appendixB

School Science Lessons
Appendix B
Updated: 2009-11-10

Table of contents
1.0 Biology media and solutions
2.0 Microscopy adhesives
3.0 Microscopy stains
4.0 Biology fixatives
4.7 Insect fixing fluids
5.0 Standard buffer solutions
5.1 Tollen's reagent, silver mirror tests for aldehydes using amoniacal silver nitrate
6.0 Culture media for routine cultivation and identification of fungi
7.0 Preparation instructions for acids and bases
8.0 Preparation instructions for salt solutions
9.0 Flammable organic chemicals
10.0 CHEMICALS PROHIBITED FOR SCHOOL USE
11.0 High toxicity chemicals
12.0 Cryogenic solids and liquids
13.0 Abbreviations
14.0 Prefixes and suffixes

15.0.0 Different classifications of hazardous chemicals
15.1 Hazard classifications EXPLOSIVE, FLAM,OXD,POISON, HARM, COR, IRR
15.2 Health effects, Very Toxic, Toxic,Harmful,Corrosive, Irritant, Carcinogenic, Mutagenic, Environment danger
15.3 Draft Australian criteria for the classification of hazardous chemicals, Physical hazards
15.4 Draft Australian criteria for the classification of hazardous chemicals, Health hazards

1.0 Biology media and solutions
1.1 Acid alcohol, biology solution
1.2 Alcohol (ethanol) absolute alcohol, biology solution
1.3 Benedict's solution, biology solution
1.4 Carbol xylol, biology solution
1.5 Fluorescein solution, biology solution
1.6 Iodine solution, test for starch, biology solution
3.12 Gram's iodine solution, microscopy stain
3.15 Lugol's iodine solution, microscopy stain
1.7 Ringer solution, biology solution
1.8 Saline solution, biology solution
1.9 Scott's blueing solution, biology solution
1.10 Sea water substitute, biology solution
1.11 Sodium thiosulfate solution, biology solution
1.12 Xylene and methylbenzoate, biology solution
1.13 Phenylthiocarbamide, PTC, phenylthiourea, PTU, C₇H₈N₂S

9.1.2.11 Media and solutions
9.1.2.12 Liquid broth media
9.1.2.13 Sterile solutions
9.1.2.14 Basal agar medium
9.1.2.15 Basal broth medium
9.1.2.16 Glucose nutrient agar
9.1.2.17 Malt extract agar medium
9.1.2.17a Malt extract broth medium
9.1.2.18 Minimal agar medium
9.1.2.19 Nutrient agar medium
9.1.2.19a Starch nutrient agar medium
9.1.2.19b Milk agar medium
9.1.2.19c Nitrogen-free mineral salts agar medium
9.1.2.19d Mannitol yeast extract agar (MYEA)
9.1.2.19e Glucose nutrient agar medium
9.1.2.20 Nutrient broth medium
9.1.2.21 Urea agar medium
9.1.2.22 Vinegar bacteria medium
9.1.2.23 MS agar medium.
9.1.2.24 BAP medium
9.1.2.25 Buffer reagent, phosphate buffer reagent
9.1.2.26 20% Domestos solution
9.1.2.27 Ringer solution
9.1.2.28 Salt solution
9.1.2.29 Appendix 4 Chemicals for microbiology
9.1.2.30 Tensides

2.0 Microscopy adhesives
2.1 Glycerine jelly, adhesive to stick sections to microscope slides
2.2 Haupt's adhesive, adhesive to stick sections to microscope slides
2.3 Meyer's albumen, adhesive to stick sections to microscope slides
2.4 Canada balsam

3.0 Microscopy stains
3.1 Acetic alcohol, microscopy stain
3.2 Aceto-orcein stain, microscopy stain
3.3 Acetocarmine, microscopy stain
3.4 Aniline hydrochloride, microscopy stain
3.5 Aniline sulfate, microscopy stain
3.6 Carmine stain, microscopy stain
3.7 Congo red, microscopy stain
3.8 DCIP (2,6-dichlorophenol-indophenol), microscopy stain
3.9 Delafield's haematoxylin, microscopy stain
3.10 Eosin, tetrabrornofluorescein, microscopy stain
3.11 Gram stain, microscopy stain
3.11.1 Crystal violet solution
3.11.2 Gram stain decoloriser solution
3.12 Gram's iodine solution, microscopy stain
3.13 Haematoxylin, microscopy stain
3.14 Heidenhain iron haematoxylin, microscopy stain
3.15 Lugol's iodine solution, microscopy stain
3.16 Karo syrup mountant, microscopy stain
3.17 Lactophenol, microscopy stain
3.18 Lactophenol cotton blue, microscopy stain
3.19 Leishmann's stain (or use ready-made Wright's stain), microscopy stain
3.20 Neutral red (0.1%), microscopy stain
3.21 Orange G, microscopy stain
3.22 Phloroglucinol (10% solution) Acid
3.23 Safranin, microscopy stain
3.24 Schulze's solution (chlor-zinc-iodine), microscopy stain
3.25 Weigert's haematoxylin, microscopy stain
9.1.2.1.6 Resazurin stain

4.0 Biology fixatives
4.1 Aceto-alcohol, biology fixative, for plant material
4.2 CRAF (chromic acid, acetic acid, formalin), biology fixative, for plant material
4.3 FAA (formalin, acetic acid, alcohol), biology fixative, for plant material
4.4 Formalin, biology fixative, for animal material
4.5 Formol-saline, biology fixative, for marine animals
4.6 Zenker's fluid, biology fixative, for animal material

4.7.0 Insect fixing fluids
4.7 Barber's relaxing fluid, insect fixing fluid
4.8 Carnoy's fluid, insect fixing fluid
4.9 KAA, insect fixing fluid
4.10 Kahle's fluid, insect fixing fluid
4.11 Oudeman's fluid, insect fixing fluid
4.12 Lacto-alcohol, insect fixing fluid
4.13 Pampl's fluid, insect fixing fluid
4.14 Sugaring mixture, insect fixing fluid

6.0 Culture media for routine cultivation and identification of fungi
6.0 Direct microscopic mounts or squash preparations
6.1 Calcofluor white with 10% KOH, to identify fungi
6.2 Cellotape flag preparations, to identify fungi
6.3 Cornmeal agar, to identify fungi
6.4 Cornmeal glucose sucrose yeast extract agar, to identify fungi
6.5 Czapek dox agar, to identify fungi
6.6 Indian ink mounts
6.7 Lactophenol cotton blue (LPCB), to identify fungi
6.8 Malt extract agar, to identify fungi
6.9 Potassium hydroxide with chlorazol black, to identify fungi
6.10 Potato dextrose agar, to identify fungi
6.11 Rice grain slopes, to identify fungi
6.12 Orcinol-Bial's Reagent

1.1 Acid alcohol, biology solution
100 mL 70% alcohol and 1 mL hydrochloric acid, used for cleaning slides and coverslips and decolorizing some stains, e.g. haematoxylin.

1.2 Alcohol (ethanol), biology solution
It is used in various concentrations for preserving and dehydrating. Absolute alcohol bottles must be stoppered at all times, since alcohol readily absorbs water from the atmosphere. Remove water from alcohol by using drying agents, e.g. anhydrous copper (II) sulfate.

1.3 Benedict's solution:
1. Solution A: Dissolve with heat 173 g sodium citrate, 100 g anhydrous sodium carbonate, Na₂CO₃, in 800 mL water. Filter and dilute to 850 mL.
Solution B Dissolve 17.3 g copper sulfate crystals, CuSO₄.5H₂O in 100 mL water.
Pour Solution B with stirring, into Solution A, and make up to 1 litre.
2. Solution A: Dissolve 17.3 g of sodium citrate and 10 g of anhydrous sodium carbonate in 60 mL of water.
Solution B: Dissolve 1.73 g of copper sulfate crystals in 20 mL of water.
Pour solution B into solution A while stirring, and make up to 1 litre.

1.4 Carbol xylol, biology solution
BE CAREFUL! Use gloves!
Mix 25 g phenol with 100 mL xylene. The phenol dissolves slowly, and cools when dissolving. It is used for dehydrating in staining techniques.

1.5 Fluorescein solution
Dissolve one gram of fluorescein in 100 mL methylated, spirit.

1.6 Iodine solution, biology solution
See 16.7.3: Iodine test for starch
Specific test for starch giving blue-black colour and general stain. Kills and fixes living material and makes cytoplasm and nucleus more visible. Lignin walls in xylem stained brown, leaving cellulose walls of parenchyma unstained.
1. Dissolve 2 g potassium iodide in water. Add 1 g iodine crystals.
2. Dissolve 1 g iodine and 4 g potassium iodide in 300 mL water.

