Section B: Analytical

Process and quality control procedures

Chemical and microbiological analyses of cheese milk, finished cheese and cheese whey are required to maintain efficient operations and to ensure food safety and quality. This chapter describes some analytical procedures relevant to cheese making operations, but it is not intended to be a comprehensive process and quality control manual. The following general comments are intended to orient the reader to the general types of analyses required in cheese operations. Subsequent chapters will identify process and quality control requirements in the context of each step in the cheese making process.

Milk Analysis

Milk composition analyses should include both fat and protein, determined by infrared milk analysers. Note that casein content rather than total protein content is the critical parameter with respect to cheese yield. Cheese makers are, therefore, advised to regularly monitor the relative amounts of casein, whey proteins and non-protein nitrogen in their milk. Monthly or bimonthly analysis of protein distribution by Rowland fractionation is sufficient to monitor seasonal trends. Alternatively, an indication of casein and whey protein distribution can be obtained by comparing protein concentration in cheese whey to the protein concentration in the initial milk. This has the advantage that infra red milk analysers can be calibrated to measure protein in cheese whey. See Chapters 6 and 12 for details on standardization of milk composition and the importance of casein to cheese yield.

Quality measurements of cheese milk should include total counts (and/or psychrophilic counts), tests for inhibitors and somatic cell counts. Depending on the types of controls in place at the producer level, cheese makers may need to monitor bacteria counts, inhibitors, and somatic cell counts of individual producer milks.

Cheese Analysis

Cheese composition analyses should include fat (by Babcock, Mojonnier, or near infra red procedures), moisture, salt and pH. Cheese pH should be measured at the time of manufacture, 3 - 4 days after manufacture and periodically during curing. Other composition parameters should be determined several days after manufacture to permit time for equilibration of soluble components. Salt in particular, requires time to become evenly distributed throughout the cheese and in the case of brine or surface ripened cheese, uniform salt distribution may never be achieved. For Cheddar cheese and other vat salted cheese, representative samples for accurate determination of salt content can be usually be obtained as early as seven days after manufacture.

With respect to process and quality control, the 'pH profile' during manufacture and curing is vital. 'pH profile is a term I use to describe the set of pH values at critical process control points in the cheese making process. Other critical process control parameters are the ratio of salt to moisture (S/M), the moisture in the nonfat substance (MNFS), and the fat in the dry matter (FDM). These ratios are normally reported as percentages and calculated as in Equations 1a, 1b, 1c, below.

Note that percent total solids is 100 minus percent cheese moisture.

Routine cheese microbial analyses should include yeasts and moulds, total coliforms and staphylococci. For raw milk cheese, all vats must be tested for the presence of Salmonella, Staphylococci, Listeria and enteropathogenic E. coli. Cheese made from heat treated but not pasteurized milk must also be considered higher risk and should be monitored on a regular basis for the presence of common pathogens. Microbial analyses should be performed at the time of manufacture and after curing. Cheese whey should be monitored for the presence of bacteriophage specific for the culture currently in use.

Analytical Quality Control

A simple but vital truism, is that inaccurate analytical results are of less value than no analytical results. The most important causes of poor quality, poor yield efficiency and poor process control are insufficient and inaccurate chemical and microbial analyses. Effective control of quality and plant efficiency requires effective quality control of analytical procedures. Smaller cheese manufacturers generally find it's more economical and reliable to have most analyses performed by an outside laboratory. But, whether the analyses are performed in house or by an outside laboratory, be certain that your laboratory services are accurate and reliable. In Canada, dairy laboratory reliability can be assured by certification with the Canadian Laboratory Accreditation Programme (LAP), Ottawa, (613) 247-1395. The LAP is able to provide ongoing certification for both milk analysis (composition and quality) and cheese composition analysis. I strongly recommend that cheese makers use LAP or a similar certified testing, whether lab services are provided from inside or outside the company.

Some analytical procedures are detailed in subsequent sections. The reader is also referred to:

  1. Standard Methods for the examination of dairy products. American Public Health Association, 1015 Eighteenth St. NW, Washington, D.C.
  2. Official Methods of Analysis of the Association of Official Agricultural Chemists, P.O. Box 540, Benjamin Franklin Station, Washington, D.C. 

Cheese Sampling

 Chemical Analysis

Depending on the size and shape, firm to hard cheese should be sampled using a cheese trier (at least 100 g sample) or by taking a sector sample. Soft cheese can be blended for sampling or sector sampled depending on its texture. Cheese samples are stored in opaque air tight containers and fragmented using a grater or other device before analysis. It is important to grind and mix the sample well before subsampling for analysis.

If the analytical procedure requires less than a 1 gm sample it is desirable to prepare a liquid cheese homogenate and a subsample from the homogenate. An homogenate suitable for most purposes can be prepared as follows.

  • Weigh 40 g cheese into a blender container
  • Add about 100 g of 7% sodium citrate solution
  • Blend until homogenous (high speed blender such as Polytron is most suitable).
  • Rinse blender shaft into container and make up to final weight of about 200g.

Note that cheese is notorious for inhomogeneous composition. Brine salted cheese have pronounced salt and moisture gradients, namely, higher salt and lower moisture near the surface. Large blocks or wheels of pressed cheese, will have moisture and pH gradients, namely, increasing moisture and decreasing pH towards the interior. In addition to moisture and salt gradients, surface ripened cheese also has pH gradients, namely, pH increases at the surface during curing. These difficulties greatly complicate the matter of obtaining accurate composition and mass balance (yield) data. A useful approach to improve yield control of large blocks is to set aside small blocks (eg., 20 kg blocks of Cheddar) for early composition and quality testing, and subsequently, conduct representative sampling of the large blocks (eg., 240 kg blocks of Cheddar) during the cut/wrap process.

Microbial Analysis

Obtain samples as described above for chemical analysis. Triers or knives used for sampling must be flame sterilized. Samples should be stored in sterile bags such as Whirl Pack bags, stored at 0-4C and analysed within 24 hours.


  1. Balance, 1,000 g capacity
  2. Blender
  3. Blender container autoclaved or sanitized with 200 ppm chlorine solution for 5 min.


  1. Break the cheese into small pieces while still in the bag. Use a pestle or similar device if necessary.
  2. Heat dilution blanks of sterile aqueous 2% sodium citrate to 40C. Transfer 30 g of cheese to sterile blender container, add 270 ml diluent and mix for 2 min. at speed sufficient to emulsify the cheese properly. If temperature exceeds 40C during blending, use a shorter mixing time or decrease initial temperature of citrate solution. This 1:10 dilution should be plated or further diluted immediately.
  3. Further dilutions can be prepared as required. Pipette 11 ml of the 10-1 dilution of the homogenate, avoiding foam, into 99 ml dilution blank (0.1% peptone) or 10 ml into 90 ml dilution blank. Shake this and all subsequent dilutions vigorously 25 times in a one foot arc. Prepare 10-1, 10-2, and 10-3 dilutions.

Total Solids

Oven Method

  1. Pre-dry aluminum dishes (105C, 1 h) and weigh to the nearest 0.1 mg on an analytical balance.
  2. Weigh quickly 3-5 g of fragmented cheese into the aluminum dish. The weight of sample is the total weight minus the weight of the dish from Step 1.
  3. Dry to constant weight (about 16 h) at 105C. To check for constant weight: weigh at least two samples, return both samples to the oven for an additional 20 minutes, and re-weigh. The difference between the weights before and after the additional drying period should be less than 1 mg.
  4. Cool in desiccator and determine total dry weight. Sample dry weight is the total dry weight less the weight of the dish determined in Step 1.
  5. Report total solids and moisture contents on weight percent basis as follows:

Note: Several rapid moisture tests based on infrared or microwave drying are available. Check with your laboratory equipment supplier.


Accurate cheese moisture analysis is critical to composition and yield control. Rapid moisture tests (e.g., microwave moisture oven) can be used to obtain early feed back (e.g., cheese moisture immediately after pressing) information to help with process control.

Titratable Acidity


See discussion of pH and acidity in Section 3.5.

Apparatus and Reagents

  1. An acidimeter equipped with a burette graduated in tenths of a ml up to 10 ml, and some means of filling the same without undue exposure of the solution to the carbon dioxide of the atmosphere.
  2. N/10 sodium hydroxide solution.
  3. A dropping bottle containing a 1% alcoholic phenolphthalein solution.
  4. White cup, glass stirring rod, 17.6 ml pipette (or 8.8 or 9.0 ml pipette)
  5. For cream, Torsion balance and 9 g weight.


  1. Mix sample thoroughly by pouring it from one container to another. The temperature of the sample should be near 20C.
  2. Pipette 17.6 ml of milk or cream into a white cup. Note: 8.8 ml pipettes may also be used but are no longer as readily available as 17.6 ml pipettes. Readily available 9 ml pipettes are also used but require application of a correction factor to the final result.
  3. Add six drops of phenolphthalein indicator solution to milk, 10 drops if the product is cream.
  4. Titrate the sample with the N/10 sodium hydroxide solution (0.1 Normal NaOH) while stirring the sample with the glass rod. Look for the appearance of a faint pink colour which signals the endpoint. Add another drop or half a drop of NaOH if the pink colour does not persist for 30 s.
  5. Record the number of ml of NaOH used to reach the endpoint. This value is called the 'titre'. Titratable acidity reported as percent lactic acid is dependent on the volume of sample.

For the 8.8 ml pipette, % Lactic acid = titre

For the 17.6 ml pipette, % Lactic acid = 0.5 x titre

For the 9.0 ml pipette, % Lactic acid = 0.98 x titre.

Note that there is practically no lactic acid in fresh milk, but it is a North American convention to report TA in terms of % lactic acid.


As described in the next section, both titratable acidity (TA) and pH are measures of acidity. However, for most process control purposes, pH is a more useful measurement. Many cheese makers, however, still use TA to monitor initial acid development (that is to check for culture activity) during the first hour after adding the culture. For this purpose, TA is a more reliable indicator because relative to pH measurement, it is more sensitive to small changes in milk acidity.

When using TA to monitor initial culture activity note that:

  1. You are looking for a measurable increase in TA to confirm that the culture is active. For example, if the initial TA taken immediately after the culture was added is 0.183% lactic acid, and the TA after one hour of ripening is 0.194 % lactic acid, the change in TA is 0.194 - 0.183 which is 0.011%.
  2. Different people will interpret the coloured endpoint differently, so it is important that the same person takes both the initial and final TA measurements.
  3. Carefully performed, it is possible to reliably measure a change in TA of 0.05% lactic acid, so if the TA increase is greater than 0.05% you can conclude that the culture is active. In most cases TA increases in the range of 0.05% to 0.10% are obtained after about 30 minutes of ripening (that is, 30 minutes after adding the culture).
  4. It is critical to take the initial TA reading after the culture is added, because the culture (especially the bulk culture) is acidic.


Concepts of Acidity and pH

All aqueous systems (including the water in you and in cheese) obey the following relationship (Equation 3) between the concentration of hydrogen ions (H+) and hydroxyl ions (OH-). Note, the square brackets indicate concentration in moles per litre. A mole is 6 x 1023 molecules, that is, the numeral six with 23 zeros after it.

[H+] x [OH-] = 10-14

Because the actual concentrations in moles per litre are small, it is customary to express the values as exponents. For example, if we know that the concentration of hydrogen ions [H+] in a sample of milk is 0.000001 moles/l which is equivalent to 10-6 moles/l, we can calculate the concentration of hydroxyl ions as 10-14/10-6 = 10-8 moles/l which is the same as 0.00000001 moles/l.

  • If [H+] = [OH-] the solution is neutral with respect to acidity.
  • If [H+] > [OH-] the solution is acidic.
  • If [H+] < [OH-] the solution is basic or alkaline.
  • Chemicals which contribute H+ or absorb OH- are acids, while bases contribute OH- or absorb H+.

The concept of pH evolved as a short hand method to express acidity. We have already seen that a hydrogen ion concentration of 0.000001 moles/l can be expressed as [10-6], an expression which defines both the unit of measurement and the numerical value. The concept of pH is a further abbreviation which expresses the concentration of hydrogen ions as the negative log of the hydrogen ion concentration in units of moles/l. This sounds complex but is quite easy to apply. For example, the log10 of hydrogen ion concentration of [10-6] is equal to -6. The final step is to take the negative of the log, that is -1 x -6 which is 6. So, 0.0000001 moles/l = [10-6] = pH 6. From the relationship expressed in Equation 3, if the concentration of one of OH- and H+ is known, it is always possible to calculate the concentration of the other. So, if the pH of a solution is 6, the pOH is 14 - 6 = 8. Because this relationship is understood, the convention is to only report pH. Note, that because the negative sign was dropped by convention, decreasing pH values mean increasing acidity, that is, increasing concentration of H+ ions. So, although both TA and pH are measures of acidity, pH decreases with increasing acidity.

All of this can be summarized by a description of the pH scale. The pH scale for most practical purposes is from 1 to 14, although a pH of less than one is theoretically and practically possible.

pH 7.0 is neutral acidity [H+] = [OH-]

pH < 7.0 = acid condition [H+] > [OH-]

pH > 7.0 = alkaline condition [H+] < [OH-]

pH Versus Titratable Acidity

TA and pH are both measures of acidity but, for most purposes, pH is a better process control tool, because the pH probe measures only those H+ which are free in solution and undissociated with salts or proteins. This is important because it is free H+ which modifies protein functionality and contributes sour taste. It is also the pH rather than titratable acidity which is the best indicator of the preservation and safety effects of acidity. It must be emphasized, that the most important factor available to the cheese maker to control spoilage and pathogenic organisms is pH control. The pH history during and after cheese manufacture is the most important trouble shooting information. Cheese moisture, mineral content, texture and flavour are all influenced directly by the activity of free hydrogen ions (i.e. pH).

Titratable acidity (TA) measures all titratable H+ ions up to the phenolphthalein end point (pH 8.5) and, therefore, varies with changes in milk composition and properties. During cheese manufacture, the pH gives a true indication of acid development during the entire process so that the optimum pH at each step is independent of other variables such as milk protein content. However, the optimum TA at each step in cheese making will vary with initial milk composition and the type of standardization procedure used.

A good practical illustration of the difference between TA and pH is the effect of cutting. Up to the time of cutting, TA of the milk increases with the development of acidity by the culture. After cutting the TA of the whey is much lower. This does not mean that acid development stopped. It simply means that titratable H+ ions associated with the milk proteins are no longer present in the whey. This leads to the concept of buffer capacity, which is an important principle in cheese making. The effect of protein removal on the TA of whey, is related to the ability of protein to 'buffer' the milk against changes in pH. That same buffer property is the reason it helps to take acidic medication, like aspirin, with milk.

Buffer capacity can be described as the ability of an aqueous system, such as milk, to resist changes in pH with addition of acids (added H+) or bases (added OH-). Specifically, buffer capacity is the amount of acid or base required to induce a unit change in pH. For example, a small addition of acid to distilled water will cause a large reduction in pH. The same amount of acid would have a small effect on the pH of milk because milk proteins and salts neutralize the acidity.

The two most important buffer components of milk are caseins (buffer maximum near pH 4.6) and phosphate (buffer maxima near pH 7.0). The buffer maximum near pH 5.0 is extremely important to cheese manufacture because the optimum pH for most cheese is in the range of 5.0 - 5.2. As the pH of cheese is reduced towards pH 5.0 by lactic acid fermentation, the buffer capacity is increasing (i.e., each incremental decrease in pH requires more lactic acid). The effect is to give the cheese maker considerable room for variation in the rate and amount of acid production. Without milk's built in buffers it would be impossible to produce cheese in the optimum pH range.

Another way to illustrate the difference between TA and pH is to consider typical ranges of pH and TA for normal milk. TA is a measure of the total buffer capacity of milk for the pH range between the pH of milk and the phenolphthalein end point (about pH 8.3). The pH of milk at 25C, normally varies within a relatively narrow range of 6.5 to 6.7. The normal range for titratable acidity of herd milks is 0.12 to 0.18% lactic acid In other words, pH is a good indicator of initial milk quality, while the traditional measurement of TA to indicate bacterial growth in milk is less precise.

pH Measurement

The pH of cheese milk, whey and soft cheese can be measured directly. Firm and hard cheese must be fragmented before analysis. Always measure cheese pH in duplicate and use extreme care in handling the electrode. Place the fragmented cheese in a 30 ml vial or small beaker and gently push the electrode into the cheese ... too much haste is likely to break the electrode on the bottom of the beaker. To ensure good contact, press the cheese around the electrode with your fingers. There is no need to rinse the electrode between cheese samples. However, if the electrode is stored in buffer it should be rinsed with distilled water before measuring cheese pH. Always store the electrode in pH 4 buffer or as directed by the manufacturer. Do not rub the electrode. The electrode should be washed with detergent and rinsed with acetone occasionally to remove fat and protein deposits.

Babcock Methods for Milk Fat

Apparatus and Materials

  1. Babcock centrifuge.
  2. Water bath at 55C.
  3. Torsion balance, 9 and 18 g weights.
  4. Babcock shaker.
  5. Glassware: 8% milk bottles, 50% cream bottles, 50% Paley bottles, 17.5 ml cylinders, 17.6 ml pipette, .
  6. Reagents: - Babcock sulphuric acid (Sp. Gr. 1.82-1.83)
  • N-butyl alcohol
  • Glymol.


  1. Temper sample to 20C and mix by pouring gently from original container to a beaker of similar capacity 4-5 times.
  2. Transfer 17.6 ml (18.0g) of milk to 8% bottle with 17.6 ml pipette. Allow pipette to drain then blow out the remaining drop into the bottle.
  3. Add 17.5 ml sulphuric acid (Sp. Gr. 1.82-1.83) in at least three increments using special cylinder. Rotate bottle between thumb and fingers while adding acid to wash milk from neck. Mix thoroughly 2 min. after each addition of acid by moving the bulb of the bottle in rapid circular motion. Final colour of mixture should be chocolate brown.
  4. Centrifuge 5 min.
  5. Add distilled water at 60C to bring contents to within one-quarter inch of base of neck. Do not mix.
  6. Centrifuge 2 min.
  7. Add water at 60C to float fat into neck of bottle. Top meniscus should be about even with the top of the graduated portion. Do not mix.
  8. Centrifuge 1 min.
  9. Temper bottles in water bath at 55C for 5 min.
  10. Measure length of fat column with dividers from top of upper meniscus to bottom of lower meniscus. Place one divider point at zero mark and read percentage fat by weight directly where other point touches the scale.

Cream and Cheese

  1. Temper cream sample to 20C and mix. Grind cheese to small particles.
  2. Weigh 9 g of cream into 50% cream bottle and add 9 ml of distilled water at 200C. Weigh 9 g of cheese into a 50% Paley bottle and add 10 ml of distilled water at 60C.
  3. Add 17.5 ml sulphuric acid in at least three increments. Mix until colour is uniform chocolate brown and all cheese particles are dissolved.
  4. Centrifuge 5 min.
  5. Add distilled water at 60C to bring contents to within one-quarter inch of base of neck. Do not mix.
  6. Centrifuge 2 min.
  7. Add water at 60C to float fat into neck of bottle. Do not mix.
  8. Centrifuge 1 min.
  9. Temper bottles in water bat at 55C, for 5 min.
  10. Place 4-5 drops glymol on the fat column letting these run down the side of the neck. Measure the length of the fat column from the demarcation between fat and glymol to the bottom of the lower meniscus.
  11. Report fat in percent by weight.

Skim milk, Buttermilk, Whey

  1. Temper sample to 20C and mix gently.
  2. Transfer 2 ml N-butyl alcohol and then a 9 ml sample to an 18 g double neck bottle. Mix thoroughly with a circular motion.
  3. Add 9 ml of Babcock sulphuric acid for skim milk or buttermilk, 7 ml for whey.
  4. Centrifuge 6 min. Place bottles in the centrifuge cup with the small neck facing the outside.
  5. Add water at 60C to bring contents 1 cm from the base of the neck. Do not mix. Centrifuge 2 min.
  6. Temper bottles in water bath at 55C for 5 min.
  7. Place a finger over the large neck and press down until the lower meniscus of fat in the small neck corresponds to a major division.

Cheese Salt

Cheese salt determination using the Volhard procedure is described below. Other methods which have proven to give accurate results are:

  1. Automatic Chloride Titraters operate on the principle of coulometric silver ion generation to titrate chloride ions in the sample. When all chloride ions are titrated free silver ions cause a conductivity change which signals the end of titration.
  2. Quantab Chloride Titrater depends on the reaction of chloride ions with silver dichromate, which is brown, to form silver chloride chromate ion and silver chloride which is white. The reaction takes place on a calibrated strip which permits direct estimation of chloride content.

Volhard procedure for salt determination

Apparatus and Materials

  1. A torsion moisture balance
  2. 250 ml erlenmeyer flask and a 500 ml beaker
  3. Two graduated cylinders, one 50 ml and the other 100 ml
  4. A 10 ml pipette and a 5 ml graduated pipette
  5. Burette graduated in ml and 1/10 ml, and burette stand
  6. An electric or gas hot plate
  7. Chemically pure concentrated nitric acid
  8. Saturated potassium permanganate solution
  9. 0.1711 N potassium thiocyanate solution (contains 16.63 g per litre) in a brown glass bottle
  10. 0.1711 N silver nitrate solution (contains 29.07 g per litre) in a brown glass bottle
  11. A saturated solution of ferric ammonium sulphate
  12. Sucrose
  13. Boiling chips such as carborundum granules or glass beads.
  14. Fume hood


  1. Prepare cheese sample as for cheese moisture test.
  2. Weigh about 3 g cheese into a clean dry 250 ml erlenmeyer flask.
  3. Add 10 ml of 0.1711 N silver nitrate solution as accurately as possible to the flask. If cheese contains more than 3% salt, add more silver nitrate.
  4. Add 15 ml of the chemically pure nitric acid.
  5. Add 50 ml of distilled water.
  6. Add a few boiling stones.
  7. Place flask on hot plate in fume hood and boil.
  8. When contents of flask are boiling uniformly, carefully add 5 ml of saturated potassium permanganate. Continue boiling until purple colour disappears, then add a second charge of 5 ml of potassium permanganate. When purple colour again disappears, add another 5 ml of potassium permanganate. Continue boiling until all cheese particles are digested. To ascertain when digestion is complete, remove flask from hot plate and allow to stand quietly for a few moments. Undigested cheese particles will float upon the surface, while the white precipitate of silver chloride will sink to the bottom of the clear liquid. When no more white particles are seen upon the surface, digestion is complete.
  9. Add sufficient distilled water to bring the volume up to approximately 100 ml. Allow precipitate to settle and very carefully pour off the liquid into a beaker. Be careful not to pour off any of the white precipitate of silver chloride.
  10. Add 100 ml of distilled water to flask and swirl contents to wash precipitate.
  11. Add 3 ml of saturated ferric ammonium sulphate as an indicator and titrate the excess silver nitrate with 0.1711 N potassium thiocyanate. A reddish colour denotes the end point.
  12. The number of ml of 0.1711 N silver nitrate originally added minus the titration value found in step 11, divided by the weight of the cheese in the sample equals the percentage of salt in the cheese.


3.00 g of cheese to which 10.00 ml of 0.1711 N silver nitrate had been added gave a reading of 4.00 ml in Step 11.

4.00 ml 0.1711 N potassium thiocyanate required to combine with excess silver nitrate.

6.00 ml 0.1711 N silver nitrate combined with salt in cheese.

Therefore per cent salt by weight = 6.00/3.00 = 2.00

Because the salt in the cheese is measured by its chloride content, it is necessary to test the reagents used for chloride, or related substances content. This is done by carrying out a test using sucrose instead of cheese. The titration value subtracted from the original amount of silver nitrate added is subtracted from the value found in Step 12 before dividing by weight to find the percentage salt in the cheese.

To check the strength of the 0.1711 N silver nitrate solution, dissolve 10 g chemically pure dry sodium chloride in sufficient water to make up one litre of solution. Each ml of this solution is equivalent to one ml of 0.1711 N silver nitrate. When the silver nitrate has been standardized, each ml of silver nitrate is equivalent to one ml 0.1711 N potassium thiocyanate.

Culture Activity Test


This simple test is useful to ensure that cheese cultures have adequate activity before inoculating the cheese vat. For most cheese a general rule of thumb is that the activity and amount of inoculum should be sufficient to produce a titratable acidity of about .34% lactic acid, in 10% reconstituted skim milk, after 4 h of incubation at 37C. The test is also useful to compare types of cultures or bulk cultures prepared under different conditions. For these purposes a pH versus time chart is quite useful . A further application is to check sensitivity of the culture to bacteriophage in the plant (see next section Detection of Bacteriophage and Figure 3.1).


  1. Mix 10 g of low-heat, antibiotic-free skim milk powder in 90 ml of distilled water in a 100 ml Erlenmeyer flask.
  2. Sterilize at 15 lb pressure (1.05 kPa.) for 10 min.
  3. Cool to 37C.
  4. Inoculate with 3.0 ml starter or other amount as appropriate. Rinse pipette twice by drawing the sterile milk into it.
  5. Incubate at 37C for at least 4 h. Longer if desired for pH versus time profile.
  6. Check pH at 30 min. intervals.
  7. Titrate 17.6 ml with N/10 sodium hydroxide (NaOH) using 1 ml phenolphthalein. Divide the required ml of NaOH by 2 the obtain titratable acidity in units of percent lactic acid.
  8. Record starter activity as follows:
    Active, over 0.34%
    Slow 0.26 to 0.30%

Figure 3.1 Culture Activity Test

Detection of Bacteriophage

The following tests are based on the principle that bacteriophage specific to the culture in use will be present in high numbers in the cheese whey. Therefore, by monitoring whey for the presence of phage "a dead vat" on subsequent days can be avoided.

Culture Activity Test

The culture activity test described above can be used to detect the presence of phage in cheese whey. Prepare 300 ml of reconstituted skim milk and place 99 ml in each of three beakers. Add 1 ml of whey to Beaker 1 (100 x dilution), then transfer 1 ml from Beaker 1 to Beaker 2 (10,000 x dilution) and finally, transfer 1 ml from Beaker 3 to Beaker 4 to make a 1 million times dilution. Add culture and monitor pH as described in section 3.8.

Bromocresol Purple (BCP) Phage Inhibition Test

This test is quite simple to perform, and produces more accurate results than the culture activity test.

1. Prepare Materials

  • BCP stock solution (1 g/100 ml water)
  • Test tubes containing 9.9 ml sterile BCP-milk (5 ml BCP stock solution/litre milk)
  • 30-32C water bath or heating block
  • 1 ml graduated pipettes
  • Membrane filter (0.45 u) -- optional
  • Disposable syringe -- optional
  • Clinical centrifuge -- optional
  • Whey sample for phage testing
  • Freshly grown culture, frozen syringe, or frozen can of each strain

2. Add Whey to BCP Milk and Make Dilutions

Transfer 0.1 ml of fresh (or filter-sterilized) whey to the first dilution tube (10-2) and mix well. Transfer 0.1 ml from the first to the second dilution tube and mix well. Repeat process for the third dilution tube. (If unfiltered whey is used, a control tube containing BCP milk and whey only, must be prepared. This control tube tests for the presence of active culture in the whey that could mask phage inhibition of a strain.) Whey samples should be refrigerated immediately after collection and held cold until tested for phage.

3. Add Culture to Control and Whey Dilution Tubes

Cheese culture (0.2 ml) is added to whey dilution tubes and to a control tube for each strain. If you are using direct-to-the-vat culture, dilute 1 ml of culture in 9 ml of milk and then add 0.2 ml of the mixture to the dilution tubes. The control tube contains only BCP milk and culture---NO whey. The control tube serves to show starter strain inhibition by colour comparison with the other tubes.

Incubate Tubes and Interpret Results. Incubate both control and dilution tubes for 6 hours at 30-32C. Compare the colour of the whey dilution tubes to that of the control tube. Ignore coagulation. An uninhibited culture will produce sufficient acid to turn the BCP dye from blue to yellow. Strains should be removed from the culture blend when full inhibition persists at the 10-6 dilution level. The following system should be used to record phage inhibition:

0 = No inhibition at any dilution

1 = Partial inhibition at 10-2 dilution

2 = Full inhibition at 10-2 dilution

3 = Partial inhibition at 10-4 dilution

4 = Full inhibition at 10-4 dilution

5 = Partial inhibition at 10-6 dilution

6 = Full inhibition at 10-6 dilution

Inhibitory Substances(1)

This section is adapted from two reports prepared by: Mark Mitchell (1995), Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario


Most jurisdictions have regulations concerning the testing methods and limits of certain antibiotics in raw milk. The Milk Act of Ontario, Regulation 761, Section 52, Subsection, states:

"The milk of every producer shall be tested at least once a month for the presence of an inhibitor by an official method."

An official method is described in a separate inhibitor policy document which states:

The minimum sensitivity of an official method to test for the presence of an inhibitor under section 52 of Regulation 761 shall be:

  1. 0.01 international units of penicillin per millilitre of milk by the Standard Disc Assay (Bacillus stearothermophilus) procedure.
  2. 10 parts per billion sulfamethazine by the High Performance Liquid Chromatography (modified Smedley and Weber) procedure.

A concentration of .01 international units of penicillin per millilitre of milk is equivalent to 6 parts per billion (ppb). Note: 1 ppb is equivalent to a single penny in $10 million or one second in 32 years.

Detection Methods

It is beyond the scope of this manual to discuss any specific methods in detail. What follows is are brief descriptions of five types of inhibitor tests which are currently used in the dairy industry. For each category one or more brand name tests are listed to indicate possible choices. For cheese manufactures seeking assistance with inhibitor testing, there are many private labs which provide suitable services. In Ontario, a wide range of expertise and methodologies are available from Laboratory Services Division, University of Guelph.

Growth Inhibition Assays

Examples: Delvotest P, Delvotest SP, BR test, BR-AS test, Charm Farm, and the Disk Assay

This test format involves a standard culture of a test organism in an agar growth media, usually Bacillus stearothermophilus, that is inoculated with a milk sample and incubated for periods of up to several hours. If the milk contains sufficient concentrations of inhibitory substances the growth of the organism will be reduced or eliminated. The presence of an inhibitory substance is indicated by zones of inhibition or a change in colour of the media (pH and redox indicators).

The major disadvantages of these tests are that they are not very specific for identification purposes, have limited sensitivities to many antibiotics and take a long time before results are available. Growth inhibition tests are only able to classify residues into either the ß-lactam (penicillin like antibiotics) or other than ß-lactam antibiotic families. A further concern is that growth inhibition tests are subject to the effects of natural inhibitors (eg. lysozyme, lactoferrin, complement and defensins) which can be found in high levels in mastitic milk and may give false positive test results, particularly when used at the cow level. These effects can be minimized by heating individual cow samples at 82C for 2-3 minutes in a microwave oven or water bath before testing to destroy natural inhibitors and allow antibiotics which are more heat stable to remain.

The advantages of these tests are that they are cheap, easy to perform and have a very broad detection range.

Enzymatic Colorimetric Assays

Example: Penzyme Test for ß-lactams

The penzyme test is based on the inactivation of an enzyme by ß-lactam antibiotics. The enzyme (DD-carboxypeptidase or penicillin binding protein) is present in all bacteria and is involved in the synthesis of the bacterial cell wall. ß-lactam antibiotics will bind specifically with this enzyme and block it's activity, thus preventing the formation of the bacterial cell wall. This enzyme has been freeze dried and placed in sealed vials to which the milk sample is added. After addition of 0.2 ml (200 µl) of milk sample to the vial the sample is incubated for 5 minutes at 47C. During this time any ß-lactams present in the milk bind to the enzyme and inactivate a certain amount depending on the concentration present.

Reagent tablets specific for the enzyme (D-alanine peptide and D-amino acid oxidase) are then added to the milk sample and the sample is incubated at 47C for 15 minutes. During incubation any remaining active enzyme will react with the reagent added. The end product of the substrate and enzyme reaction (pyruvic acid and hydrogen peroxide) is measured by a redox colour indicator and the final colour is compared to a colour chart provided with the kit.

An orange colour (reduced) indicates a negative test result.

A yellow colour (oxidized) indicates a positive test result.

Microbial Receptor Assays:

Example: Charm II

This test uses bacterial cells (Bacillus stearothermophilus), which contain natural receptor sites on or within the cells for antibiotics, and radio labelled (C14 or H3) antibiotics. Milk sample is added to a freeze dried pellet of bacterial cells (binding reagent) in a test tube and the sample is mixed and incubated. During incubation any antibiotic present in the milk will bind to it's specific receptor site. Radio labelled antibiotic (tracer reagent) is then added and the sample is mixed and incubated. Unbound receptor sites on the bacterial cell will be bound by the radio labelled antibiotic. The sample is then centrifuged to collect the bacterial cells in the bottom of the test tube and the supernatant and butterfat is discarded. The bacterial cells are then resuspended and mixed in scintillation fluid. Binding is measured with a scintillation counter and compared to a positive and negative control. The more antibiotic present in the sample the lower the scintillation counts determined by the equipment.

Charm currently has test kits in this format for ß-lactams, macrolides, aminoglycosides and sulfonamides.


Unlike other residue testing methods immunoassays are fast, sensitive, inexpensive, reproducible, reliable and simple to perform. The technique depends upon the measurement of the highly specific binding between antibodies (Ab) and antigens (Ag). Antigens are substances which are foreign to the body (eg. bacteria, viruses, toxins, pollens, drugs, hormones and pesticides) and that when introduced into the body give rise to the production of antibodies. Antibodies are proteins produced in the body by white blood cells (lymphocytes) as a result of exposure to antigens (destroy invading pathogens). The extreme sensitivity of the immunoassay is due to the development of certain labelling techniques for molecules (conjugates), enabling the measurement of very small masses (picogram or parts per trillion) of substances.

Immunoassays are classified according to the label which is attached to either the antigen (the anolyte being measured) or the antibody. The label may be a radioactive atom as in radio immunoassays (RIA), or an enzyme as in enzyme immunoassays (EIA or ELISA (Enzyme- linked immunosorbant assay)) or a fluorescent substance as in fluorescence immunoassays (FIA).

There are 3 major types of immunoassays used commonly for the detection of antibiotics in milk:

  1. Enzyme-Linked Immunoassay (eg. LacTek tests, SNAP for Tetracyclines, Single Step Block for SMZ)
  2. Enzyme-Linked Receptor Binding Assay (eg. SNAP for ß-lactams, Delvo-X-Press)
  3. Radio immunoassay (CHARM II for tetracyclines and chloramphenicol)

Rennet Activity

Coagulation Time versus Setting Time

Rennet is generally described in the industry as single, double or triple strength. Single strength is considered to be that concentration where 200 ml is sufficient to set 1,000 kg of milk in 30 - 40 min. at 30 - 32C. Setting time is the point where the curd will break cleanly and exude clear whey. Coagulation time is the point where flecks of curd first appear on a spatula or slide dipped into the milk. Coagulation time is about half of setting time, so typically, coagulation using single strength rennet requires 15-20 minutes followed by setting at 30-40 minutes. The following simple test can be used to check coagulation time which can be measured much more accurately than setting time. The test uses skim milk because the presence of fat globules makes it difficult to see the first sign of coagulation.

Measurement of Coagulation Time

  1. Prepare 200 ml samples of 10% reconstituted low heat skim milk powder in 250 ml beakers. Add 0.02% calcium chloride dihydrate (40 mg per 200 ml).
  2. Temper to 32C in a water bath.
  3. Add 1.0 ml of 5% rennet solution to each sample.
  4. Determine the clotting time by dipping a clean spatula or glass slide into the milk. When coagulation has occurred flecks of curd will appear in the milk film on the slide.

Relative Milk-Clotting Activity Test

A more rigorous test of coagulant activity is the "Relative Milk-Clotting Activity Test" (RMCAT) which measures the activity of rennet and other coagulants in "International Milk-Clotting Units" (IMCU). The method is described in International Dairy Federation standard 157:1992.

Yeasts and Moulds

Selective media for yeasts and moulds include acidified media and antibiotic media. The method described below uses acidified potato dextrose agar.

Equipment and Material

Potato dextrose agar

Equipment for plating

Tartaric acid solution (10 aqueous)

Incubator set at 22-25C


  1. Prepare cheese homogenates and serial dilutions as described in Section A.
  2. Predetermine the quantity of sterile 10% tartaric acid solution necessary to obtain a pH of 3.5 + 0.1. Put a portion of the medium in a small beaker and titrate to pH 3.5 at 45C. Check the accuracy of the titration by allowing the agar to cool to incubation temperature, place electrodes directly into the solidified medium, and read the pH. It should be 3.5 + 0.1. Calculate the amount of sterile 10% tartaric acid solution necessary for the volume of tempered agar to be used for pouring plates.
  3. Place 5 ml of the 0.1 dilution and 1 ml of additional dilutions as required into each of duplicate petri dishes.
  4. Add the tartaric acid solution to the tempered agar immediately before pouring 15 - 20 ml into each of the plates containing the sample dilutions.
  5. Mix well and let solidify before inverting the plates. Incubate at 22 - 25C.
  6. Count the plates at 3 and 5 days of incubation. Yeast cells will appear as cream coloured shiny colonies.

Presumptive Coliforms

  • Prepare cheese homogenates and serial dilutions as described in Section A.
  • Place 5 ml of the 0.1 dilution (= 0.5 g of original sample or Omega dilution) and 1 ml of additional dilutions as required into each of duplicate petri dishes and add molten VRB agar. (Note: Do not sterilize VRB agar.) When solidified, pour over layer (5 ml VRB).
  • Incubate at 35C + 10C for 18 - 24 hrs.
  • Count the dark red colonies, at least 0.5 mm in diameter, and record results as coliforms per g of sample.

Samples of cottage cheese and other acid milk products should be plated within 24 hrs. after manufacture because coliform counts decline under acid conditions. Coliforms also decrease in number during aging of ripened cheese varieties.

It must be emphasized that this method provides a presumptive count only. If presumptive counts are consistently high, colonies should be confirmed (see Standard Methods). The Canadian Food And Drug Act and Regulations permit 500 coliforms/g of cheese made from pasteurized milk and 5,000 coliforms/g of cheese made from unpasteurized milk. Permitted counts of Eshericia coli are 100 and 500/g respectively.


Procedure A

The method described here enumerates total Staphylococci by surface plating on Baird-Parker media. A coagulase test can be used to determine if individual colonies are S. aureus. Canadian Food And Drug Act and Regulations permit up to 100 coagulase positive S. aureus in pasteurized milk cheese and up to 1,000/g in cheese made from unpasteurized milk.

Equipment and Materials

Plating equipment

Glass spreaders (hockey stick-shaped glass rods)

Incubator set at 37C

Baird-Parker Agar


  1. Pour plates of B.P. agar (15 ml/plate) and dry surfaces (using sterile laminar airflow cabinet -- 2 hrs).
  2. Pipette 0.1 ml of homogenate and of subsequent dilutions onto surface of agar and spread evenly with a sterile bent glass rod until surface appears dry. Prepare duplicate plates. Use 10-1, 1--2, and 1--3dilutions.
  3. Incubate at 37C for 48 hrs.
  4. Count the number of colonies in each of the following groups:
    1. convex, shiny, black, with or without narrow gray-white margin, surrounded by clear zone extending into opaque medium.
    2. convex, shiny, black, with or without narrow gray-white margin, surrounded by clear zone extending into the opaque medium with an inner opaque zone.
    3. convex, shiny, black, with or without narrow gray-white margin, >1 mm in diameter.

Procedure B

Pipette 1 ml or 0.1 ml of homogenate and of subsequent dilutions into petri dish.

Add approximately 10 ml Baird-Parker medium. Mix well. Let stand on bench.

When solidified, invert and put in incubator at 37C (for 48 hours).

Read the same as Procedure A.

Note: Add 5 ml of well mixed Ey Tellurite, enrichment, at 5C to Baird-Parker agar prior to pouring plates.

Figure 3.1. Culture Activity Test

This section is adapted from two reports prepared by: Mark Mitchell (1995), Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario

Figure 3.1. Culture activity test: example.


Culture Lactococcus lactis subsp. lactis

Lactococcus lactis subsp. cremoris

Temperature 37 C

Inoculum 2% of mother culture prepared with 10% reconstituted skim milk powder.

Test media 1 10% skim milk powder, low heat, antibiotic free.

Test media 2 Same as one with 1% cheese whey.


Titratable acidity after 4 hours:

Treatment 1 0.34%

Treatment 2 0.25%

pH versus time

Time 0 1 2 3 4 5 6 7 8 9
Skim powder 6.62 6.59 6.5 6.4 6.15 5.74 5.39 5.08 4.92 4.87
Skim with whey 6.61 6.57 6.5 6.42 6.35 6.31 6.3 6.3 6.29 6.29


  1. Test media 1 shows normal growth. 0.34% acidity after 4 h with a 2% inoculum is adequate for most types of cheese. pH versus time plot is typical, reaching pH 5.2 between 6 and 7 hours.
  2. Test media 2, containing cheese whey, shows inadequate acid development, indicating the probable presence of bacteriophage in the cheese plant.

 pH vs Time