| Chapter 47 The Ophthalmic Microbiology Laboratory JOHN PEACOCK and MELVILLE MATHESON Table Of Contents |
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LABORATORY ORGANIZATION LABORATORY EQUIPMENT LABORATORY ADMINISTRATION INFECTION CONTROL WASTE DISPOSAL QUALITY ASSURANCE, CONTROL, AND IMPROVEMENT REFERENCES |
| LABORATORY ORGANIZATION |
| The laboratory area designated for ocular microbiology ideally should be
used exclusively for this purpose. The laboratory area must comply with
all statutory health and safety requirements pertaining to the country
or state in which the laboratory is geographically sited. The area
should be roomy, allowing for easy access to all equipment. The floors, walls, and
benching must be constructed of materials that allow for
easy cleaning and chemical disinfection. A workbench fitted with a laboratory sink and water supply for microscope slide staining is required. Gas taps should be incorporated into the bench, because these are more convenient than portable gas burners. Electric outlets should be fitted along the working area for small bench equipment. Adequate provision for floor-standing equipment and storage space for laboratory consumables must be available. SPECIMEN RECEPTION The specimen reception area must be easily accessible to portering staff and others delivering specimens. The reception area must be fitted with a sink for handwashing, and the bench surfaces should be constructed of water-resistant materials. The benching should be wiped down with an antiseptic frequently during the working day. A biohazard spill kit suitable for disinfecting blood or any other body fluid must be placed on the bench so that any spillage may be dealt with immediately. There should be an established link with a histology laboratory so that appropriate tissue specimens can be forwarded immediately for diagnostic investigation, such as histologic analysis for fungal hyphae or monoclonal staining for Acanthamoeba cysts. OFFICE The office space must be completely separate from the laboratory and specimen reception area. Procedures must be in place to ensure that no potentially clinically contaminated materials, including laboratory coats, are placed in the office. |
| LABORATORY EQUIPMENT |
INCUBATORS A carbon dioxide incubator should be of medium size and have an integrated microprocessorcontrolled system to maintain a 5% concentration of carbon dioxide. An increased level of carbon dioxide is a necessity for the isolation of Neisseria gonorrhoea and N. meningitidis on primary culture. A digital display unit on the exterior of the equipment will show the temperature and carbon dioxide concentration within the incubator chamber. Stainless steel trays in the base of the incubator are for water, which increases the humidity in the chamber. Increased humidity prevents agar plates from drying out during the long incubation time required for the isolation of slow-growing microorganisms such as Nocardia species. The isolation of Acanthamoeba also is enhanced when nonnutrient agar plates, seeded with Escherichia coli, are incubated in a moist atmosphere. A second smaller incubator, without carbon dioxide, operating at a temperature range of 26° to 28°C is used for growing some species of filamentous fungi. Many of the fungal isolates from ocular tissues have plant origins and have a lower optimum growth temperature than most bacteria. An incubator without carbon dioxide also is used for incubating antibiotic disc-diffusion plates and for the biochemical identification methods for Enterobacteria. Equipment and materials supplied to nonhospital-based clinics enable medical and nursing staff to take ocular samples and inoculate them directly onto the correct media (Table 1). The recovery rates are increased greatly using direct inoculation techniques rather than transport media. The specimens ideally should be transported directly to the laboratory, but a small 37°C incubator should be available for specimens collected during out-ofoffice hours.
