Laboratory methods for detection of pests in food

For most purposes, hand sorting or screening of a fraction of the product may be sufficient. In more special cases it is possible to use techniques that are adapted to each type of material and the animals you would expect to find in them.

Hand sorting: Use a metal tray with sides that are at least a few centimetres perpendicularly from the bottom up. The bottom and sides of the tray must be painted white, smooth and anti-dazzle. Place an architect lamp, so it can be moved over the tray. Before opening the sample you should have catching equipment ready – tweezers or a brush, and a Petri dish or a similar container with a lid. When it is opened, it is appropriate to take a look into it to decide whether one can control the pests when they are let out onto the tray. Too active samples can be calmed with an hour-long stay in an oven or freezer and can later be looked upon without risk of spread into the room. The product is spread over the tray bottom in a thin layer so that at least a third of the base paint remains visible. Everything that moves or looks like pests is picked up for further investigation. Tweezers are best for larger insects and larvae. Small larvae and beetles, less than 3 mm, are most conveniently picked up with the tip of a moist brush. If the sample is cold, let the lamp heat up the sample in the tray, while you look. This makes the pests more lively and therefore more visible. You should watch the tray for at least five minutes. Some pests will – after disturbances – play dead for a few minutes. Small whitish pests, such as mites, are most easily found if you shake the test easily in a kitchen sieve over a piece of black paper in the tray bottom.

The pests that are found may be looked upon dry or you can kill and preserve them in alcohol added glycerine (3 parts 70% ethanol to 1 part glycerine). Denatured alcohol is a workaround. The smallest pests do best as microscopic preparations.

Sieving: Pests in bulk goods can be concentrated by sieving, where you can either remove their substrate (for example to find insects, larvae and web in flour) or you sieve and remove the fraction that contains particles of the same size as the pests you expect to find. When using common analysis sieves with wire tissue or punched circular holes in the bottom, most species of the smaller beetles could pass a 2 mm sieve. A 0.5 mm sieve stops all beetles and lets all mites of importance in foods pass. Sieves with a revolving screen – called oscillating screens – are useful for comminute material and coarser materials that can be forced to roll. For special sieving in which the sample structure does not allow rolling movements (for example, by screening of hay) or where the amount of wear dust becomes too large in the oscillating screen, use a vibration screen, where the movement is up and down and the amplitude low. For grain etc. where you want to screen kilos of material in a short time and where you are looking for pests the size of granary weevils or less, a forward and backward-moving sieving in a seed sorting machine with a 1, 4 / 35 mm screen has proven effective. For these types of sieving the screening is most efficient at first where there are most pests per unit weight of dust. Extended run time beyond a few minutes increases the amount of dust without pests proportionately with time.

The Berlese extraction method: Insects and mites can be drawn out from a sample using a heat source that heats and dries the sample from above. Pests will escape downwards and end up on the wire mesh that supports the sample from which they fall into a collection container with liquid. The liquid may be an alcohol-glycerine mixture, 4% formalin, stained lactic acid or simply water with added detergent. This method can be found in many different modifications and common to them all is that only the mobile stages of the pests land in the preserving fluid. Eggs, moulting stages and dead pests remain in the sample. It takes much time to drive out the pests (hours, days and sometimes even weeks) and the period of time is extended significantly if the sample thickness exceeds a few centimetres. The pests leave the sample as the food dries out. If samples have different water content, the wettest samples give off animals the slowest, so samples that are analysed in this way are comparable after quite long time. Very finely divided samples can be held up by a neutral medium, which prevents the sample from passing the wire screen in the bottom.

The Berlese extraction method is especially good for finding mites, but when it comes to finding mites in grain, sieving is also an option. There is indeed a lot of dust, but in return you get faster results. Despite the long time is takes, the Berlese method is popular in quantitative analyses. The pests are collected, and the procedure for the analysis is of a few minutes duration. The Berlese extraction method allows more pests to be found than most other methods.

Cultivation: You cannot always see the pests and have them determined. In cereals, rice, etc. larvae can sit hidden and if you have the time for it, you can store the samples at room temperature or a little more for a month. By that time, any larvae have grown larger or become mature and can be detected more easily. The same goes for larvae and pupae, which cannot be determined. Let them be in what they were in, and wait until they are mature. Cultivation should be carried out in glass or containers of heavy plastic with vents at the top. The containers are put in a warm, not too dry area and should be inspected every day.

Staining: For detection of the “plugs” that rice and granary weevils close their egg holes with, use acid fuchsine in vinegar. It provides a red colour. A similar method uses UV alkaloid berberine, which gives the plugs a yellow-green fluorescence at 366 nm.

Radiographs: Used for detection of cavities in cereal, macaroni, etc., in which gnawings and larvae can be seen. It takes some practice to interpret what you see.

Uric acid detection: Uric acid amount in a product is largely proportional to the amount of insect excrement. It is therefore an expression of the number of insects and the metabolism; they have had in the goods, and thus the possible harmful effects. In a dry state, uric acid is crystalline and stable. In an aqueous solution it has an absorption maximum at 293 nm. By addition of uricase, a specific enzyme, absorption decreases and the content of uric acid can be determined. In the absence of a UV spectrophotometer, one may use a non-specific, non-enzymatic method with phosphotungstic acid, which is measured at 670-690 nm.

Carbon dioxide measurements: In a product that does not breathe significantly itself, for example, dry cereal, the insect respiration will be many thousand times greater than the respiration from any amount of weight of the product. The method has been used for quality control of cereals with special emphasis on granary weevil larvae inside the grains. CO2 measurements can be done using the traditional methods. For routine tests gas chromatography is probably the simplest method. The method can hardly be used in cold products or in cool warehouses where the insect respiration is barely noticeable.

Ninhydrin smash: Grains of cereals and rice pass between two rollers and are crushed. On one roller a strip of ninhydrin-impregnated paper is applied. Crushed larvae body fluids contain free amino acids, which in contact with the ninhydrin give coloured spots on the strip. In theory, this method works well enough. In practice it is a difficult method to use. It is difficult to impregnate a paper roll evenly with ninhydrin in acetone. The paper works best when it is kept moist during the analysis and the durability is only a few months.

Floating methods: Insects and mites usually have a density below 1.2. Plant products are often more dense – about 1.4. In liquids with the appropriate densities pests will thus flow, while other parts may sink to the bottom. As flotation agent NaCl or various sugar solutions is often used. Organic solvents are particularly suitable for goods – such as flour – that gelatinise in water. In some floating methods you make use of the fact that insect exoskeletons are lipophilic. Via a NaCl-phase pests are forced up in a gas phase, which is then removed and filtered. One can also make use of benzene, which is frozen in situ and removed frozen for evaporation in a Petri dish. In connection with quite extensive quality demands, where you want it all, you can use special “filth” methods where you by acid hydrolysis or enzymatic digestion break down food or just wash it thoroughly with dissolving agents. Then proceed with the flotation and filtration. The filtrate is mounted on slides in a mountant fluid. The content can be determined using specific hair and fragment keys and the results reported as the number of mouse / rat hair and as numbers that are an expression of what the fragments found correspond to, in whole insects.

Tests based on pest movement: Although pests can nibble rather loudly, the listening methods do not matter much. The ratio between signal and noise is, in most situations not satisfactory.

If you with your hands form a small peak of dust, you will, if there are many mites in the dust, discover that the top surface is flattened in a quarter of an hour. A few simple tests that can be used in flour: (1) Put the flour into a cylindrical tube and wait a few hours. If mites are present you will be able to see their tracks where the flour is in contact with the sides of the glass. Mites can probably be seen near the top side of the tube where the flour is not present, and wherein the mites are looking up when disturbed. (2) An alternative method. Pour the flour into a tray or something similar. Smooth the surface by means of a spatula. Wait an hour. Any mites and insects in the flour will then show themselves with tracks and other imperfections on the surface. If there are no crevices there may of course be animals present anyway, but hardly in such large quantities that you cannot use the flour for cooking or baking here and now.

Microscopy and microscopic preparations: When it comes to large pests, you can see the main insects with the naked eye or through a magnifying glass. If the pests are smaller than 5 – 6 mm, one should also look at them in a stereo microscope with a low magnification (a so-called stereo magnifier). It must be equipped with light and preferably with a zoom lens. With this type of lens you can keep live pests in focus while you change the magnification. Magnification beyond 4 xi zoom lens and 10 xi eyepieces is not needed since both the depth of field and brightness deteriorates significantly beyond 40 x total magnification. For very small insects, use a different type of microscope. Use a high resolution microscope with transmitted light. It has a different light path, is more expensive and can, with oil immersion, magnify up to 1250 x. A reasonable magnification for mites and booklice will be 100-400 x. If you have to determine the species of mites in food, the microscope should further be equipped with phase contrast. Although mites, booklice and other tiny, pale insects are most easily collected with transparent tape, which later is microscoped, this is just a workaround. To view the details needed in order to determine the species, one must always make a proper preparation.

In some contexts, which you only have an ordinary microscope available or where it is desired to count the animals, it may be desirable to colour them. Use concentrated lactic acid, coloured light red with the dye Lignin Pink, which is a good agent. The pests must stay in this liquid for about a day or more, and preferably in heat, 50 ° C. Then the lactic acid is removed and the insects that are now both bright and red-coloured can be transferred into alcohol – glycerol or Hoyer’s medium (page xxx).