1.0 Objective
To lay down a procedure for sampling and
microbial analysis of different grades of water.
2.0 Responsibility
Microbiologist/QC-Executive.
3.0 Accountability
Head-QC
executive
4.0 Procedure
4.1 Water
sampling
4.1.1 Carry out the sampling of water from the
sampling points as per the sampling plan.
4.1.2 Sanitize
the hand with 70% filtered Iso propyl alcohol (IPA) then wear hand
gloves.
4.1.3 Now
sanitize the hand gloves with 70% filtered IPA prior to sampling.
4.1.4 Take sterile 1Litre water sampling
container for sampling of water and label them with sampling point
reference
no.,
Date & Signature.
4.1.5 Before sampling drain the water from the
sampling point for 30 second then collect the water approximately 500mL
to 1000mL.
4.1.6 After taking the water sample quickly
close the top of the sampling container by using cotton plug or aluminium
foil.
4.1.7 Bring the water sample in microbiology
laboratory for analysis and enter the sample log book.
4.2
Water testing
4.2.1
Membrane filtration method (for purified water).
4.2.1.1 Prepare SCDA agar medium plates according to SOP.
4.2.1.2 Use membrane
filters having a nominal pore size not greater than 0.45 µm.
4.2.1.3 Take sterile
membrane filter holder and aseptically transfer1 mL water sample with the
help of filtration assembly.
4.2.1.4 Immediately
filter the water sample with the help of vacuum pump.
4.2.1.5 Wash each
filter with three quantities, each of about 100 mL peptone water solution pH
7.0.
4.2.1.6 Transfer the membrane filter on the surface of SCDA
media plates.
4.2.1.7 Incubate
the plate of SCDA medium at 30° to 35° C for five days.
4.2.1.8 Similarly,
carry out the above procedure by using Sabouraud DextroseAgar (SDA) by
incubating at 20-25ºC.
4.2.1.1 After 5 days of incubation period,
note down the observation of counts in log book.
4.2.2 Pour plate method (other than
purified water)
4.2.2.1 Pipette out 2 mL water sample and
place 1 mL each in two petriplates 9 cm diameter.
4.2.2.2 Pour 15-20 mL SCDA media whose
temperature is cooled to about 45ºC into these two petriplates.
4.2.2.3 Cover the petridishes, mix the
sample with agar by swirling and allow the media to solidify at room
temperature under laminar air flow (LAF).
4.2.2.4 Invert the petridishes and incubate
the plates of SCDA media at 30° to 35°C.
4.2.2.5 Similarly carry out the above
procedure by using SDA medium and incubate these plates at 20-25ºC.
4.2.2.6 Examine the growth, count the
number of colonies and express the average for the two plates, in terms of
Colony forming units (Cfu) per mL of specimen.
4.2.2.7 The highest number of colonies are
less than 100 for bacteria and 10 colonies for fungi.
4.2.2.8 After 5 days of incubation period,
note down the observations in Log book.
4.3 Procedure for pathogen testing of
water
4.3.1
Transfer 10 mL water sample in 100 mL soyabean casein digest medium (SCDM).
4.3.2 After incubation, observe for growth in
terms of turbidity, if it is observed perform pathogen testing.
4.4 Test for Escherichia coli (E.
coli).
4.4.1 Transfer 1 mL of 24 hour old culture]
suspension (SCDM) in 5 ml MacConkey’s Broth (MB) tube containing Durham’s
tube and incubate it at 42° to 44° for
24 to 48 hours.
4.4.2 After incubation, observe for purple to
yellow color change of the MacConkey’s broth
with gas production in the
4.4.3 Subculture 1 mL of broth on a plate of
MacConkey’s agar (MA) at 35° to 37° for 18 to 72 hours.
4.4.4 If growth of red, non-mucoid colonies is
observed after incubation, then transfer 1 mL MacConkey’s broth in 5 mL
peptone water (PW) tube and incubate at 42° to 44° for 18 to 24 hours.
4.4.5 After incubation, add 0.5 mL Kovack’s
reagent into the above peptone water and if red color ring appears the Indole
test indicates the possible presence of E.coli.
4.4.6
Perform Gram staining of the colonies observed on MA .
4.4.7
Observe for presence of Gram negative rod.
4.4.8 If Gram negative rod is present, streak loop full colony on Levin
Eosin-Methylene Blue Agar (LEMBA) plate.
4.4.9
Incubate this LEMBA plate at 35-37° C for 18 to 72 hours.
4.4.10 After incubation, observe the plate
and if none of the colonies exhibits a
characteristic metallic sheen under reflected light, and if none exhibits a
blue-black appearance under transmitted light, the test specimen meets the
requirement for the absence of E. coli.
4.5 Test for Salmonella Spp.
4.5.1 Transfer 1
mL of 24 hour old culture] suspension (SCDM) in 10 mL Fluid Selenite Cystin
Medium (FSCM) and Tetrathionate Broth (TB) and incubate at 42° to 44° C for
18 to 24 hours.
4.5.2
Subculture on at least two different agar media chosen from agar media,
Bismuth Sulphite Agar (BSA), Deoxycholate Citrate Agar (DCA) and Brilliant
Green Agar (BGA). Incubate at 35° to 37° C for 18 to 72 hours.
4.5.3
The probable presence of salmonella is indicated by the growth
of cultures having the following
appearance:
BSA: well-developed, colour less
colonies.
DCA: well-developed, red
colonies, with or without black centers.
BGA: small, transparent, colourless
or pink opaque-white colonies often
surrounded by a pink or red
zone.
4.5.4 Perform Gram staining according to
SOP/QM/14 if the above characteristic colonies are observed.
4.5.5 If Gram negative rod is present, streak loop full culture from
suspected colonies on Triple Sugar Iron (TSI) agar slant in tubes, using
surface and deep inoculation (stabbing) and incubate at 41° to 43° C for 18
to 24 hours.
4.5.6 The presence of salmonella spp. is
confirmed if there is a change of color from red to yellow and usually a
formation of gas, with or without production of hydrogen sulphide in the agar
in the deep inoculation but not on the surface culture.
4.6
Test for Pseudomonas aeroginosa
4.6.1 Strick loop full of 24 hour old culture
[refer point 4.3.1, 4.3.2] suspension (SCDM) on a plate of Cetrimide Agar
(CA).
4.6.2 Incubate this plate at 35 - 37° C for 18
- 72 hours.
4.6.3 Perform Gram staining of the colonies
observed on CA according to SOP.
4.6.4 Observe for presence of Gram negative
rod.
4.6.5 If Gram negative rod is present, streak representative suspect
colonies from the CA plate on the surface of Pseudomonas fluorescin agar(PFA)
medium for detection of fluorescin and Pseudomonas pyocyanin agar (PPA)for
detection of pyocyanin.
4.6.4 Incubate these plates at 35° - 37° C for
48 to 72 hours.
4.6.5 Examine the streaked surface of the both
agar media under UV light, and interpret the results with reference to the
following table.
4.6.6 Oxidase disc test:
4.6.7 Transfer the
colony from the PFA medium or PPA medium to disc that previously impregnated
with N, N- dimethyl-p-phenylenediamine dihydrochloride.
4.6.8
If the disc colour is changed (from white to purple) within
five to ten seconds it confirms the presence of
Pseudomonas
aeroginosa
4.7 Test for Staphylococcus aureus:
4.7.1
Strick a loop full of 24 hour old culture suspension (SCDM) on a plate
of Mannitol Salt Agar (MSA) or Vogel-Johnson Agar (VJA) or Baired Parker Agar
(BPA).
4.7.2 Incubate the plate at 35° to 37° C for 48
to 72 hrs.
4.7.3 After incubation, examine the streaked
plates and interpret the results with reference to the following table.
4.7.4 Perform Gram staining of the suspect
colonies observed according to SOP/QM/18.
4.7.5 Observe for presence of Gram positive
cocci in cluster.
4.7.6
If Gram positive cocci in cluster are present, perform coagulase test.
4.7.7
Take sterile test tubes each containing 0.5mL of mammalian, preferably
rabbit or horse, plasma with or without suitable additives.
4.7.8
Transfer representative suspect colonies from the surface of the VJA or
MSA or BPA plate to the above individual tubes and incubate in a water bath
at 35° to 37° C.
4.7.9
Examine the tubes after 3 hours and at suitable intervals up to 24
hours. Compare the negative and positive controls.
4.7.10 If coagulation is observed, the
specimen confirms the presence of Staphylococcus aureus.
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Thursday 9 March 2017
Sampling and microbial analysis of different grades of water.
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