Version date 1/2/2007 Project Number 013/06VIE Replacing Fertiliser N with Rhizobial Inoculants for Legumes in Vietnam for Greater Farm Profitability and Environmental Benefits QUALITY CONTROL OF LEGUME INOCULANTS WORKSHOP PROCEDURES SECTION Compiled by: E. Hartley G. Gemell J. Hartley Australian Legume Inoculants Research Unit An NCSI QMS ISO9001:2000 Certified Laboratory 9187-21 1 CONTENTS PAGE Preparation of sterile Yeast Mannitol Broth (YMB) 2 Autoclave times for liquids 3 Water loss through autoclaving 3 Growth assemblies for test host legumes Preparing Jensen’s nitrate-free nutrient solution (modified) 5 Plant tubes 6 Gemell Roll Tubes 7 Sub-culturing rhizobial cultures onto agar slopes in McCartney bottles 9 Sub-culturing rhizobial cultures onto agar in Petri plates 10 Gram stain of rhizobial culture 11 Serology (serological agglutination test) 13 Preparing and surface-sterilising seeds with sodium hypochlorite 15 Inoculating YM broths with rhizobia 16 Preparing Yeast Mannitol Agar (YMA) and Congo Red Yeast Mannitol Agar (CRYMA) 17 Plating out YM broth cultures 19 Measuring the moisture content of rhizobial inoculants 21 Counting rhizobia within an inoculant by Direct Plate count 24 Estimating rhizobial numbers using plant infection and Most Probable Number (MPN) 26 Isolating rhizobia from nodules 30 2 Ingredients set out before weighing into a flask containin g distilled wate r Weighing ingredients Preparation of sterile Yeast Mannitol Broth (YMB) Requirements Reagents for 500mL • Balance, accurate to 0.01g • Glass flask or beaker to hold 500mL • Disposable weigh cups • Spatula • 250mL conical flasks with cotton wool bungs or equivalent • Autoclavable paper bags • Autoclave indicator tape • 0.5 g of yeast extract powder • 0.1 g of MgSO 4 .7H 2 O • 0.25 g of K 2 HPO 4 • 5.0 g of Mannitol • 0.05 g of NaCl • 500mL of distilled water Method 1. Pour 500mL of distilled water into a clean glass flask or beaker 2. Using the balance, weigh out each of the reagents into separate disposable weigh cups 3. Add the reagents one at a time to the distilled water and mix thoroughly 4. Carefully pour approximately 50mL of broth into the 250mL conical flasks. Plug with cotton wool bungs 5. Place small paper bag over cotton wool bung and secure with autoclave tape 6. Place autoclave indicator tape on paper bag. 7. Autoclave at 121°C for 20 minutes 8. Wearing protective clothing and heat resistant gloves, remove flasks from the autoclave 9. Allow to cool before use 3 Sterilization of Single Volumes 0 1000 2000 3000 4000 5000 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Minutes at 121 degrees C Single volume (ml) from: Methods in Microbiology 3A p 289. Eds. Norris and Ribbons Autoclave times for liquids The graph below shows the autoclave time required when sterilising single volumes of liquids. Water loss through autoclaving It is important to consider the way that diluents are prepared in terms of accuracy of volumes. There are differences in accuracy between: • dispensing water in tubes or bottles before sterilisation by autoclave • dispensing sterile water into pre-sterilised tubes or bottles If water is dispensed into bottles before autoclaving the amount of water loss through autoclaving should be taken into account so that the final volume is correct. Dispensing sterile water into pre-sterilised bottles has been found to be more accurate than pre dispensed water into bottles before autoclaving. Below is an example of the variation in water loss that occurs as result of autoclaving. 18.5mL water was 4 measured gravimetrically into 15 McCartney bottles loosely capped, then sterilised at 121°C for 20 minutes. The range in water loss was between 2.43 and 7.78%. Sample No. Weight of empty McCartney's + lids before autoclaving (g) Weight of McCartney's + lids with18.5mL aliquot (g) after autoclaving Actual amount of diluent in McCartney (mL) % water loss 1 55.71 73.76 18.05 2.43 2 53.84 71.62 17.78 3.89 3 51.76 69.53 17.77 3.95 4 52.04 69.74 17.70 4.32 5 54.45 72.12 17.67 4.49 6 53.53 71.18 17.65 4.59 7 52.08 69.69 17.61 4.81 8 53.91 71.50 17.59 4.92 9 51.75 69.31 17.56 5.08 10 54.84 72.37 17.53 5.24 11 55.21 72.73 17.52 5.30 12 52.01 69.48 17.47 5.57 13 52.98 70.36 17.38 6.05 14 56.14 73.42 17.28 6.59 15 54.39 71.45 17.06 7.78 5 Growth assemblies for test host legumes Preparing Jensen’s nitrate-free nutrient solution (modified) A diverse range of legumes of different shapes and sizes need to be grown in the laboratory. When growing legumes under controlled environmental conditions such as a glasshouse or growth cabinet the plant nutrient solution must contain all the necessary elements for growth. Jensen's medium provides all nutrients and trace elements (with the exception of N) necessary for the growth of most legumes. Growing legume plants using N-free nutrients will encourage nodule formation and N 2 fixation. Jensen’s nitrate-free nutrient (modified) solution Requirements Reagents • Electronic balance (accurate to 0.01 g) • Disposable weigh cups • Spatula • Autoclavable glass flask • Test tubes • Bromothymol blue pH indicator • Glass pipette and dispenser • Heat resistant gloves • Glass stirring rod • CaHPO 4 (calcium hydrogen orthophosphate) • K 2 HPO 4 (di-potassium hydrogen orthophosphate) • MgSO 4 .7H 2 O (magnesium sulphate) • NaCl (sodium chloride) • FeCl 3 (ferric chloride) • 1M NaOH (sodium hydroxide) • Deionised water • Stock A-Z solution (Gibson) of minor elements made from: g L -1 • H 3 BO 3 (boric acid) 2.86 • MnSO 4 .4H 2 O (magnesium sulphate) 2.03 • ZnSO 4 (zinc sulphate) 0.22 • CuSO 4 .5H 2 O (cupric sulphate) 0.08 • Na 2 MoO 4 2H 2 O (sodium molybdate) 0.14 • Bromothymol blue pH indicator solution 6 Notes: Method 1. Using an electronic balance weigh out: • 1.0g of CaHPO 4 • 0.2g of K 2 HPO 4 • 0.2g of MgSO 4 .7H 2 O • 0.2g of NaCl • 0.1g of FeCl 3 2. Using a measuring cylinder, measure out 1L deionised water into heat resistant glass flask (or stainless steel bucket if preparing larger volumes) 3. Add all chemicals to water one at a time in the above order and mix thoroughly after each addition. 4. Add 1mL of A-Z stock solution 5. Cover flask with aluminium foil and autoclave at 121 °C for 20 minutes (or for the recommended length of time if a greater volume is being prepared). 6. Wearing protective clothing and heat resistant gloves remove medium from autoclave. 7. Adjust the pH. to 6.8-7.0. Plant tubes This is an enclosed aseptic growth assembly. Requirements • Electronic balance • Weigh cups • Spatula • Test tubes and foam or non absorbent cotton wool bungs (either 18 mm or 25 mm diameter depending on legume being grown) • Glass stirring rod • Autoclave indicator tape • Autoclavable glass flask / beaker or container • Agar dispenser • Wire basket to hold tubes • Agar • Jensen's nitrate-free plant nutrient solution 7 Notes: Method 1. Weigh out 12 g of agar 2. Add the agar to 1L of Jensen's nitrate-free plant nutrient solution in a container, and stir thoroughly. 3. Place solution into autoclave for the required amount of time to melt the agar. 4. Carefully remove the molten agar solution from the autoclave 5. Test and adjust the pH of the medium to pH 6.8-7.0. 6. Using a pre-calibrated dispenser, dispense the required volume of agar medium into each test tube. 7. Place sponge or cotton wool bung into each tube, and attach autoclave tape to one of the tube tops or the side of the container. 8. Place tubes in the autoclave at 121°C for 20 minutes. 9. Wearing protective clothing and heat resistant gloves remove tubes from autoclave. 10. Before agar has cooled, tip the basket of tubes at an angle (e.g. lean against a wall) so that the agar within the tubes sets on a slope. 11. Store in a cool place until required. NB: When preparing plant tubes to be used for nitrate controls , follow the above procedure and add 1.0 g KNO 3 L -1 when testing soybeans, cowpeas, mung beans and 0.5 g KNO 3 L -1 for clovers, medics and lucerne. Gemell roll tubes This is a modified hydroponic system for growing larger seeded legumes in an open aseptic growth assembly. Requirements • 2L plastic autoclavable jar with screw cap lid • 18 mm x 15 mm heat resistant test tubes • Non-bleached absorbent paper towelling • Measuring jug • Germinated seedlings • Forceps • Absolute alcohol • Gloves 8 • Jensen’s nitrate-free plant nutrient solution Method 1. Place the test tube at one end of the paper towel. 2. Wrap the paper towel around the tube leaving about 10 mm overlap at each end of the tube. 3. Tuck the over-lapping paper into the open end of the test tube, leaving the overlap at the closed end. 4. Place wrapped tube, closed end upwards into the autoclavable jar. 5. Pour 750-1000mL of plant nutrient solution into the jar, wetting the towel wrap. 6. Screw lid on loosely, lid and autoclave at 121°C for 60 minutes. 7. Using heat resistant gloves remove assemblies from autoclave and allow to cool. 8. Wearing gloves, and using pre-sterilised forceps, sow 2 germinating seedlings of the test host plant between the enclosed end of the tube and the overlapping paper towel. 9. Replace lids 10. Place assemblies in a controlled environment room at an appropriate temperature and remove the lids after 2 to 3 days. 9 Sub-culturing rhizobial cultures onto agar slopes in McCartney bottles Requirements • Authenticated source of the rhizobial strain • Freshly prepared agar slopes in McCartney bottles • Self adhesive labels • Marker pen • Spirit burner • Inoculation loop Method The following steps should be carried out in a Laminar flow, or a Class II safety cabinet. 1. Carefully write out 2 labels for each of the rhizobial strains being sub-cultured. Labels should have strain name, and date of sub-culture clearly written. 2. Place labels on the lids and side of the McCartney slopes, and place next to original source of rhizobia to be sub-cultured. 3. Sterilise the inoculation loop by holding it in a burner flame, starting at the top of the wire and slowly moving down to the loop until the loop glows red. 4. Aseptically unscrew the McCartney slope lid containing the original culture (re-check to make sure that the labels are correct), pass mouth of bottle through the flame. Cool the loop in the agar before touching the rhizobial culture. 5. Take a loop-full of culture, and quickly pass mouth of bottle through the flame before replacing the lid 6. Ensuring that the inoculation loop does not come into contact with any surface, remove the lid from the fresh McCartney slope, pass mouth of bottle through the flame, and place the inoculation loop with the rhizobia onto the surface of the agar slope. Spread the loop-full of rhizobia across the agar surface taking care not to touch the sides of the McCartney bottle. 7. Remove the inoculation loop, and pass the mouth of the bottle through the flame before replacing the lid. 8. Re-sterilise the inoculation loop. 9. Place McCartney slopes upright in a container, and incubate at 26°C for the required length of time. Ensure the loop is flamed full length Subculture onto YMA slope [...]... ranging from 1 0-7 to 1 0-1 0 Nodulation and growth of plants inoculated in a 10-fold serial dilution series, 3 tubes per dilution level MPN is calculated from this result Calculations Nodulated plants in the above example should be scored as follows: Dilution level Nodulation (+) or (-) Replications Nodulation Score at that dilution level I II III 1 0-7 + + + 3 1 0-8 + + + 3 1 0-9 + + - 2 1 0-1 0 - - - 0 25 The... Calculation of results (i) Calculate the % moisture content of the peat sample (wet weight basis): Remove the peel-off labels and calculate the moisture potential of the sample as shown in the following example Weight of empty Petri plate (a) = 49.6 g Weight of Petri plate + wet inoculant (b) = 