QUALITY CONTROL OF LEGUME INOCULANTS WORKSHOP - PROCEDURES SECTION potx

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 Prepar

Project Number 013/06VIE

Replacing Fertiliser N with Rhizobial Inoculants for Legumes in Vietnam for Greater Farm Profitability and Environmental

An NCSI QMS ISO9001:2000 Certified Laboratory 9187-21

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

Ingredients set out before weighing

into a flask containing distilled water

• Glass flask or beaker to hold 500mL

• Spatula

• 250mL conical flasks with cotton wool

bungs or equivalent

• Autoclave indicator tape

• 0.5 g of yeast extract powder

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

Sterilization of Single Volum es

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

Below is an example of the variation in water loss that occurs as result of autoclaving 18.5mL water was

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%

Actual amount of diluent in McCartney (mL)

% water loss

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 N2 fixation

Jensen’s nitrate-free nutrient (modified) solution

• 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)

• Stock A-Z solution (Gibson) of minor elements

• Na 2 MoO 4 2H 2 O (sodium molybdate) 0.14

• Bromothymol blue pH indicator solution

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

• Glass stirring rod

• Autoclave indicator tape

• Autoclavable glass flask / beaker or container

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 KNO3

L-1 when testing soybeans, cowpeas, mung beans and 0.5 g KNO3 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

• 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

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

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

Notes:

The desired results of each of

these procedures, is that less of

the initial suspension is

distributed over each consecutive

streak, resulting in isolated single

colonies in the last 2 quadrants

Sub-culturing rhizobial cultures onto agar in Petri plates

Requirements

• Rhizobial culture to be examined

• Freshly prepared YMA and CRYMA plates

2 Keep the Petri plates inverted

3 Sterilise the inoculation loop by holding it in a burner flame, starting at the top of the wire and slowly moving down the loop, until the loop glows red Cool the loop by placing it on the surface of the agar

4 Take a loopful of the rhizobia suspension to be cultured, lift the agar plate with the other hand (leaving the lid face up on the work surface of the cabinet); streak the loopful of culture across the surface of the agar backwards and forwards several times, at the top

of the plate

5 Turn the loop over, and using the other side continue

to spread the rhizobia suspension over the second quadrant of the plate, at right angles to the initial streak Place the agar plate back into the lid Re-sterilise the inoculation loop

6 Pick up agar plate and touch the loop into the agar at the edge of the plate to cool it Beginning at the edge

of the second quadrant of the streak, make 4 single streaks into the 3rd quadrant of the plate

7 Turn the loop, turn the plate, and using the clean side

of the loop, barely crossing the edges of the previous 4 streaks, do a final zig-zag streak in the 4th quadrant of the plate and replace agar plate into the lid

Notes:

Gram stain of rhizobial culture

Requirements

• Crystal violet solution

• Iodine concentrated solution

• Dilute iodine (iodinated alcohol) solution

• Safranin solution

• Cleaned glass microscope slide

• Glass marker (wax pencil)

4 Using a burner, sterilise an inoculation loop

5 Collect a small loop of culture from the sample and evenly mix it in the small drop of sterile water on the slide

6 Allow the smear to air-dry then heat fix by quickly passing the slide through a burner flame a few times

To be carried out on a slide rack over a sink with access

to cold water

7 Cover the smear with a drop of crystal violet and leave

for 1 minute

8 Wash gently with cold water for 5 seconds

9 Cover the smear with one drop of concentrated iodine

solution and leave for 1 minute

10 Pour off concentrated iodine and cover smear with

iodinated alcohol and leave for 5 minutes

11 Wash gently with water for 5 seconds

Notes:

12 Counterstain the smear with one drop of Safranin and

leave for 3 minutes

13 Wash smear gently with water, allow to air-dry

Microscopic examination of Gram stain

1 Add a drop of emersion oil onto stained smear, and examine under a microscope at a magnification of x1000 using a 100x oil emersion lens

2 If slide shows any Gram +ve cells or Gram -ve cells

which are not bacilli, the culture is not a rhizobia

3 If slide shows Gram -ve bacilli alike in size and shape, the culture fits the criteria for rhizobia

N.B Time of application of each stain should be accurate,

as leaving any stain on for either too long or too short a time may affect the results

Serology (serological agglutination test)

Requirements

• Rack containing metal capped test tube

• Pipette calibrated to dispense aliquots equal to 16 drops

• Sterile plugged pipette tips

• Vortex mixer

• 3 serology tubes and rack

• Water bath at 100°C or similar apparatus

• Water proof marker pen

• Pure culture of strain to be tested

• Frozen antisera of strain to be tested at appropriate titre (1:20 dilution) Do not use concentrated antisera

2 Using a glass pipette and dispenser, dispense 3.5mL of 0.85% NaCl (saline) into the tube

3 Pre-sterilise the inoculation loop, by placing in a flame, and allow to cool before taking a loopful of pure culture and adding it to the tubes containing NaCl

4 Mix solution using the Vortex mixer and place tubes in

the water bath set at 100°C and boil for 30 minutes to inactivate flagella `H' antigens

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 tubes at room temperature (warm) overnight

8 After 12h, check suspension for cell agglutination

Notes:

Preparing and surface-sterilising seeds with sodium hypochlorite

Requirements

• Seeds - to be surface sterilised

• Pyrex conical flask and rubber bung

• Sink with access to running water

Using sodium hypochlorite

1 Place sufficient amount of seeds in a clean conical flask stoppered with a rubber bung

2 Add absolute alcohol to cover the seeds and wash by gentle agitation for a few seconds then immediately pour off the excess into the sink

3 Pour sufficient bleach (NaOCl) into flask to cover seeds and rotate the flask to wet all the internal area of the flask including the bung

4 Set stopwatch and leave seeds soaking for 4 minutes

5 Pour off the bleach carefully into sink with running water, keep the seeds trapped inside the flask

6 Using cold sterilised water, cover the seeds and wash carefully

7 Tip the water away using the bung to stop the seeds from being washed out of the flask Repeat the rinse procedure a total of 9 times

8 After the final 9th rinse, leave seeds soaking in sterile water until the seeds fully imbibe except for soybeans which are drained immediately

Pour off excess liquids between steps

Notes:

Inoculating YM broths with rhizobia

The following steps should be carried out in a Laminar flow, or a Class II safety cabinet

1 Carefully label side of flask and the paper cap Labels should have strain name, and date of inoculation and operators name clearly written

2 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

3 Aseptically unscrew the McCartney slope lid containing the original culture, pass mouth of bottle through the flame Cool the loop in the agar before touching the rhizobia culture

4 Take a loop-full of culture, and quickly pass mouth of bottle through the flame before replacing the lid

5 Ensuring that the inoculation loop does not come into contact with any surface, remove the bag and cotton wool bung from the YM broth, pass mouth of flask through the flame, and place the inoculation loop with the rhizobia into the broth and wash the cells off the loop

6 Flame the top of the flask before replacing the cotton wool bung (Care must be taken not to wet the bung)

7 Sterilise the inoculation loop

8 Gently mix the broth, and place in an incubator shaker

at 26°C -28°C until broth becomes milky in colour

(3-7 days depending upon the rhizobia being cultured) Ensure the loop is flamed full

length