CLINICAL BACTERIOLOGY LABORATORY INTRODUCTION TO MICROBIOLOGY LABORATORY OUTLINE • Basic Biosafety in the Laboratory o What is Biosafety? o Containment Principles o What is the Hierarchy of Hazard Controls? • Microbiology Apparatus and Equipment o List of Tools and Equipment • The Microscope o The Microscope o Structural Parts of a Microscope and their Functions o How to Use the Microscope LESSON 1: BASIC BIOSAFETY IN THE LABORATORY WHAT IS BIOSAFETY? • Biosafety is defined as, “The discipline addressing the safe handling and containment of infectious microorganisms and hazardous biological materials”. • The practice of safe handling of pathogenic micro-organisms and their toxins in the biological laboratory is accomplished through the application of containment principles and the risk assessment. CONTAINMENT PRINCIPLES • Safety in the laboratory is achieved by application of layered, containment principles applied in accordance with the risk assessment to prevent exposure of laboratory workers to a pathogen or the inadvertent escape of a pathogen from the microbiological laboratory. • Containment is defined in levels that increase in complexity as the risk associated with the work in the microbiological laboratory increases. All containment levels have defined primary and secondary containment features. • Primary containment provides immediate protection to workers in the biological laboratory from exposure to chemical and biological hazards. Primary barriers include biological safety cabinets, fume hoods and other engineering devices used by laboratory technicians while working with a biological hazard. o Biological Safety Cabinets (BSC) ▪ Primary means of containment developed for working safely with infectious microorganisms ▪ Designed to contain biological hazards ▪ Supply air HEPA (High Efficiency Particulate Air) filter for product protection (except Class I) ▪ Inward airflow for personnel protection • UV Light ** ▪ HEPA filtered exhaust air for environmental protection ▪ Separated into Classes and Types • Class I • Class II (most common) o Type A1, A2 o Type B1, B2 • Class III (common in tertiary) ▪ Microbiological studies, cell cultures, pharmaceutical research, etc. ▪ • Secondary containment is intended to protect the laboratory worker, the community and the environment from unintended contamination with a biological hazard. Secondary containment consists of architectural and mechanical design elements of a facility that prevent worker contamination and escape of pathogens from the laboratory into the environment. • Personal protective equipment (PPE) such as gloves, laboratory coats and safety glasses may also be considered a primary containment, however, articles worn on the body are considered a last line of defense and are only used in conjunction with other primary and secondary containment elements when working with pathogenic organisms.
BIOSAFETY SAFETY CABINETS (BSC) • A device that encloses a working area to protect workers from aerosol exposure and infectious disease agents • The air that contains the infectious material is sterilized, either by, heat, UV light, or by passage through a high efficiency particulate (HEPA) resistant filter • Class I Cabinet o An open-fronted type of cabinet with negative pressure (ventilation cabinet) o Allows room (unsterilized) air to enter the cabinet, circulate around the area, and expose material within; only air to be exhausted is sterilized using a HEPA filter o Used for biosafety levels (BSL) 2 and 3 agents • Class II Cabinet o Also known as the laminar flow BSC o Most commonly used BSC in a clinical microbiology laboratory (Class IIA) o Sterilizes the air using a HEPA filter that flows over the infectious materials and the air to be exhausted o Used for BDL 2 & 3 agents o There are 2 types of Class II cabinet: ▪ Class IIA - w/ fixed opening; 70% of air is recirculated ▪ Class IIB - variable sass opening; used for chemicals, radioisotope and carnogens o Most hospital clinical microbiology laboratory technologists use class II** BSC • Class III Cabinet o Provides the highest level of safety to the worker o The air coming into and going out of the cabinet is sterilized using a HEPA filter and the infectious material within is handled with rubber gloves that are attached and sealed to the cabinet o Used for BSL 4 agents CLASSIFICATION OF BIOLOGIC AGENTS BASED ON HAZARD • A BSC is composed of different biosafety levels that range from BSL I to BSL 4 depending on the level of biocontainment precaution required for the specimen being studied. • Biosafety level 1 agents o Agents that have no known potential for infecting healthy people o Used in laboratory activities of students (for academic purposes) o Some examples of pathogens that require this containment level are Bacillus subtilis & Naegleria gruberi • Biosafety level 2 agents o Agents acquired by ingestion or exposure to percutaneous or mucous membrane o Include all the common agents of infectious diseases o In handling these agents, access to the laboratory is limited. o Also requires the personnel to change their clothes with the recommended laboratory clothing before going to their specific stations. o The personnel handling these agents should also receive immunization. o Some examples of pathogens that require this containment level are HIV, Bacillus anthracis, Yersinia pestis, Salmonella, and Shigella • Biosafety level 3 agents o Potential agents for aerosol transmission o In processing these lethal pathogens, the air movement in the laboratory must be controlled to contain the infectious materials o Some examples of pathogens that require this containment level are Mycobacterium tuberculosis, Francisella tularensis, Brucella spp., Coxiella burnetti, St. Louis encephalitis virus, and systemic fungi • Biosafety level 4 agents o Agents that cause life-threatening infections o In handling these organisms, maximum containment and decontamination of all personnel and materials before leaving the area are observed o Aerosol transmission with pressure is possible o Some examples of pathogens that require this containment level are arbovirus, arenavirus, filovirus, and smallpox virus CATEGORIES OF POTENTIAL INFECTIOUS AGENTS OF BIOTERRORISM • Category A agents o Agents that pose the greatest public health threat o Easily transmitted and highly infectious o Some examples are smallpox, anthrax bacillus, and Francisella tularensis • Category B agents o Agents with moderate morbidity and low mortality o Not as easily transmitted as category A agents o Some examples are Coxiella burnetti, Burkholderia pseudomallei, and Rickettsia • Category C agents o The emerging pathogens o Some examples are the viruses that cause yellow fever, and dengue hemorrhagic fever NOTES TO REMEMBER • All clinical laboratories must adhere to biosafety level 2 guidelines • Processing of samples from a possible bioterrorism event should occur within class II BSC • The agents that pose the greatest risk are those that are transmitted by aerosols • HIV and hepatitis C virus are bloodborne pathogens that may be transmitted from contaminated specimens through a needlestick injury or percutaneous route • Statistics show that males and younger employees are involved in more laboratory-acquired infections than females and older employees • The five most frequently acquired laboratory infections are shigellosis, salmonellosis, tuberculosis, brucellosis, and hepatitis • The laboratory procedures that create aerosol are pipetting, flaming loops, agar plates streaking, and centrifugation. WHAT IS HIERARCHY OF HAZARD CONTROLS? • The Hierarchy of Hazard Control seeks to protect workers by ranking the ways in which hazards can be controlled, providing employers with a framework for reducing risk to employees.
LESSON 2.1: MICROBIOLOGY APPARATUS AND EQUIPMENT LIST OF TOOLS AND EQUIPMENT • Autoclave • Microbiologic incubator • Biological Safety Cabinet • Biological refrigerator • Class II Laminar flow • Dry Heat oven • Centrifuge • Arnold’s sterilizer • Bunsen burner • Water Bath • Funnel • Alcohol lamp • Petri dish • Test tube with cotton plugs • Anaerobic jar • Triple beam balance • Erlenmeyer flask • Beaker • Graduated cylinder • Inoculating loop and needle • Culture media • Fermentation tubes/Durham tubes • Stirring rod • Colony counter • Microscope • Glass slides • Antibiotics • Antibiotic Disc Dispenser • Forceps • Electric Incinerator • Hot plate AUTOCLAVE • It is a robust, electrically heated steam vessel meant for sterilizing ‘thermostable’ culture media, glassware, and other materials that are not spoiled by moist heat. Autoclave runs on the principle of pressure cooker. The moist heat (steam) has a very good penetrating power. Microorganisms / cells are killed as a result of denaturation of cellular constituents (protein and nucleic acids). In routine process, sterilization can be achieved by operating the autoclave at 121°C (15 psig) for 15 min. • In its simplest form, the equipment has a removable lid for the delivery of materials to be sterilized. It is necessarily equipped with a gasket, pressure-cum-temperature gauge, a vent for letting out air or excess pressure, a safety valve, and a drain. INCUBATOR • This an insulated, electrically heated cabinet meant for providing microorganisms with optimum temperature for growth. The cabinet is insulated and thermostatically controlled. For routine purposes, the temperature is maintained at 28-30°C for bacteria, about 25°C for molds, and 35-37°C for mesophilic bacteria. A temperature as high as 100°C can also be maintained for extremely thermophilic organisms (stereothermophiles). A very common laboratory incubator is shown in Fig. 2. HOT AIR OVEN • This is similar to incubator in make except that it can operate at temperatures up to 300°C and has a fan for circulating hot air. Hot air oven is used for sterilization of glassware and materials that are spoiled by moist heat. The death of cells occurs due to the oxidation of cellular constituents by the dry heat. • For routine purpose, sterilization can be achieved by running the equipment at 180°C for 1.5 hours. Hot air oven is less effective than autoclave. Fig. 3 shows a typical hot air oven popular in microbiology laboratories. INOCULATING LOOP • This is a tool for transferring and streaking cultures. It consists of a thin nichrome wire whose one end is twisted into a small loop while the other end is fixed to a thermoset plastic handle. Sometimes, the looped end is straightened out to form what is called inoculating needle. Inoculating needles are used for preparing ‘stab’ cultures. Fig. 4 shows inoculating loops and needles.
VORTEX MIXER • This equipment is used for mixing liquids kept in a test tube. It has one or more cup-like depressions at the top to receive the bottom of the test tube. The machine is electrically powered. When actuated, the machine moves the bottom of the test tube in a gyratory motion, thereby affecting a thorough mixing of the solution. The speed of the mixer can be varied. Fig. 5 shows a typical vortex mixer WATER BATH / BOILING WATER BATH • Water bath is used for heating and melting of media, solutions, samples etc. at temperatures below 100°C. It can also be used to maintain constant temperature that is required in microbiology lab work. Several models and types of water bath are available. Fig. 6 shows a typical water bath commonly used in laboratories. It is electrically heated and thermostatically controlled HEATING MANTLE • It is an electrically heated and thermostatically controlled unit used to heat or melt samples and reagents. The inner lining is made of asbestos and therefore gives an indirect heat to the materials to be heated. Fig. 7 shows a typical heating mantle being used for heating water in a beaker. HOT PLATE WITH MAGNETIC STIRRER • This is an electrically powered equipment performs the dual function of heating and agitation. The agitation occurs by magnetic arrangement. Any type of glassware can be used for the heating and agitation. Magnetic beads are used for the agitation UV CHAMBER / UV VIEWING CABINET • This equipment is used for analyzing fluorescent materials, spots in thin layer chromatography, etc. The equipment has two lamps for long- and short wavelength UV radiation. Since UV radiation is genotoxic (mutagenic) its exposure to skins and eyes must be avoided. A viewport with colored glass is provided for safety. Fig. 9 shows a typical UV cabinet. INOCULATION CHAMBER / STERILE CHAMBER • This is an enclosed box in which culture transfers, plating, etc. can be carried out aseptically. The chamber is equipped with UV lamp for periodic disinfection of the chamber. While working, the UV light must be turned off and day-light bulb is turned on. Fig. 10 shows a locally fabricated inoculation chamber. pH METER • pH meter is an electrical instrument used for measuring hydrogen ion concentration of solutions and mixtures (Fig. 11). In microbiology lab, it is used for maintaining pH of the medium and diluents. The pH meter must be standardized with buffer solutions before operation. Since the instrument is very sensitive, it must not be used for stirring and it must not be dipped in hot or very cold solutions. The electrodes must always be kept immersed in suitable solutions. Read the manual carefully before using the instrument.
COLONY COUNTER • It is used for counting microbial colony (bacterial and yeast). The instrument is equipped with a backlight source, gridlines and a magnifying lens. It also has a sensor for digitally registering the number of colonies counted (Fig. 12). MICROSCOPE • It is an instrument for observing microscopic items such as cells, crystals and cell organelles. It has the dual function of magnification and resolution. For routine microbiological works, bright field compound microscope with oil immersion objective is adequate. A compound microscope is shown in Fig. 13. REFRIGERATOR • This is a common household equipment for keeping foods and beverages cool. This equipment is used in microbiology laboratory for storing / preserving cultures, media, and many sensitive materials (Fig. 14). The equipment is electrically powered and uses ammonia as the refrigerant. BUNSEN BURNER • Bunsen burner is a common tool used in science lab (Fig. 15). In microbiology lab, it is used for sterilizing inoculating loop, plating out cultures, transferring cultures, heat-fixing of smears and creating a sterile zone for aseptic operation. SPIRIT LAMP • The function of spirit lamp is the same as the Bunsen burner but is portable. It uses rectified spirit as the fuel (produces smoke-free flame). The lamp must be covered with a lid when not in use to prevent loss of spirit (Fig. 16). MICROMETERS (STAGE AND OCULAR) • These are graduated glass pieces (Fig. 17) used for the measurement of size of the cells. Stage micrometer is a slide on which etching is done with 0.001 mm spacing. The ocular micrometer, which is place on the eyepiece, has an arbitrary scale and must be calibrated against the stage micrometer. During measurements, the ocular micrometer is retained while the stage micrometer is replace with the specimen slide. BALANCE • Balance is needed in microbiology lab for weighing chemicals, samples, media, etc. Digital balances are fast to work with but needs frequent calibration (Fig. 18). • The triple-beam and 4-beam balances are robust equipment that need little care and maintenance. Beam balances run on mechanical principles while the principles on which electronic balances run is quite complicated (Fig. 19).
THERMOMETER • Thermometers are required to ensure the heating equipment is running at the correct temperature. The temperature of the medium, incubator, etc., need to be frequently checked. Mercury in glass thermometers are standard thermometers, the temperature measurement is based on the expansion of mercury present in the bulb. Digital thermometers use probes for measurement of temperatures. COLIFORM MEMBRANE FILTER • This glass equipment is used for the testing of coliforms in water (Fig. 20). 100 ml of test water is poured in the funnel and filtered through a special Millipore filter through external application of suction. The filter retains the microorganisms. The filter is then aseptically transferred to a selective-cum-differential semisolid medium kept in a petri dish. If there are coliforms, they will appear as pink dots after incubation at 35°C for 22 hrs. LESSON 2.2: THE MICROSCOPE THE MICROSCOPE • Having been constructed in the 16th Century, Microscopes have revolutionalized science with their ability to magnify small objects such as microbial cells, producing images with definitive structures that are identifiable and characterizable. • Microscopes are instruments that are used in science laboratories, to visualize very minute objects such as cells, microorganisms, giving a contrasting image, that is magnified. It can view very small specimens and distinguish their structural differences, for example, the view of animal and plant cells, viewing of microscopic bacterial cells. • Microscopes are generally made up of structural parts for holding and supporting microscope and its components and the optical parts which are used for magnification and viewing of the specimen images. This description defines the parts of a microscope and the functions they perform to enable visualization of specimens. STRUCTURAL PARTS OF A MICROSCOPE AND THEIR FUNCTIONS • There are three structural parts of the microscope i.e. head, base, and arm. o Head – This is also known as the body, it carries the optical parts in the upper part of the microscope. o Base – It acts as microscopes support. It also carriers the microscopic illuminators. o Arms – This is the part connecting the base and to the head and the eyepiece tube to the base of the microscope. It gives support to the head of the microscope and it also used when carrying the microscope. Some high-quality microscopes have an articulated arm with more than one joint allowing more movement of the microscopic head for better viewing. • The optical parts of the microscope are used to view, magnify, and produce an image from a specimen placed on a slide. These parts include: o Eyepiece – also known as the ocular. this is the part used to look through the microscope. Its found at the top of the microscope. Its standard magnification is 10x with an optional eyepiece having magnifications from 5X – 30X. o Eyepiece tube – its the eyepiece holder. It carries the eyepiece just above the objective lens. In some microscopes such as the binoculars, the eyepiece tube is flexible and can be rotated for maximum visualization, for variance in distance. For monocular microscopes, they are none flexible. • Objective lenses – These are the major lenses used for specimen visualization. They have a magnification power of 40x-100X. There are about 1- 4 objective lenses placed on one microscope, in that some are rare facing and others face forward. Each lens has its own magnification power. • Nose piece – also known as the revolving turret. It holds the objective lenses. It is movable hence it cal revolve the objective lenses depending on the magnification power of the lens.
• The Adjustment knobs – These are knobs that are used to focus the microscope. There are two types of adjustment knobs i.e fine adjustment knobs and the coarse adjustment knobs. • Stage – This is the section on which the specimen is placed for viewing. They have stage clips hold the specimen slides in place. The most common stage is a mechanical stage, which allows the control of the slides by moving the slides using the mechanical knobs on the stage instead of moving it manually. • Aperture – This is a hole on the microscope stage, through which the transmitted light from the source reaches the stage. • Microscopic illuminator – This is the microscopes light source, located at the base. It is used instead of a mirror. it captures light from an external source of a low voltage of about 100v. • Condenser – These are lenses that are used to collect and focus light from the illuminator into the specimen. They are found under the stage next to the diaphragm of the microscope. They play a major role in ensuring clear sharp images are produced with a high magnification of 400X and above. The higher the magnification of the condenser, the more the image clarity. More sophisticated microscopes come with an Abbe condenser that has a high magnification of about 1000X. • Diaphragm – its also known as the iris. Its found under the stage of the microscope and its primary role is to control the amount of light that reaches the specimen. Its an adjustable apparatus, hence controlling the light intensity and the size of the beam of light that gets to the specimen. For high-quality microscopes, the diaphragm comes attached with an Abbe condenser and combined they are able to control the light focus and light intensity that reaches the specimen. • Condenser focus knob – this is a knob that moves the condenser up or down thus controlling the focus of light on the specimen. • Abbe Condenser – this is a condenser specially designed on high-quality microscopes, which makes the condenser to be movable and allows very high magnification of above 400X. The high-quality microscopes normally have a high numerical aperture than that of the objective lenses. • The rack stop – It controls how far the stages should go preventing the objective lens from getting too close to the specimen slide which may damage the specimen. It is responsible for preventing the specimen slide from coming too far up and hit the objective lens. HOW TO USE THE MICROSCOPE • Procedure on how to use the microscope: 1. Get a microscope by taking the arm with one hand supporting the instrument at the base with the other hand. 2. Place the microscope at least six inches from the edge of the laboratory table with the microscope arm facing you. 3. Draw the compound microscope and label the parts. Briefly describe the function of each part. • Focusing the Low Power Objective (LPO) 1. Place the prepared slide on the stage and align the LPO with the eyepiece. 2. Adjust the mirror by looking at the eyepiece until a bright microscopic field is obtained. 3. Manipulate the iris diaphragm to adjust the light passing in the condenser. 4. Using the coarse adjustment knob, slowly move the body tube upward. 5. Stop when the object is clearly focused. • Focusing the High Power Objective (HPO) 1. After focusing the LPO, shift the HPO without moving the body tube. 2. Manipulate the fine adjustment knob until a clear object is seen. 3. To have a clearer object, adjust the light using iris diaphragm. • Focusing the Oil Power Objective (OIO) 1. Place one drop of cedar wood oil over the stained slide. 2. Focus with the HPO, shift to OIO, allow the cedar wood oil to touch the objective. 3. Make a proper adjustent using the fine adjustment knob until a clear view of the specimen is obtained. •