1FINAL DRAFT  
STANDARDS FOR ETHNIC MILK–BASED CONFECTIONERIES ( PASTILLAS AND 
YEMA ) 
 
1. SCOPE 
 
This standard shall apply to ethnic milk-based confectio neries, specifically pastillas  
(milk candy) and yema (custard candy), in suitable packaging materials or 
containers. 
  
2. DEFINITION OF TERMS 
 
For the purpose of this standard, the following terms sh all mean: 
 
Confectionery - a group of food items primarily made of sugar and ot her 
sweeteners, and includes candies, caramels, toffees, and choc olate bars.  It is a 
generic term for sweetened food products.  Sugar confecti onery refers to products 
such as sweets, candy and chocolates.  These products are sh elf-stable and usually 
have water activity below 0.85. (Dictionary of Food S cience and Technology, 
International Food Information Service, Blackwell Pub lishing, UK, 2005.)  
 
Container - any form of packaging material, which completely or par tially encloses 
the food (including wrappers). A container may enclose  the food as a single item or 
several units or types of prepackaged food when such is pre sented for sale to the 
consumer. 
 
Current Good Manufacturing Practices (cGMP)  - a quality assurance system 
aimed at ensuring that products are consistently manufactu red, packed or repacked 
or held to a quality appropriate for the intended u se. It is thus concerned with both 
manufacturing and quality control procedures. 
                                                              
 2Flavor and flavoring substances - substances which are added to impart flavor 
which are either natural, nature identical or artifi cial flavoring substances (A.O. No. 
88-B s. 1984; Rules and Regulations governing the Labe ling of Prepackaged Food 
Products distributed in the Philippines).   
(a) natural flavor  
flavoring substances derived through appropriate physica l processes from spices, 
herbs, fruit or fruit juices, vegetable or vegetable j uices, edible yeast, bark, bud, root, 
leaf or plant materials, meat, fish, poultry, eggs, da iry products or fermentation 
products thereof. 
(b) nature-identical flavoring substances  
substances chemically derived from aromatic materials or ob tained synthetically, 
which are chemically identical to substances present in' natural products intended for 
human consumption. 
(c) artificial flavoring substances  
substances that impart flavor but which have not been id entified in natural products 
or natural sources of flavorings. 
 
Food - any processed substance which is intended for human consump tion and 
includes drink for man, beverages, chewing gum and any su bstances which have 
been used as an ingredient in the manufacture, prepar ation or treatment of food. 
(RA 9711 Food and Drug Administration (FDA) Act of 20 09). 
 
Food additives - any substance the intended use of which results or may 
reasonably be expected to result, directly or indirectl y, in its becoming a component 
or otherwise affecting the characteristics of any food (in cluding any substance 
intended for use in producing, manufacturing, packing, p rocessing, preparing, 
treating, packaging, transporting, or holding food; an d including any source of 
radiation intended for any such use), if such substance is not generally recognized, 
among experts qualified by scientific training and exp erience to evaluate its safety, 
as having been adequately shown through scientific proce dures to be safe under the 
conditions of the intended use (R.A. 3720. Food, Drug and Cosmetic Act).   
                                                              
 3Food and Drug Administration or FDA  - formerly known as Bureau of Food and 
Drug (BFAD) of the Department of Health (DOH); which  was renamed in accordance 
to RA 9711 (Food and Drug Administration (FDA) Act of  2009). 
 
Food standard  - a regulatory guideline that defines the identity of a given food 
product (i.e. its name and the ingredients used for i ts preparation) and specifies the 
minimum quality factors and, when necessary, the require d fill of the container. It 
may also include specific labeling requirements other tha n or in addition to the 
labeling requirements generally applicable to all pre packaged foods. 
 
Ingredient - any substance including food additive, used as a component  in the 
manufacture or preparation of a food and present in the final product in its original or 
modified form.  
 
Label - includes any tag, brand, mark, pictorial, or other descri ptive script, written, 
printed, marked, embossed or impressed on, or attached to the container. 
 
Labeling - any written, printed or graphic matter (1) upon an y article or any of its 
container or wrappers and/or (2) accompanying the package d food. 
 
Lot - food produced during a period of time and under more  or less the same 
manufacturing condition indicated by a specific code. 
 
Milk - the normal mammary secretion of milking animals obtaine d from one or more 
milkings without either addition to it or extraction f rom it, intended for consumption as 
liquid milk or for further processing (CODEX STAN 206-1 999). 
 
Milk product - a product obtained by any processing of milk, which may co ntain 
food additives, and other ingredients functionally ne cessary for the processing 
(CODEX STAN 206-1999). 
                                                              
 4Packaging - the process of packing that is part of the production cycle  applied to a 
bulk product to obtain the finished product. Any materi al, including painted material, 
employed in the packaging of a product including any o uter packaging used for 
transportation of shipment. Packaging materials are ref erred to as primary or 
secondary according to whether or not they are intended  to be in direct contact with 
the product. 
 
Rancidity - formation of off-flavors in food due to lipid oxidat ion (oxidative rancidity) 
and/or release of free fatty acids by lipolysis (hydrol ytic rancidity). 
 
Water activity - the ratio of vapor pressure of water in the food substr ate to the 
vapor pressure of pure water at the same temperature ( Jay et. al. , 2005). It is also a 
measure of water available for chemical reactions and m icrobial growth (Fennema, 
1996). 
 
3.   DESCRIPTION OF PRODUCTS 
 
3.1 Product Definition 
Ethnic milk-based confectioneries are confectionery produ cts with milk or milk 
products and sugar as basic ingredients. The ethnic milk-ba sed confectioneries 
specifically covered by this standard are the following: 
 
3.1.1 Pastillas – This is also known as milk candy, or milk fudge. The produ ct is 
made from milk or milk products, and sugar. In place of f resh milk and sugar, a 
combination of powdered milk and condensed milk could al so be used. Other 
ingredients such as fruits and nuts could be added. The pr oduct mixture is formed 
into thin cylinders, sticks, or balls, and may be rolled  in sugar.  
 
3.1.2 Yema  – The product may also be called custard candy. Yema  is traditionally 
made from egg yolks cooked with sugar and milk. It is now commonly made from 
condensed milk, and eggs. Since condensed milk is already sw eetened, the 
formulation usually does not require additional sugar.  Other ingredients such as root                                                              
 5crops, fruits, and nuts could also be added. The product  mixture could be formed 
into pyramid-like shapes/forms. It could also be forme d into balls and dipped in 
caramel glaze. 
 
3.2 Process Description 
The product is prepared by mixing the ingredients toge ther and cooking over low 
heat to form a thick paste. In the case of pastillas , milk products and sugar could be 
mixed together without cooking. The product shall have undergone a process 
sufficient to ensure quality and shelf life stability a t ambient conditions and shall be 
packed in any suitable container.  
 
4 ESSENTIAL COMPOSITION AND QUALITY FACTORS 
 
4.1  Raw Materials 
  
4.1.1 Basic Ingredients 
(a) Milk and milk products – Must conform to requirement s prescribed by 
PNS/BAFPS 36:2008 (Philippine National Standard for  Fresh Milk), FDA A.O. No. 
132 s. 1970 (Regulation Prescribing the Standard of I dentity and Quality of Milk and 
Milk Products, B-4.12-01), and other applicable food sta ndards.   
 
The preparation of pastillas  and yema  may utilize two forms of milk or milk product, 
namely: 
(1) Liquid milk – This may be as fresh, evaporated, co ndensed milk, and other 
suitable types of liquid milk.  
(2) Powdered milk – This may be as full cream, skimmed po wdered milk, and other 
suitable types of powdered milk.   
(b) Sugar and other sweeteners –  May include table sugar, corn syrup, and other 
similar food items, and must conform to all applicable st andards. In some cases, 
condensed milk also acts as the source of sugar and/or sweeten er since it is already 
sweetened.                                                               
 6(c)  Eggs  – Applicable for yema  only. Must come from fresh eggs, and must comply 
with the requirements prescribed by PNS/BAFPS 35: 200 5 (Philippine National 
Standards for Table Egg), and other applicable food st andards. 
 
4.1.2 Optional Ingredients 
(a) Butter, margarine, and other similar food items  – Must comply with FDA A.O. No. 
243 s. 1975 (Regulation: B-4 Definition and Standar ds of Food; B-4.18 Margarine), 
and other applicable food standards. 
(b) Fruit, vegetables, nuts, and root crops – May includ e fresh items or preserves 
and must conform to all applicable food standards 
(c) Potable Water – water fit for human consumption 
(d) Flavor and Flavoring Substances - All flavor/flavo ring substances as defined in 
subsection 2.4 shall be certified as food grade by the F DA. 
(e) Other ingredients  – May include starch, cocoa powder, and other ingredient s. All 
other ingredients to be used shall be of food grade q uality and conform to all 
applicable food standards. 
 
4.2 Quality Criteria 
       
     4.2.1 General Requirements 
 
      (a) Water Activity 
 The water activity (a W) should not be greater than 0.85 at 25 oC  
     
  (b) Microbiological limits  
The microbiological limits of ethnic milk-based confectio neries ( pastillas and yema)  
shall conform to the standards set for Processed Foods, unde r the food description 
Chocolate products (Bureau Circular No. 01-A, s. 2004; Guidelines for the 
Assessment of Microbiological Quality of Processed Foods,). The microbiological 
limits of ethnic milk-based confectioneries ( pastillas and yema)  shall be as follows*: 
   
  
                                                              
 7   Table 1. Microbiological limits of ethnic milk-based confect ioneries  
    ( pastillas and yema) * 
Test Microorganism n c m M 
Yeast and Molds, cfu/g 5 2 10 2 10 4 
Salmonella/ 25g 10 0 0  
Coliforms, MPN/g 5 2 <1.8 10 2 
SPC/APC, cfu/g 5 2 10 4 10 6 
*Based on Bureau Circular No. 1-A, 2004; Guidelines  for the Assessment of Microbiological 
Quality of Processed Foods; under the Food Descript ion, Chocolate Products 
 
Legend: 
 n –  the number of sample units selected from a lot of  food to be examined 
m – the acceptable level of microorganism determined by  a specified method; the values 
are generally based on levels that are achievable unde r GMP 
M – the level which when exceeded in one or more sampl es would cause the lot to be 
rejected as this indicates potential health hazard or imminent spoilage 
 c – the maximum allowable number of defective or mar ginally acceptable units 
 
 
4.2.2 Types of Defects 
(a) Odor/flavor/color 
A sample unit affected by objectionable odors, flavors a nd colors which are 
indicative of rancidity and yeast and mold growth. 
(b) Presence of yeasts and molds 
The presence of yeasts and molds may signify spoilage and  may be due to improper 
handling of raw materials and finished products during processing, storage, and 
distribution. 
(c) Foreign matter 
The presence in the sample unit of any matter, which h as not been derived from 
ingredients or processing aids used, does not pose a threa t to human health and is 
readily recognized without magnification or is present at a level determined by 
magnification method or any equivalent methods that i ndicates non-compliance with 
good manufacturing practices and sanitation practices. 
 
4.2.3 Classification of “Defectives” 
A container that has any of the type of defects set in 4.2.2 shall be considered as 
“defective”. 
                                                              
 84.2.4 Lot Acceptance 
A lot shall be considered as meeting the applicable qual ity requirements when the 
number of “defectives”, as defined in sub-section 4.2.3,  does not exceed the  
acceptance number of the appropriate sampling plan. 
 
5 FOOD ADDITIVES 
 
5.1 Food additives when used shall be in accordance with the regulations 
established by the Food and Drug Administration (FDA) (Bureau Circular No. 016 
s.2006. Updated List of Food Additives) and/or the Cod ex Alimentarius Commission.  
 
The following food additives listed in, but not limit ed to, Table 2, may be used for the 
manufacture of ethnic milk-based confectioneries ( pastillas  and yema ). 
 
5.2 All others that have not been included in the abo ve list shall be allowed as carry-
over provided they are approved by FDA regulation ( B.C. No. 016 s. 2006; Updated 
List of Food Additives) and shall be in accordance to th e Section 4 of the Preamble 
of the General Standard for Food Additives (GFSA) (C odex Stan 192-1995, Rev. 5 
(2004)).  These additives include those that are used for  the raw materials and other 
ingredients.  
 
Table 2. Food Additives for Ethnic Milk-based Confectionerie s (Pastillas and Yema) * 
(B.C. No.016 s. 2006. Updated List of Food Additives)  
Function  Food Additive  Max. level of usage  
Anti-caking 
agent Polydimethylsiloxane 10 mg/kg** 
50 mg/kg*** 
Antioxidant BHA 100 mg/kg (Fat or oil basis)** 
200 mg/kg (Fat or oil basis)** 
2 mg/kg *** 
BHT 200 mg/kg (Fat or oil basis)** 
90 mg/kg (On dry ingredient, dry weight, dry mix or  
concentrate basis)  ***  
Gallate, propyl 200 mg/kg (Fat or oil basis) ** 
90 mg/kg (On dry ingredient, dry weight, dry mix or  
concentrate basis)  ***  
Tertiary 
Butylhyroquinone 200 mg/kg (Fat or oil basis)** 
Tocopherols 500 mg/kg (Fat or oil basis)** 
150 mg/kg*** 
Color Allura Red AC 348 mg/kg** 
300 mg/kg ***                                                              
 9Amaranth 100 mg/kg ** 
300 mg/kg *** 
Annatto Extracts 25 mg/kg (As total bixin or norbix in) ** 
10 mg/kg *** 
Brilliant Blue FCF 300 mg/kg** 
150 mg/kg*** 
Caramel Colour, Class 
III GMP** 
GMP*** 
Caramel Colour, Class 
IV GMP** 
GMP*** 
Fast Green FCF 100 mg/kg** 
100 mg/kg*** 
Sunset Yellow FCF 400 mg/kg** 
300 mg/kg*** 
Preservative Benzoates 1500 mg/kg (As benzoic acid)  ** 
1000 mg/kg (As benzoic acid)*** 
Hydroxybenzoates, p- 2000 mg/kg (As p-hydroxybenzoi c acid) ** 
Stabilizer Polysorbates 10000 mg/kg** 
5000 mg/kg*** 
Sorbates 2000 mg/kg (As sorbic acid) ** 
1000 mg/kg (As sorbic acid) *** 
Sorbitan Esters of 
Fatty Acids 20000 mg/kg * 
5000 mg/kg *** 
Sweetener Alitame 300 mg/kg* 
Acesulfame Potassium 3500 mg/kg ** 
350 mg/kg*** 
Aspartame 10000 mg/kg ** 
1000 mg/kg *** 
Saccharin 3000 mg/kg** 
100 mg/kg *** 
Sucralose 1500 mg/kg** 
250 mg/kg*** 
*Based on the Food Category System: 5.0 Confectione ry;  
** Based on the Food Category System:5.2 Sugar-base d confectionery including hard and soft 
candy, nougats, etc. other than food categories  05.1, 05.3, and 05.4;  
*** Based on the Food Category System:10.4 Egg-base d desserts (e.g. custards) 
 
6  CONTAMINANTS  
 
The products covered by this standard shall comply with  the maximum limits for 
contaminants and the maximum residue limits for pesticid es and veterinary drugs 
established by the Codex Alimentarius Commission. 
      
7 HYGIENE 
 
7.1    It is recommended that the product covered by the pr ovisions of this standard 
be prepared and handled in accordance with the approp riate sections of the                                                              
 10  Recommended International Code of Practice – General Principles of Food Hygiene 
(CAC/RCP 1 – 1969, Rev. 4-2003), Code of Hygienic Pr actice for Milk and Milk 
Products (CAC/RCP 57-2004), Recommended Internationa l Code Of Hygienic 
Practice for Egg Products CAC/RCP 15-1976 (amended 1978 , 1985) , and/or the 
BFAD A.O. No. 153 s. 2004 - Guidelines, Current Good Manufacturing Practices in 
Manufacturing, Packing, Repacking or Holding Food 
 
7.2    When tested by appropriate methods of sampling and  examination, the product: 
-    shall be free from filth that may pose a hazard  to health; 
-     shall be free from parasites which may represent a hazard to health; 
-  shall not contain any substance originating from micro organisms in amounts  
  which may represent a hazard to health; 
-  shall be free from microorganisms capable of develop ment under normal  
  conditions of storage; and 
-     shall be free from container integrity defects wh ich may compromise the hermetic  
     seal. 
 
8 PACKAGING AND LABELING 
 
 
8.1   Packaging 
The packaging used for ethnic milk-based confectioneries ( pastillas and yema ) 
should be made of suitable food-grade materials, and sh ould be clean, and hygienic. 
The packaging materials used should not adversely affect  product quality and safety. 
Primary packaging used for pastillas and yema may include uncolored cellophane 
paper, and wax paper. 
 
The product must also be packed and sealed in suitable co ntainers that will provide 
additional protection against contamination. The packagi ng material used should be 
able to withstand mechanical, chemical and thermal stresse s encountered during 
normal product handling and distribution. 
  
8.2 Labeling                                                              
 11  Each container shall be labeled and marked with the fol lowing information in 
accordance with FDA’s Labeling Regulation (A.O. 88-B s. 1984; Rules and 
Regulations governing the Labeling of Prepackaged Food  Products Distributed in 
the Philippines): 
(a) The name of the product shall be “ Pastillas ” (Milk Candy) or “ Yema ” (Custard 
Candy). Additional descriptors pertaining to the ingr edients used or the product form 
may also be included (e.g. “ Pastillas de Leche ”, “ Ube Pastillas ”, “Yema  balls”). Other 
local or regional names referring to products similar to  those defined in 3.1 may also 
be included, provided that these names are acceptable i n the area of distribution. 
(b) The complete list of ingredients and food additive s used in the preparation of the 
product in descending order of proportion. 
(c)  The net quantity of content by weight in the met ric system. Other systems of 
measurement required by importing countries shall appe ar in parenthesis after the 
metric system unit. 
(d) The name and address of the manufacturer, packer an d/or distributor of the food. 
(e) Open date marking  
The words “Consume Before” or ”Expiry Date” indicating end of period at which the 
product shall retain its optimum quality attributes at defined storage conditions. 
(f) Lot or code number identifying product lot. 
(g) The words “Product of the Philippines”, or the coun try of origin if imported. 
(h)  Additional requirements 
A pictorial representation of raw material or end-pro duct on the label should not 
mislead the consumer with respect to the raw material or  end-product so illustrated. 
(i) Optional information 
Storage instructions may also be indicated on the label . 
 
8.3  Nutrition Labeling  
Nutrition labeling shall conform to the established r egulations of FDA.  
 
 
9 METHODS OF ANALYSIS AND SAMPLING 
 
9.1 Method of Sampling                                                              
 12  Sampling shall be in accordance with the FAO/WHO Codex  Alimentarius Sampling 
Plans for Prepackaged Foods - CAC/RM 42-1969, Codex Ali mentarius Volume 13, 
1994. 
 
9.2 Determination of Water Activity  
According to the AOAC Official Methods of Analysis, 16th ed., 1995. Method No. 
978.18. 
  
