This protocol describes a robust method for developing pellicle biofilm. The method is scalable to different culture volumes, allowing easy adoption for various experimental objectives. The method’s design enables qualitative or quantitative assessment of the biofilm-forming potential of several mycobacterial species.
Many bacteria thrive in intricate natural communities, exhibiting key attributes of multicellularity such as communication, cooperation, and competition. The most prevalent manifestation of bacterial multicellular behavior is the formation of biofilms, often linked to pathogenicity. Biofilms offer a haven against antimicrobial agents, fostering the emergence of antimicrobial resistance. The conventional practice of cultivating bacteria in shake flask liquid cultures fails to represent their proper physiological growth in nature, consequently limiting our comprehension of their intricate dynamics. Notably, the metabolic and transcriptional profiles of bacteria residing in biofilms closely resemble those of naturally growing cells. This parallelism underscores the significance of biofilms as an ideal model for foundational and translational research. This article focuses on utilizing Mycobacterium smegmatis as a model organism to illustrate a technique for cultivating pellicle biofilms. The approach is adaptable to various culture volumes, facilitating its implementation for diverse experimental objectives such as antimicrobial studies. Moreover, the method's design enables the qualitative or quantitative evaluation of the biofilm-forming capabilities of different mycobacterial species with minor adjustments.
Bacteria are able to survive as single-celled entities; however, in most physiologically relevant conditions, they organize into community mimetics. Biofilm is a widely recognized community organization of bacteria formed by aggregated cells encased in a self-produced matrix1. Such assembly possesses signatures of early multicellularity and provides higher stress resilience to bacterial systems. Biofilms are often tolerant to antimicrobials and are estimated to be responsible for almost 80% of microbial infections2,3.
Shake flask and plate-based cultures have traditionally been the usual practices for bacterial culturing. Their enormous acceptability and success can be attributed to their ease of handling, reproducibility, and scalability. However, the lack of physiological context limits the translational potential of the knowledge generated using such systems4. Therefore, biofilms are becoming an attractive model system to study bacterial pathophysiology. Biofilms provide a dynamic model system, closely mirroring natural conditions, allowing researchers to replicate physiological aspects such as nutrient gradients and spatial heterogeneity5,6.
The biofilm lifestyle is particularly pertinent in mycobacterial studies, as mycobacteria, including the notorious Mycobacterium tuberculosis, are adept biofilm formers7. Their ability to thrive within biofilms contributes to their persistence in host tissues during infections. It poses a formidable challenge in treating mycobacterial diseases, given the inherent antibiotic resistance associated with biofilm lifestyles8. Biofilms also provide an ideal model system to study mycobacterial metabolism, as they allow for the investigation of the unique metabolic adaptations and nutrient utilization strategies employed by mycobacteria within complex microbial communities9.
While biofilm is increasingly being accepted as a better model system for mycobacterial studies10, there is a need for consistent and reproducible standard operating procedures, especially for drawing parallels among studies conducted in different laboratories. The method outlined here describes biofilm formation procedures for a mycobacterial species, M. smegmatis. M. smegmatis is a more accessible model for studying mycobacterial biofilms, given its non-pathogenicity and faster biofilm formation kinetics. The method can be modified to suit applications like antimycobacterial screening, metabolite extraction, and omics studies.
The details of all the reagents and equipment used for the study are listed in the Table of Materials.
1. Sauton's media preparation
2. Preparation of filter-sterilized 5% Tween-80
3. Preparing the primary culture
4. Preparing the secondary culture
5. Setting up the biofilm
6. Setting up biofilms for stage-specific observations
NOTE: The overall biofilm setup is the same as described in step 5. To harvest or image biofilm at different points in time, it is suggested that the same biofilm be set up in multiple sets on separate plates.
7. Estimating the biofilm development
8. Antibiotic tolerance assay in biofilms
Biofilm pellicles become visible to the naked eye from the third day onwards. Although biofilm grows on Sauton's media without 2% glucose, an improvement was observed in the reticulation when it was added. We obtained 10.48 mg ± 3.13 mg (n = 4) of biofilm dry weight from each well of a 24-well plate with 1.5 mL of Sauton's media (supplemented with 2% glucose) grown for four days. In Figure 2, biofilm development was visible from day 3 to day 6. It starts forming a film with slight reticulation on day 3, and by day 5, it is fully matured. From day 6 onwards, the biofilm starts to disintegrate. The MIC90 of rifampicin (64 µg/mL) reduced the biofilm's CFU by 25% ± 12% (n = 3) only.
Figure 1: Biofilms of M. smegmatis after 4 days of incubation in Sauton's media. (1) Without glucose and inoculated with 2% inoculum. (2) Without glucose and inoculated with 4% inoculum. (3) With 2% glucose and inoculated with 2% inoculum. (4) With 2% glucose and inoculated with 4% inoculum. (5) and (6) are media controls. Here, the samples are in standard 6-well plates. The photographs are captured without any magnification. Please click here to view a larger version of this figure.
