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Whereas the Parliament of India has set out to provide a practical regime of right to
information for citizens to secure access to information under the control of public authorities,
in order to promote transparency and accountability in the working of every public authority,
and whereas the attached publication of the Bureau of Indian Standards is of particular interest
to the public, particularly disadvantaged communities and those engaged in the pursuit of
education and knowledge, the attached public safety standard is made available to promote the
timely dissemination of this information in an accurate manner to the public.
1 +, 1 +

01 ' 5

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Step Out From the Old to the New

Mazdoor Kisan Shakti Sangathan

Jawaharlal Nehru

IS 1167 (1965): Casein (Edible Quality) [FAD 19: Dairy

Products and Equipment]

! $ ' +-
Satyanarayan Gangaram Pitroda

Invent a New India Using Knowledge

! > 0 B

BharthariNtiatakam

Knowledge is such a treasure which cannot be stolen

IS : 1'17 1111

xxx
(~l"')
2010

Indian Standard
SPECIFICATION FOR
CASEIN (EDIBLE QUALITY)

( Revised)
Fourtb Reprint MARCH 2000

C Copyrl,,,, 1966
BVaEAtJ
MAtfAIt

Gr'

OF

aRAVAN.

INDIAN

STANDARDS

, BAHADUR SHAH
NEW DBUlI 110002

%APAR MAllO

May 1966

IS: 1167 - 1965

Indian Standard
SPECIFICATION FOR
CASEIN (EDIBLE QUALITY)

(Revised)
Dairy Industry Sectional Committee, AFDC 12
Chairman
I)R K. C. SE~

Re-presenting

Indian Dairy Science Association, Bangalore

~\1 embers

AGRICULTURAL M.~RKETJNG ADDirectorate of Marketing & Inspection (Ministry

VISER TO THE GOVERN!'IENT
of Food & Agriculture), Nagpur
OF I:SDI.a.
SURI V. CHANDRAMOCLY (Alternate)
SHRI B. R. BEDF.KAR
Hindustan Milkfood Manufacturers Limited. Nabha
SHRI I. C. I~. D.a.BSON (A lIernate)
SHRI C. Y. CUA~DR'" SEKHAR
T. T. (Private) Limited, Bangalore
SURI S. S. ~L\~I (Alternate)
SHRJ H. M. I)-,L.\Y
Kaira District Co-operative Milk Producers' Union

Ltd, Anand
D. CONTRACTOR (Alternate)
C. D. D ..\ STOOR
Larsen & Toubro Ltd, Bombay

J)R ].
SURI
SJlRI

H. W.

H.AMClIANIl.o\NJ

(Alternate)

IlR N. X. DASTl'R

DR C. P.

National Dairy Research

~\:'iA~T ..\KRISHN.~N

Institute,

Kamal

f.Altt'rnale)

I>IRECTOR
Directorate of Military Farms, Army Headquarters
.\SSIST.'NT Dmxcroa, ~fILITAKY FARMS (PLANNING) (Alternatl')
EXECUTIVE J-IEALTH OFFICER
Municipal Corporation, Bombay
~UNICIPAL ANALYST (Alternate)
COl" A. G. FER~.\~DES
Food Inspection Organization, Quartennaster
LT-COL

X. G. C.

IENGAR

SHRl (i. S. GoonoLE

General's Branch, Army Headquarters

(Alternate';
Dairy Development Commissioner, Government
of Maharashtra

SURI

OR

K. K.
SMRI

P. C.

COL

Y. v.

(A lternates
Ministry of Food & Agriculture
(Alternate)
Technical Standardization Committee. (Foodstufts)
(Ministry of Food & Agriculture)

SAl_PEKAR

IVA
GOPINATH
KHANNA

G.

S. S. PHATAK (Alternate)
A. R. A. KRISHNAN
Defence

DR
SHRI

SHRI

Production
Defence (DG I)]

K. P.

SINGII

Organization [Ministry of

(Alte,nate)

DR A. P. !\IAHADE\"AN
DR K. K. G. MENON

Hindustan Lever Ltd, Bombay

(Alte"nat~)

(Continue. on pag, 2)

BUREAU

OF

INDI-AN

STANDARDS

MANAK BHAVAN, 9 BAl-IADUR SHAH ZAFAR MARG

NEW DELl-II 110002

IS: u tJ7 ,. 1965

(C"rditlwd from pal' I)
M,mbers

Representing

MII=K COMMISSIONER
Milk Commissioner, Madras
SHRI P. VISWANATHA MENON (Alternate)
SHRI S. N. MITRA
Central Food Laboratory, Calcutta
SRRI B. K. MURTHY
Indian Aluminium Co Ltd, Calcutta
SHRI N. GOPAL KRISHNAN (Alt~rnale)
SHRI E. E. NAEGELI
Nestle's Products (India) Ltd, New Delhi
SRRI F.
RVAN (Alte,.nate)
SHRI
PADMANABHAN
The A. P. V. Engineering Co Ltd, Calcut~a
SRRI
G. BaO\\rN (A /lerna!e)
SRRI S. RAMASWAMY
Directorate General of Technical Development
SaRI
H. SHAH
Vulcan Trading Co Ltd, Bombay
SRRI A. JENSEN (Altentalt)
Centra) Committee for Food Standards (Ministry
R. S. SRIVASTAVA

J.

J.

J.

v.

na

DR

of Health)
SHRI P. JANARDANA AIVAR (Alter.tJQtt')
M. SWAMINATHAl
Central

Food Technological Research Institute

(CSIR), Mysore

SRR1 M. R. CH."-NDRASEKHARA (A 1I~,,,alt)

SRRI R. H. VARIAVA
Polson Limited, Bombay
SaRI B. P. PALKHIWALLA (Alternale)
D. I. S. VERMA
Dairy Technology Division,

National

Dairy

Research Institute,' l<amal

SRRI M.
JAMES

SaRI

R.
N.

SRINIVASAN (Alternate)
WAltNER
In personal

P. H. RAMANATHAN,
Assistant Director (Agri &
Food) (S,cyel4ry)

SHRI

capacity (AllaAabtJd
IHstilule. Allahabad)
Director, lSI (Ex-officio M~",be,)

.Agricultural

Dairy Products Subcommittee, AFDC 12:.2

na v.

COIItI,,,er

National Dairy Research Institute, Kamal

R. BRALltRAO
Me",lr,s
DR C. P. ANANTAKRlSHNAN
National Dairy Research Institute, Kamal
DR A. T. DVDANJ (A lIe"" ale)
Da J. D.CONTIlACTOR
Kaira District Co-operative Milk Producers'
Union Ltd, Anand
na 1. M. PATEL (Altt,nate)
COL A. G. FERNAND~S
Food Inspection Organization. Quartermaster
General's Branch Army Headcuarters
LT"'(.OL N. G. C. IENGAR (Alte,nate)
GOVERNMENT ANALYST
Government of Madras
DR K. K. IVA
Ministry of Food & Agriculture
SHal R. GOPALAN (.Al,.,."al~)
SKRI B. R. KAPUR
All India Ice . . Cream Manufacturers' Association,
New Delhi
SOl K. L. SIBAL (Alle,"al,)
DR ZAL R. KOTHAVALLA
In personal capacity (8 Ali Aslltlr Road,
DR A. P. MAHADBVAN
Hindustan Lever Ltd, Bombay
Da K. K. G. MENON (Alle'''tlle)
SR.l S. N. MITItA
Central Food Laboratory, Calcutta
Saal S. RAMASWAMY
Directorate General of Technical Development
S I JAMES N. WAltNER
In personal capacity (..4 UlJhAbatl Agri'tdt.. ral
1n,slitute, A IlalJablJtl)

B"",.'".e)

AMENDMENT NO. 1

DECEMBER 1989

TO

IS : 1167 - 1965 SPECIFICATION FOR CASEIN

( EDIBLE (tV ALITY )
[ Pal' 4, Table I, Sf No. ( ii ), col 3 ] - Substitute' 3-0 <for .
[ Page 4, Table 1, Sf No. ( vii ), col 3 ] -

(AFDC 34)

Substitute'

2"5 '.

6"0 for' 5-6

IS: 1167 - 1965

Indian Standard
SPECIFICATION FOR
CASEIN (EDIBLE QUALITY)

(Revised)
o.

FOREWORD

0.1 This Indian Standard (Revised) was adopted by the Indian Standards
Institution on 30 September 1965. after the draft finalized by the Dairy
Industry Sectional Committee had been approved by the .Agricultural and
Food Products Division Council.
0.2 Casein is the chief protein of milk and it exists as a colloidal suspension.
It is manufactured from wholesome skim milk by selective precipitation.
and removal of whey, subsequent washing and drying of the precipitate.
0.3 Since casein is a good protein nutritionally, it is used in the manufacture
of protein foods for oral administration in cases of protein malnutrition,
gastric disorders and other symptoms which call for protein supplementation.
0.4 The Indian Standards Institution has already published a specification
for- natural sour (lactic) casein 10r glue manufacture (IS: 850-1957) which
covers the material produced by natural sour (lactic acid) precipitation,
meant specially for glue manufacture. But there has been a long ielt need for
a standard to cover caseinwhich is used for manufacture of edible products.
0.5 The Indian Standard specification for casein (edible quality) (IS: 1167:1YS7) was published in 1957. The ne~~ for revising the standard was felt
because the original version covered material prepared bv acid precipitation
method only. 'The scope has now been enlarged to ~ embrace products
prepared by acid precipitation or natural souring. The limit for moisture
content has been increased so that the standard would be 'more realistic.
Further. additional requirements, such as total acidity, free acidity and
total bacterial count, have been introduced in this revision.
0.6 For the purpose of deciding whether a particular requirement of this
standard is complied with,- the final value, observed or calculated, expressing the result of a test or analysis, shall be rounded off in accordance with
IS: 2-1960. The number of significant places retained in the rounded off
value should be the same as that of the specified value in this standard.
Rules for rounding 011 numerical values (revised).

IS: 1167 -1965

I. SCOPE
1.1 This standard prescribes the require:nents and the methods of test
for casein of edible quality.
.

2. REQUIREMENTS
2.1 Casein shall be prepared from skim milk of either cow or buffalo or a
mixture of both. Casein shall be nearly white or pale cream in colour and
shall have no undesirable odour or any foreign matter (see Appendix L).
It shall be free from any added colour or preservative.

].] Particle Size - The size of the particles shall be such that 100 percent
by weight of casein shall pass through SOO-micron IS Sieve (see IS: 4601962).
NOTE - The apejture of BS Sieve 30 is within the limits laid down for the
specified IS test sieve and may, therefore, be used as SOO-micron IS Sieve.

2.3 HYl1lenlc Requirements - Casein shall be manufactured and stored

in premises built and maintained under hygienic conditions (see IS' 24911963t)
2.4 The material shall also comply with the requirements given in Table 1.
TABLE I

SL

REQUIREMENTS' FOR CASEIN (EDIBLE QUALITY)'

CHARACTERISTIC

REgUIREMEN'l

METHOD OF
TEST (REF TO
ApPENDIX)

(2)

(3)

(4)

No.
(1)

i)
ii)

iii)

iv)

Moistuft', percent by we-ight, M.~

Total asb, percent by weight (on dry
basis), Mllx
Acid insoluble ash, percent by weight (OD
dry basis) Max
Fat, percent by weight (on dry basis),
M(J~

v)
vi)

vii)
viii)

ix)
x)
xi)

Nitrogen, percent by weight (on dry

basis), Mitt
Total acidity in terms of ml of 01 N
NaOH/g
Free acidity in terms of ml of 0'1 N
NaOH/IO I, Max
Bacterial count, per gram, Max
Coliform count, per gram, MlJ~
Mould count, per gram. Max
. Rate of solubility

100
25

01

15

145

6 to 14

56

50000
10

SO

To conform to
test

Specification for test sieves (reflisetl) .

tCode for _nitary conditions for food processing units. (Since reviled).

F
G

IS: 1167 - 1965

3. PACKING AND MARKING
3.1 Packlna - The material shall be packed in such a way as to protect
it from contamination and deterioration.
recommended.

Polyethylene lined bags are

3.1.1 Unless otherwise agreed to between the purchaser and the vendor,
the packages shall be of 20 or 50 kg nett of material.
3.2 Marking - The following information shall be marked legibly and
indelibly on each container:
a) Name of the material;
b) Name and address of the manulacturer ;
c) Batch or code number; and
d) Net weight.
3.2.1 The product may also be marked with Standard mark.

3.1.1 The use of the Standard Mark is governed by the provisions of tbe
Bureau of Indian Standards Act, 1986 and tbe Rules and Regulations made
thereunder. The details of conditions under which the licence for the use of
Standard Mlrk may be granted to manufacmrers or producers may be obtained
from the Bureau of Indian Standards.

4. SAMPLING
4.1 Representative samples of the material shall be drawn and tested for
conformity to this standard as described in Appendix M.

5. TESTS
5.1 Tests shall be carried out as prescribed in the appropriate appendices
given in col .. of Table 1.

5.2 Quality of Reagents - Unless specified otherwise, pure chemicals

shall be employed in tests, and dist.illed water shall be used where the use
of water as a reagent is intended.
NOTE - ' Pure chemicals' shall mean chemicals that do not contain impurities
which affect the results of analysis.

18:1167-1965

APPENDIX A
[Table 1, Item (i)J
DETERMINATION OF MOISTURE
A-I. PROCEDURE
A-I.I Weigh accurately about 3 g of the material, powdered if necessary,
into a flat bottomed glass or aluminium dish (with a cover) previously dried
and weighed. Heat the dish containing the material (after uncovering)
in an electric oven maintained at 100 to 102C for about 3 hours. Cool in
a desiccator and weigh wit.h the cover OIl. Repeat t.he process of drying,
cooling and weighing at half hourly intervals, until the difference between
two consecutive wcighings is less than one milligram. Preserve the dish
containing this dried material in a desiccator for the determination of total

ash (B-l.1).

A-2. CALCULATION
_'

A-2.t Moisture, percent by weight =

where
"Vt

:::::

Ii' 2

=:

=-:.:

100(W1

W 2)

----~::.'JV-

weight in g of the dish with material before drying,

weight in g of the dish with material after drying, and
weight in g of the empty dish.

APPENDIX B
[Table 1, Item (ii)]
DETERMINATION OF TOTAL ASH
B-1. PROCEDURE

B-l.1 Heat gently the crucible containing the dried material (A-l.l) on
the flame and then in a muffle furnace at 550 20C till grey ash results.
Cool the crucible in a desiccator and weigh, Repeat the process of
heating in the muffle furnace for 30 minutes, cooling and weighing until
the difference lx-twr-r-n t wo consecu t ivc \Vt'ighings is less than 1 mg.
Note the lowest weight. Preserve the dish with the contents for the
determination of acid insoluble ash (C-2.1).
0

IS: ll67 - 1965

CALCUL~~TION

B-2.

. .. .
. ,'..
B-2.l Total ash, percent by we-ight (on dry basis)

:=..

1no (1 r 1

r)
fJ' J-----

.--

-'---ni-~2

where
l-f 1 == wviuht in g of the crucible containing the ash,

lr :.- weight

in g of t he crucible, and

JI' 2 =:::-: weight in g of the crucible with the dried material taKen
for the test.

APPENDIX C
[Table 1, I tem (iii) j
DETERMINATION OF ACID INSOLUBLE ASII

c-r.

REAGENT

C-l.1 Hydrochloric A id - approximatvlv 5 ;\

C-2. PROCEDURE
C-2.1 To the ash contained 111 t lu: porcelain dl:-,h {ll-l.l, add 25 ml hydrochloric acid, cover with a \\atch~la~s and heat on a water-bath Ior 1(\
minutes. Al low to coo] and filter the conu-nt- of t lu- dish through a Whatman filter paper No. 4! or its equivalent. Wash the filter with water until
the washings arc Iree from t hv acid. I~t'tllrtl the filt--r and t hr rtsidu h,
the dish. Keep it in an electric air-oven maintuiur-d at 135" :r'C for
about 3 hours. Ignite ill a muffle furnace at 550') 2(r'C for OBt' hour
Cool the dish in a (itsircator and \veigh. Repeat the proccs'-; of ignit ing in
the muffle furnace, cooling and \vl~jghin~ at half-hour intervals UHt il t lH'
difference between two successive \veighillg.:; is ll'~s 1hall one JllilJig-';trll.
Notfl the Iowest ,,eight.
C-3.

CAl,CUI~ATION

C-3.1 Acid insoluble ash, percent by weight (on dry

basis)

100 (IF 2 ._- l.Jt",)

-..--- nil-~-fl-;--

where

It 2

lr

:..::

:::~

IV! ;:-==

weight in g of the porcelain dish with the acid insoluble ash;

wr-ight in g of tilt' empt v purerlain dish. and
\\,(light ill g of t he porcelain dish with the dried material taken
for the determination of total ash (8-t.l).

IS: 1167 - 1965

APPENDIX D
[Table 1, Item (iv)J
DETERMINATION OF FA'r

n-i.

APPARATUS

D-l.l Fat Extraction Apparatus - conforming to IS: 2311-1963*.

D-2. REAGENTS

D-2.1 Hydrochloric Acid - density 1122 g/lnl at 20C.

D-l.2 Ethyl Ether
D-2.3 Petroleum Ether - redistilled slowly at a temperature below 65C.

D-3. PROCEDURE
D-3.1 Place 10 ml of hydrochloric acid in a tOO-nIl beaker.

Carefully
transfer to the beaker about 5 g of the material accurately weighed. Add
about 10 ml of the hydrochloric acid so as to wet and wash down any particles of the material that might. have adhered to the sides. Cautiously heat
the contents over a burner making sure that all particles of the material
adhering to the sides are dissolved in the acid. Finally boil for 10 minutes.
Cool the beaker until a temperature of approximately 20(: is reached.
Quantitativl~ly transfer the contents to a Mojonnier flask or a Rohrig tube
using about 10 ml of acid as wash liquid. Add 25 rnl of ethyl ether to the
beaker and transfer the contents of the Mojonnier flask or the ]-tohrig tube.
Stopper with cork or a stopper of synthetic rubber unaffected by usual
fat solvents, and shake vigorously for one minute. First add 25 rnl of petroleum ether to the beaker and then transfer it to the extraction flask or
tube. Repeat vigorous shaking. Centrifuge the Mojonnier flask at about
600 rotations per minute. If a Rohrig tube is used, let it stand until the
upper liquid is practically clear. Decant the ether solution into a suitable
flask or metal dish. Wash the lip and the stopper of extraction flask or
tube with a mixture of equal parts of both the solvents and add the washings t.o the weighing flask or metal dish. Repeat extraction of the liquid
remaining in flask or tube twice, using 15 1111 of each solvent each time,
Evaporate the solvent completely on a hot-plate or steam-bath at a temperature that does not cause spattering or bumping. Dry the fat in an
oven at the temperature of boiling water to constant weight. Weigh the
cooled flask or metal dish, using counterpoise duplicate container handled
in the same manner. Remove the fat completely from the container
with warm petroleum ether and weigh as before.
Spccification for fat extraction apparatus fur milk and milk products l Since

reviled).

IS: 1167 - 1965

D-4. CALCULATION
,
.
"
D-4.1 Fat, percent by weight (on dry basis)

lOOOO(W.-W.)

= --'ur-;--TiO(f=--m)-'

where

U'1 = weight in g of contents in flask or metal dish before removal of

fat,
W 2 = weight in g of contents in flask or metal dish after removal of fat,
Hl a ::-::: weight in g of the material taken for the test) and

m = percentage by weight of moisture in the material (A 2.1).

APPENDIX E
[Table I, Item (,,)]
DETERMINATION OF NITROGEN
E-I. APPARATUS
E-I.l A recommended apparatus, as assembled, is shown in Fig, 1.
E-I.I.I Description - The apparatus consists of a round bottomed
flask . 1 of 1 000 m l capacity fitted with a rubber stopper through which
pa.sses one end of the connecting bulbs tube B, The other end of the bulb
tube R is connect~~d to the cor~dcn~er ~ which ,is ~ttached. by ~neans of a
rubber tube to a dip tube D which dips into the liquid contained In a beaker
E of 250 ml capacity,
.

E-2. REAGENTS

E-2.1 Anhydrous Sodium Sulphate

E-2.2 Copper Sulphate
E-2.3 Concentrated Sulphuric Acid - sp gr 184.

E-2.4 Sodium Hydroxide Solution - Dissolve about 225 g of sodium

hydroxide in 500 ml of water.
E-2.5 Standard Sulphuric Acid -

approximately 01 N.

E-2.6 Methyl Red Indicator Solution - Dissolve one gram of methyl

red in 200Inl of rectified spirit (95 percent by volume).
E-2.7 Standard Sodium Hydroxide Solution - approximately 01 N.

IS: 1167 - 1965

FIG.

1 i\PPARATCS FOR

DETER:\II~ATIO~ OF NITROGEN

E-3. PROCEDURE

E-3.1 Transfer carefully 02 g of the. material. accurately weighed. to a

Kjeldahl flask. taking precaution to see that particles of the material do not
stick in the neck of the ftask. Add about 15 to 18 g of anhydrous sodium
sulphate. about 02 g of copper sulphate and 2S ml of concentrated sulphuric
acid. Place the flask in an inclined position. Heat below "the boiling
point of the acid until frothing ceases. Increase heat until the acid boils
vigorously and digest for 30 minutes after the mixture becomes clear and

10

IS: 1167 - 1965

pale green or colonrless. Wash down with the minimum quantity of
concentrated sulc.iuric acid, particles, if any, sticking to the sides and
continue digestion for 60 to 90 minutes. Cool the contents of the
flask. Transfer quantitatively, using water, to the round bottom flask
A and dilute to 250 ml. Add with shaking, a few pieces of pumice stone
to prevent bumping. Add about 60 ml of sodium hydroxide solution or
more, if necessary (to make the solution alkaline) carefully througa the
side of the flask so that it does not mix at once with the acid solution but
forms a layer below it. Assemble the apparatus as shown in Fig. 1, taking
care that the dip tube D extends below the surface of a known quantity of
standard sulphuric acid contained in the beaker E. Mix the contents of
the flask by shaking and distil until all ammonia has passed over into the
standard sulphuric acid. Detach flask A from the condenser and shut off
the burner. Rinse the condenser thoroughly with water into the beaker
E. Wash the dip tube D carefully so that all traces of the condensate
are transferred to the beaker. When all the washings have drained into
the beaker E. add two or three drops of methyl red indicator and titrate
with standard sodium hydroxide solution.

E-3.2 Carry out a blank using all reagents in the same quantities but
without the material to be tested.

E-4. CALCULATION
E-4.1 Nitrogen percent by weight -- ~i!!J1!
AJ. N
,
W (100 - m)
where
B = volume in ml of standard sodium hydroxide solution used to
neutralize the acid in the blank determination,
A = volume in ml of standard sodium hydroxide solution used to
neutralize the excess of acid in the test with the material,
N = normality of standard sodium hydroxide,
W == weight in g of the material taken for the test, and
m = percentage by weight. of moisture in the material (A-l.l).

APPENDIX F
[Table 1, Item (vi)]
DETERMINATION OF TOTAL ACIDITY
F-t. REAGENTS
F-l.l Standard Sodium Hydroxide Solution - 01 N.
F-l.2 Standard Sulphuric Acid - 01 N.

11

IS: 1167 - 1965

1'-1.3 Bromotbymol Blue Indicator Solution - 004 percent (tv/v)
in rectified spirit.
F-l. PROCEDURE
F-2.1 Transfer 2 g of the sample to a 2S0-ml Bask. Add 100 rnl of freshly
boiled and cooled distilled water and soak for 15 minutes. Add 50 ml of
01 N sodium hydroxide solution and close the mouth of the flask. Heat
in a water-bath at SOC and shake until the sample has been completely
dissolved. Titrate with standard sulphuric acid. using 1 ml of 004 percent
solution of bromothymol blue as indicator. Carry out the determination
omitting the casein and make a suitable correction. The acidity is expressed
as the number of millilitres of 01 N sodium hydroxide solution required to
neutralize 10 g of sample.

APPENDIX G
[Table 1, Item (vii)]
DETERMINATION OF FREE ACIDITY
G-I. REAGENTS
G-l.1 Standard Sodium Hydroxide Solution - 01 N.G-l.2 Phenolphtbalein Indicator Solution - prepared by dissolving
01 g of phenolphthalein in 100 ml of 60 percent rectified spirit
(se, IS: 2263-1962*).

c-a.

PROCEDURE

G-2.l Weigh 20 g of casein sample to within 01 g and transfer to a 500-ml

beaker containing 200 ml of boiling distilled water. Cover the beaker with
a watch-glass while allowing the mixture to stand for 30 minutes with
frequent agitation. Filter the aqueous layer and pipette 50 ml of the cold
filtrate (corresponding to 5 g of casein) into a flask. Titrate with standard
sodium hydroxide solution using phenolphthalein indicator. The free
acidity is expressed as the number of rnillilitres of Ot N sod ium hydroxide
solution required to neutralize 10 g of sample.
-.Methods of preparation of indicator solutions for volumetric a nalysis,

12

IS: 116' - 1965

APPENDIX H
[Table 1, Item (viiij]
DETERMINATION OF BACTERIAL COUNT
9-1. APPARATUS

R-l.l Weiahina Scoop. Str.rtle - with count.er weight.

H-l.2 Bacterlologtcal Tr msfer Pipettes, Sterile -- accurately graduated, with cotton plug in t he upper orifice.

H-I.3 Dilution Bottles. SterIle - made of heat resistant glass (preferably borosilicate glass) of the following capacities:
a) 150 ml, with mark at 99 mllevel ; and
b) 25 rnl, with mark at 9 mllevel.
H-I.4 Petri Dishes - with outside dish diameter 100 mrn, inside dish
diameter 91 mm, and depth 15 mm. The exterior and interior surfaces of
the bottoms should be flat and free from bubbles, scratches or other defects.
H-I.5 Bacteriological Tubes, Sterile - 2S ml capacity with a mark
at the 10 ml level, with cot ton plugs.

B-2. REAGENTS
8-2.1 Dilution Waters - There shall be two types of dilut ion water,
namely, Dilution Water 1 for usc with Medium 1 (9-2.2.1) and Dilution
Water 2 for usc with Medium 2 (H-2.2.2). Sterilize dilution, water at
121C for 15 minutes before use.
H-2.1.1 Dilution Water 1 - Dissolve 34 g of potassium dihydrogen.
phosphate (KIl zP0 4 ) in 500 ml of distilled water. adjust to pH 7-2 with 1 N
sodium hydroxide solution and make up to one lit re wit h distilled water.
Dilute 125 ml offhis stock phosphate buffer solution with water to one
litre to obtain dilution water.

8-2.1.2 Dilution Wliter 2 - shall have t.he following composition :

Sodium chloride

9 g

Potassium chloride
Calcium chloride

042 g
024 g

Sodium bicarbonate
Distilled water

02 g
1 000 rnl

H-2.2 Me.dia - Either of the two media gIven

shall be used.

13

III

8-2.2.1 and H-2.2.2

Is: 116' -1965

8-2.2.1 Medium 1 - shall have the following composition:
5-0 g
2-5 g
10 g
200 g
I 000 lui

Peptone
Yeast extract
Glucose (dextrose)
Agar bacteriological grade
Distilled wat.er
I

8-2.2.1.1 Preparation - Soak the materials (B-2.2.1) for 3 to 5

minutes in cold water, then bring the mixture into complete solution with
minimum delay by boiling above an asbestos centred wire gauze over a
flame. Stir continuously and efficiently to avoid charring. Adjust the
solution to pIl 70 at SOt"lC with sodium hydroxide solution. Filter through
cotton pad till clear filtrate is obtained. Fill into bacteriological tubes to
10-1uIIuark. Sterilize in an autoclave at 121C for 20 minutes.
"-2.2.2 M edium 2 - shall have the following composition:
Yea~rcl
3 g
Peptone
5 g
Agar, bacteriological grade
Fresh whole milk
Distilled water

20 g
10 ml
1000 ml

8-2.2.2.1 Preparation - Dissolve the yeastrel and the peptone in

distilled water in the steamer and adjust the reaction at room temperature
to pH 74 using phenol red as the indicator. Wrap shreded agar in muslin
and wash in running cold water for 15 minutes, Squeeze out the excess
cold water and add the agar together with the freshly shaken milk to the
broth. Autoclave at 121(: for 20 minutes and filter through paper pulp
(see Note) in a Buchner funnel. (Egg should not be used for clearing.)
Take the agar directly from the autoclave and filter hot, the whole apparatus being kept warm by a surrounding atmosphere of steam, Test the
reaction of the filtrate at 50C and adjust, if necessary, to pH 7,0, so that
the final reaction at room temperature is pH 72.
Fill this medium into bacteriological tubes to Ifl-ml mark.
in an autoclave at 121C for 20 minutes,

Sterilize

NOTE - The paper pulp is prepared by pulping small pieces of postlip or white
heather paper in water by means of a pestle and mortar. A single layer of Chardin
filter paper should be laid on top of the Buchner funnel to prevent the pulp being
sucked through, and t.he pulp itself should then be packed down evenly on top
of it. The filter when ready for use, should have a total depth of about l'S rom.

"-3. PROCEDURE
0-3.1 Dilution - Weigh 11 g of the material from the sample marked
For Bacteriological Tests' (M-3.1) using a sterile spatula and suspend
in 99 1111 of dilution water at 45C. Agitate mildly, soak for 1 to 3 minutes
and then agitate vigorously to avoid churning out the fat. Prepare

14

IS: 1167 - 1965

dilutions of this so as to give bet wor-n 30 and .lOU colonies per plate
(see H-3.~). Add one millilit.rc of suitable dilutions in t riplicate to the
sterile petri dishes. Use at least 5 dilutions.
8-3.2 Pouring Plates - Melt agar medium (H-2.2) in bacteriological
tubes and keep at 48 to 50C. Introduce this medium aseptically, at
42 to 44C, into the petri dishea and mix by rotating and tilting dishes
without spreading over the edges. Spread the mixture evenly over the
bottom of the plate. Allow to so1i(lify.

8-3.3 Incubation - Invert the plates and incubate at 37C for 48 hours.
H-3.4 Counting - Count the colonies with the aid of magnification under
uniform and properly controlled illumination. Count only those plates
with 30 to 300 colonies, including pin point size colonies.
.

8-3.5 Computation - Compute the bacterial count per gram from the
dilutions used.
NOTE - All precautions shall be observed to prevent microbiological contamination throughout the test.

APPENDIX J
[Table 1) Item (ix)]
DETERl\fINATION OF COLIFORM COUNT

.r-i. GENERAL
J -1.1 Coliform Bacteria - Coliform bacteria includes all aerobic and
facultative anaerobic, Gram-negative. nonspore --- forming bacteria which

ferment lactose with the production of acid and gas. A positive presumptive
test is indicated by the formation of acid and gas within 48 hours at 35 to
37C in a fermentation tube containing lactose bile salt broth, Alternatively, the developmen t of dark red colon ies at least O'5 mm in diameter in a
solid medium (violet and rr-d bile agar) within 20 to 24 hours at 35 to 37C
may be considered as a positive evidence of the presence of coliform bacteria.
Violet red bile agar is one of t he standard media used for determination
of general types of coliform organisms including those of faecal origin in
water.. milk and other materials of sanitary importance.

J-l. APPARATUS
J-2.1 Weighing Scoop, Sterile - with counter weight.
J-2.2 Bacteriological Transfer Pipettes, Sterile - accurately graduated, with cotton plug in the upper orifice.

15

IS: 1167 - 1965

J-l.3 Dilution Bottles, Sterile - made of heat-resistant glass (preferably borosilicate glass) closed with rubber stoppers, preferably screw caps
with new friction-fit liners for making them leak-proof and of the following
capacities:
a) 1SO ml, with mark at 99 mllevel ; and
b) 2S ml, with mark at 9 mllevel.
J-2.4 Petri Dishes -\vith outside dish diameter 100 mm, inside dish
diameter 91 mm, and depth 15 mm, The exterior and interior surfaces of
the bottom should be flat and free from bubbles, scratches or other defects,
which would interfere with counting of colonies.

J-l.5 Baeterioloaical Tubes, Sterile - 25 ml capacity with a mark at

the lO-mllevel, with cotton plugs.

..

J-2.& Tubes having Inverted Vials - Durham tubes.

J-3. REAGENTS

J-3.1 Dilution Water - Dissolve 34 g of potassium dihydrogen phosphate

(KH.PO,) in 500 ml of distilled water. adjust to pH 7-4 with 1 N sodium
hydroxide solution and make up to one litre with distilled water. Dilute
125 ml of this stock phosphate buffer solution with distilled water to one
litre to obtain dilution water.
J-3.2 Medium - Violet red bile agar shall be the medium, the composition
of which shall be as follows:
Yest extract
30 ~
Peptone
70 g
Sodium taurocholate
15 g
Lactose
100 g
Sodium chloride
5-0 g
Agar-agar
200 g
Indicator:
Neutral red
Crystal violet
Water
Final pH

0-03 g
0002 g
1 000 ml
74Ot

J-3.2.1 Preparation and Sterilization of Medium. - Soak the materials (J-3.2) for 3 to 5 minutes in cold water, then bring the mixture into
complete solution with minimum delay by boiling above an asbestos-centred
wire gauze, over a flame. Stir continuously and efficiently to avoid charring. Adjust the solution to pH 74O-t at 50C with sodium hydroxide
solution. Filter through cotton pad till clear filtrate is obtained. Fill
the filtrate into bacteriological tubes to to-rot mark. Sterilize in an
autoclave at 121C for 15 minutes.
16

IS: I1b1 - 1965

J-4. PROCEDURE
J -4.1 Dilution - Using aseptic precautions and appropriate means
thoroughly.mix each sample until representative portions may be removed,
Heat 99 ml of sterile 2 percent sodium citrate solution into a sterile container (preferably 150 ml wide-mouth screw-cap jars) to 45C for dispersing
and diluting casein. Promptly add 11 g of casein and triturate it with
sterile glass rod until thoroughly mixed. Allow the mixture to cool at room
temperature. Prepare suitable dilutions of this and add one millilitre of
suitable dilutions in triplicate to the sterile petri dishes.

J-4.2 Pouring Plates - Melt the medium (J-3.2.1) in bacteriological

tubes and keep at 48 to 50C. Int.roduce this medium aseptically at 42
to 44C into the petri dishes and mix by rotating and tilting dishes without
spreading over the edges. Spread the mixture evenly over the bottom of
the plate. Allow to solidify, After solidification of medium in plate, add
cover layer of medium.

J ..4.3 Incubation - Invert t.he plates and incubate at 35 to 37C for

24 hours.

