Food microbiology lab manual

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LABORATORY MANUAL OF FOOD MICROBIOLOGY FOR ETHIOPIAN HEALTH AND NUTRITION RESEARCH INSTITUTE (FOOD MICROBIOLOGY LABORATORY) UNIDO PROJECT (YA/ETH/03/436/11-52) DEC. 2003 DRAFTED BY DR. CIIRA KIIYUKIA (INIDO / FOOD ANALYSIS – MICROBIOLOGY) TABLE OF CONTENTS INTRODUCTION.....................................................................................................4 MICROORGANISMS MORPHOLOGY AND STAINING.............7 MICROSCOPY.....................................................................................................................7 STAINED PREPARATIONS ...................................................................................................7 MAKING A SMEAR.............................................................................................................8 A SIMPLE STAIN.................................................................................................................8 A DIFFERENTIAL STAIN: GRAM’S STAINING METHOD.........................................................9 BACTERIAL MOTILITY.......................................................................................................9 ENDOSPORE STAINING (SCHAEFFER–FULTON OR WIRTZ–CONKLIN).................................10 FLAGELLA STAINING: WEST AND DIFCO’S SPOTTEST METHODS ......................................11 BASIC LABORATORY PROCEDURES AND CULTURE TECHNIQUES .........................................................................................................14 PREPARATION OF CULTURE MEDIA...................................................................................14 POURING A PLATE............................................................................................................14 STORAGE OF MEDIA.........................................................................................................14 STERILIZATION VS. DISINFECTION....................................................................................14 STERILIZATION OF EQUIPMENT AND MATERIALS..............................................................15 DISINFECTANTS...............................................................................................................15 INOCULATION AND OTHER ASEPTIC PROCEDURES .............................................................15 ESSENTIAL POINTS...........................................................................................................15 STREAK PLATE. ...............................................................................................................17 POUR PLATE....................................................................................................................17 SPREAD PLATE.................................................................................................................19 INCUBATION....................................................................................................................19 CLEARING UP...................................................................................................................20 PURE CULTURES...............................................................................................................20 MAINTAINING STOCK CULTURES......................................................................................20 COTTON WOOL PLUGS......................................................................................................21 ASEPTIC TRANSFER OF CULTURES AND STERILE SOLUTIONS..............................................21 TESTING SENSITIVITY TO ANTIBACTERIAL SUBSTANCES....................................................22 COMMON BIOCHEMICAL TESTS..........................................................24 1. INDOLE TEST................................................................................................................24 2. H S PRODUCTION TEST:...............................................................................................24 2 3. NITRATE REDUCTION TEST...........................................................................................24 4. METHYL RED TEST .......................................................................................................24 5. VOGES- PROSKAUER’S TEST ........................................................................................24 6. UTILIZATION OF CITRATE AS THE SOLE SOURCE OF CARBON.........................................25 7. FERMENTATION OF SUGAR:..........................................................................................25 8. GELATIN LIQUEFACTION:.............................................................................................25 9. ACTION ON LITMUS MILK:............................................................................................25 10. UTILIZATION OF URIC ACID AS THE SOLE CARBON SOURCE .........................................26 1FOOD SAMPLING AND PREPARATION OF SAMPLE HOMOGENATE .....................................................................................................28 SAMPLE COLLECTION.......................................................................................................29 SAMPLE ANALYSIS ..........................................................................................................31 CLASSIFICATION OF FOOD PRODUCTS FOR SAMPLING PURPOSES.......................................32 EQUIPMENT AND MATERIALS ..........................................................................................34 RECEIPT OF SAMPLES .......................................................................................................34 THAWING........................................................................................................................35 MIXING............................................................................................................................35 WEIGHING........................................................................................................................35 BLENDING