Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

The overall goal of this procedure is to visualize and quantify changes to biofilms in response to sputum and antibiotics. This method can be used by researchers to understand key questions, such as how the efficacy of various antibiotics against bacterial biofilms can be impacted by the presence of sputum. The main advantage of this methodology is it allows individuals to see changes occurring in biofilms in response to different factors.

To begin the experiment, record the volume of the previously obtained sputum sample. Add phosphate-buffered saline, or PBS, add two times the volume of the sample. Next, use a transfer pipette to thoroughly mix the sample.

Vortex the sample on the highest setting for one minute to mix it completely. Aliquot one milliliter of the mixture into 1.5 milliliter microcentrifuge tubes. Then spin down the tubes.

Following centrifugation, save the supernatant and discard the pellet. Filter sterilize the supernatant through a 0.22 micron filter and collect it in a clean microcentrifuge tube. Store the sputum supernatant at 80 degrees Celsius for future use.

Place 40 microliters of previously prepared culture into four milliliters of fresh Luria broth, or LB media, and grow the bacteria to obtain a culture with an optical density of 0.55 at 600 nanometers. Next, dilute 100 microliters of 0.5 OD culture into 400 microliters of prepared 10%sputum filtrates in LB.Use 200 microliters of the dilution to seed the wells of the slide chambers. Allow the bacteria to attach for four hours at 37 degrees Celsius without shaking.

After four hours, remove the media and gently wash the biofilm with 1X fresh LB.Replace the remaining media with 200 microliters of fresh LB.Allow the biofilms to grow at 37 degrees Celsius without shaking. Replace the media every 12 hours without washing until the biofilm is ready for microscopy. It’s very important to gently wash the slides so as not to disrupt any attached bacteria.

Following growth, remove the media from the chamber wells and gently wash each chamber two times with 300 microliters of sterile PBS. Next, to prepare the biofilm staining mixture, add one microliter of each dye provided in the viability kit to each milliliter of solution needed and mix the solution. The optimal amount of dye and staining time is determined empirically for each organism.

Add 200 microliters of the staining mixture to each well of the chambered cover glass. Incubate the cover glass. After incubation, remove the staining mixture from the chambers and wash each well with 300 microliters of sterile PBS.

Replace the PBS with fresh media. Finally, proceed with visualization via confocal microscopy. Representative images of Burkholderia cepacia complex, or BCC clinical isolates, following 48 hours of growth in chambered cover glass with media alone or in the presence of 10%sputum filtrates demonstrate that their presence can change the architecture of the biofilm.

The images were analyzed with Comstat software to obtain key biofilm parameters, including the average thickness of the biofilm, which was increased when treated with 10%sputum. In order to appreciate the different factors’effects on biofilm growth, clinical isolates were grown in media only or with sputum and either was treated with or without antibiotic. After watching this video, you should have a good understanding of how to form and treat biofilms on chambered cover glass and prepare them for staining and confocal analysis.