the administration of gaseous nitric oxide is viewed as a novel biocidal agent in the efficacious management of numerous biofilm - forming microorganisms with particular emphasis on antibiotic resistant bacteria . the gno can be administered through a variety of mechanisms . examples of said administration of gno are set forth in commonly assigned u . s . patent application ser . no . 10 / 658 , 665 . in that application , it was reported the examples are as follows : referring now to fig9 , a gaseous nitric oxide ( no ) delivery device 1 is shown connected to a source of infected blood from either a patient 2 or a stored blood source 3 , and a pumping system 4 , through lines 5 , 6 . the nitric oxide ( no ) source 7 , can be a pressurized cylinder containing nitric oxide ( no ) gas , and a nitric oxide flow control valve / pressure regulator 8 , delivering nitric oxide ( no ) to the gaseous nitric oxide delivery device 1 through supply tubing 9 and an optional gas blender 15 . the infected blood is then exposed to a controlled amount of nitric oxide ( no ) by the gaseous nitric oxide ( no ) delivery device 1 , and the treated blood is then returned to either a patient 2 or a stored blood source 3 , through line 100 . the treated blood can still carry the nitric oxide when it returns to the patient or the stored blood source . by carrying a sufficient quantity of nitric oxide into the patient , which is completely contrary to the teaching of igo , the nitric oxide can reduce the pathogens throughout the whole body of the patient . in fig9 , the nitric oxide ( no ) gas source 7 is a pressurized cylinder containing nitric oxide ( no ) gas . while the use of a pressurized cylinder is the preferable method of storing the nitric oxide ( no ) containing gas source 7 , other storage and delivery means , such as a dedicated feed line can also be used . typically the nitric oxide ( no ) gas source 7 is a mixture of n 2 and no . while n 2 is typically used to dilute the concentration of no within the pressurized cylinder , any inert gas can also be used . when the no gas source 7 is stored in a pressurized cylinder , it is preferable that the concentration of no in the pressurized cylinder fall within the range of about 800 ppm to about 1200 ppm . commercial nitric oxide manufacturers typically produce nitric oxide mixtures for medical use at around the 1000 ppm range . extremely high concentrations of no are undesirable because accidental leakage of no gas is more hazardous , and high partial pressures of no tends to cause the spontaneous degradation of no into nitrogen . pressurized cylinders containing low concentrations of no ( i . e ., less than 100 ppm no ) can also be used in accordance the device and method disclosed herein . of course , the lower the concentration of no used , the more often the pressurized cylinders will need replacement . fig9 also shows source of diluent gas 11 as part of the no delivery device 1 that is used to dilute the concentration of nitric oxide ( no ) for delivery to the gaseous nitric oxide ( no ) delivery device 1 through line 13 . the source of diluent gas 11 can contain n 2 , o 2 , air , an inert gas , or a mixture of these gases . it is preferable to use a gas such as n 2 or an inert gas to dilute the no concentration since these gases will not oxidize the nitric oxide ( no ) into no 2 , as would o 2 or air . the source of diluent gas 11 is shown as being stored within a pressurized cylinder . while the use of a pressurized cylinder is shown in fig9 as the means for storing the source of diluent gas 11 , other storage and delivery means , such as a dedicated feed line can also be used . the nitric oxide ( no ) gas from the nitric oxide ( no ) gas source 7 and the diluent gas from the diluent gas source 11 preferably pass through flow control valve / pressure regulators 8 , 120 , to reduce the pressure of gas that is admitted to the gaseous nitric oxide ( no ) delivery device 1 . the respective gas streams pass via tubing 9 , 13 , to an optional gas blender 15 . the gas blender 15 mixes the nitric oxide ( no ) gas and the diluent gas to produce a nitric oxide ( no )- containing gas that has a reduced concentration of nitric oxide ( no ). preferably , the nitric oxide ( no )- containing gas that is output from the gas blender 15 has a concentration that is less than about 200 ppm . even more preferably , the concentration of nitric oxide ( no )- containing gas that is output from the gas blender 15 is less than about 100 ppm . the nitric oxide ( no )- containing gas that is output from the gas blender 15 travels via tubing 160 to a flow control valve 17 . the flow control valve 17 can include , for example , a proportional control valve that opens ( or closes ) in a progressively increasing ( or decreasing if closing ) manner . as another example , the flow control valve 17 can include a mass flow controller . the flow control valve 17 controls the flow rate of the nitric oxide ( no )- containing gas that is input to the gaseous nitric oxide ( no ) delivery device 1 . the nitric oxide ( no )- containing gas leaves the flow control valve 17 via flexible tubing 180 . the flexible tubing 180 attaches to an inlet of the gaseous nitric oxide ( no ) delivery device 1 . the inlet for 1 might include an optional one - way valve that prevents the backflow of gas . in one preferred embodiment of the invention , the gaseous nitric oxide ( no ) delivery device unit 1 includes an no sensor 140 that measures the concentration of nitric oxide ( no ) in the treated blood or fluid stream . the nitric oxide ( no ) sensor 140 and nitric dioxide sensor ( 15 ) preferably report the concentrations of no and no 2 to a controller within the gaseous nitric oxide ( no ) delivery device 1 , for source gas flow control and alarm . the sensors , 140 , 15 , can be chemilluminesence - type , electrochemical cell - type , or spectrophotomentric type sensors . in a similar embodiment , the present invention takes the nitric oxide gas composition in line 18 and directs the nitric oxide gas composition into a patient &# 39 ; s breathing orifice , like a nose and / or mouth . the delivery device can be a conventional gas mask or plastic tubing . fig1 illustrates a block diagram representation of the device 220 , which can be an alternative version of item 17 . the device 220 has a power source 220 that provides sufficient voltage and charge to properly operate the device 220 . the device 220 also has a main microprocessor 240 that controls the operation of a solenoid valve 260 , also within the device 220 . the solenoid valve 260 operates in conjunction with operating parameters that are entered via a data entry keypad 201 and the input from a pressure sensor 280 . the operating parameters and the operating status of the device 220 are displayed on an lcd display 210 . the device 220 has a pressure regulator 266 . the pressure regulator 266 reduces the pressure of the nitric oxide to less than 100 psi so it can be administered to the patient 2 without damaging the patient &# 39 ; s organs , in particular the lungs , from too much pressure . calibrating the flow through the solenoid valve 264 is obtained by selecting the pressure of the pressure regulator 266 and controlling the time that the solenoid valve 264 is open . thereby , the valve 264 allows a precise amount of nitric oxide gas composition to be delivered through the gas delivery line 18 , which delivers the nitric oxide to the patient &# 39 ; s breathing orifice ( s ). the pressure sensor 280 is designed to detect a drop in pressure in the gas delivery line 18 , when the patient initiates a breath . this pressure drop signals the main processor 240 to open the solenoid valve 264 for a pre - programmed period of time . among the parameters that are programmed into the device are : total breaths , start delay , pulse time , pulse delay , and re - trigger lock . total breaths : this parameter is the number of breaths programmed into a run of the device 220 . each time a breath is detected as identified above , a pulse of nitric oxide gas composition is injected into the breath of patient 2 . breaths that occur during a locked out time of the predetermined time frame are not counted as breaths . after the programmed number of breaths are counted , the program stops automatically and nitric oxide gas composition is no longer injected into any breaths of the patient . this number can be set anywhere from 0 to unlimited number of breaths . if the number is set at 0 then the auto shutoff is disabled and breaths will be injected with nitric oxide until the user stops the device . start delay : this parameter is the programmed delay time in minutes that the user can set . the injection of nitric oxide gas composition into each breath will begin automatically after “ start delay ” minutes . it will then continue for the number of total breaths and then the device 12 stops automatically . pulse time : this parameter is the length of time that the solenoid valve 264 will open for delivery of nitric oxide gas composition . the resolution is 0 . 1 seconds and the range is 0 . 1 sec to 0 . 9 seconds . if the regulator is set at 50 psi then each second of the solenoid valve 264 opening 31 cc of nitric oxide gas composition . if the regulator pressure is set at 30 psi then each 0 . 1 sec solenoid valve 264 opening represents 21 cc of nitric oxide gas composition . for example , if the regulator is set at 50 psi and the pulse time is set at 0 . 3 seconds then each detected breath will be injected with a pulse of 0 . 3 seconds or about 90 cc of nitric oxide gas composition . pulse delay : this parameter is the length of time that the machine waits after detecting the beginning of a breath before opening the solenoid valve 264 to inject a pulse of nitric oxide gas composition . this allows the user to control the position of the bolus of nitric oxide gas composition in the breath . for example , if the user sets the solenoid valve 264 at 0 . 4 seconds , then 0 . 4 seconds after the beginning of the breath is detected the solenoid valve 264 will open to inject the nitric oxide gas composition pulse . retrigger lock : this parameter is the total time that the machine will ignore new breaths beginning at the detection of a new breath . if this parameter is set at 4 . 5 seconds then the device 220 will wait , after detecting a breath , for 4 . 5 seconds before recognizing a new breath . full or half breaths that are initiated by the patient during this lockout time will not be counted and no nitric oxide gas composition will be injected . if the breath is initiated before the lockout expires and the patient is still inhaling when the lockout expires then it will be recognized as a new breath and it will be counted and injected with nitric oxide gas composition . the data entry keypad 202 contains five active button switches defined as follows : start / pulse key : this key is used to start a run . the user is required to confirm the start by pressing an up key or to cancel by pressing a down key . when a run is in progress , pressing this key will cause the run to pause . the run is then resumed by pressing the up key or stopping the run by pressing the down key . up key : this key is used to confirm the start of the run , to resume a paused run and also to increment valve changes . down key : this key is used to cancel a started run , end a paused run and also to decrement valve changes . next key : this key is used to switch screen pages on the lcd display . purge key : this key is used to open the solenoid valve 264 for two seconds to purge the line . this key is not active during a run . the lcd display can display at least four screen pages , defined as follows : each screen page displays a status line . the status variations include not running , waiting , running , paused , purging and start pressed . the main screen page has a row of asterisks on the top line . this is the only screen available when the key switch is in the locked position . this screen displays the total breaths detected and also the total breaths that will cause the run to stop . the second page shows two valves . the first is the start delay valve . when the screen first appears the blinking cursor shows the value , which can be changed by pressing either the up or down key . by pressing the next key switch the cursor to the second value on the screen is total breaths . the third page allows the user to change the pulse delay and the pulse time . in any case , this embodiment of the invention allows the nitric oxide gas composition to be injected into a patient &# 39 ; s lung , preferably when the patient is inhaling , of a sufficient quantity that nitric oxide is capable of penetrating both the epithelial and capillary basement membranes to allow the nitric oxide to contact the numerous blood cells to reduce pathogens in the blood system and throughout the body . other embodiments of the dispenser apparatus of the nitric oxide gas are disclosed in u . s . pat . no . 6 , 432 , 077 , which is hereby incorporated by reference herein . the dispenser can be any device that can apply nitric oxide to any object that can contain a biofilm . the object being selected from a group consisting of a medical device , a conduit for industrial , home , office space , municipal , or medical purposes , and an animal for internal and / or external applications . alternatively , this latest method can provide the nitric oxide gas continuously , just not when the patient 2 inhales . in addition the gno can be directed into application systems via pressurized cylinders to the specific target interface . a number of experiments were undertaken to determine the efficacy of various dose concentrations of exogenously applied gaseous nitric oxide on the microorganism burkholderia cenocepacia . b . cenocepacia is an opportunistic pathogen that plays a role in the formation of biofilms and can cause marked lung infections in cystic fibrosis patients . b . cenocepacia is also associated with increased rates of sepsis and death . objective : to determine if exposure to gaseous nitric oxide ( gno ) affects the ability of b . cenocepacia c8963 to form a biofilm in a 96 - well microtiter dish assay . methods : b . cenocepacia c8963 , a non - mucoid isolate from a cystic fibrosis ( cf ) patient , and c9343 , a mucoid isolate from the same patient , were spotted on luria broth agar and grown at 37 ° c . overnight . luria broth containing 0 . 5 % ( w / v ) casamino acids was dispensed into 96 - well polypropylene microtiter dishes ( 100 μl per well ) and the wells inoculated with the c8963 or c9343 using a pin - inoculation device . blank wells were not inoculated . dishes were incubated in a humidified , closed plastic container for 24 hours at 37 ° c . ( experiment 1 ) or in the outer chamber of the matrix incubator ( a humidified incubator with controlled air flow ) for 27 hours at 37 ° c . ( experiment 2 ). at 24 hours ( experiment 1 ) or 27 hours ( experiment 2 ), one dish was processed for staining of the bacterial biofilms . the remaining dishes were incubated in the inner treatment arms of the matrix incubator at 37 ° c . in the presence or absence of 200 ppm gno . one dish for each of the conditions (+ gno or − gno ) was processed for biofilm staining at 32 , 36 , and 48 hours . to stain biofilm growth , planktonic bacteria were removed from the microtitre dishes by discarding media and cells . biofilms were washed to remove remaining non - adherent bacteria in two successive tap water washes . water was shaken from the wells and the dishes inverted and tapped vigorously on a stack of paper towels to remove as much water as possible . adherent growth was stained by adding 125 μlof a 0 . 1 % ( w / v ) solution of crystal violet to each well and incubating at room temperature for 15 minutes . crystal violet was discarded as for the previous washes and excess stain was removed in three successive tap - water washes . excess water was removed by vigorous tapping as before and the stained dishes allowed to air dry . to quantitate biofilm formation , 200 μl of 95 % ethanol was added to each well , incubated at room temperature for 15 minutes , and 125 μl from each well was removed to a clean flat - bottomed polystyrene microtitre dish . the absorbance at 595 nm was read on a bio - rad model 3550 microplate reader . the average reading from “ blank ” ( uninoculated ) wells containing only media was subtracted from each “ test ” well . the [ test - blank ] averages and standard errors of the mean ( sem ) were calculated for each