1.7 Ringer solution, biology solution
Prepare fresh before use. 0.9 g sodium chloride, 0.042 g potassium chloride, 0.025 g calcium chloride, 100 mL deionized water

1.8 Saline solution, biology solution
Dissolve 9 g sodium chloride in 1 litre of deionized water.

1.9 Scott's bluing solution, biology solution
It is used with haematoxylin to develop blue colour.
2 g sodium bicarbonate, 20 g magnesium sulfate, 1 litre deionized water.

1.10 Sea water substitute
Dissolve in 2 litres of water, 45.0 g sodium chloride, 3.5 g magnesium sulfate, 5.0 g magnesium chloride, 2.0 g potassium sulfate.

1.11 Sodium thiosulfate solution, biology solution
It is used to decolorize iodine and wash iodine from tissue.
Dissolve 5 g sodium thiosulfate (hypo) in 100 mL deionized water. Add drops of iodine solution. The colour of iodine vanishes. Add drops of weak solutions of acidified bleaching fluid (calcium hypochlorite) or citric acid or sulfuric acid. The colour of iodine solution returns.

1.12 Xylene and methyl benzoate, biology solution
They are used during staining procedures. These chemicals are highly flammable, toxic and readily absorbed through the skin. Care must be exercised when handling. These are not miscible with water.

1.13 Phenylthiocarbamide, PTC, phenylthiourea, PTU, C₇H₈N₂S
See 9.254.2: PTC tasting experiment
Phenyl thiocarbamide is toxic but is safe at a concentration < 0.13%. Weigh 0.13g of solid phenylthiocarbamide on a watch glass. Dissolve the solid in 100 mL deionized water. Cut strips of absorbent paper, 5cm x 1cm. Soak the strips in the solution, remove with forceps, drain and dry the papers on a drying
tray in an incubator < 50^oC. Store the indicator papers in a sealed bottle. Prepare strips of paper soaked in solutions X2 or X10 as dilute after diluting the above solution 50mL to 100mL or 10mL to 100mL. Rinse the mouth after each tasting trial. Te chemical has a very persistent bitter taste, so handle the strips of paper with forceps.

9.1.2.11 How to produce sterile media or solutions
Equipment: 1 autoclave or pressure cooker, 1 conical flask, 300 mL to 2.0 L according to quantity, culture tubes or test-tubes, sealed with cellulose bungs, Petri dishes, pipette and filler, 5 mL, sterile, pipetting aid, 1 pH meter, 1 spatula, 1 set of scales, 1 piece of weighing paper, 1 pair of insulated gloves
Procedures:
Agar media:
1. Weigh out the individual components used for the manufacture of the culture medium onto a piece of weighing paper and place them into a conical flask. The quantities referred to below are for a 1 litre solution. If less culture medium is required, reduce the proportions of the individual components accordingly. The size of the conical flask depends on the amount to be produced, 300 mL conical flasks are, for example, required for 200 mL culture medium.
2. Swirl the preparation around. until the soluble components have dissolved. Determine the pH and adjust it if necessary with 1 M NaOH or 1 M HCl, according to the values referred to below. 3. Seal the conical flask with a cellulose bung and autoclave in a pressure cooker at 121^oC and 1 bar excess pressure for 20 minutes. Remove the conical flask from the pressure cooker using protective gloves and cool the flask to 50^o.
3. This process can be accelerated by placing the flask under running water. The temperature has been reached when the bare back of the hand can touch the outside of the vessel without an unpleasant sensation.
4. Fill sterile plastic Petri dishes with the still liquid medium so that the base of the Petri dish is covered. Lift the lid of each Petri dish only for a short period of time so that no germs from the air get into the culture medium.
5. Place the filled Petri dishes in a safe place until the agar has set completely.
6. Inoculate the culture medium plates with material that contains bacteria, according to experimental needs.

9.1.2.12 Liquid broth media
1. Prepare the components required for the production of culture media according to the description for agar media, steps 1 through 3, autoclave, and cool down.
2. Fill culture tubes that have been sterilized in a drying cabinet, 180^oC, 3 hours, with 5 mL of the culture medium, using sterile glass pipette and fillers. 3. Inoculate the culture tubes with material containing bacteria, as described in the experiments.

9.1.2.13 Sterile solutions
Prepare the components required for the production of the solution according to the description for agar media, steps 1 through 4, autoclave, and cool down.

Media and solutions
9.1.2.14 Basal agar medium
Components: glucose 10.0 g / litre, casein peptone 4. 0 g / litre, meat extract 4.0 g / litre, yeast extract 0.5 g / litre, liver extract 0.5 g / litre, NaCl 2.5 g / litre, agar 11.0 g / litre (pH 7.2)

9.1.2.15 Basal broth medium
The composition as for basal agar medium above, without agar. This ia a liquid medium for overnight cultures.

9.1.2.16 Glucose nutrient agar
With ready made medium: glucose 1. 0 g / litre, nutrient agar 20.0 g / litre, or from individual component: glucose 1.0 g / litre, peptone from meat 1. 0 g / litre, meat extract 3.0 g / litre, agar 12.0 g / litre (pH 7.0)

9.1.2.17 Malt extract agar medium
Ready made: malt extract agar 48.0 g / litre, or from individual components: malt extract 30.0 g / litre, peptone from meat 3.0 g / litre, agar 11.0 g / litre (pH 1.6), autoclave carefully (10 min.)
Dissolve 15 g malt extract and 18g bacteriological agar in 1 litre of deionized water. Dispense into bottles and sterilize with an autoclave.

9.1.2.17a Malt extract broth medium
The composition is the same as malt extract agar medium, however without the agar. This is a liquid medium for overnight cultures.

9.1.2.18 Minimal agar medium
Components: K₂HPO₄ 3.5 g / litre, sodium citrate x 2 H₂O 0.5 g / litre, MgSO₄.7H₂O 0.1 g / litre, (NH₄)₂SO₄ 1.0 g / litre, glucose 2.0 g / litre, D, L-histidine 0. 2 g / litre, D, L-arginine 0.2 g / litre, thiamine-HCl 0.05 g / litre, agar 11.0 g / litre (pH 7.2)

9.1.2.19 Nutrient agar medium
Ready made: nutrient agar 20.0 g / litre,
or from individual components: peptone from meat 1. 0 g / litre, meat extract 3. 0 g / litre, agar 12.0 g / litre (pH 7.0)

9.1.2.19a Starch nutrient agar medium
Heat 4 g of soluble starch in 100 mL deionized water. Leave to cool then mix the suspension with 100 mL of hot liquid nutrient agar. Sterilize the mixture at 120^oC for 15 minutes.

9.1.2.19b Milk agar medium
Prepare sterilized nutrient agar, leave to cool to 45-50^oC, add 10% pasteurized milk, skimmed, semi-skimmed or full cream milk.
When a milk agar plate is made, it is assumed that the microbial population of the milk will not affect the experiment. However, the uninoculated area of the plate acts as a control.

9.1.2.19c Nitrogen-free mineral salts agar
Dissolve 0.50 g of FeCl₃.6H₂O in 500 mL of deionized water. Add 2 g K₂HPO₄ + 0.25 g of MgSO₄.7H₂O + 10 g glucose. Add 0.1 M NaOH until pH = 8.3. Add this solution to a mixture of 7.5 g agar and 1 g CaCO₃. Autoclave the mixture at 121^oC for 20 minutes then pour into Petri dishes to make a nitrogen-free mineral salts agar plate.

9.1.2.19d Mannitol yeast extract agar (MYEA)
Heat a mixture of 10 g agar in 1 litre of water until the agar is dissolved. Add 0.5 g K₂HPO₄, 0.2 g MgSO₄.7H₂O, 0.2 g NaCl, 0.2 g CaCl₂.6H₂O, 10 g mannitol and 0.4 g yeast extract. Autoclave the mixture at 121^oC for 20 minutes then pour into Petri dishes to make MYEA plates.

9.1.2.19e Glucose nutrient agar medium
Add 0.5% (w/v) glucose to nutrient agar, dispense in bottles and sterilize.

9.1.2.20 Nutrient broth medium
Ready made: nutrient broth 8.0 g / litre, or from individual components: The composition is the same as nutrient agar medium, however without the agar. This is a liquid medium for overnight cultures.
Nutrient broth medium may also be purchased as a powder.

9.1.2.21 Urea agar medium
Ready made medium: 1. urea agar (Christensen) 21.0 g / litre (pH 6.8)
2. urea 20.0 g / litre, deionized water 50 mL. Sterilize 2. with sterile filter, autoclave 1. and cool to 50^oC, add 1.2 to 1.1) Components of urea agar: peptone from meat 1. 0 g / litre, glucose 1. 0 g / litre, NaCl 1. o g / litre, K₂HPO₄, 1.0 g / l, phenol red 12 mg / litre, agar 12.0 g / litre (pH 6.8)

9.1.2.22 Vinegar bacteria medium
Components of vinegar medium: peptone from meat 3. 0 g / l, yeast extract 1. 0 g / litre, mannitol 21.0 g / litre. This is a liquid medium for overnight cultures.