Figure 2: M. smegmatis biofilm morphology from the 3rd day post inoculation to the 6th day. Here, the samples are in standard 24-well plates. The photographs are captured without any magnification. Please click here to view a larger version of this figure.
S. No. | Component | Working concentration (w/v) | Stock solution concentration | Volume/ Weight of stock solution | |||||
1 | Potassium dihydrogen phosphate | 0.05% | 2.50% | 20 mL | |||||
2 | Magnesium sulfate | 0.05% | 2.50% | 20 mL | |||||
3 | Ferric ammonium citrate | 0.01% | 5% | 1 mL | |||||
4 | Glycerol | 6% | 100% | 60 mL | |||||
5 | L- Asparagine | 0.40% | NA | 4 g | |||||
6 | Citric acid | 0.20% | NA | 2 g |
Table 1: Components for Sauton's media preparation.
The multicellular lifestyle of microbes was described almost a century ago; however, clinical studies remain sparse, mostly due to the lack of robust methods14. Methods described in works on biofilm biology are often difficult to adapt. Here, the detailed methodology, aided by demonstrations of critical steps, is expected to improve the reproducibility of the protocols.
The method of biofilm production described in this article is scalable, requiring a proportional increase in medium volume and inoculum size. Applications like metabolite extraction require a higher culture volume, whereas antimicrobial susceptibility assays require a smaller biomass12. Additionally, the size of the inoculum can be adjusted to tailor the rate of biofilm formation. This flexibility allows researchers to appropriately control the duration of experiments.
Growth conditions and the microenvironment play crucial roles in biofilm physiology15. Notably, the presence of glucose is not obligatory for forming a biofilm; nonetheless, the inclusion of 2% glucose has been observed to enhance the reticulation and stability of the biofilm. Another interesting observation is the biofilm enhancement potential of placing a glass coverslip at the bottom of the culture wells. The addition of Tween-80 in both primary and secondary cultures is crucial for mitigating cell aggregation.
Biofilm development involves distinct growth phases such as attachment, maturation, and dispersal. While the biofilm setup described here is typically incubated for 7 days, pellicles become visible to the naked eye from the 3rd day onwards. This setup allows for studying the temporal dynamics of biofilm development. While this article describes the estimation of dry weight to assess biofilm biomass, the setup is also amenable to conventional crystal violet staining or microscopy-based estimation12,16.
The production of biofilms by mycobacteria plays a significant role in their pathogenicity and confers enhanced resistance to drugs8,17. We observed seven to eight times higher antibiotic tolerance in biofilm cultures compared to shake flask cultures. Several groups use biofilms as a model system for studying bacterial physiology; a wider adoption of this growth mode by the mycobacterial research community can provide newer insights into the pathophysiology of various pathogenic species within this genus.
The authors have nothing to disclose.
This work was supported by the DBT-Ramalingaswami Fellowship awarded to Amitesh Anand.
0.2 µM PVDF syringe filter | Axiva | SFNY04 R | |
1 mL tips | Genetix | GXM-611000 C | |
10 µL tips | Genetix | GXM-6110 C | |
200 µL tips | Genetix | GXM-61200C | |
6-well polypropylene plates | Tarsons | 980010 | |
Amber tubes | Tarsons | 546051 | |
Autoclave | Hospharma | ||
Biosafety Cabinet A II | MSET | ||
Blotting paper | Any suitable vendor | ||
Centrifuge | Eppendorf | ||
Citric acid | Sigma | 251275 | |
Cuvettes | Bio-Rad | 2239955 | |
Ferric ammonium citrate | Sigma | F5879 | |
Gel documentation system | Bio-Rad | ||
Glass Beads | Sigma | G8772 | |
Glucose | Sigma | 49139 | |
Glycerol | Sigma | G5516 | |
Inoculation loops | Genaxy | HS81121C | |
L-Aspargine | Sigma | A0884 | |
LB-agar | Himedia | M1151 | |
LB-media | Himedia | M575 | |
M. smegmatis mc2155 cryo-stock | ATCC | 700084 | |
Magnesium sulfate | Sigma | M2643 | |
Micropipettes | Gilson | ||
Parafilm | Tarsons | ||
Petri Dish | Tarsons | 460020 | |
pH meter | Labman Scientific Instruments | ||
Plate Reader | Tecan | ||
Polypropylene test tubes | Genaxy | GEN-14100-PS | |
Potassium phosphate monobasic | Sigma | P5379 | |
Rifampicin | MedchemExpress | HY-B0272 | |
Serological pipette | SPL Life Sciences | 95210 | |
Shaker Incubator | Eppendorf | ||
Spatula | |||
Spectrophotometer | Thermo Scientific | ||
Static Incubator | CARON | ||
Sterile 10 mL syringe | Becton Dickinson | 309642 | |
Sterile 50 mL syringe | Becton Dickinson | 309653 | |
Tween-80 | Sigma | P1754 | |
Weighing balance | Sartorius | ||
Zinc sulfate | Sigma | Z0251 |