2. CONTENT
 Objective
 Preliminary testing
 Different media
 Sampling
 Methods
Total aerobic microbial count
 Membrane filtration
 Plate count
 Serial dilution
Tests for specified micro-organism

3. OBJECTIVE[1]
 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 BP, USP or IP.
 The care must be taken while performing microbial
test so that contamination from outside can be avoided.
3

4. Preliminary Testing[7]
4

5. Continue[1]
 MODIFICATIONS:
 If the organisms fail to grow in the relevant medium
the procedure should be modified by
(a) increasing the volume of diluents with the quantity
of test material remaining the same, or
(b) incorporating a sufficient quantity of a suitable
inactivating agent in the diluents, or
(c) combining the above modifications so as to permit
growth of the organisms in the media.
 If inhibitory substances are present in the sample,
0.5% of soya lecithin and 4% of polysorbate 20 may
be added to the culture medium.
5

6. Continue
 Alternatively, repeat the test as described using fluid
casein digest-soya lecithin- polysorbate 20 medium to
demonstrate neutralization of preservatives
OR
other antimicrobial agents in the test material.
6

7. Media[1]
 Baird – Parker Agar Medium
 Bismuth Sulphite Agar Medium
 Brilliant Green Agar Medium
 Buffered Sodium Chloride-Peptone Solution pH 7.0
 Casein Soyabean Digest Agar Medium
 Cetrimide Agar Medium
 Desoxycholate-Citrate Agar Medium
 Fluid Casein Digest-Soya Lecithin-Polysorbate 20
Medium
7

8. Continue:
 Fluid Lactose Medium
 Lactose broth Medium
 Levin Eosin-Methylene Blue Agar Medium
 MacConkey Agar Medium (culture of enterobacteria)
 MacConkey Broth Medium
 Mannitol Salt Agar Medium
 Nutrient Broth Medium
 Nutrient Agar Medium
 Pseudomonas Agar Medium for Detection of
Flourescein
8

9. Continue:
 Pseudomonas Agar Medium for Detection of
Pyocyanin
 Sabouraud Dextrose Agar Medium
 Sabouraud Dextrose Agar Medium with Antibiotics
 Selenite F Broth
 Fluid Soyabean-Casein Digest Medium
 Tetrathionate-Bile-Brilliant Green Broth Medium
 Triple Sugar-Iorn Agar Medium
 Urea Broth Medium
 Vogel-Johnson Agar Medium
 Xylose-Lysine-Desoxycholate Agar Medium
(selective media for salmonella)
9

10. Notes[1]
 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
10

11. Some common ingredients and its use[8]
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.
11

12. Continue:
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.
12

13. TERMS[8]
 Culture medium: A nutrient material prepared for
growth of micro-organism in a laboratory is called
culture medium.
 Culture: The microbes can grow and multiply in or on a
culture medium are referred to as a culture.
 Nutrient broth: if the complex media is in liquid form, it
is called nutrient broth.
 Nutrient agar: when agar is added to media, it is called
nutrient agar.
13

14. TYPES OF CULTURE MEDIA[8]
 Selective: suppression of unwanted microbes and
encouraging desired microbes.
 Differential: differentiation of colonies of desired
microbes from others.
 Enrichment: similar to selective but designed to
increase numbers of desired microbes to detectable
levels.
14

15. SAMPLING[1]:
 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 our 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.
15

16. 16

17. METHODS[1]:
1.TOTAL AEROBIC MICROBIAL COUNT:
Unit: cfu/ml or gm[3]
 colony-forming unit (CFU or cfu) is a measure of viable
bacterial or fungal numbers. Unlike direct microscopic
counts where all cells, dead and living, are counted, CFU
measures viable cells. For convenience the results are
given as CFU/ml (colony-forming units per milliliter) for
liquids, and CFU/g (colony-forming units per gram) for
solids.
 This technique allows the determination of the number of
CFU per ml in the sample, and thus the microbiological
load and the magnitude of the infection in humans or
animals, or the degree of contamination in samples of
water, vegetables, soil or fruits, and in industrial products
and equipment.
17

