Microbial limit test

Contents
• Definition
• Objective
• Preliminary testing
• Media
• Sampling
• Method
• Test for specified micro organisms
 Definition
This tests are designed to perform the
qualitative and quantitative estimation of
specific viable micro organisms present in
sample.
It include tests for total viable count of
bacteria and fungi.
The term ‘growth’ is used to designate
the presence & presumed proliferation of
viable micro-organism.
 Objective
• Microbial limit tests are designed to estimate the
number of viable aerobic organisms present in
pharmaceutical products and raw materials.
• The microbial limit testing of raw material as well as
finished pharmaceutical products can help to
determine whether the product complies with
requirement of IP.
• The most care be taken while performing microbial
limit test so that contamination from outside can be
avoided.
 Preliminary testing
• The method given here in are invalid unless it is
demonstrated that the specimen to which they are
applied not themselves inhibit the multiplication of
under the test condition of micro-organisms that can
be present.
• Therefore, inoculate diluted specimen of substance
being examined with separate viable culture of
1. E.coli
2. S.aures
3. S.typhi
4. Psudomonas aeruginosa
• If organism fails to grow in medium the
procedure should be modified by:
a) incresing the volume diluents with quantity
of diluents remain same, or
b) incresing a sufficient quantity of inactivating
agent in diluents ,or
c) combining aforementioned modification so
as to permit growth of organisms in media.
• If inhibitory subtances are present in
sample,0.5% soyalecithin & 4%of polysorbate
20 may be added to the culture medium.
• Repeat the same procedure using fluid casin
digest –soyslecithin,-polysorbate20 medium to
demonstrate neutralization of preservative or
other antimicrobial agent in test material.
OR
other antimicrobial agents in the test material.
 Media
• Culture Media:-Media are substance used to provide
nutrients for the growth and multiplication of
microorganism. Now a day, dehydrated media
containing all the ingredients in powdered form are
available.
• There are three types of media are required-
1) Enrichment Media- Soyabean Casien Digest Media.
2) Selective Media- MacConkey Agar for E.coli.
3) Differential Media- Sabourud Chloramphenicol
Agar for fungi.
 Required media
• BRILLIANT GREEN AGAR
• BISMUTH SULPHITE
• CETRIMIDE AGAR
• EOSINE METHYLENE BLUE AGAR
• MACCONKEY AGAR
• MANNITOL SALT AGAR
• PSEUDOMONAS AGAR
• SABOURAUD CHLORAMPHENICOL AGAR
• SOYABEAN CASEIN DIGEST AGAR
• TRIPLE SUGAR IRON AGAR
• BUFFERED PEPTONE WATER
• FLUID SELENITE CYSTEINE BROTH
• MACCONKEY BROTH
• PEPTONE WATER
• SOYABEAN CASEIN DIGEST MEDIUM
• TETRATHIONATE BRILLINT GREEN BILE BROTH
• Where agar is specified in a formula, use agar that
has a moisture content of not more than 15%.
• Where water is called for in a formula, use
purified water.
• The media should be sterilized by heating in an
autoclave at 115°c for 30 minutes.
• In preparing media dissolve the soluble solids in
the water, using heat if necessary, to effect
complete solution an add solutions of
hydrochloric acid or sodium hydroxide in
quantities sufficient to yield the required pH in
the medium when it is ready for use. Determine
the pH at 25°c ± 2°c
 Some common ingredients and its use
Agar:
• A solidifying agent which is a complex polysaccharide derived from
marine algae
• It has no nutritional value in media. It is bacteriological inert.
• It is stable at different temperature used for incubation.
Peptones:
• Protein is large, relatively insoluble molecules that a minority of
organism can utilized directly, but a partial digestion by acid or
enzyme reduces protein to shorter chain of amino acids called
peptone. These small, soluble fragments can be digested by most
bacteria.
• It should be stored in a tightly closed container as it is hygroscopic in
nature.
Meat extract:
• It is prepared from fresh meat by hot water
extraction.
• It contains water soluble constituents of
animal tissue that is carbohydrates, organic
nitrogen compound, water soluble vitamins
and mineral salts.
Yeast extract:
• It is particularly rich in vitamin B.
• It also contains carbohydrates, amino acids,
inorganic salts, growth factors.
 Sampling
• Use 10 ml or 10 g specimens for each of the tests
specified in the individual monograph.
PRECAUTION:
• The microbial limit tests should be carried out
under conditions designed to avoid accidental
contamination during the test.
• The precautions taken to avoid contamination must
be such that, they do not adversely effect any micro
organism that should be revealed in the test.
 Method of microbial limit tests
There are two types of method of microbial limit test-
1. Direct inoculation- In this method sample is directly
inoculated into the media and that are incubated.
2. Membrane filtration method- In this method sample
solution is filter with filter membrane and then this
filter membrane is transfer to the freshly prepared
sterilized media.
Above methods are performed for-
I. Total Bacterial Count
II. Test for Specified micro organisms
 Total aerobic microbial count
a) Membrane filtration method
b) Plate count method
i. Pour plate method
ii. Spread plate method

c) Serial dilution method

 Membrane filtration
 pretreatment of sample:
• Water soluble product:
10gm*/10ml** sample+70 ml buffered normal saline solution + make
up volume up to 100
• Water insoluble product:
10gm*/10ml** sample+ 70 ml buffered normal saline solution +
make up volume up to 100 + 0.1%w/v polysorbate 80 (surface active agent)
• Fatty product:
10gm*/10ml** sample +60 ml buffered normal saline solution + 5gm
polysorbate 80 or poly sorbate 20 +gentle heat + make up volume up to
100

{* solid like tablets , ** liquids like water}

Process:
1. Transfer 10ml of diluted sample though membrane
filter of 50mm diameter of 0.45µm if necessary dil.
for require colony count.
2. Wash mem.fil. With successive quantities of 100ml
of buffered Nacl. peptone broth soln./for fatty
prods. Add polysorbate 80 &20.
3. Transfer one of mem.filter to SDA plate with
antibiotics.
4. Incubate 30 -40oC /48hrs for bacteria and 20-25oC
/5 days for fungi.
5. Calculate No. of Mo’s/ml.
 Plate count method
A. Pour plate method:
 FOR BACTERIA: Use Petri dish 9 to 10 cm diameter, add to
each dish a mixture of 1ml of the pretreated preparation &
about 15ml of liquefied casein soyabean digest agar at NMT
45°c.
 If necessary dilute the preparation as described above so that
colony count NMT300 may be expected.
 Incubate the plate at 30 to 35 °c for 5 days unless more
reliable count is obtained in shorter time.
 Calculate the result using plate with greatest no. of colonies
but taking 300 colonies per plate as maximum consistent with
good evaluation.
 FOR FUNGI: Use saboraud dextrose agar with
antibiotics & incubate the plate at 20 to 25
°c for 5 days.
 Calculate the result using plate with nmt 100
colonies.

B. Spread plate method:

• Place 0.05-.2 ml of test fluid on solidified dried
surface of agar medium spread it uniformly using
spreader.
• Proceed under same condition as for the pour
plate method
 Serial dilution method (multiple tube
method)
• Use 12 test tubes: 9 containing 9 ml of soybean-casein digest
medium each and 3 containing 10 ml of the same medium
each for control.
• Prepare dilutions using the 9 tubes.
• First, add 1 ml of the test fluid to each of three test tubes and
mix to make 10- times dilutions.
• Second, add 1 ml of each of the 10-times dilutions to each of
another three test tubes and mix to make 100-times dilutions.
• Third, add 1 ml of each of the 100-times dilutions to each of
the remaining three test tubes and mix to make 1,000- times
dilutions.
• Incubate all 12 test tubes for at least 5 days at 30 -
35°c. No microbial growth should be observed for
the control test tubes.
• If the determination of the result is difficult or if the
result is not reliable, take a 0.1ml fluid from each of
the 9 test tubes and place it to an agar medium or
fluid medium, incubate all media for 24-72 hours at
30°-35°c, and check them for the absence or
presence of microbial growth.
• Calculate the most probable number of
microorganisms per ml or gram of the sample
 Test for specified micro organisms
• As per IP
i. Escherichia coli
ii. Salmonella
iii. Pseudomonas aeruginosa
iv. Staphylococcus aureus

Escherichia coli
• Place the prescribed quantity in sterile screw-
capped container, add 50ml of nutrient broth,
shake allow to stand for 1hr &incubate at 37°
for 18 to 24hr.
 Primary test: Add 1ml of enrichment culture to
tube contain 5ml MacConkey broth&incubate in
water bath at 36 to 38° for 48hr.
• If content show acid &gas carry out secondary
test.
 Secondary test: Add 0.1ml of content of tube
containing
 (a)5 ml of MacConkey broth
 (b)5ml of peptone water
 incubate in a water bath at 43.5 to 44.5° for 24hr
&examine tube for (a) acid &gas (b) indole
• For indole: Add 0.5 ml of kovac’s reagent, if red
colour is produced ,indole is present.
• That indicates presence of e.coli
• For control: Repeat primary &secondary test
adding 1.0ml of enrichment culture &volume
of broth containing 10 to 50 e.coli
organism,prepared from 24hr culture in
nutient broth,to 5ml MacConkey broth.

