 SZNS  034:2014 
 
ICS 67.100                         Edition 1              
 
 
 
 
 
 
SWAZILAND NATIONAL STANDARD  
 
 
 
Raw cow milk  - Specification  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Published by: Swaziland  Standards Authority  
SmithCo Building, 2nd Floor, 11th Street King Mswati III Avenue West  
P. O. Box 1399, Matsapha, Kingdom of Swaziland  
Telephone: +268 2518 4633, Facsimile: +268 2518 4526  
Website: www.swasa.co.sz  
E-mail: info@swasa.co.sz   
©Swaziland Standards Authority 2014  
 
 
SZNS 034:2014 
Edition 1  
 
 
ii  ©SWASA 201 4 - All rights reserved  COPYRIGHT NOTICE  
 
This Swaziland Standards Authority (SWASA) document is a Swaziland National  Standard and is 
copyright -protected by SWASA. Neither this SWASA document nor any extract from it may be 
reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, 
photocopying, recording or otherwise, without prior written permission being secured.  
 
Requests for permission to  reproduce should be addressed to : 
The Information Officer  
Swaziland Standards Authority  
P.O. Box 1399, Matsapha  
Tel. + 268 2518 4633  
Fax: + 268 2518 4526  
E-mail: info@swasa.co.sz   
Website: www.swasa.co.sz  
 
Reproduction may be subject to royalty payments or a licensing agreement.  
Violators may be prosecuted.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 SZNS 034:2014 
Edition 1  
 
©SWASA 201 4 - All rights reserved    iii Table of changes  
 
Clause Changed  Date Change  
 
   
   
 
NATIONAL FOREWORD  
 
This Swaziland National  Standard was prepared by Technical committee SWASA/TC  16 Dairy products  
in accordance with procedures of the Swaziland Standards Authority, in compliance with Annex 3 of 
the WTO/TBT Agreement.  During the preparation of this  National standard , assistance was derived 
from the following documents:  draft COMESA/East African Specification for raw cow milk , and the 
Malawi Specification  for Raw cow’s milk  MBS 73:1988 published by Malawi Standards Boa rd. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 SZNS 034:2014 
Edition 1  
 
 
iv  ©SWASA 201 4 - All rights reserved  Contents                                                                                                                  Page  
1 Scope  ................................ ................................ ................................ ................................ ....................  1 
2 Normative references  ................................ ................................ ................................ ...........................  1 
3 Definition  ................................ ................................ ................................ ................................ .............  1 
4 Principal compositional requirements  ................................ ................................ ................................ .. 1 
4.1 Chemical  ................................ ................................ ................................ ................................ ....... 1 
4.2 Bacteriological Grades  ................................ ................................ ................................ .................  2 
5 Contaminants  ................................ ................................ ................................ ................................ ....... 3 
5.1 Pesticides residues  ................................ ................................ ................................ ........................  3 
5.2 Veterinary drugs residues  ................................ ................................ ................................ .............  3 
5.3 Heavy Metals and other contaminants  ................................ ................................ ..........................  3 
6 Hygiene  ................................ ................................ ................................ ................................ ................  3 
7 Methods of sampling  ................................ ................................ ................................ ............................  3 
8 Rapid platform tests on quality of raw milk  ................................ ................................ ........................  3 
Annex A  (normative)  ................................ ................................ ................................ ..............................  4 
Annex B  (normative)  ................................ ................................ ................................ ..............................  5 
Annex C  (informative)  ................................ ................................ ................................ ...........................  6 
Annex D  (normative)  ................................ ................................ ................................ ..............................  7 
Annex E  (normative)  ................................ ................................ ................................ ..............................  8 
Annex F  (normative)  ................................ ................................ ................................ ..............................  9 
Annex G  (normative)  ................................ ................................ ................................ ...........................  10 
Annex H  (normative)  ................................ ................................ ................................ ...........................  13 
 
 SZNS 034:2014 
Edition 1  
 
©SWASA 201 4 - All rights reserved    1 Raw cow milk - Specification  
1 Scope  
This Swaziland national  standard specifies requirements, methods of sampling and test s for raw cow 
milk.  
2 Normative references  
The following referenced documents are indispensable for the application of this document. For dated 
references, only the edition cited applies. For undated references, the latest edition of the referenced 
document (including any amendments) applies. Information on currently valid national and 
international standard s can be obtained from the Swaziland Standards Authority (SWASA).  
CAC/MRL 02 -2006, Maximum residue limits for veterinary drugs in foods . 
CAC/RCP 57, Code of hygienic practice for milk and milk products  
CODEX STAN 193 -1995, Codex general standard for contaminants and toxins in foods  
ISO 2446, Milk — Determination of fat content (Routine method).  
ISO 4833, Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of 
microorganisms — Colony -count technique at 30 °C  
ISO 5541 -1, Milk and milk products - Enumeration of coliforms - Part 1: Colony count technique at 30 
OC 
ISO 5764, Milk — Determination of freezing point — Thermistor cryoscope method   
ISO/TS 6090, Milk and dried milk, buttermilk and buttermilk powder, whey and whey powder — 
Detection of phosphatase activity  
ISO 6731, Milk, cream and evaporated milk — Determination of total solids content (reference method)  
ISO 13366, Milk  Enumeration of somatic cells   
ISO 707, Milk and milk products  guidance on sampling  
3 Definitions  
For the purposes of this document, the following definition applies.   
milk   
normal, clean and fresh secretions, without any addition or subtraction, extracted from the udder of a 
healthy cow, and free from colostrum, i.e.  excluding that got during the first five days after calving.  
4 Principal compositional requirements  
4.1 Chemical  
4.1.1  When determined in accordance with ISO 2446, milk shall contain not less than 3.25 % milk fat 
and not less than 8.50 % milk -solids -not-fat when d etermined in accordance with ISO  6731. It shall 
not contain added water, preservatives or other added substances, nor shall any proportion of a natural 
constituent be removed.  
4.1.2  Density of milk at 20 C shall be within the range of 1.028 g/ml – 1.036 g/ml.  
4.1.3  When determined in accordance with ISO 5764,  the freezing point depression of milk shall be not 
less than 0.525 C and not more than 0.550 C. 
4.1.4  When tested in accordance with Annex A, milk shall have a characteristic creamy – white color, 
free from of f flavours and taints.  SZNS 034:2014 
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2  ©SWASA 201 4 - All rights reserved  4.1.5 When determined in accordance with Annex B, milk shall be free of objectionable matter.  
4.1.6  When tested in accordance with Annex D, milk shall not coagulate in the clot on boiling test.  
4.1.7  When tested in accordance with Annex E, milk shall test negative to the alcohol test.  
4.1.8  When tested in accordance with Annex F, milk shall have no more than 0.17% titratable lactic 
acid.  
4.1.9 Milk shall test positive to phosphatase when tested according to ISO/TS 6090 . 
4.1.10  Milk shall pass the 10 - minute resazurin test in accordance with Annex G and/or 4.5 hours 
methylene blue test in accordance with Annex H.  
4.2 Bacteriological Grades  
Milk shall conform to the following microbiological limits:  
4.2.1 Total plate count   
The total p late count  shall be determined in accordance with ISO 4833. The counts shall be graded as 
specified in table 1  
Table 1  Microbiological limits  
 
4.2.2 Coliform plate count  
The coliform count shall be determined in accordance with ISO 5541 part 1 . The counts shall be 
graded as specified in table 2  
Table 2 — Coliform limits  
S/N Quality  cfu/ml  
a) Very good  0 – 1000 
b) Good  1000 - 50000  
 
4.2.3  Somatic cell count  
When determined in accordance with ISO 13366, the somatic cell count shall not exceed 300 000 per 
millilitre.  
5 Contaminants  
5.1 Pesticides residues  
Milk shall conform to the maximum limits of pesticide residues as specified in Codex Alimentarius 
Commiss ion standards.  Grade  cfu/ml  
  I or A  < 200,000  
 II or B  >200,000 – 1000,000  
III or C  >1000,000 – 2000,000  SZNS 034:2014 
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©SWASA 201 4 - All rights reserved    3 5.2 Veterinary drugs residues  
The products covered by this standard shall comply with the maximum residue limits specified in 
CAC/MRL 2 -2006.  
5.3 Heavy metals and other contaminants  
The products covered by this standard shall comply with the maximum limits as specified in CODEX 
STAN 193 -1995 . 
6 Hygiene  
Milk shall be produced, processed and handled in accordance with CAC/RCP 57.  
7 Methods of sampling  
Sampling of butter for analysis sh all be done in accordance with ISO 707.  
8 Rapid platform tests on quality of raw milk  
See Annexes A to H.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 SZNS 034:2014 
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4  ©SWASA 201 4 - All rights reserved  Annex A  
(normative)   
 
Organoleptic test and temperature  
 
A.1 General  
Judging the quality of milk by its physical and sensory characteristics requires considerable skill, which 
could only be acquired by practice. Organoleptic tests are used in all dairies and an experienced person 
can pick out bad samples with a high degree of accuracy.  
A.2  Procedure to be adopted on the receivi ng platform  
A.2.1 Colour  
Observe the colour of the milk. If abnormal in colour, it should be held over for subjection to 
confirmatory tests.  
A.2.2 Odour and Taints  
Smell the milk in the container immediately after removing the lid. In case of foul or abnor mal smell, 
hold over the milk for subjection to confirmatory tests.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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©SWASA 201 4 - All rights reserved    5 Annex B   
(normative)   
 
Determination of insoluble matter  
 
B.1 Sediment test  
B.1.1 Sediment test on raw milk reveals the extent to which visible insoluble matter has gained entrance 
to the milk and the extent to which such material has not been removed from milk by single service 
strainers.  
The Sediment test represents a simple, rapid and quantitative measure of indicating the cleanliness of 
milk with respect to visibl e dirt.  
Allow a measured quantity of milk to pass through a fixed area of a filter disc and compare the 
sediment left with the prepared standards.  
B.1.2 Apparatus  
a) Sediment tester w ith filtering surface 24 mm in diameter.  
b) White lintine cotton discs , 32 mm i n diameter, expected filtration area 28 mm in diameter.  
c) Sampling dipper , of 500 ml capacity for sampling from milk cans or weighing vats.  
d) Standard sediment discs . Use commercially available standard sediment discs   
B.1.3 Procedure  
Take a milk sample from well -stirred cans or vats of milk with the sampling dipper. Measure the 
quantity used with reasonable accuracy. Filter the milk through a properly adjusted, firm lintine cotton 
disc (rough side facing milk) held in the sediment test er so that a filtration area of 28 mm is exposed. 
Compare the sediment disc with the prepared sediment standard discs and record the sediment score.  
B.1.4 Interpretation  
For the purpose of comparison, it is convenient to use about five prepared standard discs so as to classify 
the milk with respect to its sediment content in accordance with the specific requirements of the dairy 
or the milk collection depot. For the former, five discs showing 0.1, 0.2, 0.5, 1.0 and 2.0 mg may suffice. 
Under rural conditio ns, discs showing 0.0, 0.5, 2.0, 5.0 and 7.0 mg sediment may be more convenient 
to start with. In either case, no attempt shall be made to estimate the degree of sediment in milk in more 
than five classes, for example, Excellent, Good, Fair, Bad and Very B ad. No attempt shall be made to 
grade as sediment, any hair, flies, piece of hay or straw or any large particles of dirt. These shall be 
reported separately.  
The presence of appreciable sediment in unprocessed milk supplies indicates careless or insanitary  dairy 
farm practice. However, the lack of sediment is not always indicative of ideal conditions, since visible 
sediment may be readily removed by straining at the dairy farm.  
 
 
 
 
 
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6  ©SWASA 201 4 - All rights reserved  Annex C   
(informative)   
 
Determination of pH  
C.1 General  
The pH value or h ydrogen ion concentration gives a measure of the true acidity of milk. The relationship 
between pH and acidity of milk is only approximate. In normal cow milk the pH ranges from 6.6 to 6.8. 
The value is reduced by the development of acidity. On the other h and, the pH value of milk from a 
cow suffering from mastitis is alkaline in reaction, the value being over 7.0. The pH test is mainly used 
for the detection of abnormal mastitis in milk. The pH of milk may be determined rapidly by using the 
indicator strip s. 
C.2 Indicator strips  
Indicator paper strips or discs are made by soaking strips of absorbent paper in a suitable indicator and 
drying them.  
A rough estimate of pH is obtained by dipping a strip of the prepared paper in milk and observing the 
colour. Bro mocresol purple (pH range 5.2 to 6.8 colour changes from yellow to purple) and 
bromothymol blue (pH range 6.0 to 7.6 colour changes from straw yellow to bluish -green) are 
commonly used as indicators. Both narrow and wide range ready -made indicator papers a re available 
over the pH range 2.0 to 10.5.  
NOTE  Indicator paper strips shall always be kept in closed glass bottles and under dry conditions.  
C.3 Interpretation  
In normal milk the pH is well below 6.9. On an average, cow milk gives a pH of 6.6. Milk of pH  over 
6.9 should be regarded with suspicion as indication of some diseases of the udder or of late lactation 
milk.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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©SWASA 201 4 - All rights reserved    7 Annex D   
(normative)   
 
Clot-on-boiling (C.O.B.) test  
 
D.1 General  
This is a quick test  to determine developed acidity and the suitability of milk  for processing.  
D.2 Apparatus  
a) Test-tube: 15.6 cm x 1.9 cm , preferably with a mark at 5 ml.  
b) Water -bath. 
D.3 Procedure  
Transfer 5 ml of the sample to the test -tube and smell. Place the tube in a boiling water -bath and hold 
for about 5 min, and smell again for any acidic flavour. Remove the tube and rotate it in an almost 
horizontal position and examine the film of milk or side of the test -tube for any precipitated particles.  
The formation of clots is indicative of a positive test.  
D.4 Interpretation  
The principal features of the boiling test are speed and definiteness of results. Milk either remains 
unchanged or coagulates .  Milk, which gives a positive C.O.B. test, has acidity generally above 0.17 % 
(as lactic acid) and is not suitable for distribution as liquid milk or for processing.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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8  ©SWASA 201 4 - All rights reserved  Annex E   
(normative)   
 
Alcohol test  
 
E.1 General  
The alcohol test is used for rapid assessment of stability of milk to processing, particularly for 
condensing and sterilisation.  
The alcohol test is useful as an indication of the mineral balance of milk and not as much as an index 
of developed acidity. Th e test aids in detecting abnormal milk, such as colostrum, milk from animals in 
late lactation, milk from animals suffering from mastitis and which the mineral balance has been 
disturbed.  
E.2 Apparatus  
a)         Test-tubes ; 150 mm x 19 mm, preferably with  graduation marks at 5 ml and 10 ml.  
b)         Measure for alcohol ; 5 ml.  
E.3 Reagent  
Ethyl Alcohol; 68 % by weight or 75 % by volume (density 0.8675 g/ml at 27 C). 
E.4 Procedure  
Place 5 ml of milk in a test tube and add an equal quantity of alcohol. Mix the contents of the test tube 
by inverting several times.  
Note any flakes or clots. The presence of a flake or a clot denotes a positive test.  
E.5 Interpretation  
A negative test indicates low acidity and good heat stability of milk sample.  
Milk showing pos itive is not considered suitable for the manufacture of evaporated milk, which has to 
be sterilised to ensure its keeping quality.  
 
 
 
 
 
 
 
 
 
 
 
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©SWASA 201 4 - All rights reserved    9 Annex F   
(normative)   
 
Alizarin -alcohol test  
 
F.1 General  
This test is similar to the alcohol test and the incorporation of alizarin helps to indicate the approximate 
percentage of acidity.  
F.2 Apparatus  
Same apparatus as described in E.2. 
F.3 Reagent  
Alizarin Solution, 0.2 % in ethyl alcohol (6.8 % by weight or 75 % by volume, density 0.867 5 g/ml at 
27 OC). 
F.4 Procedure  
Place 5 ml of milk in a test -tube and add an equal quantity of the alizarin solution. Mix the contents of 
the test -tube by inverting several times. Note the colour of the mixture and presence of flakes or clots. 
Also note whether the flakes, if any, are small or large.  
F.5 Interpretation  
The general interpretation of the results is as indicated for the alcohol test ( see E.5 ). For acidity of 0.14 
% upwards, the graduation in size of the flakes and colour is approximately as follows:  
 
Colour  Size of flakes  Approximate acidity  
(% lactic acid)  
Lilac  --- Up to 0.14  
Pale red  --- 0.4 to 0.17  
Reddish -brown to brown       Small flakes  0.17 to 0.20  
Brownish -yellow  Large flakes  Over 0.20  
 
If acidity has not developed and yet coagulation occurs, it indicates the presence of rennet producing 
bacteria (sweet curdling). Milk from animals suffering from mastitis is alkaline in reaction and when 
mixed with alizarin -alcohol solution, violet or pur ple colour is produced. From the practical point of 
view, it is of little material difference whether milk clots through the production of acid or the 
production of rennin by bacteria as in either case it is unstable to heat.  
 
