Protocol Citation: Xiaoxuan Shi, Azhaya Cuervo, Aisha Burton 2026. Sporulation Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3r4dqg25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 11, 2026
Last Modified: June 11, 2026
Protocol Integer ID: 318968
Keywords: cleaning spores in the burton lab, cleaning spore, sporulation assay protocol, burton lab at cornell university, burton lab, cornell university
Disclaimer
This protocol will be updated as needed.
Abstract
Protocol for making and cleaning spores in the Burton lab at Cornell University.
Day 1
Streak out controls on LB plates
WT and ΔspoIIE
Place plates at37 °C Overnight in static incubator.
Day 2
Start 5 mL LB Overnight culture from a single colony of WT B. subtilis 168 in Biological Triplicates and do the same forΔspoIIE . Put in benchtop shaker at 250 rpm, 37°C .
Equipment
New Brunswick™ Innova® 42
NAME
Incubated Benchtop Shakers
TYPE
Eppendorf
BRAND
M1335-0004
SKU
Day 3
1d 3h 2m
Thaw 3 biological replicates the pooled BKK library on ice.
Add thawed cells to 10 mL LB (x 3; each replicate will get it's own test tube). Grow up for 01:00:00 in benchtop shaker at 250 rpm, 37°C
1h
Take OD600 of all samples (WT, ΔspoIIE, and Pooled Library)
Equipment
Implen OD600
NAME
Spectrophotometer
TYPE
Implen
BRAND
Back dilute all samples to an OD600 = 0.1 into 20 mL LB in a 150 ml beveled flask. Grow until cells reach an OD600 = 0.8.
Centrifuge 15 mL of cells in 15 ml conical. 3000 rcf, 25°C, 00:10:00
10m
Wash the cell pellet with 5 mL 1x PBS. 3000 rcf, 25°C, 00:10:00
10m
Resuspend cells in 10 mL of DSM in a test tube. Immediately remove 5 ml for T0 processing. Allow rest of the 5 ml of sample to shake for 250 rpm, 37°C, 24:00:00
1d
Take 100 µL of the 10 ml T0 sample and put into microcentrifuge tubes to dilute for
plating pre-spore cleanup. Take another 100 µL and put in a separate microcentrifuge tube to do microscopy.
Expected result
No spores should be present in any of the samples.
Put the rest of the 9.8 ml of sample into ThermoMixer at 80 °C for 00:20:00
Equipment
ThermoMixer® C
NAME
ThermoMixer
TYPE
Eppendorf
BRAND
5382000023
SKU
20m
Add 5 µL of 25 mg/mL lysozyme for 01:00:00 at Room temperature
1h
3000 rcf, 25°C, 00:05:00 to wash media and enzymes away. Resuspend in 5 ml cold Milli-Q H2O. Repeat 2x. Final suspension shall be 400 µL of 20% Histodenz. Then layer 400 µL of cells over 800 µL of 50% Histodenz in a microcentrifuge tube.
5m
14000 rcf, 25°C, 00:15:00
15m
Remove debris at liquid interface, then rest of histodenz. Do not disturb cell pellet at the bottom.
Water wash the cell pellets 2 X's with 1 ml water. 14000 rcf, 25°C, 00:02:00 After final wash, add 200 µL of sterile water on each pellet.
2m
Plate spores for CFU counts (serial dilution as done earlier) and microscopy.
Day 4
Go back to 10.1 and repeat all steps when culture has reached T24.
Protocol references
https://static.igem.org/mediawiki/2016/7/71/T--Freiburg--SporulationProtocol.pdf