a. mechanical stage.

b. condenser with iris diaphragm.

c. source of illumination.

d. two objectives - a 10 x (16 mm) for counting and a 20 x (8mm) for confirmation.

e. 8 x - 12.5 x oculars.

f. The 10 x objective must be calibrated with the ocular to give a field diameter of 1.382 mm (Preparation of Microscope, section 3.2.1).

g. The ocular must be equipped with a micrometer disk cross-ruled in sixths of ocular diaphragm opening (Preparation of Microscope, section 3.2.2).

a. Obtain or make a micrometer disk of suitable diameter to fit into ocular (approximately 21 mm) and 1 mm thick. The disk should be marked with a centre grid made up of 36 small squares, six to each side of such a size that the length of six squares is equal to the diameter of the ocular diaphragm which has been adjusted to give a field diameter of 1.382 mm as in step 3.2.1, (Part 6, Diagram I).

b. To make the grid, calculate the width of grid (10 - 14 mm) that will coincide with 1.382 mm on stage. Mark width on micrometer disk, place disk in ocular and check that width coincides. If not, remove disk and change lines as necessary. Once the proper width has been determined, etch grid on micrometer disk with very fine lines making certain grid is centred on the disk.

a. Locate and focus a mould filament with the microscope.

b. Focus the light source into the condenser, adjust the height of the condenser, the diameter of the iris diaphragm and the intensity of the light source to give clear uniform illumination such that there is sufficient light to see all particles but not so intense as to mask the characteristics of the mould.

c. Use a coloured filter if necessary to increase contrast of filaments.

(i) Before opening, shake container (sample unit) 60 times in 30 sec through a 30 cm arc.

(ii) Open container. If considerable foam is produced, pass the flame of a Bunsen burner lightly over the surface to disperse the foam.

(iii) Proceed as in step 3.3.4, Preparation of Howard Mould Count Cell.

(iv) Repeat procedure for remaining five sample units.

(i) Before opening, shake container (sample unit) 60 times in 30 sec through a 30 cm arc.

(ii) Open container. Drain liquid from canned tomatoes through a no. 2 sieve into a suitable clean receptacle.

(iii) Transfer liquid to a wide mouth bottle and screw lid on securely.

(iv) Continue as in step 3.3.3.a.

(i) Open container (sample unit) and mix tomato product 60 times in 30 sec with a spoon or other suitable utensil.

(ii) Transfer a small portion onto a coarse filter paper or celluwipe and measure the refractive index of the filtrate. Removal of the pulp from tomato mixture does not affect the refractive index as it is based only on the soluble solids. If the pulp is not removed, a hazy image will be formed which is hard to centre and read.

(iii) Determine amount of distilled water to add to 100 ml of sample unit from Table I to give a final refractive index of 1.3448 -1.3454 at 20°C or 1.3442 -1.3448 at 25°C.

(iv) Mix sample unit as in step (i), transfer 100 ml to a wide mouth bottle, add required amount of distilled water, secure lid and repeat mixing.

(v) Measure refractive index as in step (ii) and correct if necessary.

(vi) Proceed as in step 3.3.4, Preparation of Howard Mould Count Cell.

(vii) Repeat procedure for remaining five sample units.

(i) Open container (sample unit) and mix 60 times in 30 sec with a spoon or other suitable instrument.

(ii) Transfer a measured well mixed representative portion to a wide mouth bottle.

(iii) Dilute contents of bottle with an equal volume of distilled water, secure lid and shake 60 times in 30 sec through a 30 cm arc.

(iv) Proceed as in step 3.3.4, Preparation of Howard Mould Count Cell.

(v) Repeat procedure for remaining five sample units.

3.3.4.1 Clean Howard cell and cover glass making certain central area of cell is clean.

Rinse with distilled water, dry with a lint free cloth and pass lightly over a Bunsen flame.

3.3.4.2 Determine adequate cleanliness of slide by placing cover glass in position and pressing it firmly against the shoulders. If Newton's rings appear between each shoulder and the cover glass, and remain after pressure has been released, the slide is considered sufficiently clean. When the rings are formed they may be observed by holding the slide at such an angle that the light is reflected from the cover glass. These rings resemble a rainbow in colour and when properly formed are broken arcs of concentric circles. If Newton's rings are not formed re-wash slide and cover glass. Absence of Newton's rings indicates dirt preventing proper seating of cover glass on shoulders which results in chamber holding an incorrect volume of sample.

3.3.4.3 Clean spatula and dissecting needle, rinse in distilled water, flame and cool.

3.3.4.4 Prepare glass slide using technique (a) or (b) as follows:

a. Inclined Cover Glass Technique

b. Parallel Cover Glass Technique

a. Sufficient material to fill area used for counting.

b. Newton's rings visible.

c. Even distribution of material on slide. Ensure sample portion is taken from a thoroughly mixed sample. Otherwise, when cover glass is put in place, insoluble material, and consequently moulds, may be more abundant at the centre of the mount.

d. Absence of air bubbles.

a. Uneven distribution of material.

b. Absence of Newton's rings.

c. Liquid which has been drawn across the moat and between the cover glass and shoulder.

d. Numerous air bubbles.

3.3.5.2 From each of 2 or more mounts examine at least 25 fields taken in such a manner as to be representative of all sections of the mount. The recognized procedure for examining a mount is to examine alternate fields in alternate rows throughout the entire area of the mount. To accomplish this, examine alternate fields horizontally across the slide preparation until 5 fields have been examined. Then move the mechanical stage vertically to the next alternate row and examine 5 more alternate fields in reverse horizontal direction. Repeat this process until 25 fields have been examined. If a field with an air bubble is encountered, move to another field unless mould is seen at first glance, because the field will contain insufficient sample. Otherwise never move the slide purposely to exclude or include mould filaments.

3.3.5.3 Observe each field noting presence or absence of mould filaments as characterized in Part 6, Diagram III. If not familiar with the diverse forms of mould, examine known moulds as follows:

3.3.5.4 Count field as positive when the aggregate length of < 3 of the longest filaments present exceeds 1/6 diameter of field. These filaments may be separate or attached to each other. A clump or mass of mould has the same value as a single filament (Part 6, Diagram IV).

3.3.6.1 Calculate proportion of positive fields from results of examination of all observed fields for each sample unit.

3.3.6.2 Report results as a percentage of fields containing mould filaments individually for each sample unit:

and as an average for the whole sample: