Vous êtes sur la page 1sur 152
Training Manual on QUALITY ASSESSMENT OF FOOD PRODUCTS College of Food and Dairy Technology Tamil


College of Food and Dairy Technology

Tamil Nadu Veterinary and Animal Sciences University Koduvalli, Chennai - 52.




Page No.

Chapter 1


Total Quality Management in Food Industry


Advancements in Food Inspection Tools


Sampling of Food Products.

Chapter 2


Quality Control of Milk and Milk Products


Bacteriology of Water

Chapter 3


Quality control of Meat


Estimation of Egg Qualities


Quality Control of Fish

Chapter 4


Fruit and Vegetables Storage Guidelines


Adulterants in Foods and its Detection Methods

Chapter 5


Quality Testing of Bakery Products


Permissible Limit of Contaminants, Toxins and Residues in Various Food Products


Procedure for Packaging of Various Food Products

Chapter 6


Food Borne Diseases


Prevention and Control of Food-Borne Diseases.

Training Manual on Quality Assessment of Food Products



Training Manual on Quality Assessment of Food Products



G. Sujatha 1 , D. Baskaran 2

1 Assistant Professor, College of Food and Dairy Technology, Koduvalli, Chennai 600 052 2 Professor and Head, Department of Food Engineering, College of Food and Dairy Technology, Koduvalli, Chennai 600 052

Quality and food safety have become competitive edge in the global market for the enterprises producing and marketing foods products. Therefore, the installation of ISO 14000, ISO 22000 Quality Management Systems and Hazard Analysis and Critical Control Points (HACCP) based food safety system is extremely desirable in view of the changing scenario of food market in the international trade. With a view to motivating the food processing industries for adoption/ implementation of food safety and quality assurance mechanisms such as Total Quality Management (TQM) including, ISO 14000, ISO 22000, HACCP, Good Manufacturing Practices (GMP), Good Hygienic Practices (GHP) and prepare them to face the global competition in post WTO era, Ministry of Food Processing Industries is implementing a Plan Scheme for Setting up/ Up-gradation of Quality Control/ Food Testing Laboratory/ R &D and Promotional Activity.

The objectives of this Plan Scheme are as under

1. To motivate the food processing industries for adoption of food safety and quality assurance mechanisms such as TQM including ISO 14000, ISO 22000, HACCP, GMP, GHP;

2. To prepare them to face global competition in post WTO Regime;

3. To enable adherence to stringent quality and hygiene norms;

4. To enhance product acceptance by overseas buyers;

5. To keep Indian industry technologically abreast of international best practices;

6. To focus attention on the development of Food Processing Industries through participation, directly or indirectly, in seminar, workshop, international events etc.

The scheme has the following components

Setting Up/ Up-gradation of Quality Control/ Food Testing Laboratory

Implementation of HACCP/ ISO 22000, ISO 14000/ GHP/ GMP Etc.

Research & Development in the Food Processing Sector


The outlay under the scheme during 11th Five year plan was Rs 250.00 crore. As against this, the Budget allocation under the scheme was of the order of Rs. 140.30 crore. Of this, the Ministry utilized Rs. 126.36 crore.


Quality management is becoming increasingly important to the leadership and management of all companies for competitive advantage, thus it is important to view TQM as a distinct management

philosophy which advocates of excellent management of every department of the company. Total quality management (TQM) is a management philosophy for continuously improving quality of goods and services delivered through the participation of all organizational members; it is the process of making quality the concern of every-one in the company (Zelealem & Getachew,2002). TQM mainly focuses on

1. Customer satisfaction

2. Human resource management

3. Continuous improvement

4. Leadership and management

Effective measurement of total quality cost gives the following benefits

1. increased opportunity to make objective business decisions based on quantitative data rather than judgment

2. identification and priotization of areas and aspects of the business requiring quality improvement and quality cost reduction

3. ability to compare the performance of departments and processes as well as in some instances the performance of the company against competitors

4. improved quality in budgeting and cost control with the standardization of measures of performance and comparators

5. increase in the focus of management on quality and quality improvement as an integral part of management responsibility.

TQM philosophy champions a considerable number of concepts which are fundamental for effectively quality management, are

1. Customer Focus: TQM champions customer satisfaction, thus companies should be aware of the customer quality needs and should work to achieve them in order to retain customer goodwill and maintaining the market share

2. Quality Assurance: Food products produced should satisfy or exceed the minimum required quality standards. Furthermore products should abide to the local quality regulations as well as the international ones e.g ISO standards. Food companies use Hazard Analysis Critical Control Point (HACCP) as a quality assurance strategy.

3. Benchmarking: The TQM philosophy highlights that companies should engage in benchmarking activities to monitor control and assess the company’s products, ser-vices and processes relative to other highly performing companies. This includes product, energy, strategic, processes and financial benchmarking

4. Maintenance: TQM highlights that adequate maintenance of the companies processes should be initiated to ensure perpetual excellence of processes. Effective organization-al maintenance is achieved through Total Productive maintenance focus office TPM, quality and planned maintenance.


Human Resource management: The TQM philosophy most importantly focuses on the human resource of the company emphasis mainly on motivation and employee satisfaction through adequate salaries, training and education, empowerment and involvement in an effort to harness commitment from all employees.

Thus Total Quality Management (TQM) needs great under-standing to properly manage and achieve quality performance of any organization. The use of accounting concepts to justify investment in TQM should not be applied because the management philosophy materializes after a considerable time period. However companies and stakeholders should look at other entities to justify TQM implementation. These include

Product quality & customer satisfaction

Rate of employee absenteeism

Quality rate and O.E.E

Scrap and rework levels

Customer goodwill and market share

Due date performance

ISO certification success levels

Accidents and health risks

Market response

The Hierarchy of TQM performance indices

1. Customer Focus

2. Quality Assurance

3. Benchmarking

4. Process quality control

5. Supplier Management

6. Environmental and safety management

7. Strategic quality planning

8. Production management

9. Leadership and Management

10. Teamwork and quality circles

11. Continuous improvement

12. Maintenance

13. Employee involvement & empowerment


Training and education

15. Human resource management

16. Employee satisfaction

17. Information analysis

18. Social responsibility.

The results show that the food manufacturing and processing industry at the moment (2010-2011) is mainly focused on the customer. As shown by the hierarchy concepts of customer focus, quality assurance, benchmarking and process quality control are highly ranking. Furthermore concepts which mainly focus on the employee are sidelined as highlighted by the hierarchy. Employee satisfaction, human re-source planning, empowerment and involvement are low ranking.


Six Sigma is a project-, data- and technology-driven quality management tool and acts as a business improvement strategy in order to improve the business competitiveness through reducing the defects and improving customer-oriented quality. Six Sigma is a highly and rigorously systematic approach to greater quality based on figures and facts and it is one of the best tools to measure the performance in an organization of any size or in any process. A food distribution SME is in the so-called ‘service industry’ in which quality attribution is unlikely to be clear for the quality management team due to unclear customer assessment and customer satisfaction criteria. Therefore, Six Sigma could be a good approach, since it is a process based on performance measurement and this is a fundamental requirement to improve the Supply Chain. Arguably, Six Sigma is not always a bunch of clear and great results in the service industry. It has been suggested that implementing Six Sigma in the service industry could be more difficult in the manufacturing side, as the human recourses and ambiguity could impact on the application of Six Sigma.

and ambiguity could impact on the application of Six Sigma. Figure- complexity of food chain with

Figure- complexity of food chain with food distribution in centre

Six Sigma has a great link with other quality initiatives and highlights the values of the integrated approach of all these tools to achieve a collaborative Supply Chain in the food distribution SME. Six Sigma is a combination of the Six Sigma statistical metric and TQM, with additional innovations that enhance the program’s effectiveness while expanding its focus”. The main components of Six Sigma retained from TQM include a focus on the customer, recognition that the quality is the responsibility of

all employees, and the emphasis on employee training. The importance of the integrated view of these quality initiatives will enable the management team in food distribution SME to avoid individualistic views and, instead, enlarge their cross-functional view and increase their ability to look at ‘quality’ from different angles. It is believed that TQM is an approach to improving the competitiveness, effectiveness and flexibility of a whole organization. The impact of TQM on any organization is first to ensure that the management adopts a strategic view of quality. Six Sigma as a tail of TQM can be next step to identify the most critical aspects of quality and solve the problems through performance measurement in a systematic way. Contribution of Six Sigma in Supply Chain has been acknowledged as its methodology can be adopted to improve Supply Chain through different aspects. It has been indicated that Six Sigma has proven to be extremely valuable by reducing the cycle time, by providing new problem- solving tools and by providing improved functional and customer alignment and by allowing calculations of the value of R & D. Six Sigma has a collaborative interaction with SC as balanced score card is a top requirement for both to define the customer requirements.


The fishbone diagram, also called the Ishikawa diagram, was created in Japan. The fish bone diagrams have since become popular in many parts of the modern business world, including in manufacturing, where they can be useful in assessing what has gone wrong with a complex process.

Fishbone diagrams are composed of a main horizontal line where smaller lines branch off of the main line diagonally. This makes the chart look like a fish skeleton. The “fish bones” represent cause and effect in a situation where it’s necessary to troubleshoot a production problem or other dilemma. The fishbone diagram provides a much better “quick picture” perspective than a block of text, which is a main part of its appeal to busy executives.

Fishbone diagrams for product design, quality control, and other common uses often group different types of causal factors onto the same fish bone or category. These include people, or those who are involved in the process, as well as Method, or how the job was designed to be done. Other categories include Machines, the gear used in the process, Materials, the raw goods used, and Environment, a larger catch-all term for a variety of causal factors. Some types of fishbone diagrams use words with the same beginning letter to promote easy categorization.

with the same beginning letter to promote easy categorization. Training Manual on Quality Assessment of Food

Fishbone diagrams could be called an “aggregate model” since they incorporate smaller causes contributing to larger ones. This is represented by small diagonals attaching to the various diagonals that attach directly to the main horizontal line. This kind of model is useful in visually assessing a number of conditions or events that may have a bearing on a production outcome.

Fishbone diagrams are just one of several types of cause and effect diagrams that planners can use to minimize problems in a task. The same kinds of professionals who use fishbone diagrams may use histograms, pareto charts, scatter diagrams, control graphs, check sheets, or any number of other planning and troubleshooting tools. In more complex systems involving money or other variables, advanced planning might utilize tools that measure or quantify a variable condition in more abstract ways. Computers have made more kinds of projections in decision-making possible for human planners, and in the modern world of planning, fishbone diagrams represent a more concrete or basic planning tool.


G. Sujatha 1 , D. Baskaran 2

1 Assistant Professor, College of Food and Dairy Technology, Koduvalli, Chennai 600 052 2 Professor and Head, Department of Food Engineering, College of Food and Dairy Technology, Koduvalli, Chennai 600 052

The modern food inspector must think of himself/herself as a reviewer of the food safety control measures and a contributor to their improvement. Control measures must individually evolve continuously in response to technological advances and to the establishment‘s own experience, and as part of what should be an equally evolving national food control system. Such improvements can also be very advantageous to the processor from a marketing standpoint. Product safety and quality are characteristics that can be exploited to gain advantage in the marketplace. The food inspector is in a unique position to convey these messages to food producers and processors. Creating awareness about food safety and quality among food producers and processors is as important an element of food inspection as verifying compliance with regulations. Improvements to a quality and safety management system are almost always possible and attainable if the processor is willing to progress and the inspector is able to assist.

A generalized trend in modern food processing safety and quality assurance systems is the concept of statistical process control (SPC), which is based on continuous improvement. Although this topic has dealt with SPC only marginally, when discussing critical limits, disseminating the concept of continuous improvement of the quality and safety management system and actively contributing to such improvement must be integral parts of food inspections.Food inspectors monitor food distributors, processors and manufacturers for safety and sanitation violations that could lead to food being contamination with adulterants or bacteria. They may inspect live animals and carcasses at a slaughterhouse or examine food imports at a port for mislabeling or other food safety issues. Inspectors report violations to the appropriate regulatory agency and are responsible for ensuring that foods are suitable for human consumption.

Basic inspection tools

1. Pen and pencil

2. Writing tablet

3. Notebook

4. Inspection forms

5. Watch

6. Gloves

7. Aprons

8. Microbiological Sample Kit for Total Plate Count (by 3M) (optional)

9. Flashlight

10. UV light (optional)



12. Copies of Laws and Regulations

13. Hair nets

Nature of inspection tools

1. Portable, preferably small and lightweight, non-reactive, rugged and safe to use.

2. Disposable or easily cleaned and decontaminated or easily protected from contamination. Relatively inexpensive.

3. If powered tools, should run on “standard” batteries.

4. Easy to replace any battery readily available from most convenience stores.

5. Expendable materials used with any instrument should be non-proprietary;

6. Simple and easy to operate with one hand because our other hand is generally holding a notebook, pen and flashlight.

7. Accurate and easy to read, even in poor lighting conditions.

8. Easy to calibrate or validate in the field.

9. Capable of maintaining accuracy during hard use.

10. Not subjected to interference or damage by adverse environmental conditions or during transportation

11. Direct read or requiring only minimal interpretation of results.

12. Conforms to standards or standard protocols whenever possible or practical.

13. If any part of the instrument comes into contact with food, it should be non-toxic instruments to be versatile.


The Food Code also defines potentially hazardous foods in terms of water activity (AW) and pH, and the interaction of the two under certain conditions. Therefore, any comprehensive food safety evaluation of retail food establishments must include the monitoring of these two parameters.

Water Activity. There are a few very good portable water activity monitors offered in the marketplace. They are similar to the pen-type hygrometer and not really easy to operate. About five years ago, Decagon Devices, Inc. introduced the easy-to-use and compact Pawkit. The Pawkit is a small, lightweight, albeit rather pricey, field kit for measuring water activity in foods. Pawkit for measuring ambient relative humidity, if conditions call for it. Measuring water activity with the Pawkit is relatively simpleCalibration is also easy to check and adjust whenever necessary. The kit contains everything needed for field work including the instrument, sample cups and verification standards.

Measuring pH.

For some inexplicable reason, The pH pens do well back in the lab but whether it is the jarring or temperature extremes from transporting them to the job site, it seems that food inspectors always have to rely on some other method for pH screening.


rely on some other method for pH screening. Refractometers There is always another way to characterize

There is always another way to characterize potentially hazardous foods from those that are not potentially hazardous: refractometry. Consider purchasing inexpensive handheld sugar/Brix and salinity refractometers to measure syrups and brines. Refractometers are available through most scientific suppliers.

The Food Code guidelines necessitate the use of several types of food safety inspection instruments, including light meters, single-use paper thermometers, and sanitizer indicator papers.

Light Meter

The food inspectors are partial to the General Electric (GE) Type 217 light meter available through most scientific catalogs. It is the ideal portable instrument: smaller than the typical size of a digital unit, rugged, versatile and lightweight and it uses the light it measures as its energy source—therefore, no batteries. The only drawback is that it cannot measure illumination below 10’ candles.

is that it cannot measure illumination below 10’ candles. Single-Use Paper Thermometer It is a thermocouple

Single-Use Paper Thermometer

below 10’ candles. Single-Use Paper Thermometer It is a thermocouple thermometer with an Atkins 50415

It is a thermocouple thermometer with an Atkins 50415 dishwasher probe. However, for routine inspections and day-to- day quality assurance the paper thermometer are found to be convenient and quite accurate.

thermometer are found to be convenient and quite accurate. They have the following advantages like the

They have the following advantages like the self-adhering label works on any clean porcelain or metal surface, Thermolabel measures the temperature on a heat-sink (which implies a dwell time), the label is accurate to within 1% of the calibrated temperature and the label is removable and can be repasted on the inspection report and serve as a permanent record

Sanitizer Indicator Papers The reaction on most chlorine indicating papers is a change in the intensity of blue color. The food inspectors use Code 4250-BJ, acid-free LaMotte Chlorine Test Papers, whose end-point color is probably the easiest to interpret. Quaternary ammonium chloride sanitizers, however, are measured with pH. The pHydrion QT-40 test papers, manufactured by Micro Essential Laboratory, relies on color change. The comparator with each roll pack is already given in parts per million (ppm). Micro Essential Laboratory has a wide variety of sanitizers.


It uses the basic AA-cell type halogen flashlight featuring one-handed operation. The latest constant companion is the Streamlight 4AA ProPolymer LED flashlight. This flashlight uses four AA batteries and features seven LED bulbs that produce an extremely intense white light.

seven LED bulbs that produce an extremely intense white light. Training Manual on Quality Assessment of

While this flashlight cannot be adjusted to produce a spot, its light provides startling visual contrast, thereby making it easier to spot insect and rodent damage, water leaks and chronic wetness, and general soiling. The flashlight is ruggedly built and features a protected on/off switch. The battery life far exceeds that of the halogen lights. LED flashlight 80 lumen directed light beam of the Infinition shows everything and is indeed the best tool for any inspection

UV Light ultraviolet (UV) light to a food safety audit because of its size and weight. However, in the past year the Emissive Energy Corp. introduced theInova X5 UV flashlight. The Inova X5 is a small (4.75"), hand-held LED powerhouse that uses five super-bright UVLEDs to produce abroad smooth pattern of light.

For the first time, we now have a flashlight bright enough to detect rodent urine and mold. It is quite handy to qualify the level of cleanliness in restrooms. The Inova X5 uses two 3V lithium batteries and comes with an open-top nylon belt holster.

batteries and comes with an open-top nylon belt holster. Ground Fault Interrupter Electrical Safety to small

Ground Fault Interrupter

an open-top nylon belt holster. Ground Fault Interrupter Electrical Safety to small instruments to ensure our

Electrical Safety to small instruments to ensure our safety (and that of the employees), and to detect most electrical problems. The A/C Sensor, which is slightly larger than a pen, simply indicates the presence of electrical current when held close to a live source or piece of equipment. A ground fault circuit interrupter/receptacle tester is also needed to check the wiring configuration of outlets, as well as the Ground Fault Interrupter (GFI). Both are available in most hardware stores.

Ventilation. to characterize ventilation, particularly direction and flow, the Flowchecker. A small squeeze bottle that contains amorphous silicon dioxide powder available from Lab Safety Supply. For something a bit less expensive, try a zinc stearate powder in a small, empty nasal spray bottle is complemented with an inexpensive and compact Dwyer 460 Air Meter.

Other miscellaneous includes 8’ tape Measure, a pen-sized door pressure gauge and a 1:20/1:12 ramp-slope bubble measuring device, pressure gauge and ramp-slope bubble measuring device are manufactured by HMC International of Littleton, CO are also used in addition to all of the above, the basic food safety field kit also contains the following:


Wooden-stick cotton swabs to show where the dirt really is.


A collapsible inspection mirror (Sears Craftsman) to see where the dirt really is.


A small awl or ice pick for a thousand and one uses.


Alcohol wipes.


Self-sealing, quart-sized plastic bags.


Vinyl disposable exam gloves (sensitive to latex allergies).


A small blade and Phillips screwdriver and tweezers.


A Mellon tester pocket knife and/or an electrician’s knife.


Dr. P. Selvan

Assistant Professor, College of Food and Dairy Technology


To control food quality and acceptance within satisfactory limits, it is important to monitor the vital characteristics of raw materials, ingredients, and processed foods. This could be done by evaluating all foods or ingredients from a particular lot, which is feasible if the analytical technique is rapid and non-destructive. However, it is usually more practical to select a portion of the total product volume and assume the quality of the selected portion is typical of the whole lot. Obtaining a portion, or sample, that is representative of the whole is referred to as sampling, and the total quantity from which a sample is obtained is called the population. Adequate sampling technique helps to ensure that sample quality measurements are an accurate and precise estimate of the quantity of the population. By sampling only a fraction of the population, a quality estimate can be obtained more quickly and with less expense and personnel time than if the total population were measured. The sample is only an estimate of the value of the population, but with proper sampling technique, it can be a very accurate estimate.


It is important to clearly define the population that is to be sampled. The population may vary in size from a production lot, a day’s production, to the contents of a warehouse. Extrapolating information obtained from a sample of a production lot to the population of the lot can be done accurately, but conclusions cannot be drawn from data describing larger populations, such as the whole warehouse. Populations may be finite, such as the size of a lot, or infinite, such as in the number of temperature observations made of a lot over time. For finite populations, sampling provides an estimate of lot quality. In contrast, sampling from infinite populations provides information about a process. Regardless of the population type, that is, finite or infinite, the data obtained from sampling are compared to a range of acceptable values to ensure the population sampled is within specifications.

i. Homogeneous Versus Heterogeneous Populations

The ideal population would be uniform throughout and identical at all locations. Such a population would be homogeneous. Sampling from such a population is simple, as a sample can be taken from any location and the analytical data obtained will be representative of the whole. However, this occurs rarely, as even in an apparently uniform product, such as sugar syrup, suspended particles and sediments in a few places may render the population heterogeneous. In fact, most populations that are sampled are heterogeneous. Therefore, the location within a population where a sample is taken will affect the subsequent data obtained. However, sampling plans and sample preparation can make the sample representative of the population or take heterogeneity into account in some other way.

ii. Manual Versus Continuous Sampling

To obtain a manual sample the person taking the sample must attempt to take a “random sample” to avoid human bias in the sampling method. Thus, the sample must be taken from a number of locations within the population to ensure it is representative of the whole population. For liquids in

small containers, this can be done by shaking prior to sampling. When sampling from a large volume of liquid, such as that stored in silos, aeration ensures a homogeneous unit. Liquids may be sampled by pipetting, pumping, or dipping. However, when sampling grain from a rail car, mixing is impossible and samples are obtained by probing from several points at random within the rail car. Such manual sampling of granular or powdered material is usually achieved with triers or probes that are inserted into the population at several locations. Errors may occur in sampling, as rounded particles may flow into the sampling compartments more easily than angular ones. Similarly, hygroscopic materials flow more readily into the sampling devices than does non hygroscopic material. Horizontal core samples have been found to contain a larger proportion of smaller-sized particles than vertical ones. Continuous sampling is performed mechanically.

iii. Importance of Sample Collection

The reliability of analytical data thus obtained depends on several factors, sampling being the major factor. Current analytical methods require only few grams of food sample to analyze. Thus, it is necessary that a sample be as representative of the population as possible. There are three basic activities involved in analysis of food products:

Collection of representative sample.

Sample preparation.

Analysis using appropriate methods and instruments.

These activities, although independent in nature, yet can have decisive influence on each other. Furthermore, each of these activities have their own potential sources of variations that contribute to the uncertainty level associated with any analytical result. Thus, care must be taken to identify the sources of variation and minimize or avoid them while accomplishing any activity. On the part of the laboratories, it is therefore necessary to develop a plan for the proper performance of each activity, and then establish quality standards and written procedures in compliance with the standards. Many times, the activity of sampling falls outside the purview of a laboratory’s mandate or control. This is especially true in commercial testing laboratories where the “first contact” is the arrival of samples. To improve the overall quality of the analytical process, a laboratory must do all it can to receive appropriate, applicable, defensible samples. The development of appropriate plans will depend upon an understanding of the problems involved in each activity, and then the application of reasonable judgments in seeking solutions. It should be noted that sampling terminology and procedures used may vary between companies and between specific applications. However, the principles described in this Unit are intended to provide a basis for understanding, developing, and evaluating sampling plans and sample handling procedures for specific applications encountered. A sample should represent a population as adequately as possible. To ensure proper sampling, the analysts need to be consulted time to time concerning proper sample size, suitable containers for sampling or the use of appropriate preservatives to prevent any spoilage or transformation in a sample before analysis. One common cause of lack of precision or lab-to-lab variation in analytical results for a particular population can be traced back to erroneous sampling. For example, in case of grapes, a laboratory sample size of meager 3 kg berries represents the whole population of > 10000 kg in 1 hectare vineyard area. Thus, if the sample collected is not representative, then there will be sample-to-sample variation in results. When significant difference in results occurs among laboratories which have supposedly analyzed the same sample, a serious conflict may arise questioning the competence and credibility of the laboratories.

Many of these situations can be avoided if samples are collected according to a rational plan that gives some assurance that the sample delivered to the laboratory represents the composition of the parent lot. There are at least two ways to measure a given lot of goods: one, that we often assume to be the “proper” way, is to find its “true value”, by which we mean its average value. The other way, often discovered accidentally as a result of “poor” sampling, is to measure its variability. So called proper sampling of drug dosage forms, for example, may involve compositing 20 tablets, by which the majority of the tablets could be used to dilute and conceal the fact that several of them are severely sub- or super- potent. Similarly, two lots of grain may have been purposely, but ineffectively, mixed in an attempt to reduce the average level of a contaminant. Sampling that led to the laboratory finding inconsistent results would reveal the attempt to dilute an illegal product.

iv. Errors in Analytical Results due to Improper Sampling

Few studies have been conducted on the distribution of error among the three activities: sampling, sample preparation, and analysis. In one such study, which involved analysis of 20 nanogram/gram concentration of aflatoxin in a lot of peanuts, the error contributed by the sampling step was as high as 67% of the total variance, in comparison to 20% and 13% errors contributed by the analyst and the analytical procedure, respectively. In a field study conducted at the National Research Centre for Grapes, Pune, the pesticide residues in grape samples analyzed in 15 individual grape bunches collected out of 1 acre area showed above 50% sampling-induced variations. The results of such experimentations are not unusual and it illustrates the proportion of error that can be attributed to sampling. For peanuts, the distribution of aflatoxin can vary widely, with a few peanuts accounting for most of the contamination. Similarly, in case of the field sampling of grapes, the pesticide residues might have deposited in variable concentrations in different grape bunches and thus when they were analyzed separately, showed variable results. The important point in these examples is to show that sampling error can play a very significant part in the overall error in the analytical system.

v. Risks Associated with Sampling

There are two types of risks associated with sampling. Both should be considered when developing a sampling plan. The consumer risk describes the probability of accepting a poor quality population. This should happen rarely (<5% of the lots) but the actual acceptable probability of a consumer risk depends on the consequences associated with accepting an unacceptable lot. These may vary from major health hazards and subsequent fatalities to a lot being of slightly lower quality than standard lots. Obviously, the former demands a low or no probability of occurring whereas the latter would be allowed to occur more frequently. The second risk i.e., vendor risk is the probability of rejecting an acceptable product. As with consumer risk, the consequences of an error determine the acceptable probability of the risk. An acceptable probability of vendor risk is usually 5-10%.


Data obtained from an analytical technique are the result of a stepwise procedure from sampling, to sample preparation, laboratory analysis, data processing, and data interpretation. There is a potential for error at each step and the uncertainty, or reliability, of the final result depends on the cumulative errors at each stage. Variance is an estimate of the uncertainty. The total variance of the whole testing procedure is equal to the sum of the variances associated with each step of the sampling procedure and represents the precision of the process. Precision is a measure of the reproducibility of the data. In

contrast, accuracy is a measure of how close the data are to the true value. The most efficient way to improve accuracy is to improve the reliability of the step with the greatest variance. Frequently, this is the initial sampling step. The reliability of sampling is dependent more on the sample size than on the population size. The larger the sample size, the more reliable the sampling. However, sample size is limited by time, cost, sampling methods, and the logistics of sample handling, analysis, and data processing. It should be noted that sampling terminology and procedures used may vary between companies and between specific applications. Several standards and recommendations provide the ways and means to sample a particular lot.


Understanding a Sample Plan

Sampling is generally done for a specific purpose and the purpose may indeed suggest or dictate the nature of any sampling plan. The International Union of Pure and Applied Chemistry (IUPAC) defines a sampling plan as “a predetermined procedure for the selection, withdrawal, preservation, transportation, and preparation of the portions to be removed from a lot as samples”. A sampling plan should be a well-organized document that establishes the required procedures for accomplishing the program’s objectives. It should address the issues of who, what, where, why, and how. The primary aim of sampling is to obtain a sample, subject to constraints on size, that will satisfy the sampling plan specifications. A sampling plan should be selected on the basis of the sampling objective, the study population, the statistical unit, the sample selection criteria, and the analysis procedures. The two primary objectives of sampling are often to estimate the average value of a characteristic and determine if the average value meets the specifications defined in the sampling plan. The presence of a well designed plan is important because it provides a consistent model to guide people performing the sampling activity, and it serves as a reminder of the important elements in this part of the overall sample analysis program.

Statistical Approaches

In many sampling programs, statistical approaches are not given the requisite attention. Percentage sampling systems that specify a fixed percentage of a lot, say 5 or 10%, do not provide the quality protection that is often assumed. Statistical sampling theory furnishes the means to analyze the relationship between a lot of goods and the samples that are drawn from it. It can be used to estimate population measure, or “parameters,” such as variance and correlation, from knowledge of corresponding samples quantities. The importance of sampling is recognized in ISO 17025, which requires that test reports make reference to the sampling procedure used by the laboratory or the submitting body.


There are several sampling methods/techniques in common use. These are probability sampling, non-probability sampling, bulk sampling, and acceptance sampling. These are described in brief below:

Probability Sampling

Probability sampling is used when a representative sample is desired, and uses principles of statistical sampling and probability i.e. elimination of human bias. It is a random selection approach that tends to give each unit an equal chance of being selected.

Simple random sampling requires that the number of units in the population be known and each unit is assigned a number. A specific quantity of random numbers between one and total number of population units is selected. Sample size is determined by lot size and potential impact of a consumer or vendor error. Units corresponding to the random numbers are then analyzed as an estimate of the population.

Systematic sampling is used when a complete list of sample units is not available, but when samples are distributed evenly over time or space, such as on a production line. The first sample is selected at random and then every nth unit after that.

Stratified sampling involves dividing the population into overlapping subgroups so that each subgroup is as homogenous as possible. Group means, therefore, differ from each other as much as possible. Random samples are then taken from each subgroup. The procedure provides a representative sample because no part of the population is excluded and it is less expensive than simple random sampling.

Cluster sampling entails dividing the population into clusters or subgroups so that cluster’s characteristics are as identical as possible, that is, the means are very similar to each other. Any heterogeneity occurs within each cluster. Clusters should be small and having a similar number of units in each cluster. The clusters are sampled randomly and may be either totally inspected or sub- sampled for analysis. This sampling method is more efficient and less expensive than simple random sampling, if populations can be divided into homogenous groups.

Composite sampling is used to obtain samples from bagged products such as flour, seeds, and larger items in bulk. Two or more samples are combined to obtain one sample for analysis that reduces differences between samples. For example, FDA composite 12 and at least six subsamples, respectively, for the sample to be analyzed for compliance with nutrition labeling regulations.

Non-probability Sampling

Non-probability sampling is used when it is not possible to collect a representative sample, or a representative sample is not desired. For example, in case of adulteration such as rodent contamination, the objective of the sampling plan may be to highlight the adulteration rather than collect a representative sample of the population. The sample collector uses judgment rather than statistical considerations in the selection of the sample. The unusual or unexpected characteristics in a population could be selected to be identified. This type of sampling is done in many ways, but in each case the probability of including any specific portion of the population is not equal because the investigator selects the samples without estimating sampling error.

Judgement sampling is solely at the discretion of the sampler and therefore is highly dependent on the person taking the sample. This method is used when it is the only practical way of obtaining the sample. This method may present a better estimate of the population than random sampling if sampling is done by an experienced individual and limitations of extrapolations from the results are understood.

Convenience sampling is performed when ease of sampling is the key factor. The first pallet in a lot or the sample that is most accessible is selected. This type of sampling will not be representative of the population, and therefore is not recommended.

Restricted sampling may be unavoidable when the entire population is not accessible. For example, if sample is to be taken from a loaded truck, but the sample is not a representative of the entire population.

Quota sampling is the division of a lot into groups representing various categories, and samples are then taken from each group. This method is less expensive than random sampling but also is less reliable.

Types of Sampling

A) Bulk sampling

Bulk sampling involves the selection of a sample from a lot of material that does not consist of discrete, identifiable or constant units. Sampling may be performed in static or dynamic situations. Bulk sampling poses special problems requiring certain decisions to be made: the number of increments to be taken, the size of the increments, from where in the pile or stream they should be drawn, the sampling device to be used, and how to reduce the increments taken to a reasonable size of sample for delivery to the laboratory.

B) Acceptance sampling

Acceptance sampling differs from the previous types and involves the application of a predetermined plan to decide whether a lot of goods meet defined criteria for acceptance. The risks of accepting “bad” or rejecting “good” lots are stated in conjunction with one or more parameters, for example, quality indices of the plan. Statistical plans can be designed to regulate the probabilities of rejecting good lots or accepting bad lots.

There are two broad categories of acceptance sampling: sampling by attributes and sampling by variables.

Sampling by attributes

In sampling by attributes, the unit of product is classified as defective or nondefective, or the number of defects in a unit of product is counted with respect to a given requirement. Or, the sampling is performed to decide on the acceptability of a population based on whether the sample possesses a certain characteristic, for example, Clostridium botulinum contamination in canned goods. An example of net weight determination may serve to explain the differences between the two categories. In attribute sampling, each unit that weighs 1 pound or more is accepted, and each unit that weighs less than 1 pound is rejected. If the number of rejects exceeds a predetermined number, the lot is rejected. If the number of rejects is less than the predetermined number, the lot is accepted.

Sampling by variables

In variable sampling, sampling is performed to estimate quantitatively the amount of a substance (e.g., salt) or a characteristic (e.g., color) on a continuous scale. The estimate obtained from the sample is compared with an acceptable value (i.e., previously determined) and the deviation measured. This type of sampling usually produces data that have a normal distribution such as in the per cent fill of a container and total solids of a food sample. In general, variable sampling requires smaller sample size than attribute sampling and each characteristic should be sampled for separately when possible.

Operating Characteristic (OC) Curves

Operating Characteristic (OC) curves are used extensively in acceptance sampling. The OC curve shows the relationship between the quality and the per cent of lots expected to be acceptable for the quality characteristic inspected. In other words, the OC curve is a graph of lot defectives against the probability that the sampling plan will accept the lot.

The Operating Characteristic (OC) curve shows the probability of acceptance, Pa, for any level of lot quality. On the horizontal axis is the quality characteristic. This OC curve enables you to evaluate the probability of acceptance for any true lot quality level-on a what-if basis. This way, you can design sampling plans that perform the way you want.

Requirements of Good Sampling Methods

Samples are useful for their intended purpose when they are taken in a manner consistent with generally recognized good sampling techniques and good sampling practices. This requires the following:

Inspection of the lot before sampling.

Use of suitable sampling devices for the particular commodity and type of sample desired.

Use of suitable containers to hold the sample.

Maintenance of the integrity of the sample and associated records.

Use of adequate precautions in preserving, packing and delivery of the sample to the lab in a timely manner.

Provision of appropriate storage conditions for the sample both prior to and following analysis.

All of these factors, along with others such as cost versus benefits analysis, and a review of program objectives and regularity requirements, are to be assessed and brought together in a sampling plan that serves as a guide to management, as well as to operating personnel as a firm plan to achieve quality in sampling.

Cost of Sampling

The attention of users is drawn upon relation between the efficiency and size of sample. For a given Acceptable Quality Level (AQL), the smaller the sample size, the smaller the cost of sampling, but the worse the efficiency, that is the risk to wrongly accepting a lot increases and worsens the damage in trade.

Problems in Sampling

Analytical data never are more reliable than the sampling technique. Sampling bias, due to non- statistically viable convenience, may compromise reliability. Errors also may be introduced by not understanding the population distribution and subsequent selection of an inappropriate sampling plan. Unreliable data also can be obtained by non-statistical factors such as poor sample storage resulting in sample degradation. Samples should be stored in a container that protects the sample from moisture and other environmental factors that may affect the sample (e.g., heat, light, air). To protect against changes in moisture content, samples should be stored in an airtight container. Light sensitive samples

should be stored in containers made of opaque glass, or the container wrapped in aluminum foil. Oxygen sensitive samples should be stored under nitrogen or an inert gas. Refrigeration or freezing may be necessary to protect chemically unstable samples. However, freezing should be avoided when storing unstable emulsions. Preservatives (e.g., mercuric chloride, potassium dichromate, and chloroform) can be used to stabilize certain food substances during storage. Mislabeling of samples causes mistaken sample identification. Samples should be clearly identified by markings on the sample container in a manner such that markings will not be removed or damaged during storage and transport. For example, plastic bags that are to be stored in ice water should be marked with water-insoluble ink. If the sample is an official or legal sample the container must be sealed to protect against tampering and the seal mark easily identified. Official samples also must include the date of sampling with the name and signature of the sampling agent. The chain of custody of such samples must be identified clearly.


Another type of sampling used frequently by regulatory agencies to determine acceptance or rejection of a lot (often defined as the quality of product produced under essentially the same conditions but representing no more than one day’s production) is the three-class sampling plan. This approach is often used when assessing microbiological contamination of foods. In this case, “n”

is the number of samples, usually selected at random from the lot, the numerical value “m” represents acceptable concentrations, the numerical value “M” represents un-acceptable concentrations, and “c” is the maximum allowable number of marginally acceptable sample units such that if this number is exceeded, the lot is considered as un-acceptable. While “m” separates sample units of acceptable quality from those of marginally acceptable quality, “M” separates sample units of marginally acceptable quality from those of defective quality. For enforcement purposes, the sampling technique used should be the same as the sampling technique used to set the standard. For example, minimum reportable limits for particles are based on composite samples and not on individual lots.


The development of quality sampling plans is a science in itself and has been given consideration by a number of organizations. One plan format that deserves serious consideration, developed by the International Organization for Standardization, is shown with comments in ISO/TC 34, ISO/DIS 7002.2, “Agricultural food products- Layout for a standard method of sampling from a lot “(1988). It can serve as a starting point or check list for developing a sampling plan for most commodities. The title and headings from sections in the monograph are as below:

Model Sampling Plan

Agricultural food products- Layout for a standard method of sampling from a lot.


Title (short but appropriate for index identification)


Introduction (describing the purpose of the plan)


Scope (describing the breadth of coverage of the plan)


Field of application (products to be covered; where sampling will be done)


References (documents, the validity of the plan with reference to other requirements)


Definitions (specific terms associate with a particular matrix)


Principle (statistical basis of the method of sampling)


Administrative arrangements

a) Sampling personnel

b) Representation of parties concerned

c) Health, safety and security precautions

d) Preparation of the sampling report


Identification and inspection of the lot prior to sampling (important in survey sampling for identification, condition of the lot and selection of method of sampling).


Sampling equipment and ambient conditions (proper tools such as use of sterile equipment for aseptic sampling).


Sample containers and packing (essential to prevent contamination and damage during shipment or storage).


Sampling procedures (as dictated by the plan objectives).

a) Sampling size

b) Taking the sample.

c) Preparation of bulk samples and reduced samples

d) Selection of samples of pre-packaged products.


Packing, sealing and marking of samples and sample containers (identification of units and to establish chain-of-custody).

a) Filling and sealing sample containers.

b) Labeling or marking (including signature of sampling personnel).

c) Packing samples for storage or transportation.


Precautions during storage and transportation of samples.


Sampling report

a) Administrative details.

b) Details of unit packs or enclosure containing the lot.

c) Material samples.

d) Marking and sealing of samples.


Annexure (supplemental information, if necessary).


If the test portion analyzed does not represent the sample or the lot from which it was taken, in that

case, even the best analysis could give misleading information. Distortions introduced at this point will carry through the path of analysis and adversely affect the final results and the conclusions drawn from them. There are generally two choices in analytical sub sampling:

Preparation of a composite laboratory sample (if multiple units are submitted for analysis).

Examination of individual units.

Composite Lab Sample Preparation

A composite lab sample is one in which the individual units or representative portions of units are

mixed to form a uniform mixture. Portions are then taken from the composite for analysis. Compositing saves analytical time and in some types of contract testing it may be the procedure specified. If the results indicate that there may be a problem, it will likely be necessary to go back and analyze individual samples. Compositing is not the procedure of choice when there is a chance that an individual unit that constitutes a public health or safety threats will not be detected (there are some exceptions) or where a

unit at or outside of tolerance level will not be detected because of matrix dilutions. Multiple unit lab sampling is indicated when the possible range of values among individual units is considered significant or it is desirable to establish the variability of the lot.

Opinions of Experts

You den and Steiner (Statistical Manual of the AOAC, AOAC International, Arlington, VA, p 41) observed that, “Many materials are notoriously difficult to sample. Often the variability among samples is the controlling factor in the confidence placed in the analytical result.” They note further that

A mistake sometimes made is to composite several samples and then to run repeat determinations on this composite sample. The analyst may be happy with several results that are in close agreement because only the analytical error is involved in the results. And some may put their faith in the result admittedly, if the individual samples were of the same weight and properly mixed, the same average will result whether the samples are analyzed individually or repeats are made on the composition. Using the composite sample effectively conceals the between-sample variation. It should be mandatory to run the samples individually, for only by doing so will anybody be in a position to make any statistical statements about the results, no matter how good the analytical procedure. Somewhat similar view of sub sampling for analysis is expressed in an article published in Chemical and Engineering news by an ad hoc sub committee of The American Chemical society for “Dealing with the Scientific Aspects of Regularity Measurements.” This report observes that the number of samples to be analyzed in a given situation usually is limited by the resources available for collection of samples or for their analysis. However, the reliability of the result generally increases with the square root of the number of samples analyzed. For this reason, analysis of multiple samples are preferred over single samples since, single samples give no information on the homogeneity of the lot that was sampled. In addition, for single samples, the sampling error is also confounded with theanalytical error. As a result, if the total number of determinations must be fixed, multiple independent single samples are preferred over replicate aliquots per a single random sample. In any case, the sampling decisions should be a priori decision and should be based on the question at issue.

In addition to the number of sub samples taken for analysis, it is essential that each be prepared in a way that achieves homogeneity and is handled in a manner that prevents alteration from the original composition. Obviously, failure to prepare a homogeneous sub sample at this point will affect the results of the analysis regardless of the method used.


Every type of material that is to be prepared for analysis presents its own practical difficulties. The requirements for suitable sample preparation are dictated by the consistency and the chemical characteristics of the analyte and the matrix, and by the distribution of the analyte in the sample. Even seemingly homogeneous materials such as liquids may be subject to sedimentation or stratification. Thus, vigilance and care are the watch words to ensure homogeneity.

Precautions to be followed while Preparing a Sample for Analysis

Mixing: Single phase liquids can generally be mixed, stirred, shaken or blended. Dry particulate materials can be reduced in the volume by coning and quartering, by rolling and quartering, or by the use of a splitter, such as a refill. A variety of implements and machines are available for sample disintegration, such as mills, grinders and cutters. Care in their use is necessary to prevent loss of dust or change in composition through partial separation of components. Screening can be used to improve the efficiency of particle size reduction, followed by mixing to attain homogeneity. Sampling errors can occur even in well mixed particulate mixtures especially in trace analysis if the particles differ appreciably in size or physical properties.

Cleanliness of equipment used in process

Every piece of equipment used in the preparation of a sample must be examined critically to ensure their cleanliness, so that they do not contaminate or decompose or cause any physical loss of the sample while processing. Grinders were mentioned above as contributing to the loss of finer particles as dust. They have been known to segregate materials with in the mix by size as well, with the finer material, collecting beneath the blade e.g., Metal screens can pass fine particles, but retain powder that adheres to the screen materials. Glass containers and laboratory apparatus can adsorb certain materials and may require surface treatment. Plastic containers can retain contaminants, such as animal hairs, while the rest of the sample is transferred with apparent ease. In the other words, validation of a method of analysis, includes, most certainly validation of the method of sample preparation and storage.

Changes in physical characteristics

Loss or gain of moisture during processing can be a problem. Loss can be minimized by keeping samples covered with plastic or aluminum foil. A cold product can be protected from gaining moisture by allowing the sample to come to room temperature before preparation begins. High fat samples such as nuts may be difficult to grind without clogging up the grinder; one technique that is used is to freeze the samples prior to grinding.

Changes in chemical characteristics

When volatile organic constituents are present in any sample, processing may be difficult and needs special care, e.g. maintaining chilled condition to prevent any loss of volatile constituents. Similarly,

in case of photo-sensitive chemicals (e.g. natural product pesticides), it is required to process a sample under darkness to prevent degradation on exposure to light.

Portions for sampling

As a general guide, food samples are analyzed in the form they are commonly consumed. Inedible portions, such as stones (e.g. for mango), nutshells, or fish bones are removed and discarded prior to analysis, and suitable note made of how the sample was prepared. The technique used for setting the standard should be used to ensure comparability.

Sampling for Trace metals

Trace metals analysis can present significant problems, For example, the trace metals can be distributed unequally between liquid and solid phases in pickles, canned vegetables and canned fruits. Obviously, such irregular distribution of metals can pose problems for the analyst in establishing the level of metal residues in the product, as well as for those concerned with setting tolerances. Thus, it becomes necessary to analyze both the solid and liquid phases.


As mentioned earlier, one of the most difficult problem in sampling from a lot, and in subsequent lab subsamples, is trying to obtain a representative sample for the analysis of aflatoxins in raw agricultural commodities. Aflatoxin contamination exhibits a highly erratic distribution, with a reduction in heterogeneity as the food or feed is reduced in particle size. After it was recognized that there was a high rate of variability and within same samples from the same lot, there was a moment towards the collection of larger and larger samples. Sample sizes started, for peanuts with 1 kg, and the size increased as more reliable results were required by food procedures (increasing sample size reduces the number of good lots that are likely to be rejected and the number of bad lots accepted).

Example for Effect of Sampling on Analytical Result

At the present time in the United States, the sample taken from a lot of shelled peanuts of 144 pounds; three 48 pounds samples with portions taken at random from the lot. Examination in the lab is by sequential analysis with first 48 pounds sample ground in a subsampling mill and test portions examined in duplicate. If the average of the test portions is below the established tolerance (set by US Food and Drug Administration), the lot is passed. If the average is above the acceptance level, the lot is rejected. If the findings fall between the two figures the second 48 pound sample is comminuted and the analysis repeated. If a decision cannot be made to accept or reject the lot, the third 48 pounds sample is prepared, assayed, and the cumulative results considered. The foregoing example point out dramatically the need for attention to lot sampling, lab subsampling, and sample preparation for analysis. While this is a rather extreme case, it illustrates that sampling problems cannot be ignored or treated indifferently. In Canada, while the specified sample sizes are smaller, ranging from 12 to 20 kg. depending on the commodity and the lot size, and minimum number of sampling sites are also stipulated to address the erratic distribution of aflatoxin contamination.



A laboratory sample is generally the starting point for analytical work. The sample may be delivered

by mail, courier, flight, or directly by the collector. It may arrive in any of various containers and conditions: frozen, packed in ice, or at room temperature. The package may be sealed or unsealed, and the sample itself may be spoiled or broken. The sample may or may not be accompanied by appropriate documentation to advise the laboratory regarding purpose, test parameters and the conditions of storage, etc.

Once a sample is received, all the circumstances and conditions must be documented as they could have bearing upon the quality or the significance of the test results. It is important for appropriate quality analysis that sample arrives in proper condition with meaningful documents. Procedures for these must be established, continually reviewed, and enforced, to keep poor sample handling and delivery to a minimum level. To avoid any future legal complications, the laboratories are advised to protect themselves with the cautionary statement in the test report indicating that the results relate only to the sample that was tested.

Chain of Custody Form

In most organizations specific sampling procedures are written and the sample collectors are trained

regarding their responsibilities. The first important activity is the documentation to ensure product traceability. The sample should be easily identifiable and placed under seal and packing. Shipping and delivery instructions are followed to effect delivery to the laboratory. The documentation consists of a chain-of-custody that accompanies the sample as it moves through the laboratory and subsequent administrative handling. This form is usually prepared in multiple copies for distribution to various units in the organization, may be supplemented with affidavits, dealer’s statements, bills or other relevant information that concerns the sample, its origin, the transfers from one custodian to the next and the sample’s significance or importance. Information such as sample number, product name and identification, reason for collection, description of the sample and of the method of collection, size of the lot from which the sample was taken, codes, shipment information, collection date, name of the collector, means of transportation, and whether or not sealed are supplied with the sample. If the sample is sealed, the seal includes the sample number, date the seal was affixed, and the collector’s signature. The seal is attached to the package in such a way that it must be broken before the sample can be obtained.

Sample Receipt and Handling

The next step in the sample accountability system is receipt of the sample in the laboratory.

A dependable record of sample handling is important so that the sample is accepted by a sample

custodian who documents the action by completing a sample accountability record. This document should contain the sample number, the name of the product and date received, indicate who received

it, describe the method of shipment or delivery, describe the packages received and their condition, and

provide space for recording various storage locations before and after analyses. Deliveries of the sample

or portions of the sample to the analyst, and its return, will also be recorded on this form. There will be

a signed statement concerning the final disposition of the reserve sample. A two-part form can be used for this purpose; one copy remains with the sample custodian and the other moves with the sample through the laboratory and is used by a supervisor for sample management purposes. Some laboratories

use a sample receiving log book for sample control. The information entered in the log book is essentially the same as that described for the two-part form.

Monitoring of Samples

The sample accountability in a laboratory can be monitored by a simple computer program; a unique label should be generated and affixed to the sample container, and all the pertinent sample information should be entered into the computer database. The information entered at log–in becomes part of the data base, which is then built up through the manual or automatic addition of sample handling information and analytical data. Worksheet pages or reports can be calculated and printed, and the data base itself latter queried and manipulated for various information and reporting purposes. Regardless of the recording system used, the analytical information generally reported includes a description of the sample, subsampling procedure sample preparation methods used, deviations from methods, validation and recovery experiments (if performed), standards used, source of reference materials, raw data, calculations and description of the reserve sample and how it was prepared for storage after the completion of the analysis. In addition, pertinent supporting documents such as chromatograms, spectra, and other charts are suitably identified with instruments identification, operating conditions, analyst name, sample number and date. If the reserve sample is sealed, the information placed on the seal is shown in the report. The sample is then returned to the sample custodian to be stored for whatever future action may be necessary, or until the sample is destroyed.


After an analysis is complete and the results reported, the laboratory needs a written policy for guidance on the retention of the samples and the associated records. For samples and records that may be involved in litigation, the storage period can extend for years. For the majority of samples, fortunately, this is not usually the case. The objective should be to destroy samples as soon as it can be analyzed, with certainty that they will no longer be required for further testing or as evidence. The records may be disposed after they are no longer legally or administratively important.

Identify the Properties of Retained Samples

It is very important for the laboratory management to determine whether or not the materials being discarded are hazardous in nature. Although samples themselves may not be hazardous, acid digestions and organic solvent extractions certainly can be hazardous. Sample management includes the proper disposal of samples and laboratory preparations. Standard operating procedures for samples for sample disposal are essential.

Retention Period

Storage periods, obviously, must be determined by each facility depending on its obligations, but clear policy must be in place to prevent both the destruction of important items, and the accumulation of what is essentially junk. From a quality assurance point of view, the improper destruction of active samples or records is low quality performance in violation of policy, and the Quality Assurance (QA) program must provide a means to detect such actions in an effort to prevent their recurrence.




Professor, College of and Dairy Technology, Chennai-52


Milk may be defined as the whole, fresh, clean lacteal secretion obtained by the complete milking of one or more healthy milchy animone-sixth as sweet as sucrose. Lactose is responsible for the defect known as sandiness in ice-cream or condensed milk.

Milk Proteins : The proteins in milk consists mainly of casein, lactaglobulin, lactalbumin, milk serum albumin,fat.


milk serum albumin,fat. 1.2. CHEMICAL COMPOSITION OF MILK Water : Water constitutes the medium in which

Water: Water constitutes the medium in which the other milk constituents are either dissolved or suspended. Most of it is free and only a very small portion is in the bound form, being firmly bounded by milk proteins, phospholipids etc.

Total Solids: Total Solids constituent’s lipids (Fat) and solid not fat.

Milk Fat (Lipids): The bulk of the fat in the milk exists in the form of small globules, which average approximately 2 to 5 microns in size. This is an oil-in-water type emulsion. The fat associated substances are phospholipids, cholesterol, carotene and fat soluble vitamins (A, D, E, and K).

Milk Sugar or Lactose: This exists in milk only. It is in true solution in the milk serum. On crystallization from water, it forms hard gritty crystals. It is one-sixth as sweet as sucrose. Lactose is responsible for the defect known as sandiness in ice-cream or condensed milk.

Milk Proteins : The proteins in milk consists mainly of casein, lactaglobulin, lactalbumin, milk serum albumin, immuno globulins etc. Casein forms more than 80% of the total proteins of the milk. Casein exists only in milk and is found in the form of calcium caseinate phosphate complex.

Lactalbumin and lactaglobulin are known as ‘Whey or serum proteins’. They are also present in colloidal state and are easily coagulated by heat. Milk serum albumin is same as blood serum albumin of the blood.

Non protein nitrogenous uric acid etc.

Compounds : E.g. Ammonia, amino acids, protease-peptones, urea,

Mineral Matter or Ash : The mineral matter or salts of milk although present in small quantities, exert considerable influence on the physico- chemical properties and nutritive value of milk.

Other Constituents

Pigments: Water soluble pigments are Riboflevin and xanthophyll. Riboflavinbesides being a vitamin, is a greenish yellow pigment which gives characteristic colour to whey. Earlier it is known as lactoflavin or lactochrome.

Dissolved Gases : Milk contains gases like O 2 , Co 2 , N 2 etc.

Vitamins : Water soluble vitamins B complex and vitamin ‘C’

Enzymes : These are biological catalysts. Milk contains Amylase, Lipase, Phosphatase, protease, peroxidase and catalase enzymes.


body building proteins, bone

forming minerals and health giving vitamins and furnishes energy giving lactose and milk fat. All

these properties make milk an important food for pregnant mother, growing children, adolescents, adults, invalids, convalescents and patients alike.

Milk is an almost ideal food. It has high nutritive value. It supplies

Proteins: Milk proteins are complete proteins of high quality i.e. They contain all the essential amino acids in fairly large quantities.

Fat: Milk fat (lipid) play a significant role in the nutritive value, flavour and physical properties of milk and milk products. Besides serving as rich source of energy, fat contains significant amounts of so called essential fatty acids (linoleic and arachidonic).

Lactose: The principle function of lactose (carbohydrade) is to supply energy. However lactose also helps to establish a mildy acidic condition in the intestine (which checks the growth of proteolytic bacteria) and facilities assimilation.

Minerals: Practically all the mineral elements found in milk are essential for nutrition. Milk is an excellent source of calcium and phosphorus, both of which together with vitamin D are essential for bone formation. Milk is rather low in Iron, Copper and Iodine.

Vitamins: These are accessory food factors, which are essential for normal growth, health and reproduction of living organisms. Milk is a good source of vitamin A (provided the cow is fed with green feed and fodder), vitamin D (provided the cow is exposed to enough sun light) thiamine, riboflavine etc. However milk is deficient in Vitamin ‘C’.

Energy: The energy giving milk constituents and their individual contributions are as follows

Milk Fat

9.3°C / g

Milk proteins

4.1°C / g

Milk Sugar

4.1°C / g

The energy value of milk vary with its composition on average cow milk furnishes 75°C / 100 g and buffalo milk 100°C / 100 g of milk.



The colours of milks are

Buffalo Milk

Creamy WhiteBuffalo Milk

Cow’s Milk

Yellowish creamy whiteCow’s Milk

Skim Milk

BluishSkim Milk


Greenish yellow (This is due to pigment Riboflavin).Whey

The intensity of yellow colour of cow milk depends on various factors such as breed, feeds, size of fat globules, fat percentage of milk. The greater intake of green feed, the deeper the colour of cow milk. The larger the fat globules and the higher the fat percentage, the greater the intensity of the yellow colour.


The freshly drawn milk has certain ‘cowy’ odour which passes off by the time reaches the consumer. With the development of lactic acid the flavour also changes to‘sour’. This is due to lactic acid, butyric acid and diacetyl.


The hydrogen ion concentration of milk is about 10- 66 gms per litre that is in terms of pH value it is 6.6. Freshly drawn milk is amphoteric to litmus i.e. it turns red litmus to blue and blue litmus red. The pH of cow milk is 6.4 -6.6 and buffalo milk 6.7 – 6.8. Higher pH values in freshly drawn milk indicates mastitis.


The density of a substance is its mass (weight per unit volume). Specific gravity is the ratio of density of the substance to the density of standard substance (water). The specific gravity of milk is usually expressed at 60 o F (15.6 o C).

Milk is heavier than water. The average specific gravities are

Cow Milk -

1.028 to 1.030

Buffalo Milk -

1.030 to 1.032

Skim Milk -

1.035 – 1.037


When milk is received on the platform, it has to undergo a number of examinations and tests. These tests are carried out to determine the quality of incoming milk and it is on the basis of these tests

whether a particular lot is to be rejected. The most common of these are determination of fat%, SNF, total solids and sediments etc.

2.1. Organoleptic Test

The sensory evaluation of milk is of utmost importance to the market milk industry. The sale of fresh milk is a major activity of the Indian dairy industry. Since milk is consumed in the liquid state by all classes of people, it is judged daily be the consumers. All the five senses viz., sight, smell taste, touch and sound are used in judging and grading of milk.


To accept/reject raw milk on the basis of sensory observations.


Removal of lid of the can

Smell the milk and lid in case of any doubt, taste it by putting a small quantity on the tongue and spit it out in a spittoon.

Feel the coldness/warmth of milk from outside the can. In case of

Doubt, temperature may be observed with the help of a thermometer.

Observe for any abnormality in colour and extraneous matter in milk.







2.2. Correct Lactometer Reading (C.L.R.)

The estimates the amount of solid-not-fat (SNF) present in the milk sample. For this purpose Lactometer is used. Prior to filling the metallic cylinder upto brims with milk sample the temperature of milk (29°C) is noted. The lactometer is then inserted inside the cylinder containing milk, and the temperature of the milk is noted again. The scale of lactometer projecting above the milk gives the lactometer reading. If the temp. of the milk is 29°C then the lactometer reading itself is taken as CLR and if the temperature is below or above 29°C, then for each degree rise or fall of temperature 0.5°C is added or deducted from the observed lactometer reading.

Solid-not-fat is determined by using following expression.

SNF = CLR + 0.21 fat + 0.66


Suppose CLR is = 28.0 and Fat% = 6% then.



28 + 0.21 x 6 + 0.66


= 7 + 1.26+0.66

SNF = 8.92%

2.3. Clot on Boiling Test (COB)


COB test will be positive if acidity is more than 0.2%


Take about 5ml of well mixed sample in a test tube. Keep it in a water bath for 5 min., or rotate it almost horizontally on a spirit lamp so as to boil the milk.


Clotting of milk on the side of tube or at the bottom indicates poor keeping quality.

Keeping quality

Quality of milk COB should be –ve i.e. no clot should form on boiling.

COB + ve milk should be separated and not mixed with good milk.

For good

Natural Acidity of Milk :

Ten ml of milk is mixed well with as much amount of distilled water in a flask and the 1-2 drops of phenolphthalein indicator are added.

Then N/9 NaOH is run down from a burette to the content is the flask until a light pink color appears. The amount of N/9 NaOH utilized is noted down.

1 ml of N/9 NaOH solution is equivalent to 0.01 g of lactic acid.

Good quality milk has 0.12-0.15% lactic acid, sour milk has 0.15-0.20 lactic acid and curded milk has 0.7 to 2.0% lactic acid.


Certain indicators show change in colour with the change in pH. The pH paper or strips are impregnates with these indicators such as bromothymol blue (pH 6.0 to 7.6) and bromocresol purple (pH 5.2 to 6.8). pH papers in narrow range and wide range are available.

Objectives : To study the freshness of milk.

Reagents : pH ++paper strips


Take a small quantity of milk in the test tube.

Dip the pH strip in the milk.

Compare the colour changes with standard chart and note the pH.


The result with pH paper are not precise and for more precision pH meter is used. The pH strips should be stored in a glass bottle properly stoppered in dry conditions.


Normal milk pH is between 6.6-6.8. pH above 6.9 is indication of mastitic milk/late lactation milk.

2.5. Phosphate Test :

This test is taken as an indicator of proper pasteurization. To determine whether a lot of milk is properly pasteurized or not. 10ml of the pasteurized milk sample is mixed with 1 ml. of p-nitrophenyl disdium. This is then kept at 37°C temp. for 30 minutes and observed for appearance of yellow coloration. Under pasteurized milk give yellow color while properly pasteurized milk will give no coloration.

2.6. Alcohol Test :

The alcohol test determines the susceptibility milk of coagulate due to developed acidity or unbalance salt. This test is of prime importance in milk to detect milk which has a tendency to curdle during processing sterilization or pasteurization.

Procedure :

Five ml of raw milk is mixed with 5ml of 8% absolute alcohol, if precipitation occurs then the alcohol test is + ve i.e. milk is least heat stable. Absence of any precipitation indicates appropriate heat stability of milk.

2.7. Methylene Blue Reduction (MBRT) :

MBRT is one of the most important tests for quality assessment of milk. It is an indicator of shelf life or keeping quality of milk in addition of checking whether milk is properly pasteurized or not.

To determine the test, 10ml milk is taken in a test tube and to it 1 ml of methylene blue dye is mixed. The content is heated and kept in a waterbath at a temp. of 37 o C.

Methylene blue dye is prepared in oxidized form. Bacteria Present in milk reduce this dye in a short time. So if blue colour in the test tube (dye+milk) is changed to white in short time, this shows that larger count of bacteria is still present in the milk which is attributed to improper pasteurization.

2.8. Resazurin Reduction Test (RRT)

This test is similar to the Methylene blue test, but it uses the indicator resazurin to measure the bacteriological quality of milk. The colour of resazurin at the normal pH of milk is blue. This compound is reduced to resorufin, which is pink, the colour changing gradually during the reduction process.

The test milk is added to a screw capped vial plus resazurin to give a concentration of about 1 part of dye in 1,80,000 parts of milk. Standard tablets of resazurin are available, one tablet dissolved in 50 ml of boiled, cooled distilled water make 0.005% solution which can be directly used in the test. The tubes are then incubated in a water bath at 37 o C. After the samplus reach this temperature, they are gently inverted three times and returned to the bath, then the time of incubation begins.

For 1 hour and 10 mts RRT tests samples are compared with control tubes (milk without dye and incubated at 37 o C) is a lovibond comparator with Resazurin disc. The disc will have 6 disc (Starting 0- 6 with colours ranging from blue, and shades of purple, lavender and pink). The disc number matching is recorded and the quality of milk is graded as follows.

1 Hour RRT test

Disc No



(or) 6








& 0


10 Mts RRT Test

Disc No.



or 6


3 ½


Doubtful, requires further examination

0.5 and 0



Direct microscopic count (DMC) is one of the several methods used in quality control laboratories for direct enumeration of micro organisms in milk sample. It consists of examining fixed and stained smears of a known volume of milk and milk products under a compound micro scope. This method provides a rapid indication of quality of milk or liquid milk products.

A small quantity (0.01 ml) of the sample is spread over the outlines area of one cm square (100 mm 2 ) on a grease proof microscope slide with the help of a breed’s pipette. After making a uniform smear, it is air dried, fixed, stained with new-man’s stain and then examined under the microscope. The number of organisms per field is counted and average number per field is determined after examining at least 10-20 fields. Total number of organisms (viable as well as non viable) per ml are then calculated by multiplying the average number of organisms per field by the microscopic factor.

For determining the microscopic factor (MF) of a given compound microscope, the following formula is used.

MF = 100 x 100 r2

Where ‘r’ is equal to radius of the microscopic field. (pi) is a constant having a value of 3.14. In this formula in order to convert area of one field from Sq. mm to sq. cm field area in Sq mm has been divided by hundred.

The number of such fields to be counted depends upon the average number of organisms per field as given below.

the average number of organisms per field as given below. The average number of organism per

The average number of organism per field multiplied by the MF – yields the number of organisms per ml of milk product. It is better to count clumps instead of individuals cells because clump count agrees most with SPC.

Results Interpretation

Count / Ml

Less than 5,00,000 Good


5,00,000 – 4,000,000


4,000,0000 – 20,000000


Over 20,000,000

Very Poor


The standard plate count or pour plate method is used for estimating the viable micro organisms in milk and milk products. In view of a wide range of bacterial population in dairy products, their number can be counted only by making appropriate dilutions. An aliquot of 0.1 ml or 1 ml of the diluted samples is poured in sterilized plates and mixed with liquefied sterilized agar medium. After solidification of agar, the plates are incubated at a specific temperature and for suitable period of time depending on the type of bacteria being suspected in the food sample. After incubation, bacterial cells grows in to distinct and isolated colonies (each colony develops from a single bacterial cells) which can be counted with the help of a colony counter. The plates with 30 – 300 colonies are selected for counting to obtain plate counts or colony forming units (cfu) per ml or g of the product. In order to calculate the total number of viable bacteria / g or ml of the sample, the number of the colonies developed on each plate are multiplied by the dilution factor.

The dilutions will be 1 : 10, 1 : 1000, 1 : 10000, 1 : 1,00000, 1 : 10,00000 etc. done as shown in the figure.

1 : 1,00000, 1 : 10,00000 etc. done as shown in the figure. Fig. 5.3 Protocol

Fig. 5.3 Protocol for preparing dilutions of a milk sample, indicating volumes to be added to dilution blanks and petridishes


Count / Ml

Quality / Grade

Less than 2,00,000

Very good

2,00,000 – 1 million


1 – 5 millions


Over 5 million


2.3.11. Coliform Test:

The members of the coliform group of bacteria eg. Escherichia and aerobacter aerogenes are commonly found in dairy products, produced and handled under insanitary conditions. Their presence in milk and milk products is indicative of possible faecal contamination although some species (Aerobacter aerogenes) may be derived from feeding materials and soil.

The test for coliform organisms is based on the principle that the members of this group are capable of producing acid and gas from lactose in the presence of bile salt. A small amount of milk (1.0, 0.1 or 0.01 ml) is added to liquid or solid media containing lactose and bile salt with a suitable indicator. Production of acid and gas in liquid media and appearance of typical colonies of coliform on the plates is taken as evidence of coliform contamination. A few other bacteria such as those belonging to the genus clostridium and gencus bacillus and certain yeasts also produce acid and gas under these conditions giving rise to false positives. However even these organisms are undesirable in milk and their interference with the test therefore is not considered to be of much significance. Hence the test commonly employed to detect the presence of coliform bacteria in milk is called presumptive test and in cases of doubt the completed test in considered to confirm the presence of coliform.

Liquid Media test : Transfer 1 ml portion of milk and its dilutions (1 / 10 and 1/ 100) into macconkey’s broth tubes in triplicate. Incubate the tubes for 24 hours at 37 o C and observe for acid and gas production.

The production of acid is indicated by change of colour of medium from purple to yellow in the case of bromocresol purple and orange to pink in the case of andrade’s indicator. Production of gas is observed in the Durham’s tubes which may be partially or completely filled with gas. If no changes is observed incubate for another period of 24 hours and record the observation.

Solid Media Test

Incubate 1 ml portion of the required dilutions into sterile petridishes (in duplicate). Add to each plate 10 -15 ml of Macconkey’s agar previously melted and cooled to 45 o C. Mix the content

thoroughly by tilting and rotating the plates. Allow the agar to solidify. Pour additional layer (3 – 4

ml) of the medium completely over the surface of the solidified medium

plates at 37 o C for 24 hours. After incubation examine for typical colonies of coliform bacteria. Presence of dark red colonies measuring at least 0.5 mm in diameter constitute a positive test. Count such colonies only and express the results as coliform count per ml of milk,

Invert and incubate the

2.3.12. Yeast & Mould

For certain dairy products the yeasts and moulds count is used as an index of proper plant sanitation and high quality raw products.

Yeasts and moulds counts can be made by using potatodextrose agar or malt agar with a pH adjusted to 3.5 + 0.1. At this pH bacterial growth is inhibited although most yeasts and moulds are uninhibited of owing their preference for an acid reaction. The pH is adjusted with a predetermined amount of sterile 10% tartaric acid after the medium is melted and tempered and then plates are poured in the usual manner explained under SPC method. The medium should not be acified before sterilization or melting for the acid will hydrolyse the agar and destroy its ability to solidify. Extended holding of the acidified melted agar will prove undesirable for the same reason. The plates are incubated at 21 o C or 25 o C. for 5 days and the count is reported as yeasts and mould plate count per ml of milk or butter.

When examining butter, one should place a quantity of the product in a sterile jar and should warm this in a bath at 40 o C until the butter melts. Then 1:10 dilution is prepared by adding 11 ml of the melted butter to a 99 ml of water blank from which after dilutions can be made. It is well to have all glassware and dilution blank tempered to 45 o C, until just before use to facilitate the handling of the sample and to prevent any solidification. The pipeting of diluted sample should be done immediately subsequent to shaking while the fat droplets are evenly distributed. This will aid in preventing errors caused by the coalescing of the fat and by uneven distribution of organisms adhering to the fat droplets.


Adultration of milk may be defined as addition of any material to the milk, or removal of any constituent of milk. As per PFA adultration of milk is not allowed and it is punishable with fine and imprisonment. The common adulterants in milk are

1. Addition of water

2. Addition of starch / cereal flour

3. Addition of cane sugar


Addition of urea

5. Addition of skim milk powder

6. Addition of Vanaspati

7. Addition of Formalin

8. Addition of Gelatin

9. Addition of Ammonium sulphate

10. Addition of glucose

Detection of Adultrants in milk

1. Added Water: The presence of water can be by putting a drop of milk on a polished slanting

surface. The drop of pure milk either or flows lowly leaving a white trail behind it, where as milk adulterated with water will flow immediately without leaving a mark.

2. Addition of starch / Cereal flour

Take 3 ml of well mixed sample of milk in a test tube

Boil the milk over a bunsen burner

Cool and add one drop of 1 percent Iodine solution and observe for colour change.

Iodine solution gives intense blue colour with starch due to formation of an unstable complex starch – iodo compound. So development of blue colour indicates adulteration of milk with starch / cereal flour.

3. Addition of Cane Sugar

Take 10 ml of milk in a test tube

Add 1 ml of concentrated hydro chloric acid and mix

Add 0.1 g of resorcinol powder and mix thoroughly

Place the test tube in a boiling water bath for 5 minutes and observe for colour

Red colour obtained with resorcinol indicates adultration of milk with cane sugar.

4. Addition of Urea

Take 5 ml of milk sample in 50 ml of conical flask.

Add 5 ml of sodium acetate buffer or 24% Trichloroacetic acid solution and heat for 3 minutes in boiling water bath (no heating if Trichloroacetic acid is used).

Filter the precipitates through a what man no 42. Filter paper and collect 1 ml of filtrate in a test tube.

Add one ml of sodium hydroxide solution (2% solution) to the filtrate, followed by 0.5 ml of sodium hypochloride solution (2% solution), mix thoroughly and finally add 0.5 ml of 5% (W / V) phenol solution and observe.

A characteristic blue or bluish green colour in the filtrate from the milk with extraneous urea indicate the presence of urea. Colourless indicates no urea added. This will detect even 0.1 percent of urea addition.

5. Addition of skim milk powder

Take 50 ml of milk in each of two centrifuge tubes and balance properly in the centrifuge.

Centrifuge at 3000 RPM for 30 mts.

Decant the supernatant liquid carefully.

Dissolve the residue in 2.5 ml of concentrated nitric acid.

Dilute the solution with 5 ml of water.

Add 2.5 ml of liquid ammonia and observe for colour development.

Skim milk powder being highly proteinacious in nature gives orange colour with nitric acid, while unadulterated milk being low in protein content gives only a yellow colour.

6. Addition of Vanaspati

Take 3 ml of milk in a test tube. Add 10 drops of hydrochloric acid. Mix up one teaspoonful of sugar. After 5 minute, examine the mixture. The red colouration indicates the presence of vanaspati in the milk.

7. Addition of Formalin

Take 10 ml of milk in a tests tube and add 5 ml of con Sulphuric acid from the sides of the wall without shaking. If a violet or blue ring appears at the intersection of two layers then it shows presence of formalin.

8. Addition of Gelatin

Take 10 ml of milk in a test tube

Add an equal amount of acid mercuric nitrate solution (mercury is dissolved in twice its weight of nitric acid of sp. G. 1.422. Before use this solution is diluted with distilled water to 25 times its volumes.

Shake and add 20 ml of distilled water, shake again and allow to stand.

Filter after 5 minutes.

Add to a part of the filtrate an equal volume of picric acid reagent (Saturated solution of picric acid solution) and observe.

White cloudiness shows the presence of gelatin in milk. Yellow precipitate indicates a large amount of gelatin added. Transparent yellow solution indicates absence of gelatin.

9. Addition of Ammonium sulphate

Take 1 ml of milk in a test tube.

Add 0.5 ml of sodium hydroxide (2%) solution and 0.5 ml of sodium hypochlorite solution (2%) and mix thoroughly.

To the solution add 0.5 ml of phenol solution (5%) and heat for 20 seconds in a boiling water bath, and observe.

A bluish colour immediately forms, which turns deep blue after wards, in the sample of milk having added ammonium sulphate. In case of pure milk only a salmon pink colour forms which gradually changes to bluish in course of about 2 hours, even 0.1 % addition of ammonium sulphate can be detected by this method.

10. Addition of Glucose

Take 1 ml of milk sample in a test tube.

Add 1 ml of Bar foed’s reagent.

Heat the mixture for 3 minutes in boiling water bath and cool for 3 mts under tap water.

Add one ml of phosphomolybdic acid reagent to the turbid solution and observe.

Immediate formation of deep blue colour indicates the presence of extraneous glucose, which is stable for 24 hours. In case of pure milk only faint bluish colour due to diluted barfoeds reagent appears. By this methods as low as 0.05 % extraneous glucose in milk can be detected.

10. Addition of Detergent

Shake 5 – 10 ml of sample with an equal amount of water lather indicates presence of detergent.

Detection of Adultrants in milk products

1. Sweet curd – Vanaspati

Take 1 teaspoon full of curd in a test tube. Add 10 drops of hydrochloric acid. Mix up the contents shaking the test tube gently. After 5 minutes, examine the mixture. The red colouration indicates the presence of vanaspati in curd.

2. Rabri – Blotting paper

Take a teaspoon of rabri in a test tube. Add 3 ml of hydrochloric acid and add 3 ml of distilled water. Stir the contents with a glass rod. Remove the rod and examine. Presence of fine fibres to the glass rod will indicate the presence of blotting paper in rabri.

3. Khoa and its products - Starch

Boil a small quantity of sample with some water, cool and add a few drops of iodine solution. Formation of blue colour indicates the presence of starch.


Paneer or Chana – Starch

Boil a small quantity of sample with some water, cool and add a few drops of iodine solution. Formation of blue colour indicates the presence of starch.

5. Ghee

a. Vanaspathy or margarine

Take about 1 teaspoon full of melted sample of ghee with equal quantity of concentrated hydrochloric acid in a stoppered test tube and add to it a pinch of sugar. Shake for 1 minute and let it for five minutes. Appearance of crimson colour in lower (acid) of vanaspati or margarine.

b. Mashed potatoes, Sweet potatoes and other starches

easily be detected by

adding few drops of iodine, which is brownish in colour turns to be blue if mashed potatoes / sweet

potatoes/other starches are present.

The presence of mashed potatoes or sweet potatoes in a sample of ghee can

6. Butter

a. Vanaspathy or margarine

Take about 1 teaspoon full of melted sample of ghee with equal quantity of concentrated hydrochloric acid in a stoppered test tube and add to it a pinch of sugar. Shake for 1 minute and let it for five minutes. Appearance of crimson colour in lower (acid) of vanaspati or margarine.

b. Mashed potatoes, Sweet potatoes and other starches

The presence of mashed potatoes or sweet potatoes in butter can easily be detected by adding few drops of iodine( which is brownish in colour), turns to be blue.

7. Ghee, cottage cheese, condensed milk, Khoa and milk powder etc., - Coal tar dyes

Add 5 ml of diluted sulphuric acid or concentrated hydrochloric acid to 1 teaspoon full of melted sample in a test tube. Shake well. Pink colour (in case of H2SO4) or crimson colour (in case of HCL) indicates coal tar dyes. If HCL does not give colour, dilute with water to get the colour.


Micro organisms are susceptible to the action of chemicals which either check their growth or destroy the organisms and then keep the milk for a longer time.

1. Boric Acid or Borax

Take 5 ml of milk in a test tube.

Add 1 ml of concentrated hydrochloric acid and mix well.

Dip a strip of turmeric paper in the acidified milk.

Dry the filter paper immediately and note the change in colour. Turmeric paper turns red if boric acid or its salts are present.


Carbonates / Bicarbonates

Take 10 ml of milk in a test tube.

Add 10 ml of alcohol and shake well.

Add 3 drips of aqueous solution of rosalic acid (1%)

Mix well and observe the change of colour.

Rose red colour indicates presence of carbonate / bicarbonate in the milk. Only brownish colour indicates absence of carbonate / bicarbonate.


Formalin There are two tests


Hehnes Test

Take 10 ml of milk in a test tube

Add 0.5 ml of 1% ferric chloride solution.

Add carefully about 5 ml of concentrated sulphuric acid down the side of the test tube in such a way that it forms a separate layer at the bottom without mixing with milk.

Observe the colour of the ring formed at the junction of the two liquids.


Leech Test

Take 5 ml of milk in a test tube

Add to it equal volume of concentrate hydrochloric acid containing 1 ml 10% ferric chloride solution to each 500 ml of the acid.

Heat over a flame for 5 minutes.

Rotate the tube to break up the curd and observe the colour. Violet colour indicate presence of formaldehyde.


Hydrogen Peroxide

Take 10 ml of a sample of milk in a test tube.

Add 2 drops of paraphenylene diamine hydrochloride solution mix thoroughly and observe.

Development of an intense blue colour indicates presence of hydrogen peroxide.

5. Salicylic Acid

Mercuric nitrate is added to the milk and milk is filtered. If much salicylic acid is present the filtrate attains a red colour after some time.

6. Benzoic Acid

About 20 gms of milk is treated with equal volume of concentrated hydrochloric acid until the curd dissolves. It is now allowed to cool. About 25 ml of a mixture of either and petroleum ether is

added to the mixture of milk and hydrochloric acid and is shaken. The ether layer separation and gives a precipitate with a drop of ammonium hydroxide in the presence of benzoic acid.

7. βββββ - Naphthol

Milk is extracted with chloroform and heated with potassium hydroxide for few mts. If a deep blue colour appears it indicates the presence of â- naphthol.


3.1.1. Fat by Gerber method


a. Mix the sample by carefully stirring without causing frothing or churning. If the cream is very thick, warm it to 30 o C.

b. Weigh 5 gm of cream in the cream butyrometer (0 – 70% range) by keeping the butyrometer in a conical flask.

c. Add about 18 -20 ml of 50% Gerber acid (freshly prepared by adding equal volumes of sulphuric acid and distilled water). Take care that there is space for 1 ml amyl alchol are added. After covering it with a cork the tube is taken in centrifuge for 5 mts. Then the fat percentage is calculated.

d. Proceed as in the case of milk.

e. Record the fat %.

One more method

To the butyrometer tube 10 ml Gerber’s acid and 3 ml of the given cream is pipette out into it. Then 8.2 ml of distilled water and 1 ml of amyl alcohol are added. After covering it with a cork the tube is taken in centrifuge for 5 mts. Then the fat percentage is calculated.

For thin cream = (B.R × 4) - 0.8 = % fat

Thick cream

= (B.R × 4) – 0.8 × 2 = %


(Diluted with equal quantity of water)

3.1.2. SNF

SNF % = 100 – Fat % in cream


3.1.3. Acidity


a. Mix the sample by carefully stirring without causing frothing or churning. If the cream is very thick, warm it to 30 - 40p c and then mix it.

b. Pipette out 10ml or weigh 10 gm if it is too viscous into a beaker.

c. Add minimum amount of distilled water to make the sample sufficiently liquid for titration.

d. Proceed as in milk.

% Acidity= 9×V×N


V- Volume of 0.1N NaOH

N- Normality of 0.1N NaOH

W-Weight of cream

3.1.4 Phosphatase Test:



Transfer 5ml of buffer substrate solution in to a test tube. Bring the temperature to 37°C.


Add 1 ml of cream to the solution.


Incubate at 37°C in a water bath for 5-10 minutes.


Prepare one blank from boiled cream of same sample.


Yellow colour indicates under pasteurization. Compare the colour with that of blank.

Buffer Solutions:

3.5g of anhydrous AR Sodium Carbonate and 1.5g AR Sodium bicarbonate dissolved in water and made upto 1ltr.


P.Nitro-Phenyl-Disodium Orthophosphate

Buffer Substrate:

Transfer 0.15g of the substrate in 100 ml flask, make upto the mark with buffer solution. The solution should be practically colourless and should not be stored for longer period (1 week).


According to PFA Rules (1976), table or creamery butter should contain not less than 80% fat, not more than 1.5% curd and not more than 3.0% common salt.





Take clean dry flat bottom aluminum dish and weigh. b)Take 10g of sample into the dish and weigh it.


Put the dish on steam water bath or on hot plate with frequent stirring until no moisture is seen at the bottom of the dish and curd particles turn slightly brown in color.


Wipe the bottom of the dish and keep in an oven at 100+10C for 90 min.


Cool the dish in a dessicator and weigh.


Repeat the process of heating (30min.) cooling, weighing until the difference between two consecutive weighting does not exceed 0.1mg.


Moisture % should not exceed 16%.

3.2.2. SALT


a) Weigh 10g of butter in a beaker.

b) Add hot distilled water to melt the butter and transfer it into a separating funnel without disturbing the top fat layer, run down the water layer in a 250ml volumetric flask.

c) Wash repeatedly the fat left in the separating funnel thrice with small quantities of hot distilled water and collect the washings.

d) Cool the contents to room temperature and make up voloume to 250ml mark with distilled water.

e) Take 25ml of this sol and add 1ml potassium chromate indicator and titrate against 0.1N silver nitrate until a brownish colour persists for ½ min.


Salt % = 5.85 x N x B/A



Normality of AgNO3



Vol. of AgNO3 used



Wt. of butter i.e. 10g

3.2.3. OVER RUN

The Weight of butter obtained from a given lot of cream exceeds the amount of fat in cream. The amount of butter which exceeds the fat present in cream is called over run.

Over run may be defined as the increase in the amount of butter made from a given amount of fat. It is usually expressed in percentage.

Over run is caused by the presence of Moisture, curd particles and salt in butter. It is a source of profit to the manufacture.

% Over run =




B- Butter made (kg)

F – FAT in Cream (kg)

3.3. GHEE

Standards and Specification of Ghee



max F.F.A.









a) Weigh a dried and cooled moisture dish.

b) Weigh 10g of sample in this dish.

c) Keep the dish in hot air oven at 105+1oC for 1 hr.

d) Take out dish from oven and cool, weigh it.

e) Keep the dish in the oven for ½ hr., cool and weigh it.

f) Repeat heating, cooling and weighing until the loss of wt. between two consecutive weightings does not exceed mg.


Weight of dried sample

Moisture % by wt. =

Sample weight


Moisture should not more than 0.3%.




a) Weigh 10g of sample in a 250ml conical flask.

b) Add 50ml of neutralized alcohol in the flask and mix the contents.

c) Digest the mixture over a hot water bath at 50oC for 15 min by continuously agitating the flask.

d) Titrate immediately with 0.1N Sodium hydroxide using 1ml of phenolphthalein indicator.

e) The end point of the titratin is reached when the addition of a single drop produce a slight, but definite change, persisting for 15 sec.

f) Read the volume of Sod. Hydroxide used during titration (v).

Interpretation – Free fatty acid (%oleic acid) =

A -

Wt. of Sample taken.

2.82 V/A

Free fatty acid should not be more than 1.2%.


TEST Procedure

Take 50ml of ghee. Boil it. Add 22ml of alcoholic KOH and 25ml boiled distilled water. Wait for a minute.


If ppt. occur then adultration of mineral oil but if clear or transparent solution it indicates no adultration of mineral oil.


It ranges between 40-43.


For testing of sample by Butyro Refractometer, the apparatus is maintained at 40 o C by circulating water at least 10min. Now oil/Fat to be tested is put on the glass of B.R. and it is covered now reading is observed on the scale. That reading is B.R. value of given sample.

B.R. Value of same imp. Oil/Fat

Sr. No.

Oil Fat

B.R. Value





Parachute Coconut Oil



Dalda Ghee



Soyabean Oil



Mustard Oil



Groundnut Oil



Sunflower Oil





a. Weigh 4 gm of Ammonium sulphate with a graduated cylinder and transfer it to the same flask.

b. Measure 20 ml of milk sample with graduated cylinder and transfer it to the same flask.

c. Shake well for 1 min to completely dissolve the ammonium sulphate. Set it aside for 10 min.

d. Filter through a filter paper and collected 5 ml of clear filtrate in a test tube.

e. Keep the test tube to a beaker of cold water. Examine it for turbidity.


No sigh of turbidity indicates satisfactory sterilization

A. Fat by Gerber method

B. SNF by lactometer (including added sugar)


3.5.1. Fat


i) Weigh accurately about 3g of finely devided cheese samples into the cheese butyrometer.

ii) Add 10 ml of gerber’s acid, 10ml of distilled water and 1 ml of amyl alcohol.

iii) Close the butyrometer with the lock stopper and shake well till all the contents are mixed well.

iv) Place the butyrometer in a water bath at 65°C±2°C.

v) Shake well periodically until the solution of cheese is homogeneous.

vi) Centrifuge at 1200 rpm for 5 minutes.

vii) Read the percentage of fat by adjusting the fat column within the scale of butyrometer.


According to PFA standards, hard variety of cheeses should contain not less than 42% milk fat on dry matter basis. The processed cheese should contain not less than 40% milk fat on dry matter basis.




a) Weigh a dried and cooled moisture dish.

b) Weigh 2-3g of cheese sample in this dish.

c) Keep the dish in hot air oven at 70oC for 8 hr.

d) Take out dish from oven and cool, weigh it.

e) Keep the dish in the oven for ½ hr., cool and weigh it.

f) Repeat heating, cooling and weighing until the loss of wt. between two consecutive weightings does not exceed mg.

Moisture % by wt. =


Weight of

dried sample ×100

Sample weight

According to PFA standards, hard variety of cheeses should contain not less than 43% moisture and the processed cheese should contain not less than 47% moisture.

3.5.3. Acidity


1. Weigh accurately 2 gm of well mixed cheese in a conical flask.

2. Add 20 ml of distilled water and make a fine paste with a glass rod.

3. Add 2 drops of 0.5 % phenolphthalein solution.

4. Titrate against N/10 sodium hydroxide solution till a faint pink colour persists.



Then V ml. of N/10 Sodium hydroxide =


% acidity in the cheese sample

1 ml of N/10 Sodium hydroxide

3.4.4. Salt

0.009 gm of lactic acid.

0.009 V ml of lactic acid.

0.009 V


a. Weigh accurately 2 gm of cheese in a conical flask.

b. Add 20 ml of distilled water and make up a paste.

c. Add 10 ml of nitric acid.


Add 12.5 ml of N/10 silver nitrate solution. Digest on stove.

e. Add 50 ml of distilled water again and 2 ml of ferric alum indicator.

f. Titrate against 0.1 N Potassium thiocyanate solution till brick red colour persists (v2). (11 gm of KSCN in 1 it. Water gives 0.1 N KSCN).

g. Repeat with blank (v 1)

Salt % = (v1 – v2) × 0.292

V 1

V 2

= Blank reading

= Volume of KSCN with sample


3.5.1. Fat


1. Weigh carefully 5 gm of melted sample into a butyrometer.

2. Add 6 ml of hot water, 10 ml of Gerber’s acid and 1 ml of amyl alcohol on to the butyrometer.

3. Insert the stopper. Shake the contents and centrifuge for 5 minutes at 1100 rpm.

4. Keep the butyrometer in a water bath at 60°C for 5 minutes. Read the fat content.

Interpretation as per ISI standards

The ice cream should contain a minimum of 10% milk fat.

Softy Ice cream

The average fat percentage in a softy ice cream is 3-6%

3.5.2.Titratable Acidity


1. Weigh 20 gm of the ice cream in a porcelain dish.

2. Add 50 ml of distilled as water.

3. Add 1 ml phenolphthalein indicator.

4. Titrate against standard sodium hydroxide in the burette till a pink colour is formed and remains for 10 – 15 seconds. Note the burette reading as V 1 .


%acidity = 9 ×V 1 ×N 1

V 2

V 1 – Volume in ml of 0.1N NaOH required for titration.

N 1 – Normality of the standard NaOH solution used

V 2 – Weight in g of the product taken

Interpretation as per ISI standards

The acidity of Ice cream should not exceed 0.25% (as lactic acid)

3.5.3. Over run

Over run is defined as the volume of ice cream obtained in excess of the volume of the mix. This increased volume is mainly composed of the air incorporated during the freezing process. The amount of air which is incorporated depends upon the composition of the mix and the way it is processed.


% Over run= Volume of ice cream – Volume of mix×100


Volume of mix

The desirable percentages of over run in different types of ice cream or given as follows:


% over run

Ice cream(Packaged)


Ice cream(Bulk)


Softy Ice cream(softy)


3.6. Condensed Milk

3.6.1. Fat:


Weigh carefully 25 gm of the homogeneous sample of Condensed Milk in a clean beaker.

Add 50 ml of distilled water and mix thoroughly

Trasfer reconstituted milk into a 100 ml of volumetric flask.

Wash the beaker thrise with 100 ml of distilled water and transfer in to the volumetric flask.

Make upto 1oo ml mark with distilled water and mix the contents well.

Take 10 ml gerber’s acid in to the butyrometer.

Add 11 ml of reconstituted diluted milk and 1 ml of amyl alcohol

Stopper the butyrometer, invert the butyrometer 2-3 times and mix the contents.

Place the butyrometer in the water bath at 65°C±2°C for 5 minutes.

Keep the butyrometer in a water again bath for 5 minutes and remove.

Centrifuge for 5 minutes at 1200 rpm.

Adjust the fat column with in the scale of butyrometer and take the reading.


Fat %= butyrometer reading


Weight of condensed milk

Interpretation as per ISI standards

Condensed milk should contain not less than 9% milk fat. Skim milk and sweetened condensed milk should contain not more than 0.5%milk fat.

3.6.2. Titratable Acidity


1. Weigh 10gm of condensed milk in a suitable dish.

2. Add 30 ml of luke gram of water and 1 ml phenolphthalein indicator solution.

3. Shake well and titrate against standard sodium hydroxide solution.

4. Stir vigorously throughout. Complete the titration in 20 secounds.

5. Keep in another disk about 10 g of sample diluted with 30 ml luke warm water as a blank for comparison of colour.

6. The persistence of a slight pinkish tinge for 30 sec, indicates the end point. The titration shall be preferably made in normal light.


Titratable acidity = 9 ×V 1 ×N 1

V 2

V 1 – Volume in ml of 0.1N NaOH required for titration.

N 1 – Normality of the standard NaOH solution used

V 2 – Weight in g of the product taken

Interpretation as per ISI standards

Acidity of Condensed milk and Skim sweetened milk may contain upto a maximum of about 0.35% (as lactic acid).

3.7. Dried Milk

3.7.1. Moisture



Weigh a clean dry dish (W1).



Transfer to the dish 10 gm of milk powder sample and weigh accurately (W2).


Place the dish in an oven at 102°C for 3 hours or (125°C for 15 min).


Remove it from oven and cool it in a desiccators and weigh (W3).


Repeat it till a constant weight in two successive weightments is reached.



W2 – W3

× 100


W2 – W1


Fat by Gerber method



a. Weigh accurately 1.68 gm of powder in a beaker.

b. Dissolve it in 10 ml of water slowly and mix it well.

c. Add Gerber acid, amyl alcohol and proceed in milk test.

d. Note fat % in the butyrometer.

Fat % in powder = Fat % butyrometer × 20


3.7.3. Total solids (T.S)

T.S. = 100 – Moisture %

SNF = T.S – Fat %

3.7.4. Titratable Acidity


i) Weigh accurately about 1g of the sample in to each of two porcelain dishes.


Add 10 ml of boiling water to each dish and stir with the flat end of a glass rod until a perfectly smooth liquid is obtained and cool to room temperature.

iii) Use the contents of one dish as a blank by stirring in 2 ml of rosailine acetate.

iv) Add 1 ml of phenolphthalein indicator solution to the other dish followed by the addition of standard sodium hydroxide (NaOH) solution in drops from the burette until the colour matches the pink taint of the blank.

v) Stir the contents vigorously. The time taken for the complete titration shall not exceed 20 seconds.


Titratable Acidity =

9 ×V 1 ×N 1

V 2

V 1 – Volume in ml of 0.1N NaOH required for titration.

N 1 – Normality of the standard NaOH solution used

V 2 – Weight in g of the product taken

3.7.5. Solubility Index



Add 14 g of the sample to 100 ml of distilled water at a temperature of 24 ° C in the mixing jar. Place the jar in the mixer and stir for exactly 90 seconds. Allow the sample to stand until the foam has separated sufficiently to permit its complete removal by a spoon. The period of standing after mixing should not be more than 15 minutes. After removal of the foam, stir the sample thoroughly with a spoon for 5 seconds.

Removal of cream and soluble fraction

Fill up the centrifuge tube immediately with the reconstituted milk to the 50 ml mark. centrifuge the tube for 5 minutes, immediately siphon off the transparent liquid leaving about 5 ml above the sediment level, taking care not to disturb the sediment layer. Add about 25 ml of distilled water at a temperature of 24°C. Invert and shake to mix the contents thoroughly. Again centrifuge for 5 minutes.

Determination of Solubility Index

Hold the tube in vertical position with the upper level of the sediment in level with the eye and read the ml of sediment in the tube to the nearest graduated scale division. The sediment is easily distinguished when the tube is held between the eye and an illuminated light source in the background.


Report the solubility index as the milliliters of sediment in the tube.

Interpretation as per ISI standards

Milk Powder

Whole milk and Skim milk powder

Solubility Index (Max ml)


Roller dried powder



Spray dried powder



Solubility Percentage



Weigh accurately about 4g of the material into 50 ml boiling tube. Add 32 ml of water at 50°C. Cork the tube and shake for 10 seconds. Place the tube in a water bath at 50°C for 5 minutes and shake the tube for 1 minute.

Removal of Fat

Fill up the brim of a centrifuge tube with the reconstituted milk and centrifuge for 10 minutes at 2000 rpm. Cool in a refrigerator or in ice until the fat becomes solid and forms a cake, taking care that milk does not freeze. Remove the fat layer with a little milk as possible by running a needle around the cake of the fat and then remove it. Warm the milk at 27 ° C. Break up the deposit with the rod or wire. Cork the tube and shake well until the liquid appears to be homogeneous.

3.7.7. Total Solids


i) Transfer about 2 ml of the homogeneous liquid to a previously weighed and dried aluminium dish (No.1).

ii) Weigh the dish with the lid on and place it aside.

iii) Centrifuge the tube again for 10 minutes.

iv) Pipette about 2 ml of the upper layer of the supernatant liquid without disturbing the sediment into another aluminium dish (No.2) Cover the dish and weigh.


Solubility percent by weight =

W 4 ×W 1 ×100





W 3 ×W 2

Weight in grams of total solids in dish no.2

W 2 - Weight in grams of the liquid taken immediately after removal of fat in dish no.1

W 3 - Weight in grams of the supernatant liquid taken dish No.2

W 4 - Weight in grams of total solids in dish no.1

Interpretation as per ISI standards

Milk Powder

Whole milk and Skim milk powder

a. Roller dried powder

b. Spray dried powder

3.8. Cultured Milk products

3.8.1. Fat


Solubility % wt (Max)

85.0 ml

98.5 ml

Weigh 100 g of sample in a beaker.

Add 5 ml of strong ammonia to the weighed sample and shake well to make it homogeneous.

Take 10 ml gerber’s sulphuric acid in to the butyrometer.

Pipette out 10.75 ml of the prepared sample and transfer in to the butyrometer carefully without allowing to mix with the acid.

Add 1 ml of amyl alcohol.

Stopper the butyrometer, invert the butyrometer 2-3 times and mix the contents.

Butyrometer with the contents is centrifuged for 5 minutes at 1200 rpm.

Adjust the fat column with in the scale of butyrometer and take the reading.


% Fat =B.R+ (B.R × Dilution Factor)

B.R – Butyrometer Reading

Dilution Factor is 1/20

3.8.2. Acidity


i) Weigh accurately about 10g of well mixed dahi in dish.

ii) Add 25 ml of distilled and heat to boiling.

iii) Add 1 ml of phenolphthalein indicator.

iv) Titrate against 0.1 N sodium hydroxide (NaOH) solution.


The end point is the appearance of pink colour.

vi) Note the titre value.

vii) Repeat the experiment to get the concordant values.


Titratable Acidity = 9 ×V 1 ×N 1

V 2

V 1 – Volume in ml of 0.1N NaOH required for titration.

N 1 – Normality of the standard NaOH solution used.

V 2 – Weight in g of the product taken.

Interpretation as per ISI standards


Acidity, lactic acid (% wt)

3.9. Khoa/Channa

3.9.1. Fat


Sweet dahi


Sour dahi


i) Weigh accurately about 3g of finely devided cheese samples into the cheese butyrometer.

ii) Add 10 ml of gerber’s acid, 10ml of distilled water and 1 ml of amyl alcohol.

iii) Close the butyrometer with the lock stopper and shake well till all the contents are mixed well.

iv) Place the butyrometer in a water bath at 65°C±2°C.

v) Shake well periodically until the solution of cheese is homogeneous.

vi) Centrifuge at 1200 rpm for 5 minutes.

vii) Read the percentage of fat by adjusting the fat column within the scale of butyrometer.


According to PFA standards, Khoa should contain not less than 20% milk fat in the finished product and the channa should contain not less than 50% milk fat of the dry matter. The skim milk channa should not contain more than 13% milk fat of the dry matter.

3.9.2. Moisture


a) Weigh a dried and cooled porcelain dish.

b) Weigh 2-3g of sample in the dish.


Keep the dish in hot air oven at 70oC for 8 hr.

d) Take out dish from oven and cool, weigh it.

e) Repeat heating, cooling and weighing until the loss of wt. between two consecutive weightings does not exceed mg.

Moisture % by wt. =


Weight of dried sample

Sample weight

× 100

According to PFA standards, the channa and skim milk channa should not contain more than 70% moisture.


Water is essential for our daily life. It is transparent, colourless, odourless and tasteless liquid. Water is used for drinking, cleaning, domestic purposes and in processing of dairy products ans other industries. Water that is free of disease producing micro-organisms and devoid of chemical substances deleterious to health is called potable water. The sanitary quality of water is of very great importance whether it is meant for drinking or for use in dairy industries. As a potential carrier of pathogenic micro- organisms, water can endanger health and life. The pathogens most frequently transmitted through water (if water is polluted with faecal matter, sewage or manure) are those which cause infections of the intestinal tract viz. typhoid and paratyphoid bacteria (Salmonella typhi and salmonella paratyphi), dysentery (bacillary - Shigella and amoebic), cholera bactria (Vibrio cholera) and enteric viruses (polio virus). Natural waters (well and tanks) may also be contaminated with a variety of micro-organisms derived from soil, vegetation and other resources. Mostly gram-negative bacteria such as Psuedomonas, Flavobacterium, Acinetobacter, Chromobacterium and few gram-positive organisms such as micrococci and bacilli are present in natural water. For assessing the sanitary condition of water, it is generally examined for the total bacterial content and for the incidence of coliform bacteria. These bacteria are typical intestinal organisms and their presence in water serves as an index of pollution from sevege or faecal matter. A series of tests can be used to demonstrate the presence of coliform as an indicator of faecal contamination in water supplies.


Sterile glass stoppered / screw capped bottles.

Spirit lamp


Ethyl alcohol

Match box

Sterile petriplates

Sterile pipettes

Dilution blanks (physiological saline)

Standard plate count agar

Water bath maintained at 45 0 to 50 0 C.


1. While collecting sample from the tap, clean the tap, wipe with alcohol and flame the top (only to metal tops and not to plastic tops).

2. Allow the water to run for one minute.

3. Filed the sample bottle in one hand near the tap, remove the stopper with the other hand, flame the mouth of bottle and quickly bring the bottle below the running stream of water. When the bottle is nearly full, take it out quickly and close it with the stopper.


Transport the bottle immediately to the laboratory and refrigerate it until required for analysis.

5. Shake the water sample thoroughly by moving the bottle up and down 25 times.

6. Prepare two serial dilutions 1:10 and 1:100 using dilution blanks, mix the dilutions thoroughly by rotating the tubes between the palms or in a vortex mixer.

7. Transfer 1 ml each of 0, 1 and 2 dilution blanks to the marked SPC plates in 2 sets of duplicates.

8. Add 15-20 ml of melted SPCA to petriplates maintained at 45 0 C, mix thoroughly and add melted MacConkey agar to coliform plates.

9. After solidification, incubate the plates for 24-48 hours.

SPC I set: 37 0 C (faecal organisms grow)

SPC II set: 22 0 C (plant organisms grow)

10. Count the selected plates and tabulate the results.

10. Count the selected plates and tabulate the results. Inference SPC @ 37 0 C :


the selected plates and tabulate the results. Inference SPC @ 37 0 C : 22 0

SPC @ 37 0 C : 22 0 C should be 1:10 (the ratio changes if faecal contaminant are more). The number of organisms from plant source will always be more in good quality water.

The presumptive test is most commonly used for the detection of faecal contamination in water and evaluation of its sanitary quality. The production acid and gas in lactose broth in 24-48 hours is regarded as presumptive evidence of coliform contamination in water. The test is used to determine the most probable number (MPN) of coliform present in 100 ml water. A sample showing more than 2 coliform bacteria / 100 ml is considered unsatisfactory. As the coliform group included both faecal and non-faecol types of bacteria, it is necessary conduct confirmatory tests for the presence of faecal coliforms in water.

Presumptive coliform test

The presumptive coliform test is used to determine the gas producing lactose fermenters that are present in water sample. It is determined using MacConkey’s broth tubes. For this, the most probable number (MPN) of coliform present in 100 ml of water can be roughly estimated using MPN table.


Sample of water

MacConkey’s broth single and double strength