Arbuscular Mycorrhizal Fungi (AMF) Most Probable Number (MPN) experiment SOP-GH05/V01. Approval of the SOP. Refusal. Title. Code.

Transcription

1 Code Arbuscular Mycorrhizal Fungi (AMF) Most Probable Number (MPN) experiment SOP-GH05/V01 Pages 7 Last validated version (if any): Changes or modifications made: SOP approved by (handwritten): Signature SOP approved by (handwritten): Signature Approval of the SOP Date Date Refusal Reason for refusal: Signature Document refused by (handwritten): Date SOP-GH05/V01-28 th, February Page 1 of 7

2 Table of Contents I. Objective... 3 II. Definitions... 3 III. Abbreviations... 3 IV. Procedures... 3 A. Materials ) Plant material ) Soils... 3 B. Description of analysis... 4 C. Safety... 6 D. Quality control management... 6 E. Waste and decontamination... 7 F. Cleaning... 7 V. Bibliography... 7 SOP-GH05/V01-28 th, February Page 2 of 7

3 I. Objective Method for used for enumeration of AMF viable infective propagules in the soil. The infective propagules include spores, hyphae and infected root fragments (Daniels et al., 1981, Porter 1979 and Wilson and Trinick, 1982). II. Definitions Contaminated by microorganisms: Every instrument which contains or has been in contact with microorganisms and cannot be sterilized by the flame of a Bunsen burner is considered as contaminated. Good Laboratory Practices (GLP): The Principles of Good Laboratory Practices (GLP) have been developed to promote the quality and validity of results and of the analysis conducted in a laboratory. It is a managerial concept covering the organization and the conditions under which laboratory studies are planned, performed, monitored, recorded and reported. Its principles also include the protection of man and the environment. III. Abbreviations - o C: degree Celsius - DW: dry weight - g: gram - GLP: Good Laboratory Practices - h: hour IV. Procedures A. Materials 1) Plant material Plant is chosen depending on its mycorrhizal potential and on the aim of the study. 2) Soils Soil is serially diluted in sand previously sterilized. SOP-GH05/V01-28 th, February Page 3 of 7

4 B. Description of analysis For 4-fold dilutions - 5 reps (4 possible) g (or 50 g) per chosen small pot/ bag. - Dilute your soil in sand: 4 0, 4-1, is undiluted soil. - Mix 65 g test soil with 185 g of sterile sand (autoclaved) = 250 g mix thoroughly to get 4-1. Place 35 g in each replicate (175 g), use 65 g for the next dilution and throw away 10 g. - Calculate dry weight of the test soil per fresh weight used to give the final propagules number in gram in dry soil (72 h at 70 o C). - Sow 5 pre-germinated seeds (surface sterilized) per pot. - After set period e.g. 4-6 weeks harvest the roots carefully, don t lose the fine roots if possible. - Stain the root systems (Phillips & Hayman, 1970) and assess presence or absence of infection under a stereoscope and check if necessary infection under the compound microscope (mount roots on a glass slide). EXAMPLE OF MPN CALCULATION If using 50 g per pot set out the table of results like this: Dilution Grams Soil Per Dilution From Test Soil Infected Reps Per Dilution Formula used: Log λ = ( x Log a) K λ = number of infective propagules = mean number of plants infected = number of total infected reps Number of reps per dilution = 20 = 4 5 SOP-GH05/V01-28 th, February Page 4 of 7

5 y = number of dilution (s) = s - = 9 4 = 5 a = dilution factor K = Constant found from tables of Fishcer & Yates (1970) for the 4-fold dilution series using (x) or (y) for the number of dilutions (s). N.B. Where the neither (x) or (y) has a value in the table use the (*) value indicated. In this example it is the * value = Log λ = s λ x z n λ = no. of infective propagules s λ = for 4-fold dilutions n = no. of reps per dilution z = tabulated values of probability 90% = z= % = z= this is the level that is normally used 99% =z= Therefore in this example, Log C.L. = x = Therefore log λ upper limit = 2.19 Log λ lower limit = 1.53 λ upper limit = 8.9 λ lower limit = 4.6 Summary So in this example the number of infective propagules (λ = 6.4) in 50 g F.W. Soils falls between 8.9 and 4.6 in 95% of cases [quote as dry weight (D.W) of soil]. Points to note - The last dilution must contain no infection. - The dilutions must be well mixed. SOP-GH05/V01-28 th, February Page 5 of 7

6 C. Safety Biological hazards: Manipulating microorganisms poses a risk not only to the one who is working, but also to other people in the lab and potentially to the environment in case of dissemination. The rules of safety have to be well understood and respected in order to avoid any contamination of the staff and/or environment (Read the Hygiene and Security rules in a laboratory document for more details). In case of accidental contamination (broken test tubes, suspension spilled on the bench, direct contact with microorganisms, wounds with contaminated material...), clean and disinfect properly before the activities can be restarted. Dispose contaminated waste as indicated in the section E. For more details, consult also the Hygiene and Safety rules in a laboratory document. Heavy items to carry: Care is taken to transport heavy items (such as tubes, pots, soils...) using the necessary tools (trolleys...) to ensure that one doesn t get hurt. When necessary, care is taken to adopt the good position when lifting and carrying the items since repetitive carrying of heavy items can lead to injuries. Sharps items: Blades, scalpels or any other sharp tool used in the greenhouse is handled properly, stored in a particular place and always returned in its protective packaging. D. Quality control management A specific protocol is established before a trial is set up. It includes the number of treatments, the details about the inoculants (origin, composition, rate of application...), soils (origin, fertility level, date of collection...), plant materials (legumes, crops reference, varieties...), and any other relevant information. Calculations, weights, records (of temperature, humidity...), observations, measurements, preparation of stock solutions (nutritive solutions for example) and any other relevant information are recorded in the lab book on a daily basis. The stock solutions are labelled with the date of reception, name or initials of the person who received it, the number of the container (x of n), date of opening, name or initial of the person who opened it. The reagents are labelled with the name of the contents, date of preparation, name or initials of the person who prepared them and any other relevant information. All samples are clearly identified and records are taken about the different steps of analysis (when they have been put in the oven, grinded, sent for analysis...). Equipment maintenance: All the equipments are regularly checked in regard to the specific specifications. The results are recorded and in case of repairs, the details about the intervention are recorded. Details about maintenance services and repairs are compiled in the Maintenance file, available in the office. In case of accident: Every accident must be reported to the lab manager and the staff if needed. Lab manager must put necessary measures in place to avoid the accident to occur again. For details on what to do in case of accident, read the Hygiene and Safety rules in a laboratory document. Use of the equipments: For specific equipment, a form has to be filled when used. This may include the date and time of use (start and end), the name of the user, the notification of any deviations or problems and any relevant information about the equipment. These forms are also used to estimate the needs and the frequency of the maintenance. Books are available near the specific equipments and should not be taken away. SOP-GH05/V01-28 th, February Page 6 of 7

7 E. Waste and decontamination Non contaminated waste is eliminated in the normal bin. Plant materials can be considered as non contaminated even once inoculated. Non contaminated glass waste (Pasteur pipette, broken glassware...) are put in a separate container labelled with the mention: Broken glass. Anything contaminated by microorganisms (i.e products, pipettes, glassware...) should be decontaminated before appropriate elimination/cleaning. Waste are put in a special autoclave bag and autoclaved for 20 min at 121 o C. The autoclave bag can then be disposed off as non contaminated waste. Re used material (glassware, small tools as sieves, pestles,...) are autoclaved and then cleaned as non contaminated items. Not reused glass instruments (pipettes, broken glassware...) are put in a beaker containing Sodium hypochlorite solution (commercial Jik) for decontamination before being eliminated as non contaminated glass waste. After harvesting, soils might need to be decontaminated before elimination. Solarization may be considered. F. Cleaning In case of contamination of the bench, floor, user,..., it has to be cleaned and disinfected before the work can be continued (cf. Hygiene and Safety rules in a laboratory document). Non contaminated or decontaminated items are cleaned with soap, and rinsed with water and eventually rinsed with distilled water. Equipments are regularly cleaned to remove dust, samples or other waste. V. Bibliography Daniels B.A., McCool P.M. and J. Menge (1981). Comparative inoculum potential of spores of six vesicular-arbuscular mycorrhizal fingi. New Phytologist 89: Porter, W.M (1979). The Most Probable Number method for enumerating infective propagules of Vesicular Arbuscular Mycorrhizal Fungi in soil. Australian Journal of Soil Research 17: Wilson, JM and Trinick MJ (1982). Factors affecting the estimation of numbers of infective propagules of vesicular-arbuscular mycorrhizal fungi by the Most Probable Number method. Australian Journal of Soil Research 21: SOP-GH05/V01-28 th, February Page 7 of 7