The
three
under :
(
a) Nutrient Broth,
(
b) Cooked Meat Medium and Thioglycollate Medium, and
(
c) Sabouraud Medium.
These methods shall now be treated individually in the sections that follows :
Nutrient Broth
Importantly, it is exclusively suitable for the
‘aerobic microorganisms’.
Oxidation-reduction potential (Eh) value of this medium happens to be quite high to enablethe growth of the anaerobes specifically.
Importantly, such culture media that particularly allow the growth of festidious microorganisms,
such as :
soyabean casein digest broth, Hartley’s digest broth.*
8.2.2.2. Cooked Meat Medium and Thioglycollate Medium
These
two different types of media are discussed briefly as under :
(
a) Cooked Meat Medium : It is specifically suited for the cultivation (growth) of clostridia**.
(
b) Thioglycollate Medium : It is particularly suited for the growth of anaerobic microbes. It
essentially comprises of the following ingredients, namely :
Glucose and Sodium thioglycollate—
that invariably serve as :
an inactivator of mercury compounds,
to augment and promote reducing parameters, and
an oxidation-reduction indicator.
Agar
—to cause reduction of the ensuing ‘convection currents’.
Sabouraud Medium
It is a medium specifically meant for
fungal species. It essentially bears two vital and important
characteristic features, such as :
an acidic medium, and
contains a rapidly fermentable carbohydrate e.g., glucose or maltose.
Note : (1) All the three aforesaid media must be previously assessed adequately for their nutritive
characteristic features
i.e., in fertility tests to ascertain the growth of specified microorganisms.
(2) Duly incubated at the stipulated temperature(s).
The
direct inoculation method shall now be dealt with in a sequential manner under the following
three
categories, such as :
Quantities of sample to be employed,
Method of test, and
Observation and Interpretation of Results.
Quantities of Sample to be used :
In actual practice, the precise quantum of the substance or
pharmaceutical preparation
under investigation, that is required to be used for inoculation in the
respective culture media
usually varies justifiably as per the amount present in each particular container,
and is stated clearly in Table : 8.2 together with the exact volume of the culture medium to be
employed.
Method of Test :
The ‘method of test’ varies according to the substance to be examined, for
instance :
(
a) Aqueous Solutions and Suspensions : The actual tests for microbial contamination are
invariably performed on the same sample of the preparation under investigation by making use of the
above-stated media (Section 2.2.1 through 2.2.3). In certain specific instance when the amount present
in a single container is quite insufficient to carry out the stipulated
‘tests’, the combined contents of
either two or mroe containers may be employed to inoculate the above-stated media.
Methodology :
The various sequential steps involved are as given under :
(1) Liquid from the
‘test containers’ must be removed carefully with a sterile pipette or with a
sterile syringe or a needle.
(2) Transfer aseptically the requistite prescribed volume of the substance from each container to
a vessel of the culture medium.
(3) Mix the liquid with the medium carefully taking care not to aerate excessively.
(4) Incubate the
‘inoculated media’ for not less than 14 days (unless otherwise specifically
mentioned in the
monograph*) at : 30–35°C for ‘Fluid Thioglycollate Medium’, and 20–25°C for
‘Soyabean-Casein Digest Medium’.
Special Points :
The following special points may be noted meticulously :
(
i) In case, the substance under investigation renders the culture medium turbid whereby the
presence or absence of the actual
microbial growth may not be determined conveniently and readily by
sheer
‘visual examination’, it is always advisable and recommended that a suitable transfer of a certain
portion of the medium to other fresh vessels of the
same medium between the 3rd and 7th days after the
said test actually commenced.
(
ii) Subsequently, continue the incubation of the said ‘transfer vessels’ for not less than 7 additional
days
after the transfer, and for a total of not less than 14 days.
(
b) Oils and Oily Solutions : For carrying out the required tests for the bacterial contamination
of
oils and oily solutions it is recommended to make use of culture media to which have been incorporated
duly :
Octylphenoxy polyethoxyethanol (I) : 0.1% (w/v) [or Octoxynol]
The required test must be carried out as already described under Section (
a) above i.e., Aqueous
Solutions and Suspensions.
Precautionary Measures :
The following two precautionary measures should be taken
adequately :
(
i) Cultures essentially comprising of ‘oily preparations’ should be shaken gently every day.
