Appendix 3. Media and solutions

1. Buffer solution

Phosphate buffered saline (PBS)

Recipe to prepare five litres of PBS

Reagents

Method

1. Weigh out the reagents and place in a 5 L conical flask.
2. Add distilled water to make 5 L. Mix well.
3. Check pH. Adjust to pH 7.3 to 7.4.
4. Pour into storage bottles.
5. Autoclave at 121°C for 15 minutes. Use a slow exhaust.
6. Allow to cool, then tighten the lids and label the bottles.

Ten times (10 ×) stock solution

If you require large volumes of PBS in your laboratory, you may find it useful to prepare a stock solution that is ten times the strength of the working solution used in the laboratory procedures.

This is called a ten times stock solution, usually labeled by the abbreviation “10 × stock solution”. Before use, prepare the “working solution” by making a one in ten dilution of the stock solution.

To prepare 1 L of working solution, mix 100 mL of the stock solution with 900 mL of distilled water.

Three recipes for the stock solution are provided for total volumes of 1 L, 2 L and 5 L. Choose the recipe for the volume best suited for your laboratory's requirements.

Remember 5 L of the stock solution is enough to prepare 50 L of the working solution. This may be far more than required for the next few months. 2 L of the stock solution may be an adequate supply of the stock solution.

Reagents for varying volumes of ten times stock solution of PBS

Method

1. Weigh out reagents for the selected total volume of PBS.

2. Mix with distilled water to the required volume. Adjust pH to 7.3.

3. Dispense into sterile glass bottles, sterilize by autoclaving at 121°C for 15 minutes. Use a slow exhaust.

4. When cool, tighten lids and label.

2. Storage solution for washed red blood cells

Dextrose gelatin veronal (DGV)

Reagents

Method

1. Add the barbitone and gelatin to 250 mL of distilled water. Heat to dissolve.
2. Add remainder of the ingredients to 600 mL of distilled water and dissolve.
3. Mix these two solutions in a conical flask.
4. Make up to a total volume of 1 L with distilled water.
5. Pour into 100 mL bottles for storage.
6. Autoclave or pressure cook at 116°C for 10 minutes. Use a slow exhaust.
7. Allow to cool, then tighten the lids and label the bottles.
8. Store in the refrigerator.

3. Anticoagulants

Acid Citrate Dextrose (ACD)

Reagents

Method

1. Weigh out reagents into a conical flask.
2. Dissolve in 300 mL of distilled water.
3. Make up to 500 mL with distilled water.
4. Dispense into 100 mL bottles and put on lids. Do not tighten.
5. Sterilize by autoclaving at 116°C for 10 minutes. Use a slow exhaust
6. Allow to cool, then tighten the lids and label the bottles.
7. Store in the refrigerator.

Alsever's Solution

Reagents

Method

1. Weigh out reagents into a conical flask.
2. Dissolve of distilled water and make up to100 mL.
3. Dispense into sterile 10 mL bottles.
4. Sterilize by autoclaving at 116°C for 10 minutes. Use slow exhaust.
5. Allow to cool, then tighten the lids and label the bottles.
6. Store in the refrigerator.

4. Antibiotic Solution

The recipe given is for a solution of Penicillin, Streptomycin and Gentamycin dissolved in PBS. In this manual, this solution is given the abbreviation PSG.

Benzyl penicillin (Penicillin G) is a broad range antibiotic active against Gram positive and Gram negative aerobic cocci and most spirochaetes. It comes in various forms, which vary in solubility. Potency may be expressed in International Units (IU) rather than mg.

Streptomycin is an antibiotic affective against Gram negative bacteria.

Gentamycin is a broad spectrum antibiotic mainly affecting Gram negative aerobes. It is stable at higher temperatures. If Gentamycin is difficult to obtain it can be omitted from the solution.

Reagents

Method

1. Dissolve reagents in approximately 800 mL of PBS.

2. Make up to 1 L with PBS.

3. Cold sterilize by passing solution through a 0.2 micron filter. Dispense into 100 mL sterile glass bottles, lid and label.

DO NOT AUTOCLAVE!

If a system for cold sterilization under vacuum does not exist in your laboratory, make sure sterile PBS and aseptic technique are used to minimize the risk of contamination.

5. Bacterial culture media

Tryptic Soy Broth (TSB)

This is a general purpose broth medium prepared for the cultivation of fastidious and non fastidious organisms.

Materials for preparing 1 L of TSB

• 30 g dehydrated TSB medium
• 1 L distilled or deionized water
• Sterile 20 mL glass bottles with lids.

Method

1. Dissolve media in 1 L of water.
2. Warm slightly to dissolve completely.
3. Dispense 9 mL aliquots into the glass bottles.
4. Sterilize in the autoclave at 121°C for 15 minutes.

Sabouraud dextrose agar

Sabouraud dextrose agar is recommended for cultivating fungi. It is readily available and is supplied by Oxoid and Difco with instructions for its preparation. Choramphenicol is a bacterial inhibitor that can be added.

The following recipe has been supplied by the Diagnostic Bacteriology Laboratory at the School of Veterinary Science, University of Queensland.

Materials

Method

1. Suspend the agar in the distilled water. Heat to boiling point.
2. Add the chloramphenicol mixture to the agar. Stir thoroughly.
3. Dispense into 100 mL bottles.
4. Sterilize by autoclaving at 115°C for 10 minutes. Allow to cool to 50°C.
5. Pour into sterile Petri dishes. Allow approximately 25 mL for a 90 mm Petri dish.
6. Incubate plates at 37°C overnight and check for growth of bacterial contaminants.
7. Store at 4°C.