Swabs Campylobacter...

...still not growing. Give them another 2 days to grow. # Apparantly the "Campylobacter" cultures I was trying are problematic - they grow unresonably fast; they even grow in AEROBIC ENVIRONMENT!! Ah, I didn't notice today is Halloween until I saw the board on our home's door "Sorry, no more lollies :( ". Hilary had gone out for collecting lollies when I came home.

BBQ

As usual, it's always noisy when it comes to BBQ - kids (Hilary and her pals) are dancing and singing and yelling and couldn't be louder. Actually, yesterday was Andrew and Marivic's marriage anniversary and this BBQ is a sort of celebration of them... # Campylobacter swabs of yesterday are not growing yet. # Salmonella typhimurium MIC result looks quite good.

Yuuki gonna be South Island

with 2-3 of his (Japanese) friends. They're gonna travel around there for the whole next week (backpack).

Sample from MedLab

Finally culture samples from MedLab arrived today (obtained by Kerry though). That means I'll get some more formal data other than Salmonella from now on. The cultures (this time, Campylobacter spp. and Salmonella spp.) are dipped in charcoal media with swabs in plastic tubes. Somehow one of the Salmonella spp. are put together in the same bag of Campylobacter spp.. Is this right...?

CampyPouch (cont'd)

All testing Campylobacters are growing quite well when I see them today. But there's something strange - they all grow oddly fast. For in case (of not been campylobacter but actually been contaminated by other organisms), I redo different conditions with new storage Campylobacter culture storage beads and see what'll happen. # Incubation temperature: same as yesterday # Incubation condition: basically same as yesterday, except of adding "normal incubater (i.e. aerobic condition)".

CampyPak CampyPouch

Sometime some wierd stuff can be found in the lab. Guess what I've found today - CampyPouch, which has expired in the year 1995 and 1999(?! How old...) As this sort of GasPak is quite expensive, Kerry has been keeping them until now even though not handling Campylobacter. And since it's expired (and out of my curiosity), I'd try if it really works (although they're not supposed to be still valid any more). # Incubation temperature: 37C and 42C. # Incubation condition: CampyPouch, candle jar and CO2 incubater.

Which PCR?

If I am right, when we want to isolate microbials, we normally have to streak mixed (and unknown) cultures on appropriate agar plates and pick single colonies, followed by streaking the single colonies on other agar plates/slants/broths and incubate them (and check if they are exactly pure cultures). However, as we've discussed with Ray and Peter before, I'll have to identify Campylobacter jejuni, C. coli and C. fetus from unknown Campylobacter spp. sent from MedLabs by means of PCR. Actually, there have been several variations of PCR tests for Campylobacter identification, such as multiplex PCR and PCR-RFLP. Here comes a problem - which of them should I use? mPCR can amplify several different DNA targets at once without seperating into tubes, and thus save lots of time and tubes if there're more than one candidate gene target. However it's not perfect. One of its downsides is the possibility of "primer dimer". To eliminate it, some preventions such as "hot start" should be taken. In order to preceed "hot start PCR", normally "AmpliTaq Gold" would be used rather than other normal Taq. AmpliTaq Gold itself is good but is also extremely expensive, which would be a big problem. Besides, I have a bad memory with it during my Master course before (nothing to do with its quality though). On the other hand, PCR-RFLP requires restriction enzyme, which is quite prone under unsuitable preserving condition. Although it can be completed with normal Taq and only one (or two) restriction enzyme and identify different species (as long as their restriction maps have been recognized), the unstable enzyme itself is quite problematic. According to Ray, he'd use multiplex PCR. Hmm...so, then, I'll have to search suitable primers for each of the species and optimal PCR condition. By the way, when it comes to disposing EtBr, some people say "just soak in bleach then throw away" yet I've also heard others said "that would inversely make it more carcinogenic". I used to place the used agarose gels under sunlight (as EtBr is light-sensitive), but I never quite sure what the proper treatment is. And I'm quite surprised when Ray told me that they normally DISPOSE OF EtBr DOWN THE SINK!!

