Hi all, enjoying your SIP?? I hope you do enjoy it.. Left with 4 months!!!
Alright here to share with you my experiences during my attachment in school..
For my SIP, what I will be doing is mostly on data entry. Well seem very unrelated to what we doing right.. haha.. but it plays a major role in operating the whole lab smoothly. For examples what I dealing with is the purchasing or maintaining of equipments, chemicals products etc. So what I do is key in the amount and what is the amount use for then sending the invoices to the finance department. I will also be updating the actual prices for chemicals products for the next semester use. When lecturer wanted to buy chemical products, they will refer to the price stated to estimate the amount they going to spend on the items.
For my MP, I will be doing a project on 'Evaluation of anti-microbial activity in different tea extracts'. Tea is one of the most commonly consumed beverage(other than water) and reports have shown the benefits of tea to our health. So I will be setting up a whole cell-based assays which is(in my case) S.aureus to evaluate different tea extracts for anti-microbial activity. For now I will be focusing on what I have done in the past 4 weeks.
During the past 4 weeks I had done a series of experiments to determine the log phase of S.aureus. This is just the first part of my whole experiment.
Aims: To determine the log(exponential) phase of S.aureus.
Methods:
1. Preparing of 1L of Trypic-soy agar and 1L Trypic-soy broth.
2. Inoculate S.aureus into TSB and incubate at 37oC for 18 hours, shake with 150-200rpm, as
S.aureus is in beads form this is to dissolve the beads and allow the S.aureus to be release.
3. Streaking of S.aureus from TSB broth to TSA agar plate so as to obtain isolated colonies. The
streak plate is incubated at 37oC for 18 hours.
4. Gram stain of S.aureus, this is to check for the purity of the culture. For S.aureus, it will
appear purple and cocci in cluster.
5. Inoculation of one colony from the TSA agar plate to TSB broth and incubate at 37oC for
18 hours, shake with 150-200rpm.
6. Take OD reading at 600nm to find out the log phase of S.aureus at a specfic time(for me I
take the reading once every half an hour). The reading is to be continue until the culture
reached stationary phase.
7. Confirm OD reading and do a serial dilution. Serial dilution is necessary so that when doing
spread plate it will not be overcrowded.
8. After serial dilution do spread plate and incubate at 37oC for 18 hours. For a good plate/ good
dilution, the cells number in the plate should be in the range of 30-300 colonies.
9. Count plate to get the CFU/ml. This result will be use for reference for further experiments.
For the experiment above, I have repeated a few times so as to ensure the result is reproducible. Alright that is all I had done so far.
Cheers;)
Justina
0605950E
TG01
Cheers;)
Justina
0605950E
TG01
17 comments:
hey justina..
Why do u use tea? Why not use other herbal plants like aloe vera and etc
Andika Putra TG01
hey justina!
So long haven't seen you.. How are you doing? Anyway I've got question to ask you. You've mentioned that after serial dilution you'll do a spread plate and a good plate will produce 30 to 300 colonies right? Then what if it's not within this range? does that mean anything? eg. maybe the log phase starts later or something?
Thanks babe! :)
Sharon
Tg01
Heyos! Cant wait to see you on next friday!
anyway.. can you tell me more about Trypic-soy agar? sounds alien to me!
16 weeks to go girl! JY
Tan Zhao Rong
Tg01
hey andika
Lol her MP title is on tea ...i dun think she can choose her title ....
Hello justina
Yo... 4 months left already .. how time flies. Hope u r having a good time in the research lab :)
I have some questions I would like to ask u
1) What are some health benefits of tea? (boost immune system? detoxify toxins?)
2)What are some different tea extracts u will b using?
3)What is the storage temperature of the 'bead' form of S.aureus?
4) What is the principle of gram-staining? Is it indicative, presumptive or confirmatory?
5) What is the purpose of taking OD600? (to find out log phase?) and what is the equipment used to take OD600?
6)After performing serial dilution to what series (10^-5? , 10^-6?) than you will haf to count the colonies? (i hope u understand wat im trying to say...)
Thanks for answering my questions!!
From: Ma xianwei Benjamin
class:tg01
0606181F
hi
To Andika:
Well you can actually do on other plant too. But I think because tea is more widely consume so it will be a better choice.
To Sharon:
We need to do the spread plate in order to find the concentration of the cells. If the range fall out, the dilution is not the one you want. As in you can't use the dilution for further tests. As the result will not be 'perfect'.
To Zhao:
Trypic soy agar(TSA) is a type of agar use to isolate and cultivate nonfastidious and fastidious microorganisms. In my case I use it to isolate S.aureus.
hi
To Ben:
To start off with is 1 month(4 weeks).. haha..
1) Some health benefitd of tea: Prevent cancer, heart disease etc. Tea contain antioxidants that will help to remove free radicals from the body which can prevent healthy cells from damage.
2)I will mainly focus on tea under the plant family of Camellia sinensis. Eg. black tea, white tea, green tea and oolong tea.
3)Storage temperature of beads S.aureus is at 80oC.
4) Normally we will use gram stain to determine if the microorganisms is gram positive or gram negative. It is use to indicate whether the culture is pure or not, the result are quite reliable. For confirmatory, you can perform a coagulase test after you gram stain.
5) The purpose of taking OD600 is to find time taken for the S.aureus to reach the log phase. The equipment used is UV spectrophtometer.
6)Hmmm.. normally we will do on different dilution. Such as 10^-5, 10^-6 and 10^-7 so that eg. if at 10^-5 we got a lot of colonies then we can use 10^-6 which will have the plate count of 30-300 colonies.
I hope you get a clearer picture now.
hello there, interesting MP uve got there=)
i would like to ask
why did u choose trypic soy agar n broth? is this a specialised media?
thanks
raihana.
hello..
just wondering..why do you have to use both the trypic-soy agar?
sutiana
Hello Justina,
I have some questions to ask:
1)For your SIP, what type of equipments do you maintain and how do you maintain them?
2)What do you meant by whole cell-based assays?
3)What is the significance of evaluating anti-microbial activity in different tea extracts? Maybe you may want to describe more?
4)Why choose S.aureus as the microbe of interest?
5)How much S.aureus do you inoculate into the TSB?
6)In step 4. what do u meant by checking the purity of the culture? What indications would be shown if the culture is not pure and what subsequent actions would be taken?
7)How do you know if S.aureus has reached its stationary phase?
8)Why is such a wide range allowed (30-300 colonies) to be considered as a good plate/dilution?
9)Where do you perform your experiments at? BSC 2 or Laminar flow hood? Are there any precautions to take when doing your experments?
10)What are some of the errors you have encountered in the course of your experiments and how do you go about rectifying them (if there is any)?
Thankz!
Han Yang
TG01
Hi
To Raihana:
Actually TSA is just a general media. Unless you want to confirm the strain is it a S.aureus, you can use Baird-Parker agar for the morphology. For my case I am just using the agar to grow the culture, thus I will use a general agar.
To Sutiana:
Do you mean why I use both TSA & TSB? For TSA I use it to obtain isolated colonies and for TSB, I use it to grow my culture. The colonies in the TSA can be stored up to 3 weeks while for colonies in TSB, it will die off faster. So colonies in the TSA, I can use it to subculture whenever I need it.
To Han yang:
1)For my SIP, I am just doing data entry, so the only 'equipment' I am using is the computer. So for maintanence part I don't think I will need to do it.
3)To see what benefit the tea can do to our body and does the tea help to 'fight' off bacteria.
4)I can choose other microbe such as E.coli, B.subtilis etc. But because last year there's a student doing on S.aureus so I will just continue from there. If I got the time I will then start on other microbes.
5)I will only need to inoculate 1 colony into TSB.
6)Mean to check if the culture is free from containmination. If the culture is pure the morphology(for S.aureus) will be in cluster, gram-postive cocci shape. If the culture is not pure I guess you can just redo it.
7)If the OD reading remain constant after a period(after log phase)?!?
8)I really have no idea why. This is what I think. If the colonies is above 300 this will lead to overcrowding and eventually death. If below 30, there is not enough to react and give the optimise result.
9)As the microbe I'm dealing with is only level 1 thus I will do my work in laminar flow hood. Precaution I take is to wear glove and spray ethanol before and after the expriment.
10)Some errors I made.. I think is the problem i made is when doing the serial dilution and spread plate part. For the serial dilution I'm suppose to use the sample from the broth(when it reach the log phase) but i use colony from the TSA. For the spread plate if you still remember, you are suppose to soak the glass spreader in alcohol and heat over the bunsen burner. But I did not heat over bunsen burner. Well I redo the whole experiment from the beginning.
Thanks for your question. I hope you understand what I'm trying to say.
Hello Justina,
Thankz for your answers! I get what you are saying. =)
Han Yang
TG01
Hi Justina,
Can you share about why u want to find out the log phase of s.aureus? How does it contribute as part of your MP? (as in e.g you want to add the tea extract into the culture when it reaches the log phase or sth?) Thanks alot.
Jean Leong
TG02
hi Justina, can i ask u what is the link between tea and S.aureus?because there are a lot of microorganisms..is there a reason why S.aureus is choosen?thanks:)
Rachael
Tg01
Hi Justina
Ermm how S. aureus actually be in beads form? And may I know what is the purpose of using TSB?
Thanks
LeeJin
TG02
Hi
To Jean:
To find the log phase so that when doing the experiment my result can be optimise. Yup, I will be using the log phase of the bacteria to add into the tea extract.
To Racheal:
Erm.. there is no link of tea and S.aureus. S.aureus is use as it is a common bacteria and I'm continuing the project from last year.
To Leejin:
S.aureus are enclose in agarose to preserve it. Purpose of using TSB is to grow the S.aureus and dilute it.
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