18. Water soluble products:
Dissolve 10 g / 10 ml of the preparation being examined
in buffered sodium chloride peptone solution pH 7.0
OR
any other suitable medium shown to have no
antimicrobial activity and adjust the volume to 100 ml
with the same medium.
If necessary, adjust the pH to about 7.
18

19. Products insoluble in water (non-fatty) :
Suspend 10 g or 10 ml of the preparation
in buffered sodium chloride-peptone solution pH 7.0 or
any other suitable medium shown to have no antimicrobial
activity
adjust the volume to 100 ml with the same medium.
If necessary, divide the preparation being examined and
homogenize the suspension mechanically.
A suitable surface-active agent such as 0.1% w/v of
polysorbate 80 may be added to assist the suspension of
poorly wettable substances.
19

20. Fatty products :
Homogenize 10 g or 10 ml of the preparation with 5g of
polysorbate 20 or polysorbate 80.
If necessary heat to not more than 40°c
Add 85 ml of buffered sodium chloride-peptone solution
pH7.0 or any other suitable medium shown to have no
antimicrobial activity under the conditions of the test.
Maintain this temperature for the shortest time necessary for
formation of an emulsion and in any case for not more than 30
minutes.
If necessary adjust the pH to about 7.
20

21. Membrane filtration method
 Use membrane filters 50 mm in diameter and having a
nominal pore size of not greater than 0.45 μm the
effectiveness of which in retaining bacteria has been
established for the type of preparation being examined.
Eg. Cellulose nitrate membrane filter
 Sterilize and assemble the filtration apparatus.
 Transfer 10 ml or a quantity of each dilution containing
1 g of the preparation being examined to each of two
membrane filters and filter immediately.
 If necessary, dilute the pretreated preparation so that a
colony count of 10 to 100 may be expected.
21

22. Membrane filtration assembly and
A sterile screw-capped container[4][6]
22

23. Continue:
 Transfer one of the membrane filters, intended for the
enumeration of bacteria, to the surface of a plate of
casein soyabean digest agar and the other,
 intended for the enumeration of fungi, to the surface of a
plate of Sabouraud dextrose agar with antibiotics.
 Incubate the plates for 5 days, unless a more reliable
count is obtained in shorter time, at 30° to 35°c in the
test for bacteria and 20°c to 25°c in the test for fungi.
 Count the number of colonies that are informed.
 Calculate the number of micro-organisms per g or per ml
of the preparation being examined, if necessary counting
bacteria and fungi separately.
23

24. Laminar air flow unit[6]
24

25. Plate count method[1]
 For bacteria
 Using Petri dishes 9 to 10 cm in diameter, add to each dish a
mixture of 1 ml of the pretreated preparation and about 15 ml of the
liquefied casein soyabean digest agar at not more than 45°.
 Alternatively, spread the pretreated preparation on the surface of the
solidified medium in a Petri dish of the same diameter.
 If necessary, dilute the pretreated preparation as described above so
that a colony count of not more than 300 may be expected.
 Prepare at least two such Petri dishes using the same dilution and
incubate 30° to 35° for 4 days, unless a more reliable count is
obtained in a shorter time.
25

26. Plate showing the colony[11] colony counter[12]
26

27. Continue:
Count the number colonies that are formed. Calculate the
results using plates with the greatest number of colonies
but taking 300 colonies per plate as the maximum
consistent with good evaluation.
 For fungi –
 Proceed as described in the test for bacteria but use
Sabouraud dextrose agar with antibiotics in place of
casein soyabean digest agar and incubate the plates at 20°
to 25° for 5 days, unless a more reliable count is obtained
in a shorter time.
 Calculate the results using plates with not more than 100
colonies.
27

28. Serial Dilution Method (Multiple tube method)[9]
 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.
28

29. Continue:
 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
29

30. TESTS FOR SPECIFIED MICRO ORGANISMS
 As per IP
 Escherichia coli
 Salmonella
 Pseudomonas aeruginosa
 Staphylococcus aureus
 As per USP
 Escherichia coli
 Salmonella
 Pseudomonas aeruginosa
 Staphylococcus aureus
 Candida albicans
 clostridium sporogenes
30

31. Escherichia coli[3][6] is a Gram negative rod-shaped bacterium that is
commonly found in the lower intestine of warm-blooded organisms
(endotherms). Most E. coli strains are harmless, but some, such as
serotype O157:H7, can cause serious food poisoning in humans, and
are occasionally responsible for product recalls. The harmless strains
are part of the normal flora of the gut, and can benefit their hosts by
producing vitamin K2 and by preventing the establishment of
pathogenic bacteria within the intestine.
31

32. As per IP
Escherichia coli[1]
 Place the prescribed quantity in a sterile screw-capped
container, add 50 ml of nutrient broth, shake, and allow
standing for 1 hour and shaking again. Loosen the cap
and incubate at 37°c for 18 to 24 hours.
 Primary test:
 Add 1.0 ml of the enrichment culture to a tube
containing 5 ml of Mac Conkey broth. Incubate in water-
bath at 36° to 38° for 48 hours. If the contents of the tube
shows acid and gas carry out secondary test.
32

33. Continue:
 Secondary test:
 Add 0.1 ml of the content of the tubes containing
(a) 5 ml of MacConkey broth for acid and gas and
(b) 5 ml of peptone water for indole.
 Incubate in a water-bath at 43.5° to 44.5° for 24 hours
 Test for indole, add 0.5 ml of Kovac’s reagent, shake
well and allow to stand for 1 minute; if a red color is
produced in the reagent layer indole is present.
 The presence of acid and gas and indole in the secondary
test indicates the presence of Escherichia coli.
33

34. Continue:
 Carry out a control test by repeating the primary and
secondary tests adding 1.0 ml of the enrichment culture
and a volume of broth containing 10 to 50 Escherichia
coli organisms, prepared from a 24 hour culture in
nutrient broth, to 5 ml of MacConkey broth.
 The test is not valid unless the results indicate that
control contains Escherichia coli.
 Kovac's reagent[10]: A reagent used to detect the presence
of indole which is used in identification of bacteria.
 Indole test[10] :The indole test is a biochemical test
performed on bacterial species to determine the ability of
the organism to split indole from the amino acid
tryptophan.
34

35. Salmonella[3][6] is a genus of rod-shaped, Gram-negative,
non-spore forming, predominantly motile enterobacteria.
Flagella which project in all directions (i.e. peritrichous).
They cause illnesses like typhoid fever, paratyphoid fever,
and the food borne illness.
35

36. Salmonella[1]
 Transfer a quantity of the pretreated preparation being
examined containing 1 g or 1 ml of the product to 100 ml
of nutrient broth in a sterile screw-capped jar, shake,
allow to stand for 4 hours and shake again.
 Loosen the cap and incubate at 35º to 37º for 24 hours.
 Primary Test:
 Add 1.0 ml of the enrichment culture to each of the two
tubes containing (a) 10 ml of selenite F broth and (b)
Tetrathionate bile-brilliant green broth and incubate at
36º to 38º for 48 hours.
 From each of these two cultures subculture on at least
two of the following four agar media: bismuth sulphite
agar, brillinat green agar, desoxycholate-citrate agar and
xylosetysine desoxycholate agar.
36

37. Test for salmonella:
37

38. Continue:
 Incubate the plates at 36ºc to 38ºc for 18 to 24 hours.
 Upon examination, if none of the colonies conforms to
the description given in Table, the sample meets the
requirements of the test for the absence for the genus
Salmonella.
 If any colonies conforming to the description in Table
are produced, carry out the secondary test.
 Secondary test:
 Subculture any colonies showing the characteristics
given in Table in triple sugar- iron agar by first
inoculating the surface of the slope and then making a
stab culture with the same inoculating needle, and at the
same time inoculate a tube of urea broth.
 Incubate at 36ºc to 38ºc for 18 to 24 hours.
38

39. Continue:
 The formation of acid and gas in the stab culture and the
absence of acidity from the surface growth in the triple
sugar iron agar, together with the absence of a red color
in the urea broth, indicates the presence of salmonellae.
 Carry out the control test by repeating the primary and
secondary test using 1.0 ml of the enrichment culture and
a volume of broth containing 10 to 50 salmonella
organisms, prepared form a 24-hour culture in nutrient
broth.
 The test is not valid unless the results indicate that the
control contains Salmonella.
39

40. Pseudomonas aeruginosa[3]
 It is a gram negative bacterium which can cause disease
in humans and non-human animals. It is found in soil,
water, skin flora, and most man-made environments
throughout the world.
 It uses a wide range of organic material for food; in
animals, the versatility enables the organism to infect
damaged tissues or people with reduced immunity. The
symptoms of such infections are generalized
inflammation and sepsis. If such colonization occur in
critical body organs such as the lungs, the urinary tract,
and kidneys, the results can be fatal.
40

41. Continue[3][6]
 P. aeruginosa strains produce two types of soluble pigments, the
fluorescent pigment pyoverdin and the blue pigment pyocyanin.
The latter is produced abundantly in media of low-iron content
and functions in iron metabolism in the bacterium. Pyocyanin
refers to "blue pus", which is a characteristic of infections caused
by Pseudomonas aeruginosa.
41

42. Continue[1]
 Pretreat the preparation being examined as described above
and inoculate 100 ml of fluid soyabean-casein digest
medium with a quantity of the solution, suspension or
emulsions, thus obtained containing 1 g or 1 ml of the
preparation being examined.
 Mix and incubate at 35º to 37º for 24 hours.
 Examine the medium form growth is present, streak a
portion of the medium on the surface of cetrimide agar
medium, each plated on Petri dishes.
 Cover and incubate at 35º to 37º for 18 to 24 hours.
 If upon examination, none of the plates contains colonies
having the characteristics listed in Table for the media used,
the sample meets the requirement for freedom from
Pseudomonas aeruginosa.
 If any colonies conforming to the description in Table are
produced, carry out the oxidase and pigment tests.
42

43. Tests for Pseudomonas aeruginosa
43

44. Oxidase and pigment tests:
 Streak representative suspect colonies from agar surface
of cetrimide agar on the surfaces of pseudomonas agar
medium for detection of fluorescein and pseudomonas
agar medium for detection of pyocyanin contained in
Petri dishes.
 Cover and invert the inoculated media and incubate at
33º to 37º for not less than 3 days. Examine the streaked
surfaces under ultra-violet light. Examine the plates to
determine whether colonies conforming to the
description in previous table are present.
44

45. Continue:
 If growth of suspect colonies occurs, place 2 or 3 drops
of a freshly prepared 1% w/v solution of N, N, N1, N1 –
tetramethyl-4-phenylenediamine dihydrochloride on filter
paper and smear with colony; if there is no development
of a pink color, changing to purple, the sample meets the
requirements of the test for the absence of Pseudomonas
aeruginosa.
45

46. Staphylococcus aureus [3][6]
 S. aureus are gram positive cocci (in clusters) which can
cause a range of illnesses from minor skin infections, such as
pimples, scalded skin syndrome, to life-threatening diseases
such as pneumonia, meningitis, osteomyelitis, endocarditis,
chest pain, bacteremia, and sepsis. Its incidence is from skin,
soft tissue, respiratory, bone, joint, endovascular to wound
infections.
46

47. Staphylococcus aureus[1]
 Proceed as described under Pseudomonas aeruginosa.
If, upon examination of the incubated plates, none of
them contains colonies having the characteristics listed
in Table for the media
 used, the sample meets the requirements for the absence
of Staphylococcus aureus.
 If growth occurs, carry out the coagulase test.
47

48. Continue:
48

49. Continue:
 Coagulase test:
 Transfer representative suspect colonies from the agar
surface of any of the media listed in Table to individual
tubes, each containing 0.5 ml of mammalian, preferably
rabbit or horse, plasma with or without additives.
 Incubate in water-bath at 37º examining the tubes at 3
hours and subsequently at suitable intervals up to 24
hours.
 If no coagulation in any degree is observed, the sample
meets the requirements of the test for the absence of
Staphylococcus aureus.
49

50. TEST FOR SPECIFIED ORGANISM AS PER USP[2]
 Transfer 10 ml of preparation to 90 ml soya bean
casein digest broth and shake the container.
 Incubate at 30-35ºc for 18-24 hour and proceed
further for E.coil, S.aureus and P.aureginosa as per
follows.
 For salmonella incubate the preparation at 30-35ºc for
18-24 hour and proceed further.
50

51. Test for E.coil:
 After completion of incubation period, transfer 1 ml
enriched medium to 100 ml Mac-Conkey΄s broth, with
the help of sterile pipette and incubate at 42-44ºc for
24-48 hour.
 Subculture on the plate of Mac- Conkey agar from
above medium and incubate at 30-35ºc for 18-72 hour.
 Growth of colonies indicates possible presence of E.coil
and then performs the confirmatory test.
 Sample passes the test if colonies not observed as
mentioned above and if the growth observed perform
the confirmatory test.
51

52. Test for salmonella:
 After completion of incubation period, transfer 0.1 ml of
above preparation to 10 ml of Rappaport vassilisdis
salmonella enriched broth and incubate at 30-35ºc for
18-24 hour.
 From above enriched broth, streak on the surface of
Xylose Lysine Decarboxylate agar and inverted the petri
plate and incubate at 30-35ºc for 18-48 hour.
 The possible presence of salmonella is indicated by the
characteristics colony having the following appearance
 XLD agar: Well developed, red colonies with or with out
black centers.
 Sample passes the test, if no growth as mentioned above.
If appearance of colonies found as mentioned above,
perform the confirmatory test.
52

53. Test for pseudomonas aeruginosa:
 After incubation period streak a loopful of above
enriched medium on surface of Cetrimide agar plate and
incubate at 30-35ºc for 18-72 hour.
 After incubation period, if there is no growth observed
on plates, the sample passes the test and if there is a
growth observed in above medium, perform the
confirmatory test.
53

54. Test for staphylococcus aureus:
 After incubation period streak a loopful of above
enriched medium on surface of Mannitol salt agar.
Incubate at 30-35ºc for 18-72 hour.
 After incubation period, there is no typical growth in
above medium as mentioned below the sample passes
and if there is gram positive cocci, perform the
confirmatory test such as coagulation test.
 The probable presence of S.aureus is indicated by the
characteristic of colony having following appearance.
 Mannitol salt agar: Yellow/white colonies surrounded
by yellow zone.
54

55. Test for Candida albicans[2][5]
 Candida albicans is a diploid fungus (a form of yeast)
and a causal agent of opportunistic oral and genital
infections in humans. Systemic fungal infections have
emerged as important causes of morbidity and mortality
in immunocompromised patients (e.g., AIDS, cancer
chemotherapy, organ or bone marrow transplantation).
 Transfer 10 ml preparation to the 100 ml of Sabouraud
Dextrose broth and incubate at 30-35ºc for 24 hour.
 Sub culture from above enriched broth on the plate of
Sabouraud Dextrose agar with Chloramphenicol. Invert
the Petri plate and incubate at 30-35ºc for 3-5 days.
 The probable presence of c.albicans is indicated by the
growth of white colony.
55

56. Candida albicans[6] Clostridium sporogenes[6]
56

57. Test for clostridium sporogenes[3]
 Large Gram positive
 Straight or slightly curved rods with slightly rounded
ends
 Anaerobic bacilli
 Spore bearing
 Causes diseases such as gas gangrene, tetanus, &
pseudo-membranous colitis by producing toxins which
attack the neurons pathways
57

