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## c0ok1e's Content

There have been 22 items by c0ok1e (Search limited from 14-April 20)

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### #31212Bacteria culture

Posted by on 31 July 2009 - 07:54 AM in Microbiology

Im calculating this for my growth curve... Initially i carry out the experiment in 2ml medium just to estimate the time it takes to reach a certain OD because besides the growth curve, im also uusing it for other purpose.. kills 2 bird with 1 stone..

so I inoculate 50 microlit of OD 0.922 culture into 1ml and got the OD after 11 hrs...

This time Im trying to scale up to 100ml and I want it to reach the same OD in 48 hrs...

So I thought maybe if I half the initial amount I might get to the OD in 48 hrs..

By the way I have carried out enumeration so I know the exact number of bacteria corresponding to the OD

### #31168Bacteria culture

Posted by on 31 July 2009 - 12:19 AM in Microbiology

Just a quick question,

I carried out an experiment and got my bacteria to grow from (3.56 X 10(7)) to (8.5 X 10(8)) in 11 hrs.

My question is that if I half the initial inoculation amount, will it take double the time to reach the same number of bacteria? That is if I inoculate(1.78 X 10(7)) number of bacteria into the medium, will I get (8.5 X 10(8)) bacteria after 22 hrs?

Posted by on 22 June 2009 - 11:05 PM in Microbiology

These are mostly personal quesions regarding you husbands ability. Only he knows the answer. As for question 4 it can be read up in the internet.

### #27215OD of bacteria at different culture volume

Posted by on 21 June 2009 - 02:42 PM in Microbiology

Based on cell growth. I did the bacteria count before this. Even if its based on time, when I cultured 1 ml I cultured it for 2 days and it reached OD 1.something

WHile for the 1 liter I cultured it for 4 days already and its growing slow at 0.5something

Do you guys get this often?

### #27189OD of bacteria at different culture volume

Posted by on 21 June 2009 - 03:44 AM in Microbiology

Hi. I cultured my bacteria at 2 ml and got stationary phase at OD 1.398.

But when I cultured the bacteria at 1 liter culture, I seem to get slow growth after reaching OD 0.576

Will there be a difference in OD at stationary phase at different culture volume or is there something wring with my experiment? WHy is this happening?

Thanks

### #24979counting number of cells

Posted by on 22 May 2009 - 05:28 AM in Microbiology

Im trying to identify one of the product produced by the bacteria.. so its not important to know how many cells there are right.. only interested in the product.. then why do researcher take their time to calculate how many cels are there?

### #24960counting number of cells

Posted by on 22 May 2009 - 12:32 AM in Microbiology

yeah but wats the use of knowing the number of bacteria present?? We only need to know that there are enough viable bacteria present right? The number doesnt make a difference right?

### #24956counting number of cells

Posted by on 21 May 2009 - 11:38 PM in Microbiology

When we deal with bacteria, we take the OD and then plate on agar and see how the OD correlates with the number of bacteria. WHy is this important?

### #22231WHen you autoclave

Posted by on 19 April 2009 - 06:42 PM in General Lab Techniques

I see, thanks klinmed.

I do know that the boxes we use always seem to be wet inside. Maybe its some sort of plastic that lets steam pass ? Dont you think that the people that make those pipetboxes build them so that steam can penetrate?

however still another question: why does it need to be dry when storing it? does a wet object attrackt more micro-organisms?

I think its because wet area, microorganisms can grow on it so it will not be sterile anymore. There are microorganism in the air also so if its wet then it will be contaminated very fast.

### #22079WHen you autoclave

Posted by on 17 April 2009 - 04:54 AM in General Lab Techniques

klinmed is right.
Ignore what I said about tips (it's not a common practice to wrap them in aluminium foil anyway - some folk autoclave already 'sterile' tips and wrap the box tightly in aluminium foil as a kind of wrapping. The autoclaving, seