MCB Instrumentation (Practical) PDF

# Basic Equipment in Medical Microbiology Laboratory The basic equipment in a medical microbiology laboratory are as follows: 1. Microscopy 2. Centrifuge 3. Incubator 4. Water bath 5. Hot air oven 6. Autoclave 7. Weighing balance 8. Glass wares 9. Bunsen burner 10. Test tube racks 11. Safety Cabin...

# Basic Equipment in Medical Microbiology Laboratory The basic equipment in a medical microbiology laboratory are as follows: 1. Microscopy 2. Centrifuge 3. Incubator 4. Water bath 5. Hot air oven 6. Autoclave 7. Weighing balance 8. Glass wares 9. Bunsen burner 10. Test tube racks 11. Safety Cabinets 12. Inoculation loop 13. Petri dishes 14. Vortex mixer 15. Refrigerator 16. Deioniser ## Microscope Microscope is an indispensable piece of apparatus in all medical labs. Its purpose is to produce an enlarged image of an object too small to be observed with a naked eye. It is thus a prestigious object whose magnification depends on the quality of its lenses. Microscope can either be monocular (ie having a single eye piece) or binocular ie having two eye pieces. Essentially, a microscope consists of 3 parts namely: The stand, the body, and the trend of optical lenses. The upright stand rests on a heavy foot & bears on its upper end an inclined binocular head with two eye pieces above and a revolving nose piece bearing interchangeable lenses of various magnitude, attached through a racking mechanism to the middle of the stand is a horizontal platform or stage with a central hole over which the slide with specimen is held by clips. The stage may be of the fixed type fitted with two spring clips or of the mechanical type that can be moved in two planes by rack and pinion mechanism for searching different areas of the slide. For focusing, the stage is racked upwards or downwards by turning the milled heads of coarse and fine focusing adjustments. A built-in lamp in the foot of a microscope passes a beam of light upwards through a fitted diaphragm controls the amount of light reaching the object. The beam of light is open unto a specimen by a substage condenser which is attached beneath the stage. The height of the condenser and therefore the focus of the light can be adjusted up or downward by the rack and pinion mechanisms. Just below the iris diaphragm is a ring shaped filter holder designed to carry circular coloured glass filter required to reduce excessive red or yellow component in some type of light. ### Method of use of light microscope 1. Place the microscope at a convenient position on the bench and adjust the light and position of observer's chair. The eye pieces of the microscope should be level and close to the observers eye while sitting in upright position. 2. Check to be sure that object and eye pieces are free from dust and oil. 3. Rack up fully the substage condenser until its top surface is reading about 1mm below the upper surface of the stage. 4. Switch on the lamp at low intensity and increase the intensity. 5. Place the slide with object on stage so that it is held firmly by the stage clips. 6. First view the specimen with a low power objective & use the coarse focusing adjustment to focus the specimen on the slide. 7. Adjust the distance between the eye pieces so that a single field is seen. 8. If continued, examining at a lower power objective is required, diffuse the light uniformly over the field by defocusing the condenser. 9. Before proceeding to use a high power objective, check if the iris diaphragm is opened. The condenser is focused by fully racking up & illuminating is properly centered. 10. Before using an oil immersion objective rack, down stage to give adequate clearance between the slide & object & place a moderately large drop of immersion oil on the middle of the specimen central to the light beam focused on the condenser. Rotate the nose-piece until the immersion oil lens is in position. 11. Use the coarse focusing adjustment to make contact with the oil & a fine adjustment to get the fine image of the object. 12. Search the specimen in an orderly way by moving the adjustment in the mechanical stage. Keep a hand on a fine focusing adjustment to maintain a fine image during the movement of the slide. ### Care of Microscope 1. Microscope should be kept at a uniform temperature and not exposed to sunlight or any source of heat. 2. It should be moved with care to avoid mechanical damage and should be lifted by its fixed upright stand and foot & not by any movable part. 3. When not in use, it should be protected from dust in its box or under a plastic cover. 4. It should be cleaned at intervals. Oil immersion objective should be cleaned after use each day. 5. The objective eye pieces, condenser lenses and mirror should be wiped free from dust with a piece of cloth or fresh lense tissue. 