It seems like every time we go to a complaint there's a myth lab somewhere, but I've never seen this many at one time. And like I said, the detectives that are charged with cleaning these things up say that that's the biggest one they've ever seen, and that's something very unusual. This is not the crystal myth like you would see in Breaking Bad. This is just a homegrown one-step nasty stuff that I can't even believe people would put into their body. But this is the biggest one I've ever seen.


a pure culture study of pickles and acetic acid. Acetic acid bacteria belongs to the family acetylbacteria, which includes several species and genera. The main species that are involved for vinegar production include acetylbacter, glucobacter and gluconoacetobacter. These species have ability to oxidize ethanol to acetic acid and also have ability to the high resistance to acetic acid which is released into the fermentive media. For production of pure culture study of acetic acid, falling procedure is used. We have left grape samples and left the samples for over ripening. After over ripening, 5 cereal dilutions were made by using the similar procedure as described in previous video for cereal dilution preparation. Prepare gyc media for the inoculation of grape samples. This is already prepared gyc media plate for sterilization of media at antibiotic. The solution is prepared by measuring according to 10 mg per ml solution. For addition of antibiotic, we use syringe filter, place the syringe filter and adjust on syringe. Now take antibiotic, mix it properly, inoculate the plate with grape sample from 10 to 5 dilutions. For spreading, place the plate near the flame inoculum evenly throughout the surface of agar. When we distribute the sample on agar surface, inoculum is fully absorbed in the plate at incubator at 30 degree centigrade for 7 days. This is the aceto-vector growth on gyc media. For differentiation of aceto-vector and gluco-vector, we prepared another media known as car media. This is already prepared agar plate with inoculating loop to red hot to cool for 1 to 2 minutes. Now, colunis showing colosone, take those colunis and inoculate on car media by using zigzag manner. For incubation, place the plates at 30 degree centigrade for 4 days. This is the growth of aceto-vector and gluco-vector on car media. The basic differentiation is aceto-vector turns media into green and then into the yellow, whereas gluco-vector remains the media color green. Aceto-vector morphologically characterized and examined according to their acetic acid production with the help of bromocrysol green dye. There are differentiated on the presence of acetic acid production which turns the media color into yellow or green due to the presence of bromocrysol green dye. Aceto-vector turns the media to yellow color whereas gluco-vector turns the media into green.


Good evening everyone, I hope everyone is safe and sound at home right now. As we all know these are kind of trying times and challenging times and you know we wish we could all be together and but I think for now virtual rooms like this are how we're all going to be meeting. So actually tonight, this project Raga Makam was supposed to be playing at the Rubin Museum of Art. And unfortunately for all these circumstances we weren't able to do it but we figured the show must go on online. So, you know we decided that we're going to do this and we're really thankful to the culture hub for having us and for setting this platform up and it's something you know, it's a little experimental we haven't done this before. And the technology is not so incredible that we can all play simultaneously. We're not yet the culture hub is really together a beautiful platform for us to play together. And we're going to take turns we're going to go one by one and we're going to kind of pass the baton and and go through these two musical traditions. This project is kind of exploring the crossroads of Indian Cascaraga and Arabic Makam and it's a much larger band. We're going to have 10 or 11 at the Rubin Museum but tonight we are down to four and it'll be myself and Amir El Safar Jay Gandhi and George Zedaya. And we're really looking forward to playing for you. We hope you're all well at home and sit back and enjoy and I'll pass the baton over to Amir. And hello everybody. Good evening from my living room to yours from our living rooms to yours. Yeah, tough times living in lockdown and not being able to perform and being able to share music, the way that we all have done all of our lives and that humanity has done since the very beginning. I do believe that there are ways of still communicating and still staying in close proximity through sound. And this is a crude means of doing it through the internet. But our sound and vibrations are happening in this moment right now. And we're all playing together in various locations and sharing with you and all of your locations throughout the world. It's truly a joy to share with you this evening. And we hope that the fidelity and that the technology will be able to serve well enough to so that the message can be delivered and the sound can come through. Thank you everyone. Thank you everybody. Thank you everyone. Yeah, please I'm here. Go ahead. Thank you. Yeah, but thanks everyone for tuning in. This was a wonderful experience to play with these guys it's been so long since we all met. And it's such a nice experience and some were grateful to even have this. There are challenges of hearing each other and, you know, and being able to interact and we wanted to have the human element be a part of our music today so it was very important to us to make sure that we played together and not just separately, as we all are today so Before I forget, I just also want to thank some some folks that also helped make this happen. Of course, Culture Hub and want to thank the India Center Foundation also and Raul Bhavnani for their partners with Brooklyn Raga Massive and Lincoln Center for putting this project and getting us all together and helping kind of, you know, get some shows on the map. So we like to thank India Center, Mira, Google, David Elm Bogan, they're all watching I'm sure hey guys. Anybody else want to say anything. Yeah, thank you so much for having the opportunity to play a game together it's great to be able to hear each other and interact again I mean, since we can't do it in real life anymore. Right. For the time being, hopefully. It's really pleasant to me that I was able to play again, you know, it's been like two weeks since I've been shot at home and, you know, just playing by myself with great musicians like these guys. Thank you. I don't thank you and Jay, thank you so much. And the technical. Thank you so much. India Foundation is so much for having us and for trying this. Yes, to reach out to people without being on stage in the concert hall for the time being. Yeah. Yeah, thank you everybody and we're going to keep this up. Hopefully we'll do more performances like this. This week we were supposed to play three times together so right now with larger groups so right now we're making up for it with the four of us. And I think it's the beginning of something I actually, despite all the latencies and delays and sound issues and I really feel like I'm playing with with Arun and Jay and George in a way that I wouldn't, if we weren't, it just agreeing to be in the same space together. And I think that we're all connected. And this experience, just the rehearsals and this performance. I'm making it evident to me that we really are connected and sort of a, not just what we're doing in our, what we're able to hear and see but also just in our very existence at every moment so we're playing together through this internet connection but I think we're also playing together in a larger sense through through the ether or whatever you want to refer to it as but and that means that we're not only connected with each other as musicians but connected with all of you at home watching this with all the other people on the planet right now those who are suffering directly from the coronavirus and those were waiting us out and all of us are in the same boat every every place on the planet right now so but we really are connected and our hearts are connected throughout so let's let's let that connection grow. Yes, thank you so much, everybody.  If it transmits, it's, it's all worth it so. So, um, so stay connected, everybody, lots of love and hopefully we keep on doing this. All right. Yeah. So, thank you everyone.


Pay attention to all the training in for both sides, so four by two, one for the night time things, because even just landing on the deck can be dangerous. I started to get in stand before, whenever I landed on the deck before. It makes it easier for us to go in and out, especially for you guys to get in and out. This is for your efficiency. I'm going to sling him down so he doesn't want to get in too.


18th meeting of our 2013 Common Council 14 Common Council year would the clerk please call the roll 15 present Oliver and Dorn is excused please stand and join me pledge allegiance to the flag of the United States of America and to the Republic for which it stands one nation under God indivisible with liberty and next is approval of minutes from our last meeting Alderman Hammond thank you mr. Mayor I move with the minutes of the previous meeting approve the minutes previous of the minutes second to approve the minutes from our last meeting is there any discussion see none will the clerk please call the roll 15 ayes we have no council appointments tonight move on to public forum that would be milk storm Scott you signed right have your address please yes it's 1736 Marvin Court thank you you'll have five minutes I would like to thank the mayor and the city clerk's department for giving us citizens a time to speak at these public forums the reason I chose to speak this evening is to set the record straight when I read a letter to the editor by a former older person challenging the credibility of our present mayor she was using of course her political savvy and cleverness I have written letters to the editor for approximately about 40 years her opinions were of no value to the city of Sheboygan when the opinion editor Pat Panckers spoke to me of one of my many editorial letters I informed him that when I along with my identical twin brother we did attend the University of Wisconsin Extension Center up there in the Walsall area that was in 1947 49 48 in our English composition book written by two professors from the Columbia University of New York City was a chapter and how to write letters to the editor of the printed media I learned to use enough words to explain your comments and make sure that they are truthful and factual also I learned that truthfulness is not always beautiful but then of course there is no beauty in untruthfulness whether we are rich or poor it is through our self-discipline and a commitment to the truth that we can restore our city and our county to the number one status that we enjoyed many years ago let us celebrate the Christmas season this year as we did in the years past merry Christmas and good tidings and may of the year year be filled with prosperity and richness thank you thank you for those comments Wayne Emmer have you had this please Wayne 906 South 15th Street I am I'm representing the Tavern League of Wisconsin or Sheboygan County Tavern League here we are opposed to this public intoxication ordinance that they're trying to pass we feel it's going to hurt the individual businesses and because we have become such a tourist attraction lately it's going to hurt our waterfront all of our festivals that we have running we we actually feel that there's enough laws right now to take care of the problem that we have they've they've mentioned they mix drugs and alcohol together alcohol is a legal substance drugs is not a legal substance they have a number of remarks in here about drugs comparing them to alcohol and that it's not right so thank you Brandon Givonovich may I have your address please West 7911 Center Road Columbia Wisconsin thank you I'm here also about the public intoxication I guess I'm not questioning the good intentions that were used to construct this new ordinance but rather I question the actual need definitions and unintended consequences of this proposed ordinance the need of any new regulation law or ordinance should not be examined but scrutinized and held to the highest standard it's at the heart of American freedom to only create and enforce laws that must be passed banning big gulps may have been considered in New York but here in Wisconsin I like to believe that we appreciate personal accountability more so than they in regards to this ordinance it appears pretty redundant as there are already multiple laws against every behavior mentioned in section one a2 if there is a problem with enforcing prosecuting or escalating the penalties at any of the existing ordinances it would seem more prudent to alter the existing laws rather than fabricate a new one to address issues that are already in the books we should also wonder how much vagarity we want to codify into our laws the lack of any quantifiable test in this ordinance makes us rely on human judgment as a sole means of defining an impaired person that humans make mistakes is undeniable the problem is all humans be they police officers or priests make mistakes in both decisions and their judgments personally and professionally to believe that a vague law will always be enforced only based on its good intentions rather than the languages written in seems to be a utopian dreaming at best and a gross misunderstanding of both humans and history at worst the final question I'd like us to consider before this ordinance is voted on is the unintended consequences it may cause once again I'm not questioning the good intentions that were used to propose this new ordinance but rather the real effect on public perception and then public behavior that passing this law would create any law that seems to encourage impaired people not to walk home then by default seems to encourage them to drive by simply driving impaired people then prevent a police officer from smelling their breath looking at their eyes hearing them slur or see them stumble in the snow unless they make a moving violation I'm sure it's not the intent of this ordinance to promote drunk driving but once again we're dealing with the reality of the public's perceptions not the good intentions at the end of the day I can't see any reason why this ordinance must be passed it is repetitious vague and punishes a safe alternative to drunk driving the proposal of this ordinance might have been crafted with good intentions but we all know what good intentions pave the way to so thank you very much for your time and consideration thank you very much Don Sinclair was that Dan is that Dan you're close and your address again please 434 pen thank you you have five minutes I guess I also want to speak on public intox I think coming through here I know my clientele I have a huge clientele comes in from really all over the city or not city county right now I have Milwaukee people coming up Chicago people coming up I'm pretty much a tourist destination now I have a connection with the local hotels where people are walking back and forth my passing this law all you're going to do is encourage my people to drive why would you walk I mean you're going to go into drug and alcohol classes it's ridiculous it goes too far guys it's no common sense to it we already have enough laws and books that everyone else has said so think about what you can do just think if one person a tourist gets ticketed for walking drunk I tell you that's going to go through the tourist community like you know hotcakes and we'll be another laughing stock of the country so think about what we're voting on thank you at the end of public forum next is the mayor's announcements as the year comes to an end we see several of our long-time and well-serving employees deciding to retire it's a sad time but we have to honor some of those individuals this evening and give them some recognition the first one is chief Jeffrey Herman Jeff started his career with the Sheboygan fire department 1981 in the position of firefighter in 1987 he was promoted to acting lieutenant in charge of the fire rather the engine company in 1990 fire lead person in 1998 fire lieutenant and 2004 captain and in 2005 shift commander and on january 1st of 2010 he was promoted to fire chief the position that was the highlight of his career some of the additional highlights of his leadership within the department included hitting up the ambulance service acquisition team in 2007 and holding various leadership roles with the international association of firefighters local 483 including president from 1995 to 2005 he also coordinated defibrillators in the school fundraiser back in 2000 this past july we had a ceremony right here in the council chambers to swear in the newest first lieutenant and shortly after the ceremony Jeff was returning to his office and a call came in on his radio of a person that had fallen off a raft near to land park and was being pulled out into the lake by the current chief Herman immediately responded to the bathhouse on broken drive and ran down towards the beach and approached the water while still in his dress uniform I think he took a coat off he jumped into the water and swam out to the victims there was actually three people in the water a mother and two daughters and approximately 150 yards from shore the daughters won a raft which began to be pushed away from the shore by the west wind and while trying to get the raft back to shore the girls fell out of the raft their mother swam out to help them and held on until chief Herman arrived on the scene chief Herman took one of the girls in his arms and assisted the mother and the other girl to shore from the time of dispatch to the time the family was out on the water out of the water was six minutes chief Herman's quick action and bravery saved the lives of those three victims it's with great honor and privilege that I provide Jeff with a certificate of appreciation on behalf of the city of Sheboygan for his 32 years of service at the Sheboygan fire department I also have a special mayor's medal of bravery to present to him as well please give Jeff a hand and Jeff please come forward next second thank you mayor thank you for those kind words and thank you for this very unexpected honor appreciate it when the mayor asked me to come here tonight I wasn't quite sure if I wanted to speak and if I did I didn't know what I would say I know I know some of you couldn't wait for that day that I'd be speechless up here I've been called up to this podium so many times in the past and I do know what I really wanted to say on a lot of those times and never did so I guess this is my last chance Don you know what's coming you people you want me on that line you need me on that line I may have left out a little bit of that we've all witnessed some very interesting speeches that have taken place at this podium I can assure you this is six pages long but the font is very large and there's nothing about hot dogs in it anywhere and I've also got 745 in the pool at the back of the room for the meeting to end so I've got to make this quick truthfully this is a little bit uncomfortable for me 33 years ago I never dreamed that my career was going to end up here the other day I was cleaning out some things in my office and I came across an article from when I was inducted into the softball hall of fame and that article said there never was a ball player that got more big hits at more crucial times and more big ball games I think that ability that gift has carried me through and served me well in my career as a firefighter I've always seemed to be on duty for the worst calls the prangy fire don't a jumes landmark and what has been called the most tragic fire in shabuigan's history the day we lost three small children and their mother to an early Sunday morning house fire for some reason you always seem to remember the ones you lost and not the ones that you could save occupational hazard I guess I couldn't help but think back to all the calls on March 18th 2007 as I left the dispatch center I told the dispatchers and I think it was Kelly and Alicia don't call me tonight unless it's really something big and Alicia had a nickname for me it was commander chaos because when I was working weird stuff I always seemed to happen of course that night they called and at three fifty eight in the morning I pulled up to the landmark building just as a door and a couple of windows blew out of the third floor and landed right in front of me on 7th street there was fire across the third floor nearly a city block long we're taught to do a 360 around a building before we give any orders 7th street was now impassable I have 18 firefighters responding to a fire that's going to require way more than 50 and for the only time in my 33 year career we have no aerial ladder truck and service in the city that day and like tonight all I could think of was I've got to say something I have to give some kind of order well instincts and training took over and we stopped the fire that at one point looked like it was going to jump 6th street and burn all the way to the lake maybe because I've become so accustomed to standing up here and being grilled on ambulance issues or station closings or replacing personnel or reorganizations being up here now is a little bit out of my comfort zone I guess it's an occupational requirement to be good under fire my comfort zone has always been responding to the most critical and intense emergencies my guys constantly kid me that I always seem to be around the action whether it's being four blocks from north side beach in my dress uniform as the mayor mentioned when a drowning call comes in or having somebody suffer a heart attack right in front of me at the wild center or having a serious car accident occur a block away from my house I don't know if it's a cursory gift but I'm sure hoping this stops next year when I take this badge off the only explain explanation I have for that is that God puts the right people in the right place at the right time for a reason maybe someday there's going to be a master's degree for that 33 years ago in this very room with no gray hair I took an oath to protect and serve my community I fulfilled that promise four years ago as chief I promised to protect our citizens lives property and tax dollars I'm proud to say today we're delivering fire protection at a lower cost and we did four years ago unfortunately we have suffered two fire fatalities in the city during that time the biggest promise I made four years ago was to return each and every firefighter home to his or her family at the end of their shift I've done that although sometimes battered and bruised I don't want to stand up here and bore you with the accomplishments of the last four years I leave you with a department you can be proud of one that's headed in the right direction on a good path we've lowered our per capita cost we've maintained our excellent response times we have become a leader in the state and national fire incident reporting recently hosting a training session for other fire departments we have completely revamped our public education programs so then one of the most complete and comprehensive in the state we have developed innovative scheduling to reduce our overtime costs in 2013 I believe it will be the lowest in the state we have completely reorganized the command staff integrating the public education officer two fire inspectors a training officer and a deputy chief into a new hybrid command staff we have updated all of our fire stations to meet public health regulations and to be gender compliant we have implemented a new hiring process that has brought us the best candidates the best candidates we have ever seen while reducing city staff time and liability in the past year-end reports are four pages long last year we submitted a year-end report that was 40 pages long and covered nearly everything we do I was born and raised here I'm proud of my sorry Don's city this city has so much to offer so many positives everyone in this room is a spokesperson for this city comments on this council floor like the one I heard a couple months back that the real estate market in this city is pathetic have to stop rather statements bolstering our affordable housing are what we need to come from this body you and the leaders of this city have done an awesome job of assembling a great team of department heads as a whole the best that I've been around each and every one of them has a city's best interest at heart however they can't do it alone they need your support it takes a total team effort I have worked for seven mayors we have a good one here give him the respect he deserves and maybe even a pay raise I can't even imagine how many council members I've worked with over the years three generations of an Akron's for crying out loud man I must have been here a long time I'm not staying around for the fourth day I was remember we have been put in our positions not for our own benefit not for our own agendas but to serve the people the best part of my job has been and will always be to look on the people's faces when we showed up to help them when nobody else good I cannot leave without thanking my administrative staff the 66 firefighters underneath me and most of all my administrative assistant Lisa Horn they have supported my decisions carried out my orders and never once complained to me anyway I think they're smarter than that as I piled more and more responsibilities on them and changed their schedules three times in one year at one time I own much of my success to all of them I cannot leave without thanking my girlfriend Tony she's the one that's had to put up with me when I got home from all of those meetings when you guys called me up to this podium Tony four years ago you lost me to this city now we can spend every second of every minute of every hour of every day together we all feel your excitement back there it will be a weird adjustment for me next year I have held the job for all but five days of my life since I was 12 years old the only other time I was idle for an extended period time was when I was out with a broken foot I was so bored I rearranged the kitchen there are still some things that haven't been found so if you think I've been a pain in the butt up here for you just look at what Tony has to look forward to I've been approached to run for alderman so far I haven't had that much to drink so for now alderman born you are safe so at the end of the year time will has come for me to move on I look forward to setting my own schedule to no more speeches hopefully lower blood pressure I will not miss all the meetings not that this haven't hasn't been a lot of fun I will not miss getting up in the middle of a cold night or being on call 24-7 I am not however just riding off into the sunset I will be doing more of what a firefighters apparently love to do just driving along the lake hi dolesie I know you're watching past third in Michigan uprooting drive over to Lincoln Avenue and so on I'm sorry alderman born couldn't have been here tonight I know alderman born and versi couldn't wait for this day to come for me to go on early Christmas present please do not insult yourself or sacrifice your integrity by feeling you need to stand and applaud me it's been a real honor and a privilege to be your fire chief thank you thank you Jeff next we have an another individual who's been a long-serving member at the library Mark Zafis Mark started his career with the Mead Public Library in 1998 in a dual role of reference and librarian and cataloger in February of 2006 he was promoted to the business manager at Mead Library and responsible for budget preparation and building services and in January of 2011 he was promoted to deputy director at Mead Public Library adding responsibility for information technology circulation and technical services prior to working at the library Mark was a member of the US Marine Corps where he served as an engineering officer and followed by service as a marine reserve officer where he attained the rank of lieutenant colonel the staff at Mead will miss Mark's amazing talent they tell me that he has a very interesting talent to speak foreign foreign accents from time to time he'd start speaking Russian German Chinese French or some other accent it can be extremely funny and a great way to get everyone's attention at the beginning of a meeting and and keeping everybody in a good mood with it was great honor I provide Mark Zafis this certificate of appreciation on behalf of the city of Sheboygan for his 24 years of service at the Mead Public Library Mark please come forward and thank you very much for all that you've done I know it's a hard act to follow but you're welcome to make a few comments as well after the after the chief speech there's no sense my even trying to to follow up with that thank you very much mr. mayor council members citizens department heads it's been a long time but I've enjoyed it it's been a privilege to work in your library it's been a privilege to try to make it a better library every day I think Sheboygan's on the right track moving in the right direction now I think the library is definitely moving in the right direction thank you very much Merry Christmas I've got a very special announcement for you I want to remind you that back in in fall you passed an ordinance that's going to change the time of our meeting in January all of our meetings are going to begin at six o'clock so it's going to be a while before that becomes a habit but please stand reminded and I also want to say Merry Christmas to everyone in the council chambers here and to all of our residents I hope you have a blessed Christmas celebration and also a happy new year thank you now we're going to we're going to go back to confirmation of council council appointments there's a document that we missed and I'll turn it over to the city attorney this was dated December 2nd the members of the council pursuant to the requirements of section 7.30 the Wisconsin statutes I herewith submit for your approval the list of nominations for election inspectors for all elections in 2014 before mentioned section of the law stipulates the manner in which election officials shall be chosen and I tender my appointments as follows to retain as much seniority and experience as possible while complying with the state law respectfully submitted Mike Vanderstein mayor and then there's the attachment that has the list of election inspectors thank you Alderman Hammond thank you mr. mayor I move to approve the appointment as submitted second thank you for that motion in support is there any discussion seeing none will the clerk please call the roll on appointments 15 eyes next we'll move on to the consent agenda that'll include items 2.1 through 2.15 Alderman Hammond thank you mr. mayor I move that all our C's be accepted and adopted all our rows be accepted and placed on file on all resolutions and ordinances be put upon their passage second it's been moved and seconded the consent agenda is before us under discussion Alderman van Akron thank you honor I need to pull forward number 2.8 for a separate vote I need to abstain okay uh is there a second second okay we have a motion and a second to pull forward 2.8 any discussion on 2.8 okay the clerk will call the roll this is for 2.8 that's right there 14 eyes one abstention motion passes now we're back to the consent agenda on 2.1 through 2.15 except for 2.8 is there any discussion on on the motion to approve the consent agenda seeing none will the clerk please call the roll motion passes next we'll go on to item number three reports of officers item 3.1 will lie over items 3.2 through 3.7 will be referred to various committees under resolutions item 4.1 will be referred to the city planning commission items 4.2 and 4.3 will lie over and items 4.4 through 4.10 will be referred to various committees under reports of committees item 5.1 is an rc by public protection and safety to whom was referred rc 82 of 1314 by public protection and safety and resolution number 14 of 1314 by alderman carlsson recommends passing the substitute resolution alderman carlsson thank you mayor i move to accept and adopt and pass the substitute resolution thank you for that motion second second second second very good we have a second under discussion alderman carlsson at this point i would like to invite up a lieutenant metal stud and the chief of police to come up and talk about this document okay if there's no objection would the two of you please come forward to discuss this document good evening thanks for the opportunity to talk to you about the public impairment ordinance that's in front of you before we get to that point i think it's important that we understand how we got here starting in 2010 we really tried to drill down and look at our crime and what was driving it one of the problems that we saw is crime was constant crime and disorder were both concentrated around the many of the taverns in our city and so those are some of the things that we tried to address one of the first things that we did was start out with compliance checks to essentially test license establishments to see if they were complying with the liquor laws that were in place we had a 43 percent failure rate one of the things that we tried to do to address that was to work with tavern owners to identify business practices that would lead them into greater compliance with that and thus try to reduce some of the crime and disorder that we were experiencing another one of the things that we did was lieutenant middle stat here worked with the tavern owners to form a tavern safety coalition and the idea behind that is really to get the tavern owners a to work together and to work with the police to try to address these issues and to make sure that best practices business practices are in place one of the things that we saw was problems with over serving and and not recognizing when somebody had too much to drink and so we put tips training into place so essentially training so that bartenders would be able to recognize when people are in this situation really what one of the things that we were hearing as this was going on is that the police department was so focused on the actual businesses the people engaged in the bad conduct were not being addressed and so this is one of the attempts that we're making to try to address some of that bad conduct I think it's important to note that again there's a small very small group of people engaged in this conduct and that's really where this ordinance is trying to focus on perceptions were mentioned earlier I think one of the things that needs to be mentioned is that there's a misperception that someone can be cited under this ordinance for being impaired that is incorrect this ordinance addresses behavior of the person so it takes both things to be present somebody has to be impaired and engaged in this behavior so somebody who's impaired and walking down the street cannot be arrested or cited under this ordinance it targets behavior another one of the important things I think about that that is about this ordinance is that we tried to really approach things from a different way we've seen time and time again where someone has been cited and we haven't been able to change their behavior by simply citing them and so what we tried to do is find an evidence-based program that we could use as an alternative to refer people to and if they successfully completed that program then it was our belief that we might be able to change behavior and at least a small part of that population and if we are able to do that that I believe will be success so that's really what the ordinance is about in the steps that we've taken thus far thank you for those comments I appreciate it further discussion they should probably stay up here chief olerman carlson olerman hamlin thank you mr. mayor um maybe either chairman carlson or or chief you guys can help me out I was my understanding that the tavern league was involved in the formation of this document um or a part of the sub-sector there no no tavern safety coalition is a group that at this point is made up of about 75 percent of the tavern owners in the city like I said it's a group that was made up just so when these type of issues come up we have somebody to work with every tavern owner in this city is is welcome to join that okay next question is and just reading through the document um what are the types of behavior you know it mentioned you know originally when this was drafted it was more of a status offense and just the fact that you were so maybe some examples of what would cause this you know or just to be enacted or whatever the case may be um you know is it stuff that we already have laws on the books for um or I mean just to help me understand what somebody could it's for engaging in in conduct that is disturbing the piece including but not limited to limited to engaging in obnoxious behavior acting in an unruly or combative manner vomiting creating loud noises to the disturbance of other persons refusing to follow the instructions of a police officer refusing to follow the instructions to leave a place of business by the owner employee or other person in charge and so really what it tries to do is address those situations where somebody is causing disorder and either a business owner or a police officer is addressing them telling them it needs to stop you need to go on your way and and proceed someplace else home wherever you're going and they refuse to do that and continue to engage in that type of behavior has this been done anywhere else and if so you know do we have any measurement of whether this has been successful or not it's been done in in La Crosse monomony recently a couple months ago was all past a public intoxication ordinance many of the things that we tried to model this uh under we're from an ordinance that was created in La Crosse where they used a program that were essentially using based on motivational interviewing where they saw over a 70 success rate with people who went through the program did not then re-offend and last um and this is kind of my ignorances to the court system but if we have things like you know disorderly conducts and trespassing and you if somebody doesn't leave when they're asked to and things like that could the courts give them the option as to go into one of these programs versus a fine instead of having an ordinance on or ordinance on the books to do that in order to it's possible if if there is a program that exists to do that and so you know one of the issues that we we came into with this is we tried to find a program that we could refer people to we checked with UW Sheboygan LTC Family Resource Center there wasn't a program and nobody was was willing to put one together and make it available and so we took on that task to try to deal with the issue thank you Alderman van Akron mine's not for the chief does anyone else have a question for the chief thank you very much thank you your honor um I stand up tonight to I guess express my concerns and I guess to indicate that I will not be supporting this proposal as it's written um I have three main concerns and I'm going to highlight here shortly and I'll try to be brief um bear with me I do have a bit of a cold that I'm trying to deal with um my first concern is the redundancy of this law I feel that uh in the time that we've had to discuss this in our committee um there's been different proposals and and this has been one of my main concerns from the beginning is I do feel that the current proposal does focus on people's behavior um as indicated in number two under the definition for public nuisance uh in saying that we have laws that address pretty much all of those behaviors officers currently have the ability to address those behaviors in a manner of ways whether it's a written or verbal warning and say it's time to move on whether it rises to the level of a city ordinance um and they get a ticket or it rises to the level of a misdemeanor crime and they go in handcuffs and they go to jail we have the ability to address those behaviors officers have the ability to address those behaviors currently whether someone is drinking or not drinking um so the difference here is involving the consumption of alcohol uh as a factor in these behaviors um so I do feel it's redundant I don't feel that it's needed I feel that we have laws that address these behaviors and we need to use those in a manner that um addresses the problems that we see um in a free society we don't make laws because we can't we make laws because we need them we make laws because we have to have them this just isn't the case with this proposal um secondly it was expressed from the beginning that this proposal would be a trigger mechanism to the alcohol education program which I support I support the idea of trying to change people's mindsets trying to change their behavior I guess open their eyes to some of the things that maybe they're doing that maybe they don't realize I support the idea of the alcohol education program as an alternative outcome to um a fine however when I asked the city attorney who's here now um if the alcohol program could be an alternative to our current laws he certainly indicated it could we would have to take the time to amend our current ordinances to allow for the alcohol program to be an alternative in which again disorderly conducts trespassing damage to properties if alcohol is a factor we could amend our current ordinances that deal with that to allow for the alcohol education program as an alternative so I feel that we do have options rather than creating a new law to deal with the behavior and allow for the alcohol education program and lastly my my concern and I think the most important and which has been expressed here already tonight is the possible negative impact for our small businesses we've had several people stand up tonight and express their concerns I've had several people call and approach me and express their concerns and I think we need to remember that's who we're here to represent is the people of Sheboygan the small businesses here in Sheboygan we need to try to make things easier for them not make things harder again they've expressed their concerns that if the headline reads Sheboygan is one of the few communities in the state to have a public intoxication law or the radio puts out broadcast to that same effect what is the impact on their businesses and that's their concern and I think it's a valid concern and I think it should be a concern over this council I think they have a legitimate point and then I don't think our purpose here is should be to take the risk of damaging their businesses it's hard enough to keep a business afloat here in the city and we've seen that over the last year we've seen several small businesses close and I don't want to make it harder for them or even take the risk of making it harder for them so I guess that's that's one of my biggest concerns is that they've expressed that concern to myself they've expressed that concern here today and I think we need to really take that into effect we have all other alternatives we have different ways to deal with this problem we have an alternative to enact the alcohol education program and again I do find that it's redundant so I would I guess encourage people to not support the current proposal and that we look at amending our current city ordinances to deal with again the small group of people that seem to cause a lot of the problems so I will not support this as it goes forward and I would ask that we encourage others to not do so as well thank you for your comments any other discussion on the motion see none will the clerk please call them I'm sorry my Alderman Donahue and I'm going to speak in favor of the ordinance and I think my cold beats yours so the when this ordinance was first brought to public protection and safety I had some very very substantial concerns about it as I said to the chief you know this is bringing out this my inner civil libertarian because it did really punish people just for the offense or for the status of being drunk we have spent considerable time reworking this ordinance so that to be honest with you in its current form it does not resemble the initial proposal really at all now what's the problem that we're trying to solve that's always a good place to start the problem that we're trying to solve is in certain business areas at bar closing time in particular there are substantial numbers of people who are not only impaired but they are a public nuisance now what are the what are the tools that the police department has to deal with these folks there are certain ordinances that people can be charged with violating if their behavior is extreme enough to merit for example a disorderly conduct charge as a former prosecutor of disorderly conduct charges I can tell you that those cases can be relatively difficult to win it sounds like it's easy but it's really not what this ordinance does is take into account the behavior of people who actually are not helping our tourist community these are not the people that we want folks coming to visit our city walking down the street or congregating in certain areas engaging in really fairly unpleasant behavior now I'll refer you to the last whereas clause of the ordinance whereas it is the intention of the common council that this ordinance be applied only to the extent that other law enforcement measures are unavailable or ineffective in dealing with such behavior so it's really a pretty narrow scope of the behavior that we're trying to that we're trying to take care of here so you get people who are not probably do not reach the level of disorderly conduct which is relatively serious behavior and yet these are not the people who are probably really pretty impaired I'm the parent of now two adult children and I can tell you that in their day they probably you know walking home they would certainly have been impaired but they would not have been public nuisance at least to the best of my knowledge so these are the folks who are not only impaired but they're being troublesome they're being difficult they're congregating it's bar closing time the food stands are open people are standing around and it's difficult for police to control to control the area so this is this is one tool that we're giving our police department to try to do that now what really has persuaded me that in in addition to substantially reducing the status offense nature of this ordinance and really connecting it to bad behavior bad behavior that makes our city look bad that is is is unpleasant for the neighbors is unpleasant for the people who don't want to deal with the people who are falling down drunk and quite obnoxious the other piece of this ordinance that really helps is that before a person receives a citation under this ordinance the person will receive a warning and if the person completes the alcohol education program after receiving that warning the citation is not issued at all so in that respect people who it provides a public service and trying to point out to people we're not trying to we're not trying to cure alcoholism we're not trying to make this a dry community but we are trying to get people to understand in this way that when they're engaging in the behavior that they're engaging in this really not it's not good for it's not good for the community and it's not good for them it's not good for those taverns I would I would certainly suggest finally because I do there are measures here for us for the for the officers to see if someone is impaired and those include all of the standard in field sobriety measures of slurred speech and toxic odor of intoxicants bloodshot and glassy eyes and so forth so I think that the the the application will be narrow I think that the the results will be good the chief is going to report to us on this on a quarterly basis so we can just see what is in fact happening if this is at all helpful I will tell you I've spoken to people in La Crosse where this ordinance has been enacted a very similar ordinance and of course their situation is much different they had they do have 50 bars within a three square block area and they also have a fair student population there has been however after enacting this ordinance a similar ordinance in 2009 I've seen the statistics there's been a dramatic drop in the number of unruly and public nuisance kinds of behaviors the people who go through the program are not reoffending I mean it's it's really a fairly extraordinary statistics I know it's a different I mean we've got students it's a different population base and so forth but I think we should give it a try I think we should see if this makes bar closing time a little bit more civilized and and just see if this is a good thing not only for the people who are being public nuisances but also for our community and and for the people who live around and for our reputation as a city where you know people are relatively well behaved thank you for those comments Alderman Maddachek thank you I'm going to have to stand up and though I do appreciate much that's that's in this ordinance there is some quite a bit that is a bit concerning just simply put an ordinance as to support the small businesses and when you have this many small businesses coming out in I opposed to it that is a bit concerning as well one thing I do have a specific question regarding to the the warning that is issued could we have someone speak on behalf of that is that directly a warning given out that evening and they would receive the citation as well or is that something that's overseen by the court or is that something overseen by the class Alderman Carlson do you want to respond to that yes it would be no different from getting pulled over for perhaps a broken tail light you would receive a warning and you would have 90 days to complete the the class and then you would go into the police department show completion of the class and at that point no citation would be issued if you did not come in with the 90 within the 90 day period a citation would then be issued thanks for that clarification Alderman Maddachek thank you and how would we verify whether or not this person has already received a warning within the evening or within a short the 90 days that they have or if it would be a second warning how much staff time would be associated with that to oversee this additional response that question I would defer to the chief chief would you like to answer that warnings would be given out through our track system the same as any traffic citation it would go into the records management system and it would be able to be tracked that way so very little it's about running a query and getting back to results how long does it usually take the staff each and every time to check into that like I said very little 30 seconds maybe to punch in the query based on the the codes that are associated with it and get back to data they're ready to receive a warning within the 90 days they would receive a citation I didn't understand if they got it if they got a warning and they did not complete the program within the prescribed time then a citation would would be mailed to them should someone be causing a should someone otherwise be disturbing the piece in any public place while intoxicated and receiving a warning already they within the 90 days if they do that a second time would they receive a citation or another warning or they would receive a citation yes any other discussion alderman vinnach thank you your honor just to follow up on some of alderperson donahue's comments which I certainly applaud her effort she's certainly actually indicated was pretty vehemently opposed to this at the start and has made a lot of changes to make this more palatable some of the things I wanted to touch on again is is the intention of this and I don't think anyone at least so far tonight has stood up against the intention of this the tavern owners that have stood up and expressed their concerns again are concerned about the perception and what that perception could possibly do to their business and that's something I think we really need to take into effect when we have other options to create this alcohol education program I think the perception of this unfortunately becomes reality that's that's the world we live in and again I think it has good intentions and I support amending our current ordinances to create an alternative disposition but I don't support going ahead with this perception that could have that negative impact and I think we need to remember these small businesses pay their taxes these small businesses employ people and is it really worth risking damaging those businesses that's one of my biggest concerns and again that's one of the main reasons I can't support this I also wanted to touch on the tavern safety coalition there were members of the tavern safety coalition at our committee meeting from my understanding they've been in creation for approximately one year it again it's uh it's approximately 75 percent of the local taverns that get together and have come together to I guess start working together to solve the problem of the small group of people that seem to be a problem for everyone from my understanding they have created a basically a banned list that if you are kicked out of one of their establishments you're kicked out of all of them again they've been working together to try to solve some of these problems in conjunction with the police department and from my understanding it's been pretty successful in the last committee meeting one of the members stood up and said that they've seen a 25 percent reduction in incidents I think that's pretty successful in one year so I again I I really question whether or not we should be going forward with this at the risk of these people's small businesses and if you indulge me mr. mayor if I can quickly just ask for a show of hands if we went to amend the current ordinances to allow for this alternative alcohol class under our current ordinances and not this would you gentlemen be in favor of that rather than this can I just see a show of hands as you can see several of them would be in favor of that alternative so again I question who we're here to represent there's a large a large quantity of the people here don't want you to support this and would support an alternative so I really ask that you don't support this going forward and that we continue to work on it and come up with a different answer thank you for those comments is there any other discussion seeing none will the clerk please call the roll and five point one seven eyes eight no motions defeated next is item five point two an rc by public protection and safety to whom was referred rc 83 of 1314 by public protection and safety and general ordinance eight of 1314 by alderman carlson recommends passing the substitute general ordinance alderman carlson thank you mayor with that being said i'd move to file moved and seconded to file is there any discussion seeing none will the clerk please call the roll change was it accept n-file motion passes next is item number six ordinances item six point one will be referred to the city planning commission under matters laid over 7.1 is an ro number 196 of 1314 by the city planning commission tumors refer general ordinance 43 of 1314 amending the city zoning map to establish the use district classification of recently annexed property owned by the town of wilson and located at 4108 south 18th street to sr3 suburban residential alderman versi thank you mr. mayor i'd like to accept and file and pass the ordinance second has been moved and seconded to put the motion on the floor under discussion anyone wishing to be heard if not will the clerk please call the roll 15 i's motion passes next is 7.2 an ro number 198 of 1314 by the city planning commission to whom was referred general ordinance number 45 of 1314 by alder person versi amending subsection 15.936 sub 1 of the city zoning ordinance relating to fees alderman versi thank you mr. mayor i move to accept and file and pass the ordinance second thank for that motion and support any discussion seeing none will the clerk please call the roll on 7.2 15 i's motion passes 7.3 is a res is resolution number 86 of 1314 by alderman person hamlin authorizing entering into a contract with butine peterson and sigma environmental in order to fill the flood plain in the former shookered property alderman hamlin thank you mr. mayor i'm going to put the resolution upon its passage second it's been moved and seconded to put the resolution on its passage is there any discussion seeing none will the clerk please call the roll 15 i's motion passes we'll go on to 7.4 which is resolution number 101 of 1314 by all the person hamlin authorizing entering into agreement with segal gallagher management company for the management of the harbour senate marina alderman hamlin thank you mr. mayor i'm going to put the resolution upon its passage second it's been moved and seconded to approve the resolution under discussion alderman hamlin thank you mr. mayor as many of you guys know we had a conversation in a closed session regarding that this contract we did go back to segal gallagher with many of our thoughts and recommendations attorney mclean if you wouldn't mind just real briefly going through the changes to the contract that were made that from what we talked about in closed session and so the council understand thank you alderman rather city attorney promotion got promoted there and two two major items one is the term of the contract i think originally the discussion was three-year term and discussed and negotiated to be a five-year term and i would say that that's consistent with what we we've always had five-year contracts with skipper buds over the last 20 years they were in five years and a couple of them had possibility five-year extensions this has a possibility of an extension at the city's insistence and the incentive fee is is a change from what was discussed previously um the the management fees since it was converted to a five-year