Microbiology is an interesting field that has some specific equipment and instruments that define the profession. Though some of these equipment and instrument may be found in other fields including but not limited to Biochemistry, Biology and medicine; the field of microbiology would not have been complete without these piece of equipment especially the microscope. In this section, some of the basic equipment and instruments of microbiology are outlined with their respective functions.
The microbiology laboratory (whether in a research facility, hospital setting or in an academic institution) is usually fitted with specific working equipment and instruments that makes it unique and different from other laboratories in the biological, biomedical and medical sciences. Without these instruments and equipment, microbiological experimentation and techniques would be difficult if not impossible. For example, it was due to the serendipitous discovery and development of the microscope by some notable scientists (particularly Anthony Von Leeuwenhoek and Robert Hooke to mention a few) that the field of microbiology blossomed as one of the areas of biological sciences which specifically studies microorganisms. Without the invention of the microscope, it would be difficult if not impossible to discover the unseen world of living organisms and in this case, microorganisms– which are forms of life too small to be seen with an unaided or naked eyes. Microbiology cannot be studied in isolation without the input or application of some important equipment and instruments which aid the laboratory investigations of microbiologist’s all over the world.
Centrifuge is an equipment used to separate suspensions or particulate matter in a liquid based on their different sedimentation properties or densities. The centrifuge machine is generally used in the microbiology laboratory to sediment or separate different macromolecules (especially particulate matter) in a solution. Liquid specimens such as urine and blood for example can be spinned or centrifuged using the centrifuge machine (Figure 1). Centrifuge separates solutions into two basic forms known as the supernatant and sediment. The supernatant is decanted from the tube to obtain the sediment or deposits found at the bottom of the tube. To separate a given solution into their different components (i.e. supernatant and sediment), the solution is dispensed into a tube before it is placed in the centrifuge machine which carries out the actual separation process.
After separation, the supernatant (which is in liquid form) is usually above the sediment or pellets which always stays at the bottom of the tube because of its higher molecular weight than that of the supernatant. This process of separating the components of a solution into different parts using the centrifuge is known as centrifugation. Centrifugation occurs under a centrifugal force – which causes the particulate matter in the solution being centrifuged to move in an outward direction and towards the bottom of the tube containing the liquid while the supernatant remains above it. During centrifugation, more massive particles sediment faster than less massive particles; and tubes containing the experimental sample must always be of equal volume.
Centrifugation occurs in different revolutions and periods or time; and this is usually expressed as revolutions per minute (rpm), and with the unit revs min-1. There are different types of centrifuge machine, and each performs different functions. There is the small bench centrifuge (with a relative centrifugal field of 3000-7000 g), large capacity refrigerated centrifuge (with inbuilt refrigerated rotor chamber; and a relative centrifugal field of up to 6500 g), high speed refrigerated centrifuge (with a refrigerated rotor chamber; and a relative centrifugal field of up to 60000 g) and the ultracentrifuge (with refrigerated rotor chamber; and a relative centrifugal field of about 600000 g). Ultracentrifuge is used to separate macromolecules and other minute substances or organisms including viruses from a sample. Some of the basic components of a simple centrifuge machine include: a metallic-driven rotator or rotor, centrifuge lid or cover, tube, power source, speed controller and time controller.
Fig. 1. Centrifuge
MAINTENANCE/CARE OF THE CENTRIFUGE
- Always read the owner’s manual before operating the centrifuge machine.
- Keep the machine clean always, and ensure that spilled liquid is properly cleaned off.
- Always abide by the mass and speed levels or limits of each rotator and/or centrifuge when spinning experimental samples.
- Tubes containing the experimental sample must be properly balanced prior to spinning. Improperly balanced tubes can damage the centrifuge machine during centrifugation.
- After centrifugation, allow the rotator to stop before opening the centrifuge lid.
- After centrifugation, clean the rotors and tube properly using disinfectant and dry towel.
- Consult the laboratory technician when any problem arises.
Fig. 2. An illustration of nichrome wire loop. A=handle, B=wire loop.
Inoculating loop or wire loop is a general purpose piece of instrument that allows microbiologist to transfer microorganisms in an aseptic manner from one place to another. Colonies of microbes growing on culture media plates of slants can be transferred onto glass slides or fresh culture medium for further studies, and this is only made possible with the aid of a wire loop or inoculating (Figure 2). Several types of wire loop exist for the transfer of microorganisms in the microbiology laboratory. Wire loop can also be used to streak microbial cultures or experimental samples onto the surface of solid culture media plates.
They are also used to inoculate broth or liquid culture medium with experimental samples or microbes. The main components of the wire loop include nichrome wire and the wooden handle. Apart from the traditional nichrome wire loop used for various microbiological investigations, disposable plastic wire loops which can only be used once also exist. The nichrome wire loop is heat-stable and can be used over a long period of time without breaking down. Nichrome wire loops should be kept dry and away from moist environment to prevent corrosion or rusting.
Fig. 3. Petri dish plates
Petri dishes are shallow glass or plastic plates that are cylindrical in shape, and which are used to cultivate microorganisms in the microbiology laboratory. Glass Petri dishes are heat-stable and can be sterilized in the autoclave and re-used over a long period of time after proper washing and sterilization in an hot air oven. But plastic Petri dishes are disposable after first usage, and cannot be re-used or autoclaved because they are heat-labile, and are only used once. Petri dishes are important microbiological apparatuses, and they provide a platform for the phenotypic or physical examination of microorganisms (particularly bacteria and fungi) based on their colonial characteristics on solid growth media.
After the inoculation of microbial cultures or experimental samples, culture media plates (i.e., Petri dishes) are incubated upside down (with the part containing the agar on top) in order to ensure the sterility of the medium. Droplets of water from the other side of the plate (not containing agar) onto the agar medium could introduce contaminating organisms to the growth vessel, and thus contaminate the whole cultivation process; and this usually happens if the Petri dish is not turned upside down (with the part containing the media on the top) prior to incubation in the incubator. Petri dishes (Figure 3) are mainly used for bacteriological and mycological investigations in the microbiology laboratory; and they are quite different from those used for other investigations such as cell culture techniques.
MAINTENANCE/CARE OF PETRI DISHES
- Petri dishes should not be opened prior to usage.
- Glass Petri dishes should be properly washed and sterilized prior to their usage for microbial cultivation in the laboratory.
- Disposable Petri dishes should be autoclaved (for proper disinfection) after use before disposal.
Fig. 4. Slide rack
Slide rack is a piece of vessel used for packing and storing clean glass slides after they have been used for staining. After staining, it is important to wash the glass slides with soap/detergents under running water. The washed glass slides are carefully inserted into the slide rack (Figure 4) where the excess water on the slides is allowed to drain off. Slide racks should be kept clean and away from dusty areas. The slide rack provides an appropriate medium or environment where glass slides are held in horizontal positions until usage. They should be kept in flat surfaces or cupboards in order to prevent the breakage of the slides they are carrying. Slide racks can also be used to collect and store slides ladened with the test samples, test organisms or tissue sections prior to further investigation using the microscope.
Fig. 5. Staining rack
Staining rack is a piece of apparatus that holds several glass slides in a horizontal manner for staining techniques. It allows glass slides on which microbial smear is made to be stained all at once, and allowed to dry. Staining rack allows microbiologist to make several slides of microorganisms (inclusive of wet preparation and smears) more efficiently and in less time; and it provides a horizontal platform for keeping slides prior to staining techniques (Figure 5).
Fig. 6. Bunsen burner flame
Bunsen burner is practically a tube or hosepipe that is connected directly to a steady supply of flammable gas contained in a gas cylinder. It is primarily used to achieve aseptic technique in the microbiology laboratory. Usually, a blue flame from the Bunsen burner (Figure 6) is used to sterilize the mouth of glass wares or test tubes, needles, inoculating loop and other heat-stable instruments by passing the material to be heat-sterilized through the blue part of the flame.
MAINTENANCE/CARE OF THE BUNSEN BURNER
- Do not leave the Bunsen burner on when not in use because open flame is a safety hazard.
- Turn off the burner when it is not in use.
