Learn to make fixatives, media, glues and adhesives.

Learn to make fixatives, media, glues and adhesives. School Science Lessons
Appendix B Biology preparations
2024-03-16
Please send comments to: johnelfick@hotmail.com.

Appendix B, Biology preparations
(appendix B)
Table of contents

Prepare the following media and solutions.
1.0 Prepare agar media and nutrient solutions
2.0 Prepare biology fixatives
3.0 Prepare biology media and solutions
4.0 Prepare culture media to identify fungi
5.0 Prepare insect-fixing solutions
7.0 Prepare microscopy stains and adhesives

1.0 Prepare agar media and nutrient solutions
1.1 Prepare basal agar solution
1.2 Prepare cornmeal agar solution
1.3 Prepare cornmeal glucose sucrose yeast extract agar solution
1.4 Prepare Czapek Dox agar solution
1.5 Prepare DRBC agar solution, (food spoilage solution)
1.6 Prepare glucose nutrient agar solution
1.17 Prepare MacConkey agar solution
1.8 Prepare malt extract agar solution
1.9 Prepare mannitol yeast extract agar solution, (MYEA)
1.10 Prepare milk agar solution
1.11 Prepare minimal agar solution
1.12 Prepare MS agar solution
1.13 Prepare nitrogen-free mineral salts agar solution
1.14 Prepare nutrient agar solution
1.15 Prepare nutrient agar, Microbiological base ingredients
1.16 Prepare starch nutrient agar solution
1.17 Prepare sterile media or solutions
1.18 Prepare urea agar solution

2.0 Prepare biology fixatives
2.1 Prepare aceto-alcohol solution
2.2 Prepare Bouin's solution
2.3 Prepare Carnoy's solution
2.4 Prepare CRAF biology fixative solution
2.5 Prepare decalcifying solution
2.6 Prepare differentiation solution
2.7 Prepare ethanol solution for molecular biology
2.8 Prepare formaldehyde-acetic acid alcohol, FAA, solution
2.9 Prepare formaldehyde solution
2.10 Prepare formal saline solution
2.11 Prepare glutaraldehyde solution
2.12 Prepare Zenker's solution

3.0 Prepare biology media and solutions
3.1 Prepare acid alcohol solution
3.2 Prepare alcohol solution, absolute alcohol
3.3 Prepare BAP solution
3.4 Prepare basal broth solution
3.5 Prepare basal salt solutions
3.6 Prepare buffer reagent solution
3.7 Prepare carbol xylol solution
3.8 Prepare Domestos solution, NaOCl
3.9 Prepare fluorescein solution
3.10 Prepare Gram stain decolorizing solution
3.11 Prepare Gram's iodine solution
3.12 Prepare iodine solution
3.13 Prepare liquid broth media and solutions
3.14 Prepare Lugol's iodine solution
3.15 Prepare malt extract solution
3.16 Prepare methyl cellulose solution
3.17 Prepare nutrient broth solution
3.18 Prepare nutrient gelatin solution
3.19 Prepare phenylthiourea solution, PTU
3.20 Prepare Ringer solution
3.21 Prepare salt solutions
3.22 Prepare Scott's blueing solution
3.23 Prepare seawater substitute solution
3.24 Prepare sodium thiosulfate solution
3.25 Prepare sterile media solutions
3.26 Prepare vinegar bacteria solution
3.28 Prepare xylene and methylbenzoate solution

4.0 Prepare culture media to identify fungi
4.1 Prepare calcofluor white solution
4.2 Prepare cellotape flags
1.2 Prepare cornmeal agar solution
1.3 Prepare cornmeal glucose sucrose yeast extract agar solution
1.4 Prepare Czapek Dox agar solution
4.6 Prepare direct microscopic mounts or squash preparations
4.7 Prepare India ink mounts
4.8 Prepare lactophenol solution, (LPCB)
4.9 Prepare Loeffler serum solution
1.17 Prepare MacConkey agar solution
4.11 Prepare Orcinol-Bial's reagent
4.12 Prepare potassium hydroxide with chlorazol black solution
4.13 Prepare potato dextrose agar solution
4.14 Prepare rice grain slopes

5.0 Prepare insect-fixing solutions
4.15 Prepare Barber's insect-fixing solution
5.2 Prepare KAA insect fixing solution
4.10 Prepare Kahle's insect-fixing solution
5.4 Prepare lacto-alcohol insect fixing solution
5.5 Prepare Oudeman's insect fixing solution
5.6 Prepare Pampl's insect fixing solution
5.7 Prepare sugaring mixture insect fixing solution

7.0 Prepare microscopy stains and adhesives
7.1 Prepare acetic alcohol solution
7.2 Prepare aceto-carmine stain
7.3 Prepare aceto-orcein stain
7.4 Prepare alcian blue solution
7.5 Prepare alizarin solution
7.6 Prepare alizarin red S solution
7.7 Prepare aniline blue solution
7.8 Prepare aniline hydrochloride solution
7.9 Prepare aniline sulfate solution
7.10 Prepare basic fuchsin solution
7.11 Prepare Calberla's pollen stain
6.1 Prepare Canada balsam mounting solution
7.12 Prepare carbol fuchsin solution
7.13 Prepare carmine stain
7.14 Prepare corn syrup mountant solution
7.21 Prepare crystal violet solution
7.16 Prepare DCIP solution (2,6-dichlorophenol-indophenol)
6.2 Prepare DPX mountant solution
7.18 Prepare eosin solution
7.19 Prepare Giemsa stain
6.3 Prepare glycerine jelly solution
7.20 Prepare Gram stain
7.22 Prepare fluorescence staining of cells and tissues
6.4 Prepare Haupt's adhesive solution
7.23 Prepare haematoxylin stain
7.26 Prepare immersion oil
3.14 Prepare Lugol's iodine solution
7.29 Prepare Leishman's stain, Wright's stain
7.30 Prepare methylene blue solution
6.5 Prepare Meyer's albumen solution
6.6 Prepare Peru balsam mounting medium
3.31 Prepare nuclear fast red solution
3.32 Prepare oil red solution
7.33 Prepare orange IV solution
7.34 Prepare Papanicolaou stain
7.35 Prepare phloroglucinol solution
7.36 Prepare safranin solution
7.37 Prepare Schulze's solution
7.38 Prepare toluidine blue solution
7.40 Prepare Ziehl-Neelsen stain

1.2 Prepare cornmeal agar solution
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.

1.3 Prepare cornmeal glucose sucrose yeast extract agar solution
It is used 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.

1.4 Prepare Czapek Dox agar solution
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.

1.5 Prepare DRBC agar, (food spoilage solution)
DRBC, Dichloran Rose-Bengal Chloramphenicol Agar is a selective solution, for yeasts and moulds associated with food spoilage.
It inhibits bacterial growth and spreading moulds, e.g. Rhizopus and Mucor, but supports the growth of those species that cannot be otherwise isolated.
Inhibition of spreading moulds and the general restriction of colony size results in improved counting, and detection of mycotoxigenic moulds and food spoilage species.

1.6 Prepare glucose nutrient agar solution
Purchase: Potato glucose agar.
Prepare glucose nutrient agar:
1. Use ready made solution: glucose 1. 0 g / litre, nutrient agar 20.0 g / litre.
2. 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).