contract section 14 speaks of base management fee beginning in 2014 at 60 000 and then that goes up five thousand dollars a year for each of the five years so it's 60 then 65 and 70 75 and 80 is 2018 that's the base management fee then throughout the term of the agreement there's an incentive fee uh that was paid for 2014 and 2015 incentive fee would be based on 35 percent of the revenue increase from net new boat slips over the previous year excluding any increases that are due to cost the living type of increases for 2014 the base used to calculate that incentive is 30 373 277 dollars which is the revenue i understand revenue we received from slip rentals for 2013 so basically that incentive fee is for the first two years 35 percent of the revenue increase on the net new boat slip rentals over the previous year then for the balance of the five-year term the remaining three years the incentive fee would be 20 percent of the revenue increase from net new boat slips over the prior year there's a disincentive fee put in that would be applied if Segal Gallagher failed to meet the budgeted net operating income by 20 percent or more in that year and the disincentive is that the base management fee would be frozen on a going forward basis there are some other changes to the basically the contract otherwise is similar to what we've got had over the years with skipper buds one sort of administrative change is the way the skipper buds contract worked was skipper buds would provide invoices and get their bills and so forth and submit them all to the finance department finance department would would pay everything out of an account under this proposal basically Segal Gallagher will have the bank account they will collect the revenues in an account on behalf of the city and pay the bills and then be an accounting on a monthly basis of the revenues and expenses and then any net revenues would be turned over to the city at the end of the year so that's more of an administrative change than a substantive change but it's it should make it easier for the finance department I think I don't know if Chad or Nancy or Bernie or David has any other comments but I think those are the basic changes since this was discussed by the council previously the floor John Matheson from Segal Gallagher is here just so you guys can if there's no objection I'd like to open up the floor John Matheson moved and seconded to do so all in favor signify by saying aye aye opposed and John I understand took his life in his hands to come up here in the snowstorm today and a lot of traffic down in Milwaukee John welcome thank you very much that was no fun coming up from these two hours and 30 minutes from Milwaukee so thank you I appreciate you invited me and I'm John Matheson with Segal Gallagher management company one of the principals with the company and I've been with Segal Gallagher for about 11 years I've been a voter for 18 years we took over managing reef point marina and we're seeing in the spring of this year and it's been a very successful relationship with them so far and it's something that we're very excited about seeing here in at Harbor Center marina and think we have a real good plan to get there were there any specific questions I'd be happy to kind of give you a general overview of of our kind of our strategy to do so um that uh all right I'll just take a minute and go through that um we don't sell boats we're strictly a customer orientated uh in a service-oriented company we focus very very heavily on that we have our infrastructure and systems set up to do that using latest in technology web-based programs um internet uh social media to to not only make it um easier for voters to communicate with us and also do make payments and administrative work but also market the marina the second thing that we think is different and that helps us is that we're heavily focused on on the community and that is uh creating and fostering a better relationship with the local businesses and the marina which is as all of you know one of the main assets of this marina is that proximity to the local community and we don't feel that that has maybe been played as up as well as it could be and we think that's a major area of a focus for us going forward to help grow the marina which is really the goal here and that is to get more voters in the marina and help the financial uh the revenue and the bottom line of the marina so it can um be more profitable it can invest in itself more and also maybe even begin paying back a little bit of what the marina is a city that's that their short story of how uh what our strategy is any questions by the alderman john one of the things that's been particularly difficult about this negotiations that we've got some very strong individuals that work for skipper buds in the past and i'm just wondering what your plans are with personnel and staffing of the marina as the operator if we approve that good question chris marx has been with uh harbour center marina for 15 years and i've spent quite a bit of time with him um several hours last week and he was at our company party this past friday with him and his wife and um i think there's a real good um match in what he he can bring to us as a company what we can bring to him and and help him achieve he has a lot of good ideas that um he probably wasn't able to implement um in his position in the past the way our company works is a collaborative relationship with our staff chris would be a major part in helping define and execute that strategy as well as some other folks um in our marina side even collaborating with um reef point and that staff and working on ideas to to help uh get to our goals our company is um is nimble enough in that area that we're able to not just take a model and force it on this marina but really design a strategy very specific to harbour center marina and chris knows the marina he knows the boaters he knows the market and he would be able to with our support uh to execute maybe a better strategy to help get that done so to answer your question um the four key players in the marina are going to remain um we're going to continue to do boat to do service on boats we're going to continue to do the hall outs and storage as have been done in the past and chris will still be running the show there but he'll have um more support i think from us and it'll be a very collaborative effort with myself and and a couple other people as well thank you any other comments or questions john thank you very much for making the trip thank you very much for your comments there's no other discussion we have a motion on the floor um alderman van akron thank you your honor i guess just a question on the increase in term um from three years to five years i did appreciate the fact that there is incentive based um i guess there's incentives in place as well as disincentives in place based on the income and how it increases or decreases my question is is if and i certainly i'm hoping that it's not um and by no means am i indicating that it's going to be um but my question is if somehow this goes very badly um it's a catastrophic failure do we have any options of getting out of that uh agreement within that five years city attorney um there are termination provisions in the contract uh basically uh termination by the city uh provides if more than if one or more of the following events occur the city may terminate the agreement by notice to sequel gallagher uh basically if they're in material default in the performance of any term or in the agreement uh we would have to give them 30 days advanced notice an opportunity to cure if they fail to cure uh within a 30 day period we could uh get the contract there based on default by the by the marina manager um if they were to file bankruptcy um if they're they made any misrepresentation to induce the city to enter into the agreement that's false in a material respect uh if they fail to meet the yearly budgeted net operating income two consecutive years you have the right to terminate in the event we're able to terminate city could exercise any or all the following remedies in addition to termination city could without further notice enter onto the marina premises and expel the manager if necessary remove their effects and require the manager to turn over the slip uh leases and the boat rental agreements similar agreements so that's basically our termination rights it's similar to most contracts it's a for cause sort of termination i guess just to clarify then to follow up if if they don't meet those those budgeted net income i i guess is how you phrased it if they don't meet those two years in a row we would have that option is that correct i guess with with that being in place tonight then i would support the motion going forward and there's a lot of you know exciting new things that they bring on board and i certainly am hoping for the best and hoping that this is successful as it's being proposed and i will support it going forward with that clause thank you for those comments and questions any other discussion seeing none will occur please call the roll and passage motion passes next is item 7.5 resolution number 102 of 1314 by all the person ham and carlson approving the capital improvements recommendation for the capital improvement from the capital improvements commission for the period of 2014 through 17 and adopting the program for 2014 for implementation all of them ham and thank you mr. mayor move we put the resolution upon its passage second it's movement seconded with the resolution upon its passage is there any discussion all of them ham and thank you mr. mayor just to clarify you know to clarify a little bit we've been embarking over the last couple of years um you know what i'd like to call some future planning so as you see before you we have the capital requests from 2014 through 2017 and the idea was to kind of give the council an idea of what is coming up on the horizon from a capital standpoint so what we're approving tonight is you know again the baseline of the 20 set you know the plan but in particular the 2014 plan as indicated here for capital improvements but again just kind of gives you an idea of what we're looking at 2015 16 17 for capital improvements and some of the things that are being requested and need to be done thank you thank you for those comments is there any other discussion seeing none will clerk please call the roll-in passage motion passes next is i as an item 7.6 which is resolution 103 of 1314 by all the person Hammond Carlson Donahue Heidemann van der wheel committing the fund balance committing the fund balances in accordance with gatsby 54 and subs of resolution number 44 of 12 of 13 alderman hammond thank you mr. mary moved to put the resolution upon his passage second the movement seconded to put the resolution on its passage is there any discussion seeing none will the clerk please call the roll motion passes 7.7 is resolution number 104 of 1314 by all the person Hammond Carlson Dastler Heidemann and Bellinger to authorize a transfer of appropriations in the 2013 budget alderman hammond thank you mr. mary moved to put the resolution upon its passage second moved and seconded to put the resolution upon its passage is there any discussion on the resolution seeing none will the clerk please call the roll motion passes next is item 7.8 resolution number 105 of 1314 by all the person Hammond Carlson Donahue Heidemann and van der wheel to authorize a transfer of appropriations in the 2013 budget alderman hammond thank you again mr. mary moved to put the resolution upon its passage second moved and seconded to pass the resolution is there any discussion on the resolution seeing none will the clerk please call the roll motion passes next is other matters city attorney 8.1 it's an RO by the board of contractors examiners submitting applications for building contractor licenses already granted that will lie over 8.2 is an RO by the city clerk submitting various license applications for the period ending June 30 2014 and June 30 2015 that will be referred to law and licensing 8.3 is an RO by the city clerk submitting various license applications that will also be referred to law and licensing 8.4 is an RO by the director of planning and development submitting a request requesting that the marina parks and forestry committee make a recommendation of the public works committee relating to naming of the new interurban trail recently constructed with non-motorized transportation pilot program funding and city county funding from penn avenue to martin avenue now we refer to marina parks and forestry alderman hammond thank you mr. mary i moved to convene in a closed session on the exemption provided in section 1985 1e wisconsin statutes for the purpose of deliberating regarding the proposals for management of harbour center marina including the terms conditions of the management agreement and deliberating the possible purchase of public property and the possible sale of public property were competitive and bargaining reasons require a closed session although to that amend that we will not be discussing the harbour center marina as part of that it'll just be the sale and purchase okay and moved and seconded to go into closed session with a clerk please call the roll we'll stand adjourned for five minutes and reconvene


Hello, we're now looking at the seventh installment and the final installment of our series on mass spectrometry basics. We've talked about amino acids and proteins in the first lecture, we talked about the basic concepts of mass spectrometry and ion sources in the second lecture, third lecture was about the mass analyzers, fourth lecture was about the detectors, fifth lecture was about the Fourier transform ion cyclotron resonance instrument and the orbit trap that summed up the whole mass spec bit and then the sixth and previous lecture we talked about tandem mass spectrometry and how we fragment ions into smaller bits and then analyze these and now we're going to have a closer look at this fragmentation and that will conclude our basics of mass spectrometry. So without further ado let's have a look. Let's start with something very basic. We have a peptide and this peptide carries a positive charge. If we use electrospace sources, multiple positive charges. Now these positive charges actually are protons, so hydrogen atoms without their electron is a single proton and they're somewhere on this peptide. Now the hypothesis, the assumption is that the protons can in principle travel, so they don't have to be located very precisely in place, they have the ability to move along the entire chemical structure of the peptide. Then we make a very important hypothesis and that is that the location where the proton is most often found will be the weakest point in the whole peptide structure and so if we fragment or when we fragment with CID or HCD remember these are the fragmentation methods that are ergodic that where the energy spreads out across the whole backbone and they can find the weakest point that these weakest points will be found and will trigger a fragmentation event. So knowing which kind of fragments we can expect to see and there are usually B and Y ions so we've established that these are the weakest bonds but then which ones of these B and Y ions will fragment that we try to reduce to this very simple question where do we think the proton will be and if we know where the proton is likely to be we know where the fragmentation is likely to occur. Now how do we figure out where this proton will be? Well a proton being somewhere has something to do with the basicity of that location. If this location is very basic it means it's very ready to accept a proton which again means that the proton there is very energetically stable at that location. Now how do we figure out basicity if you think back to our very first lecture when we had this table of amino acid properties we had this the PKB so this was the minus logarithm of the equilibrium base constant of the amino group of an amino acid and we had the PKX which was the basicity or acidity of the side chain. Now I've plotted these numbers from the original first lecture in a graph this time. What we're looking at is we're looking at the acidity equilibrium constant of the carboxyl group which we're not going to bother with but it's just here for completeness and then we're looking at the amino group of each individual residue of each individual amino acid. So here are the amino acids and this is the PKB of those amino acids and then here these blue bars there are the PKXs. As you can see only a few amino acids actually have a PKX. Remember that something like valine or isoleucine or leucine do not have a PKX because they do not act as bases or acids they won't accept or lose a proton. Now what you should remember is that the side chains they're always available because of course the side chains of the amino acids are there but this amino group is only present at the amino terminal residue in the peptide. So the head of the peptide that amino acid still has its own free amine all the other amines have now been joined together in the peptide bond. What is interesting to see is of course that arginine as we know has the highest PKB so it is a location that very strongly attracts protons. The second most attractive position if you like for a proton is lysine side chain but lysine is very close in attractiveness for a proton as the proline n-terminus. So any peptide that starts on a proline will have a really nice location for a proton on the n-terminus and if that peptide should end on a lysine there will also be an equally attractive position here. Now imagine that there are two positions where a proton can be held reasonably easily and roughly equally stable. That actually means that there is a very good probability that the protons will be highly localized. They will either spend a lot of time here or they will spend a lot of time here. If there's an arginine at the end and remember arginine and lysine why are they so special? We use trypsin to cleave proteins into peptides and trypsin cleaves c-terminally or after lysine or arginine so the vast majority of our peptides will end in lysine or arginine. If there's an arginine here the proton will be very strongly localized there. Remember arginine is about two units of pkx higher than lysine which is in fact a hundred fold more basic. So a hundred fold better location for a proton. So with all of this in mind let's have a look whether we can now apply our knowledge about the specificity of all these different locations and with the hypothesis of this where the proton is that's where things will break and see if this hypothesis holds. This hypothesis in fact is called the mobile proton model. So we are assuming that we have a model of a peptide and in this model the key factor is the fact that the proton is mobile and can in principle travel around and so the ability of the proton to travel around will determine how well how completely something fragments. Now this theory was proven by a series of very elegant papers by Wiesoki et al and earned Wiesoki the ABRF award amongst many other awards and rightfully so because this is very interesting study that is very simple to understand and is very elegant in its design. Let's have a close look at how this works. What we have here on this plot is the collision energy we have put in roughly stating the collision energy is this voltage or is related to the voltage we put across the collision cell. Now the higher the voltage the faster the ions will move. The faster they will move the more energy they will have the more force they have when they smack into the collision gas. So we actually add much more energy and the question we're going to ask ourselves is how much energy do we need to invest in the collisions in order to get all possible fragments and when we get all possible fragments this means that we have a complete coverage of the sequence we have the y1 ion we have the y2 ion the y3 the y4 the y5 we have all these ions somewhere in the spectrum so we've completely blown everything sufficiently apart to see the full ladder of all possible y ions. It's a great way to see how easy it is to fragment. Now remember a more mobile proton should make for easier fragmentation so the more mobile a proton is if the model is correct of course the the less energy we need to get to a high amount of coverage. Now let's see we have two peptides here they are very specifically built for this purpose one has the arginine at the end terminus so it doesn't look like a triptych peptide but if that doesn't matter it's a matter to prove a hypothesis and it has a phenylalanine which is not very interesting from a base perspective because it's it doesn't even have a pkx at the c-terminus and here we have one with a proline at the end terminus and the arginine at the c-terminus. Now in fact we've just moved the arginine into the back and that is also shown by the way that is written we've just swapped the location of the arginine. Now if we have a singly charged version of this peptide that means there's only one proton that can possibly be mobile. The singly charged proton is going to be at the arginine remember arginine at this pkx of 12 and a bit so it's going to hold on to that proton and that proton is going to be very very happy on that arginine you want the denium group so the proton is going to stick around here or here. The result is the proton is not very mobile the proton is happy on the arginine and it's not going anywhere so we don't have a mobile proton it's going to be very difficult to break all the other bonds because the proton never gets to be at these places and so we'll need to invest a lot of energy to get these things broken and that's exactly what we see we have to start at the collision energy of 60 electron volts before we see fragments and then we need to go all the way up to almost 120 for both of them so both the triangles and the circles to get a hundred percent coverage so that's a lot of energy no surprises there so far the hypothesis holds if the proton is not mobile it's very tricky to get the thing to fragment and if you have only one location where the proton is is happy to be and if you have only one proton it doesn't even matter where that location is now things change dramatically when we go to the doubly charged version of this peptide the doubly charged version of this peptide actually has of course still one proton at the arginine side chain but the other proton now no longer feels happy on that arginine that is very simple because two positive charges even on a very basic side will start to repel each other so one proton is going to be at the arginine and the other one will have to go find a new place to be now have a look at this first peptide where is that second proton going to go there isn't a single side chain here that has a reasonable pkx in fact none of them has so the proton doesn't really find another place where it's happy and where it can just sit comfortably at a low energy state it's going to essentially wander around looking for such a place never finding any because this one is already taken and it's the only one there so we here have in a doubly charged peptide we have a very very mobile proton one that is locked down one that is mobile now look what happens this is the triangle plot we start seeing fragmentation already at about 30 electron volts and we get complete coverage really quickly this is a very very sharp curve and we get that at 60 electron volts which is where for the singly charged version we saw the first signs of fragmentation so it's very obvious the mobile proton that we have created here by adding two protons to the sequence yield a lot of coverage very quickly at low energy it still shows the mobile proton hypothesis has merit by the way these curves that you see here are the ones that we've talked about for detectors there are sigmoid curves they don't just show up in detectors now to to finish the whole story let's have a look at this other peptide the circle and doubly charged state obviously one of the protons is going to be on the arginine and it's not going anywhere i think we've agreed on that we've seen that on the singly charged version now the second proton is going to try and find the location to be to feel happy so it it's not going to be happy on any of these residues because they don't have any pkx so they're not basic in any way but look at this we have an n-terminal proline and remember that the n-terminal proline the amino group which is cyclical there that amino group is pretty basic so our second proton is going to find a happy home on the n-terminus as a result of this low energy state that can occupy here on the proline it's