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Microbiological Techniques 
To Confirm CIP effectiveness            

Introduction

The main reason for the lack of application of microbiological methods for quality assurance is one of speed. In order to be able to control a process it is necessary to obtain information in real time. Traditional microbiological procedures, relying as they do on growth of spoilage organisms, are inevitably very slow and can only provide retrospective reassurance of adequate cleaning. In contrast, techniques which measure physical or chemical characteristics can provide a response rapid enough to enable operational decisions to be made. Nowadays, microbiological techniques are becoming available which can provide information quicldy enough for process control. They can only do this by dispensing with the need for micro organisms to grow and by measuring physical or chemical changes associated with the presence of living organisms.

Methods for Ensuring Cleanliness of Plant

• cycle times
• solution temperatures
• flow rates
• pressures
  
  
  

Additionally, a wide range of chemical analyses can be used including:

• detergent concentration (using conductivity)
• alkalinity (either in-line or off-line)
•  specific chemical activities (e.g. sequestrant concentration)
   pH
• soil load of detergent solution (this can be determined by measuring colour, suspended solids, tendency to foam etc.)
• causticity

However, despite the sophisticated control and measurement systems now available for quality assurance of CIP procedures, there is still a need for ensuring that the process has actually worked. This is an area where microbiological methods can take their place alongside physical and chemical measurements.

Methods for Assessing Cleanliness of Plant
There are only four basic ways of assessing plant cleanliness:

1. Visual Inspection

This is, of course, a very simple procedure involving merely looking at the equipment which has just been cleaned. The only tools which are required are a small lens and a torch to help with the inspection. Although apparently trivial, this is a very fast and valuable approach for assessing the efficiency of CIP systems. It is possible to assess cleanliness and to detect residual soil and adsorbed microbes. If, on inspection, plant appears dirty then, without doubt, the CIP system has failed. The response is instantaneous and there is no need for any further checks. Such visual inspections can be carried out when doing the assessments or collecting samples for laboratory analysis, emphasising the importance of brewery microbiologists keeping their eyes open when working on he plant. Although it is a valuable procedure, the main problem with visual inspections is that access to the plant is required which, with modern equipment, can be difficult

2. Plant Swabbing

This technique, involving rubbing a swab over a surface in order to pickup any contaminants, can be used for both chemical and microbiological analyses. Once again, access to the plant is required. When performing a swab analysis it is extremely important that a defined area is monitored if results from different equipment and different samples are to be compared. Care must also be taken when deciding which parts of the plant to swab; monitoring only easily accessible areas can lead to optimistic results whereas the examination of poorly accessible areas alone can give pessimistic results. Again the person carrying out the collection of the swabs needs to use their eyes. If the swab becomes visibly dirty whilst the sample is being taken, inadequate cleaning is immediately indicated.

Depending upon the analysis system employed this technique can detect both residual soil and microorganisms. Two approaches can be used for taking the samples. One involves rubbing sausage-shaped agar medium over the surface being investigated and then incubating the agar to assess whether anything grows. This is not really appropriate for CIP monitoring because it leaves residues of growth medium on the freshly-cleaned surface. By far the best approach is to use conventional swabs (sticks with an area of cotton, alginate or synthetic material on one end) and then transfer any collected material into an appropriate solution which can be tested for the presence of dirt or microbes

3. Final Rinse Sampling

This is in effect a special case of swabbing. Either a pressure spray is used to rinse a vessel and the liquid draining out collected and analysed or, more usually, a sample of the final rinse from the CIP sequence is collected. The major advantage of this type of sampling is that access is not required. However for the test to be of any value it is essential that the rinse fluid makes good contact with the surfaces being examined. Additionally the liquor used for the final rinse must itself be sterile or the test will be worthless. There are a number of ways of sterilising this liquor, both physical (ultraviolet light, heat, filtration) or chemical (silver, ozone, chlorine dioxide, peracetic acid). Where terminal sterilants are added it is vitally important that these are neutralised before any tests are carried out since the sterilant will continue acting in the test medium and give a false negative result.

Final rinse samples can, of course, be assessed for the presence of product, chemical contamination or microbes but in all cases the contaminant materials must be either soluble or physically removed by the rinse if they are to be detected.

4. Testing Next Batch

Once again access is not required to carry out this type of test, but as a method for detecting CIP failure, it leaves a lot to be desired since, by its very nature, a positive result is obtained by detecting contamination and potential spoilage of the final product. Hence this is not a method to be recommended unless it is impossible to use any of the other techniques described above. "Clear" results from forcing tests of packaged beer can give a certain degree of reassurance; failures however cause a large workload for brewing and laboratory personnel since the problem may lie almost any where within the brewery

Which Method to Use?
The choice of method largely depends upon whether it is possible to gain access to the plant. If access is possible then a combination of final rinse sampling, swabbing and visual inspection should be used. If access is not possible then there is no other choice but to use final rinse. In a modern plant designed for CIP operations, final rinse sampling alone will probably be adequate. In older plant not originally intended for in-place cleaning, final rinse sampling can be misleading since pockets of contamination may go unnoticed and the benefits of visual inspection will be lost.

Thus, rinse samples are universally applicable and, in most cases, will probably be the method of choice. Swabbing should not however be ignored: it is a very valuable technique which tends to be used less frequently because of the disruption to plant operations that it causes. It is particularly useful for checks on centrifuges, heat exchangers, fillers etc. As already indicated it is more applicable to small plants than to large complex modern breweries. Finally, I will stress again the value ofvisual inspection: it is simple, quick and, if used correctly, can provide valuable information.

Methods for Assessing Samples
Once the samples have been taken they have to be analysed in some way. With final rinse samples it is possible to examine them directly for turbidity, although this will only detect major failures. Additionally, microscopic examination of rinse samples or of swabs resuspended in saline can be used. Again this will only detect large failures but is more sensitive than measuring turbidity. Normally samples are examined either microbiologically, by growing the contaminant microorganisms, or chemically, by analysing for sterilants, detergents, beer residues or microbial metabolites

Microbiological Methods
For final rinse samples where the rinse water contains a disinfectant it is necessary to neutralise before testing. With peracetic acid and halogens this is best achieved by dosing with sodium thiosulphate. The sample can then be analysed either directly or following concentration by membrane filtration. For direct analysis the method used depends upon the sensitivity required. Up to O.2ml of sample can be analysed by the spread-plate technique, volumes of l-5ml require pour plates whilst larger volumes have to be incubated as liquid forcing samples. Membrane filters are normally laid on agar plates or pads soaked with nutrient medium although they can be immersed in liquid medium.

Swab samples can be analyzed directly or after they have been resuspended in saline. Direct analysis involves either streaking the swab across the surface of an agar plate or immersing it in liquid medium. Saline suspensions derived from swabs are treated in exactly the same way as final rinse samples.

The choice of medium and incubation conditions depends to a large extent on the amount of information required and the personal preferences of the brewery microbiologist. Since the purpose of testing samples from CIP systems is to confirm that cleaning has been satisfactory, it is usually sufficient to assay for total contaminants; the presence of any micro-organisms, beer spoilers or not, indicates a failure of the cleaning system. Thus a non-specific medium capable of supporting the growth of most micro-organisms is adequate in most cases. If the aim is to detect only brewery-based microbes then media containing beer or wort are indicated. Only when it is necessary to identify specific organisms should selective growth media be employed. The types of organisms which can spoil beer are restricted to wild yeasts, lactic acid bacteria, acetic acid bacteria, Zymomonas, Pectinatus and Megasphaera. For the most part, wild yeasts and lactic acid bacteria are all that need concern the majority of brewers, certainly when considering the efficiency of CIP operations.

Once the organisms have grown, detection is normally visual, assessed either as colonies growing on membranes or as turbidity in liquid samples. Microscopy can be used to confirm the nature of the organisms, if this is required.

As already mentioned, the biggest problem with microbiological analyses of this type is that the time for detecting failures can be very slow because of the need for growth to occur. This is true even of modern systems which detect growth automatically. They shorten detection times but in operational management terms they are still not rapid enough for the results of a CIP failure to be corrected. In order for this to be possible the samples must be assessed chemically.

Chemical Methods
There are a range of chemical tests available for assessing swab and final rinse samples. Specific tests are provided by manufacturers for detecting detergent and sterilant residues, whilst beer residues can be detected by assaying for components such as sugars, oxalate, amino-acids (soluble nitrogen) or alcohol. The most sensitive chemical assays are those that look for microbial residues such as ATP or DNA. The detection systems employed do not depend on traditional analytical chemical methods but involve modern biotechnological procedures. The systems relying on ATP measurement are already commercially available and are finding widespread application in the food and drinks industries. Those based on detecting DNA are still at the research stage but will undoubtedly become available for routine use in the near future

ATP Measurement
The measurement of ATP depends upon the reaction which causes the tails of fireflies to glow. By reacting samples with luciferase enzyme from the firefly and measuring the light produced, an estimate can be made of the amount of ATP present. The quantity of light produced is directly proportional to the amount of NFP in the sample and hence to the level of contamination.

ATP bioluminescence systems can be applied in two ways. For hygiene assessment, swabs are employed and total ATP derived from both microbes and soil is measured. With rinse samples it is usual to monitor only ATP derived from microbial sources. This can be either total microbial ATP measured immediately, or ATP from specific contaminant organisms measured after an incubation period.

Hygiene assessment is very simple to perform. The whole procedure from taking swab sample to measuring the result typically takes about one minute well within the time needed to make operational decisions about the effective ness of a CIP system. The correlation between ATP results and normal microbiological analyses is usually very good. It is important to remember that the results shown here were obtained after vastly different incubation times (1 minute for ATP, 2 days for plate count).

Analysis of the total microbial ATP in rinse-water samples takes somewhat longer, typically one hour. The wash and enzyme treatment prolong the test. With such a test it is possible to monitor yeasts directly; typically 1O cells can be detected. Rather higher numbers of bacteria (ca. 1000 cells) are required for instant detection in this way. Low bacterial counts can only be detected after an incubation period of between 24 and 48 hours. The procedure is broadly the same as for the 1-hour rinse water assay except that the membrane is incubated on a selective medium for the required time prior to washing and assaying for ATP. In this way membranes harbouring as few as 6 lactic acid bacteria can be detected after 24-48 hours incubation, a saving of 3-5 days on the normal incubation time required to reveal visible colonies.

Polymerase Chain reaction
(PCR)
This new technique for detecting low levels of micro-organisms has arisen from the explosive development of recombinant DNA (genetic engineering) procedures in recent years. It is a rapid procedure for amplifying very small specific pieces of DNA so that they can be easily visualised by simple gel-staining procedures. The presence of DNA originating from a particular type of organism in a sample can be readily detected within a working day. The specificity resides in the primer DNA molecules used in the amplification stages. By varying these the presence of different organisms in the same sample can be readily detected. The PCR procedure thus fulfils the main objectives of any CIP microbiological assay of being specific, sensitive and rapid. Unfortunately the sensitivity can be a problem; since the technique is able to detect very small quantities of DNA, great care has to be taken when handling samples to avoid cross contamination. Additionally components in beer can reduce the sensitivity of the system and, since it is detecting DNA, dead cells will also be picked up. These difficulties are currently the subject of much research and within a few years PCR will surely become one of the battery of tests available to the brewery microbiologist

Conclusions
A wide range of sampling techniques and monitoring systems are available to confirm the effectiveness of CIP procedures. Current best practice can be summarised as follows

Sampling

Of the techniques available both swabs and final rinses can provide suitable samples for analysis. The value of visual inspection cannot be over-emphasized: it is a technique too often lacking in modern automated plants

Monitoring

The choice of analytical methods depends upon the data being sought. For hygiene monitoring, ATP bioluminescence is undoubtedly the method of choice with total count measurement as a backup or as an alternative if the rapid method is not available. For detecting specific spoilage organisms the use of selective media is still the only practical choice, although the incubation times can be significantly shortened by the use of ATP measurement for detecting the presence of organisms rather than waiting for the appearance of visible colonies.

The methods described in this article can form a valuable part of an efficient CIP system within a brewery. However, it is important to stress that effective control of CIP operations is of paramount importance whilst the methods described here can provide reassurance or detect failure they cannot themselves produce a clean and sterile plant.

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