by | PURPOSE: This Sop Describes the Procedure for MLT For Purified Water / Raw Water / RO Water. SCOPE: This Sop Is Applicable to the Procedure for MLT For Purified Water / Raw Water/ RO Water. RESPONSIBILITY: Microbiologist ACCOUNTABILITY: QC Manager. PROCEDURE Test for Aerobic Microbial Count (By Plate Count Method):Pipette the Water sample & input water add 1 ml sample in duplicate sterile petri dishes by sterile pipette. Pour 15 ml to 20 ml of sterile soybean casein digest agar (cooled at 45 °C). Mix thoroughly & allow solidifying the medium.Invert & incubate the plates at 30°C to 35°C for 5 days. After incubation count the colony and maintain the record .For detection of fungal, yeast & mold use Sabouraud dextrose agar & incubate the plates at 20°C to 25°C for 5 days. After incubation count the colony and maintain the record .Test for Pathogen:Escherichia coli Take 1ml of water sample and incubate with soyabean casein digest broth at 30 – 350C for 18 -24 hours.After incubation shake the broth and transfer 1ml to 100ml of MacConkey Broth, incubate at 42 – 440C for 24 -48 hours.After completion of incubation, subculture on a plate of MacConkey agar and incubate at 30 – 350C for 18 -72 hours.If Growth of pink, non-mucoid colonies indicates the presence of Escherichia coli. This should be confirmed by identification test of Escherichia coli, record the maintain Protocol for specific microorganism test.Identification test for Escherichia coli After incubation add 0.5 ml of Kovac’s reagent to the peptone tube and shake allow the stand the tube for 1 -5 minutes.Cherry red colour ring produced indicates the presence of Escherichia coli.Salmonella Take 10ml of water sample and incubate with soyabean casein digest broth at 30 – 350C for 18 -24 hours.After incubation shake the broth and transfer 0.1ml to 10 ml of Rappaport Vassiliadis Salmonella Enrichment broth medium and incubate at 30 – 350C for 24 -48 hours.After completion of incubation, subculture on a plate of Wilson and Blair’s agar and incubate at 30 – 350C for 24 -48 hours.If Growth of Green colonies with black center develop and in 48 hours the colonies become uniformity black. Colonies surrounded by a dark zone and metallic sheen indicates the presence of Salmonella.If sub cultured on plates of Xylose Lysine Deoxycholate agar and incubate at 30 – 350C for 24 -48 hours. Well developed, red colonies with or without black center indicates possibility of Salmonella.This should be confirmed by identification test of Salmonella.Identification test for Salmonella Subculture on triple sugar-iron agar incubates at 30 – 350C for 48 hours.At the same time inoculate a tube of urea broth. Incubate at 30 to 35°C for 18 to 24 hours.Upon examination, no evidence of tubes having alkaline (red) slant and acid (yellow) butt (with or without concomitant blackening of the butt from hydrogen sulfide production), the sample meets the requirements of the test for absence of genus salmonella. Shigella Take 10ml of water sample and incubate with soyabean casein digest broth at 30 – 350C for 18 -24 hours.After incubation shake the broth and transfer 1ml to 100ml of GN Broth medium and incubate at 30 – 350C for 24 -48 hours.After completion of incubation, subculture on a plate of Xylose Lysine Deoxycholate agar and incubate at 30 – 350C for 24 -48 hours.If Growth of red colour translucent colony without black centre indicates the possibility of Shigella.This should be confirmed by identification tests.Pseudomonas aeruginosa Take 1ml of water sample and incubate with soyabean casein digest broth at 30 – 350C for 18 -24 hours.After completion of incubation, subculture on a plate of Cetrimide agar and incubate at 30 – 350C for 18-72 hours.If Growth of greenish colony indicates the possibility of Pseudomonas aeruginosa.This should be confirmed by identification tests. Identification test for Pseudomonas aeruginosa Pigment Test: Streak representative suspect colonies from the agar surface of cetrimide agar on the surface of pseudomonas agar medium for detection of fluorescein and pseudomonas agar medium for detection of Pyocyanin.Cover and invert the inoculated plates and incubate at 33 to 37°C for not less than 3 days.Examine the streaked surface area under UV light and determine whether colonies confirming to the description in Table.Oxidase Test: 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 suspected colony. If there is no development of a pink color, changing to purple, the sample meets the requirements of the test for absence of Pseudomonas aeruginosa. Medium Colony characteristic Fluorescence in UV light Oxidase Gram stain Cetrimide Agar Generally greenish Greenish Positive Negative rods Pseudomonas agar for detection fluorescein Generally colorless to yellowish Yellowish Positive Negative rods Pseudomonas agar for detection Pyocyanin Generally greenish Blue Positive Negative rods. Staphylococcus aureus Take 1ml of water sample and incubate with soyabean casein digest broth at 30 – 350C for 18 -24 hours.After completion of incubation, subculture on a plate of Mannitol salt agar and incubate at 30 – 350C for 18 -72 hours.If Growth of yellow or white colony with yellow zone indicates the possibility of Shigella.This should be confirmed by identification tests. Identification test for Staphylococcus aureus Coagulation Test: Containing 0.5 ml of mammalian, preferably rabbit or horse plasma with or without additives.Incubate at 37°C and examine 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. Candida albicans Take 1ml of water sample and incubate with Sabouraud dextrose broth at 30 – 350C for 3-5 days.After completion of incubation, subculture on a plate of Sabouraud dextrose agar and incubate at 30 – 350C for 24 -48 hours.If Growth of cream colour colony indicates the possibility of Candid albicans.This should be confirmed by identification tests. Clostridia Take two equal portions corresponding to 1ml of the water sample and heat one portion 800C for 10 minutes and cool rapidly. Do not heat the other portion.Transfer 10 ml of each of the homogenized portion to two containers containing 100 ml of Reinforced medium for Clostridia. Incubate under anaerobic condition at 30 – 350C for 48 hoursAfter completion of incubation, subculture on a plate of Columbia agar and incubate under anaerobic condition at 30 – 350C for 48 hours.The accordance of anaerobic growth of Gram positive bacilli with or without endospores giving a negative catalase test indicates possibility of Clostridia.This should be confirmed by identification tests. Precaution Keep the hands clean and used strictly IPA 70% rinsed hand gloves throughout the operations.Run positive and negative control along with each test.The Microbial limit test must be carried out under LAF.For pour plate method, if necessary dilute the sample in the sample solution to obtain 100 to 300 cfu.Sample the water. Acceptance Criteria Purified Water: NMT 100 CFU/ML & Pathogen should be absent. Alert Limit: 50 cfu / ml & Action Limit: 75 cfu/ml. Raw Water: NMT 500 CFU/ML & Pathogen should be absent Alert Limit: 250 cfu / ml & Action Limit: 375 cfu/ml. RO Water: NMT 100 CFU/ML & Pathogen should be absent Alert Limit: 50 cfu / ml & Action Limit: 75 cfu/ml. TRAINING: Trainer : Manager – Quality Control Trainees : Staff of the microbiology departments DISTRIBUTION: Master Copy : QA Department Controlled Copy : Microbiology Department Display Copy : Microbiology Department (If Required) REFERENCES: In-House |
