English for biotechnology 22019

NONG LAM UNIVERSITY HO CHI MINH CITY DEPARTMENT OF BIOTECHNOLOGY ENGLISH FOR BIOTECHNOLOGY 2019 MSc TON BAO LINH TOPIC LABORATORY SAFETY The laboratory environment can be a hazardous place to work Laboratory workers are exposed to numerous potential hazards including chemical, biological, physical and radioactive hazards, as well as musculoskeletal stresses Laboratory safety is governed by numerous local, state and federal regulations Over the years, OSHA has promulgated rules and published guidance to make laboratories increasingly safe for personnel Laboratory safety guidance document is designed to make employers aware of the OSHA standards as well as OSHA guidance that is available to protect workers from the diverse hazards encountered in laboratories The extent of detail on specific hazards provided in this document is dependent on the nature of each hazard and its importance in a laboratory setting In addition to information on OSHA standards and guidance that deal with laboratory hazards, appendices are provided with information Volcabularies OSHA = Occupational Safety and Health Administration n hazard (hăz′ərd): A possible source of danger n appendix (ə-pĕn′dĭks); pl appendices: A collection of supplementary material, usually at the end of a book Discussion Discuss in group and identify potential hazards in the laboratories where you are studying Reading and comprehension Read a guideline on health and safety induction of a university and answer the questions What are potential hazards in a biological laboratory? Can you bring your laptop into laboratories? Should you take off your labcoats when going to toilets? why following the laboratory guidline is important? what you should not in the laboratory? what should you when seeing chemical spillage? where can you find information relating to hazards at your workplace? When may lab coats be worn in office areas? a Never b when there is no obvious contamination c always d for important meetings Which of the following are examples of personal protective equipment (PPE)? a safety glasses b laboratory coat c gloves d safety shoes e jumper f MP3 player 10 How would you dispose of tissue used for drying your hand? a sharp bins b clinical waste bag c black bag waste d autoclave then yellow clinical waste bag 11 The chemical you want to use does not have a substance risk assessment available on HSDMS Should you … a Use it anyway b obtain safety data sheet from the supplier and write a substance risk assessment base on this information c Read the data sheet and start your experiment d Look at the hazard warning signs on the bottle and write a substance risk assessment based on these symbols General Principles (DO) Avoid transferring contamination from laboratory areas, for example by:    Taking care if bringing lab books, laptops, tablets etc into laboratories to ensure that they not get contaminated Washing your hands when exiting laboratories: you must remove your lab coat before washing your hands Not wearing laboratory coats or gloves in office areas, eating areas or toilets Ensure you have been trained to use equipment    so you are using it safely - so you don't get hurt so you are using it correctly - and get the correct results so you don't break it Training must be recorded in your training log and on the trained user list Ensure you label solutions, flasks, tissue cultures, etc with:     Your name Contents Date Hazards - attach appropriate hazard labels for long term storage Anything not correctly labelled may be disposed of with no warning Clean up spillages immediately To avoid slips Spillage of solids on balances should be cleared up immediately to avoid exposing other users to hazardous substances   General Principles-DO NOT NO Eating and Drinking in the laboratory unless a designated area for Food Consumption (eg: North Lab/Nutritional Sciences Dietetics Lab and Clinical Skills Centre, Food Processing Facility, Food Flavour laboratory) DO NOT remove laboratory coats from the laboratory areas They must not be worn or taken into offices, toilets or eating areas Mobile phones, personal stereos, mp3 players and i-pods etc must not be brought into or used in laboratory areas where there is a high risk of them becoming contaminated with any of the hazardous substances being handled in the laboratory (including from your hands or gloves) You must always be aware of what is going on and be able to hear the fire alarm so you must not play music loudly Hazards There are many different types of hazards in a laboratory Spend a minute thinking through which ones may apply to your work here - they probably won't all apply! Before you carry out any experimental work (this should have already have been done if you are following established procedures in your research group) you need to consider what the hazards are, whether these can be eliminated eg: by substituting a chemical with a less hazardous one that does the same thing, or actually not doing that part of the work Normally this is not possible (but it is occasionally) so you then need to consider how the residual risks are going to be minimised This may be an engineering control such as working in a fumehood or microbiological safety cabinet, or wearing personal protective equipment such as gloves or eye protection, or it may require much more