KAN-TN-LP 02 - KAN Requirement for Microbiological Testing Laboratory

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KAN TECHNICAL NOTES FOR

MICROBIOLOGICAL TESTING

LABORATORY

Issue Number : 3 April 2016

Komite Akreditasi Nasional

National Accreditation Body of Indonesia Gedung BPPT 1, Lt. 14

Jl. M.H. Thamrin No. 8, Jakarta Pusat 10340 – Indonesia Tel. : 62 21 3927422 ext. 173, 194, 195, 197, 210, 211 Fax. : 62 21 3927528

Email : laboratorium@bsn.go.id Website : http://www.kan.or.id

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded i APPROVAL SHEET

Reviewed by :

Quality Manager

Approved by :

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded ii LIST OF AMANDMENT

No. Date

Part number revised

Brief description of changes

Part revision number 1. January 2008 All Change of identification 2

2. April 2016

Point 4a. Adding

xvii. colony counting (if applicable)

3 Point 5

Adding

ix. Laminar air flow x. Sterilising ovens

Point 6

Divided into 2 Point :

6. Reagent and Culture Media

Adding the information that should be available from producer.

7. Reference Material and Reference culture

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 1 SPECIFIC REQUIREMENT FOR

ACCREDITATION OF MICROBIOLOGY TESTING

1. INTRODUCTION

a. This Specific requirements are an interpretation of the general requirements of ISO/IEC 17025-2005.

b. This accreditation requirements are applicable to field of testing microbiology, testing technical, type of test, and or specific test in microbiology area, as extra information to the already generally stated requirements in each of the clauses ISO/IEC 17025-2005.

c. Microbiological testing is taken to include sterility testing, detection, isolation, enumeration and identification of micro-organisms including viruses, bacteria, fungi and protozoa and their metabolites in different materials and products, or any kind of assay using micro-organisms as part of a detection system as well as the use of micro-organisms for ecological testing.

d. This document can also provide guidance to laboratories using techniques in areas related to microbiology, such as biochemistry, molecular biology and cell culture laboratories.

2. PERSONNEL

a. The Laboratory analyst has been working in relevant test areas of a microbiology laboratory for at least two years.

b. Technical Manager, Laboratory Supervisors and Laboratory analysis shall possess a basic education in biological analysis vocation or a related science.

c. The laboratory shall be directed by persons having executive responsibility & competence to assume responsibility for the services of microbiology testing.

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 2 d. Laboratory management shall ensure that :

i. Appropriate numbers of laboratory personnel, with the required education & training, to meet the demands of the service to customers.

ii. Laboratory personnel have the knowledge, skills, and abilities based on education, experience, demonstrated skills, and training. To perform their duties.

iii. Laboratory personnel had trained in handling pathogenic agents.

iv. Full educational and professional records of all technical staffs available to confirm their competence in microbiology testing.

e. Laboratory shall establish and define an internal training program and ensure the competency of laboratory personnel.

f. Laboratory shall have training procedure that used to ensure that training has taken place with each employee for procedures and methods that the employee performs. The procedure applies to on-the-job training, in-house training and new-hire training.

g. The training procedure is applicable to new employees, for the introduction of new procedures and methods, for retraining of employees, and for re-verification of employee performance.

h. The laboratory shall maintain an up-to-date record of the training that each member of staff has received.

3. ACCOMMODATION AND ENVIRONMENTAL CONDITIONS a. The laboratories are designed to :

i. Provide space, engineering controls, and proper environmental conditions for optimal sample storage, sample handling, and analysis, in accordance with general laboratory practices.

ii. Minimize areas with cracks or fibers that could serve to accumulate debris and serve as an area for growth of microorganisms.

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 3 iii. Floors in the laboratories are constructed from a material that is resistant to

easily disinfected.

iv. Floors in the laboratories are clean, dry, and in sound condition so there are no tripping hazards.

v. Environment suitable for the tasks carried out microbiology testing.

b. Laboratory shall have :

i. Distinct space, in line with the biosafety requirements, for performing microbiology testing.

ii. Facilities that meet the required environmental conditions, including any needed separation of work areas to ensure that analyses will not be adversely affected within resources provided.

iii. Written policies for handling spills of contaminated materials.

iv. Laboratory storage areas provide proper storage of samples, reagents, microbiological media, select agents, standards and reference materials, and hazardous waste. Procedures and facilities in place for collection, storage and disposal of biohazard waste.

v. Laboratory monitored and documented the air quality of work places where clean operation is expected.

c. Laboratory shall have separate location provided for : i. Sample reception and preparation

ii. Manipulation of pathogens relevant to their hazard level iii. Cleaning of lab-wares, decontamination and sterilization