58. Continue[2]
 Take 10 ml of preparation in two separate sterile test tubes and
marked as set-1 and set-2. Heat the test tube of set-1 at 80°c for 10
min in water bath and cool rapidly & Do not heat the tube of set-2.
 Transfer 10 ml from each set to the 100 ml Reinforced medium
separately.
 Incubate both the tubes under anaerobic condition at 30-35⁰c for 48
hour.
 After incubation, make sub culture from each tube on Columbia
agar and incubate under anaerobic condition at 30-35⁰c for 48 hour.
 Sample passes the test, if anaerobic growth is not observed on
Columbia agar. If growth observed above media and it is rod with or
with out endospore giving a negative catalase reaction, indicates the
presence of cl.sporogenes.
58

59. Control[2]
 Positive control
 For E.coil, salmonella, s.aureus, p.aeruginosa, c.albican
and cl. sporogen carry out the control test by repeating
the procedure using 1 ml of inoculum containing <100
organism with out product.
 Note done the observation of positive control.
59

60. observation
No Evidence of
growth Evidence of growth
Pass
Repeat test (RT 1)
growth No growth
Repeat test (RT 2)
Isolate and identify micro organism
60

61. Retest
 If the sample shows positive result, carry sample as and
proceed as per SOP for investigation of microbial limit
test failure.
 For the purpose of confirming a doubtful result by any of
the procedures outlined in the foregoing tests following
their application to a 10.0-g specimen, a retest on a 25-g
specimen of the product may be conducted. Proceed as
directed for Procedure, but make allowance for the larger
specimen size.
61

62. Bacterial identification:
 If the test sample shows positive result in retest, finally
confirm by Bacterial identification kit as per SOP.
Attach the identification report with rejection report of
product.
62

63. APPLICATION
 MICROBIOLOGICAL ASSAY OF ANTIBIOTIC DRUGS
 DISINFECTION EFFICACY TEST OF DISINFECTANTS
AND ANTISEPTICS
 STERILITY TEST OF STERILISED PHARMACEUTICALS
 TESTS FOR MICROBIAL LIMITS FOR NON-STERILE
PHARMACEUTICAL AND BIOLOGICAL PRODUCTS
 TESTING OF WATER
63

64. MICROBIOLOGICAL ASSAY OF ANTIBIOTIC DRUGS
 This assay is to determine whether the potency of
antibiotics complies with the requirement for label
potency and associated limits stipulated in the USP/BP
monographs for antibiotics.
 Specimen required: Antibiotic preparations:
Capsules/tablets – 30 nos.
 Raw material – 2 gm
 Ampoules (injections) – 6 nos.
 Ointment tubes – 3 nos.
 Vials (injections) – 6 nos.
 Bottles (syrup) – 2 nos.
64

65. Continue:
 Collect samples of raw materials and light-sensitive
antibiotic drugs in screw-capped plastic containers or
amber-coloured glass bottles. Do not expose sample to
warm temperatures during transport.
 Method : United States Pharmacopeia (USP)/British
Pharmacopoeia (BP)
 Test results : Pass or fail potency assay limits stipulated
in the USP/BP monograph of the antibiotic drug
65

66. DISINFECTION EFFICACY TEST OF
DISINFECTANTS AND ANTISEPTICS
 The disinfectants/antiseptics are tested at the use-
dilutions or use-conditions prescribed on the product
label to determine their efficacy.
 Specimen required : 100 ml
 Method : ISO or other standard tests
 Test results : Pass or fail the standard tests
66

67. STERILITY TEST OF STERILISED
PHARMACEUTICALS
 Specimen required: Parenteral drugs and administration
devices, ophthalmic preparations, surgical dressing,
sutures and other sterilized pharmaceutical products,
packed in unopened original containers. Generally 10 –
20 sample units (containers) per batch of manufacture.
Refer to sample size stipulated in BP for various batch
sizes of production.
 Method : United States Pharmacopeia (USP) /British
Pharmacopoeia (BP)
 Test results : Pass or fail the Tests for Sterility USP /BP
67

68. TESTS FOR MICROBIAL LIMITS FOR NON-STERILE
PHARMACEUTICAL AND BIOLOGICAL PRODUCTS
Route of TAMC TYMC (cfu/ Specified micro organism
adminstration (cfu/ml or ml or
cfu/gm) cfu/gm)
Non-aqueous Absence of E.coli
preparation for oral 103 102 (1 gm or 1 ml )
use
aqueous Absence of E.coli
preparation for oral 102 101 (1 gm or 1 ml )
use
Rectal use 103 102 -
Cutaneous use Absence of S.aureus
Nasal use 102 101 (1gm or 1ml)
Auricular use Absence of P.aeruginosa
(1gm or 1ml )
68

69. Continue:
Vaginal use Absence of
102 101 S.aureus,
P.aeruginosa,
C. Albucans
( 1gm or 1 ml)
Transdermal Absence of
patches (limit for 102 101 S.aureus,
one patch P.Aeruginosa
including ( in 1 patch)
adhesive layer)
Inhalation use Absence of
(liquid 102 101 S.aureus,
preparation for P.Aeruginosa
nebulization) ( 1gm or 1 ml)
69

70. TESTING OF WATER
This can be done by following two methods:
2. Chemical Analysis
3. Microbial Analysis
TOTAL VIABLE AEROBIC COUNT (cfu/ml):
 Requirement:
Membrane filtration assembly
Forceps
Vacuum pump
Cellulose nitrate membrane filter having porosity of 0.45 μ
and 47 mm diameter.
Sterile pre-incubated glass plates
 Sample quantity: 2000 ml of water for injection
70

71. Procedure:
 Testing to be done under laminar air flow to avoid
accidental contamination of sample to be examined.
 The filtration unit, membrane and their accessories are
sterilized in autoclave.
 The LAF is cleaned with 70% isopropyl alcohol, then the
sterile filtration assembly is arranged with unit and
vacuum line is connected.
 The sterile wet membrane filter is put on holder of the
filtration unit with help of sterile forceps with actual
sample solution, the valve of assembly is opened and
vacuum is applied. The sample is allowed to filter out.
 The vacuum line is switch off. The membrane filter is
transferred aseptically to the surface of sterile agar plate
with help of sterile forceps and incubates. 71

72. Specification of different type of water
 Specification of potable water:
Total viable count (cfu/ml) : Not more then 500
 Specification of Raw water:
Total viable count (cfu/ml) : Not more then 500
Microbial Limit Test (E.coil, Salmonella, S.aureus,
p.aeruginosa) : Should be absent
 Specification of Water for Injection:
Total viable aerobic count
Normal level : Not more than 2
Alert level : 3 -10
Action level : more than 10
72

73. Bibliography:
1. The Indian pharmacopoeia, IP-1996, volume-2, Indian
pharmacopoeia commission, Ghaziabad, India, 1996.
Appendix-9.4,A-110:A-117
2. The United States Pharmacopoeia,USP-25 NF-20,
2002, p 1873-1878
3. http://en.wikipedia.org
4. http://productimage.tradeindia.com/00530916/s/1/Me
mbrane-Filtration-Assembly-PC-Single-Unit-.jpg
5. http://en.wikipedia.org/wiki/Candida_albicans
6. http://www.google.co.in/image
7. http://www.usp.org/pdf/EN/meetings/asMeetingIndia2
009/session2Track2Tirumalai_1.pdf
73

74. Continue:
1. Gerard J. Tortora, Berdell R. Funke, Christine L. Case,
Microbiology An Introduction, Eighth Edition, Pearson
Education, 2005, p 193-199
2. http://ffcr.or.jp/zaidan/FFCRHOME.nsf
3. http://en.wikipedia.org/wiki/Indole_test
4. http://biology.clc.uc.edu/fankhauser/Labs/Microbiolog
y/Yeast_Plate_Count/07_yeast_0.2mL_plate_P720118
1.jpg
5. http://chemicoscientific.com/images/Digital_Colony_C
ounter.jpg
74