From xue.xuexue from gmail.com Mon Nov 3 03:45:24 2008 From: xue.xuexue from gmail.com (xue.xuexue@gmail.com) Date: Mon Nov 3 12:34:59 2008 Subject: [Microbiology] Re: I need help in phytase activity References: Message-ID: pls. download the following doc. of inspecting phytase activity, http://www.fudabiotech.com/data/Determination_of_Phytase_Activity.pdf It is from Fujian Fuda Biotech Co., Ltd. in China, a pioneer of phytase manufacturer and supplier. On Sep 26, 4:00?pm, mohamed eda wrote: > I isolated some fungal isolates from compost sample and I want to measure the phytase activity of theses fungi in the liquid media. I want to ask about which media can I use and the method for measuring the phytase activity in this liquid media > thanks in advance ? > Mohamed Eida > ? ?Agricultural Microbiology Dept. > ? ?National Research Center, Egypt > ?Hiroshima University ? > ?Graduate School of Biosphere Science > Assessment of Environmental Dynamics Dept. > ?Plant Environmental Science Lab. ? > > phone No. 090-6405-4222 ?(SoftBank) ? From hanliat from tp.edu.sg Thu Nov 6 02:30:41 2008 From: hanliat from tp.edu.sg (Loh Han Liat) Date: Thu Nov 6 14:28:09 2008 Subject: [Microbiology] Reporting of enumeration results Message-ID: <2DD6DB1F7798C9418ED50B03C2F91C01B140C0@TAM-STFEX1.TP.EDU.SG> Hi All, I've got questions regarding the reporting of enumeration results based on plating. The same issue occurs with several testing methods from FDA-BAM as well as AOAC, and I really need some good advice. I'll use the example of enumerating for S. aureus, according to FDA-BAM. Accordingly, I'll divide 1 ml of inoculum over 3 Baird-Parker agar plates. After incubation, I'll select plates that have 20-200 colonies, add up the S. aureus colonies on the 3 plates and report as count per g X the dilution factor. Of course prior to this, I would have determined the fraction of "true" S. aureus among the presumptive colonies and all that. Now, the problem is I never get anywhere close to 20 colonies on any of my plates, for example, as in the table below. Dilution Plate 1 (0.4 ml) Plate 2 (0.3 ml) Plate 3 (0.3 ml) 10-1 5 colonies 4 colonies 5 colonies Assume that I've already confirmed all 14 colonies are S. aureus, how do I report my results? There's a clause somewhere in the manual that says if the count from the lowest dilution is < 20, I could still use the plates. Does this mean I could report the results as 140 cfu/g? If not, how should I report the results? Also how should I report the results if there're no colonies on any of the plates? Any advice, opinions or comments from anyone with any experience in this area would be greatly appreciated. Thank you very much! Regards HL From limbic_lesion from hotmail.com Thu Nov 6 18:58:51 2008 From: limbic_lesion from hotmail.com (N10) Date: Thu Nov 6 22:19:24 2008 Subject: [Microbiology] Re: Reporting of enumeration results References: Message-ID: "Loh Han Liat" wrote in message news:mailman.643.1225999747.29717.microbio@net.bio.net... Hi All, I've got questions regarding the reporting of enumeration results based on plating. The same issue occurs with several testing methods from FDA-BAM as well as AOAC, and I really need some good advice. I'll use the example of enumerating for S. aureus, according to FDA-BAM. Accordingly, I'll divide 1 ml of inoculum over 3 Baird-Parker agar plates. After incubation, I'll select plates that have 20-200 colonies, add up the S. aureus colonies on the 3 plates and report as count per g X the dilution factor. Of course prior to this, I would have determined the fraction of "true" S. aureus among the presumptive colonies and all that. Now, the problem is I never get anywhere close to 20 colonies on any of my plates, for example, as in the table below. Dilution Plate 1 (0.4 ml) Plate 2 (0.3 ml) Plate 3 (0.3 ml) 10-1 5 colonies 4 colonies 5 colonies Assume that I've already confirmed all 14 colonies are S. aureus, how do I report my results? There's a clause somewhere in the manual that says if the count from the lowest dilution is < 20, I could still use the plates. Does this mean I could report the results as 140 cfu/g? If not, how should I report the results? Also how should I report the results if there're no colonies on any of the plates? Any advice, opinions or comments from anyone with any experience in this area would be greatly appreciated. Thank you very much! Regards HL Hi Hl If you are following your protocol exactly then you are no doubt returning the correct result. The problem with microbiological assays of this type is that at low levels of isolation there is usually a high degree of " Uncertainty" associated with the result of obtained At high levels of uncertainty you really should report your result with a caveat expresing the range for upper an lower limits within which results might reside. YOu need to carry out a validation exercise to calculate the degree of uncertainty associated with this assay in your lab and possibley with respect to concensus results in ring trials. Uncertainty is a statiscally quanity related to confidence limits ( therefore functions of varience and SD) and which takes into account repeatability, reporducability , precision and bias. There are no set rules for the calculation of uncertainty but some stable guide lines do exist based on sound statiscal criteria. Thsi link might help as will many other ; google "Microbiological uncertainty of meadsurement www.ifcc.org/ejifcc/vol13no4/130401006.htm Until then report your results :- Any isolation of confirm SA is significant ..usually. Hope this helps From yjgent from nospamcox.net Fri Nov 7 11:38:45 2008 From: yjgent from nospamcox.net (John Gentile) Date: Fri Nov 7 15:07:40 2008 Subject: [Microbiology] Re: Reporting of enumeration results References: Message-ID: <2008110711384516807-yjgent@nospamcoxnet> On 