Microbiological Analysis of Purified Water, Pretreatment Water and Source Water

1.0 OBJECTIVE

To lay down the procedure for check the microbiological quality of Raw water, RO water and Purified water.

2.0       SCOPE

This procedure is applicable to microbiological analysis of Raw water, RO water and Purified water for determination of Microbial quality in Microbiology laboratory of Quality control department .

3.0   RESPONSIBILITY

Executive – Microbiologist

   4.0 DISTRIBUTION

Master copy                :           Quality Assurance Department

Controlled Copy – 1     :           Quality Control Department

 5.0 PROCEDURE 

      5.1 Required Instruments & accessories:

• Sterile petriplates (90 mm)
• Filtered 70% v/v IPA
• Colony counter
• Laminar Air Flow
•   Water bath
• Sterile forceps & Spatula
• Sterile filtration units
• Microscope
• Nichrome loop
• Glass Slide and Glass rod
• Sterile Membrane Filters (0.45μm)
• Vacuum Pump
• Forceps
• Scissor
• Burner

5.2 Required Media & Microbial cultures:

• Buffered sodium chloride peptone water or 0.1% Buffered peptone water (BPW) / Peptone water (PW).
• Soyabean casein digest medium (SCDM)
• Soyabean casein digest agar (SCDA)
• R2A agar
• Plate count agar
• MacConkey agar (MCA)
• MacConkey broth (MCB)
• Eosin Methylene Blue (EMB)
• agarMannitol Salt agar (MSA)
• Rappaport Vassiliadis Salmonella Enrichment broth (RVSEB)
• Triple Sugar Iron agar (TSI)
• Enteriobacteria Enrichment Broth Mossel (EEB)*
• Xylose lysine deoxycholate agar (XLDA)*
• Cetrimide agar (CA)
• Violet Red Bile Glucose Agar (VRBGA)
• Pseudomonas agar pyocyanin
• Pseudomonas agar
• fluoresceinStaphylococcus aureus
• Pseudomonas aeruginosa
• Bacillus subtilisEscherichia coli
• Salmonella abony
• Candida albicans
• Aspergillus niger
• Shigella boydii

* Media Do not Autoclave

5.3 Required Test Reagent:

• Oxidase Disc
• Gram Staining Kit
• Kovac’s Reagent
• Mammalian (rabbit or horse) plasma with or without suitable additives
• Schaeffer & Fulton’s Spore stain A & B

5.4 Methodology:

Media Preparation:

• The media given above are suitable for the water analysis.

5.5 Total Viable Aerobic Count (TVAC):

• The test describes the quantitative enumeration of viable aerobic microorganism present in the sample. The test procedure must be carried out as prescribed below.

5.6 RO water and Purified water Analysis:

• Mark two pre-incubated (24-48 hrs.) media plate of R2A agar with Media name, Sample Name, Sampling point no., date and sign of tested by person at the bottom side.

• Aseptically transfer 1 ml water sample to 99 ml sterile BPW or PW or purified water & filter whole 100 ml of solution to 0.45mm filter paper.

• Arrange a sterile membrane filter assembly and aseptically filter the sample through sterile 0.45mm membrane filter.

• Aseptically with the help of sterile forcep, pick up the membrane filter and place it on the surface of a marked R2A agar plate.
• Place the filter on the surface of a marked second R2A plate with the help of a sterile forcep.
• Incubate R2A plates at 30-35°C for 5 days in inverted position.
• Arrange a sterile membrane filter assembly and aseptically filter the sample through sterile 0.45mm membrane filter.
• Aseptically with the help of sterile forcep, pick up the membrane filter and place it in 100 ml sterile SCDM for pathogen testing.
• Incubate the SCDM container at 30-35°C for 18-24 hrs.
• After completion of incubation, count the colonies present on the membrane filter on duplicate plate and record it as Avg. CFU/ml as a result and record it as CFU/ml in respective format.

• 5.7 Raw water & Pretreatment water Analysis:
• Mark 2 sterile plates with media name, sample name, sampling point no., date and sign of tested by Person at the bottom side.
• Aseptically pipette out 1 ml of water sample to two sterile marked petriplates and pour 15-20 ml Soyabean casein digest agar in labeled plates.
• Media should be cooled to around 45°C.Rotate the plates in clockwise and anticlockwise directions for homogenous mixing and let the media solidify.
• Incubate SCDA agar plates at 20-25°C for 3 days for fungal observation after that transfer to at 30-35°C for 2 days for bacterial observation in inverted position.
• After completion of incubation period, Count the number of colonies of both duplicate plates and record it as Avg. CFU/ml as a result in respective format.
• Filter 100ml of raw water sample in 0.45 µ filter paper & after filtration inoculate the filter paper to 100 ml of SCDM medium for pathogen testing.
• Incubate the SCDM container at 30-35°C for 18-24 hrs.Note: – Round off to complete number wherever average counts observed is in decimal places.  For e.g., if the average count is 32.5 then report the result as 33 CFU/ml.

5.8 Negative Control:

Negative control (PW water & RO water):

• Take one pre-incubated plate of R2A and mark it as mark them as “Negative control” along with media name & date.
• Aseptically filter 100 ml of suitable sterile diluent (BPW / PW) through membrane filter assembly and transfer the membrane to the surface of R2A plate.
• Incubate R2A plates at 30-35°C for 5 days in inverted position.

5.9 Negative control (Raw Water):

• Add 1 ml of sterile diluent in to the petriplate then pour SCDA agar in one sterile petriplate and allow them to solidify, mark them as “Negative control” along with media name & date.

• Incubate SCDA plates at 20-25°C for 3 days after that transfer to at 30-35°C for 2 days in inverted position.

5.10 Positive Control:

Positive control (PW water & RO water):

• Add 0.1 ml of 10-100 cfu/ml concentration of P.aeruginosa & B.subtilis culture organism on the R2A agar plate and spread it mark them as “Positive control” along with media name & date.

5.11 Positive control (Raw water):

• Add 1 ml of 10-100 cfu/ml concentration of one bacterial & one fungal culture like S.aureus, B.subtilis, P.aeruginosa, Salmonella, E.coli, S.boydii, C.albicans & A.niger (Take one bacterial & one fungal culture alternatively) culture organism and add 15-20 ml of SCDA agar media and it mark them as “Positive control” along with media name & date.
• Incubate SCDA plates at 20-25°C for 3 days after that transfer to at 30-35°C for 2 days in inverted position.

• Test for Indicator Pathogens in Purified water, RO water, and Raw water:
• Mark One 100 ml Soyabean casein digest medium tube with the Sample Name, sampling point no., date & Analyst.Filter 100 ml of the water sample through sterile 0.45mm membrane filter using sterile filtration assembly.Transfer the membrane filter aseptically to SCDM tube with sterile forceps.If required shake the media tube to dip the membrane filter completely in the media.Proceed with SCDM tube as per below:-

• Test for Salmonella species:Incubate the inoculated SCDM tube at 30-35°C for 18-24 hrs along with a blank SCDM tube as negative control.After completion of incubation period, transfer 0.1ml to 10 ml Rappaport Vassilidis Salmonella Enrichment Broth, mix and incubate at 30-35°C for 18-24 hrs.If growth occurs, aseptically streak a loopful from RVSEB to on the surface of XLDA. Invert and incubate the petriplates at 30-35°Cfor 18-48 hrs. If characteristic growth is found (Refer Table II) then proceed for confirmatory identification test (Refer Table III).The sample complies for the absence of Salmonella if the confirmatory identification test results are found negative.Observe and record the observation in the respective format.

• Test for Pseudomonas aeruginosa:Incubate the inoculated SCDM tube at 30-35°C for 18-24 hrs..After completion of incubation period, aseptically streak a loopful of SCDM tube on the surface of cetrimide agar plate. Invert the petriplates and incubate at 30-35°C.for 18-72 hrs. If characteristic growth is found (Refer Table II) then proceed for further confirmatory identification test (Refer table III).The sample complies for the absence of P.aeruginosa if the confirmatory identification test results are found negative.Observe and record the observation in respective format.

• Test for Staphylococcus aureus:Incubate the inoculated SCDM tube at 30-35°C for 18-24 hrs.After completion of incubation period, aseptically streak a loopful from SCDM tube on the surface of Mannitol Salt agar. Invert the petri plate and incubate at 30-35°C for 18-72 hrs.If characteristic growth is observed (Refer Table II) then proceed for confirmatory identification test (Refer table III).The sample complies for the absence of S.aureus if the confirmatory identification test results are found negative.Observe and record the observation in respective format.

• Test for Escherichia coli:Incubate the inoculated SCDM tube at 30-35°C for 18-24 hrs.After incubation period, pipette out 1ml and add to 100 ml MacConkey’s broth, mix and incubate at 42-44°C for 24- 48 hrs.Examine the media for acid and gas production. If acid or gas is produced then aseptically streak a loopful from MCB on the surface of MacConkey’s agar and incubate the plates at 30-35°C for 18-72 hrs. If characteristic growth is observed (Refer Table II) then proceed for confirmatory identification test (Refer table III).The sample complies for the absence of E.coli if the confirmatory identification test results are found negative.Observe and record the observation in respective format.

  5.12 Test for Shigella boydii:

• Incubate the inoculated SCDM tube at 30-35°C for 18-24 hrs.
• After incubation, shake the container and transfer 1.0 ml of soya bean casein digest broth to
• 100 ml of GN broth (Medium 10) and incubate at 30-35°C for 24-48 hours.
• Subculture on plate of xylose lysine deoxycholate agar.
• Incubate at 30-35°C for 24-48 hours.
• If characteristic growth is observed (refer table II).       
• The sample complies for absence of Shigella boydii if the confirmatory identification test results are found negative.
• Observe and record the observation in respective format.

5.13    Acceptance Criteria:

The acceptance criteria of TVAC of different type of water are defined in following below

           Table-I.

           If the acceptance criterion is not meet then the investigation shall be done as per SOP.

 Table I: Acceptance Criteria

S. No.	Type of Water	Parameter	Alert Limit	Action Limit	Standard Limit
1.	Raw Water	TVAC (cfu/ml)	250	350	500
2.	RO Water	TVAC (cfu/ml)	50	60	100
3.	Purified Water	TVAC (cfu/ml)	50	60	100

5.14 Result Interpretation:

• There should not be any contamination in the negative control or blank.
• Positive shall be show growth.
• Record the TVAC results in the respective formats and records.

Table II – Morphological characteristics of Indicator Pathogen on selective agar

S. No.	Indicator Pathogen	Selective Media	Characteristic Growth
1.	Salmonella sps.	XLDA	Red, with or without Black centres
2.	P.aeruginosa	CA	Generally greenish and show greenish in fluorescence in UV light
3.	S.aureus	MSA	Yellow colonies with yellow zone
4.	E.coli	MA	Brick red colonies, may be surrounded by precipitated bile
5.	Shigella boydii	XLDA	Red colored translucent colony without black centre

Table –III-Confirmatory Identification Test for Indicator Pathogen

S. No.	Indicator Pathogen	Confirmatory test	Result	Gram Staining
1	Salmonella species	Streak Triple- Sugar Iron agar medium from XLDA media, first in the slant and then stabbing the wire well beneath the surface and incubate it at 35-37°C for 18-48 hrs.	Alkaline (red) slants and Acidic (yellow) butt with or without H2S production (Black colour development)	Gram Negative Rods
2	Ps. aeruginosa	Oxidase  Test– Transfer a characteristics colony from CA plate to Oxidase disc containing N, N-dimethyl p-phenylenediamine dihydrochloride (oxalate). Pigment Test – Streak out suspect colonies from 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. Incubate at 35 to 37°C for not less than 3 days.	Development of pink colour changing to purple within 5 to 10 seconds shows presence of P.aeruginosa. Examine the streaked surfaces under ultra-violet light Presence of colonies conforming to the description in Table I indicates the presence of P.aeruginosa.	Gram Negative Rods.

3

S. aureus

Coagulase test – Transfer representative colonies from agar surface into 0.5 ml of mammalian plasma preferably rabbit or horse plasma with or without suitable additives. Incubate in a water-bath at 37° ± 1°C, examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours.

If coagulation in any degree is observed, then shows the presence of S.aureus.

Gram positive cocci

4

E. coli

Streak the suspect colonies individually on the Levine EMB agar and incubate at 18-48 hrs at 30-35°C. Indole Test – Take the 5 ml of peptone water. Mix the suspected colonies and incubate in a water bath at 43.5 to 44.5oC for 24 hours. Hang the kovac’s reagent strip in the test tube, and allow to 1 minute;

Metallic sheen observed under reflected light and Blue-Black appearance under transmitted light. if strip colour is changed into red. Indole is present      medium confirms the presence of E.coli..

Gram Negative Rods.

6.0 PRECAUTIONS:

• Always use media and materials sterilized in a validated autoclaving cycle.
• Disinfect the hands with sterile 70% IPA at frequent intervals during analysis.
• Shake the sample preparation before each inoculation for homogenous mixing.
• Properly label all the tubes and plates used in analysis.