autoclave
or pressure cooker.
9. Solutions useful for decontamination include chlorine-based disinfectants, which are effective against vegetative bacteria, most viruses,
and fungi at 500 ppm.
Clorox or Purex diluted at 1:100 provide the necessary concentrations.
A dilution of 1:20 yields 2500 ppm, which will also kill bacterial spores.
Ethanol 70% in water is effective against vegetative bacteria and nonlipid-containing viruses and is effective for surface decontamination.
10. Solid trash that has been contaminated by biological waste must be collected in a separate, specially marked disposal bag, see step 8.
Package all sharp instruments, e.g. needles or scalpel blades inside the container, in separate cardboard containers or in other
available containers for disposal.
No pipette and fillers should protrude from the disposal bag.
Free liquids and gels must be absorbed by paper towels to minimize the risk of leakage.
The bags must be secured tightly, and tape that shows the word "AUTOCLAVED" after with autoclave or pressure cooker must be
clearly visible on the bags.
11. Contaminated materials that are to be decontaminated at a site away from the laboratory must be placed in a durable, leak-proof
container that is closed before removal from the laboratory.
Contaminated materials that are to be reused should be decontaminated before washing.
12. Laboratory workers, students, and teachers must clean and decontaminate work surfaces with 70% ethanol.
Those individuals also are responsible for the decontamination of biological material spills, removing contaminated supplies (such as
pipette and fillers and syringes) from floors, and decontamination of contaminated floor surfaces.

2.3.0 Ten rules for safe microbiology and biotechnology in school
See: Autoclaves, (Commercial)
In the Federal Republic of Germany, the safety of scientific teaching is governed by guidelines set out by the Ministers of Education.
The code of conduct that generally applies to scientific experiments is expounded here, e.g. no eating, drinking, or smoking in
laboratories, chemicals should not be tasted, hands should be washed, pipetting should not be done using the mouth.
The IPN, Institute for Science Education, recommends that the following ten rules should also be observed for microbiological and
biotechnical experiments:
1. Pure cultures of micro-organisms suitable for use in schools should be ordered from the DSM (international: from the ATCC:
American Type Culture Collection).
2. Enrichment plates of micro-organisms from the environment should be sealed by sticking the slit between the base and lid of the
Petri dish together with clear adhesive tape.
3. Bacteriological experiments with faecal material ("biogas from sewage sludge") should not be done in schools.
4. Work in sterile conditions, i.e. nutrient solutions should not only be boiled, but sterilized in a pressure cooker or autoclave.
Glass equipment or pipette and fillers that come into contact with sterile solutions should be sterilized for 30 minutes in a drying
cupboard at 18oC.
5. Colonies of mould that have formed spores should not be examined under the microscope without using Petri slides.
6. Do not use antibiotics from a pharmacy for school science experiments to avoid increasing resistance to antibiotics on prescription.
7. Use disposable Petri dishes instead of glass Petri dishes.
8. Mouth pipetting should never be employed.
A piston device should be used to draw the liquid up the pipette and filler instead of a rubber bulb.
9. Do not leave bacterial cultures that are no longer required lying around.
Old cultures should be destroyed in a pressure cooker and disposed of.
10. Only use microbial nucleic acids if they occur naturally in the organism (in vivo DNA).
Do not use genetically engineered material (in vitro DNA).

2.4.0 "Safety in the microbiology laboratory", by Eleanor Gough, Australian Science Teachers Journal Vol. 33, No. 3. (edited)
See: Safety, (Commercial)
See: Autoclaves, (Commercial)
See: Cleaning, (Commercial)
See: Containers, sharps, (Commercial)
See: Packaging, (Commercial)
See: Syringes, (Commercial)
1. Do not use human or other animal materials as sources of micro-organisms.
You may isolate a human pathogen and in the culturing process produce many millions of these cells.
2. Treat all organisms as "potential pathogens" by following the safety guidelines.
People who are immuno-compromised are at risk and in experiments in which you are growing cultures.
Many millions of cells are concentrated in a way not found in the environment.
3. Do not do experiments using antibiotic discs because you may succeed in producing antibiotic-resistant bacteria.
The range of antibiotics available for medical use is limited and pathogenic bacteria resistant to these could become a major problem.
4. Do not expose Petri dishes in toilet areas or allow students to cough or sneeze over them.
5. Do not eat, drink or smoke in the laboratory.
Do not touch a plate or unsterilized loop with the fingers, pencils or pens but use disposable gloves.
6. Do not pipette and filler bacterial cultures by mouth.
7. Laboratory coats or gowns or aprons should be worn by everyone working with bacterial cultures so that the coat can be removed
and sterilized if there is any accidental spillage or contamination.
8. All people working in the laboratory must wash their hands in a disinfectant solution diluted to the recommended concentration or
with a disinfectant soap upon contamination and before leaving the laboratory.
9. Swab down all working surfaces when the practical session is completed, using disinfectant diluted to the recommended concentration.
Provide this in a bucket, with a "Wettex" cloth kept for this purpose.
10. Flood spills with disinfectant and leave to stand for one minute.
Mop up with "Wettex" from the bench swab bucket.
Wash hands with disinfectant soap or solution.
11. Replace all disinfectant solutions at the end of each day.
12. When exposing microbial cultures to the atmosphere, always work within the safety zone of a Bunsen burner flame.
This zone extends 15 cm around the flame.
Any cells that may escape from the cultures, suspensions or bacteriological loop in the form of an aerosol will be drawn into the flame
by the convection currents surrounding the flame and be destroyed.
13. Incubate cultures at room temperature, or a temperature close to the original environment of the organism.
If you wish to hurry the culture along, try 30oC to 34oC .
Use disinfectant diluted to the recommended concentration.
Provide this in a bucket, with a Wettex cloth kept for this purpose.
To avoid culturing pathogens, do not use of 37oC as an incubation temperature because 37oC is the normal human temperature.
14. Seal Petri dishes after inoculation by taping the circumference with clear adhesive tape.
The lids must not be removed when the students examine the cultures.
15. Discard wastes as follows:
15.1 Discard waste paper in a bin or garbage can for burning.
15.2 Discard slides into a jar of disinfectant.
15.3 Discard pipette and fillers and swab sticks into a cylinder of disinfectant.
15.4 Discard plastic equipment by autoclave, or sterilize in a pressure cooker follow the manufacturer's instructions.
The equipment will be destroyed by the treatment and may be incinerated after being sterilized.
Sterilize plastic equipment separately from glassware.
15.5 Discard glass equipment.
Use the autoclave or sterilize in a pressure cooker before cleaning out the material, then wash normally and store until next time.