2008-11-06 18:58:51 -0500, "N10" said: > > "Loh Han Liat" wrote in message > news:mailman.643.1225999747.29717.microbio@net.bio.net... > Hi All, > > > > I've got questions regarding the reporting of enumeration results based > on plating. The same issue occurs with several testing methods from > FDA-BAM as well as AOAC, and I really need some good advice. > > > > I'll use the example of enumerating for S. aureus, according to FDA-BAM. > > > > Accordingly, I'll divide 1 ml of inoculum over 3 Baird-Parker agar > plates. After incubation, I'll select plates that have 20-200 colonies, > add up the S. aureus colonies on the 3 plates and report as count per g > X the dilution factor. Of course prior to this, I would have determined > the fraction of "true" S. aureus among the presumptive colonies and all > that. > > > > Now, the problem is I never get anywhere close to 20 colonies on any of > my plates, for example, as in the table below. > > > > Dilution > > Plate 1 (0.4 ml) > > Plate 2 (0.3 ml) > > Plate 3 (0.3 ml) > > 10-1 > > 5 colonies > > 4 colonies > > 5 colonies > > > > Assume that I've already confirmed all 14 colonies are S. aureus, how do > I report my results? > > > > There's a clause somewhere in the manual that says if the count from the > lowest dilution is < 20, I could still use the plates. Does this mean I > could report the results as 140 cfu/g? If not, how should I report the > results? > > > > Also how should I report the results if there're no colonies on any of > the plates? > > > > Any advice, opinions or comments from anyone with any experience in this > area would be greatly appreciated. > > > > Thank you very much! > > > > Regards > > HL > > > Hi Hl > > > If you are following your protocol exactly then you are no doubt returning > the correct result. The problem with microbiological assays of this type is > that at low levels of isolation there is usually a high degree of " > Uncertainty" associated with the result of obtained At high levels of > uncertainty you really should report your result with a caveat expresing > the range for upper an lower limits within which results might reside. > > YOu need to carry out a validation exercise to calculate the degree of > uncertainty associated with this assay in your lab and possibley with > respect to concensus results in ring trials. Uncertainty is a statiscally > quanity related to confidence limits ( therefore functions of varience and > SD) and which takes into account repeatability, reporducability , precision > and bias. There are no set rules for the calculation of uncertainty but > some stable guide lines do exist based on sound statiscal criteria. > > Thsi link might help as will many other ; google "Microbiological > uncertainty of meadsurement > > www.ifcc.org/ejifcc/vol13no4/130401006.htm > > Until then report your results :- Any isolation of confirm SA is > significant ..usually. > > Hope this helps Your calculation seems to be correct, and if so, direct plating of your suspension should give sufficient countable colonies in the 20 - 200 cfu range. It would confirm your dilution results. -- John Gentile MS, M(ASCP) Laboratory Information Mgr. VA Medical Center Providence, RI yjgent@cox.net From nishamirsha2002 from yahoo.com Mon Nov 10 19:31:16 2008 From: nishamirsha2002 from yahoo.com (mehru nisha) Date: Tue Nov 11 12:29:11 2008 Subject: [Microbiology] Please advise Message-ID: <700290.16628.qm@web30202.mail.mud.yahoo.com> ? ? Dear sir, Good day to you. I'm Nisha from Malaysia. I'm a Master student working on live attenuated vaccine on Shigella flexneri 2a. Sir, I have come across your article entittle "vector to knockout E. coli genes". I am working on mutating 2 genes in Shigella namely, apy and hemA gene. Currently im working on hemA gene. I have successfully mutate the gene by inserting a kannamycin marker and i have transformed the mutated gene in a suicidal vector called pWM91. ? But, i unfortunately i have problem? in deleting the wild hemA gene in Shigella. I have transformed my construct into Shigella by conjugation with E.coli and i have obtained merodiploids. I have tried sucrose selection many many times, its already 5 months now-its still a failure!!!! ? I'm getting worried and frustrated.. ? Can you please advise me sir? ? Thank you for your kindness. ? Sincerely, Nisha New Email addresses available on Yahoo! Get the Email name you've always wanted on the new @ymail and @rocketmail. Hurry before someone else does! http://mail.promotions.yahoo.com/newdomains/aa/ From monarch00 from comcast.net Sun Nov 23 22:55:59 2008 From: monarch00 from comcast.net (monarch00@comcast.net) Date: Sun Nov 23 23:37:24 2008 Subject: [Microbiology] membrane filtration technique Message-ID: What are the rules for counting and calculating for total bacteria using membrane filtration technique? From limbic_lesion from hotmail.com Mon Nov 24 19:17:27 2008 From: limbic_lesion from hotmail.com (N10) Date: Mon Nov 24 20:08:11 2008 Subject: [Microbiology] Re: membrane filtration technique References: Message-ID: wrote in message news:db9ki4ph7ffflcftg858kf7lg5licjaf1l@4ax.com... > What are the rules for counting and calculating for total bacteria > using membrane filtration technique? Heloo Re counting ;the colony count per ml is returned as colonies/volume filtered mls Obviously the lowest colony counts is 0 which rightly would be expressed as < 1 colony per volume filtered In the case of low numbers you would correcftly express the count obtained with some measure of uncertainty of measurmemnt which is usually the count obtained +/- the sd*1.96 for lower range counts. There will be upper limit to the number of colonies you can count on a membrane but that will depend on which protcol you are using and the size of filter; perhapes another can help on this one. Best N10