J-4.4 Counting - Count the dark red colonies which have a diameter of
05 rom or over.
J-4.5 Computation - Compute the coliform count per gram from the
dilutions used (J-4.1).
NOTE t - 1n case of doubt regarding the colonies developed 011 violet red
bile agar, representative colonies are picked and transferred to lactose hile salt
broth in tubes hav i ng inverted vials. Production of acid and gas is confirmatory
for coliform organisms.
NOTE 2 .._.~ 1\11 precautions shall be observed to prevent microbiological contamination throughout the test.

APPENDIX K
[Table 1, Item (x)J
DETERMINATION OF MOULIl COUNT

x-i.

GENERAL

K-l.l Mould -l-Iigh mould count is not desirable and indicates that
casein is manufactured under improper plant sanitary control. It is also
a result of improper packing and faulty storagt'. The develupment of
colonies in a solid medium (potato-dextrose-agar at pH 35) within 5 days
at 22(. should be considered as a positive evidence of moulds.

17

IS: 1161- lCJ65

K-l. APPARATUS

K-l.1 Weighing Scoop, Sterile - with counter weight.

K-2.1 Bacterlolol1lcal Transfer Pipettes, Sterile - accurately graduated, with cotton plug in the upper orifice.
K-2.3 Dilution Bottles, Sterile - made of heat-resistant glass (preferably
borosilicate glass) closed with rubber stoppers, preferably screw caps with
new friction-fit liners for making them leak-proof and of the following
capacities:
a) 150 ml, with mark at 99 mllevel; and
b) 25 ml, with mark at 9 mlleveI.

K-2.4 Petri Dishes - with outside dish diameter 100 mm, inside dish
diameter 91 rnm, and depth 15 mm, The exterior and interior surfaces of
the hottom should be flat and free from bubbles, scratches or other defects,
which would interfere with counting of colonies.
K-l.5 Bactertologlcal Tubes, Sterile - 25 ml capacity with a mark at
10 mllevel, with cotton plugs.
K-3. REAGENTS

K-3.1 Dilution Water - Dissolve 34 g of potassium dihydrogen phosphate

(KH 1P04 ) in 500 rnl of distilled water, adjust to pH 74 with 1 N sodium

hydroxide solution and make up to one litre with distilled water. Dilute
125 ml of this stock phosphate buffer solution with distilled water to one
lit.re to obtain dilution water.

K-3.2 Medium - Pot.ato glucose agar (acidified) shall be the medium,

the composition of which shall be as follows:
Potato infusion
From 200 g of white potatoes (see Note)
Agar
15 g
Glucose
20 g
Distilled Water
1 000 rnl
NOTE - Suspend 200 g of sliced potatoes in distilled water sufficient to cover
the material. Boil for one hour and filter through cheese cloth.

K-3.2.1 Preparation and ..~te,ilization of Medium - Heat the mixture (K-3.2) to boiling to dissolve the ingredients. Distribute into tubes
or flasks and autoclave for 15 minutes at 121e. Melt in flowing steam
or boiling water, and cool. Adjust reaction in each container to pH 3SOt
with sterile 10 percent tartaric or lactic acid. As remelting of acidified
medium may destroy its solidifying properties, adjust only amount needed
for immediate plating. Amount of acid required for adjustment in any
one flask of same batch of medium ordinarily will establish amount needed
in each of the other flask containing equal quantities.

18

IS: t 16'1 - 19t-5

For colorimetric adjustment, use bromophenol blue and titrate 5 m1

of medium with dilute acid prepared by adding 1 ml of sterile 10 percent

stock solution to 19 rnl of water. The number of millilitres of dilute acid
used to titrate to pH 35 will represent the amount of stock solution that
shall be added to 100 ml of medium.

K-4. PROCEDURE
K-4.1 Dilution - Using aseptic precautions and appropriate means,
thoroughly mix each sample until representative portions may be removed,
Heat lJY ml of sterile 2 percent sodium citrate solution into a sterile container
(preferably 150 111 I wide-mouth screw-cap jars) to 45(: for dispersing and
diluting cast-in, Prompt ly a<1(1 1 t g of cast-in and triturate it with sterile
glass nul until thoroughly mixed. Allow the mixture to cool at room tC111perature, Prepare suitable dilutions of this and add one millilitre of
suitable dilutions in triplicate to the sterile petri dishes.

K-4.2 Pouring Plates --1'{(-1t the medium (K-3.2.1) in bacteriological

tubes and keep at 4X') to 50('C. J ntroduce this medium aseptically at 42 0
to 44"( into the petri dishes and mix by rotating and tilting dishes without
spreading over the edges. Spread the mixture evenly over the hottom
of the plate. ....\110\\ to solidify. After solidification of the medium in
plate, add cover layer of medium.

K-4.3 Incubation - Invert the plates and incubate at 22C for 5 days.
K-4.4 CountinQ- If moulds are developing in large numbers, count plates
on the third day of incubation and then recount on the fifth day.
K-4.5 Computation - Compute the mould count per gram from the

dilutions used (see K-4.1).

APPENDIX L
[1"able 1 ItC11t (xi)]
J

DETERMINATION OF RATE OF SOI4UBILITY

t-r.

APPARATUS

L-l.1 Glass Stirring Rods -- wit tl flat tr-ued ends.