- Inflammable materials should not be passed through the flame.
- Adjust the air vents at the bottom of the Bunsen burner in order to obtain a bright blue cone of flame (Figure 6) for sterilization.
Safety tip: Always ensure that your laboratory coat or gown is not loose but body-tight before using the Bunsen burner flame. Loose laboratory gowns can easily catch fire when they come in contact with the flame from the burner.
THERMOCYCLER / PCR MACHINE
Fig. 7. Thermocycler
Thermocycler or thermal cycler is a piece of equipment that is used for the copying or amplification of specific sequences of nucleotides or genes through a controlled timing and temperature regulation. It is an important tool in the molecular biology laboratory. Thermocycler allows reaction mixtures in specialized tubes known as Eppendorf tubes (PCR tubes) to be heated and cooled at varying times and temperatures. It is generally used to achieve a controlled denaturation, renaturation and elongation of nucleotide or gene sequences of unknown or known organisms.
Thermocycler can also be called PCR machine because it is used to achieve polymerase chain reaction (PCR), a reaction that allows molecular biologists to amplify the nucleotide sequences that make up a given gene or DNA molecule. PCR is a routine technique in the molecular biology laboratory, and it is achieved with the help of the thermal cycler (Figure 7) aside other reagents such as primers and materials used in the PCR reaction process. The thermocycler is programmed to work at specified time and temperature range.
MAINTENANCE/CARE OF THE THERMOCYCLER
- Always follow and read the owner’s manual before operating the thermocycler.
- Thermocycler must be kept in clean areas of the laboratory to avoid contamination of the reaction process by extraneous DNA from the environment or from the equipment.
- The equipment should be unplugged from the power source when not in use or better still switch of power source.
- Thermocycler is and expensive piece of equipment, and it should be handled with utmost care to avoid damage.
Fig. 8. Weighing balance
Weighing balances are used to measure the weight of objects (particularly powdered agar and reagents) in the microbiology laboratory. Several weighing balances exist for measuring media and other substances in the microbiology laboratory and they include mechanical weighing balance (Figure 8) and chemical or analytical weighing balances to mention a few. They provide accurate readings or values of material required for microbiological investigations.
MAINTENANCE/CARE OF THE WEIGHING BALANCE
- Always follow the owner’s instructions for the operation of each weighing balance.
- Clean the balance pan after each use with a dry towel.
- Allow the balance pan to dry before storage.
- Do not store the equipment in moist environment.
- Lubricate (oil) the nuts and bolts parts of the weighing balance.
Fig. 9. Autoclave
Autoclave is a piece of equipment that allows materials to be heat-sterilized under pressure and with a saturated steam. It is generally used to achieve sterilization of culture media and other reagents in the microbiology laboratory; and sterilization using the autoclave is achieved under pressure at various temperatures and time limit (for example, 121oC for 15 minutes at 15 psi). The principle behind the operation of the autoclave is similar to the pressure cooker used for cooking at our homes. Sterilization using the autoclave is achieved by moist heat unlike the hot air oven which sterilizes materials with dry heat (Figure 9).
The components of the autoclave include the gasket (which carries the materials to be sterilized), the lid containing valves (especially pressure valve and the steam/air valve) and electric element (that generates the steam or heat for the sterilization process) to mention a few. In the most conventional type of autoclave, the valve(s) is usually left open during sterilization until all the air in the vessel or chamber has been displaced; and then it is closed again to allow the pressure in the chamber to build up for proper autoclaving or sterilization. Autoclaving is one of the most dependable methods of sterilizing laboratory equipment, culture media, and other reagents in the microbiology laboratory; and it is also used for the decontamination of old cultures and reagents and other biohazardous waste material before disposal.
MAINTENANCE/CARE OF THE AUTOCLAVE
- Read the owner’s manual carefully before operating the autoclave.
- Allow the pressure of the autoclave to fall to zero level before opening the lid.
- Do not sterilize combustible materials in the autoclave.
- Report any fault of the autoclave to the technician in the laboratory.
- Drain water from water chamber after use to prevent rusting.
- Always check the water level of the autoclave before use.
Fig. 10. Colony counter
Colony counter is a piece of equipment which is used in the microbiology laboratory to count individual colonies of microorganisms (particularly bacteria and fungi) on solid agar plates. Fully automated colony counters and manually-operated counters are available for the counting of microbes in samples or culture plates. The accurate count of bacterial colonies and cells from solid agar plates and/or samples is critical for most biological procedures in the microbiology laboratory (especially in clinical settings) because they give presumptive results about the infecting organism so that action can be taken to contain the situation. Apart from the traditional colony counting machine as shown in Figure 10, automated techniques also exist for the fast enumeration of bacterial cells on agar plates or even in suspensions.
In the microbiology laboratory, microorganisms growing on solid agar plates (especially for bacteria and fungi) are usually enumerated in rough estimates which are expressed as colony forming unit (CFU). CFU gives an implicit count of viable cells of bacteria and fungi growing on solid agar plates; and microbial counts (especially on solid agar plates) as counted under the colony counter (Figure 10) are expressed as CFU per ml (CFU/ml) because it is usually uncertain to conclude whether or not the individual colonies on the agar plate arose from one cell or from many other cells. Other techniques used in the microbiology laboratory to estimate microbial counts in a sample include the Most Probable Number (MPN) and microscopic techniques (which gives an estimate of both living and dead cells) using the haemocytometer or improved Neubauer counting chamber.
Fig. 11. Microscope
Microscope is a piece of instrument or equipment used by microbiologists to examine microorganisms and objects which are too small to be seen by the naked eye. They are also used by biologists and other scientists because microscope allows investigators/researchers and students alike to observe objects that are invisible to the naked eye. When materials are examined under the microscope (Figure 11); the objects being observed are magnified in such a way that they produce an image that can be seen by the eyes of the observer. There are several types of microscopes available for examining the microbial and/or unseen world of microorganisms as well as other living cells that requires better observation by the naked eyes. Examples of microscopes include light microscope, phase contrast microscope, confocal microscope, fluorescence microscope and electron microscope.
MAINTENANCE/CARE OF THE MICROSCOPE
- Read the owner’s manual carefully before operating the microscope.
- Carry the microscope with care (as a delicate object) to avoid dropping it.
- Unplug the microscope from the power source when not in use.
- Clean the microscope with clean dry towel after use, and return it to its box. In most cases especially in research laboratories, the microscopes are left standing on the laboratory bench tops, and covered with a cloth or linen after use.
Fig. 12. pH meter
pH meter is a piece of equipment that is used to maintain the actual pH of a solution i.e. the hydrogen ion concentration of a particular solution. In some instance, a pH indicator (for example, methyl red or litmus paper) is used to evaluate the actual pH of a solution, but the pH of a solution can also be evaluated with the pH meter (Figure 12). A pH indicator is a substance that changes colour over a particular pH range. Typical examples of pH indicators include methyl orange, methyl red, phenol red, Congo red, bromothymol blue, phenolphthalein, malachite green and litmus paper. The components of a solution that maintains constant pH in a particular solution is known as buffers.
Buffers usually contains a weak acid and its conjugate base in a relatively equal and large amount; and a buffer solution is a solution in which the pH is usually resistant to small additions of either a strong acid or strong base. Solutions used for microbiological investigations could either be acidic, basic or neutral; and it is the pH meter that can be used to determine the actual physiological state of the solution based on the equilibrium between its weak acid and bases. The main components of a pH meter include the voltmeter which gives the reading of the pH value of the solution and the glass electrode which is made of a special glass permeable to only hydrogen ions (H+).
MAINTENANCE/CARE OF THE pH METER
- Always follow the owner’s manual or manufacturer’s instruction when using the pH meter.
- The glass electrode must always be handled with care and kept in its holder to avoid breakage.
- The pH meter should be kept in a dry place.
- The glass electrode should always be immersed in a buffer to maintain/increase its shelf life.
HOOD / BIOSAFETY CABINET
Fig. 13. Hood
Biosafety laminar flow cabinet or hood is used to maintain a clean environment when performing microbiological investigations. The hood also protects the worker or scientist from a direct contamination with the organisms or materials they are working with. It provides the perfect environment for the handling of pathogenic microorganisms or infectious specimens. The hood excludes all forms of contamination during experimentation in the laboratory due to its inbuilt airflow mechanism. Biosafety laminar flow cabinet or hood (Figure 13) is an immovable and fragile laboratory equipment, and thus it should be sited in places within the laboratory where they will not be moved from one point to another.
MAINTENANCE/CARE OF THE HOOD
- Users of the hood should always read the owner’s manual before usage.
- The hood should be properly cleaned and disinfected with 70 % ethanol before and after use.
- The UV light in the hood should only be put on when no work is being performed in the biosafety laminar flow cabinet.
- Microbiologists working in the biosafety laminar flow cabinet must always ensure that the glass covering in the hood is kept at the recommended levels during experimentation.
- The hood should be properly covered after experimentation and the fan switched off, while turning on the UV light.
Fig. 14. Incubator
Incubator is a piece of equipment which is used in the microbiology laboratory to provide optimum temperature and oxygen concentration amongst other vital environmental factors required for the optimal growth of microorganisms. It is generally used for growing microbial cultures inclusive of fungi and bacteria. The incubator supplies and maintains optimum physical and chemical conditions required for the development or growth of microbial cells. This incubator is different from the humidified CO2 incubator used in cell culture experiment. Temperature, humidity, oxygen and carbondioxide (CO2) are some of the factors provided by the incubator for the growth of microbes in microbiology laboratory.
Petri dishes or culture plates containing microorganisms are placed inside the incubator and it is regulated and left for specific time and temperature during which the cultured organism is expected to replicate and increase in its cell size. For example, bacteria are incubated for 18-24 hours at 37oC. The incubation time and temperature varies for the different types of microorganisms; and it is important to note this so that the culture process of which the incubator (Figure 14) plays a vital role will be successful.
MAINTENANCE/CARE OF THE INCUBATOR
- Always read the owner’s manual before using the incubator.
- Keep the incubator clean; and always clean off spilled liquids using a dry towel.
- Ensure the power source of the incubator is turned on when in use, and off when not in use.
- Culture media plates and bottles should be properly stacked in the incubator to avoid them from following over.
HOT AIR OVEN
Fig. 15. Oven
Hot air oven is used for the sterilization of glassware’s such as glass Petri plates and other heat-stable materials in the laboratory. The sterilization of materials in the hot air oven is achieved using dry heat unlike the autoclave that achieves the same purpose but with moist heat. Hot air oven is operated at different time intervals and temperature levels, but this is dependent on the nature of the materials to be sterilized. It is critical that users of the hot air oven read the owner’s manual carefully in order to get optimum service from the equipment. Combustible materials and solvents such as ethanol, methanol and chloroform should not be placed inside the hot air; and inflammable materials, cotton wool and other laboratory clothing’s and plastics should not be sterilized in the hot air oven (Figure 15). The hot air oven should be turned off when not in use because heat buildup in the equipment can result to fire outbreak. Only recommended glass wares and materials should be sterilized in the hot air oven.
Fig. 16. Water bath
Water bath is a piece of equipment used to maintain culture media and other laboratory reagents at thermostatically-controlled and optimal temperatures. They are generally used to keep water at a constant temperature for incubating samples and other reagents in the microbiology laboratory. Culture media or reagents to be maintained at a particular temperature prior to their usage for further microbiological investigations are normally placed in a vessel that is suspended in the water contained in the water bath (Figure 16). Water bath is also used for incubation the same way that the incubator operates, but the former (i.e. water bath) utilizes water maintained at a particular ambient temperature level to achieve incubation. The main components of the water bath include the thermostat, water tank, heater, cooling coil and the cover or lid.
MAINTENANCE/CARE OF THE WATER BATH
- Read the owner’s manual carefully before operating the water bath.
- Culture media and reagents should not be left too long in the water bath to avoid overheating.
- Always check the water bath before use to ensure that the tank is properly filled with water.
- Also check that the temperature level is properly regulated and set at the require temperature.
- Always switch off the water bath from its power source after use.