King B Agar Lab Report

Agar and Media Preparation— Agar plates containing King’s B Agar were often used throughout the experiment to support growth of Pseduomonas fluorescens. A recipe was used that included a mixture of 10g Proteose Peptone #3, 1.5g Potassium Phosphate Dibasic (K2HPO4), 30ml 50% Glycerol, ~965ml water and 20g agar. The mixture, post- autoclave, was left to cool and 5ml 1M Magnesium Sulfate (MgSO4) was added and created about 40 plates. King’s B Medium was made using the same procedure as the King’s B Agar with the exception of adding 20g of agar. These recipes were referred to later in the protocol to create additional plates and liquid cultures when growing multiple strains. King’s B Agar and Medium served as enrichment media that enhance growth
of fluorescin bacteria (King, Ward and Raney, 1954).
Bacteria Stock Cultures, Static Cultures and Colony Growth Plates— Stock cultures were created for each strain, starting with the original SM and WS+ strains from the provided lab stock.
Single colonies from the SM and WS+ lab stock were inoculated into 500μl of sterile King’s B medium and vortexed. 250μl of each inoculated suspension were added to a sterile screw top tube and 250μl of sterile glycerol was added. This was stored in the -80oC freezer and allowed for long term use and storage of an original strain or mutated strain. The stock cultures were only removed for plate streaking and static cultures to ensure the use of consistent strains throughout the entire experiment (Capuccino and Sherman, 1983). More stock cultures were created for any mutant strains that arose throughout the
experiment.
The lab stock plates of SM and WS+ were also used to streak King’s B Agar plates for isolation of single colonies. The streaked plates were incubated at 30oC for 2-3 days to allow for growth. The morphologies of the two different strains were recorded and compared for future identification purposes when assessing strain variation. These microbial cultures that are grown on plates can be used as working stocks for about 2 weeks when kept in a 4oC refrigerator to halt additional colony growth.
Using the visual from plated colonies of SM and WS+, static cultures were grown and compared.
To construct liquid cultures of bacteria, 6ml of King’s B medium was added to a 25mm test tube. In a microcentrifuge tube, a single colony of the corresponding strain was inoculated into 200μl of medium. 10μl of the cell suspension was taken and inoculated into the 6ml of medium and vortexed, which served as the final microbial liquid culture. The static cultures were incubated at 30oC, apart from the conditions in Rainey and Travisano at 28o C, and were observed after 3 and 5 days of growth, often yielding the formation of a biofilm at the liquid interface of each culture (Rainey and Travisano, 1998). The production of biofilms in liquid microbial cultures and colony growth on agar plates allowed for the contrast and comparison of SM cells, WS+ and mutations that
arise.
Mutant Strain Isolation— After creating 3 replications of the WS+ and SM strains provided in 3 and 5 day microbial liquid cultures, the samples were vortexed and OD600 values were recorded. From the vortexed cultures, 10-fold dilutions were made and plated and incubated at 30oC. To explore the diversity among the rapidly growing Pseudomonas fluorescens, the plated dilutions were observed under stereoscopes, allowing for better viewing and quantification of the frequency of the mutant WS morphs in the SM plates. The development of WS colonies on the WS dilution plates identified the evolution of genetic diversity from the dominant SM strain in the spatially heterogeneous environment created after vortexing (Rainey and Travisano, 1998). The WS colonies that developed on the SM dilution plates were compared to the morphology of the WS+ strain provided by the original lab stock (Fig…).
Four of the different WS strains that appeared on the SM plates were chosen to serve as the new mutant WS strains, later named and referred to as PS15-13-A, PS15-13-B, PS15-13-C, and PS15-13-D. Each mutant strain (A, B, C, D) was a mutant that emerged on dilution plates from separate SM static cultures. The single colonies of the WS mutant strains from the SM plates were inoculated and streaked onto separate agar plates for further isolation of the diverse mutant strains. With the four new strains, microbial plates, static cultures and archival stocks were created.
Congo Red and Competition Assays— The Congo Red-binding assay was completed to quantify the amount of cellulose produced by the four mutant strains compared to that of the SM strain. Twelve microcentrifuge tubes were created from 3 different colonies for each of the four mutant strains and then 3 for the SM strain and 3 for the WS+ strain for future comparisons among strains. Two-step dilutions were then performed to make 0.004% Congo Red (CR) dye, beginning with 7µl of 4% CR dye with 693µl sterile water. The inoculated tubes with the corresponding strains were vortexed and the initial OD600 values were recorded. The supernatant from each tube was removed and 500µl of 0.004% CR dye was added to each tube and resuspended. The tubes were incubated at 37oC for two hours and final OD490 readings were taken. The absorbance was calculated then multiplied by 500µl and then .04 µg/µl to determine the mass (µg) of cells bound to the Congo Red in each tube. By taking the mass (µg) divided by the initial OD600 value, the final amount of Congo Red cells per strain was calculated in µg/OD600. In addition, following the completion of growing Psedomonas fluorescens on the Congo Red plates, the morphologies, dominant growth and behavior, and the visible binding of Congo Red were observed (Fig...).
In addition to the Congo Red-binding assay, a competition assay was completed to compare the competitive relationship and fitness of SM and WS+ to two of the mutant strains, PS15-13-A, PS15-13-B. We plated 100:1 dilution plates for the four strains as follows: SM:WS+, WS+:SM, SM:WS-A, SM:WS-B, WS-A:SM, WS-B:SM. The dominant strain and colony counts for the dilutions were recorded from day 0 and day 5 plates. The relative fitness was calculated from the ratio of the cell doubling time for the rare strain versus the common strain.
DNA Sequencing of PCR Products— Four PCR products were created for sequencing awsX and wspF as the provided primers: A* (strain A + awsX), AO (strain A + wspF), B* (strain B + awsX), and BO (strain B + wspF). The four PRC samples, the awsX control, wspF control and the DNA ladder were loaded into the gel electrophoresis wells for DNA amplification of the products (Fig…). The DNA amplification results were then sent for sequencing. The sequencing was compared to the given SM sequence and the SBW25 sequence on NCBI and the results were analyzed using the ApE program with the comparison of the three in order to identify true mutations and errors.
Biofilm Strength Tests— Based on the results from the Congo Red-binding assay and the biofilm growth characteristics of SM versus WS strains, test tube sizes were manipulated to further examine the relationship between diversity, cellulose production and biofilms. In the prior sections, all liquid cultures were grown in 25mm test tubes. A total of 54 test tubes were gathered: 18- 16mm, 18- 25mm, and 18- 40mm. In replicates of three, the biofilms of three strains WS+, WS-A, and WS-B were tested after 3 and 5 days of growth. To remain consistent, 10ml of King’s B medium was added to all 54 test tubes with the three different sizes. Three microcentrifuge tubes with 200µl of King’s B medium were made and one tube per strain was inoculated with single colony growth from the corresponding strain. From the WS+ microcentrifuge tube, 10µl was taken and inoculated into the proper test tubes equaling 18 tubes. This was repeated for the WS-A and WS-B strains, totaling 54 tubes. After 3 days and 5 days of growth, each biofilm produced for all 54 tubes was strength tested with 425-600µm glass beads. The weight of the beads added until the biofilm collapsed was recorded (Fig…) and the three strains were cross-referenced with the variable test tube widths.