condition and time . results : as expected , the mucoid c9343 isolate did not form biofilm under any conditions ( fig1 ). this is consistent with its previous behavior since the mucoid exopolysaccharide interferes with adherence to surfaces ( 1 ). overall , the non - mucoid c8963 isolate continued to form biofilm in the presence of gno but growth was lower than in the absence of gno . c8963 biofilm growth in the presence of gno was greater than in the absence of gno at 32 hours , but by 36 hours , growth in the presence of gno was significantly lower than in the absence of gno in both experiment 1 ( fig1 ) and experiment 2 ( fig1 ). biofilm growth remained consistently lower in the presence of gno for the remaining time points up to 48 hours in both experiments ( fig1 and 12 ). in experiment 1 , maximum biofilm growth occurred at approximately 36 hours ( fig . 11 ) but in experiment 2 , maximum biofilm growth did not occur until 48 hours or more ( fig1 ). discussion : b . cenocepacia c9343 was a mucoid pulmonary isolate from a cf patient that was previously shown to be a poor biofilm former ( 1 ). b . cenocepacia c8963 was a non - mucoid pulmonary isolate from the same cf patient and was shown to be a competent biofilm former ( 1 ). to determine if exposure to gno affected biofilm formation by these organisms , both were grown in the presence and absence of 200 ppm gno . the organisms were grown for 24 hours without gno to establish the biofilm , then exposed to gno or air only in the final 24 hours of the assay . c9343 served as a negative control since it did not form biofilm under any condition . the presence of gno did not induce biofilm formation by this organism . for this reason , c9343 was not included in experiment 2 . c8963 formed biofilm in two independent assays . in both cases , introduction of gno after 24 hours ( experiment 1 ) and 27 hours ( experiment 2 ) resulted in increased biofilm growth at 32 hours compared to biofilm growth in the presence of the carrier gas ( air ). at first , gno likely provides a source of nitrogen to the growing bacteria , and that this is advantageous while the effective concentration of gno dissolved in the media is low . at all subsequent time points , c8963 biofilm growth was lower in the presence of gno . this implies that once the concentration of gno equilibrated to 200 ppm within the biofilm system , it decreased the amount of biofilm formation by c8963 compared to the carrier gas . thus , gno acted as a nutrient when present at a low effective concentration and as a biofilm inhibitor at higher effective concentrations . the maximum amount of c8963 biofilm formation was higher in experiment 2 ( a 595 = 0 . 507 ) than experiment 1 ( a 595 = 0 . 441 ). this difference could be due to different initial inocula received or due to differences in the way the organisms were grown on the first day of the experiment . a more detailed time - course and repetition of the growth conditions from experiment 2 would answer this question . gaseous no affected the biofilm growth of b . cenocepacia c8963 in two ways : at low initial concentrations it enhanced biofilm growth and at the 200 ppm final concentration it inhibited biofilm formation in the 96 - well microtiter dish assay . chart 1 . b . cenocepacia c8963 biofilm formation in the presence and absence of gno . biofilms were allowed to grow for 24 hours in air before being grown in the presence (+ no ) and absence ( control ) of 200 ppm gno . squares represent c8963 biofilm growth and circles represent c9343 biofilm growth . average a 595s and sem appear below . for 27 hours in air before being grown in the presence (+ no ) and absence ( control ) of 200 ppm gno . average a 595 s and the term “ ambient ” refers to the gases that surround the targeted interface . if the nitric oxide is exposed to blood , the nitric oxide can work within the blood for a very brief period of time until it is modified by the hemoglobin . the modification is normally when the nitric oxide attaches to the hemoglobin . once attached , the nitric oxide is normally not able to destroy a biofilm with the present apparatus . it is appreciated that various modifications to the inventive concepts described herein may be apparent to those of ordinary skill in the art without departing from the scope of the present invention as defined by the herein appended claims .
the administration of gaseous nitric oxide as a biocidal moiety is proffered as a de novo treatment in the control and eradication of biofilms . the present invention relates to the use or methods of application of exogenous nitric oxide gas as a stand alone biocidal agent or in cohort with any or all adjunct vehicles in the control of biofilms generated by microbial organisms , i . e ., bacteria , protozoa , amoeba , fungi etc . further , the present invention introduces the concept of utilization and methods of application of gaseous nitric oxide in control and eradication of biofilm forming microorganisms . noteworthy areas of application are offered as examples . they include , and are not limited to , air and / or water heating / cooling distribution systems in facilities such as hospitals and laboratories , surfaces of medical devices , household surfaces , dental plaque , dental and / or medical water treatment lines , industrial pipelines , water treatment and distribution facilities and fluids sterilization . various specialized delivery apparatus will be designed to facilitate nitric oxide gas administration to each specific unique application .