9.1.2.23 MS agar medium
With ready made medium: MS powder (basal salts with minimal organics) 4.7 g / l, granulated sugar 30.0 g / litre, agar 8. 0 g / litre, phytohormone solution (e.g. BAP, see below) 1 M1 (pH 1.8). Regulate with 1 m KOH.

9.1.2.24 BAP Medium
ndividual components: BAP (6-benzylaminopurine) 100 mg, 1 M HCl 20 mg. Fill up to 100 mL with boiling deionized water (stock solution). Add 0.5 to 1.0 mL of this stock solution to culture medium, e.g. MS agar medium.

9.1.2.25 Buffer reagent, phosphate buffer reagent
Individual components for 1 M solution:
1. K₂HPO₄, 17.4 g / 100 mL,
2. KH₂PO_4,12.9 g / 100 mL
Composition of 1 M buffer reagent: Make a solution of bipotassium hydrogen phosphate (solution 1.) and potassium bihydrogen phosphate (solution 2.) separately, each time using 100 mL deionized water. Place 60 mL of solution 1 into a 100 mL conical flask and, using a pipette and filler, adjust the pH to 7.5 with solution 2.
Composition of 0.1 M buffer reagent: Place 5 mL of solution 1. into a 100 mL conical flask with 45 mL deionized water. Place 5 mL of solution 2. into another 100 mL conical flask with 45 mL deionized water. Adjust the pH of dilute solution 1. to 7.5 by adding dilute solution 2 with a pipette and filler.

9.1.2.26 20% Domestos Solution
Composition: "Domestos" to household cleaning fluid 20 mL. Fill up with deionized water to 100 mL. "Domestos" is composed of saturated sodium hypochlorite solution.

9.1.2.27 Ringer Solution
Composition: 1 Ringer tablet. Fill with 500 mL deionized water and autoclave.

9.1.2.28 Salt Solution
Components: K₂HPO₄, 3.8 g / litre, KH₂PO₄ 1.2 g / litre, MgSO₄.7H₂O 1.1 g / litre, NaCl 2.5 g / litre, Fe₂(SO₄)₃.4H₂O 0.05 g / litre, Mn₂(SO₄)₃ 4H₂O 0.05 g / litre, "Tween 80" 1 mL/ litre (pH 7.0)

2.1 Glycerine jelly, adhesive to stick sections to microscope slides
Soak 10 g gelatine in 60 mL water for 2 hours. Add 70 mL glycerine and 1 g phenol crystals. Heat the solution gently in a water bath and then cool. To soften the jelly before embedding heat in water bath.

2.2 Haupt's adhesive, adhesive to stick sections to microscope slides
Dissolve 1 g gelatine in 100 mL water at 30^oC. Add 2 g phenol crystals and 15 mL glycerin. Stir, cool and filter.

2.3 Meyer's albumen, adhesive to stick sections to microscope slides
Beat an egg white until well broken up, but not stiff. Pour into a tall cylinder and leave to stand overnight. Add equal volume of glycerin to liquid collected from bottom. Add a thymol crystal to prevent growth of fungus.

2.4 Canada balsam
From Abies balsamea, refractive index similar to crown glass (n = 1.55), used to conserve micropscope samples but nowadays synthetic resins used

3.1 Acetic alcohol, biology fixative
Mix 99 mL 70% ethanol with 99 mL concentrated ethanoic acid. Prepare this solution prepared immediately before use.

3.2 Aceto-orcein stain, microscopy stain
Stains chromosomes red.
1. Add l g synthetic orcein to 25 mL concentrated ethanoic acid (glacial acetic acid) and 20 mL deionized water. Boil for 4 to 5 minutes in a narrow neck flask, fitted with a glass filter funnel to act as a condenser. Filter the solution while still hot. Add 5 mL concentrated ethanoic acid and stir to dissolve any orcein appearing on the surface of the mixture after filtration. Add 4 to drops of glycerol to retard evaporation.
2. Heat 50 cc 60% acetic acid until almost boils. Add 0.5 g orcein stain. Stir, cool and filter. Use fresh.

3.3 Acetocarmine, microscopy stain
Concentrated ethanoic acid (glacial acetic acid) 45 mL, 0.5 g carmine, 55 mL deionized water.

3.4 Aniline hydrochloride, microscopy stain
BE CAREFUL! Use gloves!
Make a saturated solution in aniline hydrochloride in deionized water. Filter then add a few drops of hydrochloric acid until solution is acid.

3.5 Aniline sulfate, microscopy stain
BE CAREFUL! Use gloves!
Prepare as for the aniline hydrochloride above but use sulfuric acid.

3.6 Carmine stain, microscopy stain
4 g carmine, 1 mL concentrated hydrochloric acid, 15 mL deionized water. Boil gently in a fume cupboard for 10 minutes with continuous stirring. Cool then add 95 mL 85% ethanol (alcohol). Filter before using the stain.

3.7 Congo red, microscopy stain
Congo red is blue in acid and red in alkali and is made from coal tar.

3.8 DCIP (2,6-Dichlorophenol-indophenol), microscopy stain
This stain is used to show action of enzymes e.g. succinic dehydrogenase, chloroplasts when exposed to light and to test for vitamin C. Add 4 g carmine and 1 mL concentrated hydrochloric acid 15 mL deionized water. Boil gently in a fume cupboard for 10 minutes with continuous stirring. Cool, and add 95 mL 85% ethanol (alcohol). Filter the solution. Add 1 g 2,6-Dichlorophenol-indophenol and 1 litre water.

3.9 Delafield's haematoxylin, microscopy stain
1. Dissolve 4 g powder in 25 mL absolute ethanol. Mix gradually into 400 mL saturated aqueous alum, NH₄Al(SO₄)₂.12H₂O. Leave to stand for 3-5 days with a cotton plug in flask, exposed to direct light. Filter, then add 100 mL glycerin and 100 mL methanol. Leave to stand for at least 6 weeks.
2. Dissolve 1 g haematoxylin in 6 cc absolute alcohol. Add this drop by drop to 100 mL saturated ammonium alum. Leave in light and air for one week. Filter than add 25 mL glycerine and 25 mL methyl alcohol. Leave to stand until dark colour. Filter again.

3.10 Eosin, tetrabrornofluorescein, microscopy stain
Counter stains are used to stain cell walls and cell contents. This is an animal tissue counter stain. Eosin stains cytoplasm
1. Use 1 g Eosin Y powder, 1000 mL 70% ethyl alcohol (ethanol) 5 mL glacial acetic (ethanoic) acid. Dilute 100 mL with 100 mL 70% alcohol, Add 2-3 drops of glacial acetic (ethanoic) acid.
2. Dissolve 1 g eosin in 100 cc 70% alcohol.

3.11 Gram stain, microscopy stain
Solutions required for the Gram stain procedure:
3.12 Gram's iodine solution
3.11.1 Crystal violet solution
3.11.2 Gram stain decoloriser solution
3.23 Safranin, microscopy stain

1. Mix 2 g potassium iodide crystals and 1 g iodine crystals in 200 mL of deionized water (Gram's iodine solution). Put the heat-fixed smear on the staining rack, cover with Crystal violet solution and leave for 1 minute. Tilt the staining rack and gently wash with water for 3 seconds. Flood the slide with Gram's iodine and leave for 1 minute. A stain-iodine complex forms. Tilt and wash with water for a few seconds. Hold the slide at a 45 angle where the smear is clearly visible and run 95% alcohol, as a decolorising agent (Gram stain decoloriser solution) , down the smear until no more colour runs out after 2-10 seconds. Wash with water. Counterstain by flooding the smear with safranin for 1 minute. Wash, blot dry and examine under an oil immersion lens. Gram stain is used routinely as a differential stain so that you can classify bacteria as "gram positive" or "gram negative". Gram positive bacteria stain a blue purple colour because they retain the stain-iodine complex inside their cells. Gram negative bacteria stain a red colour because they have cell walls which allow the stain-iodine complex to be washed out of the cell by alcohol.

3.11.1 Crystal violet solution
1. Dissolve 0.4 g crystal violet in 20 mL 95% ethanol. Mix with 80 mL 1% aqueous ammonium oxalate. Leave to stand for 48 hours before use. The solution is stable and can be stored for months.
2. For Solution A, dissolve 2 g crystal violet in 100 mL absolute alcohol. For solution B, dissolve 1 g ammonium oxalate in 100 mL deionized water. Add 25 mL Solution A to 100 mL Solution B.