1.8 Prepare malt extract agar solution
Purchase Malt Extract Agar for microbiology.
Use for malt extract agar solution for routine cultivation and identification of fungi.
Prepare malt extract agar solution:
1. Use ready made: malt extract agar 48.0 g / litre.
2. Malt extract 30.0 g / litre, peptone from meat 3.0 g / litre, agar 11.0 g / litre (pH 1.6), autoclave for 10 minutes.
3. Dissolve 15 g malt extract and 18g bacteriological agar in 1 litre of deionized water.
Dispense into bottles and sterilize with an autoclave.
4. Dissolve 20 g Oxoid Malt Extract, in 1000 mL of deionized water in a plastic beaker, and pH the solution to pH 6.5 with NaOH.
5. Soak 20 g Bacto Agar in small quantity of solution.
Bring remaining solution to the boil, stirring constantly.
Add to soaking agar.
Bring to boil, stirring constantly.
6. Dispense for slopes as required.
Autoclave at 121^oC for 10 minutes, remove and slope or pour for plates as required.

1.9 Prepare mannitol yeast extract agar (MYEA)
Purchase: Yeast mannitol agar for microbiology.
Prepare mannitol yeast extract agar:
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.

1.10 Prepare milk agar solution
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.
Prepare sterilized nutrient agar, leave to cool to 45-50^oC, add 10% pasteurized milk, skimmed, semi-skimmed or full cream milk.

1.11 Prepare minimal agar solution
Purchase: Davis Minimal Agar for microbiology (Minimal Agar, Davis).
Prepare minimal agar solution:
K2HPO4 3.5 g / litre.
sodium citrate 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).

1.12 Prepare MS agar solution
Purchase: MRS Agar for microbiology.
Lactobacillus Agar, MS agar solution is used for Lactobacillus culture.
Prepare MRS Agar:
1. With ready-made solution: MS powder (basal salts with minimal organics):
4.7 g / litre, granulated sugar 30.0 g / litre, agar 8. 0 g / litre, phytohormone solution (e.g. BAP).
Regulate pH with 1 M KOH.
2. Agar 12 g / litre, diammonium hydrogen citrate 2 g / litre, dipotassium hydrogen phosphate 2 g / litre, glucose 20 g / litre.
magnesium sulfate 0.1 g / litre.

1.13 Prepare nitrogen-free mineral salts agar solution
Purchase: Nitrogen-free mineral salts agar solution.
Nitrogen free mineral salts agar is used to culture a free-living nitrogen-fixing bacterium (Azotobacter), from the soil.
Prepare 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.

1.14 Prepare nutrient agar solution
Purchase: Nutrient Agar for microbiology.
Prepare nutrient agar solution: peptone from meat 1. 0 g / litre, meat extract 3. 0 g / litre, agar 12.0 g / litre (pH 7.0).

1.16 Prepare starch nutrient agar solution
Prepare starch nutrient agar: 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.

1.17 Prepare MacConkey agar solution
MacConkey agar is a primary plating solution, used to differentiate members of the coliform group.
It is selective and differential.
It selects for gram-negative bacteria, because it contains bile salts and the dye crystal violet, which inhibit the growth of gram-positive bacteria.
It differentiates, because it contains lactose.
Gram-negative bacteria can ferment lactose to form acid end products shown by a pink colour from the neutral red indicator.
These pink staining colonies show the presence of coliform bacteria, an indicator of unsanitary food and water.

1.18 Prepare urea agar solution
Purchase: Urea Test Agar, Christensen's Urea Agar, Urea Agar Base according to Christensen.
Prepare using ready-made solution:
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. to 2.).
Components of urea agar:
peptone from meat 1. 0 g / litre.
glucose 1.0 g / litre.
NaCl 1.0 g / litre.
K2HPO4, 1.0 g / litre.
phenol red 12 mg / litre.
agar 12.0 g / litre (pH 6.8).

2.1 Prepare aceto-alcohol solution
Purchase: Ethanol, 200 proof (absolute), for molecular biology.
Aceto-alcohol fixative is used for animal material.
Prepare:
Absolute ethanol (alcohol), 30 mL.
Glacial acetic 10 mL.
Mix immediately before use and discard after 1 hour.

2.2 Prepare Bouin's solution
Purchase: Bouin's solution, Bouin's solution, biology fixative.
Bouin's solution, is a fixative for preserving soft and delicate structures, and is used as a mordant in trichrome procedures.
Prepare Bouin's solution:
Picric acid (saturated) 75 mL.
Formaldehyde (37-40%) 25 mL.
Glacial acetic acid 5 mL.
(Sold as: acetic acid 5%, formaldehyde 9%, picric acid 0.9%.).

2.3 Prepare Carnoy's solution
Carnoy's solution is an insect-fixing solution and is used for fixation of DNA, RNA, Nissl granules and glycogen.
Prepare Carnoy's solution:
1. Alcohol 95%, 75 mL.
Chloroform 30 mL.
Glacial acetic acid 10 mL, mix well.
2. Absolute alcohol 60 mL.
Chloroform 30 mL.
Glacial acetic acid 10 mL, mix well.
3. Ferric chloride 1 g.
Absolute alcohol 24 mL.
Chloroform 12 mL.
Glacial acetic acid 4 mL.

2.4 Prepare CRAF biology fixative solution
Formula: Chromic acid, 1% 40 mL, Formalin (40% methanal), 10 mL, Glacial acetic acid, 5 mL, Deionized water 5 mL.

2.5 Prepare decalcifying solution
Purchase "Decalcifying Solution-Lite:, aqueous solution, bp 208 F, light yellow colour.
Decalcifying solution is used for the decalcification of routine, immunohistochemical and bone marrow core specimens.

2.6 Prepare differentiation solution
Differentiation solution is an acidified alcohol solution for the differentiation of regressive haematoxylin stains.

2.7 Prepare ethanol solution for molecular biology
Purchase: Ethanol, 200 proof (absolute), for molecular biology.
A 50% solution of ethanol / water solution is used to preserve biological specimens.

2.8 Prepare formaldehyde-acetic acid alcohol, FAA solution
Formalin acetic acid alcohol (FAA) and formalin propionic acid alcohol, (FPA), are used as general purpose biological fixatives.
Prepare FAA solution:
(FAA = formalin, alcohol, acetic acid).
1. Formalin (40% methanal) 5 mL.
Ethanol 70% 90 mL.
Glacial acetic acid 5 mL.
Formalin-acetic acid solution.
2. Formaldehyde (37-40%), 10 mL.
Distilled water 90 mL.
Glacial acetic acid 5 mL.
3. Formal ethanol fixative solution.
5 mL formaldehyde.
45 mL 95% ethanol.

2.9 Prepare formaldehyde solution
Formaldehyde, CH2O, formalin, biology fixative.
Purchase: Formalin solution, neutral buffered, 10% histological tissue fixative.
Case containers are polypropylene.
Formalin is used as a general purpose histological fixative.
Formalin is the name used for 40% formaldehyde solution.
1% formalin = 1 mL formalin then dilute to 100 mL with water.
Formaldehyde vapour irritates eyes and delicate body tissues.
Biological specimens preserved in formaldehyde should be kept in sealed containers.
Prepare formalin solutions:
1. Formalin Solution (10%, unbuffered):
Formaldehyde (37-40%) 10 mL.
Distilled water 90 mL.
2. Formalin Solution (20%, unbuffered):
Formaldehyde (37-40%) 20 mL.
Distilled water 80 mL.
3. Formalin Solution (10%, buffered neutral):
Formaldehyde (37-40%) 100mL.
Distilled water 900 mL.
NaH2PO4 4.0 g, Na2HPO4(anhydrous) 6.5 g.
4. Formalin Solution (20%, buffered neutral):
Formaldehyde (37-40%) 200mL.
Distilled water 800 mL.
NaH2PO4 4.0 g.
Na2HPO4 (anhydrous) 6.5 g.