TABLE 1. Equipment for Microbiology Laboratories Hospital-Based Laboratory
MICROBIOLOGIC CABINETS A class II laminar flow hood that offers operator and specimen protection from contamination is used for the manipulation of biohazardous bacterial, fungal and protozoal cultures, and infected clinical samples. These hoods are fitted with a coarse prefilter before the air enters a double high-efficiency particulate filter. The cabinets can be used without ducting to recirculate the laboratory air but, ideally, the cabinets should be ducted outside the laboratory building. To disinfect the interior of the cabinet, an ultraviolet light source is fitted as a standard feature. A formaldehyde vaporizer may be fitted to the side wall of the unit. When switched on, it heats up and releases formaldehyde vapor directly into the cabinet. Decontamination of the cabinets with formaldehyde-based regimens causes no problem with externally ducted cabinets; however, with nonducted recirculating cabinets, the laboratory area has to be closed down until all formaldehyde fumes are cleared from the area. Extra options that may be incorporated into inoculating hoods are gas taps, power points, and a water supply. A class I microbiologic cabinet offers maximum operator protection. Class I cabinets have ducting fitted with an integral filter, and the air is exhausted to the outside of the laboratory building. It is mandatory that class I cabinets are used when working with viruses, tubercule bacilli, or other high-risk pathogens. CENTRIFUGES A small- to medium-sized centrifuge is needed for separating vitreous biopsy samples, vitreous washings, contact lens care solutions, and other liquids. After removal of the supernatant, culture of the deposit maximizes the recovery rate from samples that may only contain a few viable microbes. A microcentrifuge is useful for separating small samples of aqueous or vitreous humor. A cytospin centrifuge is used for preparing vitreous and aqueous smears for cytologic studies. Cytologic examination of intraocular fluids should be done routinely. LABORATORY BURNERS Bench gas or electric burners are used to sterilize wire inoculation loops. A gas flame also is used to flame the necks of sample and media bottles to ensure an aseptic technique. Smears on microscope slides may be heat fixed in the flame of a gas burner. REFRIGERATION AND FREEZING All laboratory refrigerators and freezers should be electric and spark-free. All unused bacteriologic media and diagnostic kits should be stored in a clean refrigerator at 4° to 8°C. New media should never be stored in the proximity of microbially contaminated materials. Clinical material (e.g., vitreous humor, anterior chamber fluid, evisceration specimens) should be stored at 4° to 8°C if there is a delay before the material is inoculated onto microbiologic media. If prolonged storage is required, samples should be frozen at -20°C. ANAEROBIC CULTURE Because anaerobic organisms rarely cause ocular infections, the demand for anaerobic culture is small. Anaerobic culture apparatus need only have a capacity for up to 10 Petri dishes. It is recommended that three anaerobic jars or other systems are available for use. For optimal incubation, it is a good practice not to open jars until culture plates have completed the minimum incubation time. MICROSCOPY A binocular microscope with a tungsten light source should be fitted with 10× magnifying eyepieces and 5× , 10× , 20× , 40× , and 100× magnification objectives. The 100× objective is an oil immersion lens. An ultraviolet light source and integrated ultraviolet filters can be incorporated into the microscope for fluorescent microscopy techniques. Nomarski, differential interference contrast, and phase-contrast optics are useful additional options for examining unstained tissues, contact lens care solutions, contact lenses, and intraocular lenses. A 35-mm camera should be attached for photographing clinical cases of special interest that subsequently may be used for publication or teaching. Digital cameras are commercially available and have some advantages over conventional photographic methods. Color prints are reproduced on a computer screen, and areas of the print may be color enhanced. Montages (i.e., a collection of prints) using the computer are easy to compose. Computerized indexing of the pictures makes the archiving of slide collections easy. MOLECULAR BIOLOGY Polymerase chain reaction equipment (e.g., thermal cyclers and electrophoresis equipment for agarose or acrylamide gel preparation) is standard molecular biology laboratory equipment. To minimize contamination, the initial preparation of ocular tissues for polymerase chain reaction must be performed in a clean area exclusively designated for this purpose. Airflow cabinets specifically for preparing clinical material for polymerase chain reaction are commercially available. Ocular samples for virologic examination should be referred to a specialist virus laboratory. Ultralow temperature freezers, high-speed centrifuges, and cell culture equipment for viral isolation are standard. It generally is not economical to purchase this