 
 
 SZNS 034:2014 
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10  ©SWASA 201 4 - All rights reserved  Annex G   
(normative)   
 
Ten-minute resazurin test  
 
G.1 General  
This test provides a rapid measure of the sanitary condition and keeping quality of milk. Resazurin 
reduction occurs in two stages, the first an irreversible change from the blue resazurin to the pink 
resorufin and the s econd a reversible change from the pink resorufin and the second a reversible change 
from the pink resorufin to the colourless dihyroresorufin. The first stage of reduction or colour change 
from blue to pink is fairly easily brought about so that the quali ty of milk is assessed in much shorter 
time. Taking advantage of the two -stage reduction, several procedures have been proposed for reading 
the end point of resazurin test.  
With fresh milk the observed change is resazurin reduction is due to the bacteria p resent and the 
leucocytes content. The reduction brought about by leucocytes, however, diminishes with the age of 
milk. Reduction can be assumed to be brought by the leucocytes if the colour in the down graded milk 
sample (for example, milk from animals su ffering from mastitis) remains unchanged for a longer time 
than observed normally.  
The test is intended as a platform test for detecting milk of poor keeping quality and shall be carried 
out on samples collected for bacteriological analysis.  
G.2 Apparatus  
a) Sterile test -tube without rim ,150 mm x 16 mm internal diameter 13.5 mm  0.5 mm accurately 
marked at 10 ml. If not used directly after sterilisation, they shall be kept in closed boxes protected from 
dust.  
b) Sterilised rubber stoppers , for closing the test tubes.  
The stoppers are sterilised by immersing in a boiling water bath for not less than 10 min.  
c)  Sterile 1 ml pipettes , straight -sided, blow -out delivery pipettes for measuring the dye solution ( see 
G.3) shall preferably comply with the following  specifications:  
 
Overall length  300 mm  
External diameter  7.5 mm to 8.5 mm  
Graduation  one mark only at 1 ml level  
Distance of graduation from tips  140 mm to 180 mm  
Internal diameter  2.3 mm to 3.0 mm  
 