2.10 Prepare formal saline solution
Formal saline, para formaldehyde.
Formal saline is more effective than 10% formalin, because it is isotonic so less damage to erythrocytes.
This mixture of formaldehyde in isotonic saline was formerly widely used for routine histopathology, prior to the introduction of phosphate-buffered formalin.
1. 91 % formaldehyde.
37% solution in water.
It was used as a biological fixative for marine animals.
2. Formalin (40% methanal) 100 mL.
Sodium chloride 10% solution 7 mL.
Deionized water 83 mL.
3. 40% formaldehyde 100 mL.
Sodium chloride: 9 g.
Distilled water: 900 mL.
The fixation time is 12 to 24 hours.

2.11 Prepare glutaraldehyde solution
Glutaraldehyde solution is a microscopy fixative.
Glutaraldehyde, OHC(CH2)3CHO, aqueous solution, (glutaric dialdehyde solution, pentane-1,5-dial).
Purchase, Glutaraldehyde solution, Grade I, 25% in H2O, electron microscopy fixative.
Glutaric dialdehyde solution, pentane-1, 5-dial, OHC(CH2)3CHO, aqueous solution.
Glutaraldehyde, (C7H8O2), pentanedial, a colourless liquid, with a strong odour, is an antimicrobial bactericide, fungicide and a virucide.
It is used to sterilize hospital equipment, prevent bacterial growth in water supplies, and as a fixative for tissues.
Breathing it causes headaches.

2.12 Prepare Zenker's solution
Zenker's solution, biology fixative, for animal material.
This solution is NOT suitable for use in schools.
Prepare Zenker's fixative:
1. Potassium dichromate 2.5 g.
Mercury II chloride 5.8 g.
Deionized water 95 mL.
2. Mercuric chloride.
Potassium dichromate.
Sodium sulfate.
Glacial acetic acid.
Water.
Zenker's fixative: is a rapid fixative used for trichrome stains.
The fixed tissue must be washed to remove potassium dichromate and treated with iodine solution to remove mercuric chloride.

3.1 Prepare acid alcohol solution
Purchase: Acid alcohol solution for microscopy.
Acid alcohol is used for cleaning slides and coverslips and decolorizing some stains, e.g. haematoxylin.
Prepare acid alcohol solution: 100 mL 70% alcohol + 1 mL hydrochloric acid.

3.2 Prepare alcohol solution, absolute alcohol
Purchase: Ethanol ACS reagent, 99.5% (200 proof), absolute.
Ethanol is used in various concentrations for preserving and dehydrating.
Absolute alcohol bottles must be stoppered at all times, because alcohol readily absorbs water from the atmosphere.
Remove water from alcohol by using drying agents, e.g. anhydrous copper (II) sulfate.

3.3 Prepare BAP solution
Purchase BAP, 6-Benzylaminopurine, 6-BAP, BA, N6-Benzyladenine, (C12H11N5).
100 mg, 1 M HCl 20 mg, plant cell culture-tested BAP is a synthetic cytokinin that with auxins assists plant growth and development.
It is used in plant growth media, e.g. Murashige solution, Skoog solution, Gamborg's solution, Chu's N6 solution.
BAP inhibits respiratory kinase in plants, and increases post-harvest life of green vegetables.
Prepare BAP solution:
Add 100 mg BAP to 100 mL of boiling deionized water.
Then add 1.0 mL of this stock solution to a culture solution, e.g. MS agar solution.

3.4 Prepare basal broth solution
The composition as for basal agar solution above, without agar.
This is a liquid solution for overnight cultures.

3.5 Prepare basal salt solutions
Basal salt solutions are used as an irrigating, transporting and diluting solution while maintaining intra-cellular and extra cellular osmotic balance.
They provides cells with water and certain bulk inorganic ions essential for normal cell metabolism.
They may be combined with a carbohydrate, such as glucose, provides the principle energy source for cell metabolism.
Also, they provides a buffering system to maintain the solution within the physiological pH range (7.2-7.6).
Basal salt solutions for cell culture:
1. Dulbecco's phosphate buffered saline contains no bicarbonate, so a pH shift during filtration is less likely.
2. Earle's balanced salts contains 2.2 gm / litre sodium bicarbonate, may require pH adjustment after filtration.
3. Hanks' balanced salts contains 0.35 gm / litre sodium bicarbonate, may require pH adjustment after filtration.
Prepare basal salt solution:
Dissolve 9 g sodium chloride in 1 litre of deionized water.

3.6 Prepare buffer reagent solution
Purchase: Phosphate Buffer pH 6.6 at 25 C, for use with Wright Stain.
Purchase: Phosphate buffer pH 7.2 at 25 C, for haematology and histology staining techniques.
For use as a buffer in Romanowsky type staining procedures, e.g. Wright Stain, Wright Giemsa, Giemsa, May Grunwald, Jenner and Leishman.
Individual components for 1 M solution:
Prepare phosphate buffer reagent:
1. K2HPO4, 17.4 g / 100 mL.
2. KH2PO4, 12.9 g / 100 mL.
Composition of 1 M buffer reagent:
Make separate solutions of bipotassium hydrogen phosphate (solution 1.), and potassium dihydrogen phosphate (solution 2.), using 100 mL deionized water.
Place 60 mL of solution 1 into a 100 mL conical flask and use a pipette and filler to 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.

3.7 Prepare carbol xylol solution
Purchase: m-Xylol, m-Xylene, anhydrous (1,3-Dimethylbenzene ), Metaxylene, (C8H10).
Purchase Low cost: from hardware stores and paint stores, as xylenes (mixed dimethylbenzene isomers).
Purchase: carbolic acid, phenol, hydroxybenzene, benzenol, (C6H6O, (C6H5OH).
BE CAREFUL! Use gloves!.
Carbol xylol solution is a mixture of 3 parts xylol and 1 part melted crystal carbolic acid is used for dehydrating in staining techniques.
Prepare carbol xylol solution:
Mix 25 g carbolic acid, with 100 mL xylene, (C8H10).
The phenol dissolves slowly, and cools when dissolving.

3.8 Prepare Domestos solution, NaOCl
"Domestos" is composed of saturated sodium hypochlorite solution.
Prepare 20% Domestos Solution:
"Domestos" household cleaning solution 20 mL.
Add deionized water to 100 mL.

3.9 Prepare fluorescein solution
Purchase: Fluorescein (free acid), Dye content 95 %, (C20H12O5).
Dissolve one gram of fluorescein in 100 mL methylated spirit.

3.10 Prepare Gram stain decolorizing 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.

3.11 Prepare Gram's iodine solution
Purchase: Gram's iodine solution.
Gram's iodine is used for an indicator for the presence of starch, detection of alkaloids, source of iodine for iodometric titrations, and source of iodine / iodide.
Prepare Gram's iodine solution: 2% iodine and 3% potassium iodide in 70% ethanol.

3.12 Prepare iodine solution
1. Dissolve 2 g potassium iodide in water and add 1 g iodine crystals.
2. Dissolve 1 g iodine and 4 g potassium iodide in 300 mL water.
3. Add l g iodine crystals and 5 g potassium iodide to 50 mL water, then dilute to 100 mL.
4. Dissolve 10 g of potassium iodide in 100 mL of deionized water and add 5 g of iodine crystals.
5. Dissolve 15 g of potassium iodide in 20 mL of deionized water and add 3 g of iodine crystals, then dilute to 1 litre.
6. Add iodine flakes to methylated spirit to form alcoholic iodine solution.
7. Dissolve 10 g of potassium iodide in 30 mL of water and add 6.5 g of iodine crystals.
. Swirl the solution until all the iodine crystals dissolve and make up volume to one litre with distilled water to form a solution.

3.13 Prepare liquid broth media and solutions
1. Prepare the components required for the production of culture media, according to the description for agar media, steps 1 through 3, autoclave, and let cool.
2. Fill culture tubes already sterilized in a drying cabinet at 180^oC, for 3 hours, with 5 mL of the culture solution, with a sterile glass pipette and fillers.
3. Inoculate the culture tubes with material containing bacteria, as described in the experiments.

3.14 Prepare Lugol's iodine solution
Purchase: Lugol solution, according to Lugol, Iodine / Potassium iodide solution.
Prepare Lugol's iodine 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 of deionized water.

3.15 Prepare malt extract solution
This is a liquid solution for overnight cultures.
Purchase: Malt Extract Agar for microbiology.
Preparation: As for malt extract agar solution without the agar.

3.16 Prepare methyl cellulose solution
Methyl cellulose solution, methocel (low substitution).
Purchase: Methyl cellulose, viscosity 1, 500 cP, 2 % in H2O (20C).
(Different viscosity solutions are available.).
Methyl cellulose, E461 (vegetable gum) (HEALTH intestinal problems).
Methyl cellulose, 2-methoxyethanol, methocel (low substitution), ethers based on cellulose, viscid solutions.
Methyl cellulose is not digestible, not toxic.
It dissolves in hot water not hot water, because precipitates out.
It is used as thickener, emulsifier, virus culture, electrophoresis solution, "slime" in movies, slow movement protozoa and microorganisms, by increasing viscosity.
Prepared chemically from cellulose + sodium hydroxide + methyl cellulose.
Degree of substitution (DS) up to 3 OH groups per glucose molecule, (low substitution - to high substitution).
Prepare methyl cellulose solution:
Dissolve methyl cellulose in water, to slow protozoa for study under the microscope.

3.17 Prepare nutrient broth solution
Purchase: Nutrient Broth, No. 1, for microbiology, Standard - Nutrient Broth.
Nutrient broth solution is a liquid solution for overnight cultures.
It may also be purchased as a powder.
Prepare: broth 8.0 g / litre, or from individual components.
The composition is the same as nutrient agar solution without the agar.

3.18 Prepare nutrient gelatin solution
Purchase: Nutrient Gelatin for microbiology.
Nutrient Gelatin for microbiology is used for food control media, liquid media / broth, solid media / agars, biochemical identification media tests,
gelatine liquefaction tests, identification tests & reagents, identification of proteolytic bacteria, soil / agriculture /
environmental media, and media for cocoa, chocolate, sweets, food control, meat control, sterility testing, water control.

3.19 Prepare phenylthiourea solution, PTU
Phenylthiocarbamide, PTC, phenylthiourea, PTU, N-Phenylthiourea, 1-Phenyl-2-thiourea, (C7H8N2S), C6H5NH(CSNH2).
9.24.2 PTC tasters and non-tasters.
Phenyl thiocarbamide is toxic, but is safe at a concentration < 0.13%.
Prepare PTU solution:
Weigh 0.13g of solid phenylthiocarbamide on a watch glass.
Dissolve the solid in 100 mL deionized water.
Cut strips of absorbent paper, 5 cm X 1 cm.
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 ×2 or ×10 as dilute, after diluting the above solution 50 mL to 100 mL or 10 mL to 100 mL.
Rinse the mouth after each tasting trial.
The chemical has a very persistent bitter taste, so handle the strips of paper with forceps.

3.20 Prepare Ringer solution
Ringer solution is a mixture of salts that dissolve in water to form a physiological saline solution.
Prepare fresh before use.
Purchase: Ringers solution tablets for microbiology.
Prepare Ringer solution:
Solution 1. 0.9 g sodium chloride, 0.042 g potassium chloride, 0.025 g calcium chloride, 100 mL deionized water.
Solution 2. 7.2 g sodium chloride, 0.37 g potassium chloride, 0.17 g calcium chloride.
Dissolve in deionized water, add deionized water to 1 litre, adjust pH to 7.3-7.4.
Solution 3. 2.25 g / litre sodium chloride, 0.105 g / litre potassium chloride, 0.12 g / litre calcium chloride.6H2O, 0.05 g / litre sodium bicarbonate.
One tablet makes 500 mL of quarter strength Ringer solution.
To prepare quarter strength Ringer Solution, dissolve 1 tablet in 500 mL distilled water.
Sterilize by autoclaving at 121^oC for 15 minutes.
4. Dissolve 1 Ringer tablet in 500 mL deionized water and then autoclave it.
3.21 Prepare salt solutions

3.21 Prepare salt solutions
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).

3.22 Prepare Scott's blueing solution
Purchase: Scott's tap water substitute concentrate.
Scott's blueing solution is used as a "blueing reagent" in "haematoxylin and eosin" staining procedures to develop colour.
Prepare Scott's blueing solution: 2 g sodium bicarbonate, 20 g magnesium sulfate, 1 litre deionized water.

3.23 Prepare seawater substitute solution
Prepare seawater 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.

3.24 Prepare sodium thiosulfate solution
It is used to decolorize iodine and wash iodine from tissue.
Prepare sodium thiosulfate solution:
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 solution (calcium hypochlorite), or citric acid or sulfuric acid.
The colour of iodine solution returns.

3.25 Prepare sterile media solutions
Equipment:
1 autoclave or pressure cooker.
1 conical flask, 300 mL to 2.0 L.
culture tubes or test-tubes, sealed with cellulose bungs.
Petri dishes.
pipette and filler.
5 mL, sterile, pipette aid.
1 pH meter.
1 spatula.
1 set of scales.
1 piece of weighing paper.
1 pair of insulated gloves.
Agar media:
1. Weigh out the individual components used for the manufacture of the culture solution 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 solution is required, reduce the proportions of the individual components accordingly.
The size of the conical flask depends on the amount to be prepared.
For example, 300 mL conical flasks are required for 200 mL culture solution.
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 solution 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 solution.
5. Place the filled Petri dishes in a safe place until the agar has set completely.
6. Inoculate the culture solution plates with material that contains bacteria, according to experimental needs.

3.26 Prepare vinegar bacteria solution
Vinegar bacteria solution is a liquid solution for overnight cultures.
Prepare vinegar solution:
Peptone from meat 3. 0 g / litre.
yeast extract 1. 0 g / litre.
mannitol 21.0 g / litre.

3.28 Prepare xylene and methylbenzoate solution
Xylene, (C8H10), C6H4(CH3)2, m-Xylene anhydrous, 1, 3-dimethylbenzene, solvent.
Xylene, Toxic by all routes, highly flammable.
Methyl benzoate, (C6H5COOCH3), solution almost colourless, fragrant liquid, Toxic if ingested, use < 50 mL or g per activity.
They are used during staining procedures.
These chemicals are highly flammable, toxic and readily absorbed through the skin, so care must be exercised when handling.
These are not miscible with water.

3.31 Prepare nuclear fast red solution
Purchase: Nuclear Fast Red solution, Kernechtrot Solution, (C14H8NNaO7S).
Nuclear Fast Red (4-Amino-9,10-dihydro-1,3-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt), Calcium Red.
Nuclear fast red stain is used as a red nuclear counter stain.
Prepare Nuclear fast red stain:
Nuclear fast red 0.1% in 5% aluminium sulfate.

