Lab Experiment II. Effect of a common agricultural herbicide on honey bee gut bacteria (Medium Replicates)

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Cages in incubator.
Stained Lactobacillus Bacteria.

The effect of field realistic dosages[1] of a common agricultural herbicide on honey bee gut bacteria was measured. Six groups consisting of 300 to 500 bees in each group were fed sucrose solution with Roundup™ added at the following concentrations:

Group Field Realistic Dose[1] Glyphosate
1 none (control) 0.0 mg/l
2 half minimum 0.7 mg/l
3 minimum 1.4 mg/l
4 mid range 4.5 mg/l
5 maximum 7.6mg/l
6 twice maximum 15.2 mg/l

This is a follow up to Lab Experiment I. Effect of a common agricultural herbicide on honey bee gut bacteria (Small Replicates), using larger replicates in cages and a better incubator. In addition to the procedures used in the previous experiment, the bacteria that was cultured from the bee gut was identified as Lactobacillus.

This experiment was developed and performed at Rabun-Gap Nacoochee School as part of their Education and Bee Science program.


Field corn grown with glyphosate on Rabun Gap-Nacoochee School campus, Rabun Gap, GA, USA
Local beekeeper shakes bees into a cage.

The agricultural use of fungicides, herbicides and insecticides is increasing. Concerns have been raised that individually, or through synergistic effects, these compounds are causing Colony Collapse Disorder or otherwise contributing to colony loss. Concerns have been raised that glyphosate, contained in the popular herbicide Roundup®, is affecting animal and human health.


In addition to skills from Experiment I., the follow additional skills are developed:

  1. Counting CFUs
  2. Statistical analysis
  3. Staining and identifying the bacteria.

Equipment and Supplies

Analytical Balance Ohaus Model AP110S
Micro Pipette Eppendorf
Autoclave Tuttnauer 2540M
Vortex Mixer Baxter Scientific Products SIP Vortex Mixer Catalog S8223-1
Hotplate with Magnetic Stirrer Flinn Scientific Model AP1087
Incubator for cultures Blue M Electric Co. Model 100A
Incubator for bees Refrigerator
22 watt Heat Mat


Design Experiment

The same procedures developed in Lab Experiment I. Effect of a common agricultural herbicide on honey bee gut bacteria (Small Replicates)were used with the following changes:

  1. Cages were used instead of Solo cups to solve problems with pipette feeder and removing dead bees. See Cages for medium size replicates for details,
  2. A refrigerator was used for an incubator.
  3. Regular petri dishes instead of Y-Sectioned were used.

Obtain bees from hives

Approximately 1.5 lbs of bees were shaken from outsides frames of 3 hives into a .5 lb cage.

Transfer bees to cages

Transferring bees to cages.

  1. Number and weigh each empty cage and record the cage number and weight.
  2. Gas the bees with CO2
  3. Transfer knocked out bees to the empty cage.
  4. Weigh and record cage weight with bees

Prepare Feed

Prepare 5 concentrations of Roundup™ to be mixed with sugar syrup and fed to the bees.

Calculate Glyphosate Dilutions

The concentrations are calculated based on field realistic dosages of glyphosate from the study "Effects of field-realistic doses of glyphosate on honeybee appetitive behaviour" Lucila T. Herbert, Diego E. Vázquez, Andrés Arenas and Walter M. Farina*© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 3457-3464 doi:10.1242/jeb.109520

Prepare Glyphosate Dilutions

  1. Placed 10 mL of Roundup™ (18% Glyphosate, 0.73% Diquat) in 1 L graduated cylinder.
  2. Filled with water to the 1 L mark.

Mix a 1:1 (m/m) syrup of sucrose and water

Mix feed


Remove dead bees and count

Remove and count dead bees.

Prepare Nutrient

Prepare nutrient adiquats.
Prepare nutrient adiquats.

Using the Eppendorf Pipettes, put 500 uL in each tube.

Sample bacteria from bee's gut

Pull abdomen from thorax.

Prepare 18 samples (3 from each cage) from the gut of 3 bees.

  1. Autoclave aliquots.
  2. Prepare 18 aliquots by numbering them and filling with 500 micro L of nutrient.
  3. Gas bees to knock them out.
  4. Remove 9 bees from the cage. (There will be three replicates of 3 bees each.)
  5. Using forceps and a dissecting tool, pull abdomen from thorax.
  6. Remove the gut from 3 bees and place in aliquot.
  7. Smash the gut against the side of the aliquot.
  8. Close lid on the aliquot.
  9. Record the number of the cage and aliquot.
  10. Mix for 30 seconds on Vortex Mixer.

