This SOP is provide written guidelines to sample and establish the Microbial quality of potable water obtained from BOREWELLS. To sample and establish the Microbial quality of purified water used in manufacture of non -sterile products.
2.0 Scope :
This SOP extends to Potable water from all user points, DM water from all user points & distilled water.
3.0 Responsibility :
Q.C. Microbiologist
4.0 Procedure :
4.1.0 Collection of sample:
4.1.1 Only trained and evaluated personnel should collect the water sample.
4.1.2 Sampling personnel should wear sterile surgical gloves and nose mask before sampling. Disinfect the gloved hands with 70% IPA.
4.1.3 Collection of sample is to be done using a sterile containers only. Carry atleast two extra sterile containers for exigencies. For chlorine treated water samples add 0.5ml of 5% w/v sodium thiosulphate solution to 250ml bottle and sterilize in autoclave.
4.1.4 Care must be taken to avoid accidental contamination of the sample during collection.
4.1.5 External surfaces of all sampling points are to be sprayed with 70% v/v IPA before sample is withdrawn.
4.1.6 Flush atleast 10 ltrs. of water before collection of sample.
4.1.7 Carefully open the sterile containers for collection point and to avoid contact the neck of the flask with hands and tubings.
Do not use the container if the neck or the cotton plug comes in contact with any of other surfaces.
4.1.8 Sterile conical flask fill with approximately 225ml of water from the sampling point.
4.1.9 Plug the sample container securely and labeled adequately with details like user point identification , date and identity of the sample.
4.1.10 Remove the gloves at the end of sampling.
4.1.11 Collected samples should be tested within one hour of collection. It is permitted to hold samples at 2 to 8°C for period of four hours before testing.
4.2.0 Frequency of sampling and sampling points
4.2.1 Refer Annexure – I.
4.3.0 METHOD:
4.3.1 BY MEMBRANE FILTERATION ( for TAMC & TYMC)
4.3.2 EQUIPMENT:
Petridishes Sterile Membrane filter 0.22 micron pore size.
Sterile filter cones.
Sterile forceps & Sterile 1 ml pipette.
Vacuum line and reservoir.
4.4.0
Media and Reagents: For composition and preparation refer
SOP No. QC/MB/04/1.3
4.4.1
BAIRD PARKER AGAR (BPA)
BISMUTH SULPHITE AGAR (BSA)
BRILLIANT GREEN AGAR (BGA)
CETRIMIDE AGAR (CA)
DEOXYCHOLATECITRATE AGAR (DCA)
LEVINE EOSIN-METHYLENE BLUE AGAR (EMB)
LACTOSE BROTH (LB)
MAC CONKEY AGAR (MCA)
MAC CONKEY BROTH (MCB)
MANNITOL SALT AGAR (MSA)
NUTRIENT BROTH (NB)
PSEUDOMOMAS –CN AGAR. (P-CNA)
PSEUDOMOMAS –P AGAR (P-PA)
SABOURAUD CHLORAMPHENICOL AGAR (SCA)
SOYA BEAN CASEIN DIGEST AGAR (SCDA)
SOYA BEAN CASEIN DIGEST MEDIUM (SCDM)
SELENITE F BROTH (SF)
TETRATHIONATE BILE BRILLIANT GREEN BROTH (TBBGB)
TRIPLE SUGAR-IRON AGAR (TSIA)
UREA BROTH MEDIUM (UB)
VOGEL JOHNSON AGAR (VJA)
XYLOSE-LYSINE-DEOXYCHOLATE AGAR (XLD)
All tests to be carried in clean room under LAF only.
Water sample dilution :
dm Water and Potable water: 1ml of water sample diluted to 100ml with sterile distilled water.
4.4.2 Technique: Remove the cap of filter cone with membrane filter and transfer 100 ml of diluted sample from the bottle into the sample holder.
4.4.3 Open the vacuum valve to get negative pressure and allow the sample to flow through the membrane filter and rinse with sterile distilled water. Close the vacuum valve.
4.4.4 Remove the membrane filter using sterile forceps and place it on a pre-incubated sterile SCDA for bacteria and SCA for fungi.