4.3.4 Find and grow micro-organisms
Biology, Autoclave (Commercial websites)
See diagram 9.4.10: Agar plates 1
1. Prepare and label three plastic Petri dishes containing nutrient agar plates or malt extract agar plates as follows:
Petri dish 1.1: In the laboratory or outside, take the lid off the Petri dish, keep it open for one hour, then replace the lid.
Do not expose the agar plates in toilets.
Petri dish 1.2: Lift the lid of the Petri dish and put three drops of pond water on to the surface of the agar.
Use a sterile glass spreader to spread the drops evenly over the agar, then replace the lid.
Petri dish 1.3: Lift the lid of the Petri dish, and put three drops of soil suspension on to the surface of the agar.
Use a sterile glass spreader to spread the drops evenly over the agar, then replace the lid.
2. Attach the lids and bases of the Petri dishes with four short strips of clear adhesive tape to protect against accidental opening.
Turn the Petri dishes upside down and incubate them at 20-25oC overnight.
3. The next day, compare the three agar plates.
Observe growths of fungi and bacteria.
Be careful! Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.5 Estimate the number of bacteria in a water sample
1. Collect 100 mL of pond water in a beaker or use a diluted overnight nutrient broth culture of Bacillus subtilis or Micrococcus luteus.
Use a pipette and filler to remove 2 mL of the pond water or culture.
2. Prepare and label two Petri dishes as follows:
Petri dish 2.1: Remove the lid of a Petri dish.
Put 1 mL of the removed pond water or nutrient broth culture in a Petri dish, then replace the lid.
Petri dish 2.2: Remove the stopper of a test-tube.
Put in the remaining 1 mL of the pond water or nutrient broth culture in the test-tube, then add 9 mL of deionized water using the
pipette and filler.
Remove the lid of a Petri dish.
Put 1 mL of the diluted pond water or culture in the Petri dish, then replace the lid.
3. Remove the top of a Universal bottle containing 15 mL of melted nutrient agar at 45-50oC.
Pass the neck of the bottle through a Bunsen flame three times.
Pour the contents of the bottle into Petri dish 2.1.
Mix the contents of the Petri dish with a sterile glass spreader, moving in a figure of eight, but avoiding any splashing over the edge.
Repeat the procedure for Petri dish 2.2.
4. Leave the nutrient agar to set.
Attach the lids and bases of the two Petri dishes with four short strips of clear adhesive tape to protect against accidental opening.
Invert the Petri dishes and incubate them at 20-25oC overnight.
5. The next day, examine the agar plates and record the number of colonies.
Assume that each colony has usually come from a single cell.
Be careful!
Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker, Use disinfectants, e.g. domestic
bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.6 Breakdown of starch by micro-organisms
See diagram 9.4.10: Agar plates 1
1. Two days before the experiment:
Inoculate two nutrient broths: Bacillus subtilis culture, and Escherichia coli culture
Prepare starch nutrient agar.
Prepare 6 mm diameter paper discs from filter paper or absorbent paper with a punch.
2. Invert a Petri dish containing a starch nutrient agar plate.
Divide the base into four sections, A, B, C, D, by drawing on it with a marker pen, then invert the Petri dish again so the starch
nutrient agar is up.
Pass forceps through the Bunsen burner flame, leave to cool and use them to pick the paper discs.
Open a Bacillus subtilis culture.
Flame the neck of the container and dip a paper disc into the culture.
Allow any excess culture to drain off.
Reflame the neck of the container and replace the stopper.
Transfer the disc to the middle of section A on the starch nutrient agar plate.
Repeat the procedure with a paper disc and an Escherichia coli culture on section B.
Repeat the procedure with a paper disc and a 0.1% amylase solution, on section C.
Repeat the procedure with a paper disc and sterile deionized water on section D.
Attach the lid and base of the Petri dish with four short strips of clear adhesive tape to protect against accidental opening and incubate
at 20-25oC overnight.
3. The next day, lift the lid of the Petri dish and use a dropper to put enough iodine solution to just cover the surface of the agar, then
replace the lid.
4. Measure the diameter of any clear zones around the paper discs by placing the agar plate on the graph paper.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.7 Breakdown of protein by micro-organisms
See diagram 9.4.10: Agar plates 1
1. Two days before the experiment, inoculate two nutrient broths: Bacillus subtilis in nutrient broth culture, and Saccharomyces
cerevisiae
in malt extract broth culture.
Prepare two milk agar plates.
Prepare and label two plastic Petri dishes containing nutrient agar plates or malt extract agar plates as follows:
2. Open a culture of Bacillus subtilis.
Flame the neck of the container and use a dropping pipette and filler to remove a small amount of culture.
Reflame the neck of the container and replace the stopper.
Lift the lid of the first milk agar plate.
Release one drop of Bacillus subtilis culture on to the middle of the agar plate, then replace the lid.
3. Repeat the procedure with the second milk agar plate and Saccharomyces cerevisiae culture.
4. Attach the lids and bases of the Petri dishes with four short strips of clear adhesive tape to protect against accidental opening.
Keep the Petri dishes upright until the drops have dried, then invert them and incubate at 20-25oC overnight.
5. The next day, examine the milk agar plates.
The milk agar is opaque because of the milk protein casein so clear areas on the milk agar plates indicates activity of protease enzymes.
Be careful! Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.8 Prepare alcohol using immobilized yeast cells
See diagram 9.4.10: Agar plates 1
1. Two days before the experiment, inoculate Saccharomyces cerevisiae yeast culture in malt extract broth culture.
Prepare fresh lime water.
Prepare 250 mL of 2% calcium chloride solution.
The procedure is to prepare and label two 250 mL conical flasks, fitted with one-hole stoppers and connecting tubes, as follows:
1.1 In the "free cells" flask, 100 mL apple juice + 4 mL yeast culture + 6 mL deionized water, connected to lime water in a conical flask
1.2 In the "immobilized cells" flask, 100 mL apple juice + sodium alginate solution + 4 mL yeast culture, connected to lime water in
conical flask
2. Use a syringe to transfer 4 mL of yeast culture and 6 mL of deionized water into the "free cells" flask containing apple juice.
Fit a stopper and connecting tube dipping into a 100 mL conical flask containing 50 mL of lime water.
3. Draw up 6 mL of sodium alginate solution into the syringe, followed by 4 mL of yeast culture.
Mix the contents of the syringe by inverting it.
While gently rotating the beaker of lime water to avoid spillage, release drops from the syringe with the nozzle 5 cm above the solution
in the beaker.
Filter the contents of the beaker, to separate beads of immobilized yeast cells.
Rinse the beads with deionized water then tip them into the "immobilized cells" flask containing apple juice.
Fit a stopper and connecting tube dipping into a 100 mL conical flask containing 50 mL of lime water.
4. Leave the flasks at room temperature and examine them regularly over the next two weeks for cloudiness, the tests for carbon
dioxide that indicates alcohol production by fermentation.
Later a crust may form on the lime water and the solution may become clear.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.9 Prepare Euglena culture
See diagram 9.4.9: Euglena
1. Euglena is a micro-organism that moves with light and may be found as a green scum on the surface of ponds or may be purchased
as a culture.
Gently swirl a bottle of Euglena culture.
Remove the stopper and flame the neck.
Draw some culture up into the dropping pipette and filler.
Flame the neck again and replace the stopper.
2. Put one drop of culture on a microscope slide and put a coverslip over it.
Examine the Euglena culture under low power and high power.
Observe the structure of an Euglena: Single flagellum at the front end, the "gullet" (evagination), nearby light-sensitive photoreceptor
(chromatophores, eye spot), a contractile vacuole, a nucleus, chloroplasts and paramylum carbohydrate storage bodies.
The flexible body covering allows the body to contract and elongate as the Euglena moves in the direction of the flagellum.
3. Cut a black paper to make a sleeve to fit around a boiling tube.
Cut five 1 cm2 windows in the sleeve.
Use clear adhesive tape to fix red, yellow, green, blue and colourless filters over the windows.
Roll the sleeve around the boiling tube and use clear adhesive tape to stick the ends together.
Flame the neck of the culture bottle in a Bunsen burner and pour the remaining Euglena culture into the boiling tube.
Fill the boiling tube with growth medium and insert a cotton wool stopper.
Cover the boiling tube with aluminium foil except for the five windows, and leave it in sunlight.
4. The next day, remove the foil and the sleeve from the boiling tube.
Observe where the Euglena have aggregated at the side of the boiling tube where the windows had been situated.
Note which window was the place of most aggregation.

4.3.10 Micro-organisms and cellulose
1. Add 5 g of garden soil to 30 mL of nutrient broth in a conical flask.
Rotate the flask to form a soil suspension.
Leave the suspension to settle.
Use a pipette and filler to transfer the supernatant "nutrient broth + soil".
Cut different kinds of paper into 1 × 2 cm2 strips.
Prepare and label test-tubes 1.1 to 1.6 as follows:
1.1 Use 5 mL nutrient broth only + strip of filter paper or absorbent paper
1.2 Use 5 mL nutrient broth + soil + strip of filter paper or absorbent paper
1.3 Use 5 mL nutrient broth + soil + strip of newspaper with no print on it
1.4 Use 5 mL nutrient broth + soil + strip of heavily printed newspaper
1.5 Use 5 mL nutrient broth + soil + strip of glossy magazine cover
1.6 Use 5 mL nutrient broth + soil + strip of thin cardboard
2. Leave the test-tubes for one week at room temperature, then tap each test-tube and observe what happens to the paper strip.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.11 Micro-organisms and water pollution
See diagram 9.4.11: Agar plates 2
1. Prepare and label conical flasks 1.1 to 1.6 as follows:
1.1 Use 100 mL pond water
1.2 Use 0.1 g potassium nitrate + 100 mL pond water
1.3 Use 0.1 g potassium nitrate + 0.1 g potassium phosphate + 100 mL pond water
1.4 Use 0.01 g nutrient broth powder + 100 mL pond water
1.5 Use 1 g nutrient broth powder + 100 mL pond water
1.6 Use chopped hay + 100 mL pond water
Hay is grass that has been cut and dried for animal fodder.
Straw is dried cereal stems.
2. Plug the neck of each flask with cotton wool.
Leave the flasks in a sunny room at room temperature.
During the next few weeks examine the contents of each flask.
Note the colour of the water and whether it is clear or cloudy.
Be careful! Do not open the flasks.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.12 Nitrogen-fixing bacteria
See diagram 9.4.11: Agar plates 2
1. Prepare a nitrogen-free mineral salts agar plate.
1.1 Drop 20 crumbs of soil evenly over the surface of a nitrogen-free mineral salts agar plate.
1.2 Drop 20 crumbs of soil evenly over the surface of a nutrient agar plate
2. Incubate the Petri dishes at 20-25oC overnight.
Be careful!
Do not open the Petri dishes.
Examine the Petri dishes for microbial growth.
Colonies of Azotobacter usually appear slimy and colourless around the soil particles.
Compare the growth of bacteria in the two Petri dishes.
Be careful! Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.13 Root nodules, Isolate micro-organisms from root nodules
See: Root Nodules slide TS, (Commercial)
See diagram 9.209: T.S.
Root nodule | See diagram 9.72: Root nodules
1. Prepare a mannitol yeast extract agar plate (MYEA).
Dig up leguminous plants and select a piece of root with root nodules.
Squeeze some nodules to check that the contents are living.
2. Use tap water to wash soil from the piece of root with root nodules.
Use forceps to transfer the piece of root into 1% bleach solution to clean the nodules.
Pass forceps through a Bunsen burner flame, allow to cool and use it to transfer the piece of root to sterile water to rinse off the bleach.
Repeat this procedure twice more with fresh sterile water.
Transfer a few drops of sterile water to a sterile Petri dish and add the piece of root using the flamed metal forceps.
Use a sterile glass rod to macerate the nodules to produce a milky fluid.
3. Sterilize a wire loop by flaming, cool it, then use it to form streaks of the nodule macerate on the MYEA agar in the Petri dish.
Attach the lid and base of the Petri dish with four short strips of clear adhesive tape to protect against accidental opening.
Invert the plate and incubate at 20-25oC for three days.
4. Examine the MYEA plate and note the appearance of any colonies growing on the agar.
Be careful! Do not open the Petri dish.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.14 Breakdown of pectin by micro-organisms
Pectin, a structural polysaccharide, contains many different constituent sugars.
It has no definite chemical formula.
1. Prepare a growing culture of Erwinia carotovara grown in nutrient broth incubated at 20-25oC for 48 hours.
To use the culture, remove the top from the bottle, pass the neck through a Bunsen burner flame three times, take up culture with a
sterile dropping pipette and filler, flame the neck again and replace the top on the culture bottle.
Use the pipette and filler to transfer drops of culture.
Prepare two Petri dishes as follows:
1.1 Use slice of potato or carrot + three drops of deionized water in the centre of one slice (control)
1.2 Use slice of potato or carrot + three drops of Erwinia carotovara culture
2. Attach the lids and bases of the Petri dishes with four short strips of clear adhesive tape to protect against accidental opening.
Incubate the Petri dishes at 20-25oC with the lids uppermost overnight.
3. Examine the potato or carrot slices.
Note the "soft rot" in the potato slice.
Be careful! Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.15 Micro-organisms and bread making
1. Prepare a yeast suspension by adding 15 g of dried bakers yeast + one teaspoon of sucrose sugar to 150 mL of water.
2. Weigh 25 g flour into the beaker and then add 1 g sucrose sugar.
Measure 30 mL yeast suspension in the small measuring cylinder.
Add it to the flour and sugar in the beaker.
Stir with the spatula to form a smooth paste.
Pour the paste into a large measuring cylinder but do not let the paste touch the sides.
Push the paste down a funnel with a spatula.
3. Record the volume of the paste in the measuring cylinder.
Put the measuring cylinder in a water bath.
Note the temperature.
Record the volume of the paste every 5 minutes for 30 minutes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.16 Food preservation of peas
See diagram 9.4.12: Food preservation experiment
1. Use frozen peas or fresh peas from a pea pod or dried peas
Prepare and label test-tubes as follows:
1.1 Use 3 peas only
1.2 Use 3 peas only