The pipettes shall also be calibrated to deliver 1 ml of water at 27 C when the contents are blown out 
with the tip touching the side of the vessel, three seconds allowed for drainage and the accumulated 
drop then blown out. No pipette should have an erro r of more than  2 %, i.e. the amount delivered 
should be between 0.98 ml and 1.02 ml.  
 SZNS 034:2014 
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©SWASA 201 4 - All rights reserved    11 d) Sterile 10 ml pipettes , Straight -sided, blowout type.  
e) Sampling dippers , these shall be sterilised by keeping in boiling water for 30 min. Water shall be 
changed a t frequent intervals when a series of samples are to be examined.  
f) Pipette case of metal . 
g) Water -bath , Maintained at 37.5 C  0.5 C, fitted with a cover to exclude light and containing a 
metal rack designed to hold test tubes when immersed in water. The water -bath shall preferably be 
thermostatically controlled. The level of water in the bath shall be maintained above the level of the 
milk in the tubes. The interior of the bath shall be completely dark.  
h) Stop watch . 
i)  Hot air oven autoclave  or steam-steriliser . 
j) Wire baskets , for holding test tubes.  
k) Glass marking pencil.  
l) Bunsen burner or spirit lamp . 
m) Comparator with standard resazurin disc . The comparator may be provided with artificial 
daylight source of illumination.  
n) Sterile 50 ml measuring flask or cylinder . 
o) Glass distiller , for preparing distilled water.  
G.3 Reagents  
Sterile standard resazurin solution  
Prepare 0.05 % (W/V) stock solution of resazurin in glass distilled, sterilised water. Preserve in tightly 
stoppered amber -coloured bottle in a refrigerator. Prepare a 0.005 % bench solution by diluting with 
sterile water. It shall be prepared fresh after every 8 h. When actually not in use, keep it in a cool dark 
place.  
Resazurin powder shall conform to the following require ments:  
i) It shall contain sodium resazurate equivalent to 60%  3% resazurin;  
ii) apart from traces of sodium resocefate, no other dyestuff shall be present;  
iii) the remaining part shall consist of sodium carbonate and/or sodium acetate and moisture only;  
iv) it shall give a colourless water -clear solution on reduction in alkaline solution; and  
v) at a concentration of 1 to 220 000 in fresh normal mixed cow milk of 3.4 % fat, it shall give a 
tinto-meter disc reading of not less than 6.  
G.4 Procedure  
Start the test as soon as possible after a group of samples has been taken and at least within 30 minutes.  
Shake the sample container at least 25 times, each shake being an up and down movement with an 
excursion of about 30 cm, the whole process of shaking not e xceeding 12 seconds. After shaking, take 
10 ml for the test in the test tube. Place the tubes in numerical order, with the thumb and fingers of the 
left hand, taking care not to touch the mouth of the tube. Measure 1 ml of the resazurin solution with a 
sterile pipette, insert the pipette about half an inch into the mouth of the tube and expel the solution by 
blowing.  
Replace the stopper and invert the tube twice in 4 seconds and return to the rack. When resazurin has 
been added to a batch of not more than f ive tubes, place immediately in the water -bath and note the 
time. The delivery jet of the pipette shall not touch the milk in the tube. Any pipette becoming 
contaminated shall be immediately discarded. Use a fresh sterile pipette for every group of five sa mples.  SZNS 034:2014 
Edition 1  
 
 
12  ©SWASA 201 4 - All rights reserved  At the end of 10 minutes and 30 seconds, remove the tubes from the water -bath and immediately match 
the colour with the resazurin disc in the comparator, recording the results for the tubes in the right 
section. The comparator and stand are placed o n a bench at such a height that the operator is able to 
look down on the two apertures. The disc is then revolved until the sample is matched and the disc 
reading noted. When the colour falls between two disc numbers, it shall be recorded as the half value ; 
for example, a reading between 3 and 4 shall be recorded as 3.5. Tubes giving a reading between 0 and 
1, streaky pink or very pale pink are recorded as 0.5.  
NOTE It is an advantage for two persons to work in a team when a number of samples are to be take n rapidly, 
one to take the samples and the other to handle the containers and check the iden tity of the samples. Similarly, at 
the time of reading, one person to watch the tubes and another to record . 
G.5 Precautions  
The following precautions are necessary  to get consistent results:  
a)   All testers should be trained to correctly match the  colours  in the comparator.  
b)  The control and experimental test tubes shall be of the same type and thickness of glass.  
c)  Control samples used shall be from the same consignment as milk tested to compensate for the 
natural colour of milk.  
d)  Resazurin solution, milk, and milk to which resazurin has bee n added shall not be exposed to direct 
sunlight in the laboratory.  
e)   The water -bath shall be kept covered during the test.  
f)   The temperature of the water -bath shall be checked before commencing each batch of tests.  
G.6 Interpretation   
The results shall be interpreted as  follows:  
Disc reading  Keeping quality  
4 or higher  Satisfactory  
3.5 to 1   Doubtful  
4 or higher  Unsatisfactory  
 
 
 
 
 
 
 
 
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©SWASA 201 4 - All rights reserved    13 Annex H   
(normative)   
 
