scholarly journals Development and Dynamics of Pseudomonassp. Biofilms

2000 ◽  
Vol 182 (22) ◽  
pp. 6482-6489 ◽  
Author(s):  
Tim Tolker-Nielsen ◽  
Ulla C. Brinch ◽  
Paula C. Ragas ◽  
Jens Bo Andersen ◽  
Carsten Suhr Jacobsen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Physiological State ◽  
Cell Aggregation ◽  
Flow Chamber ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Biofilm Bacteria ◽  
Fluorescent Cells ◽  
Confocal Scanning Laser ◽  
Green Fluorescent

ABSTRACT Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.

scholarly journals Survival of Salmonella Transformed To Express Green Fluorescent Protein on Italian Parsley as Affected by Processing and Storage

2005 ◽  
Vol 68 (4) ◽  
pp. 687-695 ◽  
Author(s):  
E. A. DUFFY ◽  
L. CISNEROS-ZEVALLOS ◽  
A. CASTILLO ◽  
S. D. PILLAI ◽  
S. C. RICKE ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Storage Temperature ◽  
Control Point ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Confocal Scanning Laser ◽  
Processing And Storage ◽  
Green Fluorescent ◽  
And Storage

To study the effect of processing and storage parameters on the survival of Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in suspensions that were preequilibrated to 5, 25, or 35°C and inoculated with Salmonella transformed to express enhanced green fluorescent protein. Loosely attached and/or associated, strongly attached and/or associated, and internalized and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage at 25°C and over 0, 1, 7, 14, and 30 days of storage at 4°C using surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7 days of storage were examined using confocal scanning laser microscopy. Temperature of the dip suspension had little effect on the attachment and survival of Salmonella cells on parsley. Regardless of the temperature or duration of dip, Salmonella was internalized. Immersion for longer times resulted in higher numbers of attached and internalized cells. Microscopic observations supported these results and revealed Salmonella cells near the stomata and within cracks in the cuticle. Storage temperature had the greatest impact on the survival of Salmonella cells on parsley. When stored at 25°C, parsley had a shelf life of 7 days, and Salmonella populations significantly increased over the 7 days of storage. For parsley stored at 4°C, numbers of Salmonella cells decreased over days 0, 1, and 7. After 7 days of storage, there were no viable internalized Salmonella cells detected. Storage temperature represents an important control point for the safety of fresh parsley.

Spore Formation in Bacillus subtilis Biofilms

2005 ◽  
Vol 68 (4) ◽  
pp. 860-865 ◽  
Author(s):  
D. LINDSAY ◽  
V. S. BRÖZEL ◽  
A. von HOLY
Keyword(s):  
Bacillus Subtilis ◽  
Nutrient Limitation ◽  
Fluorescent Protein ◽  
Point Of View ◽  
Spore Formation ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Confocal Scanning Laser ◽  
Green Fluorescent ◽  
Polytopic Membrane Protein

Spore formation by a Bacillus strain (Bacillus subtilis SpoIVFB-GFP) engineered with a green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVF) that fluoresces during sporulation was observed. Biofilms of B. subtilis SpoIVFB-GFP containing ca. 8 log CFU/ml vegetative cells and spores below the lower detection limit (i.e., <1 log CFU/ml) were allowed to develop on glass wool (37°C). These biofilms were subsequently exposed to nutrient limitation to stimulate spore formation, which was monitored for fluorescence by confocal scanning laser microscopy. Sporulation in corresponding planktonic cells was also monitored for comparative purposes. Planktonic B. subtilis SpoIVFB-GFP cells began fluorescing after 5 h, while B. subtilis SpoIVFB-GFP biofilm cells began fluorescing after 30 h. Results suggested that an existing biofilm of vegetative B. subtilis cells may be stimulated to form spores when exposed to conditions of nutrient limitation. From a practical point of view, it may be suggested that a window of time does exist before sporulation occurs in attached Bacillus biofilms highlighting the need for shorter operating runs between cleaning and sanitation of food-processing equipment surfaces.

scholarly journals Use of Confocal Microscopy To Analyze the Rate of Vancomycin Penetration through Staphylococcus aureus Biofilms

2005 ◽  
Vol 49 (6) ◽  
pp. 2467-2473 ◽  
Author(s):  
Kimberly K. Jefferson ◽  
Donald A. Goldmann ◽  
Gerald B. Pier
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Agents ◽  
Confocal Scanning Laser Microscopy ◽  
Planktonic Bacteria ◽  
Transcriptional Changes ◽  
Confocal Scanning ◽  
Biofilm Bacteria ◽  
Confocal Scanning Laser ◽  
Reduced Rate

ABSTRACT When bacteria assume the biofilm mode of growth, they can tolerate levels of antimicrobial agents 10 to 1,000 times higher than the MICs of genetically equivalent planktonic bacteria. The properties of biofilms that give rise to antibiotic resistance are only partially understood. Inhibition of antibiotic penetration into the biofilm may play a role, but this has not been proven directly. In this report, penetration of the glycopeptide antibiotic vancomycin into viable Staphylococcus aureus biofilms was analyzed by confocal scanning laser microscopy using a fluorescently labeled derivative of the drug. We found that while vancomycin bound to free-floating bacteria in water within 5 min, it took more than 1 h to bind to cells within the deepest layers of a biofilm. These results indicate that the antibiotic is transported through the depth of the biofilm but that the rate is significantly reduced with respect to its transport through flowing water. This suggests that, whereas planktonic bacteria were rapidly exposed to a full bolus of vancomycin, the bacteria in the deeper layers of the biofilm were exposed to a gradually increasing dose of the drug due to its reduced rate of penetration. This gradual exposure may allow the biofilm bacteria to undergo stress-induced metabolic or transcriptional changes that increase resistance to the antibiotic. We also investigated the role of poly-N-acetylglucosamine, an important component of the S. aureus biofilm matrix, and found that its production was not involved in the observed decrease in the rate of vancomycin penetration.

Location of Escherichia coli O157:H7 on and in Apples as Affected by Bruising, Washing, and Rubbing

2001 ◽  
Vol 64 (9) ◽  
pp. 1328-1333 ◽  
Author(s):  
STEPHEN J. KENNEY ◽  
SCOTT L. BURNETT ◽  
LARRY R. BEUCHAT
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Skin Surface ◽  
Confocal Scanning Laser Microscopy ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Log Reduction ◽  
Peptone Water ◽  
Confocal Scanning Laser ◽  
Green Fluorescent

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 μm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 m below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.

Computer-Assisted Analysis of Multi-Color Confocal Microscopic Datasets: Automated Light Microscopic Discrimination of Synaptic Relationships

1996 ◽  
Vol 54 ◽  
pp. 284-285
Author(s):  
M Wessendorf ◽  
A Beuning ◽  
D Cameron ◽  
J Williams ◽  
C Knox
Keyword(s):  
Nerve Fibers ◽  
Confocal Scanning Laser Microscopy ◽  
Computer Assisted ◽  
Nerve Terminals ◽  
Neuronal Somata ◽  
Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Entire Cell ◽  
Confocal Scanning Laser ◽  
Assisted Analysis

Multi-color confocal scanning-laser microscopy (CSLM) allows examination of the relationships between neuronal somata and the nerve fibers surrounding them at sub-micron resolution in x,y, and z. Given these properties, it should be possible to use multi-color CSLM to identify relationships that might be synapses and eliminate those that are clearly too distant to be synapses. In previous studies of this type, pairs of images (e.g., red and green images for tissue stained with rhodamine and fluorescein) have been merged and examined for nerve terminals that appose a stained cell (see, for instance, Mason et al.). The above method suffers from two disadvantages, though. First, although it is possible to recognize appositions in which the varicosity abuts the cell in the x or y axes, it is more difficult to recognize them if the apposition is oriented at all in the z-axis—e.g., if the varicosity lies above or below the neuron rather than next to it. Second, using this method to identify potential appositions over an entire cell is time-consuming and tedious.

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