3.11.2 Gram stain decoloriser solution
1. Acetone/ethanol (50:50 v:v) 0.1% basic fuchsin solution.
2. 50% ethanol, 50% acetone, basic fuchsin solution.
3. Dissolve 0.2 g of safranin or basic fuchsin in 10 mL 95% ethanol. Mix with 90 mL, of deionized water. The solution is stable and can be stored for months.

3.12 Gram's iodine solution, microscopy stain
Dissolve 2 g of potassium iodide crystals and 1 g of iodine crystals in 200 mL of deionized water.

3.13 Haematoxylin, microscopy stain
Stains nuclei in plant and animal cells purple, blue or black. This stain was formerly "logwood" because it was made from the heartwood of the tree Haematoxylon campechianum that has a sweet taste and smells of violets. It is similar to natural dyes natural red 24 from brazilin, C₁₆H₁₄O₅ and brazilein C₁₆H₁₂O₅, from Caesalpinia echinata. Use a stock solution of 10% haematoxylin in 95% alcohol.

3.14 Heidenhain iron haematoxylin, microscopy stain
Part 1. 4 g FeNH₄(SO₄)₂.12H₂O (ferric alum, i.e. iron (III) ammonium sulfate) 100 mL deionized water,
Part 2. 10 g haematoxylin, 100 mL 95% ethanol. Mix equal quantities of Part 1. and Part 2. The mixture is useful for a few hours only. Solution 1. is used as a mordant and 2. is for staining.

3.15 Lugol's iodine solution, aqueous iodine solution, microscopy stain
1. Dissolve 6 g potassium iodide in 1000 mL water. Add 4 g iodine crystals.
2. Dissolve 5 g of iodine crystals and 10 g of potassium iodide in deionized water and make up to 100 mL. For bacterial staining, dilute to 1 / 5 with water.
3. Dissolve 1 g iodine crystals and 2 g potassium iodide in 300 mL deionized water.

3.16 Karo syrup mountant, microscopy stain
40 mL clear Karo syrup, 40 mL deionized water. Add 2 small crystals thymol (or phenol) (about 0.2 g) as a preservative.

3.17 Lactophenol, microscopy stain
BE CAREFUL! Use gloves!
Dissolve 25 g phenol in 50 mL water. Add 25 mL lactic acid. Add 50 mL glycerine. Store away from light.

3.18 Lactophenol cotton blue, microscopy stain
100 mL lactophenol, l g cotton blue. Dilute 5 mL to 100 mL with lactophenol before use.

3.19 Leishmann's stain (or substitute ready-made Wright's stain), microscopy stain
Add 0.115 g Leishmann's stain to 100 mL of pure methanol in a flask. Plug the neck with cotton wool and warm in a water bath for 15 minutes with occasional shaking. Methylene blue (0.1%) 0.1 g methylene blue, 100 mL deionized water, Dilute 1 in 10.

3.20 Neutral red (0.1%), microscopy stain
0.1 g neutral red, 100 mL deionized water, Dilute 1 in 10.

3.21 Orange G, microscopy stain
This is a botanical tissues counter stain. Dissolve 0.5 g powder in 100 mL 95% alcohol.

3.22 Phloroglucinol (10% solution), microscopy stain
Acid phloroglucin stains lignin in plant cells bright red.
Dissolve 1 g phloroglucin in 100 mL 50% alcohol.

3.23 Safranin, microscopy stain
1. Dissolve 0.2 g safranin in 10 mL 95% ethanol. Mix with 90 mL, of deionized water. The solution is stable and can be stored for months
2. Dissolve 1 g safranin in 100 mL 50% by volume alcohol/water mixture.
3. Dissolve 0.5 g safranin in 100 mL deionized water.
4. For a general contrast stain for lignin and cell walls, dissolve 0.02 g safranin in 10 mL 5% ethanol. Add 100 mL water to form 1% solution.

3.24 Schulze's solution (chlor-zinc-iodine), microscopy stain
Dissolve 20 g zinc chloride in 9.5 mL warm water. Cool, add drop by drop 1.5 mL of the following solution until a persistent precipitate of iodine forms: 0.5 g iodine, 1 g potassium iodide, 20 mL deionized water.

3.25 Weigert's haematoxylin, Weigert's iron haematoxylin, microscopy stain
This stain is used for animal tissue
Part A: 2.5 g iron (III) chloride FeCl₃.6H₂O, 4.5 g iron (II) sulfate FeSO₄.7H₂O, 2 mL hydrochloric acid, 298 mL deionized water
Part B: 1 g haematoxylin, 100 mL 95% ethanol. Mix 1 part of B to 3 parts of A just before use. This mixture can be used for up to 3 weeks.

9.1.2.1.6 Resazurin stain
Prepare a 0.005% solution of the blue redox indicator dye resazurin by dissolving 1 tablet in 50 mL of deionized water. Resazurin is pink when oxidised and colourless when reduced.

4.1 Aceto-alcohol, biology fixative
Absolute ethanol (alcohol) 30 mL, ethanoic (acetic) acid, glacial 10 mL, mix immediately before use and discard after 1 hour, for animal material.

4.2 CRAF, biology fixative
Chromic acid, 1% 40 mL, formalin (40% methanal) 10 mL, ethanoic (acetic) acid, glacial 5 mL, deionized water 5 mL.

4.3 FAA, biology fixative
Formalin (40% methanal) 5 mL, ethanol (alcohol) 70% 90 mL, ethanoic (acetic) acid, glacial 5 mL.

4.4 Formalin, biology fixative
Commercial name for 40% formaldehyde solution. 1% formalin = 1 mL formalin then dilute to 100 mL with water. Formaldehyde vapour irritates eyes and delicate body tissues.

4.5 Formol-saline, biology fixative for marine animals
Formalin (40% methanal) 100 mL, sodium chloride, 10% solution 7 mL, deionized water 83 mL.

4.6 Zenker's fluid, biology fixative
Potassium dichromate 2.5 g, mercury II chloride 5.8 g, deionized water 95 mL.

4.7 Barber's relaxing fluid, insect fixing fluid
95% alcohol 50 mL, water 50 mL, ethyl acetate 20 mL, benzol 10 mL.

4.8 Carnoy's fluid, insect fixing fluid
95% alcohol 75 mL, chloroform 30 mL. glacial acetic acid 10 mL.

4.9 KAA, insect fixing fluid
Kerosene 10 mL (soft body insects 5 mL) 95 % alcohol 100 mL, glacial acetic acid 20 mL.

4.10 Kahle's fluid, insect fixing fluid
95% alcohol 100 mL, glacial acetic acid 7 mL, formalin 40 mL.

4.11 Oudeman's fluid, insect fixing fluid
70% alcohol 88 mL, glycerine 4 mL, glacial acetic acid 8 mL.

4.12 Lacto-alcohol, insect fixing fluid
Lactic acid 40 mL, 98% alcohol 37 mL, water 23 mL.

4.13 Pampl's fluid, insect fixing fluid
Glacial acetic acid 4 mL, water 30 mL, 40% formaldehyde solution 6 mL, 95% alcohol 15 mL.

4.14 Sugaring mixture, insect fixing fluid
500 g treacle, 1 kg brown sugar, 300 mL beer, 5 mL rum.
Boil until uniform thickness occurs.

5.0 Standard buffer solutions
The table below shows how to prepare buffer solutions for a particular pH by mixing pairs of the following four solutions.
Solution A = 0.l M boric acid, prepared by dissolving 3.09 g AR boric acid and 3.73 g potassium chloride in water and making up the solution to 500 mL in a measuring flask.
Solution B = 0.l M sodium hydroxide, prepared by diluting standard sodium hydroxide.
Solution C = 0.l M citric acid, prepared by dissolving 9.60 g AR citric acid in water and making up the solution to 500 mL in a measuring cylinder.
Solution D = 0.2 M disodium hydrogen phosphate, prepared by dissolving 17.82 g pure Na₂HPO₄.2H₂O in water, and making up the solution to 500 mL in a measuring cylinder.

pH	Solution A	Solution B	pH	Solution C	Solution D
.	mL	mL		mL	mL
10.0	25	21.8	6.0	14.6	25
9.6	25	18.0	5.6	18.1	25
9.2	25	13.0	5.2	21.6	25
8.8	25	8.0	4.8	25	24.3
8.4	25	4.0	4.4	25	19.8
8.0	1.4	50	4.0	25	15.7
7.6	3.4	50	3.6	25	11.9
7.2	3.8	25	3.2	25	8.2
6.8	7.4	25	2.8	25	8.2
6.4	11.1	25	2.4	50	3.4

5.1 Tollen's reagent, ilver mirror tests for aldehydes using amoniacal silver nitrate
1. Put 5 mL of 5% (w/w) silver nitrate solution in a test-tube. Add 5 drops of 0.4 M sodium hydroxide solution and shake gently. Add 1 M ammonium hydroxide drop by drop, with gentle shaking, until the precipitate just dissolves.
2. To prepare Tollens' reagent, add 1 drop of dilute sodium hydroxide solution to 1 mL silver nitrate solution and when a brown precipitate of silver oxide forms add drops of dilute ammonia solution, NH₃ (aq), ("ammonium hydroxide"), solution until the brown precipitate dissolves.

6.0 Direct microscopic mounts or squash preparations
Using sterile technique, remove a small portion of the colony with an inoculation needle and mount in a drop of Lactophenol Cotton Blue on a clean microscope slide. Cover with a coverslip, squash the preparation with the butt of the inoculation needle and then blot
off the excess fluid.

6.1 Calcofluor White with 10% KOH, to identify fungi
Use for the direct microscopic examination of skin scrapings, hairs, nails and other clinic specimens for fungal elements. This as a very sensitive method, however, a fluorescence microscope with the correct ultraviolet filters is required.
Solution A: Potassium hydroxide reagent.
Potassium hydroxide 10 g.
Glycerine 10 mL.
deionized water 80 mL.
Solution B: Calcofluor white reagent
Calcofluor white 0.5 g.
Evans blue 0.02 g.
deionized water 50 mL.
Mix one drop of each solution on the centre of a clean microscope slide. Place the specimen in the solution and cover with a coverslip.

6.2 Cellotape flag preparations, to identify fungi
An excellent technique for the rapid mounting of sporulating fungi because more of the reproductive structures intact.
1. Using clear 2 cm wide cellotape and a wooden applicator stick (orange stick) make a small cellotape flag (2 x 2 cm).
2. Using sterile technique, gently press the sticky side of the flag onto the surface of the culture.
3. Remove and apply a drop of 95% alcohol to the flag, to act as a wetting agent and also dissolve the adhesive glue holding the flag to the applicator stick.
4. Place the flag onto a small drop of Lactophenol cotton blue on a clean glass slide, remove the applicator stick and discard. Add another drop of stain, cover with a coverslip, gently press and mop up any excess stain.

6.3 Cornmeal agar, to identify fungi
Use for routine cultivation and identification of fungi.
Cornmeal agar (Oxoid CM 0103) 8.5 g.
deionized water 500 mL.
1. Mix dry ingredients into 100 mL water, boil remaining water.
2. Add boiling water to mixture and bring to boil.
3. Dispense for slopes.
4. Autoclave for 10 minutes at 120^oC, remove and slope.

6.4 Cornmeal glucose sucrose yeast extract agar, to identify fungi
Use for zygomycete sporulation
Cornmeal agar (Oxoid CM 0103) 17 g.
Dextrose (Glucose) 2g.
Sucrose 3 g.
Yeast extract 1 g.
deionized water 1000 mL.
1. Mix dry ingredients into 100 mL water, boil remaining water.
2. Add boiling water to mixture and bring to boil.
3. Dispense for slopes.
4. Autoclave for 10 minutes at 120^oC, remove and slope

6.5 Czapek Dox Agar, to identify fungi
Use for routine cultivation of fungi, especially Aspergillus, Penicillium, and non-sporulating moulds.
Czapek Dox Agar (Oxoid CM97) 45.4 g.
deionized water 1000 mL.
1. Soak the ingredients in small amount of water.
2. Bring remaining water to boil, add to soaking ingredients and bring to the boil again, stirring continuously.
3. Dispense for slopes as required.
4. Autoclave at 121^oC for 10 minutes, remove and slope or pour for plates as required.

6.6 Indian ink mounts
For the direct microscopic examination of cerebrospinal fluid, CSF, for Cryptococcus species, place a drop of Indian ink on the specimen, mix well with a sterilized loop, and cover with a coverslip. The best brands to use are "Pelikan" or "Talons" Indian ink.

6.7 Lactophenol Cotton Blue (LPCB), to identify fungi
Use for the staining and microscopic identification of fungi
Cotton Blue (Aniline Blue) 0.05 g.
Phenol Crystals (C₆H₅O₄) 20 g.
Glycerol 40 mL.
Lactic acid (CH₃CHOHCOOH) 20 mL.
deionized water 20 mL.
This stain is prepared over two days.
1. On the first day, dissolve the Cotton Blue in the deionized water. Leave overnight to eliminate insoluble dye.
2. On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. Place on magnetic stirrer until the phenol is dissolved.
3. Add the glycerol.
4. Filter the Cotton Blue and deionized water solution into the phenol / glycerol / lactic acid solution. Mix and store at room temperature.

6.8 Malt extract agar, to identify fungi
Use for routine cultivation and identification of fungi.
Oxoid Malt Extract 20 g
Bacto Agar 20 g
deionized water 1000 mL
1. Dissolve malt extract in a plastic beaker and pH the solution to pH 6.5 with NaOH.
2. Soak agar in small quantity of solution. Bring remaining solution to the boil, stirring constantly.
3. Add to soaking agar. Bring to boil, stirring constantly.
4. Dispense for slopes as required.
5. Autoclave at 121^oC for 10 minutes, remove and slope or pour for plates as required.

6.9 Potassium hydroxide (KOH) with Chlorazol Black, to identify fungi
Use for the direct microscopic examination of skin scrapings, hairs, nails and other clinic specimens for fungal elements.
Potassium hydroxide 10 g
Coral Azole E Black (0.1%) 10 mL.
Glycerol 10 mL.
deionized water 80 mL.
Using sterile technique, remove a small portion of the specimen with an inoculation needle and mount in a drop of KOH on a clean microscope slide. Cover with a coverslip, squash the preparation with the butt of the inoculation needle and then blot off the excess fluid.

6.10 Potato dextrose agar, to identify fungi
Use for routine cultivation and identification of fungi.
Potato dextrose agar 39 g
deionized water 1000 mL
1. Soak potato dextrose agar in small amount of the water in a stainless steel jug.
2. Boil remaining water, add to soaking ingredients, bring to the boil, stirring constantly.
3. Dispense for slopes as required.
4. Autoclave at 121^oC for 15 minutes. Remove and slope or pour for plates as required.

6.11 Rice grain slopes, to identify fungi
Use to induce sporulation and differentiation
Polished rice grains
deionized water
1. Place 1/2 teaspoon rice grains into wide neck 20 mL lass vials.
2. Add 8 mL deionized water to each vial.
3. Lid, then slope on racks ensuring rice grains are evenly distributed.
4. Autoclave racks at 121^oC for 15 minutes.

6.12 Orcinol-Bial's Reagent
Dissolve 0.2g orcinol in 100mL concentrated hydrochloric acid (caution, corrosive).

7.0 Preparation instructions for acids and bases
molarity 1 X volume 1 = molarity 2 X volume 2, M1V1 = M2V2
Dilute acids
Acetic acid 3 M: Dilute 172 mL of 17.4 M acid to 1 litre of water. (99 -100% acetic acid, ethanoic acid)
Hydrochloric acid 3 M: Dilute 258 mL of 11.6 M acid to 1 litre with water. (35% hydrochloric acid)
Hydrochloric acid 4 M: Dilute 400 mL of 10 M acid to 1 litre of water - for normal class use.
Nitric acid 4 M: Dilute 240 mL of 15 M acid. to 1 litre water - for normal class use.
Nitric acid 3 M: Dilute195 mL of 15.4 M acid to 1 litre of water. (69% nitric acid)Sulfuric acid 6 M: Dilute 168 mL of 17.8 M acid to 1 litre of water. (95% sulfuric acid)
Sulfuric acid 2 M: Dilute 112 mL of 35 M in 800 mL water, then add water to 1 litre - for normal class use.
Dilute bases
Ammonia solution 4 M: Dilute 220 mL (28% ammonia) 18 M concentrated solution to 1 litre of water ("ammonium hydroxide")
Ammonia solution 3 M: Dilute 200 mL (28% ammonia) 14.8 M concentrated solution to 1 litre of water.
Ammonia solution 2 M: Dilute 330 mL (10% ammonia) 6 M concentrated solution to 1 litre of water - for normal class use.
Potassium hydroxide 4 M: Dissolve 220 g KOH sticks in water, dilute to 1 litre of water - for normal class use
Sodium hydroxide 3 M. Dissolve 126 g the sticks, 95%, in water and dilute to 1 litre of water.
Sodium hydroxide 4 M Dissolve 160 g NaOH in 500 mL water, then dilute to 1 litre of water - for normal class use
Sodium hydroxide 8.5 M Dissolve 330 g NaOH in water, dilute to 1 litre of water. (For CO₂ absorption)
Calcium hydroxide (limewater)
1. 0.02 M. Saturated solution, 1.5 g Ca(OH)₂per litre, use some excess, filter off CaCO₃, and protect from CO₂ of the air.
2. Add 125 g of slaked lime, Ca(OH)₂, to 3 litres of water, shake, allow precipitate to settle, siphon off clear liquid, and protect from CO₂of the air.

