User talk:DHANANJAY BALGHARE

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Microbial Limit Test (MLT), Bioburden Test for Microbiological water Analysis

Microbial Limit Test (MLT), Bioburden Test for water Analysis

Introduction: The Microbial Limit Tests are designed to perform the qualitative and quantitative estimations of specific viable microorganisms present in samples. It includes tests for total viable count (bacteria and fungi) and for specified microorganism. The care must be taken in performing the tests, so that microbial contamination from the outside can be avoided. PROCEDURE Requirements Accessories • Sterile Glass Bottles • Sterilised Filtration Cups. • Sterile Micro Pipettes Tips • Sterilised Forceps • Sterile 0.45µ, 47 mm membrane filters. • Suction flask Assembly • Sterilised silicon tubing for filtration. • Sterile hand gloves Media and Reagents • Pre-incubated Soyabean Casein Digest Agar plates (SCDA) • Sabouraud Dextrose Agar (SDA) plates. • Pre-incubated R2A plates • Pre-incubated Plate Count Agar(PCA) • Pre-incubated Reinforced medium for clostridia(RMC) • Pre-incubated Columbia Aagr (CoA) • Pre-incubated Soyabean Casein Digest Medium (SCDM). • Pre-incubated MacConkey’s Broth (MCB) • Pre-incubated MacConkey’s Agar (MCA) • Kovac’s Reagent • Pre-incubated Rappaport Vassiliadis enrichment broth( RVB) • Pre-incubated Xylose, lysine, Deoxycholate Agar (XLDA) • Triple Sugar Iron Agar slant (TSI) • Pre-incubated Cetrimide Agar (CA) • Pre-incubated Mannitol Salt Agar (MSA) • Pre-incubated Hi-Chrom Agar (HC) • Rabbit or Horse Plasma • Sterile diluent (Normal Saline or 0.1 % Peptone or Buffered Sodium-Chloride Peptone Solution pH 7.0)

   Equipments:

• Sterilised Filtration Assembly • Laminar Air Flow Unit. (LAF Unit). • Vacuum pump • Micro Pipettes Sampling • Carry out sampling of the water as per the individual specification. • Bring the samples in microbiology laboratory in sample carrier. Microbial limit test for Potable Water (POT) TVAC (Total viable aerobic count) • Assemble the filtration assembly. • Add 10 ml of normal saline in filtration cup and add 1.0 ml of sample, filter through sterile 0.45 μ membrane filter in duplicate. • After filtration of sample, rinse the side walls of the filtration unit by using 50 ml sterile normal saline solution. • Remove the membrane filter with the help of sterile forceps from the holder, Place the one membrane on the surface of pre incubated R2A plate and another membrane on pre incubated SDA plate in such a way that no air bubbles form between the filter & culture medium. • Incubate the R2A plate at 30-35 °C for 5 days and SDA plates at 20-25 °C for 5 days in inverted position. Record the count as cfu / ml. • Positive control:- Spread 0.1ml of 100-1000 cfu/ml bacterial suspension on the surface of R2A agar and fungal suspension on SDA agar. Incubate the R2A plate at 30-35 °C for 48 hrs and SDA plates at 20-25 °C for 120 hrs in inverted position. Observed for growth. • Use 10-100 or 100-1000 cfu/ml bacterial and fungal suspension of any one micro organisms from following list Bacteria : 1. Pseudomonas aeruginosa (ATCC 9027)

               2. Bacillus subtilis (ATCC 6633)

Fungi  : 1. Candida albicans (ATCC 10231)

