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Part III
Laboratory Facilities, Equipment and Procedures
17.17 Introduction
Laboratory facilities at premises provide an important backup to the diagnosis by, and judgement of, veterinarians at export premises.
Meat Inspectors should also be familiar with the equipment provided and its use.
17.17.1 Facilities
The facilities provided in each laboratory are:
(a) Wash hand basin.
(b) Space for incubator, microscope, refrigerator and general laboratory hardware.
(c) Power points for incubator, microscope and heating.
(d) Work bench with cupboards, including a lockable cupboard.
(e) Space for a trichinoscope if applicable.
(f) Provision for bunsen burner if gas available.
(g) Adequate lighting and heating.
17.17.2 Equipment
The Meat Division provides the following:
Aluminium saucepan
Microscope
Glass slides
Incubator
Lovibond comparators
Trichinoscope if applicable
Koch thermometers
Intrascope if applicable
Clinical thermometers
Calibration thermometer
pH paper
Immersion oil
Pasteur pipettes
Petri dishes
Scissors
Screwcap bottles
Spirit or bunsen burner
Surgical blades/handle
Laboratory mercury thermometers
Tel Tru thermometers (metal stem)
Refrigerator
Bacteriological stains (methylene blue, carbol fuchsin, crystal violet, grams iodine, acetic acid, acid alcohol, absolute alcohol)
KOH (fungal examination)
Water testing equipment - millipore
Hand drill/bit
Hand driven centrifuge
Forceps
Hot plate
Species testing kit
Equipment for approved special projects
17.18 Thermometers
17.18.1 'Koch' Electronic Thermometer
For temperature measurement of fresh, chilled and frozen product.
(a) Do not attempt to drive the probe directly into frozen product. Use a drill bit size .75 to 1.5 mm larger than the probe diameter. Drill bits are obtainable by local requisition and are used with the hand-drill already issued. Refer 9.6.3(b) (iii) .
(b) The probe will need to be free of moisture otherwise it will freeze to the product.
(c) If any instrument malfunctions, try placing it in the incubator at 25°C overnight to remove internal condensation, or check that the batteries are charged, otherwise forward it for repair through the Regional Office to the agents.
(d) The thermometer has a digital readout, range -35°C to 150°C (switchable to Fahrenheit). A 12 cm probe is electrically connected to the meter via a mini phone plug. The power source is nickel-cadmium rechartable batteries, permanently sealed in and rechartable with the charger supplied. These batteries have a very long service life if treated correctly. Try to cycle the batteries, i.e. from complete charge to near discharge. Keeping at constant full charge will cause excessive heat to be produced when charging and may 'cook' the batteries. Conversely, long periods of low charge will shorten battery life.
(e) The thermometer will perform more consistently in cold stores if carried in an inside pocket.
17.18.2 Calibration Thermometer
A glass calibration thermometer of guaranteed accuracy has been provided. All other thermometers are to be checked regularly against this calibration thermometer.
(a) Frequency
Calibrated thermometers with known errors (as shown by a calibration chart or graph) may be routinely (weekly) checked using a single temperature. Re-calibration over several temperatures should be done bi-monthly.
(b) Method
(i) Place the sensor of the thermometer being tested alongside the calibration thermometer in the same ambient environment and record the readings.
(ii) Ice water bath (0°C). Fill a container with finely chipped ice, add water and stir for at least 2 minutes. The sensor and calibration thermometer are immersed, taking care not to touch the container side or bottom, and the temperatures recorded after stabilising for 3 minutes. The 0°C point of the scale of a glass stemmed thermometer should emerge just above the top of the container. All other sensors should be completely immersed.
(iii) Brine mixture (-18 to -21°C) consisting of one part by weight of table salt and three parts by weight of chipped ice.
(iv) Boiling water (100°C).
(v) Adjustable thermometers may be re-aligned but others e g 'koch' will need the appropriate error added or subtracted from the displayed temperature.
(c) Calibration Graph
e.g. Thermometer No.1 recording 15°C
True temperature = 9°C
Thermometer No.2 recording 15°C
True temperature = 20°C
Thermometer No.2 recording 10°C
17.19 Trichinoscope
(a) Application
The trichinoscope is used to detect Trichinella spiralis in samples of the diaphragmatic pillars from manufacturing grade pigs with a head-on dressed weight of 90 kg or more.
(b) Description and Operation
The trichinoscope is a low power stage microscope with image projecting capacity. It has a large field of view to facilitate easy search of the sample. For use, refer to the operating manual.
