2. Basic laboratory skills

Introduction

Laboratory staff should be familiar with and have practiced the following skills prior to the commencement of Newcastle disease vaccine production. This manual does not contain further details about these skills.

Aseptic technique.

Sterilization by autoclaving and hot air of glassware and discarded materials.

Mixing or stirring by:

1. Hand
2. Magnetic stirrer
3. Vortex mixer

Measuring pH

Using centrifuges

Instructions for using a daybook

The details of all technical procedures carried out to produce and test the I-2 Newcastle disease vaccine must be recorded by the technician in a daybook.

Use a bound book.

Sequentially number all the pages.

Set aside 3 to 4 pages for a “table of contents”.

Set up an “abbreviations page” at the front of the daybook. List all personalized or uncommon abbreviations.

Put the date and title of the experiment at the top of each page.

It is best to use a black pen as this colour photocopies well. Do not use pencil.

Errors should be ruled through and corrected. Do not use an eraser or cover mistakes with white “liquid paper”.

Preparation of reagents

The following reagents will be required during the production and testing of the I-2 Newcastle disease vaccine.

• Phosphate Buffered Saline (PBS)
• An anticoagulant. Acid Citrate Dextrose (ACD) or alternatively Alsever's Solution
• Dextrose Gelatin Veronal (DGV). A storage solution for 10 percent washed red blood cells
• An antibiotic solution
• Tryptic soy broth
• Sabouraud agar

Methods for preparing these media and solutions are included in Appendix 3.

Recording details of practical work

Use the following format for recording the details of your practical work.

Aim: Outline the purpose of the activity.

Materials and Methods: Record enough information that will allow another scientific or technical person to repeat the procedure. If standard procedures are used in a series of experiments, describe them as standard. Examples of such standard procedures are those described in a Laboratory Manual or a previous experiment.

Results: Record all the results and observations. Prepare a table for recording data where appropriate.

Conclusions: Comment on your results and observations. Make suggestions for further experiments.

The care and use of single and multichannel micropipettes

Manufacturer's instructions are supplied with all micropipettes. Please read and make sure you understand how to operate and care for the pipette. Micropipettes use plastic disposable tips. For ease of use, tips are usually packed into plastic boxes that can be autoclaved. Make sure the tips you are using will fit tightly onto the end of the pipette.

Treat micropipettes very gently as they are precision instruments.

Keep upright when in use to prevent liquids running inside the shaft of the pipette.

Do not leave pipettes lying on the workbench where they can be knocked off and damaged.

Do not allow pipettes to come into contact with corrosive chemicals.

Before use, make sure the volume has been correctly set. Adjust the volume before use. Most pipettes have a digital display of volume. Some brands have a micrometer setting, which can be difficult to read.

Check all tips are securely fitted to pipette.

Draw liquid up.

Check that the liquid drawn up has reached the expected level in the tip and there are no air bubbles in the tip. When using multichannel pipettes, check that the volume of liquid is the same in each tip.

If necessary, expel the liquid and manually tighten the tips onto the pipette.

Draw up the liquid and check again.

Figure 1: Single channel micropipette

Instructions for pipetting liquids using a micropipette

1. Micropipettes have 3 positions:

1. Rest position
2. First stop
3. Second stop

2. Fit the tip to the end of the shaft. Press down and twist slightly to ensure an airtight seal.

3. Hold the pipette in a vertical position. Depress the plunger to the first stop. Air equal to the volume of the setting (e.g. 100 mL) is displaced.

4. Immerse the tip into the liquid. Release the plunger back to the rest position. Wait a second for liquid to be sucked up into the tip. The volume of liquid in the tip will equal the volume of the setting of the micropipette.

5. Place the tip at an angle (10° to 45°) against the wall of the vessel receiving the liquid, for example a well of a microwell plate. Depress the plunger to the first stop, wait one second, press the plunger to the second stop to expel all the liquid

6. Move the end of the tip away from the liquid. Release the plunger to the rest position.

Figure 2: Using a multichannel micropipette

Graduated pipettes

Graduated pipettes are calibrated and marked with graduation lines that allow the measurement of more than one volume. The volume is read by eye by reading the value indicated on the scale at the bottom of the meniscus. Disposable plastic graduated pipettes are available and are useful for pipetting toxic or viscous substances. Graduated pipettes made of glass can be washed and reused. The pipettes should be plugged with cotton wool on the top and sterilized before use to minimize contamination of fluids being measured.

1 mL and 10 mL graduated pipettes are most commonly used in the laboratory practices described in this manual. Before using graduated pipettes check the volume scale and note:

Does the pipette empty from full volume to zero or from zero to full volume?

Is the pipette designed to be emptied by gravity with the tip in contact with the vessel or to be expelled by blowing out with a pipette filler?

Pasteur pipettes

Pasteur pipettes are glass pipettes used to transfer fluids from one place to another. They are not graduated and are therefore not used to measure volumes. Like graduated pipettes they should be plugged with cotton wool and sterilized before use.

Pipette fillers

Pipetting by mouth is not an acceptable laboratory practice. Fluids are drawn up into pipettes using pipette fillers. There are several options. A simple rubber bulb is suitable for a 1 mL pipette. For 10 mL pipettes, use a triple valve rubber bulb, a hand operated pump or an electronic pipette filler.

Microwell plates

Plastic microwell plates are now routinely used in laboratories to perform assays using small volumes of samples and reagents. The plates contain 96 wells arranged in an 8 × 12 format with columns labeled 1 to 12 and rows labeled A to H. Thus each well has a designation for example A 12. Plates are manufactured with different shaped wells suitable for different tests. The wells can be round bottom, flat bottom and V-bottom.

In this manual, the haemagglutination and haemagglutination inhibition tests are carried out in V-bottom microwell plates. Two-fold dilutions can be made across the plate in either orientation that is from A1 to A11, (A12 being a control well) or from A1 to G1 (H1 being a control well).

The microwell plates used at the John Francis Virology laboratory are manufactured by Nalgen Nunc International. The plates cannot be autoclaved and are disinfected after use by soaking overnight in a 2 percent chloride solution. They are then washed and rinsed three times in tap water, followed by rinsing three times in distilled water, dried and reused.

Use of microwell plates is referred to in Section 10, Section 11 and Appendix 4.

(For details about the cost and supply of microwell plates visit the Nalgen Nunc website at http://nuncbrand.com and look up the product catalogue number 442587.)