L-I.2 Centrffuge Tubes-(~nni('al, apprux imatr-lv 170 mm long and
25 1l1n1 diametr-r , graduah-d at 50 ml and at 01 ml intervals- between 0
and 1 InI and at 02 III 1 intervals between 1 and 3 rnl.
19

IS:

11" - 1965

L-l.3 Centrlfuae -:- with swing-out buckets and, capable of a speed to

give approximately 900revolutions per minute.
L-2. REAGENTS
L-].l Borax Solution - dissolve 20 g of sodium tetraborate (Na.B,O"
10B.O) in water and dilute to 1 000 mi.
L-].] Water - pH 60 at 20C.
L-2.3 Sodium Hydroxide Solution - 1-0 N_

L-3. PROCEDURE
L-3.1 Weigh 6 g of the casein sample in a test-tube containing a glass
stirring rod. Add 36 ml of borax solution in the test-tube, stir and mix
the contents with the rod. Add a volume of 0-1 N sodium hydroxide solution equivalent to the Free acidity J, (see 0-1.1). Heat the tube in a
water-bath at 70 1C with continuous mixing of the contents for the
first 5 minutes, and then at an interval of every 5 minutes for a period of
40 minutes. If the casein dissolves completely, note the time of complete
solution. If after 4S minutes the casein is not completely dissolved, transfer
the contents of the test-tube to a centrifuge tube, wash the test tube with
water at 70C and collect the washings until the volume in the centrifuge
tube becomes 50 ml. Centrifuge at a speed of about 900 rev/min for 10
minutes. Decant the supernatant liquid, including any light material
which may be present above the compact, lower layer of sediment. Add
40 ml of cold water, mix and centrifuge again for 10 minutes at 900 rev/min.
Read the volume of sediment excluding any light material above the compact
layer.

L-4. REPORT
L-4.1 For conformity, the casein sample shall not have sediment which is
indication of the presence of insoluble material.

APPENDIX M
(Clause 4.1)
SAMPLING OF EDIBLE CASEIN
M-l. GENERAL REQUIREMENTS OF SAMPLING
M-l.O In drawing, preparing storing and handling samples the following
precautions and protections shall be observed.

20

IS: 1167 - 1965

M-I.l Samples shall be taken in a protected place not exposed to damp
air, dust or soot.
M-l.2 The sampling instrument shall be clean and dry when used. When
taking samples for bacteriological examination, it shall also be sterile.
M-l.3 The samples, the material being sampled, the sampling instrument
and the containers for sampling shall be protected from adventitious contamination.
M-l.4 The samples shall be placed in clean and dry glass containers. The
sample containers shall be of such a size that they are almost completely
filled by the sample. The sample containers shall, in addition, be sterile
when they are used for samples for bacteriological examination.
M-l.!5 Each sample container shall be sealed air-tight after filling and
marked with full details of sampling, batch or code number, name of the
manufacturer and other important particulars of the lot.
M-I.6 The samples shall be stored in such a manner that the temperature
of the material does not vary unduly from the normal temperature and
that they are protected from light.
M-l.7 Sampling shall be done by a person agreed to between the purchaser
and the vendor and in the presence of the purchaser (or his representative)
and the vendor (or his representative).

M-2. SCALE OF SAMPLING

M-2.1 Lot - In a single consignment all the containers containing the
material of the same grade and the same batch of manufacture shall constitute a lot.
M-].2 Sample shall be tested for each lot for ascertaining the conformity
of the material for the requirements of this specification.

M-l.3 The number of containers to be selected from a Jot shall depend on

the size of the lot and shall be in accordance with col 1 and 2 of Table 2.
M-l.4 These containers shall be chosen at random and for this purpose
a random number table as agreed to between the purchaser and the vendor
shall be used. In case such a table is not available the following procedure
shall be adopted:
Starting from any container, count all the containers in one order
as 1,2,3-,
up to r and so on. Every rth container so counted shall
be withdrawn, where, is the integral part of N], N being the number
of containers in the lot and n the number of containers to be selected.
M-3. PREPARATION OF SAMPLES
M-3.1 Individual Samples for Baetertologlcal Examination - Draw
with in appropriate sampling instrument, which is sterile, about 300 g

21

IS: 1167 - 1965

TABLE 1

NUMBER OF CONTAINERS TO BE SELECTED

FOR SAMPLING
(CIIJ"s, M-2.3)

NUMBER OF

NUMBER OF
CONTAINERS TO
BE SELECTED

CONTAINEas IN

THB LOT

1
2 to

9"
26 II

25
50

51 ,.
301

300
500

II

1
2

3
4
5
6
8

501 and over

of the material from each selected container by taking small portions of

the material from at least 10 different parts of the container. Thoroughly
mix all Portions of the material drawn from the same container. Take
about 100 g of the material and divide this material into three equal
parts taking care not 10 bring any bacterial contamination. Each part
so obtained shall c-onstitute an individual sample representing the consignment and shan be transferred to sterile glass containers which shall be
sealed air-tight and labelled ,..."ith the particulars described in M-I.5 and
the words For Bacteriological Tests'. The individual samples so obtained
shall be divided into three sets in such a "ray that each set has a sample
representing each selected container. One of these sets shall be marked
for the purchaser, another for the vendor and the third for the' referee.
f

M-3.2 Composite Samples for Other Tests - From the mixed material
from each selected container remaining after the individual sample has
been taken, equal quantities of the material from each container, shall be
taken and mixed together so as to give material representative of the lot
and weighing not less than 200 g. This material shall be divided into three
equal parts. Each part so obtained shall constitute a composite sample
representing the lot and shall be transferred to a clean, dry container made
of glass or tinplate and labelled with the particulars given in M-l.5. One
of these composite samples shall be for the purchaser, another tor th~ vendor
and the third for the referee.

M-3.3 Referee Samples - Referee samples shall consist of a set of

individual samples (M-3.1) and a composite sample (M-3.2) marked for
this purpose and shall bear the seals of the purchaser and the vendor. These
shall be kept at a place and under conditions agreed to between the purchaser and the vendor.

22

IS: 1167 - 1965

M-4. NUMBER OF TESTS

M-4.1 Tests for the determination of bacterial count shall he conducted
separately on each of the samples constituting a set of individual samples
(M-3.1).

M-4.2 Tests for the determination of t 11(~ remaining characteristics shall

be conducted on the composite sample (M-3.2).

M-S. CRITERIA FOR CONFORMITY

M-5.1 The lot shall btl considered as conforming to the requirement of this
specification when :
a) each of the test results for bacterial count sat.sty the requirements
specified in Table 1 (Sl No. viii) and
b) all the test results on the composite sample satisfy the respective
requirements as specified in this specification.
I

23

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