6. Occasionally eyepieces may become contaminated internally with dust. When the position of the dust detected, it can be cleaned with a densed tissue, distilled water or with a soft Carmel hair brush if available. ## Centrifuge One of the best devices for the separation of particles from its suspending fluids is the centrifuge. The suspended matter is deposited in order of weight. The heaviest element being the first to settle. This is usually achieved by centrifugal force. The greater the centrifugal force, the more rapid & effective sedimentation. Centrifuges have either of two types of heads or (rotors) the swing out or fixed angled head. A. The swing-out rotor: these are those in which the buckets containing the tubes or bottles swing outwards so that the particles in the fluid are forced to the bottom of the tube to give a horizontal separation. B. Fixed angle rotors: here, the buckets are positioned at a fixed angle to the vertical axis of the centrifuge head. During centrifugation, the particles in fields are thrown to the side and bottom of fluid to give a slanted separation. ### Use & Care Of A Centrifuge 1. Tubes must be put in the centrifuges in pairs that have been accurately balanced & placed diametrically opposite each other. 2. Before putting the tubes into the buckets, make sure that cushions or sleeves (pad) are in position at the bottom of the bucket & free from debris eg. Fragments of glass otherwise breakages are liable to occur, 3. Use capped tubes or bottles when centrifuging specimen or material which may contain pathogens. 4. Close the lid and make sure it is secured. The lid must not be opened. When the centrifuge is running to avoid the danger of creating aerosols. 5. When the tubes have been centrifuged sufficiently, switch off the motor and allow the centrifuge to come to a stop. Never slow the rotating head with your hands as break. 6. Never open the centrifuge until the motor has stopped and the bucket have come to rest, otherwise infectious materials may be spread into the lab. 7. Clean regularly the buckets and inside of a centrifuge using a cloth soaked in a non-corrosive disinfectant like 70% alcohol. 8. When a breakage occurs during centrifuging, do not open the centrifuge immediately, allow at least 30 minutes for the droplets to settle. Wear protective gloves and use forceps & cotton wool swabs soaked in disinfectant to remove the broken glass. Clean the buckets and inside the centrifuge before using it again. ## Incubators Incubation at control temperature is required for bacteriological cultures of some blood transfusion, serological, haematological & clinical chemistry test. For bacteriological cultures, an incubator is required whereas for other tests, a dry heat block or a water bath is recommended. Incubators are usually fitted with thermostats and through any desired temperature must be constantly maintained. They are also fitted with the thermometers by which the desired temperature is known. Most laboratory Incubators are left working at 37°C which is the optimum good temperature of most pathogenic organisms of man. ### Use And Care Of Incubators 1. Make sure the incubator is positioned on a level surface. 2. Before incubating cultures & tests, check the temperature. 3. Clean the incubator regularly, but care must be taken to first disconnect it from the mains electricity supply. 4. Every 3 - 6 months check the incubator for sign of wear & examine the connections inside the plugs. ## Water Baths This is a bath of water kept warm all the time by electricity or gas. It is required to incubate bottles of culture media, liquids in flask or other large containers or when needing to incubate several racks of tubes. ### Use & Care Of Water Baths 1. Fill the bath and maintain its level with clean water or chemically pure water. 2. Do not use tap water because it will cause salt to be deposited and eventually damage the inner cells and before incubating the thermometer to check the temperature of the water. 3. Ensure that the level of the water is above the level of whatever is incubated. 4. Do not use a lid when incubating uncapped tubes or bottles, contamination and dilution of test can occur due to condensation. 