Regulations relating to milk and dairy products

(updated) REGULATIONS RELATING TO MILK AND DAIRY PRODUCTS Published under Government Notice No. R. 1555 of 21 November 1997 As corrected by: Government Notice No. R.1278 of 29 October 1999 Government Notice No. R. 488 of 8 June 2001 As amended by: Government Notice No. R.9 of 7 January 2000 Government Notice No. R.53 of 28 January 2000 Government Notice No. R.755 of 28 July 2000 Government Notice No. R.837 of 25 August 2000 Government Notice No. R.1052 of 27 October 2000 Government Notice No. R.489 of 8 June 2001 T he Mini s t er of Heal th has , in terms of section 15(1) of the Foodstuffs, Cosmetics and Disinfectants Act, 1972 (Act No. 54 of 1972), made the regulat i ons i n t he S chedule. S CHE DUL E Def in i tion s 1 . In t hese regulat i ons any express ion to which a meaning has been assigned in the Act shall bear such meaning and, unl ess i ncons i s t ent wi th t he context “b u tter” means t he product the fat of which consists exclusively of butter fat and the composition of which compl i es wi th t he fat -content requirements prescribed in the Regulations relating to Dai ry P roduct s and Imi t ation Dairy Products (Government Notice No. R. 2581 of 20 November 1987, as amended) made under t he Agricultural Products Standards Act, 1990 (Act No. 119 of 1990) (hereinaft er referred t o as “ the Dai ry and Imi t at i on Dairy Products Regulations”); “b u ttermi lk” means t he mi lky by-product of t he but t er-making process ; “ch eese” means t he product t hat i s obt ained from a coagulum of – (a) mi lk or mi lk cons t i t uent s ; (b) cream; (c) part l y or whol ly skimmed mi lk; (d) recons t i t ut ed (prepared) mi lk; (e) but t ermi lk; (f) concent rat ed mi lk; or (g) a combinat ion of t he above product s , by t he removal of t he whey, and that has undergone ripening to a greater or lesser extent and that may in addi t i on have been further processed; “closed con tain er” means a cl ean container that is impervious to liquid, leak proof and will prot ect t he product t herein from contamination under normal conditions of storage, handling and t ransport ; “col i f orm b acteria” means rod-shaped, gram negative aerobic and facultatively anaerobic non spore forming bact eri a t hat ferment lactose, producing gas and acid in the process, by using the mediums and methods prescribed in paragraph 4, 5 or 11 of Annex A; 2 “comp osi te d airy p rod u cts” means a product as defined in the Dairy and Imitation Dairy P roduct s R egulat i ons ; “cream” means t he fl uid dairy product with a fat content as prescribed by the Dairy and Imitation Dai ry P roduct s R egulat i ons ; “cu l tu re” means a l i quid or powder containing one or more acceptable selected micro-organisms used in t he manufacturing of cultured buttermilk, sour cream, sour milk, yoghurt or any other type of fermented mi lk product ; “cu l tu red b u ttermi lk” means buttermilk or pasteurized or reconstituted (prepared) milk which has been inoculat ed wi th a cul ture; “d airy p rod u ct” means a product as defined in t he Dai ry and Imitation Dairy Products R egulat i ons ; “Escher i chia col i” means the organism that produces gas at 440C +/- 0,250C in brilliant green 2% (m/v) bi l e broth and produces indole in tryptone water at the same temperature when incubated for 24 hours , when us ing the method described in paragraph 2 of Annex A or, alternatively, when the viol et red bi l e MUG agar method i s used, the colonies that fluoresce blue in the surrounding mediums under an ul t raviol et l i ght aft er i ncubat ion for 24 + / - 1 hour at 300 C ; “ex tran eou s” means of ext ernal origin; “f ood ad d i tive” means a subs t ance as defined in the Regulations governing the Labelling and Advert i s i ng of F oods tuffs (Government Notice No. R. 2034 of 29 October 1993, as amended) (hereinaft er referred t o as “ the Label l i ng and Advert i s i ng of F oods tuffs Regulations”); “h ermeti cal ly seal ed container” means an unopened container which cannot be opened without breaking or damaging such container or a seal, adhesive label or other part of or attachment to such container and which is intended to protect its contents against the entry of micro-organisms; “imi tation d airy p rod u ct” means a product as defined in the Dairy and Imitation and Dairy P roduct R egulat i ons ; “mi lk” means t he normal mammary gland secretion obtained from lactating cows of the bovine speci es , goat s or sheep; “mi lk p owd er” means t he product obt ained by the removal of water only from milk, partly skimmed mi lk or whol ly skimmed milk, with or without food additives permitted by the Act; “mod i f i ed d airy p roduct” means a product as defined in the Dairy and Imitation Dairy Products R egulat i ons ; “p asteu ri sation ” means t he heat t reatment, as described in Annex B, of a dairy product or an imi t at i on dai ry product so t hat (a) al l veget at i ve pathogens are des t royed; and (b) i n t he case of mi lk, t he result of the phosphatase test described in paragraph 3 of Annex A i s negat ive and, i f t he product concerned does not undergo further processing, the cooling thereof to below 50 C immediat ely aft er having been thus heat t reat ed; “p resu mp tive tes t” means a t es t t he pos i t i ve result of which invites the presumption that a subs t ance i s present aft er which the presumpt ion must be proven to be true by using more sophi s t i cat ed and accurat e t es t methods ; 3 “p rimary d airy p rod u ct” means a product as defined in the Dairy and Imitation Dairy Products R egulat i ons ; “raw cream” means cream that has undergone pas t euri sat i on, sterilization or ultra high t emperature t reatment ; “raw mi lk” means mi lk t hat has not undergone pas t euri zation, sterilization or ultra high t emperature t reatment ; “recon sti tu ted (p rep ared ) mi lk” means the product obtained by re-constituting milk powder wi th water so t hat i t compl i es wi th all the requirements for milk as prescribed in the Dairy and Imi t at i on Dai ry P roduct s R egulat i ons ; “skimmed mi lk” means mi lk t he fat of which has been removed to comply with the fat-content requi rement s prescribed in t he Dai ry and Imi t at i on Dai ry P roduct R egulat i on; “skimmed mi lk p owd er” means t he product obt ained by the drying of skimmed milk; “sou r cream or cu l tu red cream” means the product obtained from pasteurized cream that has been inoculat ed wi th a cul ture i n order for it to develop certain microbial flora under controlled condi t i ons ; “sou r mi lk or cu l tu red mi lk” means the product obtained from pasteurized milk that has been inoculat ed wi th a cul ture i n order for i t t o develop certain microbial flora under controlled condi t i ons ; “steri l i zation ” means t he heat t reatment above 100oC, after packaging, of a dairy product or an imi t at i on dai ry product so t hat t he product concerned will be resistant to microbiological det eriorat i on for a period of at l eas t 14 days i f kept at a t emperature of 30oC +/- 1oC; “th e Act” shal l means t he F oods tuffs , C osmetics and Disinfectants Act, 1972 (Act No. 54 of 1972); “UHT ” or “u l tra h igh temp eratu re treatmen t” means the process whereby milk or a dairy product i s subj ect ed t o heat t reatment above 100oC and aseptically packaged so that the end product , aft er i ncubat ion for not less than 14 days at a temperature of 30oC +/- 1oC, is free from spoi l age by micro-organi sms; and “yogh u rt” means t he product obt ained from pasteurized milk or reconstituted milk which has been inoculat ed wi th a yoghurt cul ture and which i s al l owed to ferment under controlled condi t i ons ; Restri ction s 2 . No person shal l use or sel l raw mi lk i nt ended for further process ing which (a) contains t he fol l owing: (i ) Ant ibiot i cs or other ant imicrobial subs t ances in amounts that exceed the maximum res idue l evel s s t i pulated in the Regulations governing Maximum Limi t s for Veterinary Medicine and Stock Remedy Residues that my be present i n F oods tuffs (Government Not i ce No. R. 1809 of 3 July 1992, as amended) (hereinaft er referred t o as t he Maximum Limits for Veterinary Medicines and S tock R emedy R es idues R egulations) or which virtue of a presumptive test is suspect ed t o contain ant ibiotics or other antimicrobial substances in amounts t hat exceed such maximum res idue l evel s ; 4 (i i ) pathogenic organi sms, extraneous matter or any inflammatory product or other subs t ances which for any reason what soever may render the milk unfit for human consumpt ion; (b) gives a pos i t i ve result when subjected to the clot-on-boiling test described in paragraph 6 of Annex A (c) gives a s t andard pl at e count of more than 200 000 colony forming units per 1,0ml when subject ed t o t he s t andard plate count test described in paragraph 7 of Annex A or the dry rehydrat ed fi lm method for standard colony count described in paragraph 10 of Annex A; (d) (i ) on appl i cat i on of t he t es t described in paragraph 4(4) of Annex A, exceeds the mos t probable number (MPN) of 10,0 coliform bacteria per 1,0 ml milk or, if the t es t for col i forms described in paragraph 5 or 11 of Annex A is used, the number of colony forming uni t s exceeds 20 per mi l l i l i t er of mi lk; or (i i ) on appl i cat i on of t he modified Eijkmann test, the VRB MUG agar method, or t he dry rehydrat ed fi lm method described in paragraphs 2, 5 and 11, respect ively, of Annex A, is found to contain any Escherichia coli in 0,01ml or raw mi lk. W hen the Eijkmann test is used, or any Escherichia coli in 1,0ml of raw mi lk i f t he methods described in paragraph 5 or 11 of Annex A are used; (e) when subject ed to the Standard Methods for Counting Somatic Cells in Bovine Milk*, is found to contain an average of 500 000 or more somatic cells per 1,0ml of bovine milk or an average of 750 000 or more cel l s per 1 , 0ml goat ’s or sheep’s milk after three success ive readings at i nt erval s of at least seven days during the test period, of which shows any other s igns of abnormal secret ary act i vi t y of the mammary gland(s); * T he S t andard Method for C ount ing Somatic Cells in Bovine Milk is set forth in Int ernat ional Dai ry F ederat i on (IDF ) B ul l et i n No. 114 of 1979. (f) fai l s t he ethanol s t abi l i t y t es t described in paragraph 9 of Annex A; and (g) i s not packed in a closed container. 3 . (1) No person shal l aft er two years from the date of publication of these regulations sell any raw mi lk, raw cream, raw skimmed mi lk, raw recons tituted (prepared) milk, raw recons t i t ut ed (prepared) skimmed milk or raw milk that has become sour, except in the areas of j uri sdi ct i on of t he l ocal authori t i es l i s t ed i n Annex C . (2) Any local authori t y t hat i s of the opinion that it can exercise sufficient control over the sel l i ng of t he raw dairy products referred to in subparagraph (1) may request the Minister, i n wri t i ng t hrough the rel evant provincial health department, to be listed in Annex C. (3) Any local authority that is listed in Annex C may request the Minister in writing to delete i t s name from the l i s t . 4 . (1) No person shal l sel l for consumpt ion raw milk, raw cream, raw skimmed milk, raw recons t i t ut ed (prepared) mi lk or raw reconstituted (prepared) skimmed milk which (a) contains t he fol l owing: (i ) Ant ibiot i cs or other antimicrobial substances in amounts that exceed the maximum res idue l evel s s tipulated in the Maximum Limits for Veterinary Medicine and Stock Remedy Residues Regulations or which by vi rt ue of a presumpt ive test is suspected to contain antibiotics or other ant imicrobial substances in amounts that exceed such maximum res idue l evel s ; (i i ) pathogenic organisms, extraneous matter or any inflammatory product or other subs t ance which for any reason whatsoever may render the product unfi t for human consumpt ion; (b) gives a s t andard pl at e count of more than 50 000 colony forming units (CFUs) per 1 , 0ml of t he product when subject ed t o t he s t andard plate count test described in paragraph 7 of Annex A or t he dry rehydrated film method for s t andard colony count described in paragraph 10 of Annex A. 5 (c) gives a pos i t i ve resul t when subjected to the clot-on-boiling test desribed in paragraph 6 of Annex A; (d) fai l s t he ethanol s t abi l i t y t es t described in paragraph 9 of Annex A; (e) on execut ion of t he modified Eijkmann test, the VRB MUG agar method or the dry rehydrat ed film method described in paragraphs 2, 5 and 11, respectively, of Annex A, i s found to contain any Escherichia coli in 1,0ml of fluid or 1,0g of cream; (f) (i ) on subject ion to t he S t andard R outine Method for the Counting of C ol i form B acteri a in Raw Milk of the International Dairy Federation’s Int ernat ional IDF 73:1985, or any revi sed version thereof, or on appl i cat i on of t he VRB MUG agar method described in paragraph 5 of Annex A or on us ing the dry rehydrat ed film method described in paragraph 11 of Annex A, i s found to contain more than 20 coliform bact eri a i n 1 , 0ml of fl uid; or (i i ) on subject ion to t he coliform bacteria test described in paragraph 4(4) of Annex A, exceeds t he mos t probable number (MPN) of 10,0 col i form bact eri a per 1 , 0ml of fl uid or 1,0g of semi-solid product; (g) i n t he case of raw mi lk, on subjection to the Standard Method for Counting S omat i c C el l s i n B ovine Mi lk, i s found to contain an average of 500 000 or more somat i c cel l s per 1 , 0ml of bovine milk or an average of 750 000 or more cel l s per 1 , 0ml of goat ’s or sheep’s mi lk after three successive readings at i nt erval s of at l east seven days during the test period, or which shows any other s igns of abnormal secretory act i vi t y of t he mammary gl and(s ); (h) i s not packed in a closed cnt ainer; (i ) does not bear cl early on the l abel t he words : “ Unpas t euri sed” / “ Ongepas t euri serd” or “ R aw mi lk” / “ R ou melk”; (j ) when the mi lk i s sold i n t he consumer’s own container, is tapped from a container which does not bear a l abel cl early i ndi cat i ng the words : “ Unpas t euri sed” / “ Ongepas t euri seerd” or “ R aw mi lk” / “ R ou melk”; (k) (i ) i s not derived from a herd enrolled in the Bovine Tuberculosis Scheme and the B ovine B rucel los i s Scheme which have been established in t erms of t he Animal Di seases Act , 1984 (Act No. 35 of 1984); or (i i ) i s not derived fro a herd which annually tests negative for tuberculosis and brucel los i s . 