(c) Preparation and Examination of Samples
(i) A 2 cm diameter (approx 30 g) of muscle is taken from the diaphragmatic pillars. This is cut into pieces about 3 x 2 mm. Fourteen pieces are placed in the compressor and squashed.
(ii) Lamp voltage adjustment for 40x and 80x magnification is performed automatically by a mercury switch when changing magnification. Always make sure the switch has been activated by moving the magnification changer as far as possible.
Have the potentiometer (regulatory knob for lamp intensity) adjusted so that voltmeter does not show more than 12 V (up to the red line) when using high magnification. At low magnification, the lamp voltage should be 10 V, thus keeping light intensity on the screen the same at both magnifications. Lamp life will also be prolonged.
If there is an electrical fault, call an electrician - do not attempt repair yourself.
(d) Maintenance of Optics
(i) Maintenance of lens. Clean all lenses with alcohol. Use lens cleaning tissue or soft cloth dampened with alcohol, then wipe with dry, clean cloth.
(ii) Maintenance of Surface Reproducing Mirror. Remove dust with fine hair brush, never with handkerchief or hard rag. Remove any dirt remaining with alcohol on a soft cloth. Avoid finger contact which may damage the surface.
17.20 Microscope
A good research quality microscope will resolve objects of .25 micron without causing coloured edges. (The average bacillus is about 1 micron long).
(a) Before use, ensure that:
(i) The objectives are clean and free from immersion oil;
(ii) The eye pieces are free from dust.
(b) To set up the microscope;
(i) Switch the lamp on and close the lamp iris diaphragm leaving only a small opening in the middle.
(ii) Turn the condenser adjustment screen to bring the edge of the iris into sharp focus, then open the iris to admit the desired amount of light.
(iii) If the condenser has cantering screws, centre by viewing the condensor iris down the microscope barrel with an eye piece removed - the condenser iris opening can also be adjusted when viewing down the barrel; close the iris until approx 20% of the field is occluded. (Note that not all microscopes have a condenser iris.)
(c) Locating and Viewing the Subject
(i) Place slide on the stage and locate the subject using the low power objective.
(ii) Adjust the focus until optimum.
(iii) Medium power objective
Rotate the nose piece until the medium power objective is in position. If the low power objective is correctly focused, the medium power should only require a final focussing with the fine adjustment.
(iv) High power objective
The working distance between the high power objective and the slide is very small.
The safest method of using this objective is as follows:
Locate the subject under low power (if necessary, medium power), using the coarse and fine adjustments. Rotate the high power objective into position and place a drop of immersion oil on the slide over the area to be studied. Focus using fine adjustment.
It is very easy to crank a high power objective down onto a slide and crack the coverslip, great care must be taken to avoid this. Some objectives are spring loaded to avoid such damage but it is best to avoid contact between objective and coverslip altogether. Each time the object's power is charged, the iris on the condenser and most often the light source has to be reset.
(d) Servicing and Repairs
To operate properly, microscopes should be serviced annually.
Ensure the microscope and all components are placed correctly in its carrying case, properly labelled with 'Supervising Veterinarian' and establishment name and address. Also attach a label indicating that the contents are fragile.
17.21 Incubator
Temperature range - ambient to 100°C, fluctuating less than ±0.3°C at 37°C.
Temperature control by thermostat, operating four heaters which perform at approximately 20% above interior air temperature to avoid excess temperature fluctuation and overshoot.
Provision is made for the insertion of a laboratory thermometer through a stainless steel port in the top of the incubator.
17.22 Lovibond 1000 Comparator or Equivalent
(a) For measuring total residual, free or combined chlorine in water.
The following are supplied:
(i) Lovibond 1000 comparator or equivalent
(ii) 3/40A disc range < 1.0 ppm.
(iii) 3/40B disc range 0.2-4.0 ppm.
(iv) 3 square 135 mm glass tubes.
(v) No.1 DPD test tablets.
(vi) No.3 DPD test tablets.
(b) Instructions for Use
Instructions are supplied but are not complete in all aspects particularly on how to place the disc in position.
(i) Open the face door as per instructions.
(ii) Remove the rubber screen holder from the right hand hole with flange.
(iii) Put the disc on the flange with the figures of the disc facing the operator.
(iv) Lift the spring on the left hand side of the disc off the spring stop to ensure that the "V" in the spring makes contact with the "V" in the disc.
(v) Close the door of the comparator.
