Microbiology Methods for Wastewater Water Laboratories

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Microbiology Methods for Wastewater Water Laboratories
John Allen, Saiful Islam, & Gil Dichter Thanks to Jeanine Miller-Nelson (Fairfax Water)

Critical Elements for Microbiology
• Personnel • Laboratory Equipment and Supplies • General Laboratory Practices • Analytical Methodology (Reference method) • Sample Collection, Handling and Preservation • Quality Assurance • Records and Data Reporting • Action Response to Laboratory Results

• Analyst
– Ensure all personnel meets your lab’s requirement for analytical testing.
– Recommend at least two weeks of training for each micro test method, and signed off by trainer.
– Training on all Quality Control (QC) associated with required micro testing.
– Passing of Initial Demonstrate of Capability, results on all ten samples passing before analyzing any compliance samples (before reporting data to any regulatory agency).

Laboratory Equipment and
• pH meter (if needed to prepare media)
– Accuracy with ± 0.1 standard units – Standardize before each use with pH 4.0, 7.0 and 10.0 buffers,
recorded each pH and temperature (or other buffer concentrations) – Check with 8.0 buffer (or other buffer concentrations) – Record slope and all data points and temperatures – Maintained according to manufacturer’s instructions
• Balance (if needed to prepare media)
– Readability of 0.1 g – Sensitivity of 0.1 g for a load of 150 g and 1 mg for load of 10 g or less
(record) – Maintenance and calibration performed annually
• Thermometers
– Graduated in 0.1°C increments for Incubators and Water baths. – Graduated in 0.5°C for Refrigerators – Calibration or accuracy checked annually (may require correction factor)

Laboratory Equipment and Supplies
• Incubator
– Must maintain a constant temperature of 35 ± 0.5 °C
– Record temperature twice each day in use, separated by at least 4 hours

Laboratory Equipment and
• Autoclave
– Maintain sterilization temperature during cycle
– A complete cycle is within 45 minutes, and temp ≥121 for 15 minute and psi ≥ 15 psi for sterilization used for media. The time and temp may depend on the reference method.
– Record date, contents, sterilization time & temperature, total time in autoclave each use (for media if required)
– Maintenance performed annually
– Check automatic timer each quarter with stop watch

Laboratory Equipment and Supplies
• Refrigerator – Maintain temperature for samples of >0.0 to <10.0°C (40 CFR Part 136.3 Table II) – Temperature for certain media is >2.0 to <8.0°C – Record temperature once each day in use
• Inoculating equipment – Sterile or presterilized disposable loops, sticks and swabs should be used

Laboratory Equipment and Supplies

• Pipets

– Pre-sterilized plastic or glass, 10 mL or less must be accurate to within 2.5% (if purchased sterilized pipets check accuracy for each new lot before use with 2.5%)

Glassware and Plasticware

– Borosilicate glass or clear, non-toxic plastic

– Tube closures should be screw caps with non-toxic liners, stainless steel, plastic or aluminum

• Sample Containers

– Wide mouth plastic or glass bottles or sterile plastic bags with sodium thiosulfate. Autoclave sample bottles before using, add sodium
thiosulfate if needed (for Colilert method do not need), and autoclave tape on bottle and cap. Autoclave so temp ≥121 for 15 minute and psi ≥ 15 psi.

• Method Specific Supplies

– See reference test methods for specific equipment and supplies. For

example testing for Colilert method supplies and equipment: colilert

media, quanti-trays, containers with sodium thiosulfate, UV lamp,

sealer, incubator, sterile water, andQuanti Cult Kit to check new colilert

media lot before first use.


General Laboratory Practices
• Sterilization procedures – Autoclave at 121°C for recommended time at least 15 psi – Media should be removed immediately after end of cycle
• Sample Containers – Randomly test 1 container from each batch or lot for sterility using 25 mL nonselective broth (such as Tryptic Soy Broth), incubate for 48 hours & check for growth and/or follow specific procedures from test method used.
• Reagent grade water – Quality should meet criteria for Conductivity, Metals, Total Chlorine Residual, HPC and Bacteriological Quality
• Glassware/plasticware washing – Clean with brush and, soap & water, and distilled or deionized water for final rinsing – Check for acid or alkalinity after washing a batch of glassware/plasticware (or at lest daily) using a suitable pH indicator such as bromothymol blue solution. – Inhibitory residue test should be performed prior to initial use of detergent lot (and annually), or whenever different washing procedure or detergent is used

Analytical Methodology

• Media

– Discard media by manufacturer’s expiration date

– Store prepared medium in dark at recommended temperature

– For lab-prepared media, record date prepared, type, lot #, volume, sterilization time & temperature, final pH and initials

– For commercially-prepared media, record date received, type, lot # and pH verification if specified by method

– Each batch of lab-prepared and each lot of commercially-prepared media should be checked before use for sterility and with positive & negative control cultures

• Examples of control cultures:

– Total coliforms

• Positive: Escherichia coli

Negative: Pseudomonas aeruginosa

– Fecal coliforms

• Positive: Escherichia coli

Negative: Enterobacter aerogenes

– E. coli

• Positive: Escherichia coli

Negative: Pseudomonas aeruginosa


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Microbiology Methods for Wastewater Water Laboratories