AND DILUTING OF SAMPLES REQUIRING ENUMERATION OF MICROORGANISMS..35 ENUMERATION OF MICROORGANISMS IN FOODS..............37 A. DETERMINATION OF AEROBIC COLONY COUNT IN FOODS ....................37 B. MOST PROBABLE NUMBER METHOD (MPN) ...............................................41 CALCULATION OF MOST PROBABLE NUMBERS (MPN)....................................................43 MPN TABLES..................................................................................................................45 C. ENUMERATION OF YEASTS AND MOULDS IN FOODS.................................47 D. ENUMERATION OF COLIFORMS FAECAL COLIFORMS AND E. COLI IN FOODS USING THE MPN METHOD........................................................................53 ISOLATION AND ENUMERATION OF PATHOGENIC MICROORGANISMS IN FOOD. .................................................................64 A. ISOLATION OF E. COLI 0157 IN FOODS .........................................................64 B. ENTEROCOCCUS ..................................................................................................71 C. ISOLATION OF SALMONELLA FROM FOODS .................................................75 D. ENUMERATION OF STAPHYLOCOCCUS AUREAUS IN FOODS ...................81 E. ISOLATION OF LISTERIA MONOCYTOGENS FROM ALL FOOD AND ENVIRONMENTAL SAMPLES ...................................................................................96 F. ISOLATION AND ENUMERATION OF BACILLUS CEREUS IN FOODS......111 G. DETECTION OF CLOSTRIDIUM BOTULINUM IN HONEY AND SYRUPS...121 H. ENUMERATION OF CLOSTRIDIUM PERFRIGENS IN FOODS .....................125 MICROBIOLOGY OF WATER..................................................................130 STANDARD QUALITATIVE ANALYSIS OF WATER...........................................................130 QUANTITITIVE ANALYSIS OF WATER..............................................................................133 PURPOSE........................................................................................................................133 HOWARD MOULD COUNT.........................................................................136 EXAMINATION OF CANNED FOODS ................................................148 STANDARD OPERATING PROCEDURES (SOPS).....................161 QUALITY ASSURANCE IN MICROBIOLOGY LABORATORIES.........................168 2INSTRUMENTAL MAINTENANCE, QUALITY CONTROL AND CALIBRATION ......................................................................................................................................169 LABORATORY AUDIT..............................................................................................185 MICROBIAL STANDARDS OF FOODS..............................................187 GUIDELINES FOR WRITING LAB REPORTS ..............................198 REFERENCES AND SELECTED READINGS ................................201 3Introduction The purpose of this manual is to provide the new food microbiology laboratory at the Ethiopian Health and Nutrition Research Institute with the standard methods for qualitative and quantitative detection of microorganisms in food and water. The manual contains detailed description of microbial enumeration, isolation and identification of pathogenic food-borne bacteria. Methods of estimating sanitary indicator microorganisms as well as enumeration of moulds and yeasts are documented. These methods have been adapted from methods recommended by the ICMSF, AOAC, FDA, APHA and Health Canada. The standard operating procedures and quality control guidelines relating to food sampling and methods of analysis are included. The manual is written in such a manner that it can be used for in-house training of new technicians. Description of equipment maintenance and calibration is detailed including quality control of media and internal laboratory audit. Isolation and identification of microbial food contaminants help to understand how infectious agents enter and spread through the food chain and therefore come up with procedures to prevent or minimize exposure of the consumer to such agents. There is the need to estimate the risk that foodborne pathogens pose to human health in a national and international context and to identify possible interventions to reduce or eliminate these risks. The standards, guidelines and recommendations adopted by international trade agreements, such as those administered by the WTO, are playing an increasingly important role in protecting the health of consumers. In the case of microbiological hazards, Codex has elaborated standards, guidelines and recommendations that describe processes and procedures for the safe preparation of food. The application of these standards, guidelines and recommendations is intended to prevent or eliminate hazards in foods or reduce them to acceptable levels. This requires an elaborate laboratory with equipment and personnel well trained to carry out the analysis. Most developing countries lack the resources to put up food microbiology laboratories and to man them adequately to international standards. The globalization of food trade and increasing problems worldwide with emerging and re-emerging foodborne diseases have increased the risk of cross- border transmission of infectious agents. Because of the global nature of food production, manufacturing, and marketing, infectious agents can be disseminated from the original point of processing and packaging to locations thousands of miles away. In this regard, developing countries are required to ensure that their sanitary and phytosanitary measures are based on an assessment, as appropriate to the circumstances, of the risks to human, animal or plant life or health, taking into account the risk assessment techniques developed by the relevant international organizations. The manual details microbiological risk assessment of various food categories, guidelines and recommendations related to food safety. There is a critical need for technical advice on risk assessment of microbiological hazards in foods to meet the needs of national governments, the food industry, the scientific community, trade organizations and international consumer groups. UNIDO, FAO and WHO have a direct role to play in assisting developing countries in matters related to food safety and should strengthen efforts to facilitate access to specific advice on microbiological risk assessment. This manual has been developed with the help of UNIDO inline with the above stated spirit. 