Eating outside again

...and again, not with the Turners but with the Aunt. Unfortunately, Jason, a Taiwanese going to sit IELTS and asking me some advises, wasn't available today and didn't come together with us.

Labour Day, the national holiday

...and of course there's nobody at campus. Should be a good day to do research work, I thought. However, I was wrong - the door toward -70C freezer and Autoclave room is locked and my security card DOESN'T WORK ON IT, thus I couldn't get my sample and also couldn't sterilize media and broth!! Hence, there's nothing much I can do today... -.-;;

Eating outside for dinner

...but not with the Turners but with Aunt of Buddhism community.

Auckland Museum and lab

Went to Auckland Museum and Manukau with the Turners today, which is followed by going to lab this afternoon for collecting data and subculturing. The MIC result was quite terrible anyway... :(

Long weekend

Next Monday is Labour Day, a public holiday, and thus also public facilities such as bus and uni. are on holiday as well, which is not good for me - I can't go to lab without bus! (all staff at uni. and lab will also be on holiday though...)

Tailing-off curve? (and PCR)

This time the contamination issue is resolved. However, the growth curve reveals in another strange way - the curves are tailing off which they're supposed to be level off? What I can figure out in my mind is two possibility - not enough dilution or too short incubation time. Kerry and Peter think it may probably because Salmonella typhimurium are switching their nutrition requirment from protein or sugar to other component, so that they're still keep growing. By the way, Peter finally gave me a green light to prosecute PCR after discussion with Ray. Hmmm...but I didn't realize that the price of primers are the same as (or at least not too far away from) in Taiwan. It's just NZ$ 20...I'd been expecting that it'd be much more expensive in NZ... Anyway, now I'll have to find appropriate primer sequences for identify the species. Today's growth curve setting: 37C, 48 cycles (24 hrs), dilute from 10^-2.

Growth curve on BMG plate reader

The result of growth curve is still terrible. However, look at the microplate carefully and I notice that inter-contamination between culture_wells and blank_wells can be seen. Apparently this is caused when the BioRad microplate reader shook the plate and the media in the wells splashed out and contaminated each other. Unfortunately this BioRad reader can't change its shaking speed, thus I need to try another microplate machine - BMG microplate reader. And also, for in case, I adjusted the total volume of each well to 125 ul, dilution facter 6 (can't dilute by 10-fold because we don't have suitable 8ch pipette in this case) and for rather in case, kept a fence_well between culture_wells and blank_wells. Microplate reader setting: 37C, rotate shaking, shaking diameter 3mm, 36 cycles (i.e. 18 hrs)

Growth curve 'n' Campylobacter

1. Growth curve Somehow the curves are still strange. How come even blank_wells also shows tailing-off growth curve?! Kerry suggested increasing my sample volume from 90ul to, say, 200 ul. => Repeat making growth curve. 2. Campylobacter On the other hand, some Campylobacters (well, actually only 1 in 15 on average) are forming quite big CFUs, which are the biggiest ones I've seen these days. Keep testing other isolates!

Faulty hardwares

Found couple of news about faulty hardwares - laptop batteries and digital cameras. 1. HP Mobile Battery Pack Replacement Program This claims that some models of battery packs would suffer short circuit and are advised to replace without paying any shipment or other fees. Basically the problematic models in this issues are those which barcode start with either "GC", "IA", "L0" or "L1". Although my laptop (Compaq Presario 2100 series) is in the list of suffered-model, I don't have to be concerned about it since my battery pack bar-code starts with "SS". However, the second news does annoying me. 2. Digital-camera makers to replace faulty image sensors What it says about the faulty digital cameras is that those problematic models would appear distorted virtically or even blank images owing to CCDs on them. The distort images look like this: (Pictures from Konica Minolta) The suffered models range from Sony, Canon, Konica Minolta, Fuji, Ricoh to Olympus, which is quite a long list. Unfortunately my one - Canon Powershot A70 - is also in the list and, what's more, I noticed that similar sympton has come out days ago. (But rather than virtical distort, mine one appears horizontally). It's good to see the companies would replace or fix them regardless of warranty status. The bad thing is - I haven't seen any announcement on Canon_NZ's website...

Nothing much today

As soon as seeing the result, I found the curves are strange. It seems even blank wells were also contaminated. Futhermore, somebody may had changed the setting of the computer connected to the BioRad microplate reader before I started my incubation yesterday, and so that the result wasn't printed out after I stopped the reading... :( Anyway, just redo what'd done yesterday again - adjust absorbance (A540 = 0.5), serial dilute, incubate and wait... till Monday and see what'll happen.

Rosamund's leaving

Rosamund is gonna leave Waikato next week, and this week could be the last chance to see her. Hence I've sent all folks of Lv7 CAEL this information and ask them to attend the farewell party held this afternoon. In the party, there are some unusual faces that I haven't seen for quite a long time. Besides Carol (the boss), Katherine, Kylie and also Meg (huh, really long time no see) came to join the party. Eventully, Tomoko, Suzana, Tina and Xaomin came to the party. Well, bettern than nothing. Today's exp.: Salmonella typhimurium serovar. DT104 dilution optimisation (building growth curve). Serial diluting the culture by a facter of 10 (90ul/well), incubate in Bio-Rad microplate reader (37C) and wait for 18 hrs.

Identify or not?

Peter thinks that Campylobacter is a kind of "difficult cultures" and wouldn't like me to spend too much effort (and budget) on isolation and identification of Campylobacter species. After all, isolation and identification of Campylobacter is not the point of my research work. (and also many medical papers just notify "Campylobacter spp" rather than specifically notify the species' name like "Campylobacter coli""Campylobacter jejuni" such and such) On the other hand, Ray - my another supervisor - thinks that to identify the species of Campylobacter is very important from medical point of view and suggested me to use PCR method rather than traditional biochemical methods to identify them (to his mind, PCR is cheaper than traditional biochemical tests). However, Peter thinks identify the species of Campylobacter with PCR would cost hundreds of thousands of dollars and take months of time to do, which is unaffordable and also unfavorable. Indeed, those molecular methods are normally expensive than traditional tests; and also Campylobacters are growing much slower than other bacteria (such Salmonella spp, E.coli etc.) and in most case they don't survive in -70C storage. Identify or not?...Mmm...it's really a dillema.

Testing vitality

As we have to clear out some more space in -70C freezer, I also start to check the vitality of Campylobacters in the freezer; if they're not growing then we may throw them away (not rubbish bin, of course). Consider the space in the candle jar, I inoculated 14 isolates (SC1 - SC14) onto 7 charcoal agar plates (i.e. 2 beads/plate). And also, some referable papers about "species-specific PCR" are founded. Looks like they are based on 16S rRNA, nested PCR, real-time RT-PCR or RFLP methods. Some Campylobacter-specific primers are also reported. This could be the way I can go.

Homestay meeting, Hamilton Lake Garden and night

After school time, we came back to the Turners and let my parents see them. Due to the time, we just had a short talk and faced toward Hamilton garden. Although I've been in NZ for over a year, I haven't walked around in the Garden yet. It is a maze-like green path and consists of Japan, Rome and whatever style parts. During the dinner (again, long dinner), I was asked by Aunt - one of Tzuchi members - to join Tzuchi's charity activity. What she hoped me to do is to translate some of their documents and formal e-communication. At this moment it's hard to say if this would influence my research, but here I just left my email to her.

Isolation method

As Medlab told Kerry that they couldn't identify what species the Campylobacters and Yersinia they're going to send are, I'll have to identify them by myself. As with this issue, I had a talk with Ray. According to Ray, commercial isolation systems (a.k.a. "kit") are not cost-friendly; traditional biochemical methods are time, effort and budget consuming. The latter is also hard to judge the results. To his mind, PCR would be the best choice (he said because he likes simple yes/no results). Hmm, PCR...that means I'll have to handle nucletide things again.

Meeting with Peter

One of the objectives my parents come to NZ is to see my superviser. Arriving at school and found the reserved car park (at first we're confused with Amy's idea...), we walked up to Peter's office. His office is obviously too small to pack all of his visitors (because both Peter and me didn't expect that Uncle Yeh and Amy would also come into the office... :( ) ...anyway, Dad seems to be very excited to see Peter. Before that we expected he'd just say hello to Peter, but in fact they talked quite a long time. Before out Peter's office, they exchenged their research papers each other (and Sun Cake from Dad...Peter, I think you've had enough sweet, haven't you?) After that, they visited HRU (Honey Research Unit) - my laboratory - for a short while then went to other place...Hey! They haven't walked around the campus?!

Parents arrival

In spite of the bad weather, some members of Tzuchi and I went to Auckland airport for picking my parents up this afternoon. Although the flight delayed a bit, they eventually arrived safely. Needless to say, we gave each other a big hug as soon as we got together. I see how excited my Dad was when he hugged me. However, we had to face Hamilton as soon as possible as Dad had to make a speech about health care. For this reason, I had been asked to bring my laptop as well, so that he could show several pictures to Tzuchi audiences after the speech. And, as having been expected, the questions were raised one after another and it looked like to be an endless activity. As the church where the speech was held could be lent out until 6 pm, Uncle Yeh had to stop the speech although others still would like to ask somthing to Dad. Tonight I didn't go back to the Turners but stay with parents in Aunt Liu's house. After shower, we had a loooooooooong dinner with some Tzuchi members (and some other audiences in the speech). This dinner lasted until 11 pm... -.-;;

Campylobacter 'n' other species

Campylobacter in 42C candle jar do grow much better than in 37C CO2 incubater. I'm not sure which facter matters the most; but anyway, things seem to be getting better. On the other hand, Kerry said Medlab told her that they couldn't identify what species the Campylobacter and Yersinia they're going to send me. That is, I'll have to identify what species they are. Mm...any good methods to identify Campylobacter jejuni, C. coli, C. fetus and Yersinia enterocolitica? Biochemical methods may be a way to go, as long as everything can go smooth if following Bergey's Manual of Determinative Bacteriology (the differentiation criteria look quite complicating though)

This appointment and that appointment

Checked with Peter and rang to Amy, the person made appointment with Peter, now the fact was made clear - the 10 am appointment was exactly made for my Dad ...by Amy. She is a member of Tzuchi and had been asked by Uncle Yeh to get contact with Peter... Knowing what on earch happens, the appointment was set to 9:30 am and the 10 am one was cancelled. Phew~~~~

Car park reservation and ...?!?!

Peter told me that he'll have an appointment at 10 am on next Monday, and asked me to ask my parents at what time they'd visit him. At Peter'd reserve a car parking place for us previously, I told him 9:30 am as my Dad would just say hello to him and wouldn't stay too long. However, I heard something embarrasing from Dad on telephone at night - Uncle Yeh had asked some members in Tzuchi to get contact with Peter for Dad and, what's more, the appointment was said to be 10 am ...!? I'm not quite sure, but I have a feeling...could the appointment Peter will have at 10 am on Monday be ...???

Candle jar

Some essential conditions for Campylobacter to grow: 1. CO2 5-10% 2. O2 5-10% 3. 42C 4. Humidity 5. Relatively long time -.-;; As Kerry is also using the CO2 incubater as well (for Haemophilus influenza), I can't use 42C in CO2 incubater. Hence I have to consider candle jar. Find a suitable plastic container (actually it was Virkon's container...), put agar plates and lit a short candle (make its height as low as possible so that it won't melt the plastic lid down) then put in normal (but 42C) incubater. That's it!

Another fraud mail (from eBay)

Following the yeasterday's fanky mail, here it comes another spoof mail. ########## Begin of suspicious mail ########### Update Your Account Information Within 24 Hours Valued eBay Member, According to our site policy you will have to confirm that you are the real owner of the eBay account by completing the following form or else your account will be suspended within 24 hours for investigations. Never share your eBay password to anyone! Establish your proof of identity with ID Verify (free of charge) - an easy way to help others trust you as their trading partner. The process takes about 5 minutes to complete and involves updating your eBay information. When you're successfully verified, you will receive an ID Verify icon in your feedback profile. Currently, the service is only available to residents of the United States and U.S. territories (Puerto Rico, US Virgin Islands and Guam.) ########## End of suspicious mail ################ Again, this fraud mail is still not smart enough to fool me.

Fraud mail

Today I got 2 unusual emails, which are both from Paypal(?!). The content is as following: ######### Biginning of the suspicious mail 1 ############# Dear valued PayPal® member: Due to concerns, for the safety and integrity of the paypal account we have issued this warning message. It has come to our attention that your PayPal® account information needs to be updated as part of our continuing commitment to protect your account and to reduce the instance of fraud on our website. If you could please take 5-10 minutes out of your online experience and update your personal records you will not run into any future problems with the online service. However, failure to update your records will result in account suspension. Please update your records on or before October 05, 2005. Once you have updated your account records your paypal account service will not be interrupted and will continue as normal. To update your PayPal® records click on the following link: http://www.paypal.com/cgi-bin/webscr?cmd=_login-run Thank You. PayPal® UPDATE TEAM Accounts Management As outlined in our User Agreement, PayPal® will periodically send you information about site changes and enhancements. Visit our Privacy Policy and User Agreement if you have any questions. http://www.paypal.com/cgi-bin/webscr?cmd=p/gen/ua/policy_privacy-outside The HTML graphics in this message have been displayed. [Edit Preferences - What's This?] ######### End of suspicious mail 1 ############# ######### Begin of suspicious mail 2 ############ PayPal Security Measures! We are contacting you to remind you that: on 24 August 2005 our Account Review Team identified some unusual activity in your account, one or more attempts to log in to your PayPal account from a foreign IP address. IP Address Time Country 80.53.1.130 August 24, 2005 15:05:08 PDT Poland 80.53.255.174 August 24, 2005 15:07:58 PDT Poland 141.85.99.169 August 24, 2005 15:13:09 PDT Romania 141.85.99.169 August 24, 2005 21:28:08 PDT Romania 195.61.146.130 August 24, 2005 21:33:43 PDT Romania In accordance with PayPal's User Agreement and to ensure that your account has not been compromised, access to your account was limited. Your account access will remain limited until this issue has been resolved. To secure your account and quickly restore full access, we may require some additional information from you. To securely confirm your PayPal information please go directly to https://www.paypal.com/ log in to your PayPal account and perform the steps necessary to restore your account access as soon as possible or click bellow: To continue your verification procedure click here Thank you for using PayPal! The PayPal Team Please do not reply to this e-mail. Mail sent to this address cannot be answered. For assistance, log in to your PayPal account and choose the "Help" link in the footer of any page. To receive email notifications in plain text instead of HTML, update your preferences here. ######## End of suspicious email 2 ############ Briefly, both of them are claiming that there're some security problems with my paypal account, and ask me to check id or login through the link given in the mail blah blah blah ... Bah! Stupid and ridiculous fraud mail! Too many his sort of silly phishing mail mucking around these days (and also strangely enough, much more people DO believe in or are trapped by these stupid jokes...) Unfortunately, these silly mails don't work on me~~~~ You stupid fraud~~~~ ♪~♫♫~~♪~

What a coincidence!!

IMHO I should have informed everyone I've changed my email account, and I've expected that all the mail would be sent to my new one - now it looks like not quite true. At least, Peter, my professor, sent email to my ex-mailbox several days ago which I didn't check it until now. Anyway, I borrowed several books from campus library this week (Tuesday or Wednesday, I think) as a reference of my gastro-intestinal pathogens list. So what does this do with the email mentioned above? I checked my ex-mailbox just now (which is a once-a-week job) and realized that Peter had sent me an email, which informs me that "there is a new book 'Medical Microbiology' in the library that may be interest of you". In fact, this is exactly one of the books I borrowed JUST BEFORE Peter mailed me this book information, whereas I didn't know he'd sent the information about it until now!! That's definitely a coincidence eh!!