Appropriate biological safety cabinet for handling bacteria that highly contagious by air-borne routes

d. Laboratory shall have separate chambers or enclosures provided for i. Storing of reference cultures,

ii. Storing of samples,

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 4 4. TEST METHOD AND METHOD VALIDATION

a. The test method are adequately documented are based on the latest valid edition of a published reference method, including :

i. Title and method number, ii. Scope and field of application, iii. Number of revision,

iv. Page numbering, total number of pages, v. References,

vi. Principles and definitions, vii. Reagents and materials, viii. Apparatus,

ix. Analytical methodology, x. Expression of results, xi. Performance criteria, xii. Storage and shelf-life,

xiii. Procedures for media preparation,

xiv. Procedures for maintenance of stock cultures, xv. Procedures of safety for media handling, xvi. Quality control,

xvii. Colony counting (if applicable), xviii. Reporting criteria,

xix. Issuing authority.

b. The microbiological test method number and title used in the laboratory shall the same as the scope of testing when carry out assessment for accreditation.

c. The validation of microbiological test methods should reflect actual test conditions. This may be achieved by using naturally contaminated products or products spiked with a predetermined level of contaminating organisms.

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 5 d. The validation of microbiological test methods needs for non-standard methods and laboratory-developed methods. This activity is planned and assigned to qualified personnel.

e. The method’s performance characteristics are based on the intended use of the method. Method that used for qualitative microbiological test analysis there does

no need validate the method’s linearity over the full dynamic range of the

equipment.

f. Qualitative microbiological test methods, when the result is expressed in terms of detected / not detected and confirmation and identification procedures, should be validated by determining, if appropriate, the specificity, relative trueness, positive deviation, negative deviation, limit of detection, matrix effect, repeatability and reproducibility.

g. Quantitative microbiological test methods, the specificity, sensitivity, relative trueness, positive deviation, negative deviation, repeatability, reproducibility and the limit of determination within a defined variability should be considered and, if necessary, quantitatively determined in assays.

h. Determination of microbiological test methods performance characteristics for the validation of non-standard and laboratory developed methods recommended following :

i. Meet method system suitability requirements, if applicable. The suitability of

the method is checked and confirmed by comparing with requirements typical for the intended use of the method. Such as, a filtration method for a non-filterable food, surface tests for Colony Forming Units /square area where CFU/gram is needed.

ii. Include un-inoculated medium control to assess contamination from the

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 6 iii. Prepare and analyze positive and negative culture controls. Negatives control is a typically negative or no growth and the positive control is positive or shows microbial growth.

iv. Spike positive culture control is prepared and analyzed. This assesses the

matrix effects as well as the sensitivity of the method. It is recommended that a 25 gram sample be spiked with inoculums of 30 cells or less.

v. Evaluate interferences. This assesses the selectivity and specificity of the

method.

i. Laboratory shall have:

i. Written procedures for the proper collection and handling of sample and organism including personal safety and spill handling.

ii. Preparation procedures, protocols and quality control programs for in-house media.

iii. Procedures and records of media preparation.

j. Method is validated or verified in laboratory thorough:

i. Qualitative tests are based upon performance history and media QC.

ii. Repeatability – estimated using a minimum of 10 replicates of a known positive sample, or duplicate data collected over a period of time.

iii. Estimation of measurement uncertainty.

iv. Successfully participate in participate in proficiency testing.

k. Quality control programs for in-house media i. Physical appearance

ii. pH checking

iii. Sterility results after incubation

iv. Performance checks using positive and negative control organisms

l. Supporting work instructions shall available to: i. Work instructions for sample disposal

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 7 ii. Work instructions for disinfection / sterilization and disposal of bio hazardous

material

iii. Work instructions for glassware cleaning

iv. Work instructions for equipment instruction manuals

v. Work instructions for computer software related data entry and approval.

5. EQUIPMENT AND MEASUREMENT TRACEABILITY

a. Laboratory shall have:

i. Procedure specifies the schedule and requirements for maintenance, performance, calibration, and verification of laboratory testing equipment that meet the criteria of the maintenance and calibration parameters needed to achieve the accuracy of instruments used for analytical testing.

ii. Equipment records containing description of the instrument, critical accessories and software, manufacturer's name, type identification and serial number; Laboratory number; installation qualification (IQ) and operational qualification (OQ) records obtained from the installer or manufacturer; and other related material such as instrument service and repair, warranty information, service contract conditions and specifications.

iii. Laboratory shall have operating instructions for each instrument, including starting and shutting down the instrument.

iv. pH meter at least available two buffer to cover the range of pH needed in the tests methods for determination of pH standards, buffers available within their expiry dates.

v. Probes of pH meter maintained with cap on and/or in buffer solution or distilled water.

vi. Safety cabinet shall have documented protocol and record of decontamination.

vii. Program established to check regularly the rate of airflow and particle count in the Biohazard hoods or laminar flow cabinets.

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 8 b. The effectiveness of the autoclave operation shall checked monthly with a

biological indicator.

c. Thermometers and or thermocouples used for checking temperature of incubators, water baths refrigerators, and freezers calibrated against a reference thermometer by calibration laboratory and traceable to a national standard.

d. Automatic Pipettes is there an initial verification of volume delivered checked.

e. Are there regular checks to ensure that the equipment is performing within the specifications.

f. Hygrometer shall calibrated by calibration laboratory and traceable to a national standard, when humidity is influence to the outcome of the test.

g. Equipment that is not operating properly is clearly marked to show that it is out of service.

h. When an instrument is discovered to be improperly operating, it is tagged and taken out of service. Equipment is not returned to service until performance checks and verification have been performed and documented.

i. Each instrument has an established schedule specifying performance checks, including the testing frequency and acceptable performance specifications. These performance checks ensure the instrument is operating properly and consistently prior to analysis.

j. All instruments required for the routine test in microbiology field are available: i. Bunsen burners or other flame source functioning properly,

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 9 iii. Incubators functioning properly, maintained within the specified temperature range, temperatures monitored and recorded and spatial variability in incubators checked periodically,

iv. Water baths functioning properly, maintained within the specified temperature range; temperatures monitored and recorded periodically, v. Working thermometers are traceable to national standards,

vi. Microscopes covered with dust cover when not in use, vii. Optical surfaces of microscopes cleaned after each use,

viii. Autoclave is functioning properly, monthly test of autoclave performance using a spore strip or spore suspension, capable of demonstrating a 6 log kill of Bacillus stearothermophilus,

ix. Laminar air flow, check performance and check with sterility plates also airflow monitoring,

x. Sterilizing ovens, check stability and uniformity of temperature and Monitor temperature,

xi. Regularly scheduled maintenance program for each piece of equipment, where appropriate and records of service where service was required, xii. Distilled water that producing in-house and or purchasing from external

supplier and used to make media or reagents in microbiology tests shall check the conductivity periodically,

xiii. If total heavy metal may influenced the results of tests method for specific microbiology test, distilled water that producing in-house and or purchasing from external supplier and used to make media or reagents in microbiology tests shall be check the total heavy metal periodically.

k. Laboratory shall have arrangement for avoidance of cross-contamination arising from equipment, such as:

i. Disposable equipment should be clean and sterile when appropriate; ii. Re-used glassware should be properly cleaned and sterilized when

appropriate;

iii. Laboratories should have a separate autoclave for decontamination. However, one autoclave is acceptable provided that adequate precautions

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 10 are taken to separate decontamination and sterilization loads, and a documented cleaning programmed is in place to address both the internal and external environment of the autoclave.

l. Laboratories should carry out initial verification of volumetric equipment such as automatic dispensers, dispenser/diluters, mechanical hand pipettes and disposable pipettes and then make regular checks to ensure that the equipment is performing within the required specification.

m. Verification should not be necessary for glassware which has been certified to a specific tolerance.

n. Equipment should be checked for the accuracy of the delivered volume against the set volume (for several different settings in the case of variable volume instruments) and the precision of the repeat deliveries should be measured.

o. Where centrifuges are used in test procedures, an assessment should be made of the criticality of the centrifugal force. Where it is critical, the centrifuge will require calibration.

p. Conductivity meters, oxygen meters, pH meters and other similar instruments should be verified regularly or before each use.

6. REAGENTS AND CULTURE MEDIA

a. The following information shall be available from the manufacturer or producer (commercial or noncommercial bodies supplying media to third parties):

- Name of the medium, individual components and any supplements and, if possible, their product codes;

- Technical data sheet, e.g. formulation, intended use, filling quantity if applicable references;

- Safety and/or hazard data where needed; - Batch number;

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 11 - Target pH of the complete medium;

- Storage information and expiry date; - Assigned shelf-life;

- Quality control certificate showing test organisms used and results of performance testing with criteria of acceptance.

b. Laboratory shall have sufficient & appropriate quantities of reagents /media commercial kits available to carry out the volume of work following the required test methods.

c. Raw materials (dehydrated formulations and individual constituents) should be stored under appropriate conditions, e.g. cool, dry and dark.

d. All containers, especially those for dehydrated media, should be sealed tightly.

e. Dehydrated media that are caked or cracked or show a color change should not be used.

f. Shelf life of prepared media under defined storage conditions shall be determined and verified.

g. Stored reagents, reference materials and supplies shall be under the appropriate conditions and in a secure manner to ensure the separation of incompatible materials.

h. Solutions of reagent, standard and any other such as mobile phase properly labeled with solution name, concentration, date of preparation, expiration date, and identity of person who prepared.

i. Disposed of reagents and standards solutions appropriately followed to National and or local regulation

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 12 j. Laboratory shall have documented verification or comparison of standards and/or reference materials before use and the protocol when non-conforming results are encountered.

k. Distilled or de-ionized water systems are monitored for conductivity, bacterial content, and total chlorine periodically. Heavy metals analysis, water quality test and use test are performed annually on the water systems.

l. Microbiology media preparation and sterilization areas are separated from work areas to prevent contamination of clean media.

m. Laboratories shall ensure that all reagents including stock solutions, media, diluents, and other suspending fluids are adequately labeled to indicate, as appropriate, identity, concentration, storage conditions, preparation date, and validated expiry date and / or recommended storage periods.

7. REFERENCE MATERIAL AND REFERENCE CULTURE

a. Microbiology, standards and reference materials and cultures are stored separately from samples.

b. Reference cultures are required for establishing acceptable performance of media including test kits, for validating methods and for assessing / evaluating on-going performance.

i. Traceability is necessary, when establishing media performance for test kit and method validations.

ii. To demonstrate traceability, laboratories must use reference strains of microorganisms obtained directly from a recognized national or international collection, where these exist.

iii. Commercial derivatives for which all relevant properties have been shown by the laboratory to be equivalent at the point of use may be used as alternatively.

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Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 13 iv. Reference strains may be sub-cultured once to provide reference stocks.

Purity and biochemical checks should be made in parallel as appropriate. v. It is recommended to store reference stocks in aliquots either deep-frozen or

lyophilized.

vi. Working cultures for routine use should be primary subcultures from the reference stock. If reference stocks have been thawed, they must not be re-frozen and re-used.

vii. Working stocks should not be sub-cultured unless it is required and defined by a standard method or laboratories can provide documentary evidence that there has been no change in any relevant property.

viii. Working stocks shall not be sub-cultured to replace reference stocks. Commercial derivatives of reference strains may only be used as working cultures.

8. ASSURING THE QUALITY OF TEST RESULTS

a. Laboratory Quality Control is an essential aspect of ensuring that data released is fit for the purpose determined by the quality objectives.

b. Approach of quality control is the principal recourse available for ensuring that only quality data is released.

c. The Principe of the laboratory quality control program is its internal quality control to monitoring of analytical performance, and its external quality control based on the laboratory’s performance in proficiency testing programs.

d. Laboratory Management is responsible for establishing a Laboratory Quality Control Program and ensures that quality control is performed and reviewed of quality control data for acceptability.

e. Analysts are responsible for conducting quality control analyses in accordance with The Laboratory Quality Control Program.

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KAN-TN-LP 02 Issue Number : 3 April 2016

Dokumen ini tidak dikendalikan jika diunduh/ Uncontrolled when downloaded 14 f. Internal quality control is used to measure accuracy, precision, contamination, and matrix effects. The laboratory determines, where feasible, the accuracy and precision of all analyses performed.

g. Quality Control Program for Media shall including: i. pH of medium checked after autoclaving,

ii. Positive control culture to reference culture performed per batch of media, iii. Negative control culture to reference culture performed per batch of media, iv. Checked of sterility per batch of media,

v. Media that used for quantitative methods, quality control program including compare positive control cultures on selective media and nonselective media once per batch of media.

Bibliography:

1. Eurochem Guide 2013. Accreditation for Microbiological Laboratories

2. ISO 7218; 2007, Microbiology of food and animal feeding stuffs - General rules for microbiological examinations.

3. ISO 11133; 2014, Microbiology of food and animal fedd and water – Preparation, production, storage and performance testing of culture media

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KAN-TN-LP 02 Issue Number : 3 April 2016

iv. Water baths functioning properly, maintained within the specified temperature range; temperatures monitored and recorded periodically, v. Working thermometers are traceable to national standards,

vi. Microscopes covered with dust cover when not in use, vii. Optical surfaces of microscopes cleaned after each use,

viii. Autoclave is functioning properly, monthly test of autoclave performance using a spore strip or spore suspension, capable of demonstrating a 6 log kill of Bacillus stearothermophilus,