4Microbial Food analysis 1: Reasons for microbial food analysis. • to meet certain set standards • to estimate the shelf-life of the product • to determine quality of the food • for public health purposes 2: The organisms to look for; i) Indicator organism(s); Definition: an indicator organism or group of organisms is one whose numbers in a product reflect the success or failure of "good manufacturing practices". Coliform group of microorganisms and Escherichia coli are commonly used as indicator organisms. ii) Index organism; Definition: an index organism is one whose presence implies the possible occurrence of a similar but pathogenic organism. E. coli is used an index organism and its presence indicates possible presence of pathogenic enterobacteriacea e.g. Salmonella sp. iii) Food poisoning organisms The are two types of food poisoning organisms • those which cause the decease by infection • those which produce toxin in food a)Those which cause infection must grow in food in large numbers and cause infection when consumed together with food. The most common microorganisms in this category includes Salmonella typimurium, enteropathogenic E. coli, Vibrio parahaemolyticus, Yersinia enterocolytica etc. b) Those which cause intoxication must grow in food in large numbers and produce enough toxin and when consumed together with food cause intoxication. The most common microorganisms in this category includes, Clostridium botulinum, Staphylococcus aureus and toxigenic fungi e.g. Aspergillus flavus. iv) Infectious microorganisms Definition: Organisms whose presence in small numbers in food or water and when consumed can cause infection. In this case the food acts as a vector but not necessarily as a growth medium.Infectious organisms can be transmitted by various ways including man to man and are said to be contagious. Organisms in this group includes; Vibrio cholerae O1, Salmonella typhi, Shigella sonnei, Bacillus anthracis, Hepatitis B virus etc. v) Spoilage organisms Definition: Spoilage organisms are the organisms whose growth in the food creates undesirable characteristics in that food. Any microorganism which is not intentionally added into food or intentionally allowed to grow in food so as to impart certain qualities in that food is considered a contaminant. Growth of the contaminant in that food will spoil the food making it unfit for human consumption. Some useful microorganisms e.g. lactic acid bacteria are considered as spoilage organisms when in beer, wine and fruit juices but not in milk. 53: How to analyze i) Quantitative analysis • Serial decimal dilution • Aerobic plate count • Pour plate count • Total viable count • Most Probable Number (MPN) method • Yeast and Molds count ii) Qualitative analysis presence or absence of a specified microorganism e.g. • Salmonella sp. • E. coli • V. cholerae O1 4: Culture Methods • pre-enrichment broth • enrichment broth • selective enrichment • selective agar • Differential agar Biochemical tests • sugar fermentation • amino acid decarboxylation • gelatin liquefaction • lecithinase production Serology • agglutination • precipitin • coagulation Colony morphology • shape • colour • texture • size Cell shape by microscope • bacillus • coccus • streptococcus Gram stain characteristics • gram positive • gram negative Motility motile number of flagella arrangement of flagella non-motile 6Microorganisms morphology and staining Microscopy Using the microscope The setting up of a microscope is a basic skill of microbiology yet it is rarely mastered. Only when it is done properly can the smaller end of the diversity of life be fully appreciated and its many uses in practical microbiology, from aiding in identification to checking for contamination, be successfully accomplished. The amount of magnification of which a microscope is capable is an important feature but it is the resolving power that determines the amount of detail that can be seen. Bacteria and yeast Yeast can be seen in unstained wet mounts at magnifications x100. Bacteria are much smaller and can be seen unstained at x400 but only if the microscope is properly set up and all that is of interest is whether or not they are motile. A magnification of x1000 and the use of an oil immersion objective lens for observing stained preparations are necessary for seeing their characteristic shapes and arrangements. The information gained, along with descriptions of colonies, is the starting point for identification of genera and species but further work involving physiology, biochemistry and molecular biology is then needed. . Moulds Mould mycelium and spores can be observed in unstained wet mounts at magnifications of x100 although direct observations of “mouldy” material through the lid of a Petri dish or specimen jar at lower magnifications with the plate microscope are also informative (but keep the lid on). Routine identification of moulds is based entirely on the appearance of colonies to the naked eye and of the mycelium and spores in microscopical preparations. Stained preparations A “smear” of bacteria or yeast is made on a microscope slide, fixed, stained, dried and, without using a coverslip, examined with the aid of a microsope. Aseptic technique must be observed when taking samples of a culture for making a smear. A culture on agar medium is much preferable to a liquid culture for making a smear. A smear that is thin and even enables the shape and arrangement of cells to be clearly seen and ensures that the staining procedure is applied uniformly. There are two broad types of staining method: (1) a simple stain involves the application of one stain to show cell shape and arrangement and, sometimes, inclusions that do not