3.32 Prepare oil red solution
Purchase: Oil Red O solution, 0.5% in isopropanol, Solvent Red 27, Sudan Red 5B, (C26H24N4O).
Oil Red O stain for microscopy, {1-([4-(Xylylazo)xylyl]azo)-2-naphthol}.
Oil Red O stain is used to stain fat in tissue.
Prepare Oil Red stain:
Supersaturated solution of Oil Red O in isopropanol.

4.1 Prepare calcofluor white solution
Calcofluor White with 10% KOH.
Use for the direct microscopic examination of skin scrapings, hairs, nails, and other clinic specimens for fungal elements.
This as a very sensitive method.
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.

4.2 Prepare cellotape flags
This is an excellent technique for the rapid mounting of sporulating fungi, because more of the reproductive structures remain intact.
1. Using clear 2 cm wide cellotape and a wooden applicator stick (orange stick), make a small cellotape flag (2 × 2 cm).
2. Using sterile technique, gently press the sticky side of the flag on 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 on a small drop of Lactophenol cotton blue on a clean glass slide, remove the applicator stick and discard.
5. Add another drop of stain, cover with a coverslip, gently press and mop up any excess stain.

4.6 Prepare direct microscopic mounts or squash preparations
Using sterile technique, remove a small portion of the colony with an inoculation needle.
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 solution.

4.7 Prepare India ink mounts
1. Indian ink contains lampblack, a type of carbon black, the black pigment made from soot.
Nigrosin, Acid black 2, Nigrosin water soluble, may be substituted for India ink for histological work.
For the direct microscopic examination of cerebrospinal solution, 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 are "Pelikan" or "Talons" Indian ink.
2. Prepare India ink background stain
Clean two microscope slides by wiping them with a lint free towel or a tissue soaked in ethanol.
Place a small drop of water on the microscope slide so that the drop spreads out. Use a glass rod to mix a drop of India ink with the evenly spread drop of water.
Place a coverslip at a 45^oangle on the microscope slide.
Push the coverslip evenly across the surface of the microscope slide, so that as the suspension is spread the thickness of the film of liquid decreases.
Leave the smear to air dry.
Prepare a second smear, using a drop of bacterial or yeast suspension, instead of a drop of water.
Compare both smears are then compared under a microscope set at 400 X magnification.

4.8 Prepare lactophenol solution, (LPCB)
Lactophenol, microscopy stain, Cotton Blue (LPCB).
1. Purchase: Lactophenol blue solution, for microscopy, for staining moulds.
Lactophenol Anilin Blue solution, Lactophenol Cotton Blue solution.
Prepare lactophenol stain:
Dissolve 25 g phenol in 50 mL water.
Add 25 mL lactic acid.
Add 50 mL glycerine.
Store away from light.
BE CAREFUL! Use gloves!.
2. Purchase: Cotton blue, Methyl Blue, Acid blue 93, Aniline blue water soluble, Poirriers blue, Water blue, Cotton blue, aniline blue,
Helvetia blue, acid blue 93, C.I. 42780, (C37H27N3Na2O9S3), histology and fungus stain.
Prepare lactophenol cotton blue stain:
100 mL lactophenol.
l g cotton blue.
Dilute 5 mL to 100 mL with lactophenol before use.
3. Prepare Lactophenol Cotton Blue (LPCB).
Use for the staining and microscopic identification of fungi.
1. Cotton Blue (Aniline Blue), 0.05 g.
2. Phenol Crystals ((C6H5O4), 20 g.
3. Glycerol 40 mL.
4. Lactic acid (CH3CHOHCOOH), 20 mL.
5. 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.

4.9 Prepare Loeffler serum solution
Loeffler serum solution is used for identification of Corynebacterium species, e.g. Corynebacterium diphtheriae, diphtheria.
It is also used to identify proteolytic activity, and enzymatic hydrolysis of proteins, as a grey-white background to show microorganism pigmentation.
It contains gelatin peptone, beef extract, sodium chloride, dextrose and bovine serum.

4.10 Prepare Kahle's insect-fixing solution
Formula: 95% alcohol 100 mL, Glacial acetic acid 7 mL, Formalin 40 mL.

4.11 Prepare Orcinol-Bial's reagent
Purchase: Orcinol, 97%, 5-Methylresorcinol, CH3(C6H3-1,3-(OH)2, (3,5-dihydroxytoluene).
Prepare Orcinol-Bial's reagent:
Dissolve 0.2g orcinol in 100 mL concentrated hydrochloric acid.
(Be careful! Corrosive!).

4.12 Prepare potassium hydroxide with chlorazol black solution
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 solution.

4.13 Prepare potato dextrose agar solution
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.

4.14 Prepare rice grain slopes
Use to induce sporulation and differentiation, use polished rice grains and deionized water.
1. Place 1 / 2 teaspoon rice grains into wide neck 20 mL glass 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.

4.15 Prepare Barber's insect-fixing solution
Barber's relaxing solution.
Formula: Alcohol, 95%, 50 mL, Water 50 mL, Ethyl acetate 20 mL, Benzol 10 mL.

5.2 Prepare KAA insect-fixing solution
For soft body insects, use 5 mL of KAA solution.
Prepare KAA solution.
Formula 1. Kerosene 10 mL, 95% alcohol 100 mL, Glacial acetic acid 20 mL.
Formula 2. Kerosene 8 mL, 95% alcohol 77 mL, Glacial acetic acid 15 mL.

5.4 Prepare lacto-alcohol, insect-fixing solution
Formula: Lactic acid 40 mL, 98% alcohol 37 mL, Water 23 mL.

5.5 Prepare Oudeman's insect-fixing solution
Formula: 70% alcohol 88 mL, Glycerine 4 mL, Glacial acetic acid 8 mL.

5.6 Prepare Pampl's insect-fixing solution
Formula: Glacial acetic acid 4 mL, Water 30 mL, 40% formaldehyde solution 6 mL, 95% alcohol 15 mL.

5.7 Prepare sugaring mixture insect-fixing solution
Formula: 500 g treacle, 1 kg brown sugar, 300 mL beer, 5 mL rum.
Boil until uniform thickness occurs.

6.1 Prepare Canada balsam mounting solution
Purchase: Canada balsam, Mounting solution for microscopy, Balsam Canada.
Balsams are hard varnishes and fragrant perfumes.
They have oleoresins containing volatile essential oils and nonvolatile resins.
Canada balsam, Canada turpentine oil, balsam of fir, American silver fir, balm of gilead fir.
It is a natural turpentine used in microscopy, because the refractive index is similar to the refractive index of glass,(n = 1.55).
It comes from the thin resin in bark of the balsam fir tree, Abies balsamea
It is used as a stable apolar hydrophobic mounting solution for light microscopy slide preparation, especially for long term storage.
It was used to conserve microscope samples, but nowadays synthetic resins are used.
It contains monoterpenes: | Pinene | Phellandrene | (microscopy solution).

6.2 Prepare DPX mountant
Purchase: DPX Mountant for histology.
DPX is a neutral synthetic mountant for histology.
It is a mixture of distyrene, plasticizer, and xylene, this synthetic resin mounting solution can replace xylene-balsam.
DPX dries quickly and preserves stain, suitable for haematoxylin eosin staining.
3.10.0 Poisons and First Aid (Table)- DPX mounting solution.

6.3 Prepare glycerine jelly
Glycerine jelly, adhesive to stick sections to microscope slides.
Glycerol, gelatin aqueous slide mounting solution, for histological use, must be warmed before use then returns to semi-solid state.
Prepare glycerine jelly:
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.

6.4 Prepare Haupt's adhesive solution
Adhesive to stick sections to microscope slides.
Purchase Haupt's Adhesive Concentrate.
Prepare Haupt's adhesive: Dissolve 1 g gelatine in 100 mL water at 30^oC.Add 2 g phenol crystals and 15 mL glycerine.
Stir, cool and filter.

6.5 Prepare Meyer's albumen solution
Meyer's albumen, adhesive to stick sections to microscope slides.
Purchase Albumin from bovine serum, lyophilized powder, 96% (agarose gel electrophoresis), culture-tested powder.
Prepare Meyer's albumen:
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 glycerine to liquid collected from bottom.
Add a thymol crystal to prevent growth of fungus.

6.6 Prepare Peru balsam mounting medium
Purchase: Peru balsam oil, from Myroxylon pereirae, El Salvador.
Peru balsam for microscopy, formerly from Myroxylon balsamum, Fabaceae, is used as a mounting solution for microscope specimens.

7.1 Prepare acetic alcohol solution
Purchase: Acetic acid ACS reagent, 99.7%, CH3CO2H, (CH3COOH).
Purchase: Ethanol solution, 70% in water.
Ethanolum 70 %.
Ethyl alcohol.
Prepare this solution prepared immediately before use.
Prepare acetic alcohol solution:
Mix 99 mL 70% ethanol with 99 mL concentrated ethanoic acid.

7.2 Prepare aceto-carmine stain
Purchase: Schneider's aceto-carmine, Carmine solution.
Prepare acetocarmine stain:
Glacial acetic acid 45 mL, concentrated 0.5 g carmine, 55 mL deionized water.

7.3 Prepare aceto-orcein stain
Purchase: Orcein, synthetic, Natural Red 28, orceine, ethanoic acetic, la cour, orcein acetic, Gurr's synthetic orcein, powder.
Orcein is a mixture of phenoxazone derivatives, (C28H24N2O7).
Aceto-orcein stain, acetic orcein, ethanoic orcein, dye, former food colouring now banned.
It stains chromosomes and nuclei crimson, cytoplasm pink, Harmful if ingested.
Prepare aceto-orcein stain:
1. Add l g synthetic orcein to 25 mL concentrated 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 freshly prepared solution.

7.4 Prepare alcian blue solution
Purchase: Alcian Blue 8GX, powder, Alcian Blue, Ingrain Blue, (C56H68(Cl4CuN16S4).
Purchase: Alcian Blue solution, 1% in 3% acetic acid, pH 2.5.
Alcian Blue 8GX is used as a heteroglycan stain for neutral, sulfated, and phosphated mucopolysaccharides and glycosaminoglycans in tissues.
Tissues such as cartilage, extra cellular matrices.
Prepare Alcian Blue solution:
1% in 3% acetic acid, pH 2.5.

7.5 Prepare alizarin solution
Purchase: Alizarin, Dye content 97 %, (1,2-Dihydroxyanthraquinone), Mordant Red 11.
Alizarin, (C14H8O4), (1,2-dihydroxyanthroquinone), Toxic, (aluminium ion indicator).
Alizarin crimson, Mordant Red, Turkey Red, Rose madder, Alizarin red powder (C.I. 58000) glucoside in Rubia tinctoria, Rubiaceae.
Sodium alizarin sulfonate is used in assessing sweat function, because it changes to purple discoloration on contact with sweat.
Prepare alizarin solution, in a fume cupboard:
Alizarin 2.4 g (0.1 mM).
Sodium carbonate anhydrous 2.4 g (0.2 mM).
Wheat starch BP (pharmaceutical grade), 95.2 g.
Alizarin is anthraquinone derived and therefore carcinogenic.
These chemicals are irritating to the eyes, respiratory system and skin.
They definitely should not be inhaled or placed near the eyes.

7.6 Prepare alizarin red S solution
Purchase: Alizarin Red S, Alizarin Carmine, water soluble, Alizarin sodium monosulfonate.
Alizarin sulfonate sodium, Alizarin sulfonic acid sodium salt, Sodium alizarin sulfonate, (C14H7NaO7S).
Alizarin Red S is an anthraquinone dye used to stain for calcium deposits, indicators of mature osteocytes.
A 2% Alizarin Red S Stain forms a complex with calcium during the process of chelation, causing birefringence.
Indicator pH colour change: 4.6-6.0, Acid: yellow, Base: red.
Prepare Alizarin red S solution:
0.2 g in 100 mL of 1.5% HCl.
Alizarin red, (C14H10O4), mordant red11, --> rose madder dye.

7.7 Prepare aniline blue solution
Purchase: Aniline Blue solution, 2.5% in 2% acetic acid, (C32H25N3O9S3Na2).
Aniline blue (C.I. 42755), China blue, cotton blue, water blue (alcohol soluble), Highly toxic if ingested.
Aniline blue solution is used to stain collagen fibres blue in tissue sections using the Masson's trichrome protocol for staining multiple components.

7.8 Prepare aniline hydrochloride solution
Purchase: Aniline hydrochloride, 97%, (C6H5NH2).
Aniline hydrochloride, aniline chloride, anilinium chloride.
Aniline hydrochloride is used to stain lignified tissue yellow.
Prepare aniline hydrochloride stain:
Make a saturated solution of aniline hydrochloride in deionized water.
Filter then add a few drops of hydrochloric acid until solution is acid.
Be Careful! Use gloves!.

7.9 Prepare aniline sulfate solution
Purchase: Aniline sulfate, 98%, (C6H9NO4S).
Prepare aniline sulfate stain: Make a saturated solution of aniline sulfatein deionized water.
Filter then add a few drops of sulfuric acid until solution is acid.
BE CAREFUL! Use gloves! Highly toxic if ingested, Aniline sulfate, Solution< 3%, Not hazardous.

7.10 Prepare basic fuchsin solution
Purchase: Basic Fuchsin, Dye content >85 %, Basic Parafuchsin, Basic Red 9, Magenta O.
Parafuchsin hydrochloride, Paramagenta hydrochloride, Pararosaniline chloride, Pararosaniline hydrochloride.
Rosaniline hydrochloride ((C19H17N3). HCl).
Haematology and histology stain.

7.11 Prepare Calberla's pollen stain
5 mL glycerol.
10 mL 95% ethanol.
15 mL distilled water.
2 drops of saturated aqueous solution of basic fuchsin.
Stains pollen to light pink colour.

7.12 Prepare carbol fuchsin solution
Purchase: Carbol Fuchsin, Highly toxic if ingested.
Carbol fuchsin, carbol-fuchsin, carbolfuchsin, stains mycobacteria, (acid-fast bacteria), in Ziehl-Neelsen stain, topical antiseptic.
(Castellani's paint).
Prepare carbol fuchsin:
Dissolve 1 g of fuchsin in 10 mL of ethanol.
Add this solution to 90 mL of 5% aqueous phenol, then filter the solution.

7.13 Prepare carmine stain
Carmine, (C22H20O13), carminic acid, cochinealdye (from Coccus cacti), Natural Red 4, Nacarat, anthraquinone glycoside.
It is dark red powder, deep red colour in water, yellow-violet in acidic solutions.
It is used as a biological stain and biological marker.
Purchase: Carmine powder, an alum lake of carminic acid.
Prepare carmine stain:
Preparation 1.
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.
Preparation 2.
1 g carmine + 2.5 g aluminium potassium sulfate in 500 mL distilled water.
Boil for 20 minutes.
Adjust final volume to 500 mL with distilled water.
Filter and add a crystal of thymol as preservative.
Refrigerate.

7.14 Prepare corn syrup mountant
"Karo" is a brand name of prepared corn syrup.
Karo Light Syrup contains light corn syrup, salt, vanilla.
Karo Dark Syrup contains light corn syrup, salt, molasses.
Previously, Karo Corn Syrup used to be a high fructose corn syrup.
The "Light" in "Karo Light Syrup" refers to the colour type, not low calorie syrup, although a Karo lower calorie corn syrup is also sold.
Prepare Karo syrup mountant:
40 mL clear Karo syrup.
40 mL deionized water.
Add 2 small crystals thymol (or phenol) (about 0.2 g), as a preservative.

7.15 Prepare Delafield's haematoxylin solution
Purchase: Haematoxylin, Natural Black 1, (C16H14O6.xH2O).
Prepare Delafield's haematoxylin solution:
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 glycerine 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.

7.16 Prepare DCIP (sodium) solution
Purchase: DCIP (sodium), 2,6-Dichlorophenolindophenol sodium salt hydrate, redox indicator, Tillman's reagent, (C12H6Cl2NNaO2.xH2O).
DCIP stain is used to show action of enzymes, e.g. succinic dehydrogenase, chloroplasts when exposed to light and to test for vitamin C.
Prepare DCIP stain:
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.

7.17 Prepare Ehrlich's haematoxylin solution
Purchase: Ehrlich's solution (4-(Dimethylamino) benzaldehyde - hydrochloric acid solution).
Ehrlich's haematoxylin contains the oxidant sodium iodate.
Prepare Ehrlich's haematoxylin stain:
Mix:
100 mL water.
100 mL ethanol (absolute alcohol).
100 mL glycerol.
10 mL glacial acetic acid.
2 g haematoxylin.
Then add excess alum, aluminium potassium sulfate, AlK(SO4)2.12H2O, to leave undissolved alum in the bottom.
The solution is ready to use when it has a dark red colour.

7.18 Prepare eosin solution
Purchase: Eosin Y, Dye content ~99 %, Tetrabromofluorescein, Acid Red 87, Bromo acid J. TS, XL, or XX, Bromofluorescein.
Bronze Bromo ES, Eosin yellowish, Solvent red 43, (C20H8Br4O5).
Counter stains are used to stain cell walls and cell contents.
This is an animal tissue counter stain.
Eosin stains cytoplasm.
Eosin Y solution, aqueous or alcoholic or alcoholic, with phloxine is a general purpose cytoplasmic counter stain, used with haematoxylin and eosin staining.
Prepare eosin, microscopy stain
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 mL 70% ethanol.
3. Demonstrate fluorescence with 1:500 ethanol solution in UV light.

7.19 Prepare Giemsa stain
Purchase: Giemsa Stain, Modified Solution according to Giemsa, Azure eosin methylene blue, Giemsa solution.
Purchase Giemsa stain powder of Giemsa stain (solution), contains 50% MeOH.
Giemsa stain is a mixture of methylene blue, eosin, and azure.
It is used to stain red blood cell stain and for diagnosis of malaria parasites.
Prepare Giemsa blood stain:
Dissolve 3.8 g of Giemsa powder in 250 mL of methanol.
Add 250 mL of glycerine.
Leave to stand for two months before using.

7.20 Prepare Gram stain
Purchase: Gram Staining Kit for microscopy.
Solutions required for the Gram stain procedure: 3.12
Prepare Gram's iodine solution3.11
Crystal violet solution: 3.23 Safranin
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 that allow the stain-iodine complex to be washed out of the cell by alcohol.
Prepare Gram stain:
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^o angle, where the smear is clearly visible.
Run 95% alcohol, as a decolorizing agent, (Gram stain decolorizing solution), down the smear until no more colour runs out after 2-10 seconds.
Wash with water.
Counter stain 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 bacteria can be classified: "Gram positive" or "Gram negative".

7.21 Prepare crystal violet solution
Purchase: Crystal violet solution, (C25H30ClN3).
Crystal violet solution 1%, aqueous solution is used in Brown-Hopps method, for Gram-positive and Gram-negative bacterial staining.
Crystal violet (C.I. 42555, C.I. basic violet 3), (C25N3H30ClN3), "gentian violet", "methyl violet 10B", Toxic if ingested.
Crystal violet 10% W / V alcoholic.
Use as 0.02% solution in water.
Prepare crystal violet stain:
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.
OR.
3. For solution A, dissolve 1 g of crystal violet in 20 mL 95% ethanol.
For solution B, add 0.8 g of ammonium oxalate to 80 mL deionized water.
Add solution A to solution B and filter.

7.22 Prepare fluorescence staining of cells and tissues
Secondary antibodies can be conjugated with fluorescent dyes without compromising antibody affinity or specificity.
The dyes are photostable and not pH-dependent.
Their reagent solutions come with 2.5% normal horse serum.
Sections or cells stained with these reagents can be mounted in mounting media to display their immunofluorescence.

7.23 Prepare haematoxylin stain
Haematoxylin, (C16H14O6), Hydroxybrazilin, (Haematein, hematein)white to yellow crystals that redden on exposure to light.
It is source of haematoxylin dye.
Purchase: Haematoxylin Solution
General purpose nuclear stain, progressive type, used with "haematoxylin and eosin" staining.
Haematoxylin is a counterstain with eosin, so many botanical microscope slides are "stained with haematoxylin and eosin".
Eosin: 16.3.5.3
Haematoxylin, haematoxylin, Natural Black 1, hydroxybrasilin, Toxic if ingested.
Stains nuclei in plant and animal cells purple, blue or black.
It occurs in Haematoxylum campechianum and Haematoxylum brasiletto.
Haematoxylon campeachianum, haematoxylum, logwood, source of haematoxylin dye, Fabaceae.
This stain was formerly called "logwood", because it was made from the heartwood of the tree Haematoxylon campechianum.
When Portuguese navigators first discovered what is now Brazil, they thought the only useful thing there was Haematoxylum campechianum, a commonly used dye.
It has a sweet taste and smells of violets.
It is similar to the natural dye "natural red 24" from brazilin, (C16H14O5) and brazilein (C16H12O5), from Caesalpinia echinata.
Experiments
Prepare haematoxylin stain:
Use a stock solution of 10% haematoxylin in 95% alcohol.
The main alum haematoxylin solutions are Ehrlich's haematoxylin, Harris's haematoxylin, and Mayer's haematoxylin.
7.15 Prepare Delafield's haematoxylin solution
7.17 Prepare Ehrlich's haematoxylin solution
7.24 Prepare Harris modified haematoxylin solution
7.25 Prepare Heidenhain's iron haematoxylin solution
7.28 Prepare Mayer's haematoxylin solution
7.39 Prepare Weigert's iron haematoxylin solution

7.24 Prepare Harris modified haematoxylin solution
Purchase: Haematoxylin Solution, Harris Modified.
Harris modified haematoxylin contains mercuric oxide.
Harris modified haematoxylin is a general purpose nuclear stain, regressive type, used with common "haematoxylin and eosin" staining.
Prepare haematoxylin solution, Harris modified, stain.
Heat to dissolve.
Add 50 mL of 10% alcoholic haematoxylin solution and heat to boil for 1 minute.
Remove from heat and slowly add 2.5 g of mercuric oxide (red).
Heat to the solution and until it becomes dark purple colour.
Cool the solution in cold water bath and add 20 mL of glacial acetic acid (concentrated).
Filter before use.
General purpose nuclear stain, regressive type.
Used with haematoxylin and eosin staining (H & E stain).
It is often called the "standard stain" or "routine stain", because most sections are routinely stained with it for a first inspection of the tissue being examined.

7.25 Prepare Heidenhain's iron haematoxylin solution
Heidenhain's iron haematoxylin is used to stain mitotic figures in amoeba, after fixation.
Prepare Heidenhain iron haematoxylin stain:
1. 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 Part 1. is used as a mordant and Part 2. is for staining.
2. Incubate in 2% ammonium ferric sulfate (NH4Fe(SO4)2.12H2O).
Then rinse in distilled water and then tap water.
Then incubate in 0.5% haematoxylin in 2% ammonium ferric sulfate.
Then wash in tap water and mount on microscope slide.

7.26 Prepare immersion oil
Purchase: Immersion oil for microscopy, refractive index n20 / D 1.516, viscosity 100-120 mPa.s. (20^oC),
density 1.025 g / mL at 20^oC.
Immersion oil, high refractive index, for finer resolution and brightness, (mercury e line (5461 A) specification).
Immersion oil for microscopy, is used for high resolution (1000 X) light microscopy work under an oil immersion objective lens.
By placing a drop of immersion oil with the same refractive index as glass between the cover slip and objective lens two refractive surfaces are eliminated.
So that greater magnifications can be achieved while still preserving good resolution.

7.28 Prepare Mayer's haematoxylin solution
Purchase: Haematoxylin Solution, Mayer's.
Mayer's haematoxylin contains the oxidant sodium iodate.
Mayer's haematoxylin solution is a general purpose nuclear stain, progressive type, used with common "haematoxylin and eosin" staining.
Prepare Mayor's haematoxylin stain:
1. Dissolve 50 g aluminium potassium sulfate (alum), in 1000 mL distilled water.
When alum is completely dissolved, add 1 gm haematoxylin.
When haematoxylin is completely dissolved, add 0.2 gm sodium iodate and 20 mL acetic acid.
Bring solution to boil and cool, and filter.
2. Dissolve alum in distilled water.
When alum is completely dissolved, add haematoxylin.
When haematoxylin is completely dissolved, add sodium iodate and acetic acid.
Bring to boil and cool.
Filter if necessary.

7.29 Prepare Leishman's stain, Wright's stain
Purchase: Leishman's stain, used as histology stain, Eosin-polychrome methylene blue.
It is a Romanowsky-type dye, used to identify leukocytes, malarial parasites, and trypanosomes.
Purchase: Wright Stain, Modified, a popular haematology stain used for differentially staining the cellular elements of blood.
Wright stain, 0.3%, buffered at pH 6.8 in methanol.
Contains stabilizers and surfactant.
Use Leishman's stain, Wright's stain, stain for white blood cells microscopy.
The ready made dark green crystalline powder, Wright's stain, contains the red acid dye eosin and the blue basic dye methylene blue.
Variants of Wright's stain include buffered Wright's stain, Gram's stain, Wright-Giemsa stain, Jenner's stain.
Prepare Leishman's stain.
1. Dissolve 0.15 g of Leishman's stain in 10 mL absolute alcohol in a flask.
Plug the flask with cotton wool and heat in a water bath over an electric heater for 15 minutes.
2. Add 0.115 g Leishman'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.

7.30 Prepare methylene blue solution
Methylene blue, methylthioninium chloride, (C16H18N3SCl), redox indicator, anionic surfactants, used for yeast viability.
Purchase: Methylene Blue hydrate, (C16H18ClN3S.xH2O).
Prepare methylene blue stain: Methylene blue (0.1%), dilute 0.1 g methylene blue in 100 mL deionized water, dilute 1 in 10.

7.33 Prepare orange IV solution
Purchase: Orange IV, [ 4-(p-Anilinophenylazo)benzenesulfonic acid sodium salt], Acid Orange 5, Tropaeolin OO.
Orange IV, orange G, tropaeolin OO (acid-base indicator, 1.3): 1.3
Prepare orange IV botanical tissues counter stain:
Dissolve 0.5 g Orange IV powder in 100 mL 95% alcohol.
0.1% solution as acid-base indicator, pH < 1.4 red to pH > 2.6 orange.

7.34 Prepare Papanicolaou stain
Purchase: Papanicolaou Stain, OG-6, Papanicolaou Stain, EA 50.
Papanicolaou stain, imparts a characteristic range of coloration to exfoliative cells of vaginal, cervical, prostatic and other body secretions
It allows critical examination of nuclei and cytoplasmic components.
Papanicolaou stain is used for routine diagnostic cytology to aid in the identification and classification of exfoliative cells.

7.35 Prepare phloroglucinol solution
Purchase: Phloroglucinol, 99.0%, (1,3,5-Trihydroxybenzene ), (C6H6O3).
Acid phloroglucin is used to stain lignin in plant cells bright red.
Prepare phloroglucinol solution: Dissolve 1 g phloroglucin in 100 mL 50% alcohol.

7.36 Prepare safranin solution
Purchase: Safranin O, Dye content 85 %, Basic Red 2, Cotton Red, Gossypimine, Safranin T, Safranin Y or A, (C20H19ClN4), C.I. 50240, C.I.
Basic Red 2, Toxic if ingested, a stain for lignin and cell walls.
Prepare safranin 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 0.02g safranin in 10 mL 5% ethanol.
Add 100 mL water to form 1% solution.

7.37 Prepare Schulze's solution
Schulze's solution (chlor-zinc-iodine).
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.

7.38 Prepare toluidine blue solution
Purchase: Toluidine Blue O, Basic Blue 17, Methylene Blue T50 or T extra, Tolonium chloride, (C15H16ClN3S), Toluidine blue, C.I. 52040,
(plant cells microscopy stain), Toxic if ingested.
The stain works best in fresh plant material.
Lignified plant cell walls stain blue-green.
Unlignified plant cell walls stain pink-purple.
Prepare toluidine blue stain:
Dissolve 0.05 g toluidine blue stain in 100 mL of water.

7.39 Prepare Weigert's iron haematoxylin solution
Purchase: Weigert's iron haematoxylin solution.
Weigert's haematoxylin solution is used for animal tissue for acidic stain procedures.
It resists being discolored better than aluminium-based haematoxylin formulations.
Prepare Weigert's iron haematoxylin stain.
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.

7.40 Prepare Ziehl-Neelsen stain
Ziehl-Neelsen stain (acid-fast stain).
Purchase: Carbol-Fuchsin solution according to Ziehl-Neelsen, for microscopy.
Bacteria stain bright red due to retention of carbol-fuchsin dye.
Background is methylene blue counter stain.
It stains bright red mainly Mycobacteria, e.g. Mycobacterium tuberculosis that causes tuberculosis (TB).
It contains carbol-fuchsin (aqueous solution of phenol + alcoholic solution of fuchsin), acid alcohol (ethanol and hydrochloric acid), and methylene blue.
The acid-fast mycobacteria are usually straight rod-shaped bacilli, 1to 10 cm long by 0.2 to 0.6 cm wide.
The high lipid content of their cell wall makes the bacteria resistant to penetration by many dyes and chemicals, including Gram stain.
The procedure is to stain all the bacteria on the slide with hot carbol-fuchsin, then wash the slide with acid alcohol.
The remaining bacteria that are still red are the "acid-fast bacteria".
Then counter stain with methylene blue dye (Swiss blue), so that the acid-fast bacteria remain red, and any other bacteria are stained blue.