(
ii) Importantly, when one employs the fluid thioglycollate medium for the ultimate detection of
the
anaerobic microorganisms, shaking or mixing must be restricted to a bear minimum level so as to
maintain
perfect anaerobic experimental parameters.
(
c) Ointments : The following steps may be adopted in a sequential manner :
(1) Carefully prepare the
‘test sample’ by diluting ten times in a sterile diluent, for instance :
Fluid B
or any other suitable aqueous vehicle which is capable of dispersing the test material homogeneously
throughout the
‘fluid mixture’.*
(2) Mix 10 mL of the
fluid mixture thus obtained with 80 mL of the medium, and subsequently
proceed as per the method given under Section (
a) i.e., Aqueous Solutions and Suspensions.
(
d) Solids : The various steps involved are as stated under :
(1) Transfer carefully the requisite amount of the preparation under examination to the quantity
of culture medium as specified in Table : 8.3, and mix thoroughly.
(2) Incubate the inoculated media for not less than 14 days, unless otherwise mentioned in the
monograph at 30–35°C in the particular instance of
fluid thioglycollate medium, and at 20–25°C in the
specific case of
soyabean-casein digest medium.
(
e) Sterile Devices : For articles of such size and shape as allow the complete immersion in not
more than 1 L of the culture medium test the intact article, using the suitable media ; and incubating as
stated under Section (
a) i.e., Aqueous Solutions and Suspensions.
(
f) Transfusion or Infusion Assemblies : For transfusion or infusion assemblies or where the
size of an item almost renders immersion impracticable, and exclusively the
‘liquid pathway’ should be
sterile by all means, flush carefully the lumen of each of
twenty units with a sufficient quantum of fluid
thioglycollate medium
and the lumen of each of 20 units with a sufficient quantum of soyabeancasein
digest medium
to give an ultimate recovery of not less than 15 mL of each medium. Finally,
incubate with not less than 100 mL of each of the two media as prescribed under Section (
a) i.e., Aqueous
Solutions and Suspensions.
Exception :
Such ‘medical devices’ wherein the lumen is so small such that fluid thioglycollate
medium
will not pass through easily, appropriately substitute alternative thioglycollate medium
instead of the usual
fluid thioglycollate medium and incubate that duly inoculated medium anaerobically.
Note : In such situations where the presence of the specimen under examination, in the culture
medium critically interferes with the test by virtue of the ensuing bacteriostatic or fungistatic
action, rinse the article thoroughly with the bare minimum quantum of
fluid A. Finally recover
the rinsed fluid and carry out the ‘test’ as stated under ‘Membrane Filtration’ for Sterile
Devices.
Observation and Interpretation of Results :
In the case of ‘direct inoculation’ the various
observation and interpretation of results
may be accomplished by taking into consideration the following
cardinal factors,
such as :
(1) Both at intervals
during the incubation period, and at its completion, the media may be
examined thoroughly for the critical
macroscopic evidence of the bacterial growth.
(2) In the event of a
negative evidence, the ‘sample’ under examination passes the ‘tests for
sterility’.
(3) If positive evidence of microbial growth is found, reserve the containers exhibiting this, and
unless it is amply proved and adequately demonstrated by any other means that their (microorganisms)
presence is on account such causes unrelated to the
‘sample’ being examined ; and, therefore, the tests
for sterility
are pronounced invalid. In such cases, it may be recommended to carry out a ‘retest’
employing an identical number of
samples and volumes to be tested, and the media as in the original
test.
(4) Even then, if
no evidence of microbial growth is duly observed, the ‘sample’ under investigation
precisely
passes the ‘tests for sterility’.
(5) In case, reasonable evidence of bacterial growth is observed, one may go ahead with the
isolation
and subsequent identification of the organisms.
(6) If they are found to be not readily distinguishable from those (microbes) growing in the
containers reserved in the very
First Test, the ‘sample’ under investigation fails the ‘tests for sterility’.
(7) In case, the microorganisms are readily distinguishable from the ones actually growing in the
containers reserved in the
‘First Test’, it is very much advisable to carry out a ‘Second Retest’ by
employing virtually
twice the number of samples.
(8) Importantly, if
no evidence of bacterial growth is observed in the ‘Second Retest’, the sample
under examination legitimately
passes the ‘tests for sterility’.
(9) Contrarily, if
evidence of growth of any microorganisms is duly observed in the ‘secondretest’, the sample under investigation obviously fails the ‘tests for sterility’.