consideration for some non standard activities When considering hazards, you need to consider the hazards around all activities; from preparing the samples, carrying out the experiments and disposing of waste at the end, and also how you might manage accidents eg: spillage of chemicals This must be formally captured in a procedure risk assessment before you start the work - more about this later! (Note: For many procedures this will already be in place from other researchers in your area -you must ensure that you have read this documentation and that the work you are carrying out is covered by the procedure risk assessment) Health and Safety Data Management System The Health and Safety Data Management System (HSDMS) is where most safety information written by school researchers relating to hazards is stored Hazard Information on Chemicals Before using a chemical first check on HSDMS to see whether a substance risk assessment is in place and valid (they expire after a period of time) This will inform you of the hazards associated with the chemical, safe handling and storage and first aid measures If there is not a substance risk assessment available, or it has expired, then you need to find the Safety Data Sheet (SDS) for the chemical The supplier of any chemical is legally obliged to provide this information and typically it is available from their website Information from the SDS is put into into a substance risk assessment (COSHH) template on HSDMS Chemicals have Hazards phrases (H-phrases) that define physical hazards (eg: H223: Flammable material, H261: In contact with water releases flammable gas), health hazards (eg: H330: Fatal if inhaled, H315: Causes skin irritation) and environmental hazards (eg: H401: Toxic to aquatic life) Chemicals also have Precautionary statements (eg: P403: Store in a well ventilated place, P232: Protect from moisture) which are added into the substance risk assessment Note that Hazard phrases and Precautionary statements have now replaced the risk phrases (R) and safety (S) phrases that were previously used but some older substance risk assessments may still contain them Other bits of information are gathered from the safety data sheet to complete the substance risk assessment Note: When you need to order a chemical a Substance Risk Assessment number (S-number) needs to be quoted on the order form Plant Sciences Laboratory Guidelines LABORATORY WORKERS ARE REQUIRED TO BE AWARE OF THEIR RESPONSIBILITIES This document aims to provide new laboratory members with concise information regarding Laboratory Safety and the general running of the ACGM Laboratory Suite in Plant Sciences Ensure that you know who your Lab Mentor is before you start! This person will work with you in the initial weeks to ensure you are able to work safely in the lab Please note that Technical Services Staff are Marlene Oldman & Zoe Phillips General Laboratory consumables Consumables are normally purchased by each group using their own budgets, though there are exceptions (eg pipette tips, some glassware, some plastics, microcentrifuge tubes, sterile water) that are provided centrally It is not acceptable to go and help yourself to items from another area without first asking permission from the senior person in that area Fridges, freezers and cold store ALL items placed in cold store of any sort must be labelled with your NAME (initials are not sufficient) and be correctly stored Space is allocated for each group – you may not use someone else’s space without their permission Please note that if a freezer etc breaks down, anything that is not correctly labelled will be disposed of without any warning Autoclaves Some laboratories have bench top autoclaves – you may not use these unless you have been trained (see the person in charge of the area) There is also a central autoclaving service which provides sterile items for general use, autoclaves waste as required and provides an autoclave service as required if you need sterile media etc These autoclaves may only be operated by trained users and are not available for general use Washing up Washing up is done by Technical Services staff - you should rinse out any glassware etc, put it in a bucket in your lab and then take to Technical Services (A44) for washing up Technical Services staff are not expected to collect your washing up from the lab They also are not expected to deal with any hazardous chemicals in your bottles etc – this is for you to deal with before you put your washing up in the buckets Pipettes These will be supplied by your research group as required It is your responsibility to look after them and to check at least once a month that they are accurate and working correctly Do not borrow anyone else’s pipettes without their permission Pipette tips General tips (10ul, 200ul & 1000ul) are provided ready racked & sterile in boxes Please not hoard the boxes on your bench – place boxes for re-filling into the red box in your lab The Alphalab tip boxes are for tips only and nothing else ie not for general storage If you require storage boxes, either buy them or ask Technical Service staff if there are any old tip boxes that you can use Laboratory Coats You will usually be issued with laboratory coats for your use – please see Technical Service staff You should mark them with your name and lab number (use permanent marker) and hang