Microbial Analysis of Purified Water and Water of Injection

Learn about the microbiological analysis of Purified water and water for
injection including the method for detection of pathogens.
Water is used in verious processes in pharmaceutical
manufacturing. Microbial contamination in water may
cause contamination in pharmaceutical products. It is
necessory to analyse the Purified Water and Water for
Injection (WFI) for microbial contamination.

Related: Sampling, Preservation and Storage Procedure of Water Sample

There are two parts of microbial analysis of waterA. Total Microbial Count
B. Pathogen Detection
A. Total Microbial Count:
1.0 Filtration Method:
1.1 The amount of water to be filtered depends on the
expected microbial count. For higher microbial count,
dilution series should be prepared. Assemble the filtration
unit with filtration cap & vacuum pump.
1.2 Remove the cap of a sterile filtration unit having 0.45
micron membrane filter (diameter 47 mm) and transfer
1ml of water into the filtration unit.
1.3 Start the vacuum pump & allow the sample to flow
through the membrane filter. Wash the membrane by
filtering through it about 100 ml sterile 0.1% peptone
water. Switch off the vacuum pump.
1.4 Remove the membrane filter using sterile forcep from
the apparatus, & with the filtration side uppermost place
it on the surface of sterile solidified soybean casein digest
agar medium in such a way that no air bubbles form
between the filter and the culture medium.
1.5 Invert & incubate the plates at 30C to 35C for 5 days.
1.6 After 5 days incubation examine all the membranes.
Count the total no. of colony forming units on each

membrane filter (i.e. no. of cfu/ml).

1.7 For determination of yeast & mold use Sabouraud
chloramphenicol agar/ Sabouraud dextrose agar with
antibiotic instead of soybean casein digest agar. Follow
similar procedure like total bacterial count. Incubate the
plates at 20 C to 25 C for 5 days. Examine the plates for
absence
of
growth.
Related: Purified Water Storage and Distribution System
2.0 Pour Plate Technique (Alternative Method):

2.1
In
case
of
purified water & input water add 1 ml sample in duplicate
in sterile petridishes. Pour 15 ml to 20 ml of sterile
soybean casein digest agar (cooled at 45 C). Mix
thoroughly & allow solidifying the medium.
2.2 Invert & incubate the plates at 30C to 35C for 5 days.
After incubation examine the plates for total bacterial
count. Take maximum from the two plates & calculate the
no. of cfu/ml of water sample.
2.3 For detection of yeast & mold use Sabouraud
chloramphenicol agar/Sabouraud dextrose agar with
antibiotic & incubate the plates at 20C to 25C for 5 days.
After incubation examine the plates for absence of
growth.

Related: Purified Water System Validation

B. Pathogen Detection:
Filter 100 ml of sample through 0.45 membrane filter.
Transfer membrane filter to 100 ml sterile soybean casein
digest medium and incubate at 30-35C for 24 hours.
1.0 Detection of Escherichia coli:

1.1
Streak
a
portion of enriched soybean casein digest medium on the
surface of sterile MacConkey agar medium.
1.2 Incubate the plates at 30-35 C for 18-72 hours.
1.3 After incubation presence of brick red colonies on
MacConkey agar indicate the presence of Escherichia coli.
1.4 Carry out further confirmation by streaking the
colonies on the surface of levine eosine methylene blue
agar medium.
1.5 Blue black colonies with metallic sheen confirm the
presence of Escherichia coli
2.0 Detection of Salmonella:

2.1 Transfer 0.1 ml of

enriched soybean casein digest medium to 10 ml of
Rappaport Vassiliadis Salmonella enrichment broth and
incubate at 30-35 C for 24-48 hours.
2.2 Streak above media on the surface of Wilson and
Blairs BBS agar plate and incubate at 30-35C for 24-48
hours.
2.3 Growth of green colonies with blank centre and in 48
hours the colonies become uniformly black. Colonies
surrounded by a dark zone and metallic sheen indicates
the possibility of presence of salmonella.
2.4 If sub cultured on plate of xylose-lysine-deoxycholate
agar and incubate on 30-35 C for 24-48 hours. Well
developed red colonies with or without black centre
indicates the possibility of salmonella.
2.5 Carry out further confirmation by streaking the
colonies on the surface of triple sugar-iron agar by first
inoculating the surface of the slant and then making a
stab culture with the same inoculating needle and at the
same time inoculate a tube of urea broth. Incubate at 3035C for 18 to 24 hours. The formation of acid and gas in
the stab culture (with or without concomitant blackening)
and the absence of acidity from the surface growth in the
triple sugar iron agar, together with the absence of a red
colour in the urea broth, indicate the presence of
Salmonella.
3.0 Detection of Pseudomonas aeruginosa:

