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Best PracticesMicrobiology Proficiency Testing

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Visitwww.phenova.com/microbiologyfor more information

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Open, Pour, and Go withWhole Volume Microbiological Standards

Supplying your laboratory with a whole volume format provides PT standards that by design most accurately represents an everyday field sample, with a microbiological culture already cultivated in media.

Easy To Use No Preparation Needed

Realistic Presented and Accurate As Any Other Field Sample

Saves Time Skip The Rehydration Step and Expedite Your Analysis Time

Error Free Eliminate Possible Dilution Errors and Avoid Mis-Labeling with Extra Labels

3Phenova | Tel: 866-942-2978 | Fax: 866-283-0269 | Email: [email protected] | Web: www.phenova.com

Table of ContentsOpen, Pour, and Go withWhole Volume Microbiological Standards

Phenova’s Guide helps define the best practices and guidelines to undertake when your laboratory is performing a Microbiological Proficiency Testing (PT) Study.

Sample Preparation ......................................................................... 4

Media Preparation .......................................................................... 5

Analysis and Handling .................................................................... 6

Filter Paper ...................................................................................... 7

Incubation ........................................................................................ 8

Colony Counting .............................................................................. 9

Lab Equipment ................................................................................ 9

How to Troubleshoot Effectively .................................................... 10

Microbiological QC Standards ...................................................... 11

The Phenova PT GuaranteeIf your lab receives a Not Acceptable result in a Phenova PT Study and cannot pinpoint the issue, contact our technical support group. We will guide you through the most effective corrective action process and, if necessary, we will provide a FREE Phenova QC Standard to help get you back on track!

For Technical Support Contact PhenovaPhone: 1-866-942-2978Email: [email protected]


Sample PreparationThe Forgotten Beginning

Temperature• Allow the sample to come to room temperature before using.

• Allow the broth, media and all reagents to come to room temperature.

• Make sure your incubators are at their appropriate temperatures as well.

Homogenization• Ensure adequate homogenization of the organism by vigorously

shaking and inverting the sample prior to beginning sample analysis.

• Homogenize in between taking aliquots—from original sample.

Serial Dilutions• Perform all of your serial dilutions from lowest to highest, where the

concentration decreases by the same quantity in each successive step.

What Does This Mean? If a dilution has a 1:10 dilution the number represents 1 part of the original sample added to 9 parts of the diluent.

• If you are making a series dilution from the lowest dilution ratio (1:10) to highest dilution ratio (1:100,000), then you do not need to change pipette tip in between transfer dilutions.

• Never use Deionized (DI) Water to make dilutions, the pH may harm the organisms. A great alternative is phosphate buffer or peptone water.

Room Temperature:~23°C / 74°F

2 min

1:109 mL broth in each tube

Original Inoculum

1mL

1mL

1mL

1mL

1mL

1:100

1:1,000

1:10,000

1:100,000


Media PreparationFor Optimal Growth

Check the following parameters to help assess and maintain the quality of your growth media. Checking the quality of your media can save you additional time and resources and prevent testing failures.

Quality Checks

Media needs to be stored according to the method or manufacturer recommended temperature and light conditions. PMedia has not expired before use* PPerform Sterility Testing PTest each batch or lot with negative and positive controls (see below) PInspect media for any apparent contamination PMust be used only at room temperature P

*For laboratory made media, make sure the ingredients have also not expired.

Control Microorganisms

Standard Methods and EPA methods both note that the quality of your media should be evaluated with each new lot to ensure that the microorganisms under test are responding appropriately. The best way for checking for growth supporting your analysis is with positive and negative control organisms.

• Positive control microorganisms are known organisms that are the same as your target organisms and provide you with typical colony formation.

• Negative control microorganisms need to be an organism that grows but produces an atypical colony.

• Remember that you can use microorganisms for 5 passes. After the 5th pass you must use a fresh incubation of the microorganism.


DI H20 TNI

Analysis and Handling

Microbiological FoPT Tables

Always ensure that the filter apparatus

is sterilized between sample filtering

DI water can have a pH harmful to the organisms.

Make sure to use phosphate buffer or peptone water for

rinsing.

Review the TNI FoPT tables to know

the concentration range of the PT sample you are

receiving. This will allow you to make

the appropriate dilution scheme.

Handle filters with flamed forceps

Follow the method requirements for blanks and be

sure to follow the manufacturer’s

instuctions

Use the entire sam-ple for dilutions and never use DI water

for making dilutions, only phosphate

buffer or peptone water.

