The Discovery of Microorganisms

Even before microorganisms were seen, some investigators suspected their existence and responsibility for disease. Among others, the Roman philosopher Lucretius (about 98–55 B.C.) and the physician Girolamo Fracastoro (1478–1553) suggested that disease was caused by invisible living creatures. The earliest
microscopic observations appear to have been made between 1625 and 1630 on bees and weevils by the Italian Francesco Stelluti, using a microscope probably supplied by Galileo.
However, the first person to observe and describe microorganisms
accurately was the amateur microscopist Antony van
Leeuwenhoek (1632–1723) of Delft, Holland (figure 1.1a).
Leeuwenhoek earned his living as a draper and haberdasher (a
dealer in men’s clothing and accessories), but spent much of his
spare time constructing simple microscopes composed of double
convex glass lenses held between two silver plates (figure
1.1b). His microscopes could magnify around 50 to 300 times,
and he may have illuminated his liquid specimens by placing
them between two pieces of glass and shining light on them at a
45° angle to the specimen plane. This would have provided a
form of dark-field illumination (see chapter 2) and made bacteria
clearly visible (figure 1.1c). Beginning in 1673 Leeuwenhoek
sent detailed letters describing his discoveries to the Royal
Society of London. It is clear from his descriptions that he saw
both bacteria and protozoa.


In a broader perspective the wide-spectrum of the
pharmaceutical products,
both pure and
dosage forms, may be accomplished by adopting any one of the following
two well-recognized, timetested,
and universally accepted methods, namely :
a) Membrane Filtration, and
b) Direct Inoculation.
two methods stated above shall now be treated individually in the sections that follows :
Membrane Filtration
membrane filtration method has gained and maintained its glorious traditional recognition
to not only circumvent but also to overcome the
activity of antibiotics for which there exist practically
inactivating agents. However, it may be duly extended to embrace legitimately a host of other
relevant products as and when deemed fit.
Importantly, the method emphatically requires the following characteristic features, namely :

an exceptional skill,
an in-depth specific knowledge, and
rigorous routine usage of positive and negative controls.
As a typical example of a suitable
positive control with respect to the appropriate usage of a
‘contaminated solution’ essentially comprising of a few microorganisms of altogether different
nature and types*.

Salient Features :
The salient features of the ‘membrane filtration’ method are as enumeratedunder :

(1) The solution of the product under investigation is carefully filtered
via a hydrophobic-edged
membrane filter
that would precisely retain any possible contaminating microorganisms.
(2) The resulting membrane is duly washed
in situ to get rid of any possible ‘traces of antibiotic’
that would have been sticking to the surface of the membrane intimately.
(3) Finally, the
segregated microorganisms are meticulously transferred to the suitable culture
under perfect aseptic environment.
Microorganisms for Positive Control Tests :
There are, infact, four typical microorganisms
that are being used exclusively for the
positive control tests along with their respective type of specific
enzymatic activity
mentioned in parentheses :
a) Bacillus cerreus : [Broad spectrum] ;
b) Staphylococcus aureus : [Penicillinase] ;
c) Klebsiella aerogenes : [Penicillinase + Cephalosporinase] ; and
d) Enterobacter species : [Cephalosporinase].
Interestingly, the microorganisms invariably employed for the
positive control tests together
with a particular product containing essentially an
‘antimicrobial agent’ must be, as far as possible,
explicitely sensitive
to that agent, in order that the ultimate growth of the microbe solely indicates
vital and important informations, namely :
satisfactory inactivation,
satisfactory dilution, and
satisfactory removal of the agent.
Specific Instances of Pharmaceutical Products :
Virtually all the ‘Official Compendia’ viz.,
Indian Pharmacopoea (IP) ; British Pharmacopoea (BP), United States Pharmacopoea (USP) ;
European Pharmacopoea (Eur. P),
and International Pharmacopoea (Int. P.) have duly provided
comprehensive and specific details with regard to the
‘tests for sterility’ of parenteral products (e.g.., IV
and IM injectables),
ophthalmic preparations (e.g., eye-drops, eye-ointments, eye-lotions etc.) ; besides
a plethora of
non-injectable preparations, such as : catgut, dusting powder, and surgical dressings.
Test Procedures :
In a broader perspective, the membrane filtration is to be preferred exclusively
in such instances where the substance under investigation could any one of the following
classes of
pharmaceutical preparations :
i) an oil or oil-based product,
ii) an ointment that may be put into solution,
iii) a non-bacteriostatic solid that does not become soluble in the culture medium rapidly, and
iv) a soluble powder or a liquid that essentially possesses either inherent bacteriostatic or
fungistatic characteristic features.
membrane filtration must be used for such products where the volume in a container is
either 100 mL or more. One may, however, select the exact number of
samples to be tested; and subsequently use them for the respective culture medium suitably selected formicroorganisms and the culture medium appropriately selected for fungi.
Precautionary Measures :
In actual practice, however, the tests for sterility must always be
carried out under highly specific experimental parameters so as to avoid any least possible
of the product being examined, such as :
a) a sophisticated laminar sterile airflow cabinet (provided with effective hepa-filters),
b) necessary precautionary measures taken to be such so as to avoid contamination that they do
not affect any microbes which must be revealed duly in the test.
c) ensuing environment (i.e., working conditions) of the laboratory where the ‘tests for sterility’
is performed must always be monitored at a definite periodical interval by :
sampling the air of the working area,
sampling the surface of the working area, and
perforing the stipulated control tests.
Methodology :
In usual practice, it is absolutely urgent and necessary to first clean meticulously
exterior surface of ampoules, and closures of vials and bottles with an appropriate antimicrobial
; and thereafter, the actual access to the contents should be gained carefully in a perfect aseptic
However, in a situation where the contents are duly packed in a particular container
under vacuum,
introduction of ‘sterile air’ must be done by the help of a suitable sterile device, for
instance :
a needle duly attached to a syringe barrel with a non-absorbent cotton.
Apparatus :
The most suitable unit comprises of a closed reservoir and a receptacle between
which a properly supported
membrane of appropriate porosity is placed strategically.
A membrane usually found to be quite suitable for sterility testing essentially bears a
nominal pore size
not more than 0.45 μm, and diameter of nearly 47 mm, the effectiveness
of which in the retention of microbes has been established adequately.

The entire unit is most preferably assembled and sterilised with the membrane in place prior
to use.

In case, the sample happens to be an oil, sterilize the membrane separately and, after thorough
drying, assemble the unit, adopting appropriate aseptic precautionary measures.

Diluting of Fluids :
In the ‘test for sterility’ one invariably comes across with two different
types of fluids
which will be treated individually in the sections that follows :
a) Fluid A—Digest 1 g of peptic digest of animal tissue* or its equivalent in water to make up
the volume upto 1L, filter or centrifuge to clarify, adjust to pH 7.1
± 0.2, dispense into flasks in 100 mLquantities, and finally sterilize at 121° C for 20 minutes (in an
b) Fluid B : In a specific instance, when the test sample usually contains either oil or lecithin*,
Fluid A to each litre of which has been added 1 mL of Polysorbate 80**, adjust to pH 7.1 ± 0.2,
dispense into flasks and sterilize at 121° C for 20 minutes (in an
Note : A sterile fluid shall not have either antimicrobial or antifungal properteis if it is to be considered
suitable for dissolving, diluting or rinsing a preparation being examined for sterility.
Quantum of Sample Used for ‘Tests for Sterility’ :
In fact, the exact and precise quantities of
sample to be used for determining the
‘Tests for Sterility’ are quite different for the injectables and
plus other non-injectables ; and, therefore, they would be discussed separately as under :
a) For Injectable Preparations : As a common routine practice and wherever possible always
use the whole contents of the container ; however, in any case not less than the quantities duly stated in
Table : 8.2, diluting wherever necessary to 100 mL with an
appropriate sterile diluent e.g., Fluid A.
b) For Ophthalmic and other Non-injectable Preparations : In this particular instance exactly
take an amount lying very much within the range prescribed in Column (A) of Table : 8.3, if necessary,
making use of the contents of more than one container, and mix thoroughly. For each specific medium use
the amount duly specified in column (B) of Table : 8.3. taken carefully from the mixed sample.

Method of Actual Test :
In reality, the method of actual test may be sub-divided into the
four categories, namely :
i) Aqueous Solutions,
ii) Liquids Immiscible with Aqueous Vehicles and Suspensions
iii) Oils and Oily Solutions, and
iv) Ointments and Creams.
three aforesaid types of pharmaceutical preparations shall be treated separately as under :
[I] Aqueous Solutions :
The following steps may be followed sequentially :
(1) Prepare each membrane by transferring aseptically a
small amount (i.e., just sufficient to get
the membrane moistened duly) of
fluid A on to the membrane and filtering it carefully.
(2) For each medium to be employed, transfer aseptically into
two separate membrane filter
or two separate sterile pooling vessels prior to transfer not less than the quantity of the preparation
being examined which is duly prescribed either in Table : 8.2 or Table : 8.3.
(3) Alternatively, transfer aseptically the combined quantities of the preparation being examined
prescribed explicitely in the
two media onto one membrane exclusively.
(4) Suck in the
‘liquid’ quickly via the membrane filter with the help of a negative pressure
i.e., under vacuum).
(5) In case, the solution being examined has
significant antibacterial characteristic features,
wash the membrane(s) by filtering through it (them)
not less than three successive quantities, each of
100 mL of the sterile fluid A.
(6) Precisely, the quantities of fluid actually employed must be sufficient to permit the adequate
growth of a
‘small inoculum of microorganisms’ (nearly 50) sensitive to the antimicrobial substancein the presence of the residual inhibitory material retained duly on the membrane.

(7) Once the filtration is completed, aseptically remove the membrane(s) from the holder, cut the
membrane in half, if only one is used, immerse the membrane or 1/2 of the membrane, in 100 mL of the
‘Fluid Soyabean-Casein Digest Medium’
*, and incubate at 20–25°C for a duration of seven days.
(8) Likewise, carefully immerse the other membrane, or other half of the membrane, in 100 mL
‘Fluid Thioglycollate Medium’,** and incubate duly at 30–35° C for not less than seven days.
[II] Liquids Immiscible with Aqueous Vehicles and Suspensions :
For this one may carry out
‘test’ as stipulated under [I] Aqueous Solutions, but add a sufficient amount of fluid A to the pooled
sample to accomplish fast and rapid rate of filtration.

Special Features :
These are as stated under :
Sterile enzyme preparations, for instance :
can be incorporated to
fluid A to help in the dissolution of insoluble substances.
(2) In a situation when the substance under test usually contains
lecithin, alway make use of
fluid B
for dilution.]
[III] Oils and Oily Solutions :
The various steps that are essentially involved in treating oils and
oily solutions
for carrying out the ‘test for sterility’ are as enumerated under :
(1) Filter
oils or oily solutions of sufficiently low vicosity as such i.e., without any dilution via a
dry membrane.
(2) It is absolutely necessary to dilute viscous oils as necessary with an
appropriate sterile
e.g., isopropyl myristate which has been proved beyond any reasonable doubt not to exhibitany
antimicrobial activities under the prevailing parameters of the test.
(3) Permit the
‘oil’ to penetrate the membrane, and carry out the filtration by the application of
gradual suction (with a vaccum pump).
(4) Wash the membrane by filtering through it at least 3/4 successive quantities, each of nearly
100 mL of
sterile fluid B or any other appropriate sterile diluent.
(5) Complete the test as described under
[I] Aqueous Solutions from step (7) onwards.
[IV] Ointments and Creams :
The various steps involved are as stated under :
(1) Dilute
ointments carefully either in a ‘fatty base’ or ‘emulsions’ of the water-in-oil (i.e.,
w/o) type
to yield a fluid concentration of approx. 1% w/v, by applying gentle heat, if necessary, to not
more than 40°C
with the aid of an appropriate sterile diluent e.g., isopropyl myristate previously
adequately sterilized by filtration
via a 0.22 μm membrane filter which has been shown not to possess
antimicrobial activities
under the prevailing conditions of the test.
(2) Carry out the filtration as rapidly as possible as per details given under
‘Oils and Oily Solutions’
[Section III] from step (4) onwards.
(3) However, in certain exceptional instances, it would be absolutely necessary to heat the
substance to
not more than 45°C, and to make use of ‘warm solutions’ for washing the membrane

Note : For ointments and oils that are almost insoluble in isopropyl myristate one may employ the
second method
viz., ‘Direct Inoculation’ [Section 2.2].
[V] Soluble Soids :
For each individual cultrue medium, dissolve not less the quantity of the
substance being examined, as recommended in Tables : 8.2 and 8.3, in an appropriate sterile solvent

, fluid A, and perform the test described under Section (I) i.e., Aqueous Solutions, by employing a
suitable for the selected solvents.
[VI] Sterile Devices :
Pass carefully and aseptically a sufficient volume of fluid B via each of
not less than 20 devices so that not less than 100 mL is recovered ultimately from each device. Collect
the fluids in sterile containers, and filter the entire volume collected
via membrane filter funnel(s) asdescribed under Section
(I), Aqueous Solutions.