1FINAL DRAFT  
RECOMMENDED CODE OF PRACTICE FOR THE PROCESSING AND  
HANDLING OF ETHNIC MILK-BASED CONFECTIONERIES ( PASTILLAS 
AND YEMA ) 
 
1. SCOPE  
 
This Code of Practice is concerned with the receipt of raw  materials and ingredients, 
preparation and processing of ethnic milk-based confection eries ( pastillas and yema ) 
as defined in this Code, in order to conform with the required standards stated in 
PNS/FDA No. ___. Standards for Ethnic Milk-based Confect ioneries ( pastillas and 
yema ). The product shall be prepared from a basic mixture of milk and sugar, and 
eggs (for yema  only). This Code is intended to provide guidelines to  achieve 
compliance with the standards for ethnic milk-based confecti oneries ( pastillas and 
yema ) packed in any suitable container. 
 
2.       DEFINITION OF TERMS 
 
For the purpose of this Code, the following definitio ns apply: 
 
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 partially encloses 
the food (including wrappers). A container may enclose t he food as a single item or 
several units or types of prepackaged food when such is p resented for sale to the 
consumer. 
 
Current Good Manufacturing Practices (cGMP)  – a quality assurance system 
aimed at ensuring that products are consistently manufac tured, packed or repacked  2or held to a quality appropriate for the intended u se. It is thus concerned with both 
manufacturing and quality control procedures.  
 
Flavor and flavoring substances – substances which are added to impart flavor 
which are either natural, nature identical or artifici al 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 physical  processes from spices, 
herbs, fruit or fruit juices, vegetable or vegetable juices, 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' na tural products intended for 
human consumption. 
(c) artificial flavoring substances  
substances that impart flavor but which have not been i dentified in natural products 
or natural sources of flavorings. 
 
Food - any processed substance which is intended for human consum ption 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 2009) . 
 
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 (i ncluding any substance 
intended for use in producing, manufacturing, packing, processing, preparing, 
treating, packaging, transporting, or holding food; a nd including any source of 
radiation intended for any such use), if such substance is not generally recognized, 
among experts qualified by scientific training and expe rience 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).    3 
Food and Drug Administration or FDA  - It is 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 (FD A) 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 it s 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 compon ent 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 d escriptive script, written, 
printed, marked, embossed or impressed on, or attached to the container. 
 
Labeling  – any written, printed or graphic matter (1) upon a ny 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 mo re or less the same 
manufacturing condition indicated by a specific code. 
 
Milk – the normal mammary secretion of milking animals obtai ned 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  contain 
food additives, and other ingredients functionally nece ssary for the processing 
(CODEX STAN 206-1999). 
 
Packaging  – 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,  4employed in the packaging of a product including any o uter packaging used for 
transportation of shipment. Packaging materials are refe rred 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 oxi dation (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 sub strate 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.      RAW MATERIALS, INGREDIENTS AND PACKAGING MA TERIAL 
REQUIREMENTS 
 
3.1 Raw materials and ingredients .  
Raw materials for processing shall not contain parasites, m icroorganisms, toxins, 
and decomposed or extraneous substances. 
 
3.1.1 Basic Ingredient 
 
Milk and milk products.  Must conform to requirements prescribed by PNS/BAFPS 
36:2008 (Philippine National Standard for Fresh Milk) , FDA A.O. No. 132 s. 1970 
(Regulation Prescribing the Standard of Identity and Quality of Milk and Milk 
Products, B-4.12-01), and other applicable food standa rds.  
 
Milk and milk products used in the production of ethnic m ilk-based confectioneries 
(pastillas and yema ) should come from healthy animals. Milk and milk prod ucts 
should have been produced under hygienic conditions as re quired by the regulatory 
agency and/or authority for this type of food items. 
 
The preparation of pastillas  and yema  may utilize two forms of milk or milk product, 
namely:  5(1) Liquid milk – This may be as fresh, evaporated, con densed 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.  
 
Sugar and other sweeteners.  May include table sugar, corn syrup, and other 
similar food items, and must conform to all applicable st andards.  
 
Eggs . Applicable for yema  only. Must come from fresh eggs, and must comply with 
the requirements prescribed by PNS/BAFPS 35: 2005 (Ph ilippine National Standards 
for Table Egg), and other applicable food standards. 
 
3.1.2 Optional Ingredients 
  
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.     
 
Fruit, vegetables, nuts, and root crops. May include fresh items or preserves and 
must conform to all applicable food standards 
 
Water . Only clean, potable water (Annex A) shall be used f or the preparation and for 
all the pretreatment and processing steps of confectione ry production.  
 
Non-potable water may be used only for operations not  in direct contact with the food 
materials provided that this does not pose a hazard to  health as determined and 
approved by the official agency having the jurisdiction  over it. 
 
Flavor and Flavoring Substances . All flavor/flavoring substances as defined in section 
2 shall be certified as food grade by the Food and Dru gs Administration (FDA). 
 
Other ingredients . May include starch, cocoa powder, and other ingredie nts. All 
other ingredients to be used shall be of food grade quality and conform to all 
applicable food standards.  6 
3.2     Packaging materials . The primary packaging used shall be made of suitable 
food-grade and inert materials that would not adverse ly affect product quality and 
safety. The packaging materials should be appropriate fo r the product to be packed 
and for the expected conditions of handling during dist ribution and storage. These 
should provide the products adequate protection from co ntamination and should be 
sufficiently durable to withstand mechanical, chemical an d thermal stresses 
encountered during processing and normal distribution. A ll packaging materials must 
be clean and free from defects that may affect the pro duct or package integrity. 
These shall be stored in a clean and sanitary manner.  
 
4. HYGIENE 
  
It is recommended that the product covered by the prov isions of this code of practice 
be prepared and handled in accordance with the appropr iate sections of the 
Recommended International Code of Practice – General P rinciples of Food Hygiene 
(CAC/RCP 1–1969, Rev 4 (2003)), Code of Hygienic Pra ctice for Milk and Milk 
Products (CAC/RCP 57-2004),  Recommended International 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 P ractices in 
Manufacturing, Packing, Repacking or Holding Food, cover ing the plant facilities and 
operations requirement including the construction and l ayout of processing plant, 
hygienic facilities, equipment, utensils and working surfa ces. 
 
5. PREPARATION AND PROCESSING  
 
The preparation of ethnic milk-based confectioneries ( pastillas and yema ) is 
described from the receipt of raw materials until the p acking operations. The 
production process should be supervised by personnel with  adequate technical 
training and experience.  
 
5.1  Preparation of Raw Materials and Ingredients 
 
5.1.1 Milk and Milk Products  7 
Receipt  
Milk and milk products shall only be accepted if it is sou nd and suitable for 
processing, according to the requirements stipulated in sub-subsection 3.1.1. Those 
found with contamination should be rejected. Special pr ecautions must be taken to 
reject milk and milk products showing signs of spoilage, an d deterioration. 
 
If prepackaged processed milk products are to be used as ra w material, choose only 
those contained in clean, non-toxic, and properly labe led packaging materials. 
 
Storage/holding 
Milk and milk products held for processing should be store d in any suitable type of 
closed container to protect from domestic animals, parasite s, chemical or 
microbiological contaminants, debris, and dust.  
 
The storage temperature for milk and milk products should  be sufficient to maintain 
product safety and suitability. Fresh milk and similar f luid milk products must be 
stored at temperatures less than 7°C. Canned and dried  milk products may be stored 
at a cool, dry place. 
 
5.1.2 Sugar and other sweeteners 
 
Receipt and Inspection  
Sugar and sweeteners used for the production of ethnic milk-based confectioneries 
(pastillas and yema ) shall only be accepted if it is sound and suitable f or processing, 
according to the requirements stipulated in sub-subsection  3.1.1. Those found with 
contamination which could not be removed to acceptable l evels by normal plant 
sorting or preparation procedures should be rejected.  
 
Sugars and other sweeteners  must conform to applicable , existing standard 
specifications for white sugar (PNS/BAFPS 82:2010), raw cane sugar (PNS/BAFPS 
81:2010), coconut sap sugar (PNS/BAFPS 76:2010), and o ther types of sweeteners. 
Choose only those contained in clean, non-toxic, and pro perly labeled packaging 
materials.  8 
Storage/holding 
Sugars and sweeteners held for processing should be stored in any suitable type of 
closed container to protect from domestic animals, parasite s, chemical or 
microbiological contaminants, debris, and dust. These ing redients should be stored 
at temperature and relative humidity conditions that will minimize deterioration of 
quality. The storage area should also be free from stro ng odors or aroma. Regular 
inspection of the storage facility should be done to avo id infestation. 
    
5.1.3 Eggs 
 
Receipt  
Egg used for the production of  ethnic milk-based confecti oneries ( pastillas and 
yema ) shall only be accepted if it is fresh and suitable for  processing, according to 
the requirements stipulated in sub-subsection 3.1.1. Tho se found with contamination 
which could not be removed to acceptable levels by norma l plant sorting or 
preparation procedures should be rejected. Special preca utions must be taken to 
reject eggs with broken shells, or those with signs of de gradation, abnormal color or 
odor. 
 
Inspection and sorting  
Eggs shall be inspected and sorted according to quality and size specifications 
before processing (PNS/BAFPS 35:2005. Philippine Natio nal Standard: Table Egg). 
Choose only those contained in clean, non-toxic, and pro perly labeled packaging 
materials. 
 
Storage/holding 
Eggs held for processing should be stored in any suitable type of closed container to 
protect from domestic animals, parasites, chemical or micr obiological contaminants, 
debris, and dust.  
 
Eggs should be stored at temperature and relative humi dity conditions that will 
minimize deterioration of egg quality. Eggs are kept  at refrigerated temperatures 
during storage particularly when these are not immedi ately used for processing. The  9storage area should also be free from strong odors or a roma. Regular inspection of 
the storage facility should be done to avoid infestatio n.    
 
Washing and/or sanitizing 
Eggs are washed to remove dirt, dust, soil, insect, and f ilth that might contaminate or 
affect the color, aroma or flavor of the eggs. Water u sed for washing and rinsing 
should be of potable quality. Only approved sanitizi ng agents and permissible limits 
may be used in the wash or rinse water for eggs. 
 
5.1.4 Optional Ingredients  
 
Receipt 
Optional ingredients to be used in the preparation o f ethnic milk-based 
confectioneries ( pastillas and yema ) shall conform to the requirements stipulated in 
sub-subsection 3.3.2. Whenever applicable, certificates of  analyses (COA) from 
ingredient suppliers shall be secured to confirm their  suitability for processing. 
Ingredients shall be rejected if they do not conform to  the requirements and are 
found to have signs of deterioration, decomposition, or  contamination to an extent 
which renders them unfit for human consumption.   
 
Storage/Holding 
Optional ingredients shall be in closed containers as p rotection against infestation by 
domestic animals, parasites, filth, and chemical and microb iological contaminants. 
Storage requirements such as temperature and humidity  may vary depending on the 
ingredient, and these should be provided accordingly b y the storage facilities to be 
used. 
 
Stored stocks of ingredients should be used on a “first in -first out” (FIFO) or a “first to 
expire-first to use” (FEFU) basis. 
 
5.2  Processing Operations 
 
5.2.1 Preparation  10 If raw milk is used as basic ingredient, pasteurization m ust be done to ensure the 
safety of the product. Sufficient time and temperature  combinations for 
pasteurization must be used to ensure that the milk used  does not pose a health 
hazard. Pasteurization may be done by heating the mi lk at 63–66°C for at least 30–
32 minutes.  
 
Under FDA A.O. No. 132 s. 1970 (Regulation Prescribing  the Standard of Identity 
and Quality of Milk and Milk Products, B-4.12-01), past eurized milk should have 
been subjected to a temperature not lower than 63°C a nd held continuously at that 
temperature for not less than 30 minutes. Pasteurizatio n could also be done by 
keeping the milk temperature at not lower than 72°C f or at least 15 seconds. 
 
Breaking eggs, and/or separating the egg yolk from the egg white must be performed 
in a hygienic manner to prevent contamination. Specia l precaution must be taken to 
reject eggs with abnormal odor, color, or appearance.  
 
5.2.2 Mixing and/or Cooking 
Mix the ingredients together in a saucepan or a doub le boiler. Cook over low to 
moderate heat. Do not allow mixture to boil. Constan tly stir the mixture to avoid 
burning or scorching the mixture. Stir and cook the pro duct mixture until a thick paste 
is formed. At this point, the consistency of the mixture should have thickened enough 
that it separates from the sides of the pan. Allow the  mixture to cool. 
 
For pastillas , some formulations that utilize milk products (e.g. con densed or 
powdered milk) do not require cooking. Instead, the ing redients are simply mixed 
together and then kneaded manually until firm enoug h to handle.  
 
5.2.3 Forming and Wrapping 
Divide the mixture into equal portions. For pastillas , each portion could be formed 
into sticks or cylinders, or shaped into balls. The formed mixture may be rolled in 
sugar. The formed pastillas  may then be individually wrapped in suitable primar y 
packaging such as uncolored cellophane, wax paper, and al uminum foil. 
  11 For yema,  a portion of the cooked mixture may be placed in a p iece of cellophane 
paper, and formed into pyramid-like shapes. The cooked m ixture could also be 
shaped into balls, and either rolled in sugar or dipp ed in caramel glaze. The yema 
balls may then be individually wrapped in suitable pr imary packaging such as 
uncolored cellophane, wax paper, and aluminum foil. 
 
5.3 Packing 
Packing can be done either mechanically or manually. It is important to standardize 
filling for economic reasons. Gas-packing or vacuum-packing may be done. 
 
5.4      Closing or Sealing of Containers 
Seams and other closures shall be sealed air-tight to me et the requirements of the 
processors. 
 
The seal area of flexible containers must be free of fo od material and wrinkles. 
Sealing temperature and pressure shall conform to the sealing equipment to be 
used. 
 
5.5   Coding of Sealed Containers 
Coding of sealed container shall be indelible with det ails of production date and time, 
batch code, product code, the product line in which produ ct is packed, the 
manufacturing plant and other information necessary fo r product traceability. Where 
the container does not permit the code to be embossed or  inked, the label shall be 
legibly perforated or otherwise marked, and securely a ffixed to the product container. 
 
5.6  Post-Process Container Handling 
Mechanical shocks leading to breakage of semi-rigid contain ers due to container 
abuse must be avoided. These occur by knocking against each other during 
conveying, packaging and labeling operations, among oth ers. 
 
Flexible containers/pouches shall be handled singly rather  than in bunches, and care 
must be exercised so as to prevent damage by roughened co ntact surfaces. 
 
6.   FOOD ADDITIVES  12  
6.1 Food additives when used shall be in accordance with  the regulations 
established by the Food and Drugs 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 1, may be used for the 
manufacture of ethnic milk-based confectioneries ( pastillas  and yema ). 
 
6.2 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 fo r the raw materials and other 
ingredients.  
 
Table 1. Food Additives for Ethnic Milk-based Confectionerie s ( Pastillas and 
Yema) * (B.C. No.016 s. 2006. Updated List of Food Additive s)  
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 *** 
Amaranth 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***  13 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) 
      
7. LABELLING 
 
7.1 Each container shall be labeled and marked with the following information in 
accordance with FDA’s Labeling Regulation (A.O. 88-B s. 1984; Rules and 
Regulations  
governing the Labeling of Prepackaged Food Products D istributed 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 simi lar to those defined in 3.1 
may also be included, provided that these names are accep table in the area of 
distribution. 
(b) The complete list of ingredients and food additiv es 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.  14 (d)  The name and address of the manufacturer, packer a nd/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 . 
 
7.2  Nutrition Labeling 
Nutrition labeling shall conform to the established r egulations of FDA and/or 
authority.  
 
8.   QUALITY ASSURANCE  
 
8.1    Inspection of Finished Products 
All processed products shall be inspected before labeling a nd casing and defective 
products shall be withdrawn or rejected. The company mu st have an approved policy 
and procedures based on the BFAD A.O. No. 153 s. 2004 – Guidelines on the 
Current Good Manufacturing Practices in Manufacturing, Packing, Repacking or 
Holding Food. 
 
 8.2 Record Keeping 
Permanent and legible dated records of time, tempera ture code mark and other 
pertinent  
details shall be kept concerning each load. Such records ar e essential as a check on 
processing operations. 
  15 Written records of all container closure examinations shal l specify the code lot, the 
date and time of container closure inspections, the measu rements obtained and all 
the corrective actions taken. 
 
Records shall be maintained identifying initial distrib ution of the finished product to 
facilitate, if necessary, the segregation of specific foo d lots that may have been 
contaminated or otherwise unfit for intended use. 
 
All process deviations involving failure to satisfy the minimum requirements of the 
process shall be recorded detailing those deviations and t he actions taken. 
 
 8.3  Good Manufacturing Practices (GMP) 
Processing establishments shall have developed, documented and implemented 
prerequisite programs based on BFAD’s Current Good Man ufacturing Practices 
(cGMP) and Hygiene Control. An effective GMP and Hygie ne Control program will 
decrease the number of critical control points that a ma nufacturer must face during 
the hazard analysis of the product/process. 
 
9. STORAGE AND TRANSPORT OF FINISHED PRODUCT 
 
Storage and transport conditions of the finished product  shall be such that the 
integrity of the product container, and the safety an d quality of the product are not 
adversely affected. 
 
Cases and cartons must be thoroughly dry. They must be of  proper size so that the 
containers fit snugly and are not subject to damage from movement within the case. 
They must be strong enough to withstand normal transpo rt. 
 
Extreme fluctuations in temperature and humidity duri ng storage and transport of the 
product must be avoided to prevent product deteriorati on. 
 
10.       LABORATORY CONTROL PROCEDURES  16 Each food processing establishment shall have access to labor atory control of both 
the processes used and the finished products. All food i ngredients and food products 
declared unfit for human consumption by the laborator y shall be rejected. 
 
Representative samples for each lot or batch shall be ta ken to assess the safety and 
quality of the product. 
 
Microbiological laboratory shall be separated from the processing area. No 
pathogens shall be handled within the premises of manu facturing plant. 
 
Laboratory procedures for quality control of the process es and the product must 
follow recognized or standard methods for easy interpre tation of results. 
 
11. END PRODUCT SPECIFICATIONS 
 
Appropriate methods shall be used for sampling analysis and determinations to meet 
the following specifications: 
1. To the extent possible in good manufacturing practice s, the products shall be free 
from any objectionable characteristics. 
2. The product shall not contain any toxic substances or iginating from 
microorganisms and chemicals. 
3. The product shall be free from chemical pollutants in  amounts which may pose 
hazard to health. 
4. The product shall comply with the requirements set fo rth by the Food and Drugs 
Administration, and the Codex Alimentarius Commission o n Veterinary Drug 
Residues and Food Additives. 
 
12.      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. 
  17 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.  
 
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 City, Phi lippines. 
 
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. Al abang, 
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 Organizat ion. 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. 
  18 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 
 
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. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  19 ANNEX A 
Standard Parameters and Values for Drinking Water 
Philippine National Standards for Drinking Water 2007  (DOH AO 2007-0012) 
 
 
 Table 1. Standard values for bacteriological quali ty     
Parameter Value/Unit Point of Compliance 
Total Coliform < 1.1 MPN/100 ml Service Reservoir 
Water treatment works 
Consumers’ taps 
Refilling stations 
Water haulers 
Water vending machines 
Fecal Coliform < 1.1 MPN/100 ml Service Reservoir 
Water treatment works 
Consumers’ taps 
Refilling stations 
Water haulers 
Water vending machines 
Point sources - Level 1 
Heterotrophic Plate Count  < 500 CFU/ml Service Reservoir 
Water treatment works 
Consumers’ taps nearest meter 
Refilling stations 
Water vending machines 
 
Table 2.  Standard values for Physical and Chemical  Quality for Acceptability Aspects 
for Drinking Water 
 
 
 
 
 
 Constituents  Maximum Level (mg/L)  
or Characteristic Constituents  Maximum Level 
(mg/L)  
or Characteristic 
Taste No objectionable taste Hydrogen Sulfide 0.05 
Odor No objectionable odor Iron 1.0 
Color Apparent = 10 color 
units  
True = 5 color units Manganese 0.4 
Turbidity 3 NTU pH 6.5 – 8.5 
Aluminum 0.2 Sodium 200 
Chloride 250 Sulfate 250 
Copper 1.0 Total Dissolved 
Solids 500 
Hardness 300 as CaCO 3  Zinc 5.0  20 Table 3.  Standard Values for Organic and Inorganic  Chemical Constituents of Health 
Significance in Drinking Water  
Inorganic Chemicals 
Constituents  Maximum Level (mg/L)  Constituents  Maximum Level (mg/L)  
Antimony 0.02 Fluoride 1.0 
Arsenic 0.05 Lead 1.01 
Barium 0.7 Mercury (total) 0.001 
Boron  0.5 Nickel 0.02 
Cadmium 0.003 Nitrate 50 
Chromium (Total) 0.05 Nitrite 3.0 
Cyanide (Total) 0.07 Selenium 0.01 
 
Organic Chemicals 
Constituents  Maximum Level 
(mg/L) Constituents  Maximum 
Level (mg/L) 
Benzene 0.01 Ethylbenzene 0.30 
Carbon tetrachloride 0.004 Nitrilotriacetic acid (N TA) 0.20 
1,2-Dichlorobenzene 0.1 Polyaromatic hydrocarbons ( PAHs)  0.20 
1,4-Dichlorobenzene 0.5 Polynuclear aromatic 0.0007  
1,2-Dichloroethane 0.003 Tetrachloroethene 0.02 
1,1-Dichloroethene 0.05 Styrene 0.04 
1,2-Dichloroethene 0.07 Tetrachloroethene 0.70 
Dichloromethane 1.0 Trichloroethene 0.07 
Di(2-ethyhexyl) 
phthalate 1.01 Vinyl chloride 0.0003 
Edetic Acid (ADTA) 0.001 Xylene 0.5 
 
Organic Pesticides 
Constituents  Maximum Level 
(ug/L) Status in th e 
Philippines 
Aldrin and Dieldrin (combined) 30.0 Banned 
Atrazine 0.03 Registered 
Carbofuran 2.0 Registered 
Chlordane 7.0 Banned 
DDT ** 0.2 Banned 
1,2-Dibromo-3-chloropropane (DBCP) 1.0 Banned 
2,4-Dichlorophenoxyacetic acid (2,4-D) 1.0 Register ed 
Endrin 30. Banned 
1,2-Dibromomethane (Ethylene dibromide) 0.6 Banned 
Heptachlor and Heptachlor epoxide (combined) 0.03 B anned 
Lindane 2.0 Restricted 
MCPA (4-(2-methyl-4-chloro) phenoxyl acetic acid 2. 0 Registered 
Pendimethalin 20.0 Registered 
Pentachlorophenol (PCP) 9.0 Banned  21 ANNEX B 
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 exposed. Instruments are 
checked or calibrated on basis of RH generated by standa rd salt slushes.  
 
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, Zuri ch,Switzerland; Rotronic 
Instrument Corp., 160 E. Main St, Huntington, NY 117 43. Immobilized salt sensors are 
affected by polyols such as glycerol and glycol and by vo latile amines 
(b) Change in electrical capacitance of polymer thin fi lms. – Instrument available from 
General Eastern Instruments, 50 Hunt St, Watertown, M A 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 W einsteige 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 expa nd excessively 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 l eads. 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 accom modate 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 v acuum, using Tesla coil.  
(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.   22 (g) Hydrophilic solid. – Microcrystalline cellulose, Type PH-101 (FMC Corp.,Pharmaceutical 
and Bioscience Division, 1735 Market St, Philadelphia, PA 19103, 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 a w), 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 
minimum needed to establish saturation of salt with H 2O. Slushes are ready for use upon 
completion of mixing, and are usable indefinitely (ex cept for some high a w salts susceptible 
to bacterial attack), if contained in manner to preve nt substantial evaporation losses. Some 
slushes, e.g., NaBr, may solidify gradually by crystal coa lescence, 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 instru ment readout, as in 978.18F. Plot 
readout against a w 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 r eadout of samples to a w or to check 
vapor pressure or dew point instruments for proper funct ioning.  
 
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 tem perature control device. Use 
volume of sample or slush >1/20 total volume sample con tainer plus any associated void 
volume of sensing system, but not so much as to interfere  with operation of system. Record 
instrument response at 15, 30, 60, and 120 min after t est container is placed in temperature 
control device, or record response on strip chart. Two con secutive readings, at indicated 
intervals, which vary by <0.01 a w unit are evidence of adequately close approach to 
equilibrium. Continue readings at 60-min intervals, i f necessary. Convert last reading to a w by 
calculation from physical measurements or by reference to  calibration line. Make all 
measurements within range of calibration points; do not  extrapolate calibration line. Make all 
measurements in same direction of change, and, if requir ed by properties of sensor, expose 
sensor to controlled RH below ambient before starting each measurement.  
 
 
  23 ANNEX C 
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. Th is medium 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 Pr ogram for the food 
under analysis. Add appropriate amount of 0.1% pepton e water to the weighed sample 
to achieve 10 -1 dilution, then homogenize in a stomacher for 2 min. Alternatively, 
blending for 30-60 sec can be used but is less effective.  Make appropriate 1:10 (1+9) 
dilutions 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 sw irling 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 beg in to adhere to dish bottom 
(especially if sample is high in starch content and dishes a re plastic) and 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 considered better than the pour plate method . When the pour plate technique 
is used, fungal colonies on the surface grow faster and o ften obscure those underneath 
the surface, resulting in less accurate enumeration. Surfa ce plating gives a more uniform  24 growth and makes colony isolation easier. DRBC agar shou ld 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 substantial amounts of mold 
are present, depending on the type of mold, the uppe r countable limit may have to be 
lowered at the discretion of the analyst. Report result s 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 d igit above (e.g., 456 = 460); if 4 or below, 
round off to digit below (e.g., 454 = 450). If thir d digit is 5, round off to digit below if first 2 
digits are an even number (e.g., 445 = 440); round off to digit above if first 2 digits are an 
odd number (e.g., 455 = 460). When plates from all dilutions have no colonies, report 
mold and yeast counts (MYC) as less than 1 times the lowe st dilution used. 
Isolate individual colonies on PDA or MA, if further a nalysis and species identification is 
necessary. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  25 ANNEX D 
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 mi n at room temperature with jar securely 
capped. Mix well by swirling and determine pH with te st paper. Adjust pH, if necessary, to 
6.8 ± 0.2. Add 0.45 ml 1% aqueous brilliant green d ye solution and mix well. Loosen jar caps 
1/4 turn and incubate 24 ± 2 h at 35°C. Continue as i n 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 wit hin 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 a nd let stand 60 ± 5 min at room 
temperature with jar securely capped. Mix well by swir ling and determine pH with test paper. 
Adjust pH, if necessary, to 6.8 ± 0.2. Mix well and lo osen jar cap about 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 
(circulating, thermostatically-controlled, water bath).  Incubate TT broth 24 ± 2 h at 43 ± 
0.2°C (circulating, thermostatically-controlled, water b ath). 
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. 
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 .  26 Prepare BS plates the day before streaking and stor e in dark 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 butt. Without flaming, inoculate LIA 
slant by stabbing butt twice and then streaking slant. S ince lysine decarboxylation reaction 
is strictly anaerobic, the LIA slants must have deep but t (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 dist inct yellow in butt of tube as acidic 
(negative) reaction. Do not eliminate cultures that pr oduce 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 
tests. Cultures that give an acid butt in LIA and an alkaline 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 i n LIA 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 slan ts as described, above. 
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 presum ptive 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 isolat ed, by bioche mical and serological 
tests. Examine a minimum of 6 TSI cultures for each 25 g analytical unit or each 375 g 
composite. 
 
 
 
 
 
 
 
 
 
 
 
 
  27 ANNEX E 
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 tu bes. 
2. Immersion-type thermometer, 1-55°C, about 55 cm l ong, with 0.1°C subdivisions, 
certified by National Institute of Standards and Techn ology (NIST), 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 com positing) 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 available, 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 blend er 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. S hake all suspensions 25 times in 30 
cm arc or vortex mix for 7 s. Do not use pipets to del iver <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 pipet drain 2-3 s. Not more than 15 
min should elapse from time the sample is blended unti l all dilutions are inoculated in 
appropriate media. 
NOTE:  Use 5-tube MPN for analysis of shellfish and shellfish ha rvest waters.  28 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 addi tional 24 h and examine and record 
reactions again at 48 ± 2 h. Perform confirmed test on  all presumptive 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 3 5°C and examine for gas production at 
48 ± 2 h. Calculate most probable number (MPN) of coli forms 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 appl icator stick may 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 test to calculate fecal 
coliform MPN. To continue with E. coli  analysis, proceed to Section F under Enumeration of 
Escheria coli and the Coliform Bacteria of the USFDA Ba cteriological Analytical Manual 
(2001). The EC broth MPN method may be used for seawa ter and shellfish since it conforms 
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 anal ysis, which uses an incubation 
temperature of 44.5± 0.2°C. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  29 ANNEX F 
 
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-ventilat ed, and reasonably free 
of dust and drafts. The microbial density of air in wo rking area, measured in 
fallout pour plates taken during plating, should not exceed 15 colonies/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. Avoid 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 bottle 25 
times in 30 cm arc within 7 s if it stands more tha n 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 15 min 
of original dilutionPour agar and dilution water con trol plates for each series of 
samples. Immediately mix sample dilutions and agar medi um thoroughly and 
uniformly by alternate rotation and back-and-forth motion of plates on flat level 
surface. Let agar solidify. Invert solidified petri dis hes, and incubate promptly for 48 ± 
2 h at 35°C. Do not stack plates when pouring agar or  when agar is solidifying.