Download the FoPT tables at: http://nelac-institute.org/content/NEPTP/fopt.php

Non-Potable WaterList of all approved waste water or non-potable water analytes and respective acceptance limits.

Document Name: NPW_FoPT-Eff 03-16-15.pdf

Drinking WaterList of all approved drinking water analytes and respective accep-tance limits, except radiochemistry.

Document Name: DW_FoPT_2012_01_03.pdf


Filter Paper

Using a 47mm; 0.24 (µm) micron filter has been standard practice in microbiological methods for efficient nutrient diffusion.

47 mm Diameter

0.24 Micron Pore Size

Effectiveness Indicators

Simply place the filter over reagent water and if it sinks or the membrane wets out within 15 seconds you have a great indication of an uncompromised filter paper and proper material for the filter.

Effective Application

Maintain maximum contact with the media by rolling the filter onto the media from one edge to other. Minimize air bubbles forming on the filter by slowly applying the filter to the media plate.

Positive Controls

QC test your filters with a positive control sample to ensure they provide a typical result. Use Phenova’s QC Standards as Positive Control. (Refer to page 11.)


Incubation

Water-tightPlates should be

contained in watertight containers and fully suspended in water

bath incubators.

PlacementEnsure the plates do not touch the sides of the incubator as the temperature is different than the

heat source.

TemperatureAllow sufficient time for the incubator to

equilibrate when adjusting the temperature and make

sure all thermometers are calibrated.

Sealed-ShutIncubator doors or

covers should always be used properly to maintain

heat capture.

• Be cautious for bubbles and foaming in water baths as they may affect the heat transfer and have a negative consequence on your results. If necessary use a circulator or an approved de-foaming agent.

• Monitor the temperature twice a day to make sure it sits within your methods and organisms approved range.

• If you see atypical colonies, incubate another 4 hours to ensure the organism had enough time for optimal growth.

• Equilibration times when loading incubators and water baths need to be subtracted from the regular incubation time.

What to Look Out For


Colony CountingIt’s important to keep a trained eye when counting colonies, so that atypical colonies can be distinguished from typical colonies. Study the images below to get references as to what typical colonies look like and what to expect, however it’s always best to follow any confir-mation steps in the method(s) you follow.

Lab EquipmentMembrane Filters Method

• Check that your vacuum is working correctly.

Most Probable Number Method

• On a periodic basis check that your sealer is working properly using food dye and water.

• Check that your comparators are still working properly.

• Check that your UV light if functioning properly and providing the correct light intensity.

Fecal Coliform Bacteria grown on mFC nutrient agar

Circular blue colonies with different sizes and shades

Total Coliform Bacteria grown on LES Endo nutrient agar.

Circular dark red colonies with and without metallic sheen

E. Coli Bacteria grown on LB nutrient agar

Circular non-pigmented colonies with a shiny appearance

10

How to Troubleshoot Effectively If at any point in your PT process you come across the need to troubleshoot, the most important step is to understand the problem. Taking a systematic approach to identify the root cause of the problem will be the best investment of your time/resources and it will be much easier to implement a logical solution.

Corrective Action Guide Download our guide and learn how to evaluate a non-conformance or “Not-Acceptable” and eliminate problems systematically.

4 Steps to a Stronger Quality Program 1. Root Cause Analysis

2. Corrective Action Strategy

3. Preventive Action Strategy

4. Continuous Improvement

Get Your Copywww.phenova.com/correctiveaction.

Change Nothing

Make an Educated Guess

EliminateProblems

Sytematically

Learn more about QC standards. Visit at http://www.phenova.com/Product/QCOverview For Assistance with Incorporating QC Standards in Your Quality Program call 1-866-942-2978 or email [email protected]

Microbiological QC StandardsWhere They Fit In Your Quality Program

Crucial for maintaining a rigid quality management program, make QC standards a part of your practice to refine technical skills, ensure method confidence and check the integrity of laboratory protocols and resources.

Routine Internal Quality Evaluation Verify your laboratory or instrumentation are in con-trol and meet your data quality objectives.

Train Your Analytical Staff A powerful tool in your training regimen. Staff can analyze QC standards as if they are real-world sam-ples and results can be compared with known data.

Root Cause Investigation Determine root causes for a “Not Acceptable” and identify your corrective action.

Corrective Action Measure Investigation Does your corrective action practice make sense; is it working?


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Microbiology Proficiency Testing

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