Pseudomonas Aeruginosa Biofilms Investigation of the properties of nontransgenic biofilm formation and dispersal
Biofilm Formation Two methods of biofilm formation were tested. The first involved placing a sterile slide into a corning tube, before filling it with 25ml of LB broth. The broth was then inoculated with 1Âľl per ml of Pseudomonas aeruginosa. The second method used a petri dish with two sterile tips placed in a V shape propping a sterile slide at roughly 30Â°. The dish was filled half full with LB growth media. Both the tubes and thepetri dishes were placed on a shaker and left over the weekend. Slides were sterilised by submerging them in 100% ethanol and allowing them to drip dry. Both methods were used to grow biofilms of multiple strains: E.Coli strains: DS941 and Top 10 Pseudomonas: P. aeruginosa, P. fluorescenes and P. putida Mixed biofilms: P.a. + DS914, P.a.+ Top 10 and P.a. + P.f. Controls: Unwashed slide and no bacteria Staining Biofilm Protocol for biofilm staining with minimum sheer force <http://biofilmbook.hypertextbookshop.com/public_version/contents/appendices/appendix002/pages/page008.html>
Introduction: The Gram stain devised by Christian Gram in 1882 has become one of the most important diagnostic procedures in microbiology. This stain differentiates two main categories of bacteria according to the structure of the cell wall. Bacteria with a predominantly peptidoglycan cell wall stain blue/purple with this technique and since they retain the primary stain are called Gram positive (Gram +). Cells with only a minor peptidoglycan component and possessing a lipopolysaccharide cell wall give up the primary stain during destaining and therefore exhibit the red counter stain. These cells are called Gram negative (Gram -).
A Gram stain is usually performed on a smear preparation that has been heat fixed. One function of fixation is to secure (fix) the cells to the slide. In a biofilm, however, the cells are already fixed. Furthermore, a heat fixed slide is dry, but a biofilm is mostly water. Drying alters the biofilm virtually beyond recognition. This exercise describes a method for obtaining a Gram stain on a minimally altered biofilm.
Safety Note: It is recommended that you wear a lab coat or an apron to protect your clothes from staining and gloves to protect your hands. Also, wash your hands thoroughly before leaving the lab since you are working with bacteria.
Supplies Needed: Quantity
biofilm grown on a 1 x 3 inch microscope slide
25 mm square coverslip (the thinner the better)
Gram Stain Kit (Hucker’s Crystal violet, Gram’s Iodine, Decolorizer, Safranin)
Instructions: Prepare a biofilm slide by any appropriate method 1. 2.
One could use one of the methods described in Biofilms: The Hypertextbook . Perhaps the simplest method is to simply site a clean 1 x 3 inch microscope slide at some site likely to produce a biofilm, for example a stream, pond, seep, wharf, shower, drain or aquarium. Any site where a slide can be submerged in water will serve. Flowing water is preferable.
Preparing the slide for flow-through staining 1. 2. 3.
Wipe one side of the biofilm slide clean with a paper towel. On the remaining biofilm side of the slide, wipe the top and bottom 2 mm of the slide clean (see Figure 1). Thinly spread petroleum jelly on a sheet of paper. Hold a cover slip at right angles to the petroleum jelly and scrape it in such a way as to build up a thin ridge along one edge of the cover slip. The procedure is similar to preparing a coverslip for a hanging drop mount. Repeat the process with the opposite edge of the cover slip. Carefully place the cover slip onto the biofilm slide preparation petroleum jelly side down so that the jelly is in contact with the 2 mm clean edges. This has the effect of producing a tunnel between the coverslip and the slide.
Preparing the Gram stain The Gram stain reagents are added at one open end of the slide “tunnel” and are drawn through the space under the slide by means of an absorbent paper towel. The reagent sequence is identical to that in the standard Gram stain but the time intervals are longer to compensate for any dilution effect caused by the aqueous phase of the biofilm preparation. It is important to be certain that sufficient reagent has been drawn under the coverslip so that dilution by the volume of water present or by the previous reagent is minimized. The bacteria stain as in a traditional Gram stain, but because of the thickness of the preparation and the presence of water the slides do look different from a typical Gram stain. For example, the colors are typically more muted. 1. Add the Crystal Violet dye to one edge of the coverslip “tunnel.” Draw the dye through using an absorbant paper towel applied to the opposite edge of the cover slip. Draw the dye through until all the water has been replaced by dye. Stain for 30 seconds. 2. In a similar manner, wash the slide by drawing water under the coverslip with a paper towel until no further purple color is removed. 3. Add the Gram's Iodine solution by adding several drops of the reagent to the same edge of the coverslip and drawing it through with a paper towel. Treat for about 1.5 minutes. 4. Decolorize the slide by drawing 95% alcohol under the coverslip until no further purple due is removed. 5. Wash the slide as in step b. 6. Counter stain the slide by placing Safranin dye at the same end of the “tunnel” and drawing it through with a paper towel. Stain for 1/2 minute. 7. Wash the slide as in step b. 8. Observe the slide under high dry or oil immersion microscopy. Note: The thickness of the preparation may preclude an examination under oil immersion See Figure 1, which illustrates this modified Gram staining method.
Figure 1. Gram staining method.