Half -hour methylene blue reduction (m.b.r.) test  
 
H.1 General  
The length of time taken by milk to decolourise  methylene blue is a fairly good measure of its bacterial 
content and hence its sanitary and keeping quality.  
H.2Apparatus   
Same as in G.2.  
H.3 Reagent  
Methylene blue solution   
Prepare a standard solution of methylene blue by dissolving one of the good quality methylene blue 
thiocyanate tablets in 200 ml of cold, sterile, glass -distilled water in a sterile flask. It is preferable to 
allow the mixture to stand for several hours to  ensure complete solution.  
Depending on the nature of methylene blue tablets used, sometimes the stock solution is further diluted 
with 800 ml of sterile glass distilled water A concentration of 1 part of methylene blue thiocyanate in 
300 000 parts of milk  is used to obtain satisfactory results. The solution shall be stored in a sterile glass -
stoppered amber -coloured bottle in a dark place and at no time exposed to light.  The solution remains 
stable in the dark for a considerable time but no stock solution  more than two months old shall be used.  
The amount of methylene blue required for a day's work shall be poured off from the stock bottle into 
a suitable glass container. On no account shall the pipette used for transferring the methylene blue 
solution to the tubes of milk be introduced into the stock bottle.  Moreover, if at any time during the 
filling of the tubes methylene blue solution should become contaminated with milk carried into it by a 
pipette, which has inadvertently come into contact with the m ilk, the methylene blue solution shall be 
immediately discarded and replaced by a fresh stock.  
H.4 Procedure  
Thoroughly mix the sample of the milk by inverting and shaking the sample bottle as described in 
Clause G.3 and then pour the milk in the test -tube up to the 10 ml mark.  
While doing this, remove the stopper or cap of the bottle under aseptic conditions, the pouring lip of 
the bottle and the mouth of the test -tube being flamed, and then pour the milk rapidly into the tube up 
to the 10 ml mark. While p ouring into the tube, take care to leave one side of the interior unwetted with 
milk. Add 1 ml of methylene blue solution to the tube from a pipette taking care that the pipette does 
not come into contact with any of the milk in the tube or with the wet si de of the interior of the tube. If 
this occurs, discard the pipette immediately. During delivery, hold the tip of the pipette against the dry 
side of the tube about 1 cm to 2 cm above the level of the milk and expel the methylene blue solution 
by blowing w ith the mouth or by means of a jet in the pipette. After the lapse of 3 seconds, blow out 
the solution remaining in the tip of the pipette and withdraw the pipette. Close the tube with sterile 
forceps or by the tips of the fingers on the extreme upper end.  On no account shall the fingers come into 
contact with the mouth of the test -tube or end of the stopper which comes into contact with the test -
tube.  Invert the tube slowly once or twice so that the whole column of contained air rises above the 
level of t he milk and then within 5 min, place the tube in the water -bath.  
Put up the following control tubes with each batch:  
i) 10 ml of mixed milk + 1 ml of tap water.  
ii) 10 ml of mixed milk + 1 ml of methylene blue solution.  SZNS 034:2014 
Edition 1  
 
 
14  ©SWASA 201 4 - All rights reserved  The milk for the control tubes shall  consist of a mixture of milk, preferably from several products, so 
as to have an average fat con tent and colour. Fit the control tubes at (i) and (ii) with stoppers and 
immerse for 3 minutes in boiling water in order to destroy the natural reducing syste m present in the 
milk.  
Comparison of the experimental tubes with control tube (ii) will show when decolourisation begins and 
comparison with control tube (i) will show when it is complete.  
Inspect the tube after 30 minutes. Regard the milk as decolourised when the whole column of milk is 
completely decolourised or is completely decolourised up to within 5 mm of the surface. If a trace of 
colour persists at the bottom of the tube and does not extend upwards for more than 5 mm, it may be 
ignored. Record the t ime at which decolourisation is observed. Where a tube is found not to be 
decolourised within 30 minutes, the sample conforms to the test.  
H.5 Precautions  
The following precautions shall be taken:  
a)  It is important that the methylene blue solution when n ot in use should be kept in the dark. It shall 
at no time be exposed to sunlight.  
b)  It is essential that the interior of the water -bath during the progress of the tests shall be completely 
dark since sunlight, diffused daylight and even artificial light catalyse the reduction of methylene 
blue.  
c) The sterilisation of the rubber stoppers for the test tubes and their subsequent satisfactory 
manipulation can be facilitated by employing a simple rack for holding a large number or rubber 
stoppers immersed in a suitable vessel of boiling water.  
d) The precautions against the contamination of the milk sample described in the method for carrying 
out the test shall be carefully observed.  
H.6 Interpretation  
The samples, which show complete decolourisation of blue c olour on incubation for 30 minutes or less, 
shall not be suitable for acceptance.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 SZNS 034:2014 
Edition 1  
 
©SWASA 201 4 - All rights reserved    15  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ICS 67.100 
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