               2. Aspergillus niger  (ATCC 16404)

• Negative control:- Repeat the above procedure using 100 ml of normal saline instead of sample. Test for specified micro organisms • Enrichment: Add 10 ml of sample to 100 ml of fluid soyabean casein digest medium, mix and incubate at 30 – 35 °C for 18-24 hours and observe for turbidity (Growth). A) Escherichia coli : • Examine the enrichment culture for growth and if growth is present, shake the enriched broth and transfer 1 ml into 100 ml of sterile MacConkey’s Broth medium. Incubate at 42-44C for 24-48 hours and observe for change in colour of broth medium and turbidity (Growth). • After incubation period subculture, on MacConkey’s Agar plate and incubate at 30-35C for 18-72 hours. After incubation observe growth of colonies that indicates possible presence of E. coli. This is confirmed by identification test (Indole test). • Indole test: Inoculate suspected colony in 10 ml peptone water and incubate at 30 C – 35C for18- 24 hours. After incubation add 0.5 ml of Kovac’s reagent. If red colour is produced at the junction then the test is positive (+). If no colour is produced then the test is negative (–). • Positive control:- Use 10-100 or 100-1000 cfu/ml Escherichia coli ATCC 8739 suspension at every step. • Negative control:- Keep uninoculated broth medium or agar plate as such for incubation B) Salmonella : • If growth is present in enriched culture, transfer 0.1 ml of enriched broth to 10 ml of Rappaport Vassiliadis enrichment broth and incubate at 30-35C for 18-24 hours and observe for change in colour of broth and turbidity (Growth). • After incubation, subculture on Xylose, Lysine, Deoxycholate Agar (XLDA). Incubate at 30-35C for 18-48 hours. • Growth of Well-developed, red colonies, with or without black centre indicates possible presence of Salmonella. This is confirmed by identification test • Transfer suspected colony to Triple Sugar Iron Agar slant using surface & deep inoculation and incubate at 30C to 35°C for 18 to 24 hrs and check for change of colour from red to black colour and usually a formation of gas, with or without production of hydrogen sulphide in the agar (+). The sample passes the test if colonies of the type described do not appear (–). • Positive control:- Use 10-100 or 100-1000 cfu/ml Salmonella abony NCTC 6017 suspension at every step. • Negative control:- Keep un inoculated broth medium or agar plate as such for incubation C) Pseudomonas aeruginosa: • Examine the enrichment culture for growth after incubation. Subculture on Cetrimide Agar and incubate at 30 C to 35°C for 18 - 72 hrs. • Growth of colonies indicates the possible presence of Pseudomonas aeruginosa. This is confirmed by identification test • If growth occurs transfer colony to Soyabean Casein Digest Medium & incubate at 42C to 44°C for 18 to 24 hrs. • The sample passes the test if no growth occurs at 42C to 44°C. • Positive control:- Use 10-100 or 100-1000 cfu/ml Pseudomonas aeruginosa ATCC 9027 suspension at every step. • Negative control:-Keep agar plate or broth as such for incubation D) Staphylococcus aureus : • Examine the enrichment culture for growth after incubation. Subculture on Mannitol Salt Agar (MSA) and incubate at 30-35 C for 18-72 hrs. • Growth of Yellow / White colonies surrounded by a yellow zone indicates the possible presence of Staphylococcus aureus. Perform the gram staining and confirmed by identification test. • Coagulase test: Transfer colonies from the agar surface in to test tube containing 0.5 ml of mammalian, preferably rabbit or horse plasma with or without additives. • Inoculate in water bath at 37°C, examinining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours for clot formation of the plasma. • Legend: (+ve) clot formation, (- ve) no clot formation. • Positive control:- Use 10-100 or 100-1000 cfu/ml Staphylococcus aureus ATCC 6538 suspension at every step. • Negative control:- Keep agar plate and 0.5ml of plasma as such for incubation E) Clostridia Sample treatment and enrichment: Take two equal portions corresponding to 1.0gm or 1.0ml of sample. Heat one portion to 80 ºC for 10 min and cool rapidly do not heat another portion. • Transfer the 10 ml of each of the homogenised portions to 2 containers (38X200mm or other suitable container) containing 100 ml of reinforced medium for clostridia. Incubate under anaerobic condition at 30-35 ºC for 48 hrs. • After incubation, make