5 . No person shal l sel l for consumpt ion raw mi lk t hat has become sour which (a) contains t he fol l owing: (i ) Ant ibiot i cs or other ant imicrobial subs t ances in amounts that exceed the maximum res idue l evel s s t i pul at ed i n the Maximum Limits for Veterinary Medicine and S tock R emedy R es idues Regulations or which by virtue of a presumpt ive t es t i s suspect ed t o contain antibiotics or other antimicrobial subs t ances i n amount s t hat exceed such maximum res idue l evel s ; (i i ) pathogenic organi sms, extraneous matter or any inflammatory product or other subs t ance which for any reason whatsoever may render the raw milk unfit for human consumpt ion; (b) on appl i cat i on of the modified Eijkmann test or the VRB MUG agar method described in paragraphs 2 and 5 respectively, of Annex A, is found to contain any Escherichia coli in 1 , 0ml of t he product ; (c) on subject ion to the VRB MUG agar method or the dry rehydrated film method described in paragraphs 5 and 11 of Annex A, respectively, contains more than 50 coliform bacteria per 1 , 0ml of t he product ; (d) i s not packed in a closed container; and (e) does not bear cl early on the l abel t he words : “ Unpas t euri sed sour mi lk” / “ ongepas t euri seerde suur melk” or “ R aw sour mi lk” / “ R ou suur melk”; 6 (f) when the mi lk i s sold in the consumer’s own container, is tapped from a container which does not bear a l abel cl early i ndi cat i ng the words : “ Unpas teurised sour milk” / “ Ongepas t euri seerde suur melk” or “ R aw sour mi lk” / “ R ou suur melk”. 6 . No person shal l sel l (a) pas t euri sed mi lk, pasteurised reconstituted (prepared) milk, pasteurized skimmed milk, pas t euri zed recons t i t ut ed (prepared) skimmed mi lk or pasteurized cream which (i ) contains t he fol l owing: (aa) Ant ibiot i cs or other antimicrobial substances in amounts that exceed the maximum res idue l evel s s tipulated in the Maximum Limits for Veterinary Medicine and Stock Remedy Residues Regulations or which by vi rt ue of a presumpt ive t es t (for example the Kundrat test) is suspect ed t o contain ant ibiotics or other antimicrobial substances in amount s t hat exceed such maximum res idue l evel s ; (bb) pathogenic organisms, extraneous matter or any inflammatory product or other subs t ance which for any reason whatsoever may render the product unfi t for human consumpt ion; (i i ) has been shown by the Aschaffenburg and Mullen phosphate test described in paragraph 3 of Annex A or any other test, provided its accuracy equals that of t he aforement ioned t est, to yield the equivalent of 10 micrograms or more of p ni t rophenol per 1 , 0ml ; (i i i ) (aa) on execut ion of t he VRB MUG agar method or the dry rehydrated film method i s found to contain more than 10,0 coliform bacteria in 1,0ml or exceeds t he most probable number (MPN) of 10,0 coliform bacteria per 1 , 0ml of mi lk or 1 , 0g of semi -sol id product ; or (bb) on execut ion of t he modi fi ed Ei jkmann test, the VRB MUG agar method or t he dry rehydrated film method described in paragraphs 2, 5 and 11, espect ively, of Annex A, is found to contain any Escherichia col i i n 1 , 0ml of mi lk or 1 , 0g of semi -sol id product ; (i v) gives a s t andard pl at e count of more than 50 000 colony forming units (CFUs) per 1 , 0ml of fl uid or per 1,0g of semi-solid product when subjected to the tests described in paragraph 7 or 10 of Annex A; (v) i s not packed in a hermet i cal l y seal ed container when sold to the ultimate consumer: P rovided that i n cases where the consumer supplies his or her own empty container t o be fi l l ed from a bulk tank or container, the filled container need to be hermet i cal l y seal ed; (b) s t eri l i sed cream, sterilised milk, sterilised reconstituted (prepared) milk or UHT cream or UHT mi lk which (i ) contains t he fol l owing: (aa) Ant ibiot i cs or other antimicrobial substances in amounts that exceed the maximum res idue l evel s s tipulated in the Maximum Limits for Veterinary Medicine and Stock Remedy Residues Regulations or which by vi rt ue of a presumpt ive test is suspected to contain antibiotics or other ant imicrobial substances in amounts that exceed such maximum res idue l evel s ; (bb) pathogenic organisms, extraneous matter or any inflammatory product or other subs t ances which for any reason whatsoever may render any such product unfi t for human consumpt ion; (i i ) (aa) shows an increase i n t i tratable acidity greater than 0,02, expressed as grams of l act i c acid per 100ml of mi lk, on application of the test described in paragraph 8 of Annex A after incubation at 30oC +/- 1oC for 14 days ; (bb) shows any s igns of coagulation or blown containers after incubation; (iii) i s not packed in a hermet i cal l y seal ed container when sold to the ultimate consumer. 7 7. S ubject t o t he provi s ions of the Act, no person shall sell any dairy product or composite product which (a) contains t he fol l owing: (i ) Ant ibiot i cs or other ant imicrobial subs t ances in amounts that exceed the maximum res idue l evel s s t i pul at ed i n the Maximum Limits for Veterinary Medicine and S tock R emedy R es idues Regulations or which by virtue of a presumpt ive t es t i s suspect ed t o contain antibiotics or other antimicrobial subs t ances i n amount s t hat exceed such maximum res idue l evel s ; (i i ) pathogenic organi sms, extraneous matter or any inflammatory product or other subs t ance which for any reason whatsoever may render any such product unfit for human consumpt ion; (b) i n t he case of mi lk powder or skimmed milk powder, contains more than 50 000 colony forming uni t s per gram on appl i cat i on of t he standard plate count test described in paragraph 7 of Annex A; (c) wi th t he except ion of ri pened cheese (i ) on execut ion of t he t es t described in paragraph 4 of Annex A or the test described in Int ernat ional S t andard IDF 73A:1985, contains more than 50 col i form bact eri a per 1 , 0ml of fl uid or 1,0g of solid or semi-solid product; (i i ) on execut ion of t he modi fi ed Ei jkmann test or the VRB MUG agar method described in paragraphs 2 and 5, respectively, of Annex A, is found to contain any Escher i chia col i i n 1 , 0ml of fluid or 1,0g of solid or semi-solid product; (d) i n t he case of ri pened cheese (i ) on execut ion of t he t es t described in paragraph 4 of Annex A or the test described in Int ernat ional Standard IDF 73A: 1985 contains more than 1 000 col i form bact eri a per 1 , 0g of t he product ; (i i ) on execut ion of t he modi fi ed Ei jkmann test or the VRB MUG agar method described in paragraphs 2 and 5, respectively, of Annex A, is found to contain any Escher i chia col i per 1,0 ml of fluid or 1,0g of solid or semi-solid product; (e) i s not packed in a hermet i cal l y seal ed package or i n a closed package. 8 . No person shal l sel l any dai ry product or composite dairy product which contains any food addi t i ve not permi t t ed by regulat i on. 9 . No person shal l sel l m i lk, cream or any dairy product that is not derived from the mammary gl and(s ) of l act at i ng cows of t he bovine speci es or of goats or sheep unless it is labeled in accordance wi th t he requi rement s of t he l abeling and Advertising of Foodstuffs Regulations promulgat ed under t he Act . 10. No pas t euri sed mi lk, pas t euri sed cream or pasteurised reconstituted (prepared) milk which is returned to t he mi lk process ing pl ant shal l be resold or processed for resal e. 11. In det ermining whether milk, dairy products and composite dairy products meet the requirements l aid down in regulat i ons 2, 4, 5, 6 or 7, the tests prescribed therein shall be conducted and these t es t s shal l be conclus ive for t he said purpose Rep eal of regu lation s 12. T he regulat i ons published by Government Notice No. R. 258 of 8 February 1985, as amended by Government Not i ce No. R . 2706 of 15 November 1991, are hereby repeal ed. 8 ANNE X A ME T HODS FOR T HE T E S T ING OF MIL K, CRE AM AND DAIRY PRODUCT S 1. (1) (a) T he t es t s set forth in Annex A shall be conducted in appropriate cases in order to ascert ain t he sui t abi l i t y of mi lk, cream and dai ry product s for human consumpt ion. S amples shal l not be frozen but shall be kept at a temperature below 5o C and shall be tested within 48 hours of collection: Provided that these requi rement s shall not apply to dried dairy products, sterilized milk, UHT milk and condensed dai ry product s i n t hei r unopened containers (c) F or t he purpose of Annex A “ mi lk” shal l include milk that has undergone pas t euri zat i on or s t eri l i zation or ultra high temperature treatment, and cream. MICROB IOL OGICAL T E S T S (2) (a) Al l di s t i l l ed water used in the preparation of mediums shall be glass distilled water or water of s imi l ar puri t y . (b) Al l gl assware used in t he t es t s prescribed by this Annex shall be sterile. (c) T he s t eri l i t y of al l g l assware, media and di luent s shall be checked by (i ) t es t i ng represent at i ve control tubes, control dishes and growth media used in each bat ch of t es t s ; (i i ) us ing the growth medium referred t o i n t hi s Annex. (d) Al l pipet t es of t he blow-out type shall be suitably plugged with non-absorbent cot ton wool . (e) Al l gl assware used for volumetric measurement shall have an accuracy level at l eas t equal t o Nat ional P hys i cal R esearch Laboratory Grade B . (f) Al l chemical s used in t he preparation of the solutions and media referred to in t hi s Annex shal l , except where otherwise prescribed, be of an analytical reagent grade or a grade sui t abl e for t he preparat i on of bact eriologi cal media. (g) Appropri at e dehydrat ed cul ture media, where such preparations are available, may be used ins t ead of t he media prescribed: Provided that such dehydrated media shal l conform to t he descript i on given and yield equivalent results: P rovided further t hat any peptone, bile salts, tryptone, yeast extract and ox bile used shal l be of a standard equivalent to the reference standard kept by the South Afri can B ureau of S t andards . Mod i f i ed E i jkman n tes t f or Escher i chia col i 2 . (1) T he modi fi ed Ei jkmann t es t shal l be carri ed out i n t he manner set out below. (2) T horoughly mix the sample of mi lk or cream and, if the cream is too thick for easy handl ing, heat i t t o a t emperature not higher t han 37o C . (3) Aft er t aking al l necessary precautions to prevent contamination of the sample, inoculate t hree t ubes containing 10ml (m/v) of brilliant green bile broth and fitted with an inverted Durham fermentat i on tube for t he det ect i on of gas us ing a 1 ml pipette with the equival ent of 0 , 01ml i n t he case of raw milk intended for pasteurization and 1ml in the case of pas t euri sed mi lk, reconstituted (prepared) milk, pasteurized cream and cultured dai ry product s . In t he case of sol id or semi -sol id dairy products, inoculate tubes containing double-s t rength brilliant green bile broth with 10ml of a 1:10 dilution of the dai ry product . (4) F or t he measurement of t he 0, 01ml quantities to be tested in the case of milk, prepare decimal di l ut i ons i n accordance wi th t he s tandard plate count method described in 9 paragraph 7(1)(a) and (b), subs t i t ut i ng 11, 0ml of milk for 11,0g of milk powder or skimmed mi lk powder. (5) Incubat e t he i noculated brilliant green bile broth for 48 hours in a water bath keeping the t emperature of t he water bath at 44o C + / - 0 , 15o C . (6) If t he i ncubat ion prescribed in subparagraph (5) leads to the formation of gas as seen in t he Durham tube, transfer and inoculum of 0,2ml from each brilliant green bile broth tube i n which gas has formed to a separat e t ube of t ryptone water. (7) Incubat e t he t ryptone water t ubes referred t o i n subparagraph (6) in the water bath referred t o i n subparagraph (5) at 44o C + / - 0 , 25o C for 24 + / - 2 hours . (8) Aft er t he said 24 +/- 2 hours, test the tryptone water in the tubes for indole production by adding 0, 5ml of Kovac’s reagent . (9) T he format ion of a rose-coloured ri ng at the interface of the two liquids indicates the presence of i ndole. (10) A pos i t i ve resul t of gas and indole in any of the three tubes inoculated with the prescribed volume of t he same mi lk shal l be t aken to indicate the presence of Escherichia coli. (11) P repare t he (m/v) brilliant green bile broth, the tryptone water and the Kovac’s reagent as fol l ows: (a) (i ) T he compos i t i on of t he brilliant green bile broth shall be as follows: Ox bi l e………………………………………………. . ……………20g P eptone……………………………………………………………10g Lactose……………………………………………………………. 10g 1 per cent (m/v) aqueous solution of brilliant green………….…1,3ml Di s t i l l ed water……………………………………………………….1l (ii) Di ssolve t he cons t i t uent s i n t he di s t i l l ed water. (iii) Adjus t t he pH to a value of 7 , 2 t o 7 , 4 . (iv) Di s t ri but e t he medium in 10ml quantities among test tubes containing an i nvert ed Durham fermentat i on tube and then sterilize them in an autocl ave at 121o C for at l eas t 15 minutes . (v) In order t o prepare double-strength brilliant green bile broth, use half t he quant i t y of di s t i l l ed water. (b) (i ) T he compos i t i on of t he t ryptone water shal l be as fol l ows: T ryptone…………………………………………………………….10g S odium chloride……………………………………………………..5g Di s t i l l ed water…………………………………………………..up to 1l (ii) Di ssolve t he constituents in the distilled water by warming the mixture s l i ght ly . (iii) C ool t o 20-25oC and adjust the pH with sodium hydroxide solution or hydrochlori c acid solut ion to between 7, 4 and 7, 5. (iv) Di spense t he medium in 5ml al i quots in test tubes. Autoclave the di spensed medium at 121o C for at l eas t 15 minutes . (c) (i ) T he compos i t i on of t he Kovac’s reagent shal l be as fol l ows: P aradimethylaminobenzaldehyde…………………………………….5g C oncent rat ed hydrochloric acid…………………………………....25ml Amyl al cohol (pyridine free)………………………………………75ml (ii) Di ssolve paradimethylaminobenzaldehyde in the amyl alcohol and add the hydrochlori c acid. (iii) Aft er preparat i on, t he reagent should be yel low in colour. (iv) P l ace t he reagent in an amber-coloured glass stoppered vessel and store i n a cool , dark pl ace. 10 (v) Do not use t he reagent wi thin 24 hours aft er preparat i on. Asch af f en b u rg an d