stain, e.g. bacterial endospores; (2) a differential stain involves a sequence of several stains, sometimes with heating, and includes a stage which differentiates between either different parts of a cell, e.g. areas of fat storage, or different groups, e.g. between Gram-positive and Gram-negative bacteria. The reaction of bacteria to Gram’s staining method is a consequence of differences in the chemical structure of the bacterial cell wall and is a key feature in their identification. Yeast cells can be stained by Gram’s method but it is of no value in their identification. The basis of Gram’s staining method is the ability or otherwise of a cell stained with crystal violet to retain the colour when 7treated with a differentiating agent, usually alcohol (although professionals sometimes use acetone). Bacteria that retain the violet/purple colour are called Gram-positive. Those that lose the colour, i.e. called Gram negative, are stained in the contrasting colour of a counterstain, usually pink/red. Making a smear. 1. Clean a plain microscope slide thoroughly using lens tissue. 2. Label a microscope slide with a marker pen to record the culture being used, date and initials; this is also a useful reminder of which side of the slide is being used. 3. Flame a wire loop to ensure that no culture accidentally remains from a previous operation. 4. Transfer one or two loopfuls of tap water on to the centre of the slide. 5. Flame loop and allow to cool. 6. Using aseptic technique, transfer a very small part of a single colony from a plate or slope of agar medium into the tap water. If the amount of culture on the loop is easily visible you have taken too much 7. Make a suspension of the culture in the tap water on the slide and thoroughly but gently spread it evenly over an oval area of up to 2 cm length. 8. Flame the loop. If it is necessary to use a liquid culture or sample, the use of tap water to prepare the smear will probably be unnecessary and may result in a smear with too few cells. 9. Dry the suspension by warming gently over a Bunsen burner flame and then “fix” it by quickly passing it through the flame a few times. This is called a heat-fixed smear; it should be visible to the naked eye as a whitish area. Fixing is necessary to ensure that cells adhere to the slide and to minimise any post mortem changes before staining. A simple stain. 1. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray. 2. Thoroughly cover the smear with stain and leave for, usually, 30 seconds. 3. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle over the sink. 4. Rinse off the stain with tap water. 5. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not sideways movements that might remove the smear. 6. Examine under oil immersion. 7. When finished, dispose of slides into discard jar. Suitable stains include basic dyes (i.e. salts with the colour-bearing ion, the chromophore, being the cation) such as methylene blue, crystal violet and safranin. Staining solutions (relevant to procedures described below) Crystal violet solution: A. crystal violet 2.0g dissolved in absolute alcohol 100 ml B. ammonium oxalate 1.0g in distilled/deionised water 100ml Add 25 ml A to 100 ml B Lugol’s iodine solution: iodine 1.0g, potassium iodide 2.0g distilled/deionised water 300 ml 8 A differential stain: Gram’s staining method Times of the staining periods depend on the formulation of the staining solutions which are not standard in all laboratories. Therefore, the times given here relate only to the solutions specified here. a. Put the slide with the fixed smear uppermost on a staining rack over a sink or staining tray. b. Thoroughly cover the smear with crystal violet solution and leave for 1 minute. c. Hold the slide with forceps (optional but avoids stained fingers), at a 45° angle over the sink. d. Pour off the stain, wash off any that remains (and any on the back of the slide) with iodine solution. e. Put the slide back on staining rack. f. Cover the smear with iodine solution and leave for 1 minute. Iodine solution acts as a “mordant” (a component of a staining procedure that helps the stain to adhere to the specimen), a crystal violet-iodine complex is formed and the smear looks black. g. Hold the slide with forceps at a 45° angle over the sink wash off the iodine solution with 95% (v/v) ethanol (not water); continue treating with alcohol until the washings are pale violet. h. Rinse immediately with tap water. i. Put the slide back on staining rack. j. Cover the smear with the counterstain, e.g. safranin solution, 0.5% w/v, for 30 seconds. k. Rinse off the stain with tap water. l. Blot dry the smear with filter/fibre free blotting paper using firm pressure but not sideways movements that might remove the smear. m. Examine under oil immersion. n. When finished, dispose of slides into discard jar. Always use a young culture because older cultures of Gram-positive bacteria tend to lose the ability to retain the crystal violet-iodine complex and appear to be Gram-negative; but some bacteria are naturally only weakly Gram-positive. The amount of alcohol treatment (the differential stage) must be judged carefully because over-treatment washes the crystal violet- iodine complex from Gram-positive bacteria and they will appear to be Gram-negative. Take care to make an even smear otherwise alcohol will continue to wash the violet/purple colour from thick parts of the smear while thin parts are being over-decolorised. At the end of the procedure, check that the labeling has not been washed off by the alcohol. Don’t despair if the stained smear is not visible to the naked eye; this may happen with a Gram-negative reaction. Bacterial Motility 1. Hanging drop method of motility: - use the special microscope slide with a depression 9 - a cover slip - a micropscope - immersion oil - actively growing bacterial culture Procedure Place one drop of the culture onto the cover slip Touch the corners of the cover slip with lanolin and invert it on the grooved microscope slide glass. Observe for motility using the high power lens. Motility is characterized by fast unidirectional movement as compared to the Brownian motion whereby the cells move round in one particular point. 