8.0 Preparation instructions
Dissolve amount below then dilute to 1 L with water.
Aluminium chloride, AlCl₃.6H₂O, For 0.1 M soln., 24 g of hydrated salt in 1 L water
Aluminium sulfate, Al₂(SO₄)₃.18H₂O, For 0.l M soln., 66 g of hydrated salt in 1 L water
Ammonia, NH₃(aq) or NH₄OH, For 2 M soln., dilute 330 mL of 10% soln. in 1 L water
Ammonium chloride, NH₄Cl, For 5 M soln., 270 g in water
Ammonium carbonate (NH₄)₂CO₃.3H₂O, For 2 M soln., 300 g in 450 mL 10% NH₃, then dilute in 1 L water
Ammonium iron (II) sulfate, For 0.1 M soln., 39.2 g in water, add 5 mL conc. H₂SO₄ in 1 L water
Ammonium oxalate, C₂O₄(NH₄)₂.2H₂O, For 0.1 M soln., 16 g in 1 L water
Ammonium sulfate (NH₄)₂SO₄, For 0.1 M soln., 13.2 g in 1 L water
Barium chloride, BaCl₂.2H₂O, For 0.1 M soln., 24.4 g in 1 L water
Bismuth chloride, BICl₃, For 0.17 M soln., 53 g in 1 litre of dilute HCl, 1 part conc. HCl to 5 parts water
Bismuth nitrate, Bi(NO₃)₃.5H₂O, For 0.083 M soln., 40 g in 1 litre of dilute HNO₃, 1 part conc. HNO₃ to 5 parts water
Calcium chloride, CaCl_2,anhydrous 0.l M soln., 11 g in 1 L water
Calcium chloride, CaCl₂.2H₂O, For 0.1 M soln., 14.7 g in 1 L water
Calcium hydroxide, Ca(OH)₂Limewater, 10 g in 1 L water, shake, allow it to settle, decant clear liquid
Calcium nitrate, Ca(NO₃)₂, For 0.1 M soln., 16.4 g in 1 L water
Calcium sulfate, CaSO₄.2H₂O, For 0.1 M soln., Shake 10 g in 1 L water, leave to stand, decant the clear liquid
Cobalt (II) chloride-6-water, CoCl₂.6H₂O, For 0.1 M soln., 23.8 g in 1 L water
Cobalt nitrate, Co(NO₃)₂.6H₂O, For 0.1 M soln., 29 g in 1 L water
Copper (II) nitrate, Cu(NO₃)₂.6H₂O, For 0.1 M soln., 29.6 g in 1 L water
Copper (II) sulfate, CuSO₄.5H₂O, For 0.1 M soln., 25 g in 1 L water + 5 mL conc. H₂SO₄
Iron (II) ammonium sulfate, Fe(NH₄SO₄)₂.6H₂O, For 0.5 M soln., 196 g in 1 L water + 10 mL conc. H₂SO₄, dilute to 1 litre
Iron (III) chloride, FeCl₃.6H₂O, For 0.1 M soln., 27 g in 1 L water + 20 mL HCl
Iron (III) nitrate, Fe(NO₃)₃.9H₂O, For 0.1 M soln., 40.4 g in 1 L water
Iron (II) sulfate, FeSO₄.7H₂O, For 0.1 M soln., 27.8 g in 1 L water + 1 mL conc. H₂SO₄ to clear
Iron (III) sulfate, Fe₂(SO₄)₃.9H₂O, For 0.1 M soln., 56 g in 1 L water
Lead ethanoate (CH₃COO)₂Pb.3H₂O, For 0.1 M soln., 38 g in 1 L water + dilute ethanoic acid to clear
Lead nitrate, Pb(N0₃)₂, For 0.1 M soln., 33 g in 1 L water
Magnesium chloride, MgCl₂.6H₂O, For 0.1 M soln., 20.3 g in 1 L water
Magnesium nitrate, Mg(N0₃)₂.6H₂O, For 0.1 M soln., 25.6 g in 1 L water
Magnesium sulfate, MgSO₄.7H₂O, For 0.1 M soln., 24.7 g in 1 L water
Manganese sulfate, MnSO₄.H₂O, For 0.1 M soln., 16.9 g in water
Nickel chloride, NiCl₂.6H₂O, For 0.1 M soln., 24 g in 1 L water
Potassium bromide, KBr, For 0.1 M soln., 12 g in 1 L water
Potassium carbonate, K₂CO₃, For 0.1 M soln., 13.8 g in water
Potassium chloride, KCl, For 0.1 M soln., 7.5 g in 1 L water
Potassium dichromate, For 0.1 M soln., 29.4 g in 1 L water (K₂Cr₂O₇)
Potassium dihydrogen orthophosphate, For 0.1 M soln., 13.6 g in 1 L water (KH₂PO₄)
Potassium hydroxide, KOH, For 2 M soln., 110 g of KOH sticks in 1 L water
Potassium iodide, KI, For 0.1 M soln., 16.6 g in 1 L water
Potassium nitrate, KNO₃, For 0.1 M soln., 10.l g in 1 L water
Potassium permanganate, KMnO₄, For 0.1 M soln., 15.8 g in 1 L water
Potassium sulfate, K₂SO₄, For 0.1 M soln., 17.4 g in 1 L water
Silver nitrate, AgNO₃, For 0.1 M soln., 17 g in 1 L water
Sodium borate, Na₂B₄O₇.l0H₂O, For 0.1 M soln., 38 g in 1 L water
Sodium carbonate, Na₂CO₃.10H₂O, For 0.1 M soln., 28.6 g in 1 L water
Sodium carbonate, Na₂CO₃ (anhydrous), For 0.1 M soln., 10.6 g in 1 L water
Sodium chloride, NaCl, For 0.1 M soln., 5.8 g in 1 L water
Sodium chromate, Na₂CrO₄.4H₂O, For 0.1 M soln., 23.4g in 1 L water
Sodium dichromate, Na₂Cr₂0₇.2H₂O, For 0.1 M soln., 29.8 g in 1 L water
Sodium ethanoate, CH₃COONa.3H₂O, For 00.1 M soln., 13.6 g in 1 L water (sodium acetate)
Sodium hydrogen carbonate, NaHCO₃, For 0.1 M soln., 8.4 g in 1 L water
Sodium iodide, NaI, For 0.1 M soln., 15 g in 1 L water
Sodium molybdate, Na₂MoO₄.2H₂O, For 0.1 M soln., 24.2 g in 1 L water
Sodium nitrate, NaNO₃, For 0.1 M soln., 8.5 g in 1 L water
Sodium nitrite, NaNO₂, For 0.1 M soln., 7 g in 1 L water
Sodium oxalate, Na₂C₂O₄, For 0.1 M soln., 13.4 g in 1 L water
Sodium sulfate, Na₂SO₄.10H₂O, For 0.1 M soln., 32.2 g in 1 L water
Sodium sulfide, Na₂S.9H₂O, For 0.5 M 120 g in 1 L water
Sodium sulfite, Na₂SO₃.6H₂O, For 0.1 M soln., 23.4 g in 1 L water
Sodium sulfite, Na₂SO₃ (anhydrous), For 0.1 M soln., 12.6 g in 1 L water
Sodium thiosulfate, Na₂S₂O₃.5H₂O, For 0.1 M soln., 24.8 g in 1 L water
Strontium (II) chloride, SrCl₂.6H₂O, For 0.1 M soln., 26.7 g in 1 L water
tri-Sodium phosphate, Na₃P0₄.12H₂O, For 0.1 M soln., 38 g in 1 L water
Tin (II) chloride, SnCl₂.2H₂O, For 0.5 M soln., 113 g in 170 mL conc. HCl, dilute to 1 L + add tin foil
Tin (IV) chloride, SnCl₂.5H₂O, For 0.1 M soln., 35 g in 1 L water
Zinc sulfate, ZnSO₄.7H₂O, For 0.1 M soln., 28.8 g in 1 L water

9.0 Flammable organic chemicals
Do NOT use in school laboratories
Flammable substances shown as: FLAM and Flashpoint in ^oC

Benzene, benzol	FLAM -11^oC	1114	C₆H₆, HARM
Butan-1-ol	FLAM +29^oC	1120	CH₃(CH₂)₂CH₂OH
Butan-2-ol	FLAM +24^oC	1120	CH₃CH₂CHOHCH₃, sec-butyl alcohol
Carbon disulfide	FLAM -30^oC	1131	CS₂, POISON (alcohol sulfuris)
Cyclohexane	FLAM -20^oC	1145	C₆H₁₂
Diethyl ether	FLAM -40^oC	1155	(C₂H₅)₂O, ethoxy-ethane, anaesthetic ether, ether
iso-Octane	FLAM -12^oC	1262	C₈H₁₈, 2,2,4-trimethylpentane
n-Octane	FLAM +13^oC	1262	CH₃(CH₂)₆CH₃
Methanol	FLAM +12^oC	1230	CH₃OH, methyl alcohol, wood alcohol (in methylated spirits) HARM, highly toxic, causes blindness
Petroleum spirit	FLAM <-18^oC	1271	petroleum ether, spirit, benzine
Propan-1-ol	FLAM +15^oC	1274	C₂H₅CH₂OH, isomer propan-1-ol, n-propyl alcohol, 1-propanol
Propan-2-ol	FLAM +22^oC	1219	(CH₃)₂CHOH, isopropyl alcohol, 2-propanol, iso amyl alcohol, rubbing alcohol
Toluene	FLAM +7^oC	1294	C₆H₅CH₃

Other flammable organic chemicals with low flashpoints, below 32^oC
Acetaldehyde
Acetone (nail polish remover
Acetonitrile
Acetyl chloride
Acrylonitrile
Allyl iodide
Chloromethane
1,2-Dichloroethane
Diethylamine
Diethyl carbonate
1,3-difluorobenzene
Dimethyl ether
Dioxan (dioxane)
Ethanol
Ethyl acetate
Ethyl acrylate
Ethyl chloroformate
Ethyl formate
Hexane, n-hexane
Methylated spirit
Methyl ethyl ketone
Methyl isobutyl ketone (MIBK)
Octane
Pentane
Piperidine
isopropyl alcohol
Pyridine
Tetrahydrofuran
Triethylamine
Vinyl acetate

10.0 CHEMICALS PROHIBITED FOR SCHOOL USE
Do NOT use these chemicals in school laboratories.
Glass wool is NOT recommended for use in school laboratories. Thin pieces of glass wool can get into cuts and then into the blood stream. Do not touch glass wool with the fingers and do not breathe it in. Do not reuse damaged glass wool. It may release fibre particles into the air. Wear protective gloves.

Carbon tetrachloride	POISON	1846	CCl₄, tetrachloromethane
Carbon disulfide	POISON	1131	CS₂, FLAM -30^oC
Chloroform	POISON	1888	CCl₃
Mercury	COR	2808	Hg, quicksilver
Mercury salts, Hg (I) and Hg (II) salts	POISON	1629	mercury (I) chloride (calomel) Hg₂Cl₂, mercury (II) chloride (HgCl₂), HgI₂, HgSO₄
Phenol	POISON	1671	C₆H₅OH, carbolic acid
Phosphorus, white	POISON	.	P, white phosphorus, yellow phosphorus, spontaneously combustible
Potasssium chlorate	.	.	see below
Copper arsenite	POISON	.	CuHASO3, (acidic copper arsenite)
Copper(II)acetoarsenite	POISON	.	Cu(C₂H₃O₂)₂·3Cu(AsO₂)₂

Copper arsenite
The yellow green pigment Scheele's Green is no longer used and but its use in wallpaper may have killed the exiled Emperor Napoleon Bonaparte on St. Helena Island!
Copper(II) acetoarsenite
Paris Green, is still used as a poison and for green colour in fireworks.
Potassium chlorate
Not to be used to preparation oxygen with manganese dioxide catalyst.

11.0 High toxicity chemicals
Data provided with permission from Dr Hugh Cartwright, Chemistry Department, Oxford University, UK
The following table lists some of the chemicals which present a particularly severe risk to health (high toxicity chemicals).
Acetic anhydride
Acrolein
Acrylamide
Allyl alcohol
Allyl chloride
Aniline
Antimony compounds
Anisidines
Arsenic compounds
Soluble barium salts
p-benzoquinone
Benzoyl peroxide
Benzoyl chloride
Beryllium and its compounds
Boron tribromide
Boron trifluoride
Bromine
Butylamines
Chlorine
Chloronitrobenzenes
Chromium compounds
Cresols
Cyanides
Diazomethane
N,N-dimethylaniline
Dimethyl sulfate
Epichlorohydrin
Ethanolamine
Ethylene chlorohydrin
Fluorine
Formaldehyde
Formic acid
Hydrazine
Hydrochloric acid
Hydrofluoric acid
Hydrogen cyanide
Hydrogen peroxide
Indium salts
Iodine
Iodomethane
Isocyanates
Maleic anhydride
Mercaptans
Mercury and mercury compounds
Nickel carbonyl
Nitric acid
Nitrobenzene
Nitro compounds
Osmium salts
Oxalic acid
Ozone
Phenol
Phenylene diamines
Phenyl hydrazine
Phosgene
Phosphorus pentachloride
Phosphorus pentasulfide
Phosphorus trichloride
Phthalic anhydride
Propylamines
Pyridine
Selenium compounds
Soluble silver salts
Sodium-mercury amalgam
Sulfur dioxide
Tellurium compounds
Thallium compounds
Tetrachlorethane
Organotin compounds
Toluidines
Xylidines

12.0 Cryogenic solids and liquids
These chemicals should be handled only by teachers trained in the safe storage and dispensing of cryogenic substances, e.g. liquid nitrogen, oxygen and helium, and also solid carbon dioxide, "dry ice". These substance evaporate briskly at room temperature to increase pressure to dangerous levels in closed containers. Students should be kept at least one metre away from these substances to avoid frost bite. Children have been injured when allowed to lick "dry ice" used by ice-cream sellers.

13.0 Abbreviations

alc.	alcohol	.	.
alk.	alkali	M	molar conc, e.g. 2 M (2 molar)
aq.	water	m.p.	melting point
b.p.	boiling point	max.	maximum
cryst.	crystals	min.	minimum
decomp.	decomposes	mL	millilitre
del.	deliquescent	mm	millimetre (10^-3 m)
dil.	dilute	non-vol.	non-volatile
fp	freezing point	org.	organic
g / mL	r.d.	ox.	oxidized
g	gram	polyp.	polypropylene
ign.	ignition	ppt.	precipitate
Ind.	indicator	PVC	polyvinyl chloride
inorg.	inorganic	soln.	soln.
insol.	insoluble	W / V	weight to volume ratio
kg.	kilogram	W / W	weight to weight ratio
L	litre	.	.
ox.	oxidize	.	.

14.0 Prefixes and suffixes
R = alkyl group
Acid chloride X-CO-Cl, Prefix: halo-, formyl-, Suffix: -oyl halide
Alcohol X-OH, Prefix: hydroxy-, Suffix: -ol (-OH: alcohol)
Aldehyde X-CH=O, Prefix: formyl-, Suffix: -al
Alkane alkyl RCH₃ Prefix: methyl-, Suffix: -ane
Alkanes: methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane . . .
Alkene X-C=CRR, Prefix: alkenyl-, Suffix: -ene (no principal functional group)
Alkyne X-C=_CR (triple CC bond) Prefix: alkynl-, Suffix: -yne (no principal functional group)
Amide RCONH₂ (amine + acid) Suffix: -oic, amide
Amide X-NR-COR, Prefix: acylamido- Suffix: - amide
Amide X-CONRR, Prefix: carbamoyl-, Suffix: - carboxamide
Amine X-NRR, Prefix: amino-, Suffix: -amine, amines
amyl group: C₅H₁₁, only as suffix
Azide X-N₃, Prefix: azido-
azo azo RN=N-R' azo nitrile RC=N cyano-
azo pyridyl RC₅H₄N-
benzyl group RCH₂C₆H₅-
Bromide X-Br, Prefix: bromo-
Butane, C₄H₁₀, butyl C₄H₉-, Stem name: but-
-C=O: ketone, organic compound containing the group, only as suffix
carbonyl group (C=O)
carboxylic acid RCOOH Prefix: carboxy-, Suffix: -oic acid
Carboxylic acid, X-COOH, Prefix: carboxy, Suffix: -oic acid
Chloride X-Cl, Prefix: chloro-
cis: on same side
ester RCOOR' isocyanate RNCS
Ester X-CO-OR, Prefix: R-oxycarbonyl-, Suffix: -alkyl, -oate
Ethane, C₂H₆, ethyl C₂H₅-, Stem name: eth-
ether ROR' (Alcohol names) -ether
Ether X-OR, Prefix: R-oxy-
"flowers" of a metal: oxide of the metal
Fluoride X-F, Prefix: fluoro-
Glyco-: Sugar attached
Heptane, C₇H₁₆, heptyl C₇H₁₅, Stem name: hept-
Hexane, C₆H₁₄, hexyl C₆H₁₃, Stem name: hex-
-ide: Suffix: inorganic compounds containing two elements
imide. amide group: CONH₂, only as suffix
imine primary RC(=NH)R’ imino-, -imine
imine secondary RCH=NR’ imino-, -imine
-ine: 1. organic base, 2. amino acid, 3. halogen element, only as suffix
Iodide X-I, Prefix: iodo-
-ium: Suffix: metals or groups with metal-like properties, e.g. ammonium, only as suffix
ketone RCOR' keto-, -one
Ketone X-COR, Prefix: oxo-, Suffix: -one
-lysis: Breaking down, decomposition, only as suffix
Methane, CH₄, methyl CH₃-, Stem name: meth-
muriate of a metal: chloride of the metal
NH: imine, a compound of =NH is an imino, only as suffix
-NH₂: amine group, a compound of -NH₂ is amino, amide, only as suffix
Nitrile X-CN, Prefix: cyano-, Suffix: -nitrile
Nitroso X-NO, Prefix: nitroso-
Nitro X-NO₂, Prefix: nitro-
Octane, C₈H₁₈, octyl C₈H₁₇, Stem name: oct-
Ortho-: Regular form.
Para-: Alongside, beyond, near, contrary to
Pentane, C₅H₁₂, pentyl C₅H₁₁-, Stem name: pent-
phenol: organic compounds with functional group -OH, but attached to aromatic ring
Phenol X-C₆H₄-OH, Prefix: hydroxy, Suffix: -ol
phenyl group RC₆H₅
primary amine: RNH₂ amino-, -amine amines
Propane, C₃H₈, propyl C₃H₇-, Stem name: prop-
secondary amine: RNHR’ amino-, -amine amines
Sulfide X-SR, Prefix: R-thio-
Sulfonic acid X-SO₂-OH, Prefix: sulfo-, Suffix: -sulfonic acid
tertiary amine: RNR’R’’ amino-, -amine
Thio-: A prefix used when one atom is replaced by a sulfur atom
thiol sulfhydryl group RSH, only as prefixes
Thiol X-SH, Prefix: mercapto, Suffix: -thiol
trans: on opposite sides
trans fatty acid: fatty acid formed as a result of hydrogenation.

15.1 Hazard classifications EXPLOSIVE, FLAM, OXD, POISON, HARM, COR, IRR
The classification is based on IMO (International Maritime Dangerous Goods Code) and the (ACTDG) Australian Code for the Transport of Dangerous Goods by Road and Rail. In this document, hazard classification is shown (in square brackets) and is followed by the United Nations Chemical Number to identify all classified dangerous goods.

(EXPLOSIVE)	Class 1 Explosive, can explode due to flame shock or friction
(FLAM)	Class 2.1 Inflammable gases, can readily burn or catch fire
.	Class 3.1 Inflammable liquids, flashpoint below -18^oC
.	Class 3.2 Inflammable liquids, flashpoint between -18^oC and 23^oC
.	Class 3.3 Inflammable liquids, flashpoint between 23^oC and 61^oC
.	Class 4.1 Inflammable solid, can readily burn or catch fir
.	Class 4.2 Inflammable solid, liable to spontaneous combustion
.	Class 4.3 Inflammable solid, emits inflammable gases when wet
(OXD)	Class 5.1 Oxidizing agents, can react with substances to produce great heat
.	Class 5.2 Organic peroxides
(POISON)	Class 6.11. Toxic substances, severe risk if absorbed
(HARM)	Class 6.12. Harmful substance, some health risk if absorbed
(COR)	Class 8 Corrosives, can destroy living tissue on contact
(IRR)	Non-corrosive, but can damage skin if prolonged contact

15.2 Health effects, Very Toxic, Toxic, Harmful, Corrosive, Irritant, Carcinogenic, Mutagenic, Environment danger
Very toxic
Substances and preparations which in very low quantities cause death or acute or chronic damage to health when inhaled, swallowed or absorbed via the skin.
Toxic
Substances and preparations which in low quantities cause death or acute or chronic damage to health when inhaled, swallowed or absorbed via the skin.
Harmful
Substances and preparations which may cause death or acute or chronic damage to health when inhaled, swallowed or absorbed via the skin.
Corrosive
Substances and preparations which may, on contact with living tissues, destroy them.
Irritant
Non-corrosive substances and preparations which, through immediate, prolonged or repeated contact with the skin or mucous membrane, may cause inflammation.
Sensitising (Sensitising by inhalation) (Sensitising by skin contact)
Substances and preparations which, if they are inhaled or if they penetrate the skin, are capable of eliciting a reaction by hypersensitization such that on further exposure to the substance or preparation, characteristic adverse effects are produced.
Carcinogenic
Substances and preparations which, if they are inhaled or ingested or if they penetrate the skin, may induce cancer or increase its incidence
Mutagenic
Substances and preparations which, if they are inhaled or ingested or if they penetrate the skin, may induce heritable genetic defects or increase their incidence.
Environment danger
Substances and preparations which, were they to enter into the environment, would present or might present an immediate or delayed danger.

15.3 Draft Australian criteria for the classification of hazardous chemicals, Physical hazards
4.0 Physical hazards
4.1 Explosives (An explosive substance or mixture is a solid or liquid substance which is in itself capable by chemical reaction of producing gas at such a temperature and pressure and at such a speed as to cause damage to the surroundings. Pyrotechnic substances are included even when they do not evolve gases.
4.2 Flammable Gases (Gas having a flammable range with air at 20ºC and a standard pressure of 101.3 kPa.)
4.3 Flammable Aerosols (A non-refillable receptacle made of metal, glass or plastics and containing a gas compressed, liquefied or dissolved under pressure, and fitted with a release device allowing the contents to be ejected
Category 1 Aerosol contains ≥ 85 % of flammable components and the chemical heat of combustion is ≥ 30 kJ/g
Category 2 Aerosol contains ≥ 1 % flammable components, or the heat of combustion is ≥ 20 kJ/g).
4.4 Oxidising Gases (Gas by providing oxygen, may cause or contribute to the combustion of other material more than air does.
4.5 Gases Under Pressure (Gase in a receptacle at a pressure not less than 200 kPa (gauge), or is liquefied, or is liquefied and refrigerated.
4.6 Flammable Liquids (Liquid having a flash point of not more than 60ºC.)
4.7 Flammable Solids (Solid which is readily combustible, or may cause or contribute to fire through friction.
4.8 Self-reactive Substances and Mixtures (Are thermally unstable liquid or solid substances or mixtures liable to undergo a strongly exothermic decomposition even without participation of oxygen or air.).
4.9 Pyrophoric Liquids (Liquid catches fire spontaneously if exposed to air.)
4.10 Pyrophoric Solids (Solid catches fire spontaneously if exposed to air.)
4.11 Self-heating Substances and Mixtures (Substance react with air and without energy supply and is liable to self-heat and ignite but only when in large amounts (kilograms) and after long periods of time (hours or days).
4.12 Substances and mixtures which, in contact with water, emit flammable gases
4.13 Oxidising Liquids (Liquid not necessarily combustible which may contribute to the combustion of other material by yielding oxygen.)
4.14 Oxidising Solids (Solid not necessarily combustible which may contribute to the combustion of other material by yielding oxygen.)
4.15 Organic Peroxides (Organic substance which contains the bivalent -O-O- structure, where one or both of the hydrogen atoms have been replaced by organic radicals. They are thermally unstable substances which can undergo exothermic self-accelerating decomposition.)
4.16 Corrosive to Metals (A substance or a mixture which by chemical action will materially damage or destroy metals.)

15.4 Draft Australian criteria for the classification of hazardous chemicals, Health hazards
5.0 Health Hazards
5.1 Acute Toxicity (Acute toxicity refers to those adverse effects occurring following oral or dermal administration of a single dose of a substance or a mixture, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours.
5.3 Serious Eye Damage / Eye Irritation (Serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application. )
5.4 Respiratory or Skin Sensitisation (Respiratory sensitiser means a substance that will lead to hypersensitivity of the airways following inhalation of the substance. Skin sensitiser means a substance that will lead to an allergic response following skin contact.)
5.5 Germ Cell Mutagenicity (A mutation means a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term “mutagenic” and “mutagen” will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms.
Carcinogen means a substance or a mixture of substances which induce cancer or increase its incidence. Substances and mixtures which have induced benign and malignant tumours in well performed experimental studies on animals are considered also to be presumed or suspected human carcinogens unless there is strong evidence that the mechanism of tumour formation is not relevant for humans.)
5.7 Reproductive Toxicity (Reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females,
5.8 Specific Target Organ Toxicity, Single Exposure
5.9 Specific Target Organ Toxicity, Repeated Exposure
5.10 Aspiration Hazard (Aspiration is the entry of a liquid or solid substance or mixture directly through the oral or nasal cavity, or indirectly from vomiting, into the trachea and lower respiratory system. Aspiration toxicity includes severe acute effects such as chemical pneumonia, varying degrees of pulmonary injury or death following aspiration. )