4.4.5 Incubate the SCDA plates for 72 hours at 30° to 35°C.
4.4.6 Incubate the SCA plates for 120 hours at 20° to 25°C.
4.4.7 After completion of incubation count the number of colonies per ml.
4.4.8 Record the results in the prescribed format.
4.4.9 TAMC Limits:
Standard Limit : NMT 50 cfu/ml.
Alert Limit : MT 10 cfu/ml.
Action Limit : MT 25 cfu/ml.
Action plan microbial count exceeding alert limit
During routine monitoring if the microbial count exceeds alert limit the following steps are to be taken.
QA department, Production department and Maintenance department should be informed.
Increase the frequency of monitoring.
If the contaminant levels are seen consistent or increase, advise for sanitizing water system as per general SOP for sanitization.
Monitor the bioload of water till it comes below alert limit.
During this period water collection will be continued for production.
Action plan count exceeding action limit
During routine monitoring if the microbial count exceeds action
limit the following actions are to be taken.
QA department, Production department and maintenance department should be informed.
Carry out the sanitization of water system as per cleaning schedule & increase the frequency of monitoring till the count comes below alert limit.
During this period water collection will be continued.
If the microbial count is increasing, the frequency of sanitization must be increased and continues monitoring for microbial count must be carried out till the level comes below alert limit.
Action plan count exceeding standard limit
During routine monitoring if the microbial count exceeds standard limit the following actions are to be taken QA department, Production department and maintenance department should be informed.
Inform manufacturing department to stop collection of water.
Increase the frequency of monitoring, investigate the cause and carry out the sanitization of water system as per SOP for sanitization.
Monitor the microbial count till the bioload level was below alert level. Recheck the bioload of all products, which were manufactured during the last two checks drawing extra samples in order to evaluate the effect on product. Thorough investigation must be carried out for the route cause of increase in Bioload of water.
4.6.0 TEST FOR PATHOGENS
4.6.1 Transfer 100ml of water sample filtered through 0.22mm membrane filter and transfer to 100ml of SCDB and incubate for 24 to 48 hours at 30°to 35°C.
4.6.2 If SCDB is turbid the following tests for Staphylococcus aureus & Pseudomonas aeruginosa, Enterobacteriaceae ( to include E.coli & Salmonella sps) and other objectionable must be performed.
TESTING FOR E.Coli:
4.6.3 Add 1ml of the enrichment of SCDB to a test tube containing 10ml
of MacConkey broth . Incubate at 37° + 1°C for 48 hours. If the contents of the tube shows acid or gas carry out the secondary test.
4.6.4 SECONDARY TEST – (a) Add 0.1ml of the enrichment to 10ml of MacConkey broth, and (b) 10ml of peptone water or SCD broth.
4.6.5 Incubate 43.5°C to 44.5°C for 24 hours
Tube (a) for acid or gas and tube (b) for indole.
4.6.6 For indole test add 0.5ml of Kovac’s reagent, shake well and allow to stand for 1 minute. If the upper layer shows red colour then indole is positive.
4.6.7 The presence of acid and gas and of indole indicates the presence of E.Coli.
4.7.0 ALTERNATIVE TEST
4.7.1 By means of an inoculation loop, streak a portion of SCDB enrichment onto the surface of MCA.
4.7.2 Incubate at 37° + 1°C for 18 to 24 hours.
4.7.3 At the end of incubation observe the plates for growth. If no growth is observed then report as absence of Escherichia Coli.
4.7.4 If growth is observed and colonies are matching with the description given below the proceed with further identification.
4.7.5 Subculture the suspect colonies individually on to EMB Agar the plate invert &Incubate at 37° + 1°C for 24 to 48 hours.
4.7.6 If none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue black appearance under transmitted light, the specimen meets the requirements of the test for the absence of Escherichia Coli.
SELECTIVE MEDIUM
CHARACTERISTIC COLONIAL MORPHOLOGY
GRAM’S STAIN
MCA
Brick red; may have surrounding zone of precipitated bile.
Negative rods
EMB
Characteristic metallic sheen under
Negative rods
reflected light and blue black apperance under transmitted light.
Limit: E.coli should be absent/100ml.
4.8.0 TESTING FOR SALMONELLA SPECIES
4.8.1 Add 1.0ml of portion of LB or SCDB enrichment to the tube containing 10ml of TTBGB.
4.8.2 Incubate at 37° + 1°C for 18 to 24 hours.
4.8.3 At the end of the incubation streak from TTBGB onto any two of the following four agar media’s BGA, BSA,DCA &XLDA.
4.8.4 Incubate inverted plates at37° + 1°C for 24 to 48hours
4.8.5 At the end of incubation period observe the plates.
4.8.6 If none of the colonies confirms to the description given below then the specimen meets the requirements for the absence of Salmonella species.
SELECTIVE MEDIUM
CHARACTERISTIC COLONIAL MORPHOLOGY
GRAM’S STAIN
BSA
Black or green.
Negative rods
BGA
Small transparent, colorless or pink to
Negative rods
white opaque (Frequently surrounded by
pink to red zone).
DCA
Colourless translucent, round and
Negative rods
glistening colonies.
XLDA
Red, with or without black centres
Negative rods
4.8.7 If colonies are matching the description in above table, proceed with further identification by transferring suspect colonies individually, by means of an inoculating wire to a butt-slant tube of triple sugar- iron Agar Medium by first Streaking the surface of slant and then stabbing the wire well beneath surface.
4.8.8 Incubate the tubes at 37°c to 1°c for 18 to 24 hours.
4.8.9 At the end of the incubation examine the slants.
4.8.10 If examination discloses no evidence of tubes having alkaline (red)slants and Acid (yellow) butts (with or without contaminant blackening of the butt from Hydrogen sulfide production). The specimen meets the requirements of the test for the genus of Salmonella.
Limit: SALMONELLA SPECIES should be absent/100ml.
4.9.0 TESTING FOR STAPHYLOCOCCUS AUREUS
4.9.1 Streak the enrichment SCDB onto MSA, VJA or BPA.
4.9.2 Incubate 37°c +1°c for 24 hours.
4.9.3 At the end of incubation observe the plates for growth. If no growth is observed the report as absence of Staphylococcus aureus.
4.9.4 If growth is observed then identify the suspected colonies.
Morphologic Characteristic of Staphylococcus aureus on selective agar media.
SELECTIVE MEDIUM
CHARACTERISTIC COLONIAL MORPHOLOGY
GRAM’S STAIN
VJA
Black colonies surrounded
by yellow zone
Positive cocci
(in clusters)
MSA
Yellow colonies with yellow zones
Positive cocci
(in clusters)
BPA
Black, shiny, surrounded by clear zones of
2 to5mm
Positive cocci
( in clusters)
4.9.5
COAGULASE TEST:
With the aid of an inoculating loop, transfer representative suspect colonies from the agar surfaces of the VJA, BPA or MSA to individual tubes each containing 0.5 ml of mammalian plasma, preferably rabbit, horse or human plasma with or without suitable additives. Incubate 37°c.
4.9.6 Examine the tubes at 3 hrs and subsequently at suitable intervals upto 24hrs.
4.9.7 Test positive and negative controls simultaneously with the unknown specimens is taken. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of staphylococcus aureus.
Limit: STAPHYLOCOCCUS AUREUS should be absent/100ml.
5.0 TESTING FOR PSEUDOMONAS AERUGINOSA
5.1.0 Streak the SCDB enrichment onto CA.
5.1.1 Incubate the plates at 30 to 35°c for 18 to 24 hours.
5.1.2 At the end of incubation observe the plates for growth. If no growth is observed the report as absence of Pseudomonas aeruginosa.
5.1.3 If growth is observed then identify the suspected colonies.
5.1.4 Morphologic characteristics of Pseudomonas aeruginosa on selective and diagnostic agar media.
5.1.5 With the aid of an inoculating loop, streak representative suspect colonies from the agar surface of CA on the agar surfaces of Pseudomonas CN Agar Medium for Detection of Fluorescein and Pseudomonas P.Agar for detection of Pyocyanin contained in petri dishes.
5.1.6 If numerous colonies are to be transferred, divide the surface of each into Quadrants, each of which may be inoculated from a separate colony.
5.1.7 Cover and invert the inoculated media and incubate at 37+1°c for not less than 3 days. Examine the streaked surfaces under ultraviolet light. Examine the plates to determine whether colonies having the characteristic listed in the table below are present.
5.1.8 Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test.
5.1.9 Upon the colonial growth place or transfer colonies of filter paper that previously has been impregnated with freshly prepared 1%w/v solution of N,N,N1,N1- tetramethyl-4-phenylenediamine dihydrochloride.
5.1.10 If there is no development of a pink colour, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa.
5.1.11 The presence of Pseudomonas aeruginosa may be confirmed by the other suitable cultural and biochemical tests, if necessary.
Limit: PSEUDOMONAS AERUGINOSA should be absent/100ml.
5.2.0 PREPARATION OF CULTURE SUSPENSION
5.2.1 Freshly sub-cultured (18-24 hours ) culture slant is taken.
5.2.2 Add about 5ml of sterile normal saline 0.9% w/v and shake gently to get the cultures into the suspension.
5.2.3 Count the number of cells in the suspension.
5.2.4 Dilute the suspension to obtain a concentration of 100 organisms/ml using sterile normal saline 0.9% w/v.
5.2.5 Pipette out 1ml of the above suspension into a sterile petriplate and then pour 15ml to mix the contents and allow to solidify. POUR PLATE TECHNIQUE.
5.2.6 Incubate the plates at 30°c to 35°c for 24 to 48 hours.
5.2.7 At the end of the incubation period count the number of colonies.
5.2.8 The culture suspension is suitable for use if the number of colonies observed are 10 to 100 cfu/ml.
5.2.9 If a higher count is observed, then dilute the culture suspension to obtain the desired range.
5.2.10 Store the culture suspension at 2°c to 8°c for not more than a week and use it whenever needed.
5.2.11 If the growth promotion test fails the tests are not valid.
5.2.12 Negative control: Incubate one plate of each type of agar media from each batch of autoclaved media at respective incubation temperature.
5.2.13 Media should not show any microbial contamination.
6.0 Destruction of the used media and plates :
The exposed plates should be autoclaved in an Autoclave for destruction after the results are taken and then disposed off.
7.0 Trends to be plotted with standard, action and alert limits are defined.
Identify the organism based on the high counts and colony morphology by Gram’s staining.
Frequency of Identification: Once in a week.
In case any objectionable organism found it should be continued to species level in an out side approved laboratory.
SAMPLING POINTS & LOCATIONS
FREQUENCY OF SAMPLING
DW 1
MIXED BED OUTLET
Once in a week.
DW 2
FILTER (2) OUTLET
Once in a week.
DW 3
UV LIGHT – 1 OUTLET
Once in a week.
DW 4
DM – WATER STORAGE TANK
Once in a week.
DW 5
BOTTLE WASHING M/C
(POINT 1 – DM WATER TANK)
Once in a month.
DW 6
BOTTLE WASHING M/C
(POINT 2 – DM WATER TANK)
Once in a week.
DW 7
BOTTLE WASHING M/C (POINT 3)
Once in a month.
DW 8
EQUIPMENT WASHING AREA – 1
Once in a month.
DW 9
BULK LIQUID STORAGE AREA
Once in a week.
DW 10
LIQUID MFG – POINT 1
Once in a month.
DW 11
LIQUID MFG – POINT 2
( DM WATER STORAGE)
Once in a month.
DW 12
EQUIPMENT WASHING AREA – 2
Once in a month.
DW 13
LIQUID MFG – POINT 3
Once in a month.
DW 14
GRANULATION AREA – 1
Once in a month.
DW 15
EQUIPMENT WASHING AREA – 1
Once in a month.
DW 16
PASTE PREPARATION AREA – 1
Once in a month.
DW 17
GRANULATION AREA – 2
Once in a month.
DW 18
EQUIPMENT WASHING AREA – 2
Once in a month.
DW 19
EQUIPMENT WASHING AREA – 3
Once in a month.
DW 20
EQUIPMENT WASHING AREA – 4
Once in a month.
DW 21
COATING SOLUTION PREPARATION AREA
Once in a month.
DW 22
DISTILLED WATER PLANT
Once in a month.
DW 23
RETURN LOOP OUTLET
Once in a week.
DISTILLED WATER (Q.C.DEPARTMENT)
Once in a week.
MONTHLY SCHEDULE FOR DM WATER, POTABLE WATER , DISTILLED WATER AND BORE WELL WATER ALALYSIS SHOULD BE RECORDED IN A SUITABLE CHART
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