2. Semi-solid agar method The agar medium is prepared with the agar content of 0.2%. The medium is put into test tubes. Inoculation is done by stabbing the medium at the center. The inoculated medium is incubated at appropriate temperature for 24 hr. motility is detected by observing turbidity at the line of inoculation. Endospore staining (Schaeffer–Fulton or Wirtz–Conklin) Materials 24-to 48 hours nutrient agar slant cultures of Bacillus megaterium (ATTC 12872) and Bacillus macerans (ATCC 8244), and old (more than 48 hours) thioglycollate cultures of Clostridium butyricum (ATCC 19398) and Bacillus circulars (ATCC 4513) Clean slides Microscope Immersion oil Wax pencil Inoculating loop Hot plate or boiling water bath with staining rack or loop 5 % malachite green solution Safranin Bibulous paper Paper toweling Lens paper Slide warmer Forceps Principle Bacteria in genera such as Bacillus and Clostridium produce quite a resistant structure capable for surviving of long periods in an unfavorable environment and then giving rise to a new bacterial cell. This structure is called an endospore since it develops within the bacterial cell. This location and size of endorspores vary with the species; thus, they are often of value in identifying bacteria. Endospores are spherical to elliptical in shape and may be either smaller or larger than the parent bacterial cell. Endospore position within the cell is characteristic and may be central, subs terminal, or terminal. Endospores do not stain easily but, once stained, they strongly resist decolorization. This property is the basis of the Schaeffer-Fulton or Wirtz-Conklin method of staining endospores. The 10endorspores are stained with malachite green. Heat is used to provide stain penetration. The rest of the cell is then decolirized and counterstained a light red with safranin. Procedure 1. With a wax pencil, place the names of the respective bacteria on the edge of four clean glass slides. 2. Aseptically transfer one species of bacterium with an inoculating loop to each of the respective slides, air dry (or use a slide warmer), and heat-fix. 3. Place the slide to be stained on a hot plate or boiling water bath equipped with a staining loop or rack. Cover the smear with paper toweling that has been cut the same size as the microscope slide. 4. Soak the paper with the malachite green staining solution. Gently heat on the hot plate (just until the stain steams) for 5 to 6 minutes after the malachite green solution begins to steam. Replace the malachite green solution as it evaporates so that the paper remains saturated during heating. 5. Remove the paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds. 6. Counterstain with safranin for 60 to 90 seconds 7. Rinse the slide with water for 30 seconds. 8. Blot dry with bibulous paper and examine under oil immersion. A coverslip is not necessary. The spores, both endospores and free spores, stain green; vegetative cells stain red. Flagella staining: West and Difco’s SpotTest Methods Materials Young, 18-hour tryptic soy agar slants of Alcaligenes faecalis (ATCC8750, peritrichously flagellated) and Pseudomonas fluorecens (ATCC 13525, polarly flagellated) Wax pencil Inoculating loop Acid-cleaned glass slides with frosted ends Clean distilled water Microscope Immersion oil Lens paper Boiling water bath (250 ml beaker with distilled water, rind stand, wire gauze pad, an Bunsen burner or hot plate) Pasteur pipettes with pipettor West stain Solution A 11Solution B Difco’s SportTest Flagella stain Principle Bacterial flagella are fine, threadlike organelles of locomotion. They are slender (about 10 to 30 nm in diameter) and can only be seen directly using the electron microscope. In order to observe them with the light microscope, the thickness of the flagella are increased by coating them with mordants like tannic acid and potassium alum, and staining them with basic fuchsin (Gray method) or crystal violet (Difco’s method). Although flagella staining procedures are difficult to carryout, they often provide information about the presence and location of flagella, which is of great value in bacterial identification. Difco’s SportTest flagella stain employs an alcoholic solution of crystal violet as the primary stain, and tannic acid and aluminum potassium sulfate as mordants. As the alcohol evaporates during the staining procedure, the crystal violet forms a precipitate around the flagella, thereby increasing their apparent size. Procedure 1. With a wax pencil, mark the left-hand corner of a clean glass slide with the name of the bacterium. 2. Aseptically transfer the bacterium with an inoculating loop from the turbid liquid at the bottom of the slant to 3 small drops of distilled water in the center of a clean slide that has been care fully wiped off with clean lens paper. Gently spread the diluted bacterial suspension over a 3cm area using the inoculating needle. 3. Let the slide air dry for 15 minutes 4. Cover the dry smear with solution A (the mordant) for 4 minutes 5. Rinse thoroughly with distilled water 6. Place a piece of paper toweling on the smear and soak it with solution B (the stain). Heat the slide in a boiling water bath for 5 minutes in an exhaust hood with the fan on. Add more stain to keep the slide from drying out. 7. Remove the toweling and rinse off excess solution B with distilled water. Flood the slide with distilled water and allow it to sit for 1 minute while more silver nitrate residue floats to the surface. 8. Then, rinse gently with water once more and carefully shake excess water off the slide. 9. Allow the slide to air dry at room temperature 10. Examine the slide with the oil immersion objective. The best specimens will probably be seen at the edge of the smear where bacteria are less dense. 12 Procedure (Difco) 1. Draw a border around the clear portion of a frosted microscope slide with a wax pencil. 2. Place a drop of distilled water on the slide, approximately 1 cm from the frosted edge. 3. Gently touch a colony of the culture being tested with an inoculating loop and then lightly touch the drop of water without touching the slide. Do not mix. 4. Tilt the slide at a slight angle to allow the drop preparation to flow to the opposite end of the slide. 5. Let the slide air-dry at room temperature. Do not heat-fix. 6. Flood the slid with the contents of the Difco SportTest flagella stain ampule. 7. Allow the stain to remain on the slide for approximately 4 minutes. (Note: the staining time may need to be adjusted from 2 to 8 minutes depending on the age of the culture, the age of the stain, the temperature, and the depth of staining solution over the culture) 8. Carefully rinse the stain by adding water from a faucet or wash bottle to the slide while it remains on the staining rack. Do not tip slide before this is done. 9. After rinsing, gently tilt the slide to allow excess water to run off and let the slide air-dry at room temperature or place on a slide warmer. Examine the slide microscopically with the oil immersion objective. Begin examination at thinner areas of the preparation and move toward the center. Look for fields which contain several isolated bacteria, rather than fields which contain clumps of many bacteria. Bacteria and their flagella should stain purple 13Basic laboratory procedures and culture techniques Media, Sterilization and Disinfectants Media Preparation of culture media Re-hydrate powder according to manufacturer’s instructions. Before sterilization, ensure ingredients are completely dissolved, using heat if necessary. Avoid wastage by preparing only sufficient for either immediate use (allowing extra for mistakes) or use in the near future. Normally 3 allow 15-20 cm medium/ Petri dish. Dispense in volumes appropriate for sterilization in the autoclave/pressure cooker. Agar slopes are prepared in test tubes or Universal/McCartney bottles by allowing sterile molten cooled medium to solidify in a sloped position. Pouring a plate 1. Collect one bottle of sterile molten agar from the water bath. 2. Hold the bottle in the left hand; remove the lid with the little finger of the right hand. 3. Flame the neck of the bottle. 4. Lift the lid of the Petri dish slightly with the right hand and pour the sterile molten agar into the Petri dish and replace the lid. 5. Flame the neck of the bottle and replace the lid. 6. Gently rotate the dish to ensure that the medium covers the plate evenly. 7. Allow the plate to solidify. 8. Seal and incubate the plate in an inverted position. The base of the plate must be covered, agar must not touch the lid of the plate and the surface must be smooth with no bubbles. Storage of media Store stocks of prepared media at room temperature away from direct sunlight; a cupboard is ideal but an open shelf is satisfactory. Media in vessels closed by cotton wool plugs that are stored for future use will be subject to evaporation at room temperature; avoid wastage by using screw cap bottles. Re-melt stored agar media in boiling water bath, pressure cooker or microwave oven. Sterile agar plates can be pre-poured and stored in well-sealed plastic bags (media-containing base uppermost to avoid heavy condensation on lid). Sterilization vs. Disinfection Sterilization means the complete destruction of all the micro-organisms including spores, from an object or environment. It is usually achieved by heat or filtration but chemicals or radiation can be used. Disinfection is the destruction, inhibition or removal of microbes that may cause disease or other problems e.g. spoilage. It is usually achieved by the use of chemicals. Sterilization Use of the autoclave The principle of sterilization in an autoclave is that steam under pressure is used to produce a temperature of 121ºC which if held for 15 minutes all micro-organisms including bacterial endospores will be destroyed. 14Sterilization of equipment and materials Wire loop Heat to redness in Bunsen burner flame. Empty glassware and glass (not plastic) pipettes and Petri dishes Either, hot air oven, wrapped in either grease proof paper or aluminum and held at 160ºC for 2 hours, allowing additional time for items to come to temperature (and cool down). Note: plastic Petri dishes are supplied in already sterilized packs; packs of sterile plastic pipettes are also available but cost may be a consideration. Culture media and solutions - Autoclave/pressure cooker. Glass spreaders and metal forceps - Flaming in alcohol (70% industrial methylated spirit). Disinfectants Choice, preparation and use of disinfectants Specific disinfectants at specified working strengths are used for specific purposes. Commonly available disinfectants Hypochlorite (sodium chlorate I) used in discard pots for pipettes and slides At 2500 ppm (0.25%, v/v) available chlorine Ethanol 70% (v/v) industrial methylated spirit When preparing working strength solutions from stock and dealing with powder form, wear eye protection and gloves to avoid irritant or harmful effects. Disinfectants for use at working strength should be freshly prepared from full strength stock or powder form. Use working strength hypochlorite on day of preparation. Inoculation and other aseptic procedures Essential points There are several essential precautions that must be taken during inoculation procedures to control the opportunities for the contamination of cultures, people or the environment. - Operations must not be started until all requirements are within immediate reach and must be completed as quickly as possible. - Vessels must be open for the minimum amount of time possible and while they are open all work must be done close to the Bunsen burner flame where air currents are drawn upwards. - On being opened, the neck of a test tube or bottle must be immediately warmed by flaming so that any air movement is outwards and the vessel held as near as possible to the horizontal. - During manipulations involving a Petri dish, exposure of the sterile inner surfaces to contamination from the air must be limited to the absolute minimum. - The parts of sterile pipettes that will be put into cultures or sterile vessels must not be touched or allowed to come in contact with other non-sterile surfaces, e.g. clothing, the surface of the working area, outside of test tubes/bottles. Using a wire loop Wire loops are sterilized using red heat in a Bunsen flame before and after use. They must be heated to red hot to make sure that any contaminating bacterial spores are destroyed. The handle of the wire loop is held close to the top, as you would a pen, at an angle that is almost vertical. This leaves the little finger free to take hold of the cotton wool plug/ screw cap of a test tube/bottle. Flaming procedure 15The flaming procedure is designed to heat the end of the loop gradually because after use it will contain culture, which may “splutter” on rapid heating with the possibility of releasing small particles of culture and aerosol formation. 1. Position the handle end of the wire in the light blue cone of the flame. This is the cool area of the flame. 2. Draw the rest of the wire upwards slowly up into the hottest region of the flame, (immediately above the light blue cone). 3. Hold there until it is red hot. 4. Ensure the full length of the wire receives adequate heating. 5. Allow to cool then use immediately. 6. Do not put the loop down or wave it around. 7. Re-sterilize the loop immediately after use. If a loop does not hold any liquid the loop has not made a complete circle. To correct the problem, first ensure that the loop has been sterilized and then reshape the loop with forceps. Do not use your fingers because of the possibility of puncturing the skin. Using a pipette Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media and sterile solutions. 1. Remove the pipette from its container/ wrapper by the end that contains a cotton wool plug, taking care to touch no more than the amount necessary to take a firm hold. 2. Fit the teat. 3. Hold the pipette barrel as you would a pen but do not grasp the teat. The little finger is left free to take hold of the cotton wool plug/lid of a test tube/bottle and the thumb to control the teat. 4. Depress the teat cautiously and take up an amount of fluid that is adequate for the amount required but does not reach and wet the cotton wool plug. 5. Return any excess gently if a measured volume is required. The pipette tip must remain beneath the liquid surface while taking up liquid to avoid the introduction of air bubbles which may cause “spitting” and, consequently, aerosol formation when liquid is expelled. 6. Immediately put the now contaminated pipette into a nearby discard pot of disinfectant. The teat must not be removed until the pipette is within the discard pot otherwise drops of culture will contaminate the working surface. A leaking pipette is caused by either a faulty or ill-fitting teat or fibres from the cotton wool plug between the teat and pipette. Flaming the neck of bottles and test tubes 1. Loosen the lid of the bottle so that it can be removed easily. 2. Lift the bottle/test tube with the left hand. 3. Remove the lid of the bottle/cotton wool plug with the little finger of the right hand. (Turn the bottle, not the lid.) 4. Do not put down the lid/cotton wool plug. 5. Flame the neck of the bottle/test tube by passing the neck forwards and back through a hot Bunsen flame. 6. Replace the lid on the bottle/cotton wool plug using the little finger. (Turn the bottle, not the lid.) 16Label tubes and bottles in a position that will not rub off during handling. Either marker pens or self-adhesive labels are suitable. Occasionally cotton wool plugs accidentally catch fire. Douse the flames by immediately covering with a dry cloth, not by blowing or soaking in water. Streak plate. The loop is used for preparing a streak plate. This involves the progressive dilution of an inoculum of bacteria or yeast over the surface of solidified agar medium in a Petri dish in such a way that colonies grow well separated from each other. The aim of the procedure is to obtain single isolated pure colonies. 1. Loosen the top of the bottle containing the inoculum. 2. Hold the loop in the right hand. 3. Flame the loop and allow to cool. 4. Lift the bottle/test tube containing the inoculum with the left hand. 5. Remove the lid/cotton wool plug of the bottle/test tube with the little finger of the left hand. 6. Flame the neck of the bottle/test tube. 7. Insert the loop into the culture broth and withdraw. At all times, hold the loop as still as possible. 8. Flame neck of the bottle/test tube. 9. Replace the lid/cotton wool plug on the bottle/test tube using the little finger. Place bottle/test tube on bench. 10. Partially lift the lid of the Petri dish containing the solid medium. 11. Hold the charged loop parallel with the surface of the agar; smear the inoculum backwards and forwards across a small area of the medium 12. Remove the loop and close the Petri dish. 13. Flame the loop and allow it to cool. Turn the dish through 90º anticlockwise. 14. With the cooled loop streak the plate from area A across the surface of the agar in three parallel lines. Make sure that a small amount of culture is carried over. 15. Remove the loop and close the Petri dish. 16. Flame the loop and allow to cool. Turn the dish through 90º anticlockwise again and streak from B across the surface of the agar in three parallel lines. 