SOP: Mould Counting in Tomato Products by the Howard Method

GIFSQ — Global Institute for Food Safety & Quality

Principle

Mould growth breaks down fruit and vegetable tissue to form rot, so the presence of mould filaments in a finished product is a reliable index of rotten raw material having gone into manufacture. Because tomato products are comminuted and cooked during manufacture, visual inspection and the plate count (which reveals only living microorganisms) are of no value. The Howard direct microscopic count overcomes these drawbacks: the proportion of microscope fields containing mould filaments is reported as a percentage. A high mould count always indicates bad stock or improper handling, though a low count does not necessarily indicate sound stock.

Apparatus required

• Compound microscope (monocular or binocular) giving a flat field, with a 10x eyepiece and an achromatic 10x objective to give a magnification of 100 times; in addition, either a 20x objective with a preferably revolving nosepiece, or a 15x eyepiece
• Stage micrometer (for field calibration)
• Spotlight with a mirror, or preferably a sub-stage lamp with “daylight” glass
• Howard mould counting plate: a one-piece glass slide with an unruled central disc about 19 mm in diameter, surrounded by a moat and flanked by shoulders on each side 0.1 mm higher than the plane surface; optically worked central plane, cover glass and shoulders. Engraved calibration circle 1.382 mm in diameter on the right-hand rail of the slide (or, for the square cell, two fine parallel engraved lines 1.382 mm apart)
• Cover glass for the Howard cell
• Eyepiece micrometer disc, ruled into squares with each side equal to one-sixth of the diameter of the eyepiece diaphragm opening (desirable for estimating filament lengths); alternatively an ocular drop-in disc cross-ruled in sixths of the ocular diaphragm opening
• Blood counting cell (depth 0.1 mm), preferably ruled in the Thoma or the old Neubauer system; the so-called “improved” Neubauer system with optically worked cover glass may also be used
• Graduated stoppered cylinders
• Glass rods
• Scalpel and needle
• Centrifuge (for the tomato soup modification)

Reagents

a. Sodium carboxymethylcellulose solution (for the tomato ketchup modification): place 500 ml of hot water in a high-speed blender and cover. Start the blender, add 2.5 g of sodium carboxymethylcellulose and 10 ml of formaldehyde, and blend for 10 min.

b. 50° NaOH solution (for the tomato soup modification, when starch is present): 3 ml is added to the soup sample.

Sample preparation

For tomato juice and ketchup, use the product as it comes from the container. For routine counts, tomato puree may be used as it is; diluting the puree gives only slightly lower counts and is unnecessary except for samples whose count is near the 40% limit or higher. Dilute tomato puree and paste with water until the refractive index is 1.3448–1.346 (8.0–8.8° Brix). If the paste contains salt or any other substance that materially influences the specific gravity, take this into consideration in making the dilution.

Procedure — Calibration and preparation of the cell

Clean the Howard cell and the cover glass thoroughly before use

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Dip a piece of cotton wad in concentrated HCl and clean the surfaces

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Rinse thoroughly with distilled water, then with alcohol, and finally with acetone

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Remove any adhering particles of dust or lint with a camel-hair brush

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Adjust the microscope so the field diameter is 1.382 mm (field area 1.5 sq mm): with the 16 mm objective and 10x eyepiece, focus on the etched 1.382 mm calibration circle and adjust the draw tube length until the circumference of the microscope field coincides with the outside of the circle

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If such adjustment is not possible, fit an accessory drop-in ocular diaphragm with an aperture accurately cut to the necessary size; the field diameter may also be determined with a stage micrometer. When properly adjusted, the quantity of liquid examined per field is 0.15 cu mm

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If an Abbe, achromatic or variable focus condenser is used, remove the top lens

Procedure — Mounting the sample

Thoroughly mix the sample and place a drop on the central disc

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Spread it evenly over the disc with a needle, avoiding squeezing the more liquid parts out around the edge of the disc when the cover slip is placed (if the liquid is squeezed out, the insoluble solids and consequently the moulds may be more abundant in the centre of the mount)

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Do not use more sample than is sufficient to fill the space between the central disc and the cover slip

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Rest the cover glass against the edge of one of the shoulders and tilt it gently down onto the liquid; no liquid should penetrate between the shoulders and the cover glass, as this interferes with proper contact

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Check for Newton’s rings between the contacting surfaces; these coloured bands are the accurate criterion of proper contact

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Discard any mount showing uneven distribution, absence of Newton’s rings, or liquid drawn across the mount and under the cover glass

Procedure — Counting

Place the slide under the microscope and examine at least 25 fields from each of two or more mounts

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Observe each field, noting the presence or absence of mould filaments, and record each as positive or negative

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Examine suspected pieces at 200x (8 mm objective), then change back to 100x and move the field to the original position before continuing

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Mark a field positive only if the aggregate length of not more than three filaments present exceeds approximately one-sixth of the field diameter; mark it negative if no mould is found or the mould present is less than one-sixth of the field diameter

Procedure — Identifying mould filaments

Moulds generally show up as long, slender tubes with two walls running parallel and looking alike; the tubes are often broken into segments, sometimes show granules, and end in round or square tips. Where branches occur, they stand clear of one another, take irregular bends that may be empty of cytoplasm in places, and stay open where they leave the main tube. The distinguishing features against tomato filaments are tabulated below:

Procedure — Revised method for tomato ketchup

Place 500 ml of hot water in a high-speed blender and cover

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Start the blender, add 2.5 g of sodium carboxymethylcellulose and 10 ml of formaldehyde, and blend for 10 min (see Reagents above)

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Transfer 50 ml of the sodium carboxymethylcellulose solution so prepared to a 100-ml measuring cylinder

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Add 50 ml of well-mixed ketchup and mix thoroughly

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Use the material so prepared for counting by the procedure above

Procedure — Tomato soup

Heat and mix the soup; transfer 5 ml (thick soup) or 10 ml (thin soup) to a centrifuge tube

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If starch is present, add 3 ml of 50° NaOH solution

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Mix and fill the tube with water

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Centrifuge for about 20 min at 1600 rpm, until the liquid layer is clear

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Decant the clear liquid

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Add sufficient water to the residue to make up to the original volume

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Proceed for mould examination by the Howard mould count procedure above

Procedure — Products containing tomato sauce

Heat the product and drain it on a 6 mesh sieve

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Treat the drained sauce as in the case of tomato soup above

Calculation

Count the positive fields over all the fields observed on all the slides, and report the result as the percentage of fields containing mould filaments:

• Percentage of positive fields = (Number of positive fields × 100) / Total number of fields examined

Where:

• Use the counts of all slides in calculating the average, even if the results are widely varying
• At least 25 fields from each of two or more mounts must be examined

Precautions

• Use only a mount that shows Newton’s rings between cover glass and shoulders; discard any mount with uneven distribution or liquid under the cover glass
• Do not exceed the 1.382 mm field diameter; the calibration must be checked before counting
• Do not count a field positive unless the mould present exceeds about one-sixth of the field diameter
• A low mould count does not necessarily indicate sound stock, but a high count always indicates bad stock or improper handling

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) — Howard mould count method, cited in the book.

Howard, B.J. & C.H. Stephenson, The Sanitary Control of Tomato Canning Factories, USDA Bull. 569 (1917); Microscopical Studies on Tomato Products, USDA Bull. 581 (1917) — cited in the book.