Is there an established protocol to measure the permeability of bacterial membranes?
Thank you
Yes, there are several established protocols to assess bacterial permeability, which is crucial for understanding the effectiveness of antibiotics, the physiology of bacteria, and the interaction between bacteria and their environment. Here are a few common methods:
1. N-Phenyl-1-naphthylamine (NPN) Uptake Assay:
NPN is a hydrophobic fluorescent dye that increases in fluorescence upon interacting with the bacterial membrane.
When bacterial membranes are compromised, NPN can penetrate the outer membrane and increase fluorescence.
The procedure involves incubating bacteria with NPN and measuring the fluorescence increase using a fluorometer.
2. Antibiotic Uptake Assays:
In this method, bacteria are exposed to radiolabeled or fluorescently labeled antibiotics.
The uptake of the antibiotic by the bacteria is then measured using scintillation counting or fluorescence measurement.
This method provides a direct measurement of the permeability to specific antibiotics.
3. Flow Cytometry:
Flow cytometry can be used to assess membrane permeability by staining bacteria with fluorescent dyes that differentiate between intact and permeabilized cells.
Common dyes include propidium iodide (PI) and SYTOX Green, which only penetrate cells with compromised membranes.
4. Disk Diffusion Assay (Kirby-Bauer Test):
Although primarily used to determine antibiotic susceptibility, this method can indirectly indicate membrane permeability.
Bacteria are spread on an agar plate, and antibiotic-impregnated disks are placed on the surface.
The zones of inhibition around the disks indicate the effectiveness of the antibiotic, which can be related to membrane permeability.
5. Electron Microscopy:
Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) can provide visual evidence of changes in the bacterial membrane structure.
While not quantitative, these methods can show morphological changes indicative of altered permeability.
6. Potassium Efflux Assay:
This method measures the release of potassium ions from bacterial cells, which occurs when the membrane is damaged.
The potassium concentration in the surrounding medium is measured using ion-selective electrodes or atomic absorption spectroscopy.
7. Beta-Lactamase Assay:
This assay measures the permeability of the bacterial outer membrane to beta-lactam antibiotics.
Bacteria are exposed to a beta-lactam antibiotic, and the amount of beta-lactamase enzyme released into the medium is measured.
8. Lactate Dehydrogenase (LDH) Release Assay:
LDH is an intracellular enzyme that leaks out when the bacterial membrane is damaged.
The amount of LDH in the culture medium is measured using a colorimetric assay.
General Protocol for an NPN Uptake Assay:
1. Preparation of Bacterial Suspension:
• Grow bacterial cultures to the desired phase (e.g., mid-log phase).
• Harvest cells by centrifugation and resuspend in an appropriate buffer (e.g., PBS).
2. NPN Addition:
• Add NPN to the bacterial suspension to a final concentration of 10 µM.
3. Incubation:
• Incubate the mixture at room temperature or 37°C for a specified time (e.g., 5-10 minutes).
4. Fluorescence Measurement:
• Measure the fluorescence using a fluorometer with excitation and emission wavelengths set to 350 nm and 420 nm, respectively.
5. Data Analysis:
• Compare the fluorescence intensity of treated and untreated bacterial samples.
• An increase in fluorescence indicates increased membrane permeability.
These methods can be adapted and optimized depending on the specific bacterial strain and the experimental conditions. Always refer to detailed protocols and safety guidelines specific to the dyes and antibiotics being used
I also found this assay:
"Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid-base properties of microbial cells" (Article Microbial adhesion to solvents: A novel method to determine ...
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases