Lab. operations
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The document provides guidelines for designing and operating a microbiology laboratory. It discusses objectives for laboratory design which are to provide a safe environment for personnel and allow flexibility for research and teaching while anticipating health and safety hazards. Standards are outlined for containment of hazardous biological agents classified into four groups. Considerations for laboratory design include building design, internal structure and services, work flow, equipment used, and operation practices like documentation, standard operating procedures, and ensuring aseptic techniques.
Lab. operations
- 3. MAIN OBJECTIVE BEHIND DESIGNING A LABORATORY The primary objective in laboratory design should be to provide a safe, accessible environment for laboratory personnel to conduct their work. A secondary objective is to allow for maximum flexibility for safe research and teaching use. Therefore, health and safety hazards shall be anticipated and carefully evaluated so that protective measures can be incorporated into the design wherever possible.
- 4. Standards required for designing a laboratory Employees should be protected from exposure to substances and biological agents that may be hazardous to health. The advisory Committee on Dangerous pathogens (ACDP), UK defined four hazardous grou for biological agents: 1. Group 1: a biological agent that is unlikely to cause human disease. Eg. Lactobacillus spp. Saccharomyces spp. 1. Group 2: a biological agent that can cause human disease and may be hazard to employee it is unlikely to spread to the community and there is effective prophylaxis or effective treatment available. Eg. B. Cereus, Clostridium spp., Enterobacter spp., E.coli (Non-toxige Klebsielaa spp., L. monocytogenes, Salmonella, S. aureus, Streptococcus spp. 3. Group 3: Can cause severe human disease and may be a serious hazard to employees; it may spread to the community, but there is effective prophylaxis or treatment available eg. Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Ehrlichia sennetsu (Rickettsia sennetsu), Escherichia coli, verocytotoxigenic strains (eg O157:H7 or O103), Mycobacterium africanum, Mycobacterium tuberculosis , Salmonella typhi, Shigella dysenteriae 4. Group 4: Causes severe human disease and is a serious hazard to employees; it is likely to spread to the community and there is usually no effective prophylaxis or treatment availa Eg. very infectious viruses such as Ebola virus and Lassa fever viruses
- 5. The most common basic microbiology tests carried out in most food industry laboratories are the TCC and enumeration tests for coliforms yeasts and moulds and S. aureus. However, some Hazard group 2 organisms will occasionally occur on the plates, especially if the samples examined are unprocessed raw food materials. Therefore, for practical purposes, food microbiology laboratory desig and construction takes account of the guidance given by ACDP for th containment of Hazard Group 2 organisms. ORGANISMS PREVALENT IN FOOD MICROBIOLOG LABORATORY
- 6. Containment levels required for organisms in Hazard Group 2 affecting laboratory s and equipment, adapted from The Advisory Committee on Dangerous Pathogens (A 1. The laboratory should be properly ventilated and engineering controls and working arrangements must be devised so as to counter the risk of air borne transmission to othe areas. 2. When undertaking procedures that are likely to give rise to infectious aerosols, a Class I microbiological safety cabinet should be used. 3. There must be safe storage of biological agents (microorganisms). 4. An autoclave for the sterilisation of waste materials should be readily accessible in the same building as the laboratory, preferably in the laboratory suite. 5. Materials for autoclaving should be transported to the autoclave in robust and leak proof containers without spillage. 6. There should be means for the safe collection, storage and disposal of contaminated was
- 7. Biosafety cabinets are used to provide primary containment in the laboratory when the investigator is using potentially infectious materials. But which direction should the laminar flowmove: vertically (downward from a filter positioned above the work surface) or horizontally (forward from a filter positioned behind the work surface)? 1. Clearance requirements, 2. Process location, 3. Work surface design, and 4. Operator safety In vertical laminar flow hoods, fan/filter units are typically positioned in the ceiling. By directing the laminar flow downward, vertical laminar flow reinforces the effect of gravity and sweeps particles out of the enclosure. Micro-contaminants may not have substantial mass, but most particles do eventually settle on a work surface or the floor of a room, and vertical low helps get them there faster.
- 8. Vertical laminar air flow cabinet Horizontal laminar air flow cabinet Room air enters the system from above the HEPA filter; 99.99% particle-free air is forced downward toward the work surface. Room air enters the system from behind the HEPA filter; 99.99% particle-free air is forced in a back-to-front direction across the work surface.
- 9. There are three types of biological safety cabinets: Class I: The Class I biological safety cabinet is an open-front negative pressure cabinet. The exhaust air from the cabinet is filtered by a high-efficiency particulate air (HEPA) filter. The Class I biosafety cabinet will provide personnel and environmental protection, but not product protection as dirty room air constantly enters the cabinet front to flow across the work surface. Suitable for work involving low to moderate risk agents where there is a need for containment but not product protection. Hepa filter protect the env. By filtering air before it is exhausted. Personnel protection by constant movement of air into the cabinet and away from the user. Class II: The Class II vertical laminar-flow biological cabinet is an open-front, negative pressure vertical cabinet. hEpa filtered, vertical, unidirectional airflow within the work area. Hepa filtered exhaust air to the room or exhaust to a facility exhaust system. The exhaust air from the cabinet is also filtered by HEPA filters. Thus, the Class II biosafety cabinet will provide personnel, environment and product protection. While HEPA filters are effective for trapping particulates and infectious agents, these filters will not capture volatile chemicals or gases .
- 10. Class III: The Class III cabinet is a totally enclosed ventilated cabinet of gas- tight construction. Operations within the Class III cabinet are conducted through attached rubber gloves. When in use, the Class III cabinet is maintained through negative air pressure of at least 0.5 inches water gauge. Supply air is drawn into the cabinet through HEPA filters. The cabinet exhaust air is filtered by two HEPA filters, installed in series, before discharge outside of the facility. The exhaust fan for the Class III cabinet is generally separate from the exhaust fans of the facility's ventilation system.
- 11. BUILDING DESIGN ISSUES Because the handling and storage of hazardous materials inherently carries a high risk of exposure and injury, segregate laboratory and non-laboratory activities to the extent possible. 1. Laboratory should be isolated and separated from food production areas, preferable housed in a separate building 2. The fabric of the building must be ‘biosecure’ (no holes and cracks) 3. Large enough to accommodate all equipment and staff 4. Drainage from the lab. Should not flow through food production areas and should enter the main flow downstream of the factory drains 5. Emergency exits at suitable points 6. Windows should be designed that cannot be opened, and if ledges are present, they should be sloping to prevent them from being used as storage areas 7. If windows require shading, externally fitted blinds or films should be used.
- 12. Internal structure, fittings and services Hot and cold water supplies and electric power points must be provided Gas is required for bunsen burners Dedicated hand washing facilities with hot and cold water supplies and supplied with bactericidal soap and paper towel Dedicated double sink and drainer with hot and cold supplies and appropriate cleaning chemicals for equipment washing Suitable steam extraction and ventilation systems for autoclaves, hot air ovens. Any extraction system provided must safely exit to the external environment and not to the manufacturing areas A water treatment system. Eg. Distillation unit, reverse osmosis The laboratory should be temperature controlled to facilitate incubator control and provide a pleasant working environment All work surfaces (e.g., bench tops, counters, etc.) should be impervious to the chemicals and materials used in the laboratory. The laboratory shall be designed so that it can be easily cleaned. Bench tops should be of a seamless one-piece design to prevent contamination. Design laboratory workstations to accommodate the range of body dimensions that may be using the workstations. For example, computer and microscopes workstations may require height-adjustable work surfaces and chairs.
- 13. Work flow A. Plan for microbiology laboratory in which no specific bacterial pathogen work is underta (The hygienic integrity of lab. Should not be breached by staff , eg. From area 1, through are 7 to area 4, or from area 1 through areas 5 and 8 to areas 9 or 10 E Entrance lobby 1 Cloak Room 2 3 Laboratory Coat change/ handwash 4 Sample booking in 7 Sample store 9 Main laboratory 6 Office 5 General store 8 Media preparation 10 Wash up/decontamination
- 14. Work flow B. Plan for microbiology laboratory that includes a bacterial pathogen handling facility (The hygienic integrity of lab. Should not be breached by staff , eg. From area 1, through are 7 to area 4, or from area 1 through areas 5 and 8 to areas 9 or 10 E Entrance lobby 1 Cloak Room 2 3 Laboratory Coat change/ handwash 4 Sample booking in 7 Sample store 11 Main laboratory 6 Office 5 General store 8 Media preparation 10 Wash up/decontamination 9 Coat change/ handwash Pathogen laboratory 12 Hatch/ undercounter
- 15. EQUIPMENTS USED IN THE LABORATORY Autoclave Incubator Hot air oven Inoculating loop Vortex mixer / shaker Water bath Heating mantle Hot plate with magnetic stirrer UV chamber Inoculation chamber pH meter Colony counter Microscope Refrigerator Bunsen burner Spirit lamp Micrometer (stage and ocular) Balance (Digital and 4-beam) Thermometer Membrane filter set
- 16. LABORATORY OPERATION AND PRACTIC
- 17. LABORATORY PERSONNEL & STAFF Microbiological testing should be performed and supervised by an experienced person, qualified in microbiology or equivalent. Staff should have basic training in microbiology and relevant practical experience before being allowed to perform work. The laboratory should also maintain records of all technical personnel, describing their qualifications, training and experience. Personnel should be trained in necessary procedures for containment of microorganisms within the laboratory facility. Personnel should be trained in safe handling of microorganisms.