 9.3 Enumeration of Yeast and Mold Count 
 According to the USFDA Bacteriological Analytical Manua l (2001) 
 
 9.4 Isolation of Salmonella  
 According to the USFDA Bacteriological Analytical Manua l (2001) 
 
 9.5 Enumeration of Coliform and E.coli 
 According to the USFDA Bacteriological Analytical Manua l (2001) 
 
9.6 Enumeration of Standard Plate Count 
 According to the USFDA Bacteriological Analytical Manua l (2001) 
  
10 REFERENCES 
 
Abiva, C. C. 2001. A Quick Guide to Filipino Food and Cooking . Anvil Publishing, 
Inc. Pasig City, Philippines. 
 
A.O. No. 88-A s. 1984. Regulatory Guidelines Concerning Food Additives . 
Bureau of Food and Drugs. Department of Health. Alab ang, Muntinlupa City, 
Philippines. 
 
A.O. No. 88-B s. 1984. Rules and Regulations governing the Labeling of 
Prepackaged Food Products distributed in the Philip pines . Bureau of Food and 
Drugs. Department of Health. Alabang, Muntinlupa Cit y, Philippines. 
 
A.O. No. 132 s. 1970. Regulation Prescribing the Standard of Identity and   
Quality of Milk and Milk Products (B-4.12-01). Bureau of Food and Drugs. 
Department of Health. Alabang, Muntinlupa City, Phi lippines. 
                                                              
 13  A.O. No. 243 s. 1975. B-4 Definition and Standards of Food; B-4.18 Margar ine. 
Bureau of Food and Drugs. Department of Health. Alab ang, Muntinlupa City, 
Philippines. 
 
A.O. No. 153 s. 2004. Guidelines, Current Good Manufacturing Practice in 
Manufacturing, Packing, Repacking or Holding Food. Bureau of Food and 
Drugs, Department of Health. Alabang, Muntinlupa Cit y, Philippines. 
 
Association of Analytical Chemists. Official Methods of Analysis Manual.  16 th  ed., 
1995. AOAC International. 481 North Frederick Ave., S uite 500, Gaithersburg, MD 
20877-2417. U.S.A. 
 
B.C. No. 01-A s. 2004. Guidelines for the Assessment of Microbiological Qu ality 
of Processed Foods . Bureau of Food and Drugs. Department of Health. A labang, 
Muntinlupa City, Philippines. 
 
B.C. No.016 s. 2006. Updated  List of Food Additives. Bureau of Food and Drugs, 
Department of Health. Alabang, Muntinlupa City, Phi lippines. 
 
Belitz, H.-D., W. Grosch, and P. Schieberle. 2009. Food Chemistry . 4 th  revised and 
extended ed. Springer-Verlag, Heidelberg, Germany.   
 
Codex Alimentarius Commission. 1995. FAO/WHO Codex Alimentarius 
Commission Manual . Food and Agriculture Organization. Viale delle Ter me di 
Caracalla, 00100 Rome, Italy. 
 
Dictionary of Food Science and Technology , International Food Information 
Service, Blackwell Publishing, UK, 2005  
 
FNRI. 1997. The Philippine Food Composition Tables . Food and Nutrition 
Research Institute, Department of Science and Technology , Bicutan, Taguig, 
Philippines. 
 
Food, definition . ALINORM 04/27/41, para. 88 and Appendix VI. 2005.  Codex 
Alimentarius Commission. Food and Agriculture Organiza tion. Viale delle Terme di 
Caracalla, 00100 Rome, Italy. 
 
Konkel, P. J. 2001. Confectionery Products . Chapter 56. In Compendium of 
Methods for the Microbiological Examination of Food s . 4 th  ed. edited by F.P. 
Downes and K. Ito. American Public Health Association, Wa shington, DC, USA. 
 
PNS/BAFPS 35:2005. Philippine National Standard: Table Egg.  Bureau of 
Product Standards, Department of Trade and Industry, Makati City, Philippines. 
 
PNS/BAFPS 36:2008. Philippine National Standard: Fresh Milk.  Bureau of 
Product Standards, Department of Trade and Industry, Makati City, Philippines 
                                                              
 14  PNS/BAFPS 76:2010. Philippine National Standard: Coconut Sap Sugar.  Bureau 
of Product Standards, Department of Trade and Indust ry, Makati City, Philippines 
 
PNS/BAFPS 81:2010. Philippine National Standard: Raw Cane Sugar.  Bureau of 
Product Standards, Department of Trade and Industry, Makati City, Philippines 
 
PNS/BAFPS 82:2010. Philippine National Standard: White Sugar.  Bureau of 
Product Standards, Department of Trade and Industry, Makati City, Philippines 
 
R.A. 3720. Food, Drug and Cosmetic Act. Bureau of Food and Drugs. Department 
of Health. Alabang, Muntinlupa City, Philippines. 
 
R.A. 9711. Food and Drug Administration (FDA) Act of 2009 . Bureau of Food and 
Drugs. Department of Health. Alabang, Muntinlupa Cit y, Philippines.      
 
Vaclavik, V. and E. Christian. 2008. Essentials of Food Science . 3 rd  edition. 
Springer Science+Business Media, LLC., New York, USA. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                              
 15   
ANNEX 1 
 
FAO/WHO Alimentarius Sampling Plan for Prepackaged Foods 
(AQL=6.5) CAC/RM 42-1969  
 
1. Sampling Plan 1 (Inspection Level I, AQL = 6.5) 
1.1 Net Weight is equal to or Less Than 1 kg (2.2 lb) 
 
 
1.2 Net Weight is Greater than 1 kg (2.2 lb) but not more than 4.5 kg (10 lb) 
 
Lot size (N)  Sample size (n)  Acceptance No. (c)  
2,400 or less 6 1 
2,841 –15,000 13 2 
15,001 – 24,000 21 3 
24,001 – 42,000 29 4 
44,001 – 72,000 48 6 
72,001 – 120,000 84 9 
More than 120,000 126 13 
 
 
1.3 Net Weight Greater Than 4.5 kg (10 lb) 
 
 
 
 
 
 
 Lot size (N)  Sample size (n)  Acceptance No. (c)  
4,800 or less 6 1 
4,801 –24,000 13 2 
24,001 – 48,000 21 3 
48,001 – 84,000 29 4 
84,001 – 144,000 48 6 
144,001 – 240,000 84 9 
More than 240,000 126 13 
Lot size (N)  Sample size (n)  Acceptance No. (c)  
600 or less 6 1 
601 –2,000 13 2 
2,001 – 7,200 21 3 
7,201 – 15,000 29 4 
15,001 – 24,000 48 6 
24,001 – 42,000 84 9 
More than 42,000 126 13                                                              
 16  2.Sampling Plan 2 (Inspection Level II, AQL = 6.5) 
 
2.1 Net Weight is Equal To or Less Than 1 kg (2.2 lb) 
 
Lot size (N)  Sample size (n)  Acceptance No. (c)  
4,800 or less 13 2 
4,801 –24,000 21 3 
24,001 – 48,000 29 4 
48,001 – 84,000 48 6 
84,001 – 144,000 84 9 
144,001 – 240,000 126 13 
More than 240,000 200 19 
 
2.2 Net Weight is Greater Than 1 kg (2.2 lb) but not more than 4.5 kg  (10 lb) 
 
Lot size (N)  Sample size (n)  Acceptance No. (c)  
2,400 or less 13 2 
2,841 –15,000 21 3 
15,001 – 24,000 29 4 
24,001 – 42,000 48 6 
44,001 – 72,000 84 9 
72,001 – 120,000 126 13 
More than 120,000 200 19 
 
2.3  Net Weight Greater Than 4.5 kg (10 lb) 
     
 
 
 
 
 
 
 
 
 
 
 Lot size (N)  Sample size (n)  Acceptance No. (c)  
600 or less 13 2 
601 –2,000 21 3 
2,001 – 7,200 29 4 
7,201 – 15,000 48 6 
15,001 – 24,000 84 9 
24,001 – 42,000 126 13 
More than 42,000 200 19                                                              
 17  ANNEX 2 
Determination of Water Activity (AOAC 978.18) 
  
A. Principle 
Water activity, a w, is ratio of vapor pressure of H 2O in product to vapor pressure of pure H 2O at 
same temperature. It is numerically equal to 1/100 of relative humidity (RH) generated by 
product in closed system. RH can be calculated from direct measurement of  
partial vapor pressure or dew point or measured indire ctly by sensors whose physical or electric 
characteristics are altered by RH to which they are expo sed. Instruments are checked or 
calibrated on basis of RH generated by standard salt slush es.  
 
B. Instruments and Systems 
(Select 1 of following instruments or systems to perform  test. Each has different application 
limitations because of interferences from other volatile  components of products being 
measured. check with instrument manufacturer for more specific limitations.)  
 
(a) Change in electrical conductivity of immobilized sa lt solution. – Instrument available from 
Beckman Industrial, Rosemount Analytical Div., 89 Comm erce Rd, Cedar Grove, NJ 07009; 
Nova Sina AG, Andreastrasse 7-11, CH 8050, Zurich,Switz erland; Rotronic Instrument 
Corp., 160 E. Main St, Huntington, NY 11743. Immobi lized salt sensors are affected by 
polyols such as glycerol and glycol and by volatile amin es 
(b) Change in electrical capacitance of polymer thin fi lms. – Instrument available from General 
Eastern Instruments, 50 Hunt St, Watertown, MA 02172.  Polymer thin film sensors are 
affected by CH 3COOH.  
(c) Dew point by chilled mirror technique. – Instrument  available from EG&G, Environmental 
Equipment Division, 217 Middlesex Turnpike, Burlingto n, MA 01803 or General Eastern 
Instruments. Dew point measurements can be affected by co ndensables with lower critical 
temperature than H 2O.  
(d) Longitudinal change in dimensions of water-sorbing fiber. – Instrument available from G Lufft 
Metallbarometerfabrik, D-7, Postfach 692, Neue Weinste ige 22, Stuttgart, Germany.  
(e) Partial water vapor pressure by manometric system. – Partial H 2O vapor pressure 
measurements can be made useless by living products that re spire, such as grains or nuts; 
by active fermentation; or by products that expand e xcessively when subjected to high 
vacuum.  
(f) Relative weight of moisture sorbed by anhydrous hy drophilic solid, e.g., microcrystalline 
cellulose.-see J. Agr. Food chem. 22, 326(1974).  
 
C. Apparatus and Reagents 
(As needed for instrument or system selected.)  
(a) Dew point instrument. – Equipped to measure temp erature to ±0.1°. See 978.18B(c).  
(b) Forced-draft cabinet. – Constant temperature, set t o maintain 25 ± 1°; capacity ≥0.06 m 3 (2 cu 
ft); with access port to accommodate instrument sensor lea ds. Use in conjunction with (c).  
(c) Insulated box with cover. – Large enough to hold t est container, (e), and small enough to fit 
in forced-draft cabinet, (b); with access port to accommod ate instrument sensor leads. 
Protect test container from short-term temperature flu ctuations.  
(d) Manometric system. – Sensitive to pressure differenti al of ± 0.01 mm Hg (1.33 Pa). See 
978.18B(e).  
(e) Test containers. – 120 or 240 mL (4 or 8 oz) wide- mouth or Mason glass jars with Al- or 
Teflon-lined screw caps and gaskets. Check integrity of  cap seals and sensor leads by any 
means available, e.g., ability of system to hold vacuum , using Tesla coil.                                                               
 18  (f) Water bath. – Capable of maintaining temperatur e constant within 0.1° at 25±1°; capacity 
sufficient to hold measuring chamber of selected apparatu s.  
(g) Hydrophilic solid. – Microcrystalline cellulose, Type PH-101 (FMC Corp.,Pharmaceutical and 
Bioscience Division, 1735 Market St, Philadelphia, PA 19 103, or equivalent).  
(h) Reference salts. – ACS reagent grade, fine crystal.  see Table 978.18.  
 
D. Preparation of Reference Salt Slushes 
 
Place selected reference salt in test container to depth of ca 4 cm for more soluble salts (lower 
aw), to depth of ca 1.5 cm for less soluble salts (higher a w), and to intermediate depth for 
intermediate salts. Add H 2O in ca 2 mL increments, stirring well with spatula afte r each addition, 
until salt can absorb no more H 2O as evidenced by free liquid. Keep free liquid to m inimum 
needed to establish saturation of salt with H 2O. Slushes are ready for use upon completion of 
mixing, and are usable indefinitely (except for some h igh a w salts susceptible to bacterial 
attack), if contained in manner to prevent substantial  evaporation losses. Some slushes, e.g., 
NaBr, may solidify gradually by crystal coalescence, with no effect on a w.  
 
E. Calibration 
 
Select ≥ 5 salts to cover a w range of interest or range of sensor being used. Measu re humidity 
generated by each salt slush in terms of instrument read out, as in 978.18F. Plot readout against 
aw values given in Table 978.18 for selected salts, using cross-section paper scaled for reading 
to 0.001 a w unit. Draw best average smooth line through plotted points. Use this calibration line 
to translate sensor instrument readout of samples to a w or to check vapor pressure or dew point 
instruments for proper functioning.  
 