5. Clean the water bath regularly taking care not to damage the heating unit. Follow the manufacturer's instructions. ## Hot Air Oven This is used for sterilizing materials that are unaffected by temperature of 160°C - 180°C and for which autoclaving is unsuitable eg: test tubes, Petri dishes, flask, pipettes and instruments such as forceps, scissors, throat swab, carpets etc. The hot air oven is used for sterilizing dry materials in sealed containers ie powder, fats, oils & greases that are impermeable to moisture. These materials are penetrated very slowly by heat and must therefore be sterilized in small quantities. ### Use & Care Of A Hot Air Oven 1. Glassware should be perfectly dry before being placed in a sterilizing oven. Wet glasswares are likely to crack & should first be dried in a dry oven. 2. Before sterilizing, plug test tubes & flask with cotton wool stoppers, other glasswares like pipette may be wrapped in craft paper or sterilized in metal cans. 3. Oven may be cold or warm when loaded and is then heated up to the sterilizing temperature in the course of 1 - 2 hours. Holding time starts where the thermometer shows when the oven has reached 160°C. Holding period of 1 hour at 160°C or 30 minutes for at least 180°C is usually sufficient for certain material like glasswares. However, heavy loads in an oven without a fan and slowly heating material such as powder, oils & greases should be exposed at 160°C for 2 to 2½ hours. ## Autoclaves This is an apparatus used for moist sterilization at increased pressure and temperature above 100°C. The simplest form of laboratory autoclave is the pressure cooked type. It is a vertical or horizontal cylinder of stainless steel in a supporting frame. The lid or door is fastened by screw clamps and is rendered air tight by means of an asbestos washer. The cylinder contains water up to a certain level and this is heated by a gas burner or electric heater below the cylinder. ### Directions For Usage 1. There should be sufficient water in the cylinder. 2. Insert the material to be sterilized and turn on the heater. 3. Place the lid in position, see that the discharge tap is open and the screw down the lid. 4. As steam rises from the boiling water, it mixes with air in the chamber and carries this out through the discharge tap. Allow the stream and air mixture to escape freely until all the air has been eliminated from the autoclave. 5. Close the discharge tap. The steam pressure rises until it reaches the desired level eg 121°C when the safety valve opens and allow the excess steam to escape. 6. At exactly this point, begin to time the holding period. Continuing exposure at 151b pressure for the appropriate time which is usually 15 minutes in aqueous media for quantities of up to 100ml and longer for more values that heat up more slowly. 7. Turnoff the heater and allow the autoclave to cool until the pressure gauge indicates that inside is at 0 atmospheric pressure. At once, open the tap slowly to allow air to enter the autoclave. ### Importance Of Air Discharge All the air must be removed from the autoclave chamber and articles of the load so that the later are exposed to pure steam during the period of sterilization. There are three (3) reasons for this: 1. The admixture of air & steam results in lower temperature being achieved at a chosen temperature. 2. The air hinders penetration of steam into the interstices of porous material. Surgical drdressin especially narrow opening of containers, syringes etc. 3. The air being denser than the steam tends to form a separate and cooler layer in the lower part of the autoclave and so prevents adequate heating of the articles there. ## Weighing Balances These are used to weigh materials in the lab & it is essential to accurately weight & prepare basic reagents & culture media used in the lab. There are different types of chemical balance, some that need separate weights, others don't need and the direct readout electrical balance. ### Use And Care Of A Weighing Balance 1. Read carefully the manufacturer's instructions and always handle the balance with care. 2. Position the balance on a firm bench away from sunlight. 3. Make sure that the balance is level, adjusting if necessary, with screw & stands. 4. Before starting to weigh, zero the balance as directed by the manufacturer. 5. Weigh chemicals at room temperature in a weighing scoop, a paper or small beaker never directly on a balance pan. If using a box of weight, always use the forceps provided for adding or removing weights. Protect the weights from dust or moisture. 6. After completing the weighing, return the balance to zero weight. ## Glassware These are widely used in the medical laboratory and there are large varieties of them. Some glassware & their uses include: 1. Petri dishes - These are flat containers which are predominantly used for the cultivation of microorganisms on solid media. 2. Pipettes - These are used to measure liquid volumes of up to 50ml. There are several types of pipettes and all types must always be used with a rubber teat. 3. Biurettes - These are long graduated tubes of uniform bulb and closed at the lower end by means of a glass stop cork. Biurettes are used for measuring variable quantities of liquids. 