- 18. Laboratory housekeeping 1. The laboratory must be kept tidy at all time. 2. Boxes of disposables etc. should be stored off the floor, and levels o stocks held in the laboratory should be kept minimum. Bulk stocks should be held in a suitable dedicated microbiology store. 3. A good disinfectant (for example hypochlorite based) must always b available. Benches should be disinfected at the end of each working period and as necessary to maintain a safe working environment. 4. A regular laboratory cleaning and disinfection programme must be maintained to prevent floors, walls, ceilings, fitments and equipme such as water baths and interiors of incubators and refrigerators fro becoming source of infection for personnel, samples and media. 5. Soiled laboratory coats should preferably be autoclaved before bein sent for laundering.
- 19. Laboratory waste disposal 1. Safe waste disposal systems must be provided, understood and use efficiently by all staff. Waste must never be taken from the laborato through food manufacturing areas. 2. Used pipettes, tips for pipettes and microscope slides should be discarded into containers of disinfectant until they can be autoclav prior to disposal. 3. Samples entering the laboratory for examination must be disposed of through the laboratory and never returned to the factory. Unuse food samples that have entered the laboratory should not be consumed. Samples in the laboratory must be kept secure to preven unauthorised removal. 4. Autoclave processes for decontaminating waste should normally achieve a minimum of 121°C for atleast 30 minutes and preferably 45 minutes, or an equivalent heat process. THE RESPONSIBILITY FOR SAFE DISPOSAL LIES WITH THE WORKER RESPONSIBLE (THIS MEANS YOU !!!!)
- 20. LABORATORY SAFETYWaste materials awaiting disposal should be safely stored. Flammable & halogenated solvents & other contaminated waste liquids should not be poured into the drains carrying the factory effluent. They should be collected separately & disposed off suitably . There should be separate waste containers for broken glass.
- 21. ENVIRONMENTAL MONITORING Bench surfaces used for sample and culture processing, hand was basins, taps, handles and door seals, overhead structures, laminar air flow cabinets, balances, laboratory blenders used be monitored for their hygienic status: Visual assessment Swabs Contact plates Settle plates Records must be kept of the results
- 22. DOCUMENTATIONThe aim of documentation is :- To define specifications for all materials, method of manufacture & control. To inform all concerned manufacturers, personnel how when & why a batch is rejected. To provide audit trail for to permit investigation of any suspected defective batch.
- 23. STANDARD OPERATING PROCEDURES SOPs are basically required for :- • Analysis of drugs • Sample handling & accountability • Receipt, identification, storage & sampling of test & control articles. • Cleaning, maintenance and calibration of equipments • Responsibilities of audit team personnel. • Healthy & safety precaution • Storage & maintenance of microbial cultures. • Maintenance of animal rooms. • Use & storage of reference standards • Training programs & their frequency • House Keeping • Document control • Retention & disposal of control samples A SOP is a procedure that has been Determined, usually by a senior laboratory staff, as the most appropriate means for Carrying out a specified activity in the lab.
- 24. Fundamental requirements for achieving good aseptic and gen microbiological practices: 1. All containers, tools, media and other test equipment/ materials used for directly handlin test samples must be sterile. 2. Spreading and streaking techniques used for dispersing an inoculum over the surface of a agar in a Petri dish (plate) must be undertaken with care to avoid contact with the edge of plate which can lead to microbial growth that is difficult to assess and can lead to inaccura results. 3. Mixing procedures for each inoculum with an agar, in the pour plate technique must be undertaken with care to avoid splashing the sides and lid of the plates. 4. When a water bath is used for tempering bottles of molten agar, the bottles must be prope dried using disposable paper towels prior to pouring the agar into Petri dishes. This preve un-sterile water from the water bath from contaminating the inoculum, which would otherwise add microorganisms to the test sample under examination and lead to inaccura results.
- 25. Steps in sample processing procedure at which specific aseptic practice is required Process steps Aseptic handling specifically required (YES) Good microbiological practice required (GMP) Receipt of factory sample and storage of sample GMP Weighing of test sample Yes Addition of diluent Yes Blending/homogenization GMP Further dilution(s) Yes Inoculation (media/petri dish) Yes Addition of molten media Yes Mixing/dispersion Yes Incubation GMP Assessment of growth GMP Sub-culture for confirmation Yes Incubation GMP
- 26. Test sample preparation and handling 1. All factory samples taken for microbiological tests should be examined within a timescale appropriate to the sample, so that changes in the microbial composition of the sample between sample collection and testing are minimised. (a) A chilled product taken from a manufacturing line should be examined ideally within 1 of sample collection to ensure the sample’s condition adequately reflects the manufacturing circumstances. (b) All samples must be packed cleanly for transport taking care to keep raw or ‘dirty’ samp e.g. dusty and environmental samples well separated from clean samples. (c) When microbiological testing is sub-contracted to an external laboratory, ‘same day tes should be achieved for all factory samples held in refrigerated storage. (d) A specified quantity of test sample e.g. 10 g or 25 g is removed from the factory sample using sterile implements and good aseptic practices. 2. (a) For solid food materials, test sample treatment must ensure good dispersion of the sam in the diluent, e.g. using a laboratory blender with a min. blending time of 30 seconds, max. of 1 minute. (b) Heat (35-40°C) is used to aid dispersion, e.g. for high fat (>30%) samples; heat should b used with caution where samples are to examined for psychrotrophic bacteia
- 27. Contd.. 3. Care is required in test sample treatment to ensure that there is minimum shock to organism and maximum recovery by using, for example, Resuscitation techniques where necessary, e.g. dried products should be rehydrated gently using a method appropriate for the sample type Extra dilution, quenching agents or special diluents to inactivate inhibitory substan such as sanitizers or essential oils naturally present in foods such as onion, garlic and some herbs and spices, as well as some components of cocoa materials alone. 4. Diluted test sample must be inoculated into growth media within 20 minutes of their initia dilution and, preferably, should be re-mixed immediately before being used to inoculate m 5. Where diluents for primary dilution are pre-prepared in specific volumes, e.g. 90 ml and 18 , care must be taken to ensure the accuracy of these volumes. 6. A laboratory should have suitable control systems to ensure that contaminated diluents or media will be recognized e.g. Sterility checks of incubated uninoculated plates. 7. Surface plating media and broth media must not be used directly from the refrigerator. Med should be allowed to equilibrate to room temperature before use. 8. As a general rule, all prepared ‘pour plate’, ‘spread plate’ and ‘streak plate’ petri dishes shoul be incubated in an inverted position to prevent condensation from wetting the agar surface causing colonies to merge. However, the main exception to this are the plates used to grow m colonies, where mould spores may be dislodged by turning the plates over and over.
- 28. 9. Efficacy check of prepared media: Positive control: particularly useful for selective-diagnostic media to ensure the ‘target’ organisms, e.g. Salmonella, can grow well and produce the relevant diagnostic colony characteristics. Negative control: These, again, are particularly useful for selective diagnostic media to ensure that competitor organisms are either inhibited or produce colonies that are distinguishable from the target organism. For e.g. E. coli on selective culture media for Salmonella.
- 29. Sampling and sample terminology Factory sample Laboratory Weigh test sample, e.g. 10g, 25 g For detection tests Add pre-enrichment/ Enrichment medium Blend to produce test sample suspension Incubate Assess growth Confirmatory test as required For enumeration tests Add primary diluent Blend to produce test sample suspension Inoculation of medium Incubate Assess growth
- 30. LABORATORYACCREDITATION Accreditation is an approved procedure by which regulatory authorities or authorised body accord formal recognition to a laboratory to undertake specific task, provided that the pre-defined standards are met by. It involves re-assessment and surveillance at regular intervals so as to ensure that commitment to quality is a continuous process.
- 31. The organisation providing accreditation to the laboratories in India is the National Board Accreditation for Testing and Calibration Laboratories (NABL), Department of Science and Technology, Government Of India. The process of accreditation takes into account all the parameters as per ISO 15189, which is an International Standard. The Technical parameters of the standard include requirements of manpower and their competence, equipment, environment, material and use of standard laboratory methods.
- 32. In these schemes, an annual inspection of the laboratory's quality systems procedures and practices is carried out and reported by independent inspector In addition to an annual audit by an independent body, laboratories also particip in one or more External Quality Assessment (EQA) schemes. These tests provide a challenge to the whole laboratory operation and procedures including: Sample receipt Media preparation and quality control Sample handling procedures and traceability Staff practices Method efficacy Subscription by the laboratory to these schemes can provide a useful method f helping to demonstrate the credible operation of the laboratory.