Sampling Protocols

1. Introduction
Representative and correct sampling is an essential requirement to obtain valid results in laboratory testing.
Tests to determine safety, quality, contents and compliance with official requirements are of little or no value if the sampling is done without care. Sampling is effective if an appropriate range of sampling devices for different products is available and if the personnel involved in sampling are well trained. «International PBI»’s years of experience in servicing applied microbiology, agriculture, education and industry give the Company excellent insight into the needs of laboratories and associations involved in extensive testing programs.
Together with the knowledge of several highly qualified health inspectors and chemists, who acted as consultants, a complete set of simple, practical, economical and useful sampling devices have been produced and are illustrated in this booklet.
The aim of this booklet is to supply technicians in the food, feed, dairy, agricultural, pharmaceutical, cosmetic and chemical fields with a wide range of effective devices to improve their sampling methods.

2. Sample
An example of standards for microbiological testing is reported.

2.1 Sampling plan
It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage.
Sampling is a part of the general guidances for microbiological examinations. See the specific International Standard dealing with the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject.

2.2 Transport
The transportation of the samples to the laboratory shall ensure that they are kept under conditions wich prevent any modification in the number of microorganisms present. Preference will be given to those means of transport which offer the fastest transportation.

Particular attention will be paid to observing the storage temperature and to the conditions of transport:

- stable products: ambient temperature
- fresh and refrigerated products: between 0 °C and +4 °C
- frozen or deep-frozen products: below –18 °C
- pasteurized and similar products: between 0 °C and +4 °C
- spoiled units: between 0 °C and +4 °C

2.3 Receipt and storage

- spoiled units in closed packaging for protection against possible bursting open

On receipt of the samples, the laboratory will check the condition of the latter. If the condition is unsatisfactory or if there is an insufficient quantity of samples, the laboratory can: either refuse the samples, or have reservations about the signification of the results.
The samples admitted into the laboratory are referenced in such a matter that can be tracked up until the time of drafting the test report.
The following information will be noted:

- date of receipt
- the characteristics of the sampling operation (sampling date, sampling conditions, etc)
- the requesting party’s name and address
- the characteristics of the product

Samples awaiting examination shall be stored under conditions which prevent any modification in the number of microorganisms present.
Particular attention will be paid to observing the storage temperatures and to the examination deadline:

- stable products: as early as possible and before the storage limit date
- fresh and refrigerated products: within 24 h after receipt (if a longer storage period cannot be avoided, freeze the sample as soon as
possible at a temperature below -18 °C and mention this in the test report), because, in certain products, freezing modifies the composition of the flora
- pasteurized or similar products: as early as possible and before the storage limit date
- spoiled units: 48 h

2.4 Test portion
In order to avoid the contamination of the environment and the test portion, it is recommended to work in special premises or in safety cabinets. Failing this, those products containing very few microorganisms, e.g. pasteurized products, cooked dishes, shall always be examined first of all, followed by the more highly contaminated ones.
The protection of the environment against contamination is of particular importance for the weighing and sampling of the test portion of highly contaminated powdered products which shall be carried out under a vertical laminar flow safety cabinet.
Handle the samples in such a manner as to avoid any and all risk of contamination. To achieve this, take the following precautions:

- When it is a question of a packaged product, clean the outside of the packing at the place where it will be opened with 70 % alcohol. Flame is possible.
- Any instrument which is used for opening the packaging (tin-opener, scissors, etc...) shall be sterile.
- Carefully mark the reference of the laboratory sample on the containers, plastic bags, etc...enclosing the test sample.

The laboratory sample shall be as representative of the product as possible.
Laboratory samples are kept until the results are obtained or longer if necessary.
Deteriorated laboratory samples are decontaminated prior to being discarded.
Undeteriorated laboratory samples are either discarded directly or packed in a sterile container (plastic bag for example), and re-stored to the 1 month maximum storage temperature.

3. Sample submission instruction report
Correct sampling is essential to obtain reliable results in subsequent analytical steps. To achieve this, it is important that technicians involved in the sampling program are well trained and equipped with the necessary sampling devices and information on how to obtain an accurate sample. Furthermore the analyst in the laboratory who will receive the sample must know the details of the sample to be analysed. For these reasons it is necessary that each sample is accompanied with a “Sample Submission Instruction Report” (see following example for microbiological analysis).

Sample Submission Instruction Report for Microbiological Analysis

NOTES:
1 - Subsample size and container format
Except in case of swabs, contact plates and environmental scraping, subsamples should consist of a minimum of 100 grams (or 100 ml in case of liquids).
“RET”: each subsample should consist of a minimum of one intact retail package contains less than 100 grams (or 100 ml), enough retail packages must be provided to furnish at least 100 grams (or 100 ml) per subsample.
“WHP”: Whirl-pak or other pre-sterilised self-closing bags.
“CUP”: pre-sterilised plastic or glass containers with leak-proof lids.
“BAG”: for use with Contact-Plates. Plates should be taped shut individually and then placed inside a plastic bag. The plastic bag should be taped shut or heat-sealed.
2 - Physical State
Solid, liquid, powder, paste, frozen, pellets, water.
3 - Sampling devices
Solid: “Multipro” (cereals, grains); “Multimaxi” (grains).
Liquid: “Dip-New”, “Probe”, “Acid”.
Powder: “Pic”, “Mipaw”.
Paste: “Cebu” (hard pastes); “Stuve” (thick pastes).
Frozen: “Iceprobe” (ice cream); “Surgel” (frozen products).
Water: “Mare-Lacus” (subsurface water);"Aqua-composite" (vacuum system), “Swing” (waste water system)
4 ­ Added chemicals
Sodium tiosulphate for sampling either chlorinated or non-chlorinated water.
5 - Shipping conditions
Temperature conditions for shipment of samples:
“REF”: product should either be shipped in a refrigerated truck or else packed in a cooler containing freezer-packs.
“FRZ”: product should be hard-frozen when packed, and shipped in a cooler containing freezer packs (for delivery within 4 hours) or dry ice (for longer time lapse beetween packing and delivery).
“AMB”: product may be shipped without refrigeration, but should be protected from excessive heat or humidity.
6 - Requested analysis
“TVC”: total viable count.
“YMC”: yeast and mould count.
“COC”: coliform count.
“ECO”: Escherichia coli count.
“SAC”: St. aureus count.
“ECC”: Enterococcus count.
“SAL”: Salmonella.
7 - Number of subsamples
Number of subsamples on which the analysis is to be performed.
8 - General Acceptability Limit
Level of microorganisms per gram (or ml) that should not be exceeded under good manufacturing and storing conditions.
9 - Subsample Acceptability Limit
Maximum number of microorganisms per gram (or ml) permitted in any single subsample.
10 - Maximum number of Critical Subsamples
Maximum number of subsamples permitted to have bacterial counts included between “8” and “9” without the sample being considered unsatisfactory.

4. Glossary
• Single or spot sample: material taken from the bulk quantity in a single sampling procedure.
• Random sample: one or more samples taken successively in short intervals.
• Short-time mixed sample: composed of a mixture of at least five random samples gathered in intervals of not less than two minutes.
• Raw, mixed, or composite sample: different designations of the sum of all single/spot samples processed collectively.
• Intermediate sample: created through reduction of the collective sample, and ready for processing.
• Sample set: parts of the sample that accumulate during sample preparation, and need to be kept separate (for example, metallic - oxidic, fine - coarse, gaseous - liquid - solid).
• Final sample: the sample amount obtained from the sum of all single/spot samples (not necessarily identical to raw, mixed, or collective sample, since every single sample may have been separately processed), from which the analysis sample is taken.
• Analysis sample: the end product of the sampling and sample preparation, which has been prepared for the intended investigations.
• Time-dependent sampling: single samples of identical volume are taken in previously established time intervals (in case of continuous material flux this corresponds to mass- or volume-proportional sampling).
• Volume-dependent sampling: single samples of identical volume taken in variable time intervals which are corresponding to the flux (flow rate).
• Flow-dependent sampling: single samples taken in identical time intervals with volumes corresponding to variable flow rate.
• Continuous sampling at fixed flow rates: continuous sampling of a constant volume.
• Continuous sampling at variable flow rate: sampling of variable volumes which correspond to the flow rate.
• Surface samples: sum of all spot samples taken at the surface of, for example, stagnant waters.
• Depth-integrated samples: sum of all spot samples taken at the same place at different depths of, for example, stagnant waters.
• “Isokinetic” sampling: used in heterogeneous systems (chimneys, tubes, rivers) where the sampling is done through a suction tube and at a rate that the velocities in the tube and in the main stream are equal.

5. Sampling plan in check-list format

6. How to "Take” Samples from large lot of products in particles size (grains, cereals, seeds,soil, etc..)
Great care must be taken to secure samples that are truly representative. One shovelful of material taken at random will not be representative. At the apex of a conical pile the proportion of fines is excessive. At the base the proportion of coarse materials is excessive. In the shoveling process every fifth shovel, or every tenth shovel, etc.. should be
taken depending upon the amount of sample desired. The sample should consist of small quantities taken at random from as many parts of the pile as are accessible, in such a manner the proportion of coarse and fines in the small amount will be the same as in the larger amount.
Greater accuracy in the sampling can be obtained where material is flowing in a chute or launder (either with or without water). The ideal place to take the sample is just where the material drops from the cute. When taking the samples, use a pail or a specially built receptacle which can be swung completely across the flowing stream in a brief interval of time, so as take “all of the stream part of the time”. Under no circumstances should the sampling receptacle be allowed to overflow.

Coning and Quartering
The sample should be piled in a cone, each shovelful going to the centre of the cone and allowed to run down equally in all direction - this will mix the sample. Then spread out in a circle and walk around the pile gradually widening the circle with a shovel until the material is spread out to a uniform thickness. Mark the flat pile into quarters, and
reject two opposite quarters. Mix again by shoveling the material into a conical pile, taking alternate shovelfuls from the two quarters saved. Continue the process of piling, flattening and rejecting two quarters until the sample is reduced to the required size.

References
1 - ISO/IDF/AOAC/BS Standards
2 - Environmental Sampling for trace Analysis - edited by Bernd Markert VCH Verlagsgesellshaft mbH, D-69451 Weinheim, 1994
3 - Determination of Trace Elements - edited by Zeev B. Alfassi VCH Verlagsgesellshaft mbH, D-69451 Weinheim, 1994

Sample Temperature Control
Sample temperature control is a critical point in the sampling program. This includes the storage conditions on sampling location during transport and at the laboratory before analysis begins.
The “ZANZIBAR” portable thermometer presented on this page gives the possibility to control the temperature of the sample during all these stages in a simply and fast way.

“Zanzibar” portable Thermometer
For temperature control of samples - temperature range: –50°/+150 °C - accuracy ± 0,3 °C in the range -20/+90 °C (± 0,5 °C in other ranges) - dimensions: 106x58x19 mm - weight 80 g - supplied with a stainless steel penetration probe and 1 meter cable.

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