SOP: Separation and Counting of Insect Fragments by Oil Flotation
GIFSQ — Global Institute for Food Safety & Quality
Principle
Insects, insect fragments and rodent hairs are often lighter than food and may be floated out in liquids heavier than water; mammalian hairs behave in the same way as the insect cuticle and may be separated by oil flotation. With the exception of fly larvae and maggots, which are not wetted by oils, other insect materials and fragments float because the cuticle is wetted by oil and not by water: when the oil adheres to the cuticle and other external parts, they become lighter and are buoyed up, while the plant tissue becomes wet with water and settles down. The recovered material is counted microscopically on ruled filter paper.
The oil flotation technique as described in the general procedure can be used for the examination of apple butter, apple sauce, apricots, asparagus, berry fruits, corn, citrus juices, fruit juices and nectars, canned tomato products and vegetable juices; product-specific modifications follow below.
Apparatus required
- Wildman trap flask: a 1- or 2-litre flask fitted with a close-fitting rubber stopper mounted on a stiff brass rod (5/32–6/32 in. in diameter and approximately 4 in. longer than the height of the flask); the rod is threaded (10-32) at the lower end and furnished with nuts and washers to hold it in place on the stopper; the lower nut and washer must be countersunk in the rubber to prevent striking the flask. (A rod of greater diameter is not desirable because of its greater displacement of liquid.)
- 400-ml beaker
- Wash bottles
- Stirring rod
- Büchner flask with Büchner funnel which can accommodate 7-cm filter paper
- Rapid-acting filter paper
- Petri dish, or thick glass plate
- Stereoscopic microscope: binocular microscope with 2 objectives and 10x or 12x oculars, giving an uninverted stereoscopic image and sufficient clearance above the sample to manipulate the probe
- Spotlight with condenser and iris diaphragm
- Sharp probe
Reagents
a. Oil: castor oil (for tomato products and some other foods), light medicinal mineral oil, white petrol (not leaded), or kerosene.
b. Hot and cold water, free from air bubbles.
c. Filter paper (Whatman No. 4, 7 cm in diameter), ruled with 8 to 10 parallel lines in pencil or water-proof and solvent-proof ink.
d. Glycerine — used as a 20% solution in water for moistening the filter paper.
Sample preparation
For tomato products, use 100 g of tomato paste or 200 g of other tomato products; express the results as fragments per 200 g. For other products, recommended sample sizes vary, but often 100 g is used. If the product is in brine or syrup, drain the contents, take a known weight of the solid material and add a proportionate amount of the liquid.
Procedure — Oil flotation
Clean the extraction flask thoroughly by scrubbing with a soapy brush and rinsing several times with water
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Transfer the sample into the flask using plenty of water; bring the volume of the liquid to about 900 ml in a 2-litre flask, or 600 ml in a 1-litre flask
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Add about 35 ml of oil; tilt the flask to an angle of about 45° and move the plunger disc quickly up and down for 2 min below the surface of the liquid, to draw the oil into the sample (do not beat in any air)
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Add hot water (49°C) down the side of the flask, almost up to the neck; allow to stand for several minutes, stirring occasionally
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Scrape the drops of oil from the sides of the flask with the plunger, without disturbing the oil layer excessively
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When the oil and water phases have completely separated, add hot water slowly until the oil comes to the neck of the flask
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Raise the plunger disc slowly until it is below the oil layer, then pull it up tightly until it is about 0.25–0.5 in. below the lower surface of the oil layer
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Wash the oil from the plunger rod into the flask using hot water
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Decant the oil into a beaker; holding the flask tipped, wash the neck of the flask thoroughly with hot water
Procedure — Second extraction (products other than tomato products)
Return the flask to the upright position
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Work the disc loose with the help of a stirring rod and lower it into the extraction flask; rinse the stirring rod with water and collect the rinsing in the flask
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Add about 15 ml of fresh oil and stir gently for about a minute to mix it with the contents of the flask
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Allow the layers to separate and decant the oil into the beaker; decant as many times as necessary to completely recover the oil, scrapings and settlings (two to three decantings should be sufficient)
Procedure — Cleaning the extract
Discard the contents of the conical flask, rinse, and fill about three-fourths with hot water free from air bubbles
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Pour the collected oil and washings from the beaker into the flask
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Repeat the scraping, settling and decanting into the beaker as before, to obtain a cleaner filter paper — but do not perform this cleaning more than once on any sample
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In the final decanting, petrol followed by hot water may be used to rinse the plunger rod and the neck of the flask
Procedure — Filtration
Place a moistened ruled filter paper into the funnel, apply suction, and pour the oil and water from the beaker onto it gently with the help of a rod
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Rinse the beaker thoroughly with petrol followed by hot water, and filter through the filter paper
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Transfer the filter paper to a Petri dish or thick glass plate and moisten it with the minimum amount of 20% solution of glycerine in water
Procedure — Counting
Examine the filter paper under the microscope, using a light strong enough to show all details on the paper
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Count over the whole surface of the paper, lane by lane
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With the help of the probe, turn over each piece of material and ensure that it is an insect fragment; do not count materials which are doubtful
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Mark the paper alongside each counted fragment with a marking pencil, to avoid miscounts
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Use higher magnification (30x) if necessary to count some pieces
Procedure — Fruit paste
Mix the sample thoroughly; weigh 100 g of paste into a 2-litre Wildman trap flask
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Add about 400 ml of water; heat to boiling and continue boiling for about 10 min, or until the material is disintegrated
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Cool to room temperature; add 30 ml of kerosene oil, fill the flask with water, and allow to stand for about 30 min, stirring the lower layer 5 or 6 times during this period
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Trap off the oily layer and transfer to a beaker
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Make a second extraction with 30 ml of kerosene; mix the extracts and strain through a No. 20 sieve; rinse the sieve
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Transfer the material remaining on the sieve into a beaker, filter, and examine; similarly filter the material passing through the sieve and examine
Procedure — Jam
Mix the contents thoroughly; weigh 100 g into a beaker
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Add 200 ml of warm water (50°C), mix, and transfer to a 1-litre Wildman trap flask
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Add 10 ml of HCl and boil for 5 min; cool to room temperature
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Add 25 ml of petrol, stir thoroughly, trap off, filter and examine as before
Procedure — Jelly
Mix the jelly thoroughly; weigh 100 g into a 1,000-ml beaker
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Add 300–400 ml of hot water; warm the beaker and mix by stirring until the jelly dissolves
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Filter and examine microscopically
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If the jelly contains fruit tissues which do not filter through filter paper, proceed as in the case of jam
Procedure — Strained and chopped fruits and vegetables
Use 100 g of sample
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For separation from all fruit and vegetable pulps (asparagus, beets, carrots, green beans, peas and similar), use light medicinal mineral oil and water at a temperature of about 21°C; in the case of sweet pumpkin, use castor oil and water at a temperature of about 50–55°C
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Transfer the contents to a 1-litre trap flask with water; mix thoroughly with 20 ml of the oil and maintain the appropriate temperature
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Allow the mixture to stand for 30 min, stirring 4–6 times during the period
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Trap off and examine microscopically; save the residue in the trap flask for the examination of fly eggs and maggots
Procedure — Canned citrus and pineapple juices
Pour 250 ml of juice into a Wildman trap flask
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Add 15 ml of castor oil and fill the flask with hot water (approximately 70°C) with vigorous stirring
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Add hot water till the oil layer comes into the neck of the flask and allow to stand for 30 min
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Trap off and pour into a beaker; rinse out the neck of the flask with alcohol and add the rinsings to the beaker
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Filter as before and rinse the beaker with alcohol
Procedure — Peas
Macerate the peas through a coarse screen
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Extract with oil in the Wildman trap flask
Procedure — Canned leafy vegetables and strained or chopped spinach
Drain the contents of the can and note the drained weight; cut the drained leaves into 1 in. square pieces (do not discard the brine)
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Place 100 g of leaves in a 2-litre conical flask and add a proportionate amount of brine
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Add 300–400 ml of solution containing 20 g of lead acetate and 10 ml of acetic acid; boil for 5 min, stirring occasionally
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Cool to 50°C; add 40 ml of light mineral oil and mix thoroughly for 2 min without incorporating air
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Fill the flask slowly with hot water and proceed with the extraction as described in the general method, decanting at least twice and adding 20 ml to the full flask before the second decanting
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Count whole aphids, other large insects and their parts separately by examining microscopically at 10–30x; cast skins (which appear collapsed and are split between the antennae) should not be included in the whole aphid count
Procedure — Potato chips
Transfer 100 g of the chips to a 2-litre beaker; crush the chips into small pieces and cover with petroleum ether
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Allow to stand for 5 min, decant and filter; make a second extraction of the fat; drain any residue on the filter paper to the flask
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Allow the petroleum ether to evaporate from the chips
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Transfer the chips to a 2-litre Wildman trap flask, add 500 ml of 60% alcohol, and boil for 30 min, replacing alcohol lost by evaporation
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Cool, add 35 ml of petrol, mix, allow to stand for 5 min and fill with 60% alcohol
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Allow to stand, trap off twice, filter, and examine microscopically
Calculation
For tomato products, express the results as fragments per 200 g:
- Fragments per 200 g = (Fragments counted × 200) / Weight of sample taken (g)
Where:
- For tomato paste the sample weight is 100 g; for other tomato products it is 200 g
- For products other than tomato products, the book fixes no reporting basis — report the total fragments counted for the sample weight stated
Precautions
- Work the oil thoroughly into the water-food mixture without incorporating air, and agitate intermittently while separation is taking place
- If an emulsion is formed, capryl alcohol or alcohol may be useful in breaking the emulsion
- If the food is coated with an oil (pickles) or fat (curried vegetables, guava cheese), remove this coating material before separation
- Considerable entomological knowledge and experience are required to identify insect fragments; intensive training under a competent person is considered essential to ensure correct examination
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.
Official Methods of Analysis, 11th edn., Association of Official Analytical Chemists, Washington (1970) — cited in the book.
National Canners Association, Laboratory Manual for Food Canners and Processors, Vol. 1, The AVI Publishing Co., Westport, Connecticut, USA (1968) — cited in the book.