Microbiological Testing Techniques

CONTENTS

•

Water Testing

•

Moisture content determination

•

TOC Analysis

•

Bacterial Endotoxin Test

•

Environment Monitoring

Water Testing Water plays a very crucial role in pharmaceutical industries and is divided into 3 types based on its applications : 3.

Raw water (RW)

5.

Demineralized water (DMW)

7.

Water For Injection (WFI)

Raw Water is potable water filtered through gravel contains ions and suspended particles is further processed into DM water and WFI

Analysis Samples of raw water are collected and analyzed for the following parameters – • •

Chemical Microbiological

Chemical Analysis 1.

Odour - Examine the taste of raw water sample physically. It should be odourless

3.

pH - Measure by suitable and calibrated pH meter. pH range is 6.58.5.

5.

Conductivity - Measure suitable and calibrated conductivity meter. Limit – NMT 750µs/cm

4.

Hardness - To 100 ml water add 2 ml of ammonia buffer pH 10, 50 mg of mordant black II and 0.01M disodium edetate until a pure blue colour is produced. Measure the volume of disodium edetate used and calculate the hardness by the following formula: Hardness (mg/liter) = EDTA used (ml) 1000 / Sample Vol.

Microbial Analysis 1.

Total Plate Count

c)

Pour plate method - Dispense 1 ml of each sample into two sterile petridishes and add 15-20 ml of melted Plate Count Agar at 40- 45C. Invert the petridishes and incubated at 30-35C for 48-72 hours. Count the number of colonies and express in term of CFU/ml . Limit of viable microbial count is NMT 500 CFU/ml

h)

Membrane Filtration Method -Load the membrane(.45 m) onto the filter holder.Sterilize this apparatus by autoclaving. Pour 1ml of sample directly into funnel Apply vacuum to filter sample and transfer immediately to plate count Agar.Incubate plates in inverted position at 30-35C,48-72hrs . Count no. of colonies and express as cfu/ml .

Pathogens The sample is mainly tested for the following four pathogens : a) Salmonella species b) Escherichia coli c) Pseudomonas aeruginosa d) Staphylococcus aureus First filter 100 ml sample through sterile funnel and inoculate membrane in Soyabean Casein digest Medium (SCDM) and incubate at 30 -35 C,24-48 hrs. After incubation inoculate sterile 10-ml foll. enrichment broth using 1ml incubated SCDM. i.) Selenite F. Broth for Salmonella species ii) Mac Conkeys Broth for E. coli iii) Cetrimide Broth for Pseudomonas aeruginosa

Test for Salmonella species: If growth is present in Selenite F. Broth, presumptive test is done by inoculating and incubating the following selective media at 30-35C for 24 hrs. After incubation detect colour of colonies.

Test for Escherichia coli : If acid and gas production occur in MacConkey’s Broth tube, then presumptive test using the foll. selective media is done :

Test for Pseudomonas aeruginosa : If the inoculated Cetrimide Broth tube shows growth with the greenish/ bluish pigmentation, inoculate the following selective media plates and incubate at 30-35C for 24-28 hours for presumptive identification of the pathogen.

Test for Staphylococcus aureusIf black settled growth is present at the bottom of the Giolitti Cantoni Broth under anaerobic condition. Inoculate the following selective media and incubate at 30-35C for 24-28 hours for presumptive identification of pathogen .

Demineralized Water also called Purified water prepared by means of ion exchange total viable count is NMT 100 cfu/ml Not suitable for preparations intented for parenteral administration Analysis Samples of DM water are collected and analyzed for the following parameters – • •

Chemical Microbiological

Chemical Analysis 1.

Description - When physically examined (Organoleptic test) DMW should be clear, colourless, odourless, and tasteless liquid .

3.

Residues on evaporation- Evaporate 100 ml of sample to dryness on a water bath dry to a constant weight at 105C. The residues weight is not more than 1mg (0.001%).

5.

Total Organic Carbon- Analyze the sample for total organic carbon in a calibrated TOC analyzer Limit: NMT 500 ppb

10.

Conductivity- Measure by suitable conductivity meter. Limit: NMT 1.3µS/cm at 25C.

Water for Injection (WFI) apyrogenic distilled water intended for use in the preparation of medicines for parenteral administration obtained by distilling demineralised water from a neutral glass , quartz or suitable metal still fitted with a device for preventing moisture packaged in sterile vials / ampoules and is terminally sterilized Total viable microbial count limit is NMT 10 cfu/100 ml

MOISTURE CONTENT DETERMINATION Karl-Fischer Titration : a method for quantifying water content in a variety of products. Principle– it is based on rxn. between iodine and sulfur dioxide in nonaqueous medium . KF reagent consists of foll. components * methanol (containing excess SO2) – used as a solvent * pyridine – buffering agent * iodine – oxidizing agent

Theory Reaction :

• •

ROH + SO2+ R’N

•

[alcohol]

•

[base]

[R’NH]SO3 + H2O + I2 + 2R’N [alkylsulfite salt]

[iodine]

2[R’NH]I + [R’NH]SO4R

[hydroiodicAcid Salt]

[alkylsulfate salt]

•

Alcohol reacts with SO2 and base to form intermediate alkyl sulphite which

•

then oxidizes into alkyl sulphate salt . That signals the end-point of the titration

•

titrator’s indicator electrode. Water and iodine are consumed in a 1:1 ratio in the

•

above reaction. Once all of the water present is consumed, the presence of excess

•

iodine is detected voltametrically by the titrator’s indicator electrode. That signals the

•

end-point of the titration .

• •

Titration Monitoring – The use of specially formulated water standards enables the

•

efficient monitoring of titrator performance .

•

•

TOC Analysis Total organic carbon (TOC) is the amount of carbon bound in an organic comp. and is often used as a nonspecific indicator of water quality or cleanliness of pharmaceutical manufacturing equipment . A typical analysis for TOC measures both the total carbon present as well as the inorganic carbon (IC). Subtracting the inorganic carbon from the total carbon yields TOC. Another common variant of TOC analysis involves removing the IC portion first and then measuring the leftover carbon. This method involves purging an acidified sample with carbon-free air or nitrogen prior to measurement, and so is more accurately called NPOC

Theory 2 types of methods are used for calculation of TOC •

TC – IC :It measures the amount of inorganic carbon (IC) evolved from an

acidified aliquot of a sample and also the amount of total carbon (TC) present in the sample. TOC is calculated by subtraction of the IC value from the TC the sample

•

IC – NPOC : employs acidification of the sample to evolve CO2 and measuring it as IC ,then oxidizing and measuring the remaining NPOC

Acidification The removal and venting of IC and POC gases from the liquid sample by acidification and sparging occurs in the following manner ---

•

Oxidation

The second stage is the oxidation of the carbon in the remaining sample in the form of carbon dioxide (CO2) and other gases. Modern TOC analyzers perform this oxidation step by one several processes . •

•

•

•

Non-dispersive infrared (NDIR)

The non-dispersive infrared analysis (NDIR) method offers the only practical interference-free method for detecting CO2 in TOC analysis. The principal advantage of using NDIR is that it directly and specifically measures the CO2 generated by oxidation of the organic carbon in the oxidation reactor, rather than relying on a measurement of a secondary effect . •

•

•

•

Applications

important in detecting contaminants in drinking water, cooling water, water used in semiconductor manufacturing, and water for pharma use. •

•

•