Biofilm Results Biofilms were visualised using the "Staining Biofilms" protocol. This method is a Gram's stain that has been modified to reduce the sheer force applied to the biofilm by the repeated washes that are required. It uses capillary action to minimise disruption of the biofilm. Mixed cultures seem to form biofilms more effectively than single colonies with P.a. + DS941 being most effective. As expected P.a. formed biofilms effectively but neither other strain of pseudomonas formed a biofilm. Top10 and DS914 did not form biofilms in isolation. The bacteria seemed to form sheets of biofilm that were clearly defined. The method of sterilisation that was used to clean the slides prior to them being used in these experiments was ineffective as there was contamination in the negative control. NOTE (5/7/11): When the slides were viewed a day later the overall structure had degraded with the sheets of biofilm that were previously well defined retracting. This was probably due to desiccation. LOV Domain On 5/7/11 a single suspected transformant was found on the LOV domain plate. It was cultured over night. A mini-prep was performed to isolate the plasmid that contained the LOV domain. This plasmid was digested with EcoRV and BamHI to determine if the LOV domain was present in the plasmid.
Lane 2 and 4 seem to not have had any DNA added causing significant problems in determining the size of the LOV domain insert. Biofilm Formation 2 It has been determined that the corning tube method of biofilm formation is far more effective than the method involving petri dishes (which leaked during the night). Further biofilms have been set-up with corning tubes placed vertically as in "Biofilm Formation" as well as some tubes that have been angled at 30째 in an attempt to form larger biofilms. A new method for cleaning the slides before insertion into the corning tubes was used as the previous method was ineffective. The slides were submerged in 100% ethanol before being wiped down with blue roll that had been saturated in 100% ethanol. This led to the slides drying more quickly and has eliminated contamination. Caffeine Biofilm Caffeine inhibits the actions of PDEs, so it is hypothesised that adding caffeine to the LB broth of a biofilm forming set-up should encourage faster or denser formation of biofilm. Tubes were set-up with no caffeine, 0.5mg/ml, 1.0mg/ml and 2.0mg/ml of caffeine. Caffeine Biofilm Results The caffeine biofilms were stained to try to determine if there were any differences between biofilm formation in the presences of caffeine. There seemed to be a distinct difference in the appearance of the biofilm depending on caffeine concentration.
Figure 1: Biofilm formed in the absence of caffeine, that has been visualised with a Gram's stain following the "Staining Biofilms" protocol.
Figure 2: Biofilm formed in presence of 0.5mg/ml of caffeine, that has been visualised with a Gram's stain following the "Staining Biofilms" protocol.
Figure 3: Biofilm formed in presence of 1.0mg/ml of caffeine, that has been visualised with a Gram's stain following the "Staining Biofilms" protocol.
Figure 4: Biofilm formed in presence of 2.0mg/ml of caffeine, that has been visualised with a Gram's stain following the "Staining Biofilms" protocol. Biofilms will be quantified in future experiments using the "Biofilm Quantification" protocol. Biofilm Quantfication To determine a base rate of dispersal of cells from a biofilm, slides from the "Biofilm Formation 2" experiment were transferred to 50ml fresh media (dissociated cells) and left for 4 hours. The slides
were then transferred to 50ml of fresh media (biofilm cells) and then the biofilm was scraped into the LB, these tubes were sonicated at an amplitude of 6 microns for 30 seconds to break down the biofilm, and release the bacteria inside. Serial dilutions are performed down to a 10000 fold dilution which were then plated overnight. This method would determine whether the angled or vertical method provides a denser biofilm.
Biofilm Time Series To determine a base rate of dispersal of cells from a biofilm, slides from the "Biofilm Formation 2" experiment were transferred to 50ml fresh media (dissociated cells) and left for 4 hours. The slides were then transferred to 50ml of fresh media (biofilm cells) and then the biofilm was scraped into the LB, these tubes were sonicated at an amplitude of 6 microns for 30 seconds to break down the biofilm, and release the bacteria inside. Serial dilutions are performed down to a 10000 fold dilution which were then plated overnight. This method would determine whether the angled or vertical method provides a denser biofilm. Biofilm Quantification Results The serial dilutions did not dilute the culture enough to be able to count colonies and give a definite rate of dissociation from the bio film. Dilution
A = angled, V = Vertical.
Biofilm Time Series
As we were trying to specifically disperse areas of biofilm it was necessary to establish a base rate of dispersal of the biofilm without using any of the dispersal mechanisms we designed. This would allow us to show quantitativly the increase in rate of dispersal that our different dispersal biobrick could generate when compared to a biofilm of non-transformed bacteria. To measure the base rate of dispersal glass slides were put into 50ml tubes containing 20ml of LB broth. The LB covered around a third of the glass slide, this is the area where the biofilm would form. The LB was then inoculated with 20Îźl of overnight culture of Pseudomonas aeruginosa. These tubes
were then left on a bench top shaker at room temperature for a set amount of time (time points ranged from 1hr to 48hrs). After the biofilm had grown its alloted time the glass slide was carefully removed and placed into a fresh 50ml tube with 25ml of LB (which completely covered the biofilm) and left to allow the bacteria to disperse for 1hr. The slide was then transferred to a fresh 50ml tube with 25ml of LB. The biofilm is scraped off the slide using a thin flexible spatula. At this point both the dispersed cell and the biofilm scrapings were sonicated to stop clumping and plated in serial dilutions.
Biofilm Time Series Results