1.3 Use 3 peas + deionized water
1.4 Use 3 peas + dilute salt solution
1.5 Use 3 peas + concentrated salt solution
1.6 Use 3 peas + concentrated sugar solution
1.7 Use 3 peas + vinegar
1.8 Use 3 peas + sodium nitrite solution
2. Plug each test-tube with cotton wool.
Keep test-tube 1.1 in a refrigerator.
Incubate test-tubes 1.2 to 1.7 at room temperature, 20-25oC for two days
Be careful! Do not open the test-tubes.
3. Record the appearance of the peas and the cloudy solutions in the test-tubes.
Note the differences in turbidity caused by micro-organisms.
4. Repeat the experiment using cubes of fresh meat or pieces of bacon.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.17 Prepare yoghurt, test milk quality
See diagram 9.4.12: Food preservation experiment
1. Heat 3 teaspoonfuls of live yoghurt (not pasteurized and containing no preservatives) in a beaker until bubbles form.
Leave to cool, and label as "heated yoghurt".
Live yoghurt contains the lactic acid bacteria, lactobacilli, Lactobacillus bulgaricus and Streptococcus thermophilus.
Prepare two glucose nutrient agar plates.
The glucose provides a fermentable substrate for the lactic acid bacteria.
Prepare and label test-tubes as follows: 1.1 - 10 mL UHT milk + 1 mL resazurin solution.
test-tube 1.2 contains 5 mL UHT milk + 5 mL heated yoghurt + 1 mL resazurin solution
test-tube 1.3 contains 5 mL UHT milk + 5 mL unheated yoghurt + 1 mL resazurin solution
Insert stoppers, invert each test-tube gently three times to mix the contents and put them in a 37oC water bath for 10 minutes.
Record the colour of the contents of each test-tube by selecting the nearest colour in the table below.
2. Heat a wire loop in a Bunsen burner flame.
Leave to cool then and dip it into the heated yoghurt.
Lift the lid of Petri dish 1 and spread the contents of the loop over a glucose nutrient agar plate.
Attach the lid and base of the Petri dish with four short strips of clear adhesive tape to protect against accidental opening.
Invert and label Petri dish 1.
Repeat the procedure using unheated yoghurt.
Invert and label Petri dish 2.
Incubate the Petri dish 1 and 2 at 20-25oC overnight.
Examine the contents of Petri dishes and note the colours.
Be careful! Do not open the Petri dishes.
Table 4.3.17
Colour of sample Milk quality
1. Blue - no colour change Excellent
2. Lilac Good
3. Deep pink / mauve Fair
4. Pink Poor
5. White Bad
3. Prepare and label two beakers as follows:
Beaker 3.1 contains UHT milk + 5 mL heated yoghurt + 1 mL resazurin solution.
Beaker 3.2 contains UHT milk + 5 mL unheated yoghurt + 1 mL resazurin solution
Cover each beaker with cling film ("Glad Wrap"), incubate them at 43oC for one day, then store them in a refrigerator.
4. Record the appearance and smell of the contents of the beakers.
Test the pH of the contents with Universal indicator solution.
Be careful!
Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.18 Micro-organisms and food spoilage
See diagram 9.4.18: Series dilution
1. Use a pipette and filler to add 5 mL of deionized water to a test-tube containing freshly defrosted peas.
Use a glass rod to gently crush the peas and mix them with the water.
Allow the mixture to settle then transfer it to test-tube 1.1.
Prepare and label five test-tubes as follows, mixing each test-tube thoroughly by filling and emptying the pipette and filler several times
when preparing the dilution series.
Test-tube 1.0 contains 9 mL of deionized water + 5 mL of crushed freshly defrosted peas
Test-tube 1.1 contains 9 mL of deionized water + 1 mL of contents of 1.0 transferred with a pipette and filler
Test-tube 1.2 contains 9 mL of deionized water + 1 mL of contents of 1.1 transferred with a pipette and filler
Test-tube 1.3 contains 9 mL of deionized water + 1 mL of contents of 1.2 transferred with a pipette and filler
Test-tube 1.4 contains 9 mL of deionized water + 1 mL of contents of 1.3 transferred with a pipette and filler
Test-tube 1.5 contains 9 mL of deionized water + 1 mL of contents of 1.4 transferred with a pipette and filler
2. Use a marker pen to mark equidistant positions 1.0 to 1.5 around the disc.
Use the calibrated dropping pipette and filler to draw up a small amount of the contents of test-tube 1.5.
Lift the lid of the agar plate dish and release one drop close to the surface of the agar at position 1.0.
Repeat the procedure with the contents of test-tubes 1.4, 1.3, 1.2, 1.1 and 1.0 in that order.
Let the drops to soak into the agar.
Tape the agar plate and invert it.
Attach the lid and base of the Petri dish with four short strips of clear adhesive tape to protect against accidental opening, then invert
the agar plate and incubate at 20-25oC overnight.
3. Repeat the experiment (steps 1. and 2.) with one day old peas instead of defrosted peas.
4. Examine the two agar plates and count the number of colonies visible at positions 1.0 to 1.5 on each agar plate.
Be careful! Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.19 Micro-organisms and milk quality
See diagram 9.4.12: Food preservation experiment
1. Prepare and label test-tubes as follows:
Test-tube 1.1 contains 2 mL resazurin stain + 10 mL fresh pasteurized milk
Test-tube 1.2 contains 2 mL resazurin stain + 10 mL 24 hours pasteurized milk
Test-tube 1.3 contains 2 mL resazurin stain + 10 mL 48 hours pasteurized 1.4 - 2 mL resazurin stain + 10 mL fresh UHT milk
Test-tube 1.5 contains 2 mL resazurin stain + 10 mL 24 hours UHT milk
Test-tube 1.6 contains 2 mL resazurin stain + 10 mL 48 hours UHT milk
2. Stopper and invert each test-tube three times.
3. Record the colour of the contents of each test-tube by selecting the nearest colour in the table below.
4. Put the test-tubes in a water bath and record the colours every 5 minutes for 30 minutes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.
Table 4.3.19
Colour of sample Quality of milk
1. Blue - no colour change Excellent
2. Lilac Good
3. Deep pink / mauve Fair
4. Pink Poor
5. White Bad


