AS 5013.28:2009 Food microbiology
A6.1 Preparation of smear for microscopic examination
The procedure shall be as follows:
(a) Thoroughly rinse the pipette or micropipettor with the mixed sample and discharge to waste.
(b) Deliver the 0.01 mL of sample onto a clean microscope slide, making sure that the surface of the slide is horizontal. Spread immediately over the outlined area of 1 cm 2 , using a cool rake.
(c) Dry the film at 40°C to 45°C in a dust-free atmosphere, in a horizontal position.
A6.2 Staining
The procedure shall be as follows:
(a) Flood the slide with the methylene blue staining solution (see Paragraph A3.1) and leave for 2 min.
(b) Drain the slide of excess solution by resting the edge on absorbent paper.
(c) Dry the slide, using a warm air draught if available.
(d) Rinse the slide by dipping into three successive lots of warm tap water at 35°C to 45°C. Drain and air dry.
NOTE: Fresh rinse water should be used for each slide.
A7.1 Mounting the slide
The slide shall be mounted in the microscope (see Paragraph A4.2).
A7.2 Number of fields to be counted
The number of fields to be counted shall be determined by conducting a preliminary microscopic survey of the mounted smear. Table A1 specifies the number of fields to be counted according to the number of organisms per field.
A7.3.1 General
Having determined the number of fields to be counted (see Paragraph A7.2), select fields at approximately equidistant intervals over the smear. Do not consciously select the field to be counted while looking down the microscope as this may introduce a subjective bias.
A8.1 Microscope field area
The area of the field (mm 2 ) is determined as follows:
(a) Place a stage micrometer under the microscope, and using the oil immersion lens,determine the diameter of the field. The field diameter should be in the range 0.146 to 0.206 (146 μm to 206 μm).
(b) Calculate the area of one field using the formula Area = 3.14 R 2 (where R is the radius, or half the diameter, in mm).
(c) Recalculate the field area after serving of the microscope or if the lens system is changed.