Mu l l en p h osp h atase tes t 3 . (1) T he phosphatase t es t shal l be carri ed out i n t he manner set out below. (2) T es t each sample as soon as poss ibl e aft er i t s arri val at t he l aboratory. (3) If t he sample i s not t es t ed immediat ely on i t s arrival at the laboratory, keep it at a t emperature below 5o C , but not frozen, unt i l i t i s t es t ed. (4) R ai se t he t emperature of t he sample t o 20-25o C immediat ely before it is tested. (5) T ake the fol l owing precaut ions during or in connection with he testing of a sample; (a) Except i n t he case of cul tured dairy products, do not test a sample that shows s igns of spoi l i ng or souring. (b) Use a cl ean pipet t e for each sample of milk or cream and ensure that no pipette i s contaminat ed wi th sal i va. (c) Do not perform the t es t i n di rect sunl ight . (d) Use only di s t i l l ed water t hroughout t he t es t . (6) W henever pract i cabl e, use reagents of analytical quality for this test. Prepare the buffer subs t rat e solut ion as fol l ows: (a) B uffer solut ion: Di ssolve 3, 5g of anhydrous sodium carbonate and 1,5g of sodium bicarbontat e i n distilled water and fill up with water to 1l solution in a volumet ri c fl ask. (b) Keep the sol id subs t rat e, disodium p-nitrophenyl phosphate, in a refrigerator. (c) B uffer subs t rat e solut ion: (i ) P l ace 150mg of the substrate in a standard 100ml volumetric measuring fl ask and fi l l t o t he 100ml mark wi th t he buffer solut ion. (i i ) S tore t he solut ion in a refri gerator and prot ect from l i ght . (i i i ) W hen di s t i l l ed water is used for purposes of comparison, the solution must give a reading of less than the standard 10 on the comparator disc AP T W 5 or APTW 7 when viewed in transmitted light through a 5mm cel l i n t he al l -purpose comparator. (i v) Do not use t he solut ion for l onger t han one week. (7) Use t he fol l owing apparatus for t he t es t : (a) A lovibond al l -purpose comparator wi th a stand for work in reflected light. (b) A Lovibond comparator di sc AP T W 5 or AP T W 7. (c) T wo fused-gl ass cel l s , 25mm deep, or test tubes of colourless lass, 13,5mm int ernal di ameter, conforming to B . S . 625, fitted with non-p-nitrophenol containing s toppers , for use i n t he Lovibond all purpose 1000 comparator. (d) A waterbath capable of being maint ained at 39o C + / - 0 , 5o C . (e) A pipet t e t o del iver 5 , 0ml . (f) A supply of 1 , 0ml s t raight -s ided pipet t es . (g) A 1l volumet ri c fl ask. (h) A 100ml s t andard volumet ri c fl ask. (8) (a) Aft er use, empty each tube, rinse it in water, wash well in hot water containing soda, ri nse i n hot water and then in distilled water and dry, or clean by some other equal ly effect i ve method. (b) If, aft er t reatment i n accordance with (a) of this subparagraph, a test tube does not appear t o be clean, repeat the treatment but, in addition, after rising it in hot water, pl ace i t i n hydrochloric acid and then rinse it again in hot water and then in di s t i l l ed water and dry it, or clean it by some other equally effective method. (c) C lean new gl assware by dipping it in a solution of chromic acid consisting of fi ve volumes of 8% (m/v) pot ass ium di chromate and four volumes of concent rat ed sulphuri c acid added s lowly and carefully to the mixture of di chromate and water. (d) Keep the solut ion referred to in (c) of this subparagraph covered and discard it when i t t urns green. 11 (e) Aft er cl eaning new gl assware i n t he manner described above, rinse it in hot water, t hen ri nse i t i n di s t i l l ed water and dry. (f) P ipet t es should be rinsed in cold water and then cleaned by soaking for 24 hours i n a solut ion of chromic acid i n a 250ml gl ass cylinder or other suitable container, and thereafter well rinsed in hot water and then in distilled water and dri ed, or cl eaned by some other equal ly effect i ve method. (g) Glassware used for t he t est shall not be used for any other purpose and shall be kept separat e from al l other apparatus i n t he l aboratory. (9) T he t es t shal l be carri ed out i n t he manner as set out below: (a) T ransfer 5ml of t he buffer subs t rat e solut ion to a test tube using a pipette, s t opper t he t es t t ube and bring the contents to a temperature of 37oC +/- 0,5oC. (b) Add 1 ml of t he mi lk or cream to be tested, replace the stopper of the test tube and mix the content s wel l by shaking. (c) Incubat e t he t es t t ube for 2 hours + / - 1 minute at 37o C + / - 0 , 5o C . (d) W i th each seri es of samples, incubate one control sample prepared from 5ml of buffer subs t rat e solut ion and 1ml of boiled milk or cream of the same type as t hat undergoing the t es t . (e) Aft er i ncubat ion, remove the test tube from the water bath and mix the contents wel l . (f) P l ace t he cont rol sample on the left-hand ramp of the stand and the test sample on the ri ght . (g) T ake the readings in reflected light by looking down onto the two apertures with t he comparator facing a good source of dayl ight . (h) If art i fi ci al l i ght i s needed for matching, use a daylight type of illumination. (i ) R evolve t he di sc unt i l t he colour of the test sample matches that of the control sample. (j ) R ecord readings fal l ing between two standards by affixing a plus or minus sign to t he fi gure for t he neares t s t andard. Col i f orm B acteria T est 4 . (1) T he col i form bact eri a t es t for mi lk, reconstituted (prepared) milk, pasteurised milk, pas t euri sed cream and dairy products shall be carried out in the manner set out below or by us ing VR B MUG agar method described in paragraph 5 of Annexure A. (2) Mix the mi lk, ream or dai ry product s t horoughly before sampl ing from bulk. (3) (a) T horoughly mix samples of milk, skimmed milk, buttermilk or cream. If it is t oo t hi ck for easy handling, cream may be heated to a temperature not exceeding 37o C . P repare t he 1:10 dilution (m/m) by adding 1ml of the product to 9ml of t he s t eri l e di l uent (phosphate buffer or peptone saline solution) or 11ml of the product t o 99ml of t he di l uent (paragraph 7). (b) T horoughl t m ix vi scous or semi-solid cultured dairy products and place 11g of t he mixed product i n a s t eri l e wide-mouthed container. Add 99ml of heated (40o C ) s t eri l e 2% (m/v) sodium citrate solution and shake the mixture until homogeneous dispersion is obtained. This constitutes the 1:10 dilution (m/m) of t he product . F urther t enfold di l ut i ons are prepared in the sterile diluent (paragraph 7). (4) T he most probable number (MPN) of coliform bacteria shall be determined as follows: (a) Inoculat e t hree test tubes each containing 10ml of double-strength brilliant green bi l e broth as described in paragraph 2(11)(a)(i) to (v) and a Durham tube with 10ml of t he 1:10 dilution of the product. This inoculation corresponds to 1g or 1ml of t he product sample i n each tube. (b) Inoculat e t hree t ubes containing 10ml single-strength brilliant green bile broth and a Durham tube wi th 1 ml of t he 1:10 di lut i on of t he product. This i noculat i on corresponds t o 0 , 1g or 0 , 1ml of sample i n each tube. (c) Inoculat e t hree tubes each containing 10ml of single-strength brilliant