Prepare cultures

Pour petri dishes (plates)

Prepare agar.
  • 200 ml distilled H2O
  • 14 g Agar
  • 18 sterile disposable petri dishes
  1. Put Magnetic stirrer in 300 ml beaker.
  2. Add 200 ml distilled water.
  3. Place on hot plate.
  4. Set Heat to 10. Set Stir to 10.
  5. Slowly add agar.
  6. Allow to boil (3 to 5 minutes).
  7. Lower stir and Heat to 1 to 2 for another minute.

Using gloves to handle the beaker, pour the plates.

Dilute Samples

A serial dilution of the samples is carried out using standard bacteriological techinques based on sample trials and experience

Inoculate Plates

Incubate cultures

Count Culture forming units

Record data

Repeat steps

Identify Bacteria

Lactobacillus Bacteria.
Stained Lactobacillus Bacteria.

Use stain.


Nutrient became contaminated.


  1. All the bees in Group 6 fed twice the realistic field dose died after 10 days.
  2. Bees in the groups fed higher concentrations were stumbling around like they were drunk.


The raw data is available on Google Drive:

A LibreOffice ods spreadsheet with the graphs is available for download: Glyphosate Medium Replicates

Mortality per mass

It was expected that the bees used in the experiment were mature and had about two weeks to live. This was confirmed by the control group, Box 1. As can be seen on Graph 1, the mortality of the control group started up around day 10.

All the bees fed twice the maximum field realistic dose (Box 6) had died by day 10.

What was surprising was the mortality per mass was lowest for boxes 2 and 3, the bees fed half minimum field realistic dosages and minimum field realistic dosages.

Mortality per mass straight line.jpg

  • All the bees fed at the highest dosage (twice the maximum field realistic dosages) died before the experiment ended.
  • Bees fed higher dosages of glyphosate in the formulation Roundup Plus had a higher mortality rate than the control group.
  • Bees fed lowest dosages had lower mortality rates than the control.

CFUs per gram

At the beginning of the experiment, on February 1, the Colony Forming Units (CFUs) were about the same for samples from each box, a little over 400,000 per gram. The CFUs of Lactobacillus for bees fed half minimum realistic field dosages peaked dramatically on February 12 This peak may have been caused by glyphosate killing other bacteria species and allowing the opportunistic growth of Lactobacillus. Lactobacillus is considered a beneficial bacteria. After this peak of beneficial bacteria, the mortality of this group began to rise, although stayed below the control. It may be possible that the increase of Lactobacillus contributed to the low mortality of this group.

At the end of the experiment, all the groups were trending toward about 200,000 CFUs.

Cfu per gram straight line.jpg


At the highest dosages (twice maximum field realistic dose), glyphosate as fed in the formulation RoundUp Plus™, killed all the bees after 10 days. Since the levels of Lactobacillus on this group did not fall below the control, their deaths were not due to glyphosate killing Lactobacillus. From this one experiment it can not be determined whether the deaths were due to the active ingredients or the inert ingredients in RoundUp Plus™.

At the higher dosages (maximum field realistic dose and average field realistic dose) the mortality rate was higher than the control. But, at the two lowest doses (1/2 minimum field realistic and minimum field realistic), the mortality rate was lower than the control. At low dosages, glyphosate may actually be beneficial to bee health.

No significant changes in Lactobacillus concentrations in the bee gut were observed except for the lowest dosage (half minimum field realistic dosage) where Lactobacillus concentrations increased dramatically. All the groups fed Roundup Plus™ had Lactobacillus concentrations higher than the control. Based on this one experiment, there is no evidence that glyphosate, in the formulation RoundUp Plus™, kills Lactobacillus in the bees' gut. This also supports the idea that at low dosages, glyphosate may actually be beneficial to bee health.

How weird is that?


Additional Research

Glyphosate perturbs the gut microbiota of honey bees Erick V. S. Motta, Kasie Raymann, Nancy A. Moran Proceedings of the National Academy of Sciences Oct 2018, 115 (41) 10305-10310; DOI: 10.1073/pnas.1803880115

Contrarian View

Viewpoint: New glyphosate study suggesting danger to honeybee microbiota is detailed, sophisticated—and wrong Josh Bloom | American Council on Science and Health | October 1, 2018

  1. 1.0 1.1 Lucila T. Herbert, Diego E. Vázquez, Andrés Arenas and Walter M. Farina Effects of field-realistic doses of glyphosate on honeybee appetitive behaviour