• The test is not valid unless the result indicate

that the control contain e.coli.
• OTHER TEST:
• streak a portion from enrichment culture on surface of
MacConkey agar medium.cover the dish &incubate.
• If none of the colonies are brick-red in colour, sample
meet the requirement of test for absence of e.coli
• If colony described above are found, transfer the
suspect colony to surface of Levine eosin ethylene
blue agar medium. cover &incubate.
• Upon examination, none of colony exhibit both
metallic sheen under reflected light & blue-black
under transmitted light ,sample meet requirement test
for absence of E.coli.
• SALMONELLA:
• Transfer a quantity of pretreaed prepration being examined
containing 1g or 1ml of product to 100ml of nutrient broth
in sterile screw capped jar ,shake & incubate at 35 to 37 for
24hr.
• Preliminary test:
Add 1.0 ml of enrichment culture to each of two tubes
containing
(A)10ml of selenite F broth & (B)tetrathionet -bile- briliant
green broth &incubate at 36 to38° for 48 hr.
• From each of this two cultures subculture on following four
agar medium & incubate at 36 to 38°for24 hr
• if none of colonies conform to description given in table,
sample meet requirment for absence of salmonella.
Medium Description of colony

Bismuth sulphite agar Black or Green

Brilliant green Small, transparent and

colorless, or opaque, pinkish
or white (frequently
surrounded by a pink or red
zone)
Deoxycholate-citrate agar Colorless and opaque, with or
without black centres

Xylose-lysine-deoxycholate Red with or without black

agar centers
• Secondary test:
• subculture any colonies showing
characteristics given in table in triple sugar-iron
agar by first inoculating the surface of slope &
at the same time inoculate a tube of urea broth
&incubate both at 36 to 38 for 18 to 24 hr.

• The absence of acidity from the surface growth

in triple sugar iron agar & together with
absence of red colour in urea broth, indicates
the presence of salmonella.
• FOR CONTROL:

• Repeat the primary &secondary test using

1.0ml of enrichment culture& volume of broth
containing 10 to 50 salmonella organism,
prepare from 24hr broth culture in nutrient
broth, for inoculation of tubes (a) &(b).

• The result is not valid unless the result

indicate that the control contains salmonella.
• PSUDOMONAS AERUGINOSA:
• Inoculate 100ml of fluid soyabean casein digest
medium with quantity of solution ,suspension or
emulsion thus obtained containing 1g or 1ml of
preparation being examined &incubate at 35 to
37for 24 to 48hr
• If upon examination none of colonies having
characteristics listed in table for media used,
sample meet requirement for absence of micro-
organisms.
• If colony conform to description in table ,carry out
oxidase & pigment test.
Selective Characteristic Fluorescence Oxidase test Gram stain
medium colonical in UV light
morphology

Cetrimide agar Generally greenish positive Negative rods

medium greenish

Pseudomonas Generally yellowish Positive Negative rods

agar medium colourless to
for detection of yellowish
fluorescin
Pseudomonas Generally Blue positive Negative rods
agar medium greenish
for detection of
pyocyanin
• OXIDASE &PIGMENT TEST:
• Streak representative suspect colony from
cetrimide agar medium on surface of
pseudomonas agar medium for
• detection of florescein &pseudomonas agar
medium for detection of pyocyanin contained
in Petri dish & incubate at 33 to 37 °for NLT 3
days.
• Examine the streak surface under u.v light.
• Examine plate to determine whether colonies
conforming to description in table are present.
• If, growth of suspect colonies occur ,place 2 or
3 drops freshly prepared 1%w/v solution of
N,N- tetramethyl-4-phenylenediamine
dihydrochloride on filter paper &smear with
colony.
• If there is no development of pink colour
changing to purple, the sample meet
requirements of test for absence of
pseudomonas aeruginosa.
• STAPHAYLOCOCCUS AURES:
• Proceed as described under psudomonas
aeruginosa ,if upon examination of incubate
plate. none of them contain colony having
characteristic listed table sample meet
requirment for absence of S.aures.
• If, growth occurs ,carry out COAGULASE test.
Transfer repsentative suspect colony from agar
surface to individual tubes, each contain 0.5ml
of mammalian,
• preferably horse or rabbit plasma with or
without additive.

• Incubate in water bath at 37 °examine tubes

at 3hr & subsequently at suitable interval up
to 24hr.

• If no coaggulation is observed sample meet

requirment of test for absence of S.aures
Selective medium Characteristics colonical Gram stain
morphology

Vogel-johnson agar Black surrounded by yellow Positive cocci(in cluster)

medium zone

Mannital-salt agar medium Yellow colonies with yellow Positive cocci(in cluster)
zones

Baird-parker agar medium Black, shiny, surrounded by Positive cocci(in cluster)

clear zones 2 to 5 mm
• REFERENCES:
• (1)INDIAN PHARMACOPIEA
• (2)U .S. P
• (3) BY: JAMES SWARBRICK
• ENCYCLOPEDIA OF PHARMACEUTICAL
• TECHNOLOGY,THIRD EDITION ,VOL-1
• (4) BY: GILBERT S. BANKER
• MARTIN M . RIGER
• PHARMACEUTICAL DOSAGE FORM DISPERSE
SYSTEM, VOL-2