In vitro antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin

doi:10.1038/nprot.2007.466

Method Article

In vitro antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin

https://doi.org/10.1038/nprot.2007.466

This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.

Version 1

posted 31 Oct, 2007

You are reading this latest protocol version

Biological techniques

Microbiology

Chemical biology

Drug discovery

in vitro antimicrobial assay

PPO

ROS

Here we describe the chemically-reconstituted system for evaluating the antimicrobial activity of the microbial protease-mediated ROS production from the hemocyanin-prophenoloxidase (HMC/PPO)or hemoglobin. The antimicrobial activity is examined by endpoint measurement of the remnant microbial population or by real-time observation of the bactericidal process. To demonstrate the specificity of PO-induced ROS-production which causes the antimicrobial activity, PTU is used here as a specific inhibitor of PO activity.

Prepare the bacterial culture as described in protocol #4, and then adjust the bacterial population to 10⁶ - 10⁷ cfu/ml.
For the end-point measurement of HMC/PPO: (a) incubate the bacteria under test at 10⁶ cfu/ml with 60 μg purified HMC plus 100 nmol 4-methylcatechol (4ME) in 100 μl PBS at 37 °C for 1 h.

(b) then enumerate the remnant bacterial population in the reaction mixture by plating 100 μl of serially diluted samples on nutrient agar plates and incubating at 37 °C overnight. Perform the colony count and calculate the bacterial cfu/ml.

(d) also set up controls by incubating bacteria with HMC/PPO or 4ME or PTU separately, or in combinations.

For real-time imaging of the bacterial clearance elicited by HMC/PPO: (a) mix each bacterial strain at 10⁷ cfu/ml with 60 μg HMC/PPO and 100 nmol 4ME in 100 μl PBS.

(b) apply one μl of the mixture to fluorescence microscopy and examine it at magnification of 63 x 1.6, with time-elapse method.

For the end-point measurement of metHb: (a) incubate the bacteria under test at 10⁶ cfu/ml with 36 μg metHb and 1.6 μmol H₂O₂ in 200 μl PBS at 37 °C for 1 h.

(c) to further prove that the antibacterial activity was indeed attributable to ROS, apply 2 μmol GSH to the incubation mixture to quench the superoxide ions.

(d) set up controls by incubating bacteria with metHb, H₂O₂ or GSH separately or in combinations.

In the presence of the HMC/PPO and its substrate, more of the protease-producing bacteria are cleared compared to those of the protease-inactive strains. This difference can be reduced when ROS quenchers (eg: GSH for both quinone and superoxide anion or SOD for superoxide anion) are applied. When supplemented with exogenous proteases, the antimicrobial effect of the protease-inactive strains is increased dose-dependently. (Refer to the example shown in figure 5 as a typical result obtained using HMC).

Download PDF

Version 1

posted 31 Oct, 2007

You are reading this latest protocol version

In vitro antimicrobial assay using chemically reconstituted system with hemocyanin or hemoglobin

Status:

Version 1

Figures

Introduction

Procedure

Anticipated Results

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Cookie Settings