Nematode Paste in Horticultural Crops and their Management

Practical - 6

Exercise: Counting and Picking of Nematodes.
Objectives:

i. To estimate the number of the nematodes in a given suspension.
ii. To transfer nematodes from nematode suspension to other solution to be used for further investigations.

Materials Required:

• Counting dish, tally counter, nematode suspension, pipette, stereo zoom microscope, nematode pick, etc.

i. Nematode counting
a) Direct counting method: This method is used when the nematode population in the suspension is very low.

Procedure:

• Measure the volume of total nematode suspension with a measuring cylinder.
• Bubble the suspension with pipette and transfer five ml of suspension to the counting dish (Fig. 11) without disturbing the dish.
• Place the counting dish on the object stage of the stereo microscope and allow the nematodes to settle at the base of the dish without disturbing. This will take a few seconds.
• Adjust one corner most small square of the counting dish from one side under stereo microscope and record the nematode number using tally counter.
• Proceed to the next square and likewise count from all the squares having nematode suspension.
• Repeat the process with another five ml nematode suspension until nematodes are counted in whole suspension.
• Aggregate of all the counts will give nematode population in the quantity of soil washed.

b) Dilution method: This method is used when the nematode population in the suspension is very high.
Procedure:

• Take the nematode suspension in a beaker and dilute it by adding water so as to make the final volume of 100ml.
• Bubble the diluted suspension vigorously with a pipette.
• Transfer 5ml of nematode suspension to a counting dish and count the nematodes as suggested in the direct counting method.
• Repeat the process three times by taking another 5 ml of suspension after rigorous bubbling.
• Calculate the average number of nematodes per ml.
• Multiply with 100 to compute the total nematodes count in the sample. This is the nematode population in the given quantity of soil.

Fig. 11.Counting dish and Fig.12 Nematodes as seen under stereoscopic microscope

Precautions:

• Suspension should be bubbled properly before pipetting out the suspension for nematode counting.
• Nematodes should be allowed to settle at the bottom of counting dish for a few seconds before initiating counting.
• While counting, all the squares of the counting dish having any amount of suspension should be viewed.
• There should be no duplicity in viewing of the squares while counting.
• Counting from a sample should be repeated atleast three times and average be taken to calculate number of nematodes per sample size.

ii. Nematode picking
Procedure:

• Place the nematode suspension in counting dish/cavity block/ Petri-plate and focus the nematode under a low magnification of stereo zoom microscope.
• Gently give a slight jerk to the nematode intended to be picked with a pointed nematode pick (Fig. 12) so that the nematode starts floating in the water column.
• Refocus the floating nematode under microscope.
• Immediately bring the pick underneath the nematode and lift it with a jerk.
• Transfer it to a drop of water placed over a glass slideor to a few ml of clean water in the cavity block as per the requirement.

Fig. 13. Nematode pick

Precautions:

• Picking of the nematodes should be done at lower magnifications.
• The focus of microscope should not be lost until nematode is picked out of suspension.