Colorimetric Detection of Cortex Degradation During Bacterial Spore Germination

Abstract

Source: Francis, M. B., et al. Detecting Cortex Fragments During Bacterial Spore Germination. J. Vis. Exp. (2016).

This video demonstrates a colorimetric method to detect cortex degradation during bacterial spore germination by measuring the release of reducing sugars over time. Using N-acetylglucosamine standards and time-point samples, the assay quantifies sugar concentration through a purple chromogenic reaction, indicating the extent of cortex hydrolysis.

Protocol

1. Color Reagent

1. While samples are on ice, prepare the color reagent. Dissolve 0.320 g of p-dimethylaminobenzaldehyde (DMAB; Ehrlich's reagent) in 1.9 ml of glacial acetic acid.
   Note: The reagent is time, temperature, and light sensitive and should not be made in advance.
2. After the DMAB completely dissolves, add 100 µl of 10 N HCl and vortex.
3. Add 5 ml of glacial acetic acid and vortex.

2. Colorimetric Reaction

1. Transfer 100 µl of each cooled cortex sample to a new 1.5 ml microcentrifuge tube.
2. Add 700 µl of the freshly-prepared color solution to each sample.
3. Immediately transfer the samples to a 37 °C water bath and incubate for EXACTLY 20 min.
4. Transfer 200 µl of each sample to a clear 96-well plate.
5. Quantify the samples at 585 nm on a plate reader. Samples should start at a yellow color and a positive reaction will shift to purple. The more cortex present, the deeper the purple color.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2.0 mL screw cap tube	USA scientific	1420-3700
p1000 eppendorf pippet	Eppendorf
p200 eppendorf pippet	Eppendorf
p20 eppendorf pippet	Eppendorf
100-1250uL pipet tips	VWR	89079-486
1-200uL pipet tips	VWR	89079-458
N-acetyl-D-glucosamine	Sigma	A3286-25G
Acetic Anhydride	Alfa Aesar	L04295
Sodium Bicarbonate	BDH	BDH0280-500G
Potassium Tetraborate tetrahydrate	Alfa Aesar	39435
Sodium Hydroxide	BDH	BDH8019-500G
4-(Dimethylamino)benzaldehyde	Sigma	156477-100G
SpectraMax M3	Molecular Devices
Acetic Acid, Glacial	BDH	BDH3098-3.8LP
Heated, Circulating Water Bath	VWR Scientific	Model 1136
Microtest 96	Falcon	353072
Culture tubes	VWR	89000-506	96 well clear tissue culture plates
Heat Blocks
Vortex Machine