green bile broth and a Durham tube wi th 1ml of the 1:100 dilution or 0,1 dilution of the 12 product . T hi s inoculation corresponds to 0,01g or 0,01ml of the sample in each tube. (d) Mix careful ly , making sure t hat no ai r bubbles are shaken into the Durham tubes . (e) Aft er preparing the initial dilutions, proceed without delay with the preparation of further di l ut i ons and inoculat i ons . (f) Incubat e t he i noculat ed t ubes for 48 + / - 2 hours at 30o C + / - 1o C . (g) A tube containing sufficient gas to fill the concavity of the Durham tube shall be recorded as pos i tive. A positive result shall also be recorded if the Durham tube contains l ess t han the said amount of gas but effervescence occurs when the side of t he t es t t ube i s t apped. R ecord t he number of pos i t i ve resul t s . (h) In t he case of frui t yoghurt and other product s containing a fermentable subs t ance other t han l actose, confi rm the presence of lactose fermenters by t ransferring one loop full of the contents of each tube showing gas production to fresh t ubes of single-strength billiant green bile broth, incubating these tubes for 48 + / - 2 hours at 30o C + / - 1o C and examining them for gas production. (i ) T he number of pos i t i ve t ubes (aft er confi rmat ion, in the case of products containing fermentabl e substances other than lactose) for each dilution is used for det ermining the MPN of coliform bacteria per 1,0g or 1,0ml of the product in accordance wi th t he fol l owing t abl e: Number of pos i t i ve MP N of col i form in Number of pos i t i ve t ubes MP N of col i forms in 1 , 0g or 1 , 0ml 0, 1g or 0 , 1ml 0, 01g or 0 , 01ml 1, 0g or 1 , 0ml 1, 0g or 1 , 0ml 0, 1g or 0 , 1ml 0, 01g or 0 , 01ml 1, 0g or 1 , 0ml 0 0 0 0, 0 2 2 2 3, 5 0 0 1 0, 3 2 2 3 4, 0 0 1 0 0, 3 2 3 0 3, 0 0 1 1 0, 6 2 3 1 3, 5 0 2 0 0, 6 2 3 2 4, 0 1 0 0 0, 4 3 0 0 2, 5 1 0 1 0, 7 3 0 1 4, 0 1 0 2 1, 1 3 0 2 6, 5 1 1 0 0, 7 3 1 0 4, 5 1 1 1 1, 1 3 1 1 7, 5 1 2 0 1, 1 3 1 2 11, 4 1 2 1 1, 5 3 1 3 16, 0 1 3 0 1, 6 3 2 0 9, 5 2 0 0 0, 9 3 2 1 15, 0 2 0 1 1, 4 3 2 2 20, 0 2 0 2 2, 0 3 2 3 30, 0 2 1 0 1, 5 3 3 0 25, 0 2 1 1 2, 0 3 3 1 45, 0 2 1 2 3, 0 3 3 2 110, 0 2 2 0 2, 0 3 3 3 110, 0 2 2 1 3, 0 3 3 3 100, 0 (5) C ul tured product s wi th developed acidi ty shal l be tested within 48 hours of their manufacture. Violet red b i l e (MUG) agar meth od f or col i f orms an d Escher i chia col i 5 . (1) T he col i form organi sm t es t and the t es t for Escherichia coli in milk, reconstituted (prepared) mi lk, pas t eurised milk, pasteurised cream and dairy products shall be carried out i n t he manner set out below. 13 (2) P repare t he samples as fol l ows: (a) T horoughly mix samples of milk, skimmed milk, buttermilk or cream. If it is t oo t hi ck for easy handling, cream may be heated to a temperature not exceeding 37o C . P repare t he 1:10 dilution (m/m) by adding 1ml of the product to 9ml of s t eri l e di l uent or 11ml of t he product t o 99ml of di l uent . (b) T horoughly mix viscous or semi-solid cultured dairy products and place 11g of t he product i n a s t eri l e wide-mouthed container. Then add 99ml of heated (40o C ) s t eri l e 2% (m/v) sodium citrate solution and shake the mixture until homogeneous dispersion has been obtained. This constitutes the 1:10 dilution of t he product . P repare further t enfold di l ut i on i n t he s t eri l e di l uent . (3) T he viol et red bi l e agar i s prepared as fol l ows: g / l B rain heart i nfus ion………………………………………….………………………….7,0 P eptone………………………………………………………………. …………………4,0 Lactose……………………………………………………………………….……….…9,0 B i l e sal t s No. 3……………………………………………………………………….….1,5 Neut ral red……………………………………………………. ……………………….0,03 C rys t al viol et…………………………………………………………………………0,002 MUG (4-methylumbel l i feryl B-D-glucuronide) ……………………………………….0,1 S odium chloride…………………………………. .………………………………….…4,5 Di sodium phosphate……………………………………………………………………1,0 Agar…………………………………………………………………………………. 13,0* * W hen t es t i ng for Escher i chia col i, add the MUG reagent, if not already included in t he media, according to t he manufacturer’s instructions Note: (i ) T he preparat i on of t he samples should not be carried out in direct sunl ight ; and (i i ) normal asept i c precaut ions should be t aken when necessary. (4) T he t es t shal l be conducted as fol l ows: (a) P repare di l ut i ons so as t o obtain plates with colony counts of more than 10, if poss ibl e, and fewer t han 150. In the case of milk and liquid dairy products, make sure t hat t he micro-organisms in the test sample are distributed as evenly as poss ibl e by invert i ng t he sample container 25 times. If foam is formed, it should be al l owed to disperse. The interval between mixing and removing the t es t port i on should not be longer than three minutes. Remove 1ml of the test sample wi th a s t eri l e pipet te and add to 9ml of the diluent (or 10ml of the test sample t o 90ml of the diluent or 11ml of the test sample to 99ml of the diluent). S hake thi s primary dilution thoroughly. In this way, a 10-1 dilution is obtained. (b) Now prepare further dilutions by transferring, using a sterile pipette, 1ml of the primary di lut i on to another test tube containing 9ml of sterile diluent, avoiding contact between the pipette and the diluent. A fresh pipette should be used for each di lut i on. Al t ernat ively, t ransfer 10ml of the primary dilution to a bottle containing 90ml of t he s t eri l e di l uent , or 11ml of t he primary dilution to 99ml of the sterile di l uent . Mix thoroughly ei t her by aspirating 10 times with a fresh pipette or by mixing mechanical ly for 5 t o 10 seconds to obtain the 10-2 dilution. The frequency of rot at i on i n the case of mechanical mixing shall be such that the liquid moves two or t hree cent imeters up the side of the vessel while being mixed. If necessary, repeat t hi s procedure, us ing the 10-2 and further dilutions to obtain 10-3, 10-4, et cet era, di l ut i ons unt i l t he appropriate number of micro-organisms has been obtained. Note: T he t ime l apse between the initial measurement of the test portion, the preparat i on of t he primary dilution and the mixing of the dilutions and mediums shal l not be l onger t han 15 minutes . 