Semi-Automated Method for Multi-Tasking Measurement of Microbial Growth, Capsule, and Biofilm Formation

Abstract

The majority of bacterial infections involve the formation of biofilms. Biofilm formation is nutrient and growth dependent. Determination of the effects of nutrients on exopolysaccharide production and bacterial growth is labor and time intensive. We tested whether the Bioscreen C (Growth Curves, Inc.) would have utility as a high-throughput tool in the measurement of fundamental phenotype expression, as it relates to growth conditions. Within 48 -72 hr, reproduceble, statistically significant data on the affects of growth conditions on generation time, capsule production and biofilm formation (maximally for 25 different conditions per 24 hr run cycle; n = 4) were obtained. Although all S. aureus strains produced similar amounts of capsule, sarA -and agr -strains grew significantly slower than parent strain (1.6 fold slower) and produced significantly (p < 0.05) less biofilm (~2 fold). E. coli growth rate, biofilm and capsule production in simulated nephropathic urine medium was similar for urine with insulin (20 µU). Addition of insulin to urine medium with proline increased generation time, capsule and biofilm production. Findings from this study show that the Bioscreen C is a rapid, reproducible, and easily manipulated system to concurrently measure bacterial growth, biofilm formation, and capsule production. In addition, there is the potential for further applications of this system by expanding the types of detector dyes used.

Menaquinone (Vitamin K2) Enhancement of &lt;i&gt;Staphylococcus aureus&lt;/i&gt; Biofilm Formation

“…Biofilm formation on plastic was significantly increased at the lowest bacterial density tested (10 4 CFU/ml). As had been previously reported for S. aureus ATCC 25923, menaquinone, regardless of concentration, had no significant effect on doubling time and maximum absorbance for any of the strains tested ( Table 1; data not shown) [20]. Thus, the effect menaquinone had on biofilm formation was independent of growth rate or final bacterial density.…”

“…The ability to recognize the host environment and alter phenotype expression so as to establish an infectious process is controlled through recognition of a variety of environmental conditions, including the presence of chemical signaling compounds [1] [8]. Similar to other host-derived exogenous chemical signaling molecules, menaquinone's effects on biofilm formation on plastic (measure of surface hydrophobicity; alteration in S. aureus cell surface architecture) were population size dependent [2] [20]. Since there was no significant difference in total growth, regardless of menaquinone concentration, the differences in biofilm levels were the result of menaquinone-induced cell surface changes, and not differences in total growth levels.…”

“…S. aureus parent strain 8325-4, and agr − , sarA − and agr − sarA − strains, which are highly characterized, were a generous gift from S. Foster. Bacterial growth rate and maximal growth were determined using a semi-automated system as previously described [20]. Briefly, overnight cultures of S. aureus strains in Mueller Hinton (MH) broth were added to fresh MH (10 4 and 10 6 CFU/ml final concentration).…”

“…After incubation (24 hr, 37˚C, static, humid chamber) the plates were emptied, washed, and air dried. After drying, the plates were stained with 300 µl of commercially prepared Gram's crystal violet (biofilm formation; Troy Biologicals), or with 300 µl of Alcian blue (1% w/v DDW) which stains acidic polysaccharide, e.g., capsule [20]- [22]. After the plates were thoroughly rinsed and dried, the stain was dissolved in absolute alcohol (300 µl).…”

Antimicrobial Susceptibility and Sub-MIC Biofilm Formation of &lt;i&gt;Moraxella catarrhalis&lt;/i&gt; Clinical Isolates under Anaerobic Conditions

“…The effect sub-MICs have on M. catarrhalis biofilm formation was determined, as previously described, by the simple expedient of emptying, washing and staining the 96 well plate wells after growth in the presence and absence of antibiotics (PBS, 3x wash; crystal violet, stain; absolute ethanol elutant) [21]. Biofilm levels were determined as a measure of crystal violet staining (Beckman EIA reader; Abs 540 ).…”