SAMPLE COLLECTION

It is vitally important that samples are collected in a manner that ensures the integrity of the product be it water, soil, or food. The introduction of any microbiological or chemical contaminant at the time of sample collection can cause the results to be incorrect and therefore any remedial action that may be taken may at best be unnecessary or at worst be detrimental.We have included some information that will assist in the collection of various sample types. It is strongly recommended that this sampling advice be followed.

INTRODUCTION
The importance of surface hygiene has long been recognised in the medical field but it is now becoming increasingly important in food safety, both in food production and distribution (retail shops in particular). The relatively recent emphasis on the need to assess the hygiene of surfaces in food production and preparation, has largely come about because of the relationship of surface hygiene to human health and food spoilage. Surface hygiene has also become integrated in the development of the Hazard Assessment Critical Control Point system (HACCP) - in this system, all stages of food production are monitored on a systematic basis, rather than on a periodic or random assessment of the final product - the HACCP system also includes records of visual inspection and process temperatures.
There are several ways available for hygiene to be monitored. The scheme of choice depends upon regulatory requirements, the type of food/s involved and the operational aspects of the enterprise, plus overall cost effectiveness.
Reliable methods, both new and old, are available for hygiene assessment - the common methods in practice are the SWAB METHOD which uses either POUR PLATES, or CONTACT PLATES (in-house or commercial preparations) and INDIRECT METHODS such as ATP-luminometry, or direct epifluorescent technique (DEFT). Rapid methods such as ATP luminometry can be of particular value in production processes, but the more traditional surface swab methods continue to provide the cornerstone for less critical processes and for retailing facilities, chiefly because of their relative simplicity.
Other methods which have been examined include those based on turbidimetry, conductance, calorimetry, fluorimetry, radiometry, microcalorimetry, catalase reaction, LAL (lymnocyte amoebic lyase) endotoxin test. These have received impetus from the development of the HACCP system.

SAMPLING SITES
Hygiene samples are generally taken from surfaces that have been cleaned on the previous day and before the start of the next working day. If this is not practicable, they can be taken directly after cleaning. Microorganisms adhere to surfaces in 2 phases - a reversible phase is followed by an irreversible phase. The adhesive strength of any particular organism depends on the type of surface e.g. adhesion to glass is not as strong as adhesion to steel or rubber. Reports vary in their assessment of the relative adhesion of bacteria to plastics such as nylon, PVC and polypropylene, and to aluminium and steel. Stainless steel appears to be the easiest material to clean.
A polysaccharide layer known as a biofilm is formed as the result of bacterial adhesion to surfaces. This helps to protect the underlying bacteria and the thickness of this film can depend on (a) the temperature difference between the surface and the bacteria, (b) the acidity of the material, and (c) the extent to which it repels water (hydrophobicity). Bacteria can multiply within the biofilm layer. One school of thought believes that biofilm formation lies at the base of all hygiene problems, whilst another school of thought believes that there is undue importance attached to biofilms. Whatever the case, there is no doubt that the longer a surface remains unclean, the greater the amount of bacterial proliferation, and the more difficult it will be to clean. Thus any cleaning procedure must begin with physical removal of visible matter before using a cleaning agent or disinfectants. Hygiene checks in food establishments tend to concentrate on easily-accessible, flat areas. However, the critical points may well be elsewhere in the system - this is particularly so in retail establishments. The hygiene of cleaning has attracted greater interest as a result of pathogenic bacteria not only being found in cleaning equipment, but even in disinfectant solutions that have either become too diluted or have been reused rather than renewed.
In most cases hygiene assessment is based on the total count of viable bacteria. In some circumstances specific bacteria are targeted e.g. E. coli (indicator of faecal contamination), Staphylococcus aureus (commonly found on hands), Enterococci, Listeria, Yersinia, Salmonella. It is sometimes necessary to check surfaces for the presence of a single species which presents a particular health hazard e.g. Listeria monocytogenes.
Visual inspection is a sound preliminary step in a general assessment of hygiene, as it will detect areas of gross contamination which can degrade an entire preparation chain or production area, irrespective of how clean or sterile it may be in other areas (a case of the weakest link breaking the chain). Whilst visual appearances do not necessarily correlate with microbiological findings, especially if the visual inspection overlooks critical points, overall they do tend to correlate with the results of contact examination. Contact methods of examination tend to measure those contaminants that are relatively loosely bound, in contrast to swabbing which brings off more tightly bound matter from thin, less visible layers.
Surfaces must be in a hygienic condition if they come into contact with foods, especially finished products, and more especially those products that will be consumed directly without any further heating.
As an example, the types of surfaces of interest in the meat industry would include cutting boards, working surfaces, transportation hooks, cutting saws, feed belts, knives, trolleys, skinners, salting equipment, mincers, sharpening steels and stones, and plastic packing crates. There are also other surfaces of interest that are not in direct contact with products yet provide a potential vehicle for transmission of microorganisms – these include door handles, door flaps, packing equipment, weighing scales, aprons, mesh gloves, and the surfaces of storage areas such as chiller rooms.


METHODS OF SURFACE SAMPLING

Contact plates: molten agar medium is poured into a round or square petri dish (usually a small dish with an area of approximately 25 cm2); the amount of agar being sufficient to bring the surface above the edge of the petri dish. After the agar has set, the plate is pressed against the surface being examined, the lid is then replaced and the plate is incubated. Colonies of microorganisms develop on the agar and these are then counted - the results are expressed as a count per square centimetre (i.e. the plate count divided by the area of the agar) or other designated area. Recovery rate is reported to be from 70-80%.
Petrifilm ? : This proprietary medium comes as a dried form on squared polyethylene paper, with the upper layer covered by a polypropylene membrane. The medium is moistened with 1 mL of sterile water which is spread over the film and allowed to stand for 1 minute. The flexible film can now be applied to a surface (flat or curved). Various media types are available e.g. for total count, coliforms, E. coli, yeasts and moulds. The film is conveniently marked in 1 cm squares (total area about 20 cm2
Paddle or Dip slide: (Hygicult?is taken as an example of this type of contact method - there are several other proprietary brands available): This consists of a hinged plastic "paddle" covered on both sides with an agar medium (the area of each side is approximately 9.6 cm2 ) The paddle is attached to the lid of a protective tube. The paddle can either be pressed on to the surface being examined or a swab can be taken from a known area and rubbed over the whole surface of the agar on one side of the paddle - if the examination needs to be made on a larger area, both sides of the paddle can be used. After the sample has been taken, the paddle is reinserted in the tube and incubated. Different agar preparations are available for different groups of organisms e.g. total count, coliforms, E. coli, yeasts and moulds.
Luminescence: As cultural methods such as contact plates require at least one day to obtain measurable results, there has been considerable activity devoted to more rapid methods, of which luminometry is one. This method is based on the luciferin-luciferase reaction in which light is produced form the reaction of the adenosine triphosphate (ATP) present in all living cells.
ATP + luciferin + O2 ????? oxyluciferane + AMP + Ppi + light .
Luciferin and luciferase are extracted from fireflies. The amount of light formed is directly proportional to the amount of ATP and this in turn is related to the number of cells present. The amount is about 10-12 g per cell in animal, yeast and mould cells, and about 10-15g (i.e. 1 000 times less) in bacterial cells. The amount per cell varies with the cell structure, the stage of growth and growth temperature.
The sample for luminometry is taken with a swab moistened with a sterile saline solution. The swab is rubbed over a specified area, and the swab tip is then transferred to a reagent capsule and rotated for about 20 seconds. The ATP in the cells is released by a detergent. The next reagent added contains the components for the luciferin-luciferase reaction. The light produced from this is then measured in a very sensitive instrument called a luminometer (based on a photomultiplier), and the result expressed as relative light units (RLU).
The sensitivity of this method is 0.1 to 0.5 picoqrams (pg) ATP which is equivalent to about 500 bacterial cells. The method is limited to situations in which the cells present are all bacteria - it is not suitable for food areas because animal and plant cells also contain ATP. However, the method can be used as a measure of total organic cells (sometime referred to as "total hygiene") which gives an indication of the amount of food residues that would provide a substrate for bacterial growth. In larger production facilities the luminometry method can be useful in assessing the efficiency of cleaning procedures, in detecting areas of contamination buildup, or determining the effectiveness of CIP cleaning (in-place cleaning) by measuring the ATP in rinse water.
As cells shed from skin can have a significant affect on ATP measurements, disposable gloves must be worn when carrying out the test, and sampling equipment must be sterilized. As general procedures have not been standardized, those using this method generally rely on suggested schemes provided by the test-equipment manufacturer. The following summary gives the advantages and disadvantages of luminometry in hygiene assessment:
Advantages Disadvantages
rapid results (minutes)high cost
detects soil as well as microbescare required to avoid errors
simple to use (after training)not standardised
result given as a figuregeneral test only - not for specific organisms
future potential 
  
  
  

Swab Method
Swabbing is the oldest and most popular method for assessing the microbiological condition of surfaces - it is used in hospitals and in public health as well as in many areas of the food industry. Swab or contact methods only recover a certain fraction of the total number of microbes on a surface - less than 1% with the contact method and from 10 to 40% (some reports give higher recoveries) with the swab method (the latter recovers more because the mechanical action releases the microbes from the biofilm on the surface). However, the microbes that are released by these methods tend to be those that will be most readily transferred to any products that come into contact with these surfaces. The CONTACT METHOD is best suited to flat, even surfaces, whereas the SWAB METHOD is best for rounded, uneven surfaces.
The swab-rinse method uses either cotton or alginate swabs and a prescribed area of the surface is examined e.g. 100 square cm, and the count made of the microorganisms per unit area. This is usually carried out with the aid of a sterile template which is placed over the area which is then swabbed thoroughly with a moistened swab. The swab is then placed in a specified volume of diluent and the microorganisms are then dislodged by shaking or vortexing. Swabs intended for larger surface areas can come in the form of rectangular sponges or cylindrical sponges in the end of a plastic stick.

Some Other Methods

Sticky film
This consists of pressing sticky film or tape against the surface then pressing the exposed side on to an agar plate. It appears to be less effective than swabbing but is useful for monitoring microorganisms in surfaces such as bottles. Recovery rate reported as being between 8 and 22%.

Ultrasonic devices
Useful for surfaces that are small and detachable such as small cutting blades – these are placed in a container of sterile diluent which is then subjected to ultrasonic treatment. This releases the microorganisms into the diluent from which a bacterial count is made. Ultrasonics may be of use in releasing a greater proportion of bacteria from cotton swabs.

Spray Gun
This involves impinging of a wash solution against a prescribed area of a surface – the solution is collected and examined. This method has been shown to be more effective than swabbing for removing bacteria from meat surfaces.

CULTURE OF BACTERIA RECOVERED FROM SWABS etc
The total bacterial count is normally obtained after culturing of the agar for 2-3 days at 30oC but counts can still be obtained at room temperature (at temperatures below 30oC it may take a longer time for colonies to develop to a size that can be easily counted (3-5 days). Room temperature (20-25oC/5 days) is used for culturing yeasts and moulds (a different, selective, agar is required for this) as these generally do not grow well at higher temperatures. If culturing coliform bacteria and other enterobacteriaciae, incubation at 35oC is preferred. It should be noted that if disinfectant residues such as hypochlorite are on the surface to be tested, a neutraliser such as sodium thiosulphate must be incorporated into the sampling system.

SAMPLING FREQUENCY
This will depend on such things as regulatory requirements, the nature and risk factors in the food-handling operation. As an example, the following table gives suggested frequencies for some common food-handling situations in Europe.

Sampling pointNo. of samplesNo. of times per year
Small shops4-62
Large shops  
- Meat counters4-64
- Working surfaces4-64
- Selected pieces of equipment (3-6 pieces)6-104
- Meat mincers4-66
Small fast food premises6-102
Large fast food premises6-104
Large kitchens10-154
Catering establishments6-103
Meat industry premises8-1652
Fish industry premises8-1652
Dairy industry premises8-1652

The advantage of regular surface monitoring lies in pin-pointing critical areas of poor hygiene and in detecting failures/ omissions in routine cleaning procedures. This enables corrective action to be correctly targeted. Monitoring programs can then be adjusted to ensure the most effective use of a finite number of surface samplings. Whatever the method used, it is important to have a uniform technique so that valid comparisons can be made between test sites.

EXAMPLES OF GUIDELINE STANDARDS
Note: These may vary from industry to industry and from sample type to sample type. The following tables describe a few examples of assessment values for a commercial plastic agar paddle

cfu* per 10 sq. cm. [approx. paddle area]
 Kitchen surfacesMeat industry/ processingMeat industry/ slaughteringHairdressing salons
Good<20<20<18<16
Satisfactory  18-4016-49
Acceptable20-10020-10041-10050-100
Unacceptable>100>100>100>100
As seen in the above tables, a cleaned surface is regarded as unsatisfactory if more than 100 colonies grow on one side of the paddle i.e. more than about 10 colonies per sq. cm..

The following table shows the comparative acceptability levels for some common sampling methods
EvaluationNo. colonies (cfu)
Contact plate
[26 sq.cm.]
Petrifilm
[20 sq.cm.]
Hygicult contact
[10 sq.cm.]10
Hygicult swab
[10 sq.cm.]
Good<50<40<20<200
Acceptable51-25041-19021-200200-1000
Unacceptable>250>190>100>1000
As another example, the following table shows proposed standards for surface counts of dairy equipment. Note that for pasteurised products, the acceptable levels are more stringent as even small numbers of bacterial contaminants can significantly decrease the shelf life of milk products.
GradeCount per 100 sq. cm.
For storage of raw productsFor storage of pasteurised products
Good<200<10
Satisfactory200-100010-50
Unsatisfactory1000-10 000>50
Heavily contaminated>10 000 
TYPICAL PROTOCOL FOR ROUTINE SWABS
A moistened swab is rubbed over a designated area e.g.100 sq. cm. The swab is then either returned to its sleeve for transport to the laboratory, or (under suitable conditions) may be applied directly in the field to the surface of an agar plate which is then sent to a laboratory. Separate swabs may be recommended for each type of micoorganism. The agar plate is incubated at a suitable temperature e.g. for Aerobic (Total) Count: plate count agar, 30°C for 3 days; for E.coli, EMB agar, 36°C for 24h; for yeasts and moulds, Sabouraud agar, 25°C for 5 days. The plates are then examined and the colonies counted. The following is a typical grading system. Note that for meaningful assessments and comparisons, the swabbing procedure needs to be consistent.

KEY TO GRADING

A= no growth
B= 1-25 cfu
C= 26-50 cfu
D= 51-100 cfu
E= 101-250 cfu
F= > 250 cfu
G= TNTC (too numerous to count)
 

WATER SAMPLING

SAMPLING AND CARE OF WATER SAMPLES FOR BACTEROLOGICAL EXAMINATION

It is important to take water samples carefully because this can be vital to the deductions that are drawn from the results. Every care must be taken to avoid contamination during sampling.
At the sampling site, if there are other samples being taken ( e.g. for chemical testing), the sample for bacteriological testing should always be taken first.


GENERAL POINTS ON SAMPLING PROCEDURE
• DO NOT open the bottle (or alternative sample container) until immediately before filling.
• DO NOT rinse out the bottle before taking a sample, especially when sampling chlorinated water, as it contains a chlorine neutraliser (sodium thiosulphate) - N.B. if a test for residual chlorine is required, a sample must be taken in a separate bottle that does not contain neutraliser (25 mL is sufficient for this test)
• When removing cap or stopper from the sample bottle, hold it in such a way that the fingers do not come into contact with its inside surface or with the neck of the bottle. Do not put down the cap or stopper in such a way that will allow it to become contaminated.
• Hold the bottle near the base rather than near the neck.
• Fill the bottle immediately with sample and replace the closure, observing the same precautions as for opening.
• DO NOT COMPLETELY FILL THE BOTTLE but leave about 2.5 cm (1 inch) headspace.

SAMPLING FROM A TAP
a) Remove external fittings such as rubber tubes and hoses.
b) Clean both outside and inside of the tap with a clean rag.
c) Turn the tap full on and allow to run to clear service lines (2 or 3 minutes may be required).
d) Optional step - turn off the tap and sterilise by soaking cotton wool etc in methylated spirits, igniting it and allowing flame to heat metal for 30 seconds.
(Note: This step is optional because of controversy over the necessity to flame taps. The critical factors are care in the selection of the sampling tap, that it is clean and in good repair, and allowing water to run before sampling.)
e) Adjust flow to a gentle stream and fill the sample bottle to the required level. Avoid splashing.

SAMPLING FROM A RIVER, SPRING, LAKE, RESERVOIR, OR WELL
a) Take a sample which is representative of the water used by the consumer. Therefore do not sample too near the bank, or too far from the draw-off point.
b) Hold the bottle near its base and plunge it, neck downwards, to about 30 cm below surface.
c) Turn the bottle until the neck points slightly upwards with the mouth directed towards the current. If there is no current, move the bottle forward away from the hand. If the bank is likely to be disturbed during sampling, or there is some other difficulty in taking a sample at the edge of the bank, attach the bottle to a rigid pole (1 to 1.5 m long).
d) When the bottle has filled, remove it from the water and immediately replace the closure.
e) If it is not practicable to collect samples this way, a weight can be attached to the bottle which is then lowered into the water attached to the end of a line. If this needs to be done frequently, it can be more convenient to have a weighted frame made to hold the bottle.

SAMPLING FROM A HAND PUMP

a) Pump for about 5 minutes before collecting the sample.
b) Optional step - (see note in Sampling from a Tap) Flame mouth of pump, then flush again.
c) Collect the sample directly into the bottle from the pump.

SAMPLE SIZE
Samples should be sufficient for all tests required plus an extra 50%, but preferably not less than 250 mL for bacteriological testing, except for waters known to be highly polluted. At least 500 mL is required for a full examination of drinking waters and pool waters.

CHLORINATED WATER SAMPLES
As any residual chlorine should be neutralised at the time samples are taken, 0.1 mL (2 drops) of a 10% solution of sodium thiosulphate is added to each 500 mL sample bottle (1 drop per 250 mL bottle) before sterilisation. This gives a concentration of about 20 mg/L in the sample, which is sufficient to neutralise more than 2 mg/L of free and combined chlorine in all except highly chlorinated waters. Unchlorinated samples will not be affected by the thiosulphate.

PRESERVATION AND STORAGE OF SAMPLES
• Samples should be transported to the laboratory with the least possible delay so that they can be examined before there are any changes in bacterial levels. The maximum delay is 6 hours for preference, but if this is not feasible, samples should be chilled (ideally, below 5°C but not frozen). It is best not to delay testing beyond 24 hours as the total number of bacteria is likely to change after this time - tests on samples more than 30 h old should be limited to indicator bacteria.
• Samples more than 48 hours old are normally unsuitable for routine potability tests because the levels of indicator bacteria may have changed enough to give a misleading interpretation of the actual condition of the water. In such circumstances, clients are normally informed and given the option of re-sampling or continuing with the test.

DATA WHICH SHOULD ACCOMPANY SAMPLES
• All samples should be labelled and identified, and accompanied by adequate descriptive data - bottle labels have spaces for the basic sample details. It is advisable to fill these in before sampling - use a ballpoint pen or a pencil (avoid felt pens or other ink pens that are liable to run when wet - these make the labels illegible).
• Except for routine testing, it is advisable to notify the laboratory by phone, fax, or E-mail when forwarding samples - this will help to ensure that the sample type and quantity is suitable for the tests required
• The laboratory request form should also be completed (in case labels become detached or impossible to read). Other details to include with the sample/s, are the name of the sender /collector, the type of water, the source of the sample, the date and time of collection, and the reason for examination (e.g. suitability for drinking, suspected cause of illness etc). Other relevant information (e.g. weather conditions, accessibility to animals etc) is also useful if an interpretative report is required.

Cautionary note: A single laboratory examination, no matter how favourable the result, does not justify the conclusion that a water is suitable for drinking purposes. It merely indicates whether or not it has been subject to recent faecal pollution, and this does not exclude the possibility of dangerous pollution in the future, or in the more distant past.