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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 water-
A. 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 30°C to 35°C 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):
Bacterial Colonies2.1 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 30°C to 35°C 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 20°C to 25°C 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 µ membrane filter. Transfer membrane filter to 100 ml sterile soybean casein digest medium and incubate at 30-35°C for 24 hours.
1.0 Detection of Escherichia coli:
E.coli1.1 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:
Salmonella-colonies2.1 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 Blair’s BBS agar plate and incubate at 30-35°C 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 30-35°C 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:
Pseudomonas-colonies3.1 Streak a portion of the enriched soybean casein digest medium on the surface of cetrimide agar medium and incubate at 30-35°C 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.No
Medium
Characteristic
morphology
Fluorescence
in UV light
Oxidase Test
Gram Stain
1
Pseudomonas agar medium for detection of fluorescein
Generally colourless to Yellowish 
Yellowish
Positive
Negative rods

2
Pseudomonas agar medium for detection of pyocyanin.
Generally
Greenish
Blue
Positive
Negative rods

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 30-35°C 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:
Staphylococcus aureus colonies
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 30-35°C 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 30-35°C for 48 hours.
Table 2 Tests for Staphylococcus aureus.
No.
Selective Medium 
Characteristic Colonial Morphology
1
Vogel-Johnson agar
Black surrounded by yellow zones
2
Baird-Parker agar
Black, shiny, surrounded by 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 30-35ºC for 24-48 hours.
5.2 If growth observed streak on the plate of violet red bile glucose agar. Incubate the plate at 30-35ºC 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 35°C 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 30-35°C 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 30-35°C for 48 hours in anaerobic condition.
7.6 If no growth of microorganisms is seen, the sample passes the test.

Also see: Purified Water Testing
Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
Email: .moc.enilediugamrahp@ofni Need Help: Ask Question


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