subcultures from each tubes on Columbia agar with gentamicin (Add gentamicin sulphate corresponding to 20 mg of gentamicin in plate before plate pour) and incubate under anaerobic condition at 30-35°C for 48 hrs. If no growth of microorganism is detected. • The occurrence of anaerobic growth, Subculture each distinct colony on Columbia agar without gentamicin, under aerobic and anaerobic condition at 30-35°C for 48 hrs. • If growth of anaerobic micro organisms is detected on Columbia agar perform catalase test. Catalase test positive it indicate that sample complies. • Positive control:- Use 10-100 or 100-1000 cfu/ml of Clostridium sporogenes ATCC 19404 suspension at every step. • Negative control:- Keep uninoculated broth medium or agar plate as such for incubation. F) Candida albicans. Enrichment for Candida albicans. • Take 1gm or 1ml sample in 100 ml of Sabourads dextrose broth and incubate at 30-35°C for 3 to 5 days. • Subculture on sabourads dextrose agar and incubate at 30-35°C for 24 -48 Hrs. Growth of white colonies may indicate the presence of candida albicans. This is confirmed by identification test • Positive control:- Use 10-100 or 100-1000 cfu/ml of Candida albicans ATCC 10231 suspension at every step. • Negative control:- Keep uninoculated broth medium or agar plate as such for incubation. • If growth of colonies occurs subculture on Hi-chrom agar and incubate at 30-35°C for 24 -48 Hrs. Growth of colonies may indicate the presence of Candida albicans. Demineralised Water after Cation and Anion Exchange Microbial limit test for Purified Water • Follow the above steps (Under A,B,C,D.) Microbial limit test for WFI and PSC • Assemble the filtration assembly. • Filter 200 ml of sample, through sterile 0.45 μ membrane filter. After filtration of sample rinse the side walls of the filtration unit by using 50 ml sterile normal saline solution. • Remove the membrane filter with the help of sterile forceps from the holder and place the one membrane on the surface of pre incubated R2A plate in such a way that no air bubbles form between the filter & culture medium. Incubate the R2A plates at 30-35 °C in inverted position for 5 days. • Positive control: - Spread 0.1ml of 100-1000 cfu/ml bacterial suspension on the surface of R2A agar. Incubate the R2A plate at 30-35 °C for 48 hrs in inverted position and observe for growth. • Negative control:- Repeat the above procedure using 100 ml of normal saline instead of sample. Microbial limit test for water for injection: • Assemble the filtration assembly. • Filter 200 ml of sample, through sterile 0.45 μ membrane filter. After filtration of sample rinse the side walls of the filtration unit by using 50 ml sterile saline solution. • Remove the membrane filter with the help of sterile forceps from the holder and place the membrane on the surface of pre incubated plate count agar plate in such a way that no air bubbles form between the filter & culture medium. • Incubate the Plate count agar plates at 30-35°C in inverted position for 5 days. • Positive control:- Spread 0.1ml of 100-1000 cfu/ml bacterial suspension on the surface of Plate count agar and incubate at 30-35 °C in inverted position for 48 hrs. • Use 10-100 cfu/ml bacterial suspension of any one microorganism from following list. 1) Bacillus subtilis (ATCC 6633) 2) Pseudomonas aeruginosa (ATCC 9027) • Negative control: - Repeat the above procedure using 100 ml of normal saline instead of sample. Bio-burden Raw material samples • Perform sampling of the raw material as per individual specification. 1)Sample Preparation for Water-Soluble Products Unless otherwise specified, dissolve or dilute 10 g or 10 ml of the product to be examined in 100 ml sterile diluent or it may be further diluted using sterile diluent. • If the product is known to have antimicrobial activity, an inactivating agent or neutralising agent may be added to the diluent. • Common Neutralising agents for interfering substances Interfering Substance Potential Neutralising Method Glutaraldehyde, mercurials Sodium hydrogensulphite (Sodium bisulphite) Phenolics, alcohol, aldehydes, sorbate Dilution Aldehydes Glycine Quaternary Ammonium Compounds (QACs), Parahydroxybenzoates (Parabens), bis-biguanides. Lecithin QACs, Iodine, Parabens Polysorbate Mercurials Thioglycollate Mercurials, Halogens, Aldehydes Thiosulphate EDTA (edetate) Mg++ or Ca++ ions. Acid Alkali (NaOH) Alkali Acid (HCl, H3PO4)

2)Sample Preparation for Non-Fatty Products Insoluble In Water • Unless otherwise specified, suspend 10 g or 10 ml of the product to be examined in sterile buffered sodium chloride-peptone solution or sterile diluent. • A suitable surface-active agent such as 1 g/l of sterile polysorbate 80 may be added to assist the suspension of poorly wettable substances. • If the product is known to have antimicrobial activity, an inactivating agent may be added to the diluent 3)Sample Preparation for Fatty Products • Unless otherwise specified, homogenise 10 g or 10 ml of the product to be examined with not more than half its weight of sterile polysorbate 80 or another suitable sterile surface-active agent, heated if necessary to not more than 40°C, in exceptional cases to not more than 45°C. • Mix carefully and if necessary maintain the temperature in a water-bath or in an incubator. Add sufficient pre-warmed sterile diluent to make a one in ten dilution of the original product. • Mix carefully whilst maintaining the temperature for the shortest time necessary for the formation of an emulsion and in any case for not more than 30 minutes. • Further serial tenfold dilution may be prepared using sterile buffered sodium chloride-peptone solution or sterile diluent containing a suitable concentration of sterile polysorbate 80 or another sterile surface-active agent. Procedure for analysis of raw material: • Per-treatment: Add 1gm or 1ml in 10 ml (1:10 proportion or specified quantity of the sample) of sterile diluent and mix to release the micro-organisms in to the solution. • Assemble the filtration apparatus aseptically. • Pre-wet the membrane add 50 ml of the sterile diluent and filter it. • Filter 10 ml of pre-treated sample solution or volume equivalent to 1 gm of pre-treated sample solution, aseptically through the membrane filter in duplicate. • After filtration of the sample, rinse the inside walls of the filtration cup with 3 X 100 ml sterile diluent and filter it. • Detach the cup and remove the membrane filter aseptically with the help of sterilised forceps from the holder and place one membrane on the surface of sterile pre-incubated SCDA plate & another on SDA plate in such a way that no air bubbles should form between the filter and the culture medium. • Express the result of the total aerobic microbial count as sum of the bacterial and fungal count in cfu per gram or cfu/10 ml. • Spread 0.1 ml of 100-1000 cfu/ml of one bacterium and one fungus on the surface of agar plates from the list below for +ve control. 1) Bacillus subtilis (ATCC 6633) 2) Staphylococcus aureus (ATCC 6538) 3) Candida albicans (ATCC 10231) 4) Aspergillus niger (ATCC 16404) 5) Pseudomonas aeruginosa(ATCC 9027) • Negative control:- Filter 100 ml of sterile diluent in duplicate for SCDA & SDA. • Incubate the SCDA plates in inverted position at 30-35 °C (for enumeration of bacteria) and SDA plates at 20 - 25°C (for enumeration of fungi) for 5 days. Take the count. In-process Samples, Finished product samples, Validation samples: • For analysis of liquid samples, directly use sample solution as per quantity specified in the individual specification. • For Solid Devices Unwrap / open the devices aseptically in LAF unit and transfer the quantity specified in specification with sterilised forecep in a sterilised container. Select the container according to the size of the sample. Avoid touching of gloved fingers to the device. Add 10 ml per device or as per quantity specified in specification in sterile diluent in sterile container. Swirl the container gently for approximately 4 to 5 minutes. Pour the diluting fluid in another sterilised container and use this fluid for testing. Procedure for Bioburden: • Assemble the filtration apparatus aseptically. • For pre-wetting the membrane add 50 ml of the sterile diluent onto the membrane filter and filter it. • Filter 1 ml (or as specified in individual specification) of sample solution aseptically through the membrane filter. After filtration of the sample, rinse the inside walls of the filtration cup with 3 rinses of 100 ml sterile diluent and filter it. • For analysis of solid devices, filter volume of the sample solution equivalent to quantity of device specified in the specification, aseptically through the membrane filter. • After filtration of the sample, rinse the inside walls of the filtration cup with 3 rinses of 100 ml sterile diluent and filter it. • Detach the cup and remove the membrane filter aseptically with the help of sterilised forceps from the holder and place membrane on the surface of sterile pre-incubated SCDA plates in such a way that no air bubbles should form between the filter and the culture medium. • Negative control:- Filter 100 ml of sterile diluent instead of sample. • Positive control:- Spread 0.1 ml 100-1000 cfu/ml suspension on the surface of sterile SCDA plates, list of micro organism mentioned below. • Incubate the SCDA plates in inverted position at 20 - 25°C (for enumeration of fungi) for 3 days. Take the count. Transfer & incubate at 30-35 °C (for enumeration of bacteria) for 2 days. Take the count. • Express the result of the total aerobic microbial count as sum of the bacterial and fungal count in cfu/ml. • For Solid devices express the result of the total aerobic microbial count as sum of the bacterial and fungal count in cfu/10 ml or cfu/device or as per solid device analysed. • Use at least one bacterium and one fungus from the list below for +ve control. 1) Bacillus subtilis (ATCC 6633) 2) Staphylococcus aureus (ATCC 6538) 3) Escherichia coli (ATCC 8739) 4) Candida albicans (ATCC 10231) 5) Aspergillus niger (ATCC 16404) 6) Salmonella abony (NCTC6017) 7) Pseudomonas aeruginosa(ATCC 9027) • Incubate the +ve control plates of bacteria SCDA at 30-35 °C for 3 days or less than 3 days. • After incubation note down the results as Growth (+) or No growth (-). • If growth is not observed in positive control or growth is observed in negative control then the test is not valid. <Dhananjay B.> — Preceding unsigned comment added by DHANANJAY BALGHARE (talk • contribs) 09:29, 11 September 2017 (UTC)