4.3.20 Micro-organisms and antiseptics
| See diagram 9.4.18: Dilutions 1.1 to 1.3
| See diagram 9.4.10: Agar plates 1
1. Two days before the experiment, inoculate a culture of Bacillus subtilis in nutrient broth and incubate at 25oC.
Remove the top from the bottle containing the culture of Bacillus subtilis.
Pass the neck through a Bunsen burner flame three times.
Remove a few drops of culture with the sterile dropping pipette and filler.
Flame the bottle neck again and replace the top.
Have in store a Universal bottle of melted nutrient agar, kept at 45-50oC.
Prepare and label Universal bottles 1.1 to 1.3 and beaker 1.4
Bottle 1.1 contains 10 mL of antiseptic solution, e.g. TCP, Dettol
Bottle 1.2 contains 9 mL deionized water + 1 mL from bottle 1.1
Bottle 1.3 contains 9 mL deionized water + 1 mL from bottle 1.2
Beaker 1.4 contains deionized water
2. Lift the lid of the Petri dish and place 5 drops of the culture in the centre of the dish and replace the lid.
Remove the cap of the bottle of melted nutrient agar.
Pass the neck of the bottle through a Bunsen burner flame three times, pour the contents into the Petri dish and replace the lid.
Mix the contents of the Petri dish with a sterile glass spreader, moving in a figure of eight, but avoiding any splashing over the edge.
Let the agar set to form a pour plate.
3. Use a pipette and filler and filler to transfer 9 mL of deionized water to empty bottles 1.2 and 1.3.
Use the same pipette and filler to transfer 1 mL of antiseptic, e.g. TCP or Dettol, from bottle 1.1 to bottle 1.2.
Gently shake bottle 1.2 to mix the contents, then transfer 1 mL from bottle 1.2 to bottle 1.3
When the agar has set, invert the agar and use a marker pen to divide the base into four sections by drawing a cross.
Label the sections 1.1, 1.2, 1.3 and 1.4 then invert the Petri dish again so the starch nutrient agar is up.
Using flamed forceps, dip a paper disc into 1.4, the beaker of deionized water.
Drain excess liquid from it, then place it on section 1.4 of the agar.
Flame forceps.
Replace the lid as soon as possible.
Repeat for the other three sections using the samples in the order 1.3, 1.2, 1.1.
Do not invert the Petri dish.
Incubate the Petri dish at 20-25oC overnight.
4. Examine the agar plate.
Use a sheet of graph paper to record the diameter of clear areas around the discs.
Be careful! Do not open the Petri dish.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.21 Micro-organisms and personal hygiene
1. Label and prepare three sterile malt extract agar plates as follows:
Plate 1.1 contains sterile malt extract agar plate + touched with fingers that had wiped Saccharomyces cerevisiae culture
Plate 1.2 contains sterile malt extract agar plate + touched with fingers that had wiped Saccharomyces cerevisiae culture when
wrapped in toilet paper
Plate 1.3 contains sterile malt extract agar plate + touched with washed fingers that had wiped Saccharomyces cerevisiae culture
when wrapped in toilet paper
1.1 Wash the hands thoroughly with hot water and soap, then dry them on a clean paper towel.
Open the first plate of Saccharomyces cerevisiae, wipe two fingers lightly over the surface, lift the lid of dish 1.1, touch the agar
surface lightly with the same two fingers, replace the lid.
Wash the hands thoroughly.
1.2. Wrap two fingers in a layer of toilet paper.
Open the second plate of Saccharomyces cerevisiae, wipe the wrapped fingers lightly over the surface as in the previous procedure,
remove the toilet paper, lift the lid of plate 1.2, touch the agar surface lightly with the same two fingers, replace the lid.
Wash the hands thoroughly.
1.3. Wrap two fingers in a layer of toilet paper.
Open the third plate of Saccharomyces cerevisiae, wipe the wrapped fingers lightly over the surface as in the previous procedure,
remove the toilet paper, wash the hands thoroughly with soap and dry them on a clean paper towel, lift the lid of plate 1.3, touch the
agar surface lightly with the washed fingers, replace the lid.
Wash the hands thoroughly.
2. Attach the lids and bases of the Petri dishes with four short strips of clear adhesive tape to protect against accidental opening.
Invert the Petri dishes and incubate them at 20-25oC overnight.
3. Examine the agar plates without opening them.
Be careful! Do not open the Petri dishes.
Sterilize contaminated materials and equipment by incineration or with autoclave or pressure cooker.
Use disinfectants, e.g. domestic bleach, for pipettes, syringes, swabbing benches and accidental spills.

4.3.22 Sensitivity of micro-organisms to antiseptics
See diagram 9.4.10: Agar plates 1
1. Prepare a nutrient agar lawn plate (Bacillus subtilis or Micrococcus luteus or Escherichia coli) or malt agar lawn plate
(Saccharomyces cerevisiae).
2. Use a marker pen to draw a cross on the bottom of the Petri dish to divide the plate into four sections (a) toothpaste (b) mouthwash,
(c) antiseptic (d) sterile water control
3. Use forceps sterilized in an oven to dip an absorbent paper disc into the sample (a), drain it on the side of the container and place it
firmly on section (a) of the plate.
Then wash the forceps free of the sample.
4. Repeat the procedure for samples (b) and (c) and the control (d).
Use sterile forceps for each sample.
Open the plate for the minimum possible time.
5. Seal the Petri dish with four short sections of clear adhesive tape.
6. Invert the plate and incubate at room temperature or 20-30oC for 48 hours.
7. Examine the plate without opening it and record the size of any zones of inhibition around the paper discs.

9.1.2.2 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.

9.1.2.3 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.

9.1.2.4 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.

9.1.2.5 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.

9.1.2.7 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.

9.1.2.8.0 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 procedure 9
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.

9.1.2.8.1 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.

9.1.2.9 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.
9.1.2.10 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.
1. 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.

9.1.2.29 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)

9.2.30 Tensides, alkylphenol ethoxylates
Tensides are surface active substances, which by reducing the surface energy of water mediate the wetting of surfaces or promote that
substances insoluble in water can be emulated or dispersed in it.
Besides washing and cleaning agents, important fields of application of tensides are the use as additive in paints/ varnishes, in metal
processing fluids, in construction products as well as in paper manufacturing and processing, polymer materials and plant protection
products.
Synthetic alkylphenol ethoxylates, commonly used as non ionic tensides, are a group of non-ionic surface active substances.
During degradation in wastewater treatment plants and in the environment, nonylphenols are formed, which are only slowly degradable,
bioaccumulative and very toxic for aquatic organisms.
Besides that, hormone like effects have been observed in fish.
Nonylphenol ethoxylates have been widely substituted in household and industrial cleaners as well as in flocculates.

4.2.0 Acknowledgements
Files UNBiology 4 and UNBiology 4a are based on "Teaching Biotechnology in Schools", UNESCO / International Union of
Biological Sciences (IUBS), Germany / USA.
The above experiments have been edited from Chapter 4 "Suggestions for teaching biotechnology" and 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 (IUBS).
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
ATCC: American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA
DSM: Deutsche Sammlung von Mikroorganismen und ZelIkulturen GmbH, Mascheroder Weg 1B, D-3300 Braunschweig, Germany
IPN: Institut fur die Pedagogik der Naturwissenschaften to Institute for Science Education, Olshausenstrabe 62, D-2300, Kiel 1,
Germany
NCSB: National Centre for School Biotechnology, Department of Microbiology, London Road, Reading, RG1 5AQ, England
MERCK GmbH, P.O.(b) 4119, D-6100 Darmstadt, Germany
SERVA Fine Bioch. Inc. Westbury, New York 11590, 200 Shames Drive, USA
SIGMA Chemical Company Ltd. Fancy Road, Poole, Dorset BH17 7NH, England
1. Division of micro-organisms into large groups by observing colonies (Gliesche and Lucius)
2. Enrich wild yeast strains (R. Wistful, 1989)
3. Prepare fixed slide preparations (after R. Westphal, 1989)
4. Prepare 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. Prepare yoghurt and sauerkraut (Primary grade four students) (after H. Bayrhuber, M. Fries, and Th. Heineken, 1990)
11. Prepare lactic acid in sourdough (E.R. Lucius and L. Rohweder)
12. Prepare wine from grape juice and make vinegar from wine (E.R. Lucius)
13. Microbial decomposition of cigarette paper (E.R. Lucius)
14. Why apple juice gels when it is boiled? (after Ch. Labahn-Lucius, 1990)
11. Pectinase enzyme decomposes pectin (after Ch. Labahn-Lucius, 1990)
12. Split lactose from milk or whey by using immobilized lactase (after Ch. Labahn-Lucius and 8. Plainer)
13. In vitro culture in the ornamental Usambara violet (IPN)
14. Production of Usambara violets from pieces of leaf (from Nellen, 1988)
15. Cultivation of Gerbera by using in vitro culture (after H. Bayrhuber, 1990)
16. Isolating DNA from sweetbread (calf thymus gland) (from H. Bayrhuber, Ch. Gliesche, 1. R. Lucius, 1990)
17. Observe conjugation in bacteria (from Ch. Gliesche, 1990)