1. MICROBIAL LIMIT TEST

2. • This test are designed to perform qualitative
quantitative estimation of the no of viable
aerobic micro-organisms present or
detecting the presence of designated
microbial species in pharmaceutical product.
• The term ‘growth’ is used to designate the presence
& presumed proliferation of viable micro-organism.
• The most care must be taken while performing
microbial test so that contamination from outside can
be avoided.

3. Preliminary testing:
• The method given herein are invalid unless it is
demonstrated that the test specimen to which they
are applied do not themselves inhibit the
multiplication of under the test condition of micro-
organism 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

4. • 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.

5. • 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.
• Where inhibitory substance are contained in
product & latter is soluble, the Membrane filtration
method may be used.

6. • If inspite of incorporation of suitable inactivating
agent & a substantial increase in volume of diluents
it is still not possible to recover the viable culture
described above & where article is not suitable for
applying the membrane filtration method it can be
assumed that the failure to isolate the inoculated
organism may be due to the bactericidal activity of
product.
• This may be indicated that the article not likely
to be contaminated with the given species of
organism.
• Monitoring should be continued to establish the
spectrum of inhibition & bactericidal activity of
product.

7. Media:
• culture media may be as per procedure & they have
similar ingredient &/or yield media comparable to
those obtained from the formula.
• Where agar specified in formula, use agar that has
moisture content nmt15 %.
• Where water is mention in formula, use purified
water.
• Media should be sterilized by heating in autoclave at
115°c for 30 min.

8. • In preparing media as per formula, dissolve the
soluble solid in water, using the heat if necessary
to effect complete solution & add HCL & NaOH in
sufficient quantities to yield require pH in medium.
• SAMPLING: USE 10ML OR 10G
SPECIMEN FOR EACH OF TEST.

9. METHODS
(1)TOTAL AEROBIC MICROBIAL COUNT
MEMBRANE FILTRATION -
METHOD
PLATE COUNT METHOD
POURED PLATE
SPREAD PLATE
MUTIPLE OR SERIAL –
DILUTION METHOD

10. ( 1) TOTAL AEROBIC MICROBIAL
COUNT
PREPARATION OF TEST FLUID:
• Water soluble product: Dissolve 10g or 10ml
of the sample in buffer or fluid medium & adjust
volume to 100ml.
• Product insoluble in water(non fatty): Take
10g of sample, grind to fine powder & suspend it in
buffer or fluid medium & adjust the volume to
100ml.

11. A suitable surface-active agent such as 0.1%w/v of
polysorbate 80 may be added to assist the suspension
of poorly wettable substance.
• Fatty product: Homogenise 10g or 10ml of sample
with 5g ofpolysorbate20 or polysorbate80.
-If necessary ,heat to nmt 40`c for 30min.
-Add 85ml of buffer or fluid medium.
-Adjust the PH to about 7.
Membrane filtration:
• Use membrane filter 50mm in diameter &
having nominal pore size NGT 0.45 um or
less.

12. • Sterilized the filters,filteration apparatus,media &
other apparatus used.
• Transfer 10ml or quantity of each dilution contain 1g
of preparation being examined to each of two
membrane filter & filter immediately.
• If necessary dilute the pretreated preparation so that
10-100 colony count may be expectd.
• After filtration wash the each filter three or more
time with appropriate fluid such as phosphate
buffer,sodium chloride-peptone buffer or fluid
medium.
• For fatty susbtance add polysorbate20 or
polysorbate80 to washing.

13. • Transfer one of the membrane filter, intended for
enumeration of bacteria to surface of plate of casein
soyabean digest agar & intend for enumeration of
fungi to surface if sabouraud dextrose agar with
antibiotics.
• Incubate the plate for 5 days, unless more reliable
count is obtanied in shorter time,at 30 to 35°c in test
for bacteria & 20 to 25°c in test for fungi.
• Count the number of colonies that are formed.
• Calculate the no of organism per g or ml of
preparation being examined.

14. 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.

15. • 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.
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.

16. 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.(100ul)
• 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.(10ul)

17. • 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 (1ul)
• 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 the9 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.

18. • Test for specified micro organism
• - E.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.

19. • 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.

20. • 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.

21. • 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.

22. • 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.

23. • 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.

24. Characteristic Colonial
Morphology
Selective Medium
Brilliant Green Small, transparent, colorless or pink to white
Agar Medium opaque (frequently surrounded by pink to red
zone)
Xylose-Lysine- Red, with or without black centers
Desoxycholate
Agar Medium
Bismuth Sulfite Black or green
Agar Medium

25. • 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.

26. • FOR CONTORL:
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.

27. • 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

28. Characteristic Fluorescence
Selective Colonial in
Medium Morphology UV Light Oxidase Test Gram Stain
Centrimide Generally Greenish Positive Negative rods
Agar Medium greenish
Pseudomonas Generally Yellowish Positive Negative rods
Agar Medium colorless to
for yellowish
Detection of
Fluorescin
Pseudomonas Generally Blue Positive Negative rods
Agar Medium greenish
for
Detection of
Pyocyanin

29. • 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.
• OXIDASE &PIGMENT TEST:
• Streak representative suspect colony from
cetrimide agar medium on surface of
pseudomonas agar medium for

30. 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.

31. • 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.

32. • 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,

33.
• 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

34. Characteristic
Selective Medium
Colonial Morphology Gram Stain
Vogel-Johnson Black Surrounded by yellow Positive cocci
Agar Medium zone (in clusters)
Mannital-Salt Yellow colonies with yellow Positive cocci
Agar Medium zones (in clusters)
Baird-Parker Black, shiny, surrounded by Positive coccid
Agar Medium clear zones 2 to 5 mm (in clusters)

35. • 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