Diagnostic Microbiology (For DMLT Students) Ranjan Kumar De
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Guidelines of Laboratory Safety and Quality Assuranceone

Personnel, working in diagnostic laboratory should have an adequate educational background with an extensive practical training in all aspects of routine diagnosis of medically important bacteria, viruses and parasites. Side by side all health care workers, especially laboratory technicians should be aware of the risks involved in their daily works.
The risk of laboratory-acquired infection with HIV or HBV is primarily from contamination of the hands and mucous membranes of the eyes, nose and mouth by infectious blood and other body fluids. The risk of HIV infection following percutaneous needle-stick exposure to HIV contaminated blood is estimated to be between 0.13 percent and 0.5 percent. In contrast, the risk of HBV infection following similar exposure to that virus is 45–120 times as great. HBV can survive for long periods in dried blood at room temperature.
Health care workers (HCW), especially those who routinely handle body fluids, hazardous samples mixed with blood or blood samples, should be aware of the risks involved their daily works. As there is no vaccine of HIV/AIDS, so prevention is the cornerstone of control of this epidemic disease and for that purpose all HCW including students should follow the universal biosafety precautions with procedures.
Biosafety Procedures
These are the combination of sophisticated work culture, personal hygiene habits, safe and skilled laboratory techniques, use of well-maintained and calibrated laboratory equipments with provision of enough illumination in laboratory area and facilities for carrying out diagnostic procedures with potentially hazardous materials.
Modes of Exposure of Blood Borne Pathogens in the Laboratory
Universal Blood and Body Fluid Precautions for Laboratory Workers
(Recommended by CDC guidelines, 1987)
Body Fluids to which Universal Precautions apply
  • Blood
  • All other body fluids containing blood
  • Semen
  • Vaginal secretions
  • Cerebrospinal fluid
  • Synovial fluid
  • Pleural fluid
  • Pericardial fluid
  • Amniotic fluid.
Body Fluids to which Universal Precautions do not apply
  • Faeces
  • Nasal secretions
  • Sputum
  • Sweat
  • Tears2
Table 1.1   Mode of transmission of blood borne disease
Lab. procedure
HCW at risk
Source/mode of transmission
Collection of blood/body fluid
Lab. technician/Nursing staff/Intern/House-staff
Needle-stick injury, Broken specimen container. Blood contaminaion of hand with skin lesion/breach.
Transfer of specimen
Lab. technician/Transport worker
Contaminated exterior of the container/requisition slip.
Processing of specimen
All laboratory personnel
Puncture of skin or contamination of skin/mucous membrane from:
• Contaminated work surface.
• Spill/Splash of specimen.
• Broken specimen container.
• Faulty techniques.
Cleaning and washing
Support staff
Puncture/contamination of skin from:
• Contaminated glassware.
• Sharps.
• Contaminated work surface.
Disposal of waste
Transport of specimen to distant laboratory
Lab. personnel/Support staff
Transport/Postal staff
Contact with infectious waste specially sharps. Broken/leaking container.
  • Urine
  • Vomits
To be or not to be in Microbiological Laboratory
  • Inspect the sample container about any leakage/breakage before receiving.
  • Discard heavily spoiled sample and advice to the patient to repeat it.
  • Avoid unnecessary delay to process the sample after receiving it.
  • After processing the sample it must be discarded in specified discard container containing suitable disinfectant.
  • Always use calibrated wire loop in case of urine culture.
  • Inoculation or other processings of suspected hazardous specimens should be done in Bio-safety cabinet.
  • Avoid the direct skin/hand contact with specimens or any spillage.
  • All samples must be labelled with patient's ID number, name and date before processing.
  • Sterility checking of incubator and air culturing of laboratory room to be done periodically.
  • Floor and working surfaces should be cleaned and disinfected after daily works.
  • Avoid to produce invisible aerosols which may be generated by—
    • withdrawal of loopful from broth culture.
    • the vibration of a wire loop during inoculation procedure.
    • the sputtering of a charged loop during flaming.
    • the removal of a wet stopper or cotton-wool plug.
  • Handle carefully the cultured mediums in petridishes if water of condensation on the medium or in the lid occurs.
  • Always flame the wire loop to sterilise by keeping the loop vertically as the total portion of the wire with handle tip may properly flamed.
  • After inoculation or making the smear of M. tuberculosis suspected samples; dip the inoculating wire loop at first in a test tube 3containing 2 percent phenolic and then flame it.
  • Avoid forceful ejection of contents from a syringe. Needle is blown off the syringe carefully.
  • Check the syringe before using to avoid leakage or back flow.
  • If there is any breakage of sample in the centrifuge tube at the time of centrifuging → first turn off the switch → wait for sometimes → with gloved hands thoroughly clean the test tube holders and inside of the machine with freshly prepared 1 percent Sodium hypochlorite solution → wait for 30 minutes → sweep with dry towel.
  • Working with each hazard group, according to their infective character the respective containment must be maintained.
Universal Biosafety Precautions
The following guidelines has been taken from Bio-safety Guidelines for Diagnostic and Research Laboratories working with HIV, 1991, WHO AIDS series 9, World Health Organisation, Geneva.
  • Wear gloves when handling infectious materials or where there is a possibility of exposure of blood or other body fluids.
  • Discard gloves whenever they are thought to have become contaminated, wash your hands, and put on new gloves.
  • Do not touch your eyes, nose or other exposed membranes or skin with gloved hands.
  • Do not leave the workplace or walk around the laboratory wearing gloves.
  • Wash your hands with soap and water immediately after any contamination and work is completed. If gloves are worn, wash your hands with soap and water after removing the gloves.
  • Wear a laboratory gown or uniform when in the laboratory. Remove this protective clothing before leaving the laboratory.
  • When work with material that is potentially infected with HIV is in progress, close the laboratory door and restrict access to the laboratory. The door should have a sign: ‘Biohazard. No admitance.’
  • Keep the laboratory clean, neat and free from extaneous materials and equipments.
  • Disinfect work surfaces when procedures are completed and at the end of each working day. An effective all purpose disinfectant is a freshly prepared hypochlorite solution with a concentration of 0.1 percent available chlorine (1 gm/litre, 1000 ppm).
  • Whenever possible, avoid using needles and other sharp instruments. Place used needles, syringes and other sharp instruments and objects in puncture-resistant container. Do not recap used needles and do not remove needles from syringes.
  • Never pipette by mouth. Use automatic mechanical pipette devices.
  • Perform all technical procedures in a way that minimizes the risk of creating aerosols, droplets, splashes or spills.
  • Do not eat, drink, smoke, apply cosmetics or store food or personal items in the laboratory.
  • Make sure that there is an effective insect and rodent control programme.
Universal Precautions Include (Recommended by CDC guidelines, 1987)
Barrier Protection
Including gloves and laboratory gowns some other protective barriers reduce the risk of exposure of the laboratory worker's skin or mucous membrane to potentially infective materials. These are:
Facial protection
  • Simple and cheap deflector masks and protective glasses may be worn if splashing or spraying of blood/body fluids is expected.4
Occlusive bandage
  • All skin cuts, scratches or other breaks must be covered with waterproof dressing before patient care.
Hand washing
  • Hand washing is mandatory and after completion of works that should be done with soap and water and if necessary with 70 percent ethanol as: wash palms and fingers → wash back of hands → wash fingers and knuckles → wash thumbs → wash fingertips → wash wrist.
Safe Techniques
  • Biological safety cabinet class 2 should be used for handling materials containing higher concentration of infectious agent that may present in clinical specimens.
  • Centrifugation should be done in tubes with safety caps.
Safe Handling of Sharps
To avoid auto-inoculation:
  • Broken glass should be picked up with a moist cotton ball.
  • Disposable needles should never be recapped/bend. Syringe-nozzle to be shredded by needle-destroyer to avoid recycling.
  • One should always dispose the sharps in a puncture-proof rigid discard container.
Safe Handling of Specimens
  • Specimens should be collected in screw capped leak-proof plastic container.
  • Autoclaved/Sterilised disposable syringes, needles or lancets should be used.
  • Requisition slip contaminated with blood/body fluids should be rejected otherwise that can be handled with gloved hands.
  • All hazardous samples should be labelled as “Biohazard Precaution”.
Safe Handling of Blood/Body Fluid Spills
In case of blood/body fluid spill in the laboratory.
Area should be flooded with freshly prepared sod. hypochlorite solution with a concentration of 1 percent available chlorine (10 gm/litre, 10,000 ppm) or 1–2 percent clear soluble phenolic.
The area should be covered with paper towel/absorbent material. Wait for 10–30 minutes.
The mixture of disinfectant and spilt material should be cleaned up with absorbent material.
Place the absorbent material in a contaminated waste container.
The area should then be wiped again with disinfectant.
Health and Medical Surveillance of Employees
  • Medical examination of all laboratory workers is necessary.
  • A baseline serum sample should be obtained and stored frozen for future reference. All findings should be kept confidential.
  • Record should be kept of all illness and absences of laboratory workers.
  • All laboratory workers including students should be immunised by Hepatitis B vaccine as recommended by WHO.
Disposal of Laboratory Waste
  • Every hospital, nursing home, blood bank, diagnostic centre should install an appropriate biomedical waste facility in the premises or should set up a common facility in accordance with the directions given by 5the appropriate authority of waste management programme.
  • Biomedical waste should not be disposed of on municipal dustbin.
  • All biomedical wastes should be properly treated before disposal.
  • Untreated liquid waste should not be let into sewers.
  • All precautions and personal safety measures should be taken in handling and disposing biomedical wastes.
  • Waste segregation should be done at the site of generation by placing different types of wastes in different colour-coded bags.
Strategy of Disposal
First Aid in Laboratory Accidents
Accidents in a laboratory may caused by:
  • Burns with acids, alkalies, toxic substances and heat.
  • Cut by broken glassware.
  • Electric shock.
  • Needle-stick injury.
Acid Burns
  • If acid splashes on skin:
    • Wash repeatedly and thoroughly with water.
    • Bathe the affected skin with cotton wool soaked in 5% aqueous sodium carbonate.
  • If acid splashes in the eyes:
    • Wash the eyes immediately with large quantities of cold water sprayed from wash bottle. Alternatively, hold the eyes under running tapwater.
    • After washing, put 4 drops of 2% aqueous sodium bicarbonate solution into the eyes → refer the individual to a physician and continue to apply biocarbonate solution into the eyes pending the physician's arrival.
  • In case of swallowing acids:
    • Call immediately a physician.
    • Make the patient drink some 5% soap solution. Alternatively, give 2 whites of eggs mixed with 500 ml of water.
    • Make the patient gargle with soap water.
Alkali Burns
Alkali burns are as serious as, and often more serious than acid burns.
  • If alkali splashes on the skin:
    • Wash thoroughly and repeatedly with cold water.
    • Bathe the affected skin with cotton soaked in 5% acetic acid or undiluted vinegar.
  • If alkali splashes in the eyes:
    • Wash the eyes immediately with large quantities of water sprayed from a wash bottle; squirt the water into the corner of eyes near the nose.
    • After washing, wash the eyes with a saturated solution of boric acid repeatedly.
    • Refer the patient to a physician.
  • In case of swallowing alkalies:
    • Call immediately a physician.
    • Make the patient drink at once: 5% solution of acetic acid/lemon juice/1:3 diluted vinegar with water.
    • Make the patient gargle with the same acid solution.
    • Give the patient 3–4 glasses of ordinary cold water.
Burn caused by Heat
  • In case of severe burns:
    • If the victim on fire, roll him/her in a blanket and lay the victim on ground.
    • Inform a physician urgently.
    • Do not remove the blanket or apply any treatment. It must be left to the physician.
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Flow chart 1.1: Strategy of disposal
Cuts by broken glassware
  • By clean glass
    • Disinfect the area using tincture of iodine etc.
    • Cover with antiseptic ointment and adhesive dressing.
    • Report the incident to the laboratory incharge.
  • By glass containing infected material
    • Squeeze hard to make the cut bleed for few minutes.
    • Bathe the whole area with a surgical antiseptic.
    • Wash thoroughly with soap water → bath again with surgical antiseptic.
    • Report immediately to the higher authority about the incident.
Bodily damage by Electric Shock
  • Before doing anything else, cut off the electricity at the main fuse.
  • Begin giving artificial respiration (mouth to mouth) immediately and massage the heart externally if necessary.
  • Patient to be sent for a physician.
Management of Occupational Exposure to HIV in Laboratory
Though every occupational exposure do not lead to HIV infection but it may place the HCW at risk of HIV infection. The risk of infection with the type of exposure and factor such as:
  • The amount of blood involved in the exposure.
  • The amount of viruses in patient's blood at the time of exposure.
  • Whether post-exposure prophylaxis (PEP) was taken by HCW within recommended time or not.
On Exposure to HIV Infected Blood/Body Fluid or Contaminated Sharps, etc.
If such an exposure occurs → encourage bleeding for few minutes by squeezing the effected area. Do not put the pricked finger/area into mouth reflexly → wash thoroughly with soap and water → cover the area with waterproof antiseptic dressing → report immediately regarding the incident to the higher authority → source material (Blood/Body fluids) should be tested for the presence of virus and/or antibody.
If the source material is positive for HIV antibody, virus, or antigen or is not available for examination → HCW should be serologically tested and pre/post test counsellings are needed → seek medical evaluation of any acute febrile illness that occurs within 12 weeks after the exposure → seek also medical advice that whether PEP is needed or not → If HCW is seronegative, he or she should be retested, periodically at 6 weeks, 3 and 6 months after the exposure → during follow-up the HCW should take general precautions for HIV transmission and appropriate counselling.
Type of Occupational Exposures to HIV for Which PEP is Recommended
All occupational exposures do not lead to HIV infection but if it occurs and reported by HCW, the decision must be taken by authority immediately. Because, if PEP is needed it should be started as soon as possible, preferably within few hours. Although, any cut off time is arbitrary initiating treatment more than 72 hours after the exposure is not recommended.
PEP is made on the basis of degree of exposure to HIV and HIV Status of the source from whom exposure has occurred.
Determination of the Exposure Code (EC) See Flow chart 1.2.
Determination of the HIV Status Code (HIV Sc) See Flow chart 1.3.
Determine the PEP Recommendation See Table 1.2.
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Flow chart 1.2: Determination of the exposure code (EC)
zoom view
Flow chart 1.3: Determination of the HIV status code (HIV Sc)
Table 1.2   Determine the PEP recommendation
PEP recommendation
PEP may not be warranted.
Consider basic regimen. Negligible risk.
Recommended basic regimen. Most exposures are in this category; no increased risk for HIV transmission has been observed but use of PEP is appropriate.
Recommended expanded regimen. Exposure type represents an increased HIV transmission risk.
1 or 2
If EC is 2 or 3, consider PEP basic regimen.
It means the sum of all those activities in which the laboratory is engaged to ensure that the test results are good quality. It must be:
  • cover every quality step in the cycle from collecting of the sample to despatch the final report to the patient.
  • concentrated on the most critical steps in the cycle.
  • regular, i.e. have a continuous monitoring of test procedures.
Quality assurance are two types:
  • Internal; Which is called Quality control and each laboratory should have a programme to check the quality of its own tests by–
    • Continuous monitoring of the quality of tests.
    • Comprehensive checking of all steps, from collection of sample to delivery the reports.
  • External; Which is named Quality assessment, where laboratory performance is controlled by an external agency by–
    • Periodic monitoring of test quality.
    • Spot checking of different tests and isolation techniques.
Requirements of Quality Control Programme
Laboratory or organisation shall
  • have a legal identity and responsibility to carry out the standard testing and calibration activities which can satisfy the needs of a client (Patient).
  • have managerial and technical personnel with the authority and resources needed to carry out their duties.
  • have policies and procedures to ensure the protection of its client's confidential information including procedures for protecting the electronic storage and transmission of results.
  • specify the responsibility, authority and interrelationships of all personnel who manage, perform or verify work affecting the quality of the tests and/or calibrators.
  • appoint a member of staff as quality manager, who, irrespective of other duties and responsibilities shall have defined responsibility and authority for ensuring that the quality system is implemented and followed at all times.
Quality Manual (QM)
  • Laboratory's quality system policies and objectives are defined in the QM.
  • Management structure shall also be defined in QM.
  • Document preparation, approval, issue and changes are strictly accordance with QM.
  • A standard operating procedures manual (SOPM), prepared by centralised agency of laboratory, should available in all sections of the laboratory.
Work Instruction
Apart from SOPM, work instructions for common tests should be prepared and displayed in all sections as a ready reference.
Purchasing Services and Suppliers
  • A register of approved suppliers should be maintained.
  • Laboratory should have a policy and procedure for purchasing and storage of reagents and consumables.
  • Qualities of reagents and consumables purchased are to be checked and recorded.
Service to the Patient
  • If require, prescriptions are clarified or explained to the patient.
  • The patients should have direct access to the doctor-in-charge.10
Control of Records
Laboratory should maintain quality and technical records with indexing, filing system or electronic system for easy identification.
Internal Audit
An internal audit by authority should be undertaken in regular interval.
Technical Requirements
  • All technical personnel should be well educated with laboratory technology.
  • Competency criteria of laboratory personnel should be recorded.
  • Training programme (in-house) among technical personnel should be done in regular interval by experienced authority and recorded.
  • Job description of pesonnel is to be filled.
Accommodation and Environmental Condition
  • Access to the working area should be controlled.
  • Environmental condition such as humidity and temperature of the laboratory room should be recorded regularly.
  • Sterilisation of laboratory room and Bio-safety cabinet or Laminar air flow must be done at regular interval and immediately after that aerobic and anaerobic culture of swabs from room's floor, wall, table and also air culturing of room and inside of Bio-safety cabinet are necessary with documentation.
Calibration and Maintenance of Equipments
  • General laboratory equipments should be calibrated at regular occasion and that must be documented.
  • Certain items of equipment may be calibrated by laboratory itself without the service of external calibration bodies, provided the laboratory has the necessary reference standards and materials.
  • The performance of temperature-controlled equipments such as water bath, incubator, oven and refrigerator, etc. shall be monitored routinely to ensure compliance with the temperature requirements of test methods. Accordingly daily recorded checks of the temperature within the load space of these items of equipment shall be maintained.
  • A separate Biological safety cabinet, certified at least annually to ensure that filters are functioning properly and that air flow rates meet specifications, must be available for mycobacteriological work and for mycological work.
  • The laboratory performing fungus culture shall be equipped with heating and cooling incubator like Biological oxygen demand (BOD) incubator to meet with the environmental conditions of the isolation of fungi.
Test Methods
  • The laboratory must maintain the stock of reference organisms. These should be used to test the prepared media. Monitoring of performance of the different identification test procedures and prepared stains is done also by the organisms.
  • The number of antibiotic disc applied on the petridish to test antibiotic sensitivity shall be as per CLSI (formerly NCCLS) recommendation.
  • The laboratory located in the hospital shall test against the antibiotics as per the hospital antibiotic policy. The stand-alone laboratory shall have an antibiotic sensitivity testing policy on the basis of site of infection, antibiotic susceptibility pattern.
  • Enrichment and selective media should be used for isolation of organisms from different clinical specimens.11
  • Control strains of known susceptibility should be used along with the test sample while performing drug susceptibility testing.
  • Efficiency testing of any equipment should be done on each run.
  • pH of the prepared media must be tested.
Internal Quality Control
Quality control programme (internal) in a laboratory has some important objectives:
  • To ensure the highest possible standards of performance.
  • To assess the top accuracy of test results.
  • To help other laboratories assess their own standards.
These types of high level tasks are done by two ways:
  • By replicate testing: A sample with known result should be coded and given to the technical personnel who is not aware of it's final report. The same test will be done by the personnel with same technique, same batch of reagents or medium/s on same day with known standards. The results of test of such sample should be recorded and discussed with concerned person and any shortcomings corrected and documented.
  • By retained sample testing: Same sample (of known result) with confidential code will be done by another technical personnel on next day as an unknown sample with same technique and same batch of reagents or medium/s with known negative or positive standards. Any short comings should be corrected and documented.
External Quality Assessment (EQA)
  • Known positive or negative samples are to be exchanged between two or three laboratories within or outside of the state and results of different laboratories be compared.
  • In EQAs, an organizing agency sends material to participants who return their results to the agency. Each participant analyses the same material so that there should be no significant difference between the results of both qualitative and quantitative analysis from participitants.
  • EQAs should be supplemented by an organized system of supervision within the laboratory network.
  • Materials for EQAs should be received and treated in the same way as the routine samples are analysed by the personnel who is involved with the testing of routine samples and the data should be kept confidential, results should not be divulged to third party.
  1. Bio-safety Guidelines for Diagnostic and Research Laboratories working with HIV, WHO AIDS series 9, 1991, Geneva.
  1. Basic Laboratory Procedures in Clinical Bacteriology. WHO, Geneva,  2nd edition.
  1. Cheesbrough M. Medical Laboratory Manual for Tropical countries, vol-II, ELBS/Churchill Livingstone,  1985.
  1. Doc no. NABL 112, 215, Issue-02, National Accrediation Board for Testing and Calibration Laboratories.
  1. HIV Testing Manual; Laboratory Diagnosis, Biosafety and Quality control. National AIDS control organisation, Nirman Bhawan,  New Delhi. 
  1. HIV Testing Manual; Laboratory Diagnosis, Biosafety and Quality control. National Institute of Communicable Diseases, Samnath Marg,  Delhi.  (F.C-1.2, 1.3 and T-1.2 are reproduced here in toto).
  1. Ross PW, Peutherer JF. Clinical Microbiology, Churchill Livingstone,  1987.
  1. WHO Managing Medical Waste in Developing countries, 1994, WHO, Geneva.