68.5 g Weight of Petri plate + dry inoculant (c) = 58.0 g b-c x 100 = 68.5 - 58.0 x 100 b-a 68.5 - 49.6 = 10.5... content of the sample is 55% (ii)Calculate the moisture potential of the sample: Moisture Potential Curve of Carrier "a" 8 Calculate the percent moisture of the inoculant on a dry weight basis (the ratio of water to peat in the sample) 7 6 -Log Pa 5 4 3 2 1 0 20 40 60 80 100 120 140 160 180 200 220 240 260 % Moisture Moisture Potential Curve of Carrier "b" b-c x 100 = 68.5 - 58.0 x 100 c-a 58.0 - 49.6... nodulation of seedlings at the initial dilution levels and negative nodulation of seedlings at the highest dilution level (e.g positive nodulation at 1 0-7 and 1 0-8 levels and negative nodulation at 1 0-9 or 1 0-1 0 for most peat inoculants) 3 To inoculate the plants at the correct dilution levels to be counted, e.g 1 0-7 , take 3 x 0.1mL aliquots from the 1 0-6 and add to the 3 plant tubes labelled for 1 0-7 dilution... growth of other micro-organisms which may inhibit the growth and survival of the rhizobia On the other hand, if the moisture content of the inoculant is too low, the rhizobia become stressed resulting in death Therefore, checking the moisture potential of inoculants is one of the key tests in quality evaluation Requirements • • • • • • • Electronic balance Glass Petri plates Marking pen Peel-off sticky... on Commercial Inoculants Measuring the moisture content of rhizobial inoculants One of the principal factors affecting survival of rhizobia in a legume inoculant is the moisture potential of the inoculant Basically, the inoculant requires an amount of moisture to compensate for losses during the expected shelf life High moisture content within a carrier has little effect on the survival of rhizobia,... amount of moisture required to achieve similar moisture potentials Counting rhizobia within an inoculant by Direct Plate count Set up the equipment as for “plate out YM broth procedures 1 Surface sterilise the corner of the packet and cut the corner off with a pair of sterilised scissors 2 Weigh 10g of inoculant into a labelled sterile plastic bag 3 Add by weight or measure an aliquot of 90mL of sterile... conjunction with, or instead of, direct plate counts where there are likely to be high numbers of other microorganisms in the sample, making identity and counting of the rhizobia impossible The plant infection method of counting depends on the ability of specific rhizobia to produce nodules on a chosen species of legume, and assumes that a single rhizobial cell added to a test-plant is sufficient to cause... a rack 3 Using water proof marker pen, label 3 serology tubes with strain names and the third tube labelled # as the control 4 Dispense 16 drops of the boiled suspension into the three serology tubes 5 Dispense 2 drops of a 1:20 dilution of antiserum of the nominated strain in the first 2 serology tubes 6 Dispense 2 drops of 0.85% saline into the 3rd serology tube labelled # (control) 7 Leave serology... yeast extract 0.2 g of MgSO4.7H2O 0.5 g of K2HPO4 0.1 g of NaCl 10.0 g of mannitol 15.0 g of agar If preparing YMA with added dye prepare • 10.0mL of congo red* 2 Measure out 1L of distilled water 3 Empty the 1L of distilled water into a 2L heat resistant glass flask Weighing congo red into Pour 10mL yeast powder a measuring cylinder ready for use 4 Add each of the above chemicals one at a time, mixing . with Rhizobial Inoculants for Legumes in Vietnam for Greater Farm Profitability and Environmental Benefits QUALITY CONTROL OF LEGUME INOCULANTS WORKSHOP PROCEDURES SECTION . tape • 0.5 g of yeast extract powder • 0.1 g of MgSO 4 .7H 2 O • 0.25 g of K 2 HPO 4 • 5.0 g of Mannitol • 0.05 g of NaCl • 500mL of distilled water Method 1. Pour 500mL of distilled. • 1.0 g of yeast extract • 0.2 g of MgSO 4 .7H 2 O • 0.5 g of K 2 HPO 4 • 0.1 g of NaCl • 10.0 g of mannitol • 15.0 g of agar If preparing YMA with added dye prepare • 10.0mL of congo