Part III: Culture
quality control

Quality assurance with regard to tuberculosis culture is a system designed to continuously improve the reliability, efficiency and use of culture as diagnostic and monitoring option. The purpose of a quality assurance programme is to improve the efficiency and reliability of culture services. The components of a quality assurance programme are:

• quality control
• quality improvement
• proficiency testing

The following section will focus on aspects of quality controlin the culture laboratory. For a discussion on quality improvement and proficiency testing please refer to the Management Series.

Quality control of culture is a process of effective and systematic internal monitoring of the performance of bench work in the culture laboratory. Quality control ensures that the information generated by the laboratory is accurate, reliable and reproducible. This is accomplished by assessing - against acceptable established limits - the quality of specimens, the performance of decontamination, digestion and culture procedures, the quality of reagents, media and equipment, by reviewing culture results and by documenting the validity of culture methods.

Quality control should be performed on a regular basis in the culture laboratory to ensure reliability and reproducibility of laboratory results. For a quality control programme to be of value, it must be practical and workable.

Quality control must be applied to:

• laboratory arrangement
• equipment
• collection and transport of specimens
• handling of specimens
• reagents and media
• culture methods
• reporting of results

The keys to successful quality control are:

• adequately trained, interested and committed staff
• common-sense use of practical procedures
• a willingness to admit and rectify mistakes
• effective communication

Quality control measures which must be in place in all tuberculosis culture laboratories include:

Laboratory arrangement and administration

• Ensure that doors in the laboratory are  always closed. Work areas, equipment and supplies should be arranged for logical and efficient work flow. Work areas should be kept free of dust. Benches should be swabbed at least once a day with an appropriate disinfectant (eg. 5% phenol)
• Every procedure performed in the laboratory must be written out exactly as carried out and be kept in the laboratory for easy reference. Any changes must be dated and initialised by the laboratory supervisor
• All records should be retained for two years
• Laboratory procedures used routinely should be those that have been published in reputable microbiological books, manuals or  journals

Laboratory equipment

• Equipment should meet the manufacturers  claims and specifications
• Written operating and cleaning instructions must be kept in a file for all equipment
• Dated service records must be kept for all equipment
• Equipment must be monitored regularly to ensure the constant accuracy and precision necessary.
• Biological safety cabinet: The BSC is the primary containment device that protects the worker, product and/or environment from exposure to tuberculosis and its performance needs to be  verified at the time of installation and annually thereafter. The purpose and acceptance  level of the performance tests are to ensure the balance of inflow and exhaust air, the  distribution of air onto the work surface and the integrity of the cabinet. Other tests  check electrical and physical features of the BSC.

Daily checks of the BSC include the following:

• Ensure that the rate of airflow across the front opening is 75 linear feet/minute (22.86  meter/second) for Class I and 75 to 100 linear feet/minute (22.86 to 30.48 meter/second) for Class II cabinets.
• Check  the magnetic gauge in the exhaust duct for any pressure drop across the filters and  replace the filters when the gauge indicates that the airflow across the front opening has  dropped below optimal levels.

The following tests should be performed annually on Class I and  Class II cabinets:

Downflow velocity and volume test

This test is performed to measure the  velocity of air moving through the cabinet workspace.

• Inflow velocity test

This test is performed to determine the calculated or directly measured  velocity through the work access opening, to verify the nominal set point average inflow velocity and to calculate the exhaust airflow volume rate.

An electronic vane type anemometer should be used to measure airflow.  The airflow into a Class I cabinet should be measured in at least five places in the plane  of the working face and an average calculated. At no place should there be a reading that is 20 linear feet/minute (0.1meter/second) more or less than any of the others. If there  is such a difference there will be turbulence within the cabinet.

In Class II cabinets the airflow is greater at the bottom than at the  top of the working face. The average inward flow is calculated by measuring the velocity of air leaving the exhaust and the area of the exhaust vent. From this the volume per minute is calculated, which is also the amount entering the cabinet. Divided by the area  of the working face it gives the average velocity. The downward velocity of air should be measured at 18 points in the horizontal place, 10cm above the top edge of the working face. No reading should differ from the mean by more than 20%.

• Airflow  smoke patterns tests

This test is performed to determine if the airflow along the entire perimeter of the work access opening is inward, if airflow within  the work area is downward with no dead spots or refluxing, if ambient air passes onto or  over the work surface, and if there is refluxing to the outside at the window wiper gasket  and side seals. The smoke test is an indicator of airflow direction, not of velocity.

Commercial airflow testers are recommended. They are small glass tubes, sealed at each end. Both ends are broken off with the gadget provided and a rubber bulb fitted to one end. Pressing the bulb to pass air through the tube causes it to emit white smoke.

• HEPA filter leak test

This test is performed to determine the  integrity of supply and exhaust HEPA filters, filter housing, and after-mounting frames  while the cabinet is operated at the nominal set point velocities. An aerosol in the form  of generated particulates of dioctylphthalate (DOP) or an accepted alternative is required  for leak-testing HEPA filters and their seals. Although DOP has been identified as a  potential carcinogen, competent service personnel are trained to use this chemical in a  safe manner. The aerosol is generated on the intake side of the filter, and particles passing through the filter or around the seal are measured with a photometer on the  discharge side. This test is suitable for ascertaining the integrity of all HEPA filters.

• Cabinet  leak test

The pressure holding test is performed to  determine if exterior surfaces of all plenums, welds, gaskets, and plenum penetrations or seals are free of leaks. It is performed just prior to initial installation when the BSC is in a free-standing position in the room in which it will be used, after a cabinet has  been relocated to a new location, and again after removal of access panels to plenums for repairs or a filter change. This test may also be performed on fully installed cabinets.

• Electrical leakage and ground circuit resistance and polarity tests

These safety tests are performed to  determine if a potential shock hazard exists by measuring the electrical leakage, polarity  ground fault interrupter function, and ground circuit resistance to the cabinet connection. The polarity of electrical outlets are checked using a polarity tester. The  ground fault circuit interrupter should trip when approximately 5 milliampere (ma) is  applied.

• Lighting intensity test

This test is performed to measure the light  intensity on the work surface of the cabinet as an aid in minimising cabinet operator?s fatigue.

• Vibration  test

This test is performed to determine the  amount of vibration in an operating cabinet as a guide to satisfactory mechanical  performance, as an aid in minimising cabinet operator?s fatigue, and to prevent damage to delicate tissue culture specimens.

• Noise  level test

This test is performed to measure the noise  levels produced by the cabinet, as a guide to satisfactory mechanical performance and an  aid in minimising cabinet operator?s fatigue.

• Centrifuge: Check brushes and bearings every 6 months
• Incubator  35E-37EC: Record the temperature daily, preferably in the morning. Test the temperature at several sites within the  incubator by placing a thermometer in a water reservoir (eg. Erlenmeyer flask). Control the light within the incubator by covering the glass front of the incubator door and by  restricting the use of any lights inside the incubator
• Inspissator: Check temperature daily. Clean after each batch of culture media prepared
• pH meter: Compensate for temperature with each run. Date buffer solutions and discard when unsatisfactory. Standardise with pH 4.0 and 7.0 buffers before each test or series of  tests
• Water  baths:Check temperature before and during use. Clean monthly
•  
• Refrigerator 2E-8EC: Check temperature  daily. Clean monthly. Defrost or check refrigerator and freezer compartment every 3 months
• Freezers: Check daily. Clean every 6 months
• Glassware: Discard chipped or etched glassware. Ensure that glassware are free of detergents. Do not store sterile glassware for more than three weeks before it is used

Specimens and request forms

• Perform tests  only upon written request of authorised persons and do not allow oral requests without follow-up written instructions
• Insist on  specimen request forms being kept separate from the specimens themselves. Forms that have  been contaminated by specimens should be sterilised by autoclaving
• Insist on  adequately completed request forms and proper labelling of specimens to ensure positive  identification of patients. Reject specimens that cannot be properly identified
• Evaluate the quality of sputum specimens and make a note if a specimen resembles saliva. The report  should state ?specimen resembled saliva - treat a negative result with caution? (to facilitate reporting a rubber stamp of the comment can be made)
• Discard  leaking and broken specimen containers by autoclaving and request a repeat specimen
• Document the arrival time of specimens in the laboratory and note any delays in delivery on the report  form, particularly with negative/contaminated results

Reagents and stains

• All containers of stains and reagents should show the date received and the date  first opened. Any material found to be unsatisfactory should be recorded as such and  removed from the laboratory immediately. Stocks should be limited to six months? supply and regular stock rotation should take place to avoid unnecessary expiry.

Digestion and decontamination

• Process  sputum specimens in batches according to centrifuge capacity
• Keep a monthly record of the percentage of clinical specimens contaminated: the acceptable range is 2-5%. Contamination rates <2% indicates overly harsh decontamination, which means  that too many tubercle bacilli are killed. If the laboratory is experiencing delays in delivery of specimens the contamination rate may be greater than 5%. If a rate of >5%  persists, ensure that specimens are completely digested, since partially digested specimens may not be completely decontaminated. Thoroughly mix the contents of centrifuge tubes to ensure that the inside surfaces have been well decontaminated

Culture media

• Use fresh  eggs (< seven days) for preparation of Löwenstein-Jensen media
• Control  coagulation time and temperature for egg-based medium. Discard media that are discoloured or have bubbles following inspissation
• Check all  batches of media for sterility by incubation at 35E-37EC for 24 hours
• Keep all media in the dark in the refrigerator and discard unused media after four weeks

Culture procedures

• Avoid  cross-contamination of cultures by using individual pipettes or loops and strict aseptic  techniques
• Be suspicious  of several successively positive specimens or of cultures with few colonies that follow a heavily positive culture

Biochemical tests

Prepare reagents as indicated and check the expected biochemical test response by using appropriate positive and negative controls

Water

Check both distilled and tap water regularly for the presence of acid-fast contaminants. If water appears cloudy or dirty, centrifuge 200-250ml in multiple tubes and make a smear of the combined sediment. Alternatively, filter 1 000ml of water through a sterile 0.22Fm pore size membrane filter, cut the filter into strips with a sterile scissor and place on Löwenstein-Jensen culture medium.