14 (c) Use a pipet t e t o transfer 1ml of the liquid product or the appropriate dilutions to t he cent er of two pet ri dishes. Touch a dry area in the petri dish with the tip of t he pipet t e. Use a fresh pipet t e t o i noculat e each di lut i on. (d) P our about 15ml of t he VR B MUG agar at 45oC +/- 1oC into each petri dish. Mix immediat ely aft er pouring by rotating the petri dish sufficiently to obtain evenly di spersed colonies aft er i ncubat ion. Al low to solidify on a cool hori zontal surface. Aft er complet e sol idification, pour about 4ml of the VRB agar at 45oC +/- 1oC onto t he surface of t he i noculat ed medium and allow to solidify. Prepare a cont rol di sh wi th 15ml of t he medium to check i t s s t eri l i t y . Note: In order t o ensure t hat the temperature of the medium is 45oC +/- 1oC before pouring, pl ace a thermometer into a 1,5% agar solution portion i n a separat e container i dent i cal to that used for the medium. This cont rol port i on should be exposed to the same heating and cooling as t he medium. (e) Incubat e t he pl at es i n an adverted position. Do not stack them more than six high. S t ack of pl at es should be separated from one another and from the sides and top of t he i ncubator. Incubat e 30oC +/- 1oC into for 24oC +/- 2 hours. (f) Examine the pl at es under a 366 nm ultra violet light. All colonies showing a blue fl uorescence i n t he surrounding medium are counted. Then examine the pl at es under normal l i ght and count the coliform organisms. Select the plates wi th more t han 10 and fewer than 150 colonies. Count the dark red-coloured colonies wi th a diameter of at least 0,5mm, characteristic of coliform organisms. T hese dark pink to red colonies are usually surrounded by a red zone in the medium. C onfi rm the count by fol l owing the procedure described in subparagraph (g). C al culate the number of coliform organisms per gram or per mi l l i l i t er, t aking into account the result of the confirmatory test. Five or more fl uorescent colonies are regarded as pos i t i ve for Escher i chia col i . (g) T he confi rmatory t es t i s done by inoculat i ng five colonies of each type, if avai l abl e, i nto t ubes of brilliant green lactose bile broth containing a Durham tube and incubating at 30oC +/- 1oC for 24oC +/- 2 hours. Consider colonies that show gas format ion in t he Durham tube to be col i form organi sms . T h e clot-on -b oi l in g tes t 6 . (1) T horoughly mix the mi lk before sampl ing. (2) P our 5ml of mi lk i nto a t es t t ube. (3) P l ace t he t ube i n boi l i ng water. (4) Ensure t hat t he l evel of t he boi l i ng water i s higher t han the mi lk l evel . (5) S t and the t es t t ube of mi lk i n t he boi l i ng water for fi ve minutes . (6) R emove the t est tube from the water and tilt the tube almost horizontally without shaking the mi lk i ns ide. (7) W ai t unt i l a t hin fi lm i s formed on the mi lk. (8) T he resul t i s pos i t i ve i f al l t he mi lk clots or if floccules are seen to be adhering to the s ides of t he t ube when i t i s returned to t he vert i cal pos i t i on. Note: C olos t rum in milk will result in a positive clot-on-boiling test result. The heat stability of t he mi lk i s al so affect ed by other factors . S tan d ard p late cou n t 7 . (1) Mix raw mi lk or pas t euri zed milk thoroughly immediately before sampling from bulk: (a) T he 1:10 di lut i on (m/m) of raw or pateurised shall be prepared in the manner set forth i n paragraphs 4(3)(a) and (b) of t hi s Annex. (b) In t he case of mi lk powder and skimmed milk powder the 1:10 dilution (m/m) shal l be prepared as fol l ows: 15 P lace 99ml of s t eri l e di l uent* into a sterile wide-mouthed container equipped wi th a rubber stopper or a screw top and heat it to 47oC +/- 2oC by placing it in a water bath at t hi s t emperature. W eigh 11g of t he powder into a sterile aluminium weighing boat or glass container equipped with a rubber stopper or a screw top and heat i t t o 47o C + / - 2o C by pl acing it in a water bath at this t emperature. Quickly add the powder to the warm diluent and turn the diluent bottle slowly in order t o wet t he powder. T hen shake the bottle 25 times using up and down movement s of 300mm. R eplace the bottle in the water bath for an additional fi ve minutes and shake it at intervals. In order to facilitate the reconstitution of t he powder, a few grams of s t eri l e gl ass beads may be added to the diluent. P repare addi t i onal t enfold dilutions in sterile diluent (at room temperature) as requi red. (2) Us ing a fresh pipet t e, t ransfer 1ml of each of the dilutions at least in duplicate to sterile pet ri di shes , beginning wi th t he highes t concentration and ending with the lowest. (3) T o each di sh add 10ml of t he s t andard pl at e count agar** which has been melted beforehand and cooled to 45o C + / - 1o C . (4) Mix the contents of each dish thoroughly using horizontal rotational movement while the medium i s s t i l l fl uid. (5) Once the medium has set, invert the dishes and incubate at 30oC +/- 1oC for 72+/-2 hours. (6) At t he end of t he i ncubat ion period remove the dishes from the incubator and count the colony-forming uni t s (C F U) wi th t he aid of magnification under uniform artificial i l l uminat ion. (7) T o count t he C F Us of each di sh, spreader-free dishes containing 30 – 300 CFUs are sel ect ed; count al l t he C F Us and cal cul at e the number of CFUs per ml or per gram. (8) If t he number of C F Us of each dish exceeds 300, count the CFUs in portions of the dish represent at i ve of t he C FU distribution and use this count to determine the total number per di sh. P roceed as i n (7) above, but record as an “ es t imated” pl at e count. * Di lu en ts : Phosphate buffer solut ion: P otass ium dihydrogen orthophosphate………..……………….……………………..5,08g Di sodium hydrogen orthophosphate………………………………………………..13,63g In 2l di s t i l l ed water OR Peptone sal ine solut ion P eptone………………………………………………………………………….….…1,0g S odium chloride…………………. ……………………………………………………8,5g In 1l di s t i l l ed water Di ssolve t he component s i n t he water, heat ing i f necessary. Adjust the pH so that, after s t eri l i zat i on, i t i s 7 , 0+ / -0, 1 at 25o C . ** Plate cou n t agar T ryptone (pancreat i c diges t i ve product of casein)…………………..…………………5g Yeas t ext ract………………………………………………………………….………2,5g Glucose………………………………………………………………………………….1g Agar (bact eri al grade)……………………………………………………..…………..15g Di s t i l l ed water……………………………………………………………………..……1l F inal pH of s t eri l i zed medium………………………….………………………..7,0+/-0,1 S t eri l i se for at l eas t 15 minutes at 121o C . T i tratab le acid i ty 8 . (1) P ipet t e 9ml of mi lk i nto a whi t e di sh. (2) Add ei t her 10 drops or 0 , 5 ml of a 1 , 6% phenolphthalein indicator solution in 50% ethanol t o t he mi lk. 16 (3) T i t rat e wi th 0, 1N NaOH solution until the first tinge of pink appears, that persists for 30 seconds . (4) T o express t he t i t rat able acidity of the milk as the percentage of lactic acid, divide by 10 the number of mi l l i l i t ers of 0 , 1 NaOH used in t he t es t . S tab i l i ty tes t wi th eth an ol 9 . Mix one volume of 68% (v/v) aqueous ethanol with one volume of milk or cream. If there are no s igns of coagulat i on, t he milk or cream shall be deemed to have passed the ethanol stability test. Dry reh yd rated f i lm meth od f or s tan d ard colon y cou n t 10. (1) Mix mi lk or cream thoroughly before sampl ing from bulk. (2) P repare a 1:10 di lut i on by adding 1ml to 9ml of sterile phosphate buffer. Mix well. P repare a 1:100 di lut i on by adding 1ml of the 1:10 dilution to 9ml of sterile phosphate buffer. Mix wel l . Prepare a 1:1 000 dilution by adding 1ml of the 1:100 dilution to 9ml of s t eri l e phosphate buffer. T he fi nal pH should be between 6, 6 and 7, 4. (3) P l ace t he fi lms for aerobic bact eri al counting on a flat surface and label them. Lift the t op fi lm and careful ly transfer 1ml of the 1:1 000 dilution to the center of the bottom film by holding the pipet t e perpendicular to the film. Release the top film to drop onto the sample. R epeat t he process wi th t he 1:100 di lut i on of t he sample. (4) Di s t ri but e t he sample evenly on the film by applying gentle downward