Appendix D 1. MICROBIOLOGICAL EXAMINATION OF PLANT AND EQUIPMENT Control measures for checking plant hygiene, such as the sampling of containers and working surfaces, should be carried out regularly. The following methods are recommended: 1.1 Swab technique (1) 1.11 Apparatus The follo ving apparatus is required: Test tubes, 250/25 mm of heavy borosilicate glass. Stainless steel wire, BS 1554, rust, acid and heat resisting steel wire, 350 mm of 2.64 mm diameter, formed into a loop at one end leaving a straight length of 300 mm and notched at the other end to hold the ribbon gauze. Ribbon gauze, unmedicated, 50 mm wide. 1.12 Preparation of swab The swab should be 50 mm in length and consist of 175 mm of gauze wound round the notched end of the wire, secured with thread. 1.13 Sterilization of swab Place the swab in 25 ml of Peptone Water Diluent in the test tube, plug with cotton wool or suitable rubber closure, cover the plug with aluminium foil or greaseproof paper, and sterilize by autoclaving at 121ø for 15 min. To obtain a final quantity of 25 ml of solution, it is necessary to start initially with a larger amount to allow for evaporation during autoclaving. The actual quantity should be found by trial and error with each individual autoclave. The Peptone Water Diluent should contain sodium thiosulphate or other suitable inactivator. 1.14 Swabbing technique Where possible, examine an area of 1 000 cm2 (approx. lft2). Press the swab with a rolling motion against the side of the test tube to remove excess liquid. Remove the swab and with heavy pressure rub back and forth over the area to be examined so that all parts of the surface are treated twice. Rotate the swab so that all parts of it make contact with the 787

788 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS surface under test. Return the swab to the test tube, and insert the cotton wool plug or rubber closure. 1.15 Testing Test the swab samples as soon as possible. After not less than 5 min contact of swab and liquid, mix the sample by twirling the swab vigorously in the Peptone Water Diluent six times. Remove the swab, taking care to express the liquid by pressing against the side of the tube. After thorough mixing by rotation of the tube between the palms of the hands, plate out 1 ml and 0.1 ml quantities of the swab solution using Plate Count Agar and incubate at 30 + 1 o for 72 + 2 h. Carry out the preparation of the dilutions, inoculation, pouring, and incubation of the Petri dishes, and the counting of the colonies as previously described. 1.16 Recording results Record the results as the colony count per 1 000 cm2 of surface tested (colony count per ml X 25 X area factor, if not 1 000 cm2). 1.2 Agaroid sausage technique The principle introduced by Ten Cate (2) is to use an agar-filled plastic casing which, when the ends are sealed, resembles a sausage. The end of the agar and casing is cut off and the exposed agar surface is used to take an impression of the surface to be tested. 1.21 Sampling technique (3) Swab the outside of the Agaroid casing with alcohol in the area to be cut and sterilize a very sharp, broad-bladed knife by swabbing with alcohol and flaming. Cut off the end of the Agaroid approximately 12 mm distal to the coloured band. Do not remove the coloured band it is provided to prevent the agar slipping out of its casing during use. Applying light pressure at the base, push out about 1 cm of the agar column. Sample by pressing the cut end of the agar firmly on the test surface. Cut off a slice 4-6 mm thick and transfer, inoculated side uppermost, to a petri dish by supporting the agar slice on the blade of a knife. Do not put the exposed end of the Agaroid down on a bench or other contaminated surface. Once the plastic casing has been cut, do not re-seal. Incubate the agar slices in closed petri dishes at 37 o for 18-24 h and then count colonies. A petri dish will hold 3 to 4 samples. The area of each sample is approximately 8.5 cm 2.