4. Beakers - These are usually made of heat resistant glass and are of different shapes. 5. Flasks - These have capacities of 25 - 6000ml and there are different types. They include conical flasks, flat bottom flasks, round bottom, volumetric flasks. 6. Bottles These come in 5 - 20ml capacities and are of various types. Their bottom may be round, flat or conical. 7. Funnels - They are generally of two types; the filter funnels used for pouring liquids into narrow mouthed containers & for supporting filter papers during filtration. The separating funnels which are used for separating immiscible liquid of different densities eg: ether & water. 8. Dessicators - These are used to dehydrate substances or to keep them in anhydrous state (dry state). 9. Measuring Cylinders - used in measuring liquid. 10. Pestle & mortar - used for grinding. 11. Evaporating basins - These are shallow vessels used in facilitating evaporation. 12. Slides - These are flat rectangular glasses used in making smears & wet mounts for microscopic examination. ### Care of Glassware 1. Flasks & beakers should be placed on a guaze material when they are heated over a Bunsen flame. 2. Test tubes exposed to a naked flame should be made of heat resistant glass like Pyrex. 3. If liquids are to be heated in a bath of boiling water, the glass container should be heat resistant. Sudden plunging of cool bottles into boiling water & vice versa should be avoided. 4. When diluting concentrated acids, thin walled glass ware should be used. The heat evolved by the procedure often cracks thick glass. 5. If screw capped tubes or flasks are to be heated, the caps should be loosely screwed to avoid their cracking due to expansion. ### Cleaning of Glassware 1. New glass ware - This requires special attention because of the resistant spores which may be present in the straw & other packaging material & also because it tends to give off free alkali which may be sufficient to interfere with the growth certain organism. Consequently, it should be placed in 1% HCl overnight washed in tap water & distilled water & then autoclaved. 2. Glassware for general lab use - Glass containers with discarded cultures can be placed in 3% lysol after use or transfer directly to boiling soap solution. Containers with infections materials like the tubercle bacilli or spore bearing organisms must be autoclaved. The glassware is then boiled for 1 hour in a 5% soap solution. They are cleansed with the test tube brush and then rinsed well in hot or cold water. 3. Cleaning of glassware for biochemical work - Chemical cleaning is necessary for the following reasons: A. Traces of reagents left in the tubes and containers may interfere with later chemical investigations. B. Air bubbles may be trapped between greasy surfaces and contain liquids resulting in inaccurate volumetric readings. Potassium dichromate/acid solution is used for cleaning glassware for chemical chemical work and this is made up of 10g of potassium dichromate, 25ml of conc H2SO4, 75ml of distilled water. This fluid is highly corrosive and should be handled with care. As an alternative to the above solution detergents can be used to clean biochemical glasswares. 4. Cleaning of pipettes A. If contaminated with infective material discard the used pipette into a 3% lysol solution and leave until convenient to wash. B. Rinse in tap water. C. If necessary, soak overnight in dichromate sulphuric acid cleaning fluid. D. Wash with tap water. ## Deioniser Deionisation can be defined as a process by which impure water is passed through anion and cation (Deioniser) to produce ion free water. The apparatus used to produce deionized water is called Deioniser. (Ion Exchange Resins) A. Anion exchange Resins) B. Cation Exchange Resins) But in practice, the 2 are usually mixed in one column as mixed bed or mixed column Deioniser. To obtain pure water, simply pour the water to be purified onto mixed polystyrene resins and the water imergin after passing through the column is pure. NB: On exhaustion, the resin will change colour. It should then be changed. It should be protected from sunlight. ## Principle of autoclave. When the steam meets the cooler surfaces of the article to be sterilized, it condenses into a small volume of water and liberates its considerable latent heat to that surface. The large concentration in volume brings more steam to the same site and the process continue rapidly until the articles temperature is raised to that of the steam. The condensation water ensures moist condition for killing of the exposed microbes. An autoclave is an apparatus used for moist sterilization at increased pressure and temperature above 100°C.