17. Remove the loop and close the Petri dish. 18. Flame the loop and allow to cool. Turn the dish through 90º anticlockwise and streak loop across the surface of the agar from C into the centre of the plate 19. Remove the loop and close the Petri dish. Flame the loop. 20. Seal and incubate the plate in an inverted position. Label the half of the dish that contains medium; use abbreviations and keep them to the edge of the plate so as not to interfere with the later observation of colonies. The same applies to the pour and spread plates described below. Either marker pens or self-adhesive labels are suitable. There are two approaches to making a streak plate: (1) with the base (containing medium) placed on the working surface, lift the lid vertically (i.e. still covering the base) the least amount that will allow access of the loop; (2) with the lid placed on the working surface, lift out the base, invert it and inoculate the upwards - facing agar surface. Pour plate A pour plate is one in which a small amount of inoculum from broth culture is added by pipette to a molten, cooled agar medium in a test tube or bottle, distributed evenly throughout the medium, 17thoroughly mixed and then poured into a Petri dish to solidify. Pour plates allow micro-organisms to grow both on the surface and within the medium. Most of the colonies grow within the medium and are small in size; the few that grow on the surface are of the same size and appearance as those on a streak plate. If the dilution and volume of the inoculum, usually 1 cm³, are known, the viable count of the sample i.e. the number of bacteria or clumps of bacteria, per cm³ can be determined. Pouring the pour plate 1. Roll the bottle gently between the hands to mix the culture and the medium thoroughly. Avoid making air bubbles. 2. Hold the bottle in the left hand; remove the lid with the little finger of the right hand. 3. Flame the neck of the bottle. 4. Lift the lid of the Petri dish slightly with the right hand and pour the mixture into the Petri dish and replace the lid. 5. Flame the neck of the bottle and replace the lid. 6. Gently rotate the dish to ensure that the medium covers the plate evenly. 7. Allow the plate to solidify. 8. Seal and incubate the plate in an inverted position. (The base of the plate must be covered, agar must not touch the lid of the plate and the surface must be smooth with no bubbles). Pouring the inoculated medium If pipettes are not available then a wire loop can be used. Several loopfuls of culture must be added to the cooled molten nutrient agar to ensure that there is enough inoculum present for growth. Using a spreader Sterile spreaders are used to distribute inoculum over the surface of already prepared agar plates. Wrapped glass spreaders may be sterilized in a hot air oven. They can also be sterilized by flaming with alcohol. It is advisable to use agar plates that have a well-dried surface so that the inoculum dries quickly. Dry the surface of agar plates by either incubating the plates for several hours, e.g. overnight, beforehand or put them in a hot air oven (ca 55-60ºC) for 30-60 minutes with the two halves separated and the inner surfaces directed downwards. Sterilization using alcohol 1. Dip the lower end of the spreader into a small volume of 70% alcohol contained in a vessel with a lid (either a screw cap or aluminium foil). 2. Pass quickly through a Bunsen burner flame to ignite the alcohol; the alcohol will burn and sterilize the glass. 3. Remove the spreader from the flame and allow the alcohol to burn off. 4. Do not put the spreader down on the bench. Flaming a glass spreader Ensure that the spreader is pointing downwards when and after igniting the alcohol to avoid burning yourself. Keep the alcohol beaker away from the Bunsen flame. 18 Spread plate Spread plates, also known as lawn plates, should result in a culture spread evenly over the surface of the growth medium. This means that they can be used to test the sensitivity of bacteria to many antimicrobial substances, for example mouthwashes, garlic, disinfectants and antibiotics. The spread plate can be used for quantitative work (colony counts) if the inoculum is a measured 3 volume, usually 0.1 cm , of each of a dilution series, delivered by pipette. 1. Loosen the lid of the bottle containing the broth culture. 2. Hold a sterile pipette in the right hand and the bottle/test tube containing the broth culture in the left. 3. Remove the lid/plug of the bottle/test tube with the little finger of the right hand and flame the neck. 4. With the pipette, remove a small amount of broth. 5. Flame the neck of the bottle/test tube and replace the lid/plug. 6. With the left hand, partially lift the lid of the Petri dish containing the solid nutrient medium. 7. Place a few drops of culture onto the surface about 0.1 cm3 (ca 5 drops, enough to cover a 5 pence piece). 8. Replace the lid of the Petri dish. 9. Place the pipette in a discard jar. 10. Dip a glass spreader into alcohol, flame and allow the alcohol to burn off. 11. Lift the lid of the Petri dish to allow entry of spreader. 12. Place the spreader on the surface of the inoculated agar and, rotating the dish with the left hand move the spreader in a top-to-bottom or a side-to-side motion to spread the inoculum over the surface of the agar. Make sure the entire agar surface is covered. This operation must be carried out quickly to minimize the risk of contamination. 13. Replace the lid of the Petri dish. 14. Flame spreader using alcohol. 15. Let the inoculum dry. 16. Seal and incubate the plate in the inverted position. HINT Consider the calibrated drop method for colony counts of pure cultures of bacteria and yeast as a more economical method than the pour plate and spread plate. The procedure is as for the spread plate but fewer plates are needed because: (1) the inoculum is delivered as drops from a dropping pipette that is calibrated (by external diameter of the tip) to deliver drops of measured volume e.g. 0.02 cm³; (2) many drops (six or more) can be put on one plate. The method is not suitable for use with cultures that produce spreading growth including mixed cultures in many natural samples such as soil although yoghurt and cheese are among the exceptions. Incubation The lid and base of an agar plate should be taped together with 2-4 short strips of adhesive tape as a protection from accidental (or unauthorized) opening during incubation. (Although tape is the preferred method Parafilm could be used as an alternative for sealing the plates.) Agar plates must be incubated with the medium-containing half (base) of the Petri dish uppermost otherwise condensation will occur on the lid and drip onto the culture. This might cause colonies to spread into each other and risk the spillage of the contaminated liquid. 19

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