BAM: Food Sampling/Preparation of Sample

Homogenate
April 2003

Bacteriological Analytical Manual

Chapter 1
Food Sampling and Preparation of Sample Homogenate

Authors: Wallace H. Andrews and Thomas S. Hammack

Revision History: April 2003 Revised section A.1.a, Salmonella species sample collection.

The adequacy and condition of the sample or specimen received for examination are of primary
importance. If samples are improperly collected and mishandled or are not representative of the
sampled lot, the laboratory results will be meaningless. Because interpretations about a large
consignment of food are based on a relatively small sample of the lot, established sampling
procedures must be applied uniformly. A representative sample is essential when pathogens or
toxins are sparsely distributed within the food or when disposal of a food shipment depends on
the demonstrated bacterial content in relation to a legal standard.

The number of units that comprise a representative sample from a designated lot of a food
product must be statistically significant. The composition and nature of each lot affects the
homogeneity and uniformity of the total sample mass. The proper statistical sampling procedure,
according to whether the food is solid, semisolid, viscous, or liquid, must be determined by the
collector at the time of sampling by using the Investigations Operation Manual (5). Sampling
and sample plans are discussed in detail in ref. 6.

Whenever possible, submit samples to the laboratory in the original unopened containers. If
products are in bulk or in containers too large for submission to the laboratory, transfer
representative portions to sterile containers under aseptic conditions. There can be no
compromise in the use of sterile sampling equipment and the use of aseptic technique. Sterilize
one-piece stainless steel spoons, forceps, spatulas, and scissors in an autoclave or dry-heat oven.
Use of a propane torch or dipping the instrument in alcohol and igniting is dangerous and may be
inadequate for sterilizing equipment.

Use containers that are clean, dry, leak-proof, wide-mouthed, sterile, and of a size suitable for
samples of the product. Containers such as plastic jars or metal cans that are leak-proof may be
hermetically sealed. Whenever possible, avoid glass containers, which may break and
contaminate the food product. For dry materials, use sterile metal boxes, cans, bags, or packets
with suitable closures. Sterile plastic bags (for dry, unfrozen materials only) or plastic bottles are
useful containers for line samples. Take care not to overfill bags or permit puncture by wire
closure. Identify each sample unit (defined later) with a properly marked strip of masking tape.
Do not use a felt pen on plastic because the ink might penetrate the container. Whenever
possible, obtain at least 100 g for each sample unit. Submit open and closed controls of sterile
containers with the sample.

Deliver samples to the laboratory promptly with the original storage conditions maintained as
nearly as possible. When collecting liquid samples, take an additional sample as a temperature
control. Check the temperature of the control sample at the time of collection and on receipt at
the laboratory. Make a record for all samples of the times and dates of collection and of arrival at
the laboratory. Dry or canned foods that are not perishable and are collected at ambient
temperatures need not be refrigerated. Transport frozen or refrigerated products in approved
insulated containers of rigid construction so that they will arrive at the laboratory unchanged.
Collect frozen samples in pre-chilled containers.

Place containers in a freezer long enough to chill them thoroughly. Keep frozen samples solidly
frozen at all times. Cool refrigerated samples, except shellfish and shell stock, in ice at 0-4°C and
transport them in a sample chest with suitable refrigerant capable of maintaining the sample at 0-
4°C until arrival at the laboratory. Do not freeze refrigerated products. Unless otherwise
specified, refrigerated samples should not be analyzed more than 36 h after collection. Special
conditions apply to the collection and storage of shucked, unfrozen shellfish and shell stock (1).
Pack samples of shucked shellfish immediately in crushed ice (no temperature specified) until
analyzed; keep shell stock above freezing but below 10C. Examine refrigerated shellfish and
shell stock within 6 h of collection but in no case more than 24 h after collection. Further details
on sample handling and shipment may be found in the Investigations Operation Manual (5) and
the Laboratory Procedures Manual (3). The Investigations Operation Manual (5) contains
sampling plans for various microorganisms. Some of those commonly used are presented here.

A. Sampling plans
1. Salmonella species
a. Sample collection

Because of the continuing occurrence of Salmonella in foods, sampling

plans for these organisms have received the attention of committees of
national and international organizations (6,7). Each of these committees
has recommended varying the number of samples from a particular lot of
food according to the sampling category to which a food is assigned.
Generally, the assignment to a sampling or food category depends on 1)
the sensitivity of the consumer group (e.g., the aged, the infirm, and
infants); 2) the possibility that the food may have undergone a step lethal
to Salmonella during the manufacturing process or in the home; and 3) the
history of the food. The selection of a sampling plan depends mainly on
the first 2 criteria cited. The history of the food would be important in
deciding whether to sample, i.e., whether there was a past history of
contamination. For the Salmonella sampling plan discussed here, 3
categories of foods are identified.
Food Category I. - Foods that would not normally be subjected to a
process lethal to Salmonella between the time of sampling and
consumption and are intended for consumption by the aged, the infirm,
and infants.

Food Category II. - Foods that would not normally be subjected to a

process lethal to Salmonella between the time of sampling and
consumption.

Food Category III. - Foods that would normally be subjected to a process

lethal to Salmonella between the time of sampling and consumption.

In certain instances, it may not be possible to fully conform to the

sampling plan. Nonetheless it is still important to ascertain whether or not
Salmonella is present in the suspect food. Therefore, the analyst should
still try to analyze as many analytical units as is required for the food of
interest, i.e., 60 analytical units for Category I foods, 30 analytical units
for Category II foods, and 15 analytical units for Category III foods.
Individual 25 g analytical units may be combined into 375 g composites as
described above unless otherwise indicated in Chapter 5 or the OMA.
Below are examples of situations that might confront the analyst.

1. 1) The number and weights of the sample units is correct.

Each sample should be mixed to ensure homogeneity before

withdrawing a 25 g analytical unit. The analytical units can be
composited (fifteen 25 g units into a 375 g composite), unless
otherwise indicated in Chapter 5 or in the OMA. Samples should
be preenriched at a 1:9 sample-to-broth ratio.

2. 2) The number of sample units is correct, but several of the sample

units have been damaged and are unusable.

For example, fifteen 1 lb bags of pasta have arrived for testing, but
5 of the bags are torn and unusable. In this case, the analyst should
only sample from the 10 intact bags. The contents of each intact
bag should be mixed to ensure homogeneity before the analytical
units are withdrawn. Since the analyst needs one 375 g composite,
ten 37.5 g analytical units, from the remaining 10 intact bags,
should be used to form the composite. The composite should be
combined with its preenrichment medium at a 1:9 sample-to-broth
ratio (375 g sample/3375 ml preenrichment) as directed in Chapter
5 or the OMA.
 3. 3) The number of sample units is incorrect, but the total weight of
the sample unit(s) is greater than what would be necessary to
perform the sample analysis.

For example, a single 10 lb wheel of cheese has arrived for testing.

Since cheese is a Category II food, thirty 25 g analytical units must
be analyzed. These analytical units should be taken randomly from
a wide variety of locations around the wheel. If Salmonella is
present in a food, then the odds of detecting it will be enhanced if
two 375 g composites are analyzed rather than a single 25 g
analytical unit, as would be the case if the analyst were to treat the
entire wheel as a single sample.

4. 4) There is less sample available than is necessary to form the

required number of composites.

For example, an 8 oz (226.8 g) bag of almonds has arrived for

testing. Almonds are a Category II food. Category II foods require
thirty 25 g analytical units (750g), so it is impossible to analyze the
amount of almonds required by the sampling plan. In this case, the
analyst should analyze all of the almonds at a 1:9 sample-to-broth
ratio (226.8g sample/2041 ml preenrichment medium).

If, in the above example, the total weight of the almonds had been
less than 2 composites (750 g), but more than 1 composite, then the
analyst should analyze both a whole and a partial composite. The
analytical units comprising these composites should be taken
randomly from a wide variety of locations in the lot of almonds.
Both composites, should be preenriched at a 1:9 sample-to-broth
ratio.

This sampling plan applies to the collection of finished products under

surveillance and/or for determination of compliance for regulatory
consideration. It also applies to the collection of factory samples of raw
materials in identifiable lots of processed units and/or finished products
where regulatory action is possible. It does not apply to the collection of
in-line process sample units at various stages of manufacture since those
samples do not necessarily represent the entire lot of food under
production. The actual techniques involved in sampling are covered in the
Investigations Operation Manual (5).

A sample unit consists of a minimum of 100 g and is usually a consumer-

size container of product. Take sample units at random to ensure that a
sample is representative of the lot. When using sample containers, submit
a control consisting of one empty sample container that has been exposed
to the same conditions as those under which the sample was collected.
 Collect more than one sample unit from large institutional or bulk
containers when the number of sample units required exceeds the number
of containers in the lot. A sample unit will consist of more than one
container when containers are smaller than 100 g (e.g., four 25 g
containers could constitute a sample unit).

The numbers of sample units to be collected in each food category are as

follows: Food Category I, 60 sample units; Food Category II, 30 sample
units; Food Category III, 15 sample units. Submit all samples collected to
the laboratory for analysis. Advise the laboratory in advance of perishable
sample shipments.

b. Sample analysis

The laboratory will analyze each sample for the presence of Salmonella
according to methods described in this manual, or in Official Methods of
Analysis (2). Take a 25 g analytical unit at random from each 100 g
sample unit. When a sample unit consists of more than one container,
aseptically mix the contents of each container before taking the 25 g
analytical unit. To reduce the analytical workload, the analytical units may
be composited. The maximum size of a composite unit is 375 g or 15
analytical units. The minimum number of composite units to be tested for
each food category is as follows: Food Category I, 4 composite units;
Food Category II, 2 composite units; Food Category III, one composite
unit. For each 375 g composite, the entire amount of 375 g is analyzed for
Salmonella.

Keep the remainder of the sample unit in a sterile container for compliance
requirements as per section 702(b) of the Federal Food, Drug, and
Cosmetic Act as amended through February, 1993. Refrigerate perishable
samples and samples supporting microbial growth. An analytical control is
required for each sample tested. The sampled lot is acceptable only if
analyses of all composite units are negative for Salmonella. If one or more
composite units are positive for Salmonella, the lot is rejected, provided
that the analytical control is negative for Salmonella. A lot will not be
resampled unless the environmental control for Salmonella is positive. For
all samples positive for Salmonella, determine the somatic group. See
Chapter 5 for information on further handling of these cultures.
Recommendations for regulatory action may be based on the identification
of the Salmonella somatic group and will not require definitive serotyping
before initiation of regulatory action.

c. Imports.

These sampling plans apply to imported food products intended for human
consumption.
d. Classification of food products for sampling purposes

Foods that have been classified into the 3 categories described above for
regulatory sampling are listed in the categories according to the Industry
Product Code sequence and nomenclature (4). Listing does not necessarily
mean that these products are probable sources of Salmonella. Food
Category I. - Foods that would not normally be subjected to a process
lethal to Salmonella between the time of sampling and consumption and
are intended for consumption by the aged, the infirm, and infants. Food
Category II. - Foods that would not normally be subjected to a process
lethal to Salmonella between the time of sampling and consumption.
Examples are as follows:

Industry Product Code

2 Milled grain products not cooked before consumption (bran and

wheat germ)
3 Bread, rolls, buns, sugared breads, crackers, custard- and cream-
filled sweet goods, and icings
5 Breakfast cereals and other ready-to-eat breakfast foods
7 Pretzels, chips, and other snack foods
9 Butter and butter products, pasteurized milk and raw fluid milk and
fluid milk products for direct consumption, pasteurized and
unpasteurized concentrated liquid milk products for direct
consumption, dried milk and dried milk products for direct
consumption, casein, sodium caseinate, and whey
12 Cheese and cheese products
13 Ice cream from pasteurized milk and related products that have been
pasteurized, raw ice cream mix and related unpasteurized products
for direct consumption
14 Pasteurized and unpasteurized imitation dairy products for direct
consumption
15 Pasteurized eggs and egg products from pasteurized eggs,
unpasteurized eggs and egg products from unpasteurized eggs for
consumption without further cooking
16 Canned and cured fish, vertebrates, and other fish products; fresh
and frozen raw shellfish and crustacean products for direct
consumption; smoked fish, shellfish, and crustaceans for direct
consumption
17 Meat and meat products, poultry and poultry products, and gelatin
(flavored and unflavored bulk)
20- Fresh, frozen, and canned fruits and juices, concentrates, and
22 nectars; dried fruits for direct consumption; jams, jellies, preserves,
and butters
23 Nuts, nut products, edible seeds, and edible seed products for direct
consumption
24 Vegetable juices, vegetable sprouts, and vegetables normally eaten
raw
26 Oils consumed directly without further processing; oleomargarine
27 Dressings and condiments (including mayonnaise), salad dressing,
and vinegar
28 Spices, flavors, and extracts
29 Soft drinks and water
30 Beverage bases
31 Coffee and tea
33 Candy (with and without chocolate; with and without nuts) and
chewing gum
34 Chocolate and cocoa products
35 Pudding mixes not cooked before consumption, and gelatin products
36 Syrups, sugars, and honey
37 Ready-to-eat sandwiches, stews, gravies, and sauces
38 Soups
39 Prepared salads
54 Nutrient supplements, such as vitamins, minerals, proteins, and
dried inactive yeast

Food Category III: Foods that would normally be subjected to a process

lethal to Salmonella between the time of sampling and consumption.
Examples are as follows:

Industry Product Code

2 Whole grain, milled grain products that are cooked before

consumption (corn meal and all types of flour), and starch products
for human use
3 Prepared dry mixes for cakes, cookies, breads, and rolls
4 Macaroni and noodle products
16 Fresh and frozen fish; vertebrates (except those eaten raw); fresh and
frozen shellfish and crustaceans (except raw shellfish and
 crustaceans for direct consumption); other aquatic animals (including
frog legs, marine snails, and squid)
18 Vegetable protein products (simulated meats) normally cooked
before consumption
24 Fresh vegetables, frozen vegetables, dried vegetables, cured and
processed vegetable products normally cooked before consumption
26 Vegetable oils, oil stock, and vegetable shortening
35 Dry dessert mixes, pudding mixes, and rennet products that are
cooked before consumption

2. Aerobic plate counts, total coliforms, fecal coliforms, Escherichiacoli (including

enteropathogenic strains), Staphylococcus spp., Vibrio spp., Shigella spp.,
Campylobacter spp., Yersinia spp., Bacilluscereus, and Clostridium perfringens
a. Sample collection

From any lot of food, collect ten 8-oz subsamples (or retail packages) at
random. Do not break or cut larger retail packages to obtain an 8-oz
subsample. Collect the intact retail unit as the subsample even if it is larger
than 8 oz.

b. Sample analysis.

Analyze samples as indicated in current compliance programs.

B. Equipment and materials

1. Mechanical blender. Several types are available. Use blender that has several
operating speeds or rheostat. The term "high-speed blender" designates mixer
with 4 canted, sharp-edge, stainless steel blades rotating at bottom of 4 lobe jar at
10,000-12,000 rpm or with equivalent shearing action. Suspended solids are
reduced to fine pulp by action of blades and by lobular container, which swirls
suspended solids into blades. Waring blender, or equivalent, meets these
requirements.
2. Sterile glass or metal high-speed blender jar, 1000 ml, with cover, resistant to
autoclaving for 60 min at 121°C
3. Balance, with weights; 2000 g capacity, sensitivity of 0.1 g
4. Sterile beakers, 250 ml, low-form, covered with aluminum foil
5. Sterile graduated pipets, 1.0 and 10.0 ml
6. Butterfield's phosphate-buffered dilution water (Rll), sterilized in bottles to yield
final volume of 90 ± 1 ml
7. Sterile knives, forks, spatulas, forceps, scissors, tablespoons, and tongue
depressors (for sample handling)
C. Receipt of samples
1. The official food sample is collected by the FDA inspector or investigator. As
soon as the sample arrives at the laboratory, the analyst should note its general
physical condition. If the sample cannot be analyzed immediately, it should be
 stored as described later. Whether the sample is to be analyzed for regulatory
purposes, for investigation of a foodborne illness outbreak, or for a bacteriological
survey, strict adherence to the recommendations described here is essential.
2. Condition of sampling container. Check sampling containers for gross physical
defects. Carefully inspect plastic bags and bottles for tears, pinholes, and puncture
marks. If sample units were collected in plastic bottles, check bottles for fractures
and loose lids. If plastic bags were used for sampling, be certain that twist wires
did not puncture surrounding bags. Any cross-contamination resulting from one
or more of above defects would invalidate the sample, and the collecting district
should be notified (see C-5, below)
3. Labeling and records. Be certain that each sample is accompanied by a
completed copy of the Collection Report (Form FD-464) and officially sealed
with tape (FD-415a) bearing the sample number, collecting official's name, and
date. Assign each sample unit an individual unit number and analyze as a discrete
unit unless the sample is composited as described previously in this chapter.
4. Adherence to sampling plan. Most foods are collected under a specifically
designed sampling plan in one of several ongoing compliance programs. Foods to
be examined for Salmonella, however, are sampled according to a statistically
based sampling plan designed exclusively for use with this pathogen. Depending
on the food and the type of analysis to be performed, determine whether the food
has been sampled according to the most appropriate sampling plan.
5. Storage. If possible, examine samples immediately upon receipt. If analysis must
be postponed, however, store frozen samples at -20°C until examination.
Refrigerate unfrozen perishable samples at 0-4°C not longer than 36 h. Store
nonperishable, canned, or low-moisture foods at room temperature until analysis.
6. Notification of collecting district. If a sample fails to meet the above criteria and
is therefore not analyzed, notify the collecting district so that a valid sample can
be obtained and the possibility of a recurrence reduced.
D. Thawing

Use aseptic technique when handling product. Before handling or analysis of sample,
clean immediate and surrounding work areas. In addition, swab immediate work area
with commercial germicidal agent. Preferably, do not thaw frozen samples before
analysis. If necessary to temper a frozen sample to obtain an analytical portion, thaw it in
the original container or in the container in which it was received in the laboratory.
Whenever possible, avoid transferring the sample to a second container for thawing.
Normally, a sample can be thawed at 2-5°C within 18 h. If rapid thawing is desired, thaw
the sample at less than 45°C for not more than 15 min. When thawing a sample at
elevated temperatures, agitate the sample continuously in thermostatically controlled
water bath.

E. Mixing

Various degrees of non-uniform distribution of microorganisms are to be expected in any

food sample. To ensure more even distribution, shake liquid samples thoroughly and, if
practical, mix dried samples with sterile spoons or other utensils before withdrawing the
 analytical unit from a sample of 100 g or greater. Use a 50 g analytical unit of liquid or
dry food to determine aerobic plate count value and most probable number of coliforms.
Other analytical unit sizes (e.g., 25 g for Salmonella) may be recommended, depending
on specific analysis to be performed. Use analytical unit size and diluent volume
recommended for appropriate Bacteriological Analytical Manual method being used. If
contents of package are obviously not homogeneous (e.g., a frozen dinner), macerate
entire contents of package and withdraw the analytical unit, or, preferably, analyze each
different food portion separately, depending on purpose of test.

F. Weighing

Tare high-speed blender jar; then aseptically and accurately (± 0.1 g) weigh unthawed
food (if frozen) into jar. If entire sample weighs less than the required amount, weigh
portion equivalent to one-half of sample and adjust amount of diluent or broth
accordingly. Total volume in blender must completely cover blades.

G. Blending and diluting of samples requiring enumeration of microorganisms

1. All foods other than nut meat halves and larger pieces, and nut meal. Add
450 ml Butterfield's phosphate-buffered dilution water to blender jar containing
50 g analytical unit and blend 2 min. This results in a dilution of 10-1. Make
dilutions of original homogenate promptly, using pipets that deliver required
volume accurately. Do not deliver less than 10% of total volume of pipet. For
example, do not use pipet with capacity greater than 10 ml to deliver 1 ml
volumes; for delivering 0.1 ml volumes, do not use pipet with capacity greater
than 1.0 ml. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml
of previous dilution, unless otherwise specified. Shake all dilutions vigorously 25
times in 30 cm (1 ft) arc in 7 s. Not more than 15 min should elapse from the time
sample is blended until all dilutions are in appropriate media.
2. Nut meat halves and larger pieces. Aseptically weigh 50 g analytical unit into
sterile screw-cap jar. Add 50 ml diluent (G-l, above) and shake vigorously 50
times through 30 cm arc to obtain 100 dilution. Let stand 3-5 min and shake 5
times through 30 cm arc to resuspend just before making serial dilutions and
inoculations.
3. Nut meal. Aseptically weigh 10 g analytical unit into sterile screw-cap jar. Add
90 ml of diluent (G-l, above) and shake vigorously 50 times through 30 cm arc to
obtain 10-1 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to
resuspend just before making serial dilutions and inoculations.

References

1. American Public Health Association. 1985. Laboratory Procedures for the Examination
of Seawater and Shellfish, 5th ed. APHA, Washington, DC.
2. AOAC INTERNATIONAL. 1995. Official Methods of Analysis, 16th ed. AOAC
INTERNATIONAL, Arlington, VA.
3. Food and Drug Administration. 1989. Laboratory Procedures Manual. FDA, Rockville,
MD.
4. Food and Drug Administration. 1978. EDRO Data Codes Manual. Product Codes:
Human Foods. FDA, Rockville, MD.
5. Food and Drug Administration. 1993. Investigations Operations Manual. FDA,
Rockville, MD.
6. International Commission on Microbiological Specifications for Foods. 1986.
Microorganisms in Foods. 2. Sampling for Microbiological Analysis:Principles and
Specific Applications, 2nd ed. University of Toronto Press, Toronto, Ontario, Canada.
7. National Academy of Sciences. 1969. An Evaluation of the Salmonella Problem.
National Academy of Sciences, Washington, DC.