This document summarizes research on mass producing the beneficial nematode Heterorhabditis bacteriophora using solid state fermentation technology. The objectives are to upscale the surface area of solid agar media to increase nematode production. Once harvested, the nematodes will be sanitized, packaged, and stored for agricultural use. The research procedures involve isolating the symbiotic bacteria Photorhabdus luminescens, sanitizing the nematodes, inoculating various surface areas of solid media, harvesting the nematodes after growth, and counting the nematode yields which increased 16-25 times over smaller surface areas. The conclusion is that using natural media can provide an easy way for farmers to grow large quantities of nemat

1. Mass Production of Heterorhabditis bacteriophora
Using Solid State Fermentation Technology
Mary Tess Johnson
The University of North Carolina at
Pembroke, NC 28372
Biotechnology Research and Training
Center

2. Project Background
 The focus of our research is to mass produce,
on solid media, the beneficial
nematode, Heterorhabditis bacteriophora
that in essence will create a cost effective
way to redistribute these nematodes for
agricultural purposes.
 Photorhabdus luminescens is a biphasic,
Gram-negative, bioluminescent bacteria that
maintains a very mutualistic relationship
with the nematodes
Photorhabdus luminescens H. bacteriophora

3. Research Objective
 The purpose of growing these
nematodes is to each time upscale the
surface area of a solid agar media to
also increase the amount of nematodes
produced.
 Once harvested from the media, these
nematodes are sanitized, packaged and
then stored for further use.

4. Variables
How to Produce P. luminescens?
 Galleria mellonella is a insect considered
to be a model host used for studies of
entomoparasitic nematodes (EPNs)
 If the larva is infected, the carcass should
turn to a brownish-red color, reflecting the
presence of P. luminescens.
 Once infection is verified, P. luminescens
is extracted from the intestinal tract of the
Galleria to produce and grow multiple
cultures.

5. Variables
What are the life stages of
Heterorhabditis bacteriophora?
Developmental Stages: (J1, J2, J3
(IJ), J4)
Once endotokia has been
verified, harvesting of nematodes
can begin.
J4
Egg J1 Endotokia

6. Materials
TotalMediaVolume 2XNutrientBroth 2%Agar 1%Oil pHLevel
Small Tray(400mL) 6.4g 8g 4mL 7.5
MediumTray(500mL) 8.0g 10g 5mL 7.5
LargeTray(600mL) 9.6g 12g 6mL 7.5
CookieSheet(800mL) 12.8g 16g 8mL 7.5
Total Media Volume Total Surface Area P.lum Inoculated
Petri plates (30 mL) 56 cm² 30 µL
Small Tray (400 mL) 400 cm² 200 µl
Medium Tray (500 mL) 490 cm² 250 µL
Large Tray (600 mL) 742.5 cm² 400 µL
Cookie Sheet (800 mL) 1218 cm² 600 µL
Media Concentrations used
throughout experiment.
Surface area and amount of P.lum
added to solid media

7. Procedures
 Isolating P. luminescens: Transferred and isolated pure cultures to be used
during inoculation.
 Sanitization of H. bacteriophora: After approximately ten cycles of sanitation
using the centrifuge at 500 RPMs for five minutes, nematodes are sanitized with
sterile water and decanted to reduce volume to a desired amount of 20 µl
 Preparation of Solid Media: To create a baseline of growth, solid media of 2X
nutrient broth and 2% agar is added to large surface area trays.
 Inoculation of P. luminescens: To provide a optimal enviorment for
Heterorhabditis bacteriophora.
 Inoculation of sanitized H. bacterophora: Once nematodes have been sanitized
with a hymine treatment (to sterilize and remove any external bacteria) they are
added to the plates and grown.
 Harvesting, Counting and Packaging:. Once nematodes are sanitized, they are
added to solid media for redistribution.
Figure: (A) Solid media before nematode inoculation (B) After nematode growth, 7 days incubation (C) Nematode Harvesting
B C

8. Counting Nematodes
Example of How to Count Nematodes
11 Nematodes / 0.1 mL / 100X Dilution
1,100 / 0.1 mL
11,000 / 1 ml X 325 mL of harvested volume
3,757,000 / 325 mL / 8 Petri plates
446,875 / Per Plate / 56 cm²
= 7,979 cm²
≈ 8,000 cm²

9. Data/Observations
Significant increase in nematode growth of 16-25 times fold. Nematode
count is increased as solid media surface area is increased as well

10. Conclusion
Our goal is to use natural media products during nematode
growth to provide a easy and convenient way for agriculturalist to
also grow nematodes at a large scale on solid media.
Benefits of this Research Include:
 Withdraw from traditionally relied on insecticides
 High and more reliable efficacy with greater understanding of
products
 Desire for more environmentally sensitive growing.

11. Any Questions?