F. Determination 
 
Place calibration slush or sample in forced-draft cabinet , (b), or H 2O bath, (f), until temperature 
is stabilized at 25±1°. Transfer salt slush or sample to test container, (e), seal container with 
sensing device attached, and place in temperature control  device. Use volume of sample or 
slush >1/20 total volume sample container plus any associa ted void volume of sensing system, 
but not so much as to interfere with operation of syst em. Record instrument response at 15, 30, 
60, and 120 min after test container is placed in tem perature control device, or record response 
on strip chart. Two consecutive readings, at indicated i ntervals, which vary by <0.01 a w unit are 
evidence of adequately close approach to equilibrium. Continue readings at 60-min intervals, if 
necessary. Convert last reading to a w by calculation from physical measurements or by 
reference to calibration line. Make all measurements wi thin range of calibration points; do not 
extrapolate calibration line. Make all measurements in  same direction of change, and, if required 
by properties of sensor, expose sensor to controlled RH b elow ambient before starting each 
measurement.                                                               
 19  ANNEX 3 
Enumeration of Yeast and Mold Count 
Enumeration of Yeasts and Molds in Food--Dilution P lating Technique (USFDA, 2001)  
A. Equipment and materials   
1. Basic equipment (and appropriate techniques) for pr eparation of sample homogenate 
2. Equipment for plating samples 
3. Incubator, 25°C 
4. Arnold steam chest 
5. pH meter 
6. Water bath, 45 ± 1° C 
 
B. Media and reagents  
 
1. Dichloran rose bengal chloramphenicol (DRBC) agar 
2. Dichloran 18% glycerol (DG18) agar 
3. Plate count agar (PCA), standard methods; add 100 mg chloramphenicol/liter when this 
medium is used for yeast and mold enumeration. This me dium is not efficient when 
"spreader" molds are present. 
4. Malt agar (MA) 
5. Malt extract agar (Yeasts and Molds) (MEAYM) 
6. Potato dextrose agar (PDA), dehydrated; commerciall y available 
 
C. Procedures  
Sample preparation  
Analyze 25-50 g from each subsample; generally, larger sample sizes increase 
reproducibility and lower variance compared with small samples. Test individual subsamples 
or composite according to respective Compliance Program f or the food under analysis. Add 
appropriate amount of 0.1% peptone water to the wei ghed sample to achieve 10 -1 dilution, 
then homogenize in a stomacher for 2 min. Alternativ ely, blending for 30-60 sec can be used 
but is less effective. Make appropriate 1:10 (1+9) di lutions in 0.1% peptone water. Dilutions 
of 10 -6 should suffice. 
Plating and incubation of sample  
Spread-plate method . Aseptically pipet 0.1 ml of each dilution on pre- po ured, solidified 
DRBC agar plates and spread inoculum with a sterile, bent glass rod. DG18 is preferred 
when the water activity of the analyzed sample is less than 0.95. Plate each dilution in 
triplicate. 
Pour-plate method . Use sterile cotton-plugged pipet to place 1.0 ml por tions of sample 
dilution into prelabeled 15 x 100 mm Petri plates ( plastic or glass), and immediately add 20-
25 ml tempered DG18 agar. Mix contents by gently swir ling plates clockwise, then 
counterclockwise, taking care to avoid spillage on dish l id. After adding sample dilution, add 
agar within 1-2 min; otherwise, dilution may begin t o adhere to dish bottom (especially if 
sample is high in starch content and dishes are plastic) an d may not mix uniformly. Plate 
each dilution in triplicate. 
From preparation of first sample dilution to pouring  or surface-plating of final plate, no more 
than 20 min (preferably 10 min) should elapse. Note: Spread plating of diluted sample is                                                              
 20  considered better than the pour plate method. When th e pour plate technique is used, fungal 
colonies on the surface grow faster and often obscure tho se underneath the surface, 
resulting in less accurate enumeration. Surface plating gives a more uniform growth and 
makes colony isolation easier. DRBC agar should be used for spread plates only. 
Incubate plates in the dark at 25°C. Do not stack pl ates higher than 3 and do not 
invert. Note:  Let plates remain undisturbed until counting. 
Counting of plates  
Count plates after 5 days of incubation. If there is no growth at 5 days, re-incubate for 
another 48 h. Do not count colonies before the end of the incubation period because 
handling of plates could result in secondary growth fro m dislodged spores, making final 
counts invalid. Count plates containing 10-150 colonies. If mainly yeasts are present, plates 
with 150 colonies are usually countable. However, if su bstantial amounts of mold are 
present, depending on the type of mold, the upper cou ntable limit may have to be lowered at 
the discretion of the analyst. Report results in colony  forming units (CFU)/g or CFU/ml based 
on average count of triplicate set. Round off counts to two significant figures. If third digit is 6 
or above, round off to digit above (e.g., 456 = 460 ); if 4 or below, round off to digit below 
(e.g., 454 = 450). If third digit is 5, round off t o digit below if first 2 digits are an even number 
(e.g., 445 = 440); round off to digit above if firs t 2 digits are an odd number (e.g., 455 = 460). 
When plates from all dilutions have no colonies, repor t mold and yeast counts (MYC) as less 
than 1 times the lowest dilution used. 
Isolate individual colonies on PDA or MA, if further a nalysis and species identification is 
necessary. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                              
 21  ANNEX 4 
Isolation of Salmonella (USFDA, 2001) 
A. Sample Preparation  
For candy and candy coating (including chocolate) . Aseptically weigh 25 g sample into 
sterile blending container. Add 225 ml sterile, reconst ituted nonfat dry milk  and blend 2 min. 
Aseptically transfer homogenized mixture to sterile, w ide-mouth, screw-cap jar (500 ml) or other 
appropriate container and let stand 60 ± 5 min at ro om temperature with jar securely capped. 
Mix well by swirling and determine pH with test pap er. Adjust pH, if necessary, to 6.8 ± 0.2. Add 
0.45 ml 1% aqueous brilliant green dye solution and mix well. Loosen jar caps 1/4 turn and 
incubate 24 ± 2 h at 35°C. Continue as in B., below. 
 
For egg-containing products (noodles, egg rolls, ma caroni, spaghetti), cheese, dough, 
prepared salads (ham, egg, chicken, tuna, turkey), fresh, frozen, or dried fruits and 
vegetables, nut meats, crustaceans (shrimp, crab, c rayfish, langostinos, lobster), and 
fish . Preferably, do not thaw frozen samples before analy sis. If frozen sample must be 
tempered to obtain analytical portion, thaw below 45 °C for <15 min with continuous agitation in 
thermostatically controlled water bath or thaw within 18 h at 2-5°C. 
Aseptically weigh 25 g sample into sterile blending con tainer. Add 225 ml sterilelactose 
broth  and blend 2 min. Aseptically transfer homogenized mi xture to sterile, wide-mouth, screw-
cap jar (500 ml) or other appropriate container and l et stand 60 ± 5 min at room temperature 
with jar securely capped. Mix well by swirling and det ermine pH with test paper. Adjust pH, if 
necessary, to 6.8 ± 0.2. Mix well and loosen jar cap ab out 1/4 turn. Incubate 24 ± 2 h at 35°C. 
Continue as in B., below. 
 
B. Isolation of Salmonella   
1. Tighten lid and gently shake incubated sample. 
Guar gum and foods suspected to be contaminated wit h S. Typhi . Transfer 1 ml mixture to 
10 ml selenite cystine (SC) broth and another 1 ml mixture to 10 ml TT broth . Vortex. 
All other foods . Transfer 0.1 ml mixture to 10 ml Rappaport-Vassiliadis (RV) medium  and 
another 1 ml mixture to 10 ml  tetrathionate (TT) b roth . Vortex. 
2. Incubate selective enrichment media as follows: 
Foods with a high microbial load . Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulati ng, 
thermostatically-controlled, water bath). Incubate TT broth 24 ± 2 h at 43 ± 0.2°C (circulating, 
thermostatically-controlled, water bath). 
Foods with a low microbial load (except guar gum an d foods suspected to be 
contaminated with S. Typhi) . Incubate RV medium 24 ± 2 h at 42 ± 0.2°C (circulati ng, 
thermostatically controlled, water bath). Incubate TT b roth 24 ± 2 h at 35 ± 2.0°C. 
Guar gum and foods suspected to be contaminated wit h S. Typhi . Incubate SC and TT 
broths 24 ± 2 h at 35°C.                                                              
 22  3. Mix (vortex, if tube) and streak 3 mm loopful (10 µl) incubated TT broth on bismuth sulfite 
(BS) agar , xylose lysine desoxycholate (XLD) agar , and Hektoen enteric (HE) agar . Prepare 
BS plates the day before streaking and store in dar k at room temperature until streaked.  
4. Repeat with 3 mm loopful (10 µl) of RV medium (f or samples of high and low microbial load 
foods) and of SC broth (for guar gum). 
5. Refer to 994.04 in Official Methods of Analysis  (1) for option of refrigerating incubated 
sample preenrichments and incubated sample selective enri chments (SC and TT broths only) 
of low moisture foods. This option allows sample analyses to be initiated as late as Thursday 
while still avoiding weekend work. 
6. Incubate plates 24 ± 2 h at 35°C. 
7. Examine plates for presence of colonies that may be Salmonella . 
8. Lightly touch the very center of the colony to be p icked with sterile inoculating needle and 
inoculate TSI slant by streaking slant and stabbing bu tt. Without flaming, inoculate LIA slant by 
stabbing butt twice and then streaking slant. Since lysin e decarboxylation reaction is strictly 
anaerobic, the LIA slants must have deep butt (4 cm). Store picked selective agar plates at 5-
8°C. 
9. Incubate TSI and LIA slants at 35°C for 24 ± 2 h. C ap tubes loosely to maintain aerobic 
conditions while incubating slants to prevent excessive H 2S production. Salmonella  in culture 
typically produces alkaline (red) slant and acid (yellow ) butt, with or without production of H 2S 
(blackening of agar) in TSI. In LIA, Salmonella  typically produces alkaline (purple) reaction in 
butt of tube. Consider only distinct yellow in butt of  tube as acidic (negative) reaction. Do not 
eliminate cultures that produce discoloration in butt of  tube solely on this basis. Most 
Salmonella  cultures produce H 2S in LIA. Some non- Salmonella  cultures produce a brick-red 
reaction in LIA slants. 
10.  All cultures that give an alkaline butt in LIA,  regardless of TSI reaction, should be 
retained as potential Salmonella  isolates and submitted for biochemical and serological te sts. 
Cultures that give an acid butt in LIA and an alkalin e slant and acid butt in TSI should also be 
considered potential Salmonella  isolates and should be submitted for biochemical and 
serological tests. Cultures that give an acid butt in LI A and an acid slant and acid butt in TSI 
may be discarded as not being Salmonella  . Test retained, presumed-positive TSI cultures as 
directed in D-11 , below, to determine if they are Salmonella  species, including S. arizonae . If 
TSI cultures fail to give typical reactions for Salmonella  (alkaline slant and acid butt) pick 
additional suspicious colonies from selective medium plate not giving presumed-positive 
culture and inoculate TSI and LIA slants as described, a bove. 
11.  Apply biochemical and serological identification t ests to: 
a. Three presumptive TSI cultures recovered from set o f plates streaked from RV medium (or 
SC broth for guar gum), if present, and 3 presumptive  TSI agar cultures recovered from 
plates streaked from TT broth, if present. 
b. If 3 presumptive-positive TSI cultures are not isolat ed from one set of agar plates, test other 
presumptive-positive TSI agar cultures, if isolated, by bioche mical and serological tests. 
Examine a minimum of 6 TSI cultures for each 25 g ana lytical unit or each 375 g composite. 
 
 
 
 
 
 
 
