tag:blogger.com,1999:blog-5376717865258108402.post4409999265725547331..comments2023-10-30T22:03:09.199+08:00Comments on TG01 Group 2: Week 14 MP - Inoculation and 2nd inoculation of Stenotrophomonas maltophiliatg01 group 2http://www.blogger.com/profile/15191373009020650145noreply@blogger.comBlogger14125tag:blogger.com,1999:blog-5376717865258108402.post-15889483426150251262008-10-03T16:20:00.000+08:002008-10-03T16:20:00.000+08:00Hi Justina, Different strains of S. maltophilia ha...Hi Justina, <BR/><BR/>Different strains of S. maltophilia have different growth rate and this is most likely due to the different environments the bacteria are isolated from. For example, if the bacteria is isolated from human body, it would adapt to 37 degree celcius much faster and better and have a faster higher growth rate. If the bacterium is isolated from the environment, naturally the bacterial will be used to the environmental temperature and thus if you grow the bacteria at 37 degree celcius, it would take a much longer time to adapt to 37 degree celcius and have a slower growth rate. <BR/><BR/>Hope you can understand!<BR/><BR/>Han Yang<BR/>TG01tg01 group 2https://www.blogger.com/profile/15191373009020650145noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-77610310047383556832008-10-03T16:17:00.000+08:002008-10-03T16:17:00.000+08:00Hi Jean, Haha, you think too much le...Warming the...Hi Jean, <BR/><BR/>Haha, you think too much le...Warming the LB broth in either incubator or water bath is perfectly alright and there are no special reasons...<BR/><BR/>Han Yang<BR/>TG01tg01 group 2https://www.blogger.com/profile/15191373009020650145noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-19631985872699774122008-10-03T15:59:00.000+08:002008-10-03T15:59:00.000+08:00Hi Ivan,After further clarification, orbital is NO...Hi Ivan,<BR/><BR/>After further clarification, orbital is NOT the brand of the shaker incubator but is the way how the shaker shakes. Orbital means revolving shaking motion. The brand of the shaker incubator is MRC. <BR/><BR/>My apology...<BR/><BR/>Han Yang<BR/>TG01tg01 group 2https://www.blogger.com/profile/15191373009020650145noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-74802114703709369392008-10-02T17:43:00.000+08:002008-10-02T17:43:00.000+08:00Hi Hanyang, just to confirm..we can also warm the ...Hi Hanyang, <BR/>just to confirm..we can also warm the LB broth in the 37 degree celcius water bath ya? I thought it would be must faster than the incubator. hmmz, so no special reasons for warming in incubator rite?<BR/><BR/>Jean Leong <BR/>TG02De Incredibleshttps://www.blogger.com/profile/01827248181408586035noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-78293906676408539112008-10-02T09:04:00.000+08:002008-10-02T09:04:00.000+08:00Hi hanyangWhy is there different growth rate for S...Hi hanyang<BR/><BR/>Why is there different growth rate for S.maltophilia?? Isn't all the growth rate for bacteria the same??<BR/><BR/>Justina<BR/>TG01SIPhttps://www.blogger.com/profile/01787954963215536276noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-17659104663264289182008-09-26T20:23:00.000+08:002008-09-26T20:23:00.000+08:00Wah, that was fast...! 8 comments received within ...Wah, that was fast...! 8 comments received within a day... sweat...!<BR/><BR/>Hi Shihui,<BR/><BR/>Homogeneous growth means equal or uniform growth.<BR/><BR/>Eh, We must first culture the S. maltophilia on LB agar, get the isolated colonies before inoculating them into the LB broth. Culturing is first step and inoculation and 2nd inoculation are second and third step of extraction of periplasmic proteins of S. maltophilia respectively. If you want to ask me which is the better way, I would say it is the conventional method which is streaking on LB agar. I feel that any bacteria should first be allowed to grow and multiply on suitable agar before they are used for any application. I have never tried directly inoculating the fresh S. maltophilia stock into the LB broth directly. Chances are the bacterium won't grow.<BR/><BR/>Hi Li Ping,<BR/><BR/>The washing step is done TWICE so as to ensure most of the dead cells, debris and toxic metabolic waste products are removed from the cells. Make sure that all cell pellet are resuspended in LB broth and centrifuge at the condition I have mentioned. It should do the trick!<BR/><BR/>Eh, we do not have to subculture the broth onto any agar. Following 2nd inoculation, we will perform chloroform shock which is mentioned by Benjamin's post in the earlier weeks. Please refer to his post on more details on how to extract the periplasmic proteins from S. maltophilia.<BR/><BR/>Hi Ying Chee,<BR/><BR/>LB agar is generally suitable for the growth of gram-negative bacteria such as S. maltophilia. It is rich in nutrients (e.g. peptides, vitamins, trace elements and minerals) that will provide optimum growth for the bacterium. Yes, S. maltophilia is a non-lactose fermenter.<BR/><BR/>We do not use sodium hypochloride or virkon solution. We simply autoclave the waste.<BR/><BR/>Hi Xin Ni,<BR/><BR/>For us, we do not do optimisation as to whether 20ml of LB broth is the optimum volume for inoculation. Perhaps for previous batches of MP students or through research, they have found out that 20ml LB broth is sufficient and good enough to support the growth of S. maltophilia. <BR/><BR/>Hi Ivan, <BR/><BR/>Orbital is the brand of the machine. LOL...! As the name implies, it is a 2-in-1 incubator which has a shaker function as well as allowing incubation at desired temperatures. It is just as simple as swtiching on the incubator and adjust the speed of shaking motion (rpm)and start incubating the LB broth...! The shaker function can be turned off. Heat is generated from the fan/motor and provide the temperature you need. When we are incubating the LB broth, it is good to tape the cap of the tube as we have loosened it to prevent spiilage. <BR/><BR/>Hi Yu Mei,<BR/><BR/>The chance of contamination is very, very slim as we are performing the experiment within the BSC 2. Since we abide to strict aseptic techniques, there has not been any contamination thus far. Strict aseptic techniques should always be followed when doing microbiology work. One precaution you could take is that if you feel that the inoculating loop has touched the work surface of BSC 2 which is non-sterile, do not dip it into the LB broth. This will definitely introduce contamination into the LB broth, thus growing unwanted bacteria. <BR/><BR/>The cell density meter works similarly as a spectrophotometer but just at different wavelength from the spectrophotometer(595nm -for Bradford assay), which is at 600nm. The results obtained from the cell density meter is comparable to spectrophotometer and it is very convenient to use it. <BR/> <BR/>We are culturing 11 strains of S. maltophilia, of which some are clinical isolates while some are environmental isolates. The environmental isolates would take a longer time to adapt to 37 degree celcius (be it growing in LB broth or LB agar) as it is obtained from the environment where the temperature is at around 28 degree celcius. Thus, these environmental isolates tend to grow slower than the clinical isolates which are obtained from patient specimens at 37 degree celcius.<BR/><BR/>Hi Huimin,<BR/><BR/>The S. maltophilia are obtained from ATCC (American Type Culture Collection) and from patient samples such as cerebrospinal fluid and blood culture. The S. maltophilia isolated from patient samples are processed by ATCC and we just have to purchase from them. <BR/><BR/>During Basic Microbiology, we have learnt that cloudy media indicates growth of bacteria. In order to ensure you are inoculating just S. maltophilia but not any other bacteria into the LB broth, you have to ensure your culturing step is done properly (e.g. follow strict aseptic techniques). This will assure you that what you grow on the LB agar plate are purely S. maltophilia but not any other bacteria and when during inoculation, you will be assured that what you pick up from the LB agar and inoculate into LB broth is S. maltophilia only. Thus, the cloudy LB broth can only indicate the growth of S. maltophilia but not any other bacteria. In addition, the LB broth is sterile as it is autoclaved before use. Hence, this provides more assurance that the LB broth is close to 99% free of any organism and pure.<BR/><BR/>Hi Ernest,<BR/><BR/>You can refer to my reply to Yumei and Huimin for the reason I am assured that the cloudy LB broth indicates the growth of S. maltophilia. <BR/><BR/>Since each strain of S. maltophilia is contained in individual vials and they are obtained from a trustworthy source such as the ATCC, you can be rest assured that what you pick up from the vial and culture on the LB agar is purely the strain of S. maltophilia you want and what you pick up (the colonies) from the LB agar and inoculate into the LB broth is 100% S. maltophilia UNLESS aseptic techniques are not followed. <BR/><BR/>We do not perform any gram-staining but gram-staining is one of the method to detect S. maltophilia. The purpose of our project is not so much on identification but more on protein work. <BR/><BR/>I hope my reply to everyone is satisfactory...! Phew...! =)<BR/><BR/>Han Yang <BR/>TG01tg01 group 2https://www.blogger.com/profile/15191373009020650145noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-59610447258975887572008-09-26T15:58:00.000+08:002008-09-26T15:58:00.000+08:00HiWhat are the methods of detecting Stenotrophomon...Hi<BR/><BR/>What are the methods of detecting Stenotrophomonas maltophilia?<BR/><BR/>Because you simply assumed when the LB broth turns cloudy, it indicates the growth of S. maltophilia.<BR/><BR/>Do you do a gram stain? Like to find out if it is gram negative?<BR/><BR/>Thank you<BR/>Ernest<BR/>0606330itg01 group 2https://www.blogger.com/profile/15191373009020650145noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-26129205993387266122008-09-26T15:10:00.000+08:002008-09-26T15:10:00.000+08:00hey hanyang!Can i know where you get the source of...hey hanyang!<BR/><BR/>Can i know where you get the source of S.maltophilia? (eg.urine, sttool, swab)<BR/><BR/>And you mentioned that if LB broth turns cloudy, it indicates the growth of S. maltophilia... Can it indicates other possiblities? (eg like what yumei mentioned contamination?)<BR/><BR/>Thanks!<BR/><BR/>Cheers,<BR/>Huimin<BR/>Tg01 =)SIPhttps://www.blogger.com/profile/01787954963215536276noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-61767376413506173662008-09-26T14:55:00.000+08:002008-09-26T14:55:00.000+08:00Hi hanyang!is there a chance of contamination? if ...Hi hanyang!<BR/><BR/>is there a chance of contamination? if yes, how do you know if it's contaminated? <BR/><BR/>i notice that you use cell density meter to check the concentration of the cells. does cell densiy meter works like spectrophotometer? <BR/><BR/>u mention "not all strains of S. maltophilia have simialr growth rate" what do u mean by other strains? <BR/><BR/>Thanks!<BR/>Yu Mei<BR/>TG01group1https://www.blogger.com/profile/02375953879442727626noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-22336123317675074962008-09-26T14:23:00.000+08:002008-09-26T14:23:00.000+08:00Hi,i jus wanna know what is a Orbital shaker incub...Hi,<BR/><BR/>i jus wanna know what is a Orbital shaker incubator? what is its function? i dun mind knowing the principle of the equipment/machine...<BR/><BR/>From Ivantg01 group 2https://www.blogger.com/profile/15191373009020650145noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-23040770535224066032008-09-26T14:12:00.000+08:002008-09-26T14:12:00.000+08:00Hi thereHow u noe "20ml is the optimal volume for...Hi there<BR/>How u noe "20ml is the optimal volume for growth of S. maltophilia"....do u do optimisation? like try out with different volume of LB broth?<BR/><BR/>Lim Xin Ni<BR/>TG02De Incredibleshttps://www.blogger.com/profile/01827248181408586035noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-78059748575585225042008-09-26T13:34:00.000+08:002008-09-26T13:34:00.000+08:00Hi Hanyang,I'm just curious why is LB agar chosen ...Hi Hanyang,<BR/><BR/>I'm just curious why is LB agar chosen instead of MacConkey or the others?Is it because S.maltophilia is a non-lactose fermenter? And for the waste bottle, do u use sodium hypochloride or virkon solution and what is the concentration of the solution u use and why?<BR/><BR/>Thanks<BR/>Ying Chee<BR/>TG01SIPhttps://www.blogger.com/profile/01787954963215536276noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-72653276281149685492008-09-26T13:05:00.001+08:002008-09-26T13:05:00.001+08:00Hello.How do you ensure that 'dead cells, debris a...Hello.<BR/><BR/>How do you ensure that 'dead cells, debris and toxic metabolic waste products from cells of S.maltophilia' are not present after 2nd inoculation of the organism into the broth?<BR/><BR/>And after 2nd inoculation into the broth, do you have to subculture the broth onto any agar?<BR/><BR/>Thanks!<BR/><BR/>-Li Ping-<BR/>0607498C<BR/>TG o2Fluid collectorshttps://www.blogger.com/profile/09173340508225590352noreply@blogger.comtag:blogger.com,1999:blog-5376717865258108402.post-42571235107409135292008-09-26T13:05:00.000+08:002008-09-26T13:05:00.000+08:00HI hanyang!What do u mean by homogeneous growth? A...HI hanyang!<BR/>What do u mean by homogeneous growth? <BR/><BR/>And which is the better way to grow S. maltophilia? broth or agar? <BR/>Thanks!<BR/>Shihui<BR/>0607135AFluid collectorshttps://www.blogger.com/profile/09173340508225590352noreply@blogger.com