not going to travel around so we've got two lockdown protons one here and one there and that and that situation leads to poorer fragmentation because we don't have a wandering proton like we had in the first case in the trial case and look what happens to the collision energies needed to get good fragmentation for this second peptide with the arginine at the c-terminal and the proline at the n-terminus the circle it is very high and in fact you only reach full coverage at the same collision energies we had for the singly charged state so it shows very conclusively that the mobile proton hypothesis is very very valuable it the paper actually goes beyond that and then takes the next step which is to take the same peptide sequences and add an additional arginine now the two arginines are sufficiently widely spaced that each of them can harbor a proton without the protons deflecting each other in fact in peptide backbones usually the side chains point opposite directions which means that the protons are actually pretty far apart and they're perfectly happy where they are and as we would expect a doubly charged version of these peptides fragments exactly as badly as the singly charged version of the one with the single arginine because again one proton is going to live on the first arginine the second proton is going to live on the second arginine and the same here and none of these two sequences will have a mobile proton so this is quite convincing evidence of the mobile proton hypothesis now i have to tell you something else about this which is that arginine and lysine are very different kinds of amino acids because arginine is a hundred times more basic what you will see in an arginine ending peptide is that it's very heavily biased towards y ions if you have a lysine at the end to see terminus it will also have y ions but it will probably have a few more b ions and it will typically fragment more easily because the proton on a lysine is more mobile than the proton on an arginine the energy well the energy valley in which the proton sits in arginine is much deeper and much more comfortable than the one in lysine it's a hundred times deeper a hundred times more comfortable so it's easier to dislodge that proton from a lysine and therefore these peptides tend to fly apart a little bit easier and a little bit more comprehensively so lysine peptides are shifted a little bit back compared to arginine peptides and of course anything with a proline at the end terminus is harder to fragment because that creates a second valley for a proton to sit and not be mobile so you will see these patterns if you care to look for them another thing that you can notice is strange patterns when you have an internal lysine a mist cleavage or an internal arginine that too will localize the charge and you will see a very prominent break a very strong signal for the fragment right adjacent to the internal arginine lysine and you will see fewer fragments outside of that range these kind of things are very typical another typical thing that you tend to see with fragmentation and again look at a lot of spectra which is so easy because there are so many in the public domain a great software to look at them you can see that a proline somewhere internally is usually a site that gives a very very prominent fragment ion the reason for this is that the proline bond is actually very weak so a proline to something else bond we talked about how this is a helix breaker and how this is a special kind of bond with assist trans location so it can act a bit like a hinge that bond is super weak which means that it's very easy to break the peptide there which means that in the majority of cases the peptide will break at the proline bond so if there is an internal proline expect a really big peak there for the fragment spectrum because that's the place where the backbone will break most likely finally we talked about hcd very briefly at the end of the last lecture and hcd this higher energy collisional induced association is a very special kind of fragmentation when it comes to the kind of fragments we expect for the simple reason that b ions which are generated whenever you split a peptide into you get a bi and a y ion these bi ions are actually very unstable if you go back to the previous lecture you have a look at the structure again of these ions you'll see that they end on a co but usually we don't see these kind of bonds usually our carbon has a double bond to the oxygen and then has another bond to another group and then of course it has the bond that links it to the alpha carbon as well so the carbon atom needs four bonds and the four electrons to share and the oxygen only needs two so what people draw is usually a carbon atom connected to an oxygen atom by three bonds so in exchange of three electrons which puts a positive charge of the oxygen and is extremely unlikely to happen because oxygen is quite electronegative and really likes to to have electrons closely located to it as a result this is a very unstable kind of compound a y ion on the other hand you will see that it is stuck with an nh group but that nh group if you put an additional proton there becomes nh2 which is like a mini peptide and that is a far more stable configuration so what we see is when we break a peptide according to by breakage so according to the peptide bond we will get a very stable y ion relatively speaking and a very unstable bi ion so bi ions if you give them a little bit of energy they will refragment and they will they will very quickly break themselves down in hcd where we have this higher energy most of the bi ions are gone they don't show up in the spectrum anymore with a very notable exception which is the b2 ion so what is special about the b2 ion it starts with an amine an nh2 of the original n-terminus of the peptide alpha carbon so the first central carbon of the first residue the carboxyl carbon of the first residue the amine of the second residue the central carbon of the second residue and then finally the carboxyl carbon of the second residue so let's count we've got one two three four five six carbon atoms carbon nitrogen atoms in a row that actually cyclizes and becomes a six ring structure and that six ring structure is very very stable so what you see there is a cyclization of the b2 ion which keeps it intact it becomes strong enough to withstand further fragmentation all the other bi ions the longer bi ions it's very hard to cyclize because the the ring structures will be very big and also because it would be very difficult for the ends to meet up and join together anyway and these will deteriorate through subsequent fragmentation steps into the b2 which will then cyclize and lock in so all the bi ions get compressed if you like into this b2 ion sometimes you can see a b3 ion as well the b3 ion is already a lot bigger it's nine residues big ring but it can happen and you see some of it so that is something to look out for in hcd spectra very few bi ions but a pretty prominent b2 ion nonetheless because the b2 ion is special it is much more stable than all the other ions it's really interesting literature on that but we just don't have time to go into it but if you're interested if you just look on gas phase chemistry of the b2 ion you will find a lot of interesting studies on that so remember the mobile proton hypothesis allows us to explain a lot of the observed patterns and fragmentation spectra based on the sequence which is very valuable when you're trying to interpret a sequence of a sequence to spectrum assignments or a peptide to spectrum matches more commonly referred to it actually allows you to say based on the sequence do i expect this is there an internal protein do i expect a big peak and is there a big peak you can also look at a lysine at the c-terminus we should see more b ions and a better spread of y ions if we see an arginine at the enter at the c-terminus we can say ah we would expect a lot of y ions especially the ones close to the arginine and so forth so this can help you interpret things if you understand this and if you have a basic notion of this and of the pk axis and the pkb's you should be able with a few example data sets to quickly learn what kind of spectra you're supposed to see for certain analytes this is very interesting you get control of your data and you get to learn how the mass spectrometry actually functions on your sample now i'll just finish up with this last consideration remember that in maldi we had singly charged peptides so that was really great because we didn't have to worry about the charge state assignment in electrospray we could have two plus three plus four plus charge states and that would be really difficult to sort out later then again we learned that with high resolution instruments which is essentially anything about apart from an ion trap these days we can look at the isotopes and the distances between the isotopes will reveal the charge state so it wasn't quite a bit of quite as big a problem anymore now the one plus thing from maldi comes back to create a little problem for us if our peptide has a single charge and we fragment the peptide somewhere we now have a b ion and a y ion the problem is we have only one charge so where does the charge go it has to either go to the b ion making a b ion or it has to go to the y ion and whichever part b or y gets the charge becomes an ion the other thing becomes an uncharged molecule and disappears from the mass spectrometry so the efficiency of fragment recovery in maldi is necessarily half because you will definitely lose one of the two fragment ions you will not be able to see both this is really problematic because it creates less dense less informative spectra i say problematic it's let's say it's annoying but rather than problematic and it's less efficient as a result mind you what would you expect a b ion or a y ion to capture the charge that you can figure out from the sequence and from the pkx if we have a lysine on this end and a proline on that end i would say the chances are roughly 50 50 and it might depend on which of the fragments is the bigger fragment and the bigger the fragment the more chance for the proton to be happy essentially if however you have an arginine here and an apolar amino acid or a negative amino acid with a really low pkb for the n-terminus there then the proton is going to make an energetic choice it's going to end up here on the y ion this is one of the reasons as well why you see a lot of y ions with arginine containing peptides because it is more likely that the charges stay on the y ion on the other hand if we have c id sorry electoral spray it has nothing to do with the fragmentation per se if we have electoral spray ionization when we have two plus three plus four plus charges obviously we can split the charge over the y and the b ion and in fact this is not such a silly idea because if we have a fragment here which is say four or five amino acids long and we squeeze two charges there remember one can be on the lysine or arginine but the other one has to find another nice place to live and that may be hard on such a short peptide to find another place but on the b ion we still have the original amino terminus which is a very nice place in general for the proton to live so it is more likely to yield two singly charged ion ions rather than a single two plus ion on either side nevertheless this can be possible and it really depends on the sequence and you can probably figure it out if you look at the pkx and pkb's you can have a good good idea of whether or not this pattern that you see on the fragment ions makes sense this could be very hydrophobic and negatively charged at the end terminus and it will not be very interesting for a charge this one could have a proline at the end terminus and an arginine here and so two charges would definitely end up on this side so again knowing about the basicity of the side chain and of the end terminus and knowing a little bit about the mobile proton model and thinking a little bit about the composition of your ions in the gas phase can help you a lot with interpreting your spectrum so with that we come to the end of this lecture and we actually come to the end of the entire series on mass-petrometry basics I do hope you've enjoyed it and it was educational for you and I would like to thank you for staying with us throughout the entire course bye