3.1 Streak a portion of

the enriched soybean casein digest medium on the surface
of cetrimide agar medium and incubate at 30-35C for 18
to 72 hours.
3.2 A greenish coloured colony indicates the possibility of
presence of Pseudomonas aeruginosa.
3.3 Carry out further confirmation by pigment and oxidase
tests.
Table 1 Test for Pseudomonas aeruginosa
S.N Medium
Characteri Fluoresc Oxid Gram
o
stic
ence
ase
Stain
morpholog in
UV Test
y
light
1 Pseudomonas Generally
Yellowis Posit Negativ
agar medium
colourless h
ive
e rods
for detection
to
of fluorescein Yellowish
2 Pseudomonas Generally
Blue
Posit Negativ
agar medium
Greenish
ive
e rods
for detection
of pyocyanin.
3.4 Streak representative suspect colonies from the
surface of cetrimide agar on to the surfaces of
pseudomonas agar medium for detection of fluorescein
and pseudomonas agar medium for detection of
pyocyanin.
3.5 Cover and invert the inoculated plate & incubate at 3035C for not less than 3 days.
3.6 Examine the streaked surfaces under ultra-violet light.
Examine the plates to determine whether colonies
conforming to the description in Table 1 are present.
3.7 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 the colony;
if there is no development of a pink colour, changing to
purple, the sample meets the requirements of the test for
the absence of Pseudomonas aeruginosa.
4.0 Detection of Staphylococcus aureus:

4.1 Streak a loop of enriched culture fluid soybean casein

digest medium on the surface of mannitol salt agar plate.
Incubate the plate at 30-35C for 18-72 hours. Examine
the plates after incubation.
4.2 If, upon examination of the incubated plate, none of
them contains yellow colonies with yellow zones the
sample meets the requirements for the absence of
Staphylococcus aureus.
4.3 If growth occurs further confirm by streaking the
colonies on the surface of media given in Table 2 and
incubate the plates at 30-35C for 48 hours.
Table 2 Tests for Staphylococcus aureus.
N Selective
Characteristic
Colonial
o. Medium
Morphology
Vogel-Johnson
Black surrounded by yellow
1
agar
zones
Black, shiny, surrounded by
2 Baird-Parker agar
clear zones of 2 to 5 mm.

4.4 Examine the plates to determine whether colonies

conforming to the description in Table 2. If none of colony
exhibits characteristics mentioned in Table 2 the sample
meets the requirements of the test for the absence of
Staphylococcus aureus.
5.0 Detection of Enterobacteriaceae:
5.1 Transfer 10 ml of enriched fluid soybean casein digest
medium in a flask containing 90 ml of sterile
Enterobacteria enrichment broth-Mossel, mix and incubate
at 30-35C for 24-48 hours.
5.2 If growth observed streak on the plate of violet red
bile glucose agar. Incubate the plate at 30-35C for 18-24
hours.
5.3 Observe the plate and if any colony found perform the
gram staining. If no colonies observed the sample passes
the test for absence of Enterobacteria.
5.4 If gram negative bacteria found in gram staining the
sample contains the Enterobacteria.
6.0 Detection of Shigella:
6.1 Transfer 1 ml of enriched fluid soybean casein digest
medium into 100 ml GN broth mix and incubate at 30 to
35C for 24 to 48 hours.
6.2 Streak a loop from GN broth on the plate of xylose
lysine deoxycholate agar.
6.3 Incubate the plates at 30-35 C for 24-48 hours.
6.4 A red coloured translucent colony without black centre
indicates possibility of presence of Shigella.
6.5 If no growth of microorganisms is observed, the
sample passes the test
7.0 Detection of Clostridia:
7.1 Heat 100 ml of sample at 80 C for 10 min. and cool
rapidly.
7.2 Filter 100 ml water sample through the sterile
membrane filtration unit.
7.3 Remove the filter from the apparatus with the help of
sterile forcep and transfer it to 100 ml Reinforced medium
and incubate it at 30-35C for 48 hours in anaerobic
condition.
7.4 After incubation streak a portion of enriched
Reinforced medium on the surface of Columbia agar
plates.

7.5 Invert & incubate the plates at 30-35C for 48 hours in

anaerobic condition.
7.6 If no growth of microorganisms is seen, the sample
passes
the
test.