on a labelled peg in one of the lab coat rooms You should send them for laundry regularly Please note that lab coats should be of the ‘Howie’ type only! Laboratory coats must be worn (fastened up) at all times in laboratory areas Lab coats must not be worn outside laboratory areas Visitors must use the light blue lab coats hanging in the lab coat room – they must be returned there after use and not left elsewhere in the building Safety glasses, gloves and other PPE If required you will be issued with a pair of safety glasses which must be worn when carrying out some procedures e.g when working with liquid nitrogen (see risk assessments for advice) Prescription safety glasses are available if required, please see the Buildings Safety Officer Gloves should be worn as necessary and are provided by each research group Other PPE (Personal protective equipment) will be provided as required FOOTWEAR – Must be boots or shoes ensuring that feet are completely covered No sandals or flipflops! Legs should be covered by trousers or tights No shorts or skirts without tights! pH meters Most laboratories will have a pH meter Please note that unless the pH probe is labelled as such, it must not be used to pH solutions containing Tris as this will mean that the probe will have to be replaced The pH meter in Technical Services may also be used – this does NOT have a Tris electrode Anyone found using this meter to pH Tris will be asked for a project code so that the electrode can be replaced 10 Balances Again, these are in most laboratories You are responsible for cleaning up any spillages of chemicals onto the balance Spillages can be hazardous for the next user who will not know what the chemical is and the spill may also damage the balance if not cleaned up You must be trained in SAFE WEIGHING techniques if you intend to weigh hazardous substances! Disscussion Which tools and equipment are needed for plant tissue culture laboratory? Which tools and equipment are needed for microbiological laboratory? Which tools and equipment that a molecular biology laboratory should have? Hereunder is the subculturing procedure of microbes, match the steps with its appropriate figures a Label the tube to be inoculated with the name of the organism and your initials b Place the tubes in the palm of your hand, secure with your thumb, and separate to form a V c Flame the needle or loop until thte entire wire is red d With the sterile loop or needle in hand, uncap the tubes e Flame the necks of the tubes by rapidly passing them through the flame once f Slant-to-broth transfer: dislodge inoculum by slight agitation Broth-to-slant transfer: Following insertion to base of slant, withdraw the loop in a zigzag motion Slant- to-agar deeptransfer: insert the needle to the bottom of the tube and withdraw along the line of insertion g Flame the necks of the tubes by rapidly passing them through the flame h Recap the tubes i Reflame the loop or needle 18 Choose the appropriate description for these following tools Tools Erlenmeyer flasks Filtering flasks Rubber bulb Lab burners Mortar and pestle Graduated cylinders Beaker Description A are used to mix chemicals, dissolve into solutions, heat or cool solutions, and hold sand or water They typically have graduations, but they are not very accurate B are used for precise and accurate measurement Smaller sizes can be more accurate, but hold less volume, while larger sizes sacrifice accuracy for volume C like beakers, can be used to mix, dissolve into solutions, and heat or cool solutions In addition, they can be plugged with stoppers and used to catch vapor or condensed liquid Graduations are not very accurate D A …… is used to crush up solid chemicals into smaller pieces, or to grind solids into fine powder This makes dissolving solids into solutions much easier E are used to filter mixtures through a funnel and filter paper A tube near the top prevents unwanted pressure buildup The process can be accelerated with a vacuum pump F come in many different styles and usually run on alcohol, propane, or butane They are used to heat or boil solutions, burn or melt solid chemicals, or form glass tubing G are used in chemistry laboratories, by placing them on top of a glass or plastic tube It serves as a vacuum source for filling reagents through a pipette or pasteur pipette and also help control the flow of liquid from the dropping bottle 19 Units of Mesurement The International System of Units (from French Système International d’Unités and abbreviated SI) establishes the fundamental quantities measured in scientific work The SI system replaced the metric system about 1960 There are seven fundamental quantities, which are shown below followed in parenthesis by the standard unit and its abbreviation for each quantity Unit prefixes Power of Ten Term 10-12 10-9 Prefix Name picop nanon Prefix Symbol P N 10-6 10-3 micro milli Μ M 10-2 centi C 10-1 deci D 10+3 kilo K 10+6 mega M 10+9 10+12 giga tera G T length (meter, m) electric current (ampere, A), mass (kilogram, kg) amount of substance (mole, mol), time (second, s) luminous intensity (candela, cd) temperature (kelvin, K) Source: Kirksey, H.G 2007 Science Curriculum Inc., Lakewood, Colorado Fill in the blanks with the appropriate words Long Weight Minute Times Diameter length Bacillus subtilis cells are typically rod-shaped, and are about 4-10 micrometers (μm) _ and 0.25–1.0 μm in _ Eukaryotic cells are typically 10 _ the size of prokaryoticcells Animal cells range from 10 to 30 micrometers in _, while plant cells range from 10 and 100 micrometers in length Measuring the fresh of plants is tricky and should probably be saved as a final measure of growth at the end of the experiment Centrifuge cells for _ at 15,000 rpm at °C 20 TOPIC3 BASIC TECHNIQUES IN MICROORGANISM & ANIMAL CELL CULTURE Reading text Techniques for Isolation of Pure cultures In nature, microbial populations not segregate themselves by species but exist with a mixture of many other cell types In the laboratory, these populations can be separated into pure cultures These cultures contain only one type of organism and are suitable for the study of their cultural, morphological, and biochemical properties In this experiment, you will first use one of the techniques designed to produce discrete colonies Colonies are individual, macroscopically visible masses of microbial growth on a solid medium surface, each representing the multiplication of a single organism Once you have obtained these discrete colonies, you will make an aseptic transfer onto nutrient agar slants for the isolation of pure cultures Isolation of discrete colonies from a mixed culture Purpose To perform the spread-plate and/or the streak-plate inoculation procedure for the separation of the cells of a mixed culture so that discrete colonies can be isolated Principle The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inoculum be reduced The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to effect a separation of the different species present The following are techniques that can be used to accomplish this necessary dilution: The streak-plate method is a rapid qualitative isolation method It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate Although many types of procedures are performed, the four-way, or quadrant, streak is described Refer to Figure 2.1, which schematically illustrates this procedure a Place a loopful of culture on the agar surface in Area Flame and cool the loop and drag it rapidly several times across the surface of Area b Reflame and cool the loop and turn the Petri dish 90o Then touch the loop to a corner of the culture in Area and drag it several times across the agar in Area The loop should never enter Area again 21 c Reflame and cool the loop and again turn the dish 90 o Streak Area in the same manner as Area d Without reflaming the loop, again turn the dish 90 o and then drag the culture from a corner of Area across Area 4, using a wider streak Don’t let the loop touch any of the previously streaked areas The reflaming of the loop at the points indicated is to effect the dilution of the culture so that fewer organisms are streaked in each area, resulting in the final desired separation The spread-plate technique requires that a previously diluted mixture of microorganisms be used During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent rod while the Petri dish is spun on a “lazy-Susan” turntable (Figure 2.2) The step-by-step procedure for this technique is as follows: a Place the bent glass rod into the beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion b With a sterile loop, place a loopful of Micococcus luteus culture in the center of the appropriately labeled nutrient agar plate that has been placed on the turntable Replace the cover 22 c Remove the glass rod from the beaker and pass it through the Bunsen burner flame, with the bent portion of the rod pointing downward to prevent the burning alcohol from running down your arm Allow the alcohol to burn off the rod completely Cool the rod for 10 to 15 seconds d Remove the Petri dish cover and spin the turntable e While the turntable is spinning, lightly touch the sterile bent rod to the surface of the agar and move it back and forth This will spread the culture over the agar surface f when the turntable comes to a stop, replace the cover Immerse the rod in alcohol and reflame g In the absence of a turntable, turn the Petri dish manually and spread the culture with the sterile bent glass rod The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette The diluted inoculum is then added to a molten agar medium in a Petri dish, mixed, and allowed to solidify The serial dilution and pour-plate procedures are outlined in Experiment 20 MATERIALS Cultures 24- to 48-hour nutrient broth cultures of a mixture of one part Serratia marcescens and three parts M luteus and mixture of one part Escherichia coli and ten parts M luteus For the spread-plate procedure, adjust the cultures to an optical density (O D.) of 0.1 at 600 µm Media Two trypticase soy agar plates per designated student group for each inoculation technique to be performed Equipment Bunsen burner, inoculating loop, turntable, 95% ethyl alcohol, 500 mL beaker, L-shaped bent glass rod, and glassware marking pen PROCEDURE Following the procedures previously described, prepare a spread-plate and/or streakplate inoculation of each test culture on an appropriately labeled plate 23 Incubate all plates in an inverted position for 48 to 72 hours at 25oC EXCERCISE Choose the appropriate equipment for each of culture technique: A spread plate B streak plate C pour-plate i inoculating loop ii bent glass rod iii needle iv pipette Answer the following questions a Why is reflaming the loops required between streaks in streak-plate method? b Does a culture need to be diluted prior to streak-plating? c What is the requirement for a microorganism mixture to be proceeded with spread-plate method? d Are there any differences in media preparation for the three mentioned techniques? Explain Reading text Animal cell culture Depending on their origin, animal cells grow either as an adherent monolayer or in suspension Adherent cells are anchorage-dependent and propagate as a monolayer attached to the cell culture vessel This attachment is essential for proliferation — many adherent cell cultures will cease proliferating once they become confluent (i.e., when they completely cover the surface of cell culture vessel), and some will die if they are left in this confluent state for too long Most cells derived from tissues are anchorage-dependent 24 Suspension cells can survive and proliferate without being attached to a substratum Hematopoietic cells (derived from blood, spleen, or bone marrow) as well as some transformed cell lines and cells derived from malignant tumors can be grown in suspension Primary cells, finite cultures, and continuous cell lines differ in their proliferative potential (see below) Different cell types vary greatly with respect to their growth behavior and nutritional requirements Optimization of cell culture conditions is necessary to ensure that cells are healthy and in optimal condition for downstream applications Primary cell cultures Primary cell cultures come from the outgrowth of migrating cells from a piece of tissue or from tissue that is disaggregated by enzymatic, chemical, or mechanical methods Primary cultures are formed from cells that survive the disaggregation process, attach to the cell culture vessel (or survive in suspension), and proliferate Primary cells are morphologically similar to the parent tissue These cultures are capable of only a limited number of cell divisions, after which they enter a non-proliferative state called senescence and eventually die out Adherent primary cells are particularly susceptible to contact inhibition, that is, they will stop growing when they have reached confluency At lower cell densities, however, the normal phenotype can be maintained Primary cell culture is generally more difficult than culture of continuous cell lines Primary cell cultures are sometimes preferred over continuous cell lines in experimental systems Primary cells are considered by many researchers to be more physiologically similar to in vivo cells In addition, cell lines cultured for extended periods of time can undergo phenotypic and genotypic changes that can lead to discrepancies when comparing results from different laboratories using the same cell line Furthermore, many cell types are not available as continuous cell lines Finite cell cultures Finite cell cultures are formed after the first subculturing (passaging) of a primary cell culture These cultures will proliferate for a limited number of cell divisions, after which they will senesce The proliferative potential of some human finite cell cultures can be extended by introduction of viral transforming genes (e.g., the SV40 transforming-antigen genes) The phenotype of these cultures is intermediate between finite cultures and continuous cultures The cells will proliferate for an extended time, but usually the culture will eventually cease dividing, similar to senescent primary cells Use of such cells is sometimes easier than use of primary cell cultures, especially for generation of stably transfected clones Continuous cell lines Finite cell cultures will eventually either die out or acquire a stable, heritable mutation that gives rise to a continuous cell line that is capable of unlimited proliferative potential This 25 alteration is commonly known as in vitro transformation or immortalization and frequently correlates with tumorigenicity Rodent primary cell cultures form continuous cell lines relatively easily, either spontaneously or following exposure to a mutagenic agent In contrast, human primary cell cultures rarely, if ever, become immortal in this way and require additional genetic manipulation to form a continuous cell line However, cell cultures derived from human tumors are often immortal Continuous cell lines are generally easier to work with than primary or finite cell cultures However, it should be remembered that these cells have undergone genetic alterations and their behavior in vitro may not represent the in vivo situation Essential protocols for animal cell culture Maintaining cell cultures Establishment and maintenance of animal cell cultures require standardized approaches for media preparation, feeding, and passaging (or subculturing) of the cells Cultures should be examined regularly to check for signs of contamination and to determine if the culture needs feeding or passaging The cell culture protocols below have been adapted from the following sources: Culture of Animal Cells; a Manual of Basic Technique (1), Current Protocols in Molecular Biology (4), and Cells: A Laboratory Manual (2) These protocols are examples of methods for general cell culture, and have not been rigorously validated and optimized by QIAGEN There are many alternative protocols in current use IMPORTANT: Potentially biohazardous materials (e.g., cells, culture medium, etc.) should be sterilized before disposal, and disposed of according to your institution’s guidelines Cell thawing Heat a water bath to 37°C, and warm the growth medium into which the cells will be plated Add prewarmed growth medium to an appropriately sized cell culture vessel Remove a vial of frozen cells from liquid nitrogen, and place in the water bath until thawed IMPORTANT: Wear protective goggles and gloves when thawing vials that have been stored in liquid nitrogen Vials may explode when removed from liquid nitrogen IMPORTANT: Proceed to step as soon as the cells have thawed Do not allow the cells to warm up before transferring them into growth medium Wash the outside of the vial with 70% ethanol or another suitable disinfectant 26 Slowly pipet the thawed cell suspension into the cell culture vessel containing prewarmed growth medium Swirl the vessel gently to mix the cells with the medium Note: Immediate removal of DMSO may sometimes be necessary, especially for suspension cells, primary cells, and sensitive cell types For such cell types, pipet the thawed cell suspension into a sterile centrifuge tube containing prewarmed medium, centrifuge at 200 x g for min, aspirate the supernatant, resuspend the cells in fresh growth medium, and then transfer to an appropriate cell culture vessel IMPORTANT: Thoroughly mix the cells in the cell culture vessel to ensure even distribution of the cells throughout the vessel Incubate cells overnight under their usual growth conditions The next day, replace the growth medium Cell counting using a hemocytometer It is often necessary to count cells, for example, when plating cells for transfection experiments One method for counting cells is to use a hemocytometer A hemocytometer contains chambers (see figure Counting cells using a hemocytometer) Each chamber is ruled into major squares (volume of 0.1 mm3 or x 10–4 ml each) Cell concentration is determined by counting the number of cells within a defined area of known depth (volume) This protocol is adapted from references 1, 2, and It should be noted that there are many other protocols also in use Clean the surface of the hemocytometer with 70% ethanol or another suitable disinfectant, taking care not to scratch the surface of the central area Dry with lens paper Clean the coverslip, wet the edges very slightly, lay it over the grooves and central area of the hemocytometer and gently press down It is important that the coverslip is properly attached to obtain the correct chamber depth The appearance of Newton’s rings (bright and dark rings caused by interference in the air between the coverslip and the glass surface of the hemocytometer) will confirm that the coverslip is attached properly Harvest the cells, either by trypsinization (adherent cell cultures; see Trypsinizing cells) or by centrifugation at 200 x gfor (suspension cell cultures) Resuspend the cells in an appropriate volume of prewarmed growth medium At least 106 cells/ml are required for accurate counting 27 Tip: It may be necessary to centrifuge cells and resuspend in a smaller volume to obtain the desired cell concentration for counting For adherent cells, it is important to produce a single-cell suspension after trypsinizing Cell clumping will make counting difficult and inaccurate Mix the cell suspension sample thoroughly Using a pipet, immediately transfer 20 µl to the edge of one side of the coverslip to fill one chamber of the hemocytometer Repeat for the second chamber The cell distribution should be homogeneous in both chambers The cell suspension is drawn under the coverslip and into the chamber by capillary action The cell suspension should just fill the chamber Blot off any surplus fluid without disturbing the sample underneath the coverslip Transfer the slide to the microscope, and view a large square ruled by lines using a 10x objective and 10x ocular Count the total number of cells in of the major squares Count cells that overlap the top and left border of squares but not those overlapping bottom and right borders This prevents counting overlapping cells twice If the cell density is too high, the cell suspension should be diluted, noting the dilution factor Repeat the counting for the second chamber to give a total of 10 squares Add the number of cells counted in all 10 squares together to give the number of cells in x 10–3 ml.Multiply by 1000 to give the number of cells/ml IMPORTANT: If the original cell suspension was diluted for counting, multiply by the dilution factor to obtain the number of cells/ml Clean the hemocytometer and coverslip by rinsing with 70% ethanol and then with distilled water Dry with lens paper 28 TOPIC PLANT TISSUE CULTURE A Introduction A whole plant can be regenerated from a small tissue or plant cells in a suitable culture medium under controlled environment The plantlets so produced are called tissue-culture raised plants These plantlets are a true copy of the mother plant and show characteristics identical to the mother plant For example, if the mother plant is a high yielding plant the plantlets will also be high yielding Many plant species are presently being propagated through tissue culture successfully This capacity of a single cell to grow into a complete plant is termed as Totipotency, which was first put forward by a German Botanist Haberlandt in 1902 Tissue culture is the propagation of plants wherein a part/tissue of the plant is placed in nutrient media that favors the production of shoots, roots following which they are hardened and transferred to soil Quality planting material of economically important species can be produced in a large scale/desired quantity through tissue culture Plant tissue culture can be initiated from almost any part of a plant, however, for micropropagation or direct shoot regeneration, meristemetic tissue such as shoot tip is ideal The physiological state of the plant does have an influence on its response to tissue culture The mother plant must be healthy and free from obvious signs of disease or pest The shoot tip explants being juvenile contain a higher proportion of actively dividing cells It is important to use quality mother plant stock to initiate cultures The cultural conditions required to initiate and sustain plant cells in culture, or to regenerate intact plants from cultured cells, are different for each plant species Each variety or clone of a species often have a particular set of cultural requirements B Stages of Tissue Culture Process Preparation of nutrient medium: A semi-solid medium is prepared in double distilled water containing macro-elements, micro-elements, amino acids, vitamins, iron source, carbon source like sucrose and phyto-hormones The medium is heated for dissolving the agar and 25 to 50 mL is dispensed into each wide mouth bottles The vessels containing culture media are then sealed and sterilized by autoclaving Establishment of aseptic culture: The starting material for the process is normally an actively growing shoot tip of axiliary or terminal bud or shoot tip of a plant The process of tissue culture starts from the selection of mother plants having the desired characteristics.Ex-plant preferably the meristematic tissue of the selected mother plant is isolated The excised tissue/explant is washed with water and then rinsed witha disinfectant such as savlon or detol solution followed by a sterile-water wash The tissue is then dipped in 10% bleach solution for ten minutes for disinfecting the plant tissue material, killing most of the fungal and bacterial organisms Sterilization process of explants depends on the plant species and types of explants 3.Inoculation: Inoculation is carried out under aseptic conditions In this process explants or micro shoots are transferred on to the sterilized nutrient medium 29 Development of plants in growth room: After the inoculation of the plant tissue, the bottles are sealed and transferredinto growth room to trigger developmental process under diffused light (fluorescent light of 1000-2000 lux) at 25 ± oC and 50 to 60% relative humidity Light and temperature requirements vary from species to species and sometimes during the various stages of developments.The cultures are observed daily for growth and any signs of infection/contamination Cultures, that not show good growth or infected, are discarded The healthy cultures grow into small shoot buds These are sub-cultured on the fresh medium after weeks The number of subcultures required is specific to the plant species, which are standardized The shoots generally develop after weeks After enough number of shoots is developedin each container (10 to 15), to a minimum height of cm they are transferred to another medium for initiating the process of rooting The constituent of rooting medium for each plant species are specific Roots are generally formed within to weeks Plants at this stage are delicate and require careful handling Hardening of micro plants: Due to very high humidity inside the culture vessel and artificial conditions ofdevelopment, the plantlets are tender and are therefore are not ready forcoping up with the filed conditions The plants removed from the sterilemedium are washed and are maintained under intermittent mist or arecovered with clean transparent plastic After 10 to 15 days under highhumidity, the plants are transferred to green house and maintained foranother to weeks They are then ready to be transferred to net house orthe field Normally, the tissue culture plants are sold either as ex-agar plantsor hardened plants from the green house Ex-agar plants: Depending on the parameters such as location/the site of planting, soil quality and the climatic conditions defined by the customer, the ex-agar plant for sale could be in vitro rooted plants or only the shoots When the tissue cultureplants are sold at this stage, the plants are washed in sterilized water toremove the agar medium The washed plants are sorted into to grades and packed in corrugated plastic boxes lined with sterilized tissue paper as per specifications of the Plant Quarantine Authority, Government of India for exports The number of plants per box depends on the customer’s requirement Depending on the final destination and the preference of the customer, the plants are treated with specific fungicides and antibiotics to avoid infection.The ex-agar plants are preferred for export or for destinations where hardening facility are available The plants after being removed from nutrientmedia should preferably be transplanted within 72 hours Hardened plants: The plants are transferred to net pots/ pro tray for acclimatization after they fully develop shoots and roots in the bottles The rooted plantlets are transferred to