                                                              
 23  ANNEX 5 
Enumeration of E.coli  and Coliform Count 
(Enumeration of Escherichia coli  and the Coliform Bacteria (USFDA, 2001)) 
Conventional Method for coliforms, fecal coliforms and E. coli  
A. Equipment and materials 
1. Covered water bath, with circulating system to maint ain temperature of 45.5 ± 0.2°C. Water 
level should be above the medium in immersed tubes. 
2. Immersion-type thermometer, 1-55°C, about 55 cm l ong, with 0.1°C subdivisions, certified 
by National Institute of Standards and Technology (NIS T), or equivalent 
3. Incubator, 35 ± 1.0°C 
4. Balance with capacity of > 2 kg and sensitivity of 0.1 g 
5. Blender and blender jar 
6. Sterile graduated pipets, 1.0 and 10.0 mL 
7. Sterile utensils for sample handling 
8. Dilution bottles made of borosilicate glass, with pol yethylene screw caps equipped with 
Teflon liners. Commercially prepared dilution bottles containing sterile Butterfield's 
phosphate buffer can also be used. 
9. Quebec colony counter, or equivalent, with magnifyi ng lens 
10. Longwave UV light [~365 nm], not to exceed 6 W. 
11. pH meter 
B. Media and Reagents  
Brilliant green lactose bile (BGLB) broth, 2% (M25 ) 
Lauryl tryptose (LST) broth (M76 ) 
EC broth (M49 ) 
Levine's eosin-methylene blue (L-EMB) agar (M80 ) 
Butterfield's phosphate-buffered water (R11 ) or equivalent diluent (except for shellfish) 
Lauryl tryptose MUG (LST-MUG) broth (M77 ) 
Peptone Diluent, 0.1% (R56 ) 
MPN - Presumptive test for coliforms, fecal colifor ms and E. coli  
Weigh 50 g food into sterile high-speed blender jar. (see Chapter 1 and current FDA compliance 
programs for instructions on sample size and compositing) Frozen samples can be softened by 
storing it for < 18 h at 2-5°C, but do not thaw. Add 450 mL of Butte rfield's phosphate-buffered 
water and blend for 2 min. If <50 g of sample are av ailable, weigh portion that is equivalent to 
half of the sample and add sufficient volume of sterile  diluent to make a 1:10 dilution. The total 
volume in the blender jar should completely cover the blades. 
Prepare decimal dilutions with sterile Butterfield's p hosphate diluent. Number of dilutions to be 
prepared depends on anticipated coliform density. Shake  all suspensions 25 times in 30 cm arc 
or vortex mix for 7 s. Do not use pipets to deliver <10% of their total volume. Transfer 1 mL 
portions to 3 LST tubes for each dilution for at least  3 consecutive dilutions. Hold pipet at angle 
so that its lower edge rests against the tube. Let pip et drain 2-3 s. Not more than 15 min should 
elapse from time the sample is blended until all dilut ions are inoculated in appropriate media.                                                              
 24  NOTE:  Use 5-tube MPN for analysis of shellfish and shellfish ha rvest waters. 
Incubate LST tubes at 35°C. Examine tubes and record r eactions at 24 ± 2 h for gas, i.e., 
displacement of medium in fermentation vial or efferv escence when tubes are gently agitated. 
Re-incubate gas-negative tubes for an additional 24 h and examine and record reactions again 
at 48 ± 2 h. Perform confirmed test on all presumptiv e positive (gas) tubes. 
MPN - Confirmed test for coliforms  
From each gassing LST tube, transfer a loopful of suspen sion to a tube of BGLB broth, avoiding 
pellicle if present. Incubate BGLB tubes at 35°C and ex amine for gas production at 48 ± 2 h. 
Calculate most probable number (MPN) of coliforms based  on proportion of confirmed  gassing 
LST tubes for 3 consecutive dilutions. 
MPN - Confirmed test for fecal coliforms and E. coli  
From each gassing LST tube from the Presumptive test, tr ansfer a loopful of each suspension to 
a tube of EC broth (a sterile wooden applicator stick ma y also be used for these transfers). 
Incubate EC tubes 24 ± 2 h at 45.5 °C and examine for  gas production. If negative, reincubate 
and examine again at 48 ± 2 h. Use results of this tes t to calculate fecal coliform MPN. To 
continue with E. coli  analysis, proceed to Section F under Enumeration of Esch eria coli and the 
Coliform Bacteria of the USFDA Bacteriological Analyt ical Manual (2001). The EC broth MPN 
method may be used for seawater and shellfish since it con forms to recommended procedures 
(1). (Caution: see Note below). 
NOTE:  Fecal coliform analyses are done at 45.5± 0.2°C for al l foods, except for water testing 
and in shellfish and shellfish harvest water analysis, whi ch uses an incubation temperature of 
44.5± 0.2°C. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                              
 25  ANNEX 6 
Enumeration of Standard Plate Count 
Conventional Plate Count Method (USFDA, 2001) 
A. Equipment and materials 
1. Work area, level table with ample surface in room t hat is clean, well-lighted (100 
foot-candles at working surface) and well-ventilated, and reasonably free of dust 
and drafts. The microbial density of air in working a rea, measured in fallout pour 
plates taken during plating, should not exceed 15 coloni es/plate during 15 min 
exposure. 
2. Storage space, free of dust and insects and adequate for protection of equipment 
and supplies 
3. Petri dishes, glass or plastic (at least 15 x 90 mm) 
4. Pipets with pipet aids (no mouth pipetting) or pip ettors, 1, 5, and 10 ml, 
graduated in 0.1 ml units 
5. Dilution bottles, 6 oz (160 ml), borosilicate-resista nt glass, with rubber stoppers 
or plastic screw caps 
6. Pipet and petri dish containers, adequate for prote ction 
7. Circulating water bath, for tempering agar, therm ostatically controlled to 45 ± 1°C 
8. Incubator, 35 ± 1°C; milk, 32 ± 1°C 
9. Colony counter, dark-field, Quebec, or equivalent , with suitable light source and 
grid plate 
10. Tally register 
11. Dilution blanks, 90 ± 1 ml Butterfield's phosphat e-buffered dilution water (R11 ); 
milk, 99 ± 2 ml 
12. Plate count agar (standard methods) (M124 ) 
13. Refrigerator, to cool and maintain samples at 0-5° C; milk, 0-4.4°C 
14. Freezer, to maintain frozen samples from -15 to -20°C 
15. Thermometers (mercury) appropriate range; accuracy checked with a 
thermometer certified by the National Institute of S tandards and Technology 
(NIST)  
 
B. Procedure for analysis of frozen, chilled, precoo ked, or prepared foods 
Using separate sterile pipets, prepare decimal dilution s of 10 -2, 10 -3, 10 -4, and others as 
appropriate, of food homogenate ( see  Chapter 1  for sample preparation) by transferring 
10 ml of previous dilution to 90 ml of diluent. Avoi d sampling foam. Shake all dilutions 25 
times in 30 cm (1 ft) arc within 7 s. Pipet 1 ml of each dilution into separate, duplicate, 
appropriately marked petri dishes. Reshake dilution bo ttle 25 times in 30 cm arc within 7 
s if it stands more than 3 min before it is pipetted into petri dish. Add 12-15 ml plate 
count agar (cooled to 45 ± 1°C) to each plate within 1 5 min of original dilutionPour agar 
and dilution water control plates for each series of sam ples. Immediately mix sample 
dilutions and agar medium thoroughly and uniformly b y alternate rotation and back-and-
forth motion of plates on flat level surface. Let agar  solidify. Invert solidified petri dishes, 
and incubate promptly for 48 ± 2 h at 35°C. Do not s tack plates when pouring agar or 
when agar is solidifying.