(c) Frequency of Use
Chlorine testing is done daily. Refer Manual 3.5.8(c) .
17.23 Millipore Water Testing for Coliform Organisims
17.23.1 Introduction and Description
The SV is responsible for regular monitoring of potable water supplies within export premises under his supervision.
For definitions of potable water and Meat Division control measures
refer Meat 3.5.2
Fish 13.23.1
Game 14.23.1
The Millipore system is designed to detect coliform organisms which include the genera Escherichia, Aerobacter, (Klebsiella, Enterobacter) and Paracolobactrum. Positive results are sent to the nearest Animal Health Laboratory for confirmation.
(a) Brief Description
The test entails drawing a water sample through a fine (0.45um) filter, to trap any bacteria present. A selective medium is added which suppresses the growth of non-coliform organisms. The coliform colonies exhibit a characteristic green sheen.
(b) Equipment
(i) Millipore water testing kit.
(ii) sterile bottle for sample collection.
(iii) an incubator stabilised at 35°C.
(c) The Millipore kit consists of:
(i) A two-piece plastic monitor unit
This has a blue plug in the inlet hole, a red plug in the outlet hole and contains a plain white pad supporting a gridded filter pad.
(ii) A plastic syringe.
(iii) Stainless steel forceps
Note: these forceps should be used only in the manner described in this manual.
(iv) MF-Endo Ampoules - 0.8 ml sterile growth medium.
(v) Plastic/Rubber tubing with nylon adaptor.
17.23.2 Preparation of Sample
(a) Monitor Unit
The Monitor units are provided pre-sterilised, with support pad, filter pad and plugs in place, ready for use.
(b) Preparation of Sampling Bottle
(i) Use the plastic bottle provided. Mark the 100 ml level on the outside of the bottle.
(ii) Sterilise the bottles before leaving the laboratory. Sterilisation sufficient to kill coliform organisms can be achieved by immersing the bottle and lid (lid off bottle) in boiling water for 2 to 3 minutes. Remove the bottle and lid from the water, making sure they are handled by their exterior surfaces only i.e. not the neck of the bottle or the inside of the lid.
(iii) Immediately, shake out as much water as possible and replace lid loosely.
(iv) Tighten the lid when the bottle has cooled.
Note: For chlorinated water supplies, the residual chlorine in the water sample must be neutralised. To do this, add a few drops of 10% sodium thiosulphate solution to the sampling bottle.
Preparation of 10% sodium thiosulphate: Make small quantities at a time as the solution loses effectiveness with time. If the solution is kept in a dark place it should last for up to six months. Add 1 gm sodium thiosulphate to 10 mls distilled water. In a small beaker, heat the solution and boil for 2 to 3 minutes.
Add a few drops of this solution, either pouring directly from the beaker or using a sterile dropper, to the sampling bottle. It should be added to the sampling bottle at step 3 in the procedure above i.e. when the sampling bottle is still hot with the lid on loosely.
(c) Collecting the Water Sample
(i) Identify the water outlets to be sampled.
(ii) Any external fittings such as anti-splash nozzles or rubber tube should be removed.
(iii) The outside and inside of the nozzle should be carefully cleaned, with particular attention to collections of grease inside the nozzle.
(iv) Turn the tap on full and allow the water to run for several minutes to flush the interior of the nozzle and discharge any stagnant water in the pipe.
(v) Turn off the tap and wipe the outer surface dry with a clean cloth.
(vi) If a pair of tongs is available, use these to hold a piece of cotton wool soaked in methylated spirits. Alternatively, use a piece of wire with cotton wool wrapped round one end. Light the soaked cotton wool and flame the nozzle thoroughly.
(vii) Cool the nozzle by running water to waste.
(viii) Remove the lid from the sampling bottle.
(ix) Collect 100 mls water in the sampling bottle. Do not allow the sampling outlet to come into contact with the inside of the bottle.
(x) Replace the lid on the sampling bottle.
(d) Preparation for Filtration
(i) Carefully remove the red plug from the outlet hold of the Monitor unit. Place the plug outside down, on a clean surface for later use.
(ii) Make sure the syringe plunger is fully compressed. Press the tip of the syringe firmly into the outlet hole of the Monitor unit. A twisting action will help to seal the joint.
(iii) Carefully remove the blue plug from the inlet hole of the Monitor unit. Place the plug, outside down, on a clean surface for later use. Handle it carefully to avoid contaminating the Monitor unit when later replaced.
First use of sterile plastic tube and nylon adaptor
(iv) Without touching the sterile sampling tube, burst the nylon adaptor through the plastic envelope.
(v) Insert the nylon adaptor snugly into the inlet side of the Monitor unit while holding the tube by it's envelope. Leave the rest of the tube in it's sterile envelope until you are ready to use it.
Second and subsequent tests with nylon adaptor attached to either plastic or rubber tubing.
(iv) Immediately, before filtering, boil the adaptor and length of tubing for two minutes. Drain off water and shake off excess drips.
(v) To avoid contamination of the sampling tube, handle it by the adaptor end of the tubing. Attach the adaptor to the inlet hole of the Monitor unit.
(e) Filtration of the Sample
(i) Remove the sterile envelope from the sampling tube (if necessary).
(ii) Take the lid off the sampling bottle and immerse the sampling tube in the water sample.
(iii) Hold the Monitor unit steady so the sample will flow evenly through the filter.
(iv) Draw out the syringe plunger very slowly on the initial stroke so that the water fills the Monitor unit completely and begins to flow into the syringe.
(v) When the syringe plunger is fully out, disconnect the syringe carefully from the Monitor unit, and expel the water to waste. It is advisable to leave the tube in the bottle while the syringe is removed.
(vi) Join the syringe to the Monitor unit again and slowly draw out the plunger. Continue this 'pumping' until all the water sample has passed into the Monitor unit.
(vii) Invert the Monitor unit and use the syringe to draw out the last few millilitres of water left in the Monitor unit. Hold the plunger at the 50 ml mark for 10 seconds to make sure all remaining water has been removed.
(viii) The filtration step is now complete. Disconnect the syringe and the sampling tube from the Monitor unit. Do not replace the plugs yet.
(f) Introducing the Medium
(i) Shake the Monitor unit sharply to discharge any bubbles of water remaining in the inlet and outlet holes.
(ii) Place the Monitor unit on the table with the outlet side up.
(iii) Hold the medium ampoule vertically with the plastic sleeve at the bottom.
(iv) Holding the ampoule by the plastic sleeve, lightly flick the top end of the ampoule several times to shake as much medium as possible out of the tip. A few bubbles may remain at the tip.
(v) Break off the ampoule tip at the file mark.
(vi) Place the broken end of the ampoule into the outlet hole. Flick lightly again to discharge the medium from the tip at the plastic sleeve end of the ampoule.
(vii) Crush the plastic sleeve portion of the ampoule between the plastic handles of the forceps. The medium should now flow into the Monitor unit. If the medium ceases to flow, tap the Monitor unit sharply on the table to dislodge any air bubbles.
(viii) Remove the ampoule and replace the plugs - the blue plug in the inlet hole, and the red plug in the outlet hole.
(g) Incubation
(i) Place the Monitor unit in an incubator with the grid-marked side of the filter down i.e. the red plug up.
(ii) Incubate at 35°C ± 0.5°C for 22-24 hours.
17.23.3 Reading the Results
(a) Remove the Monitor unit from the incubator.
(b) Examine the surface of the gridded filter paper. It may help the examination if the blue plug is removed for a short time.
(c) Coliform organisms grow sharply contoured dark red colonies with a green metallic sheen (Fuchsin green). The sheen may be evident in the centre of the colony or around the periphery. Moving the unit in different directions to the light source may help to distinguish the green sheen. MF.Endo medium suppresses the growth of most non-coliform organisms, but a few closely related organisms may produce red colonies without the characteristicgreen sheen.
(d) If a positive result is indicated, immediately send the Monitor unit to your regular Animal Health Laboratory for confirmation. If the unit cannot be send immediately, it should be kept under chill conditions.
Warn the company.
(e) If the test gives negative results (i.e. no green colonies), discard the assembly.
17.23.4 Recording the Results
(a) Fill in the form supplied for every water test or series of water tests.
(b) Interpretation of the form
Most of the form is self-explanatory.
Premises: Name and official number of establishment.
Water supply: e.g. town supply; bore; etc.
Reason for sampling: e.g. routine; cloudy water; flooding; etc.
Chlorine test: ppm reading where chlorination is practised.
Presence or absence of coliforms: where green colonies are found, write present.
(c) Where coliforms are indicated and the Monitor unit is sent to the Animal Health Laboratory for confirmation, attach their report form to the Meat Division form.
If any unsatisfactory result is obtained, refer to the following manuals for action,
Manual 3.5.8
or Manual 13.23.7
or Manual 14.23.7
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