pots filled with suitable substrate and are watered This operation is carried out on an open bench These pots are then transferred to the green house for to weeks During this process, they are given fertilizers and treated like plantlets obtained by any other means of propagation After the plants are acclimatized fully, they are transferred to poly-bags At this stage the plants are completely hardened and are ready tobe planted in the field for cultivation Hardening units can be set up in sites away from the micropropagation unit 30 C Advantages of Micro-propagation Technology Micropropagation has several advantages over conventional methods of propagation such as: Rapid multiplication: Micro-propagation offers rapid multiplication of desired plant species Requirement of only limited number of explants: small pieces of plant (explants)/tissue can be used to produce a large number of plants in a relatively small space Uniform or true to type plants: Micropropagation provides a high degree of phenotypic/physical uniformity Since the production cycle takes place under controlled conditions, proper planning and scheduling based on the market demand is possible The resulting product has very high degree of uniformity compared with traditionally propagated plants Germplasm storage: Plants can be stored in vitro in a small space and less labour is required for maintenance of stock plants Disease free planting material: Plantlets produced by tissue culture are usually disease free.With proper diagnosis and treatments, elimination of fungus, bacteria and virus prior to large scale propagation is possible With the help of seroloical and molecular technique it is possible to index virus of mother plant/explant which is to beused for mass multiplication Growth manipulation: Nutrient levels, light, temperature and other factors can be more effectively controlled to manipulate the growth, multiplication and regeneration Round the year production: Micro-propagation is independent of season As micro propagation could be carried out throughout the year; production cycle can be scheduled to meet peak demands For species that have long generation time, low levels of seed production, or seeds that not readily germinate, rapid propagation is possible through tissue culture.The time required is much shortened, no need to wait for the whole life cycle of seed development Commercially propagated plants through micropropagation in India Mitigating Risks of commercial plant tissue culture: The utilization of plant tissue culture for commercial production is limited by two major risks e.g., spread of diseases especially those caused by viruses, and variations The movement of plants also involves accidental risk of introducing plant disease Pathogens that are often symptom less, such as viruses, pose a risk The risk of distribution of inferior micropropagated plants has posed a major threat to the ever-increasing agribusiness industry In order to prevent these risks, effective testing (indexing) procedures are required prior to bulking up culture for commercial propagation.Standardprocedure should be adopted such as: 31 • Carefully selection of mother plants • Ensuring establishment of virus free culture through indexing of 100 %explants • Proper package and practices to be adopted such as limited number of cycles of multiplication, grading of cultures as well as plants, insect, pest monitoring in hardening area D Need for Certification of tissue culture raised plants Micropropagation is effectively used for producing quality planting materialfree from disease Yet there is threat of inadvertent propagation of virus infected plants which will not only result in loss or poor performance of the crop but also spread of virus Further failure to used standard crop specific guidelines can lead to variations in the plants produced The most deleterious variants in tissue culture raised plants are those that affect yield through somaclonal variations and carry viruses and other pathogens which are difficult to diagnose This is an area of great concern and requires a well structured system to support the tissue culture industry to ensure virus-free quality planting material for commercial production With the objective of production and distribution of quality tissue culture planting materials Department of Biotechnology (DBT), Government of India has established National Certification System for Tissue Culture Raised Plants (NCS-TCP) For details about NCSTCP, please refer the manual on “National Certification System for Tissue Culture Raised Plants (NCSTCP): An Overview or log in to www.dbtncstcp.nic.in DBT is the Certificationagency for the purpose for certification of Tissue culture raised plants/propagules up to laboratory level and to regulate its genetic fidelity as authorized vide the Gazette of India Notification dated 10 th March 2006 of Ministry of Agriculture under section of the Seeds Act References Bhoite, H.A and Palshikar, G.S 2014 Plant Tissue Culture: A Review World Journal of Pharmaceutical Sciences 2(6): 565-572 QIAGEN Molecular Biology Methods - Animal Cell Culture Protocols & Applications https://www.qiagen.com/br/resources/molecular-biology-methods/animal-cell-culture/ 3.OSHA Laboratory Safety Guidance www.osha.gov The University of Nottingham Safety Inductions and Plant Sciences Laboratory Guidelines 32