pressure with a spreader. R emove the spreader and leave the film undisturbed for one minute to solidify. (5) S t ack the fi lms in pi l es of not more than 20 and incubate the films, with the clear sides up, at 32o C + / - 1o C for 48+ / -2 hours . (6) R emove the fi lms from the i ncubator at the end of the incubation period and count the colony forming uni t s (C F Us) wi th t he aid of magnification under uniform artificial i l l uminat ion as fol l ows: (a) Al l t he red colonies , regardless of their size and intensity, should be counted. F i lms wi th 25-250 C F Us should be counted. Calculate the number of viable bact eri a per mi l l i l i t er of mi lk. (b) An es t imated count can be made on films with the CFUs exceeding 250 by count ing at l eas t four squares or 20 per cent of the growth area. Calculate the number of vi abl e bact eri a per mi l l iliter of milk and record as an “estimated” amount . (c) T he presence of very high concentrations of colonies results in the entire growth area of t he fi lm becoming red or pink in colour and/or numerous bacteria growing on the edges of t he growth zone. Report these as too numerous to count (T NT C ). Ph osp h ate b u f f er P ot ass ium dihydrogen orthophosphate…………………………………………………………5,08g Di sodium hydrogen orthophosphate in 2l distilled water…………………………………….13,63g S t eri l i ze for 15 minutes at 121o C Dry reh yd rated f i lm f or s tan d ard colon y cou n t % sol ids on film C old water solubl e gel……………………………………………………………………..…1-10% T et razol ium indi cator dye…………………………………………………………………….<1% S tandard method nut ri ent s……………………………………………………………………1-5% Dry reh yd rated f i lm meth od f or col i f orm an d Escher i chia col i cou n t 11. (1) Mix mi lk t horoughly before sampling from bulk. The pH should be between 6,6 and 7,4. 17 (2) P l ace t he fi lms for Escher i chia col i and coliform counting on a flat surface and label t hem. Li ft t he t op fi lm and transfer 1ml of the milk to the center of the bottom film, by holding the pipet t e perpendicul ar t o t he fi lm . (3) S lowly rol l t he t op fi lm onto the sample to prevent air bubbles being trapped under the t op fi lm . (4) Di s t ri but e t he sample evenly on the film by applying gentle downward pressure with a spreader. R emove the spreader and leave the film undisturbed for one minute to solidify. (5) S t ack the fi lms in piles of no more than 20 and incubate the films, with the clear sides up, at 32o C + / - 1o C for 24+ / -2 hours . (6) At t he end of t he i ncubat ion period remove the films from the incubator and count the colonies wi th t he aid of magni fication under uniform artificial illumination as follows (R e-incubate fi lms for a further 24+/-2 hours to detect any additional Escherichia coli growth): (a) B lue colonies associ at ed wi th gas are Escher i chia coli and red colonies associ at ed wi th gas are coliform colonies. Colonies that are not associated with gas are not counted as coliform colonies. All the red and blue colonies with gas represent t he col i form colony count . (b) F i lms wi th 15 - 150 colonies should be counted. An estimated count can be made on fi lms where t he colonies exceed 150 by counting at least 4 squares or 20 per cent of t he growth area. Calculate the number of viable coliform colonies per mi l l i l i t er of mi lk and report i t as an “estimated” coliform colony count. (c) T he presence of very high concentrations of colonies causes the entire growth area of t he fi lm to become purple blue (Escherichia coli) or reddish (coliforms) and/or many smal l colonies and/or small gas bubbles to be present. This must be recorded as t oo numerous t o count (T NT C ). Dry reh yd rated f i lm f or col i f orm an d Escher i chia col i cou n ts % of solid on plate Viol et red bi l e nut ri ent s…………………………………………………………………...…1-5% C old water solubl e gel…. ………………………………………………………………….1-10% T et razol ium indi cator dye……………………………………………………………………<1% Glucuronidase i ndi cator……………………………………………………………………..<1% ANNE XURE B PAS T E URIS AT ION 1 . T he pas t euri zat i on of mi lk shal l be performed (a) by heat ing every part i cl e of t he milk to as temperature of at least 63oC (not exceeding 65, 5o C ) and keeping it at that temperature for at least 30 minutes, which heating shall be fol l owed by cool ing wi thin 30 minutes to a temperature lower than 5oC (this process is referred t o as t he “ holder method” or t he “ bat ch method”); or (b) by heat ing every part i cl e of t he milk to a temperature of at least 72oC and keeping it at t hat t emperature for at least 15 seconds, which heating shall be followed immediately by cool ing to a t emperature l ower t han 5o C (this process is hereinafter referred to as the “ high-t emperature short -t ime method”); or (c) by any other method prescribed by regulat i on: P rovided that mi lk shal l i n no instance be deemed to have been pasteurized if it fails to pass t he Aschaffenburg and Mullen phosphate test descried in paragraph 3 of Annexure A or any other t es t , provided the accuracy thereof equals that of the Aschaffenburg and Mul l en phosphatase t es t . 2 . C ream and mi lk or dai ry products containing added sweeteners shall be pasteurized as follows: (a) by heat ing every particle of the product to a temperature of at least 66oC and keeping it at t hi s t emperature for at l eas t 30 minutes ; or 18 (b) by eat i ng every part i cl e of the product to a temperature of at least 74oC and keeping it at t hi s t emperature for at l eas t 15 seconds ; or (c) by any other method prescribed by regulat i on: P rovided that such product shall in no instance be deemed to have been pasteurized if it fai l s t o pass t he Aschaffenburg and Mullen phosphatase test described in paragraph 3 of Annexure A or any other t es t , provided the accuracy thereof equal s that of the Ascaffenburg and Mul l en phosphatase t es t . 3 . T he process of pas t eurization, if carried out according to the high-temperature short-time method, shal l be mechanical ly cont rolled with regard to the temperature range of the milk and the period for which mi lk i s kept at t he prescribed t emperature, and the apparatus concerned shall be cal i brat ed monthly t o ensure t he correctness of t he pas t euri zat i on process . 4 . T hermographic recording of t emperatures of pasteurization byany method shall be made and ret ained for at l eas t four weeks . ANNE XURE C L OCAL AUT HORIT IE S IN WHOSE AREAS OF JURISDICTION RAW DAIRY PRODUCTS L IS T E D IN RE GUL AT IONS (1) MAY B E S OL D Albert i ni a Al iwal North B el fas t B enoni B ethal B oksburg C arol ina C enturion C locolan C olesberg De Aar El l i s ras /Marapong Ermelo Excel s ior F i cksburg Great B rak R iver Great er W armbaths Hanover Harri smi th James town Kos t er Ladi smi th Ladybrand Ladysmi th Lady Grey Machadodorp Malmesbury Marquard Mashae-F ouri e Middelburg Nyl s t room P aul R oux P arys P et rusvi l l e P et rus S t eyn/Mamafubedu 19 P halaborwa P hi l l i ps town P iet ermari t zburg-Msunduzi P i et ersburg/P olokwane P i et R et i ef P rince Albert R eddersburg R ichmond S enekal S t eynsrus /Mat lwagt lwang S t rydenburg S wart ruggens Volksrus t W akkers t room W aterval -B oven W epener W est C oas t W inel ands T zaneen Zeerus t