equipment exclusively for ocular microbiology. EQUIPMENT MAINTENANCE AND QUALITY CONTROL Major equipment items must be placed on manufacturers' or qualified agents' preventive maintenance contracts. Servicing of incubators, refrigeration equipment, laminar flow hoods, centrifuges, ovens, and autoclaves must be performed every 6 months. The servicing agents must provide certificates for each item of equipment, including the equipment's model and serial number, the date examined, and comments regarding the working efficiency of the apparatus. Any faults found and any new parts that were fitted must be listed in the report. Laboratory technologists should check the temperature of incubators and refrigeration equipment daily with thermometers that are independent of the equipment's integrated instrumentation. Incubators should have all surfaces cleaned and disinfected with a noncorrosive antiseptic weekly. Microbiologic cabinets should have all internal and external surfaces cleaned and disinfected thoroughly each time they are used. Equipment stored in microbiologic cabinets decreases the efficiency of the air flow within the hood, and the air flow must be checked using a portable anemometer twice weekly. Centrifuge rotors must be checked for corrosion, and the internal surfaces of the centrifuge must be cleaned weekly. HOMOGENIZER Small Wheaton tissue homogenizers are useful for the microbiologic examination of corneal or scleral tissue. These homogenizers have two pestles. The loose-fitting pestle will not disrupt keratocytes, inflammatory cells, or amoebae when homogenizing the tissue. The tight-fitting pestle should be used only for bacterial and viral isolation techniques as the homogenate is very fine and all tissue cells and amoebic trophozoites are crushed. |
| LABORATORY ADMINISTRATION |
| Medical laboratory services have had to adapt to the ever-changing needs
and demands of purchasers and patients. Health and safety issues, control
of substances hazardous to health, laboratory accreditation, and
other regulatory issues greatly increase the responsibilities of laboratory
administration.1–3 Because clinical services have had to become more cost-effective, laboratory
staff also must be involved in the daily administration of departmental
budgets and cost accounting. Medical laboratory services' primary function is to supply accurate diagnostic reporting on patient samples. Education, research, and development also are central functions of pathology services. To ensure effective communication at all levels, a microbiology laboratory users' committee must be convened. The committee should include members of senior hospital management and medical, laboratory, and nursing staff. Pharmacy, transport, information technology, and infection-control personnel should be co-opted onto the committee when required. LABORATORY ACCREDITATION Medical laboratory accreditation is administered by governmental commissions and peer review. It is mandatory in some countries and voluntary in others. Accreditation setse the minimum standards acceptable for laboratory space, furnishings, and equipment. The quality and numbers of staff employed (including their past training and continuing education) also are assessed.4 The diagnostic laboratory methods must be stated clearly in the form of a standard operating procedures manual. Laboratory staff at all levels must strictly adhere to the methodology as written in this document. Ophthalmic microbiology techniques have some unique features, particularly in the investigation of corneal specimens, anterior chamber fluid, vitreous, ocular implants, and contact lenses. Therefore, it is essential that standard operating procedures are available and easily accessible in the laboratory. Microbiology technologists who process ocular specimens infrequently can refer to the relevant procedure, therefore ensuring uniformity and quality control of all investigative methods used. LABORATORY PERSONNEL The staff will include a medical microbiologist experienced in ophthalmic microbiology, registered laboratory technologists, and laboratory assistants. Clerical and administrative staff also are needed to process laboratory reports, update computer data bases, and supply finance departments with information for correct invoicing of services rendered. The microbiologist's duties include clinically assessing patients along with ophthalmologists and giving advice on antimicrobial therapy and infection control. The microbiologist, along with senior technical staff, has the responsibility of training junior medical staff in ophthalmic specimen collection techniques, interpretation of results, and how to maximize the use of the microbiology laboratory. Qualified medical laboratory technologists have to be registered practitioners and belong to a biomedical professional body. Assessment, audit of individual competence, and continuing competence should be ongoing requirements. LABORATORY HANDBOOK A comprehensive users' guide to the services provided by the ophthalmic microbiology laboratory must be available to all users of the service. The handbook will include details of the techniques to use for collecting specimens, the correct microbiologic culture media for inoculating patient samples, and the types of specimen containers to use.5 Instructions on labeling the containers, completing specimen request forms, and transporting specimens to the laboratory should be given. The clinical significance of bacteria, fungi, viruses, and other microorganisms should be stated. The commensal and pathogenic microorganisms to be found at any ocular site should be tabulated. The antimicrobial therapy policy in force for any given hospital or area health authority must be included. Laboratory staff, ophthalmologists, physicians, infection-control committees, pharmacists, and hospital management will have agreed on this policy. Hospital infection-control policies and procedures for reporting possible nosocomial infections should be decided along with a list of members on the infection-control committee. LABORATORY REQUEST FORMS Full patient identification and patient clinical history, including any antimicrobial treatment that has been given, must be entered onto the specimen requrest form. A contact name for laboratory staff to convey urgen reports along with the requesting doctor's name must be included on the form. On receipt in the laboratory, the patient's details are transferred to a daybook. Laboratories occasionally receive incorrectly or inadequately labeled specimens or specimen request forms. These errors or omissions can cause erroneous laboratory reports to be issued. Laboratory staff time is wasted attempting to contact the requesting clinician or the clinic administrator to clarify the clinical or patient details. Laboratories should not issue verbal or written reports if there is any doubt about the identity of the patient or specimen. Unlabeled or leaking specimens typically are destroyed on receipt. SPECIMEN HANDLING AND REPORTING Any material for microscopy (e.g., Gram stain, periodic acid-Schiff) is processed as soon as possible after arrival in the microbiology department. The results are given verbally to the clinician responsible for the case. A primary written report on the outcome of solid-phase culture is issued 24 to 48 hours after receipt of the specimen. Broth cultures are subcultured after a suitable incubation period. A final written report, including sensitivity testing results, is issued at the completion of this culture period. Additional verbal reports are given during the culture period as required. Pathways of responsibility for reporting and acting on the results of laboratory investigations should be identified. DATA MANAGEMENT Information obtained from request forms is the basis for storing information electronically. These data are used for hospital accounting purposes, patient information, and medical audits. The information also is invaluable along with the laboratory results for research, education, epidemiologic, and quality-control purposes. The results of microbial cultures also should be stored in an electronic database or spreadsheet containing patient clinical details as appropriate, the date, the origin and site of the sample, the organism isolated, and the medium from which it was grown. For each isolate, space should be available to record the sensitivity testing results. Of clinical importance for audits are the types of organisms isolated and any changes in the frequency of isolates or their in vivo sensitivity to antibiotics used therapeutically. This information enables the laboratory staff to monitor changes in antibacterial resistance. As with any database, identify the questions that need to be answered before choosing the data entry fields. |
| INFECTION CONTROL | ||||||||||||||||||||||
| The ophthalmic microbiology laboratory, like any general microbiology facility, presents
a number of problems regarding infection control. Good
laboratory practice demands the safest possible processing methods,6 including the safe disposal of any waste materials and recycling of any
nondisposable equipment. Disinfection and sterilization are the two
important components of infection control. DISINFECTION Disinfection is the removal of most types of pathogenic microorganisms, usually excluding spores, rather than the removal of all organisms. Disinfection reduces the number of pathogenic organisms present in a potential source of infection to a level below that required to cause an infection. Spores are particularly resistant to physicochemical processes. There are three main types of disinfection: cleaning, heating, and using chemical agents. It is essential to select an appropriate method of disinfection relative to the materials being disinfected. Cleaning This is the most economical and common method of disinfection. Cleaning methods also are used to remove organic matter, biofilm, dirt, and grease that might otherwise protect the microorganism. Ensure that surfaces and materials are dried after cleaning to inhibit the multiplication of any surviving organisms. Good cleaning procedures should be used regularly for hands, bench surfaces, glassware, and equipment in general use. Heating Pasteurization (“Holder process” or “flash process”) is used to decontaminate instruments, fluids, and tubing. The Holder process involves heating items to 63°C for 30 minutes. The flash process involves heating items to 73°C for 20 seconds. Boiling involves heating items to 100°C to kill most microorganisms in less than 1 minute. Some bacterial spores (e.g., Bacillus stearothermophilus) are capable of resisting boiling for several minutes. Low-temperature steam disinfection occurs when steam is generated at 75°C in a specially designed autoclave. This is extremely effective because it is performed within a sealed unit that allows total heat penetration of the contents of the chamber. Items must be dried thoroughly after disinfection by this process. Dry-heat disinfection (hot-air oven) is used when moisture would otherwise damage the materials being disinfected. An appropriate temperature setting and time exposure may be selected for the materials being processed. Using Chemical Agents Chemical disinfection must be considered carefully before being used. Factors to be taken into consideration include the following:
The main types of chemical agents available are as follows: Alcohols (ethyl or isopropyl): These kill most bacteria on nonporous surfaces, such as laboratory benches, in
less than 30 seconds. A detailed, cost-effective policy should be used for any of the methods of disinfection. A suitable range of chemicals may be selected according to departmental and regulatory guidelines. STERILIZATION Sterilization is the complete removal of all organisms, including vegetative bacteria/bacterial spores/viruses/fungi, and protozoa. The concept of sterility depends on statistical rather than absolute considerations. The aim, in practice, is to render an item “safe” for the procedure to be undertaken, and a sterilization process that reduces the number of spore-forming organisms by more than a factor of 106, in a short period, is considered satisfactory (see Table 2).
Moist Heat (Autoclave) An autoclave is a sealed chamber that uses saturated steam at elevated temperatures. Heat kills bacteria by coagulating the cytoplasm, and the efficiency is enhanced by steam under pressure. At a pressure of 15 lb/in2, water will boil at a temperature of 121°C. Autoclaves are used to sterilize culture media, laboratory equipment, and supplies, and they are invaluable in the disposal of biologic waste from the microbiology laboratory. It may be necessary to thoroughly dry items processed by this method before use. The efficiency of the autoclave must be monitored by the use of thermocouples and chart recorders. Dry Heat (Hot-Air Oven) The method of destruction is by coagulation and oxidation. It is essential to use this method if materials are impervious to steam penetration and when materials may be damaged by the use of moist heat. Materials such as oils, glassware, powders, metal instruments, and substances that would be denatured by the use of moist heat can be sterilized in this way. A cycle of 160°C for 1 hour or 140°C for 2 hours is sufficient to ensure sterilization. The use of a thermocouple records that the process is working efficiently. Ionizing Radiation Gamma rays are lethal, noncharged, ultra-short-wavelength rays with great penetrating power produced from a radioactive isotope such as cobalt 60 (dose = 2.5 M rad). The mode of action is by ionization of the organisms' DNA. They are used to sterilize disposable items such as plastic, latex products, and sealed packs that are deemed unsuitable for sterilization by dry or moist heat. Ethylene Oxide This process is used when low-temperature sterilization is required. Ethylene oxide is a highly toxic inflammable gas that kills all types of microorganisms, including bacterial spores. Conditions under which ethylene oxide is used must be monitored carefully, as strict control of temperature and humidity is critical to the process. Carbon dioxide is mixed with the ethylene oxide to reduce the risk of an explosion. The gas diffuses through items such as plastic, linen, paper, and other heat-sensitive materials. Spore strips (viable spores of Bacillus globegii on filter paper) are used as a biologic indicator to ensure the effectiveness of the process. If the spore strips, when cultured under normal incubation conditions, should produce a positive growth of organisms, the process must be repeated before the items included in that particular cycle are regarded as safe to use. |
| WASTE DISPOSAL |
| The laboratory must be a safe environment that minimizes the risk of injury
or infection or both from biologically hazardous materials. All infectious
or potentially infectious waste must be rendered harmless before
it leaves the laboratory by using any one of or a combination of
autoclaving, incineration, and chemical disinfection. Each department
should have a designated safety officer who can advise on all issues regarding
waste disposal procedures. Any waste disposed of in an autoclave must be labeled clearly with respect to origin so that the source can be traced. Items for autoclaving must be placed inside an autoclave bag. These bags must not be overfilled or contain glass or sharp objects. Items for autoclaving must not be chemically reactive, radioactive in nature, or combustible. The top of the bag should be folded loosely with autoclave indicator tape. After processing, all autoclaved materials must be allowed to cool and the autoclaved bags transported to the central disposal unit in a sealed container. Items should be placed in appropriate containers designated for incineration and transported to the incinerator. Tubes or bottles containing body fluids should have their lids removed and be immersed totally in an appropriate disinfectant solution (e.g., hypochlorite) for at least 18 hours before being processed for disposal. Then, the disinfectant may be drained away and the contents autoclaved. Fresh tissue specimens must be fixed, usually in 10% formaldehyde solution, for at least 24 hours. This is performed as soon as possible after the specimen is processed. After fixation, the specimen is rinsed to remove excess fixative and transferred to a bag for autoclaving and incineration. Disposable consumables must be submerged for at least 18 hours. Reusable items must be rendered safe before submission for routine cleaning and then they may be autoclaved. Syringes and sharps must be placed in a “sharps” bin or an equivalent designated container. When the container is almost full (never overfill these containers), it is sealed and autoclaved before being incinerated. All glass waste must be rendered safe before being disposed. Bottles that may have contained biologically hazardous material must be autoclaved before being disposed. Separate disposal bins for glass should be used before being disposed by the appropriate local authority. Noninfective biologic waste should be disposed of as soon as possible after processing. Waste of this nature should be placed in appropriate containers pending disposal. Redundant chemicals must be packaged safely and labeled clearly using appropriate hazard warning tape. The originating laboratory should be indicated on the tape attached to the container. All such chemicals, sealed in their containers, then should be taken to the waste chemical storage unit pending disposal. Halogenated and nonhalogenated chemical waste must be stored separately. A record of all chemical waste should be maintained. Domestic (noninfective) waste should be disposed in designated bins placed within the laboratory area. Care should be taken not to dispose of any written material that may be confidential or contain written documentation falling within regulatory legislation or both. Such waste should be shredded before being disposed. |
| QUALITY ASSURANCE, CONTROL, AND IMPROVEMENT |
| Quality assurance is monitoring the performance of equipment and reagents
and ascertaining the value and relevance of services and patient information
given. Related to quality assurance are quality control and
quality improvement. Quality control is a continuous monitoring of the efficiency of all equipment, reagent microbiologic media test identification kits, and laboratory methods used. Quality improvement is used to evaluate all laboratory methods and to identify any problems that occur over time. Informed adjustments to methods and working practices then can be made, resulting in improvement to the overall laboratory performance. The ophthalmic laboratory microbiologist must provide reliable and useful patient clinical diagnostic data for the diagnosis and treatment of ocular infections and as archival data for subsequent clinical research, hospital antimicrobial treatment policies, infection control information, and epidemiologic studies. Consult the chapters by Sewell and Schifman, and McGowan and MacLowery in Manual of Clinical Microbiology7 for detailed quality assurance and improvement procedures and for details of the relevant federal legislature in the United States. To keep laboratory costs down by reducing staff levels and administration from a laboratory accreditation point of view, many laboratories now purchase commercially produced, ready-made microbiologic media, stains, reagents, and test kits. Most laboratory supplies are required to supply goods that are manufactured to approved standards. Governmental, federal, professional, biomedical, and independent groups such as the National Accreditation of Measurement and Sampling and the British Standards Institute in the United Kingdom and the National Committee for Clinical Laboratory Standards, the U.S. Food and Drug Administration, set agreed-on standards and actively monitor products to ensure that agreed-on quality standards are maintained. Commercially produced, ready-made microbiologic media are quality assured by the suppliers. New batches of media have batch numbers, expiry dates, supplier details, and certificates of analysis recorded. All laboratories must keep laboratory records that include these data on file. Laboratories should test samples from each new batch before use. Media are inoculated with selected American Type Culture Collection (ATCC) strains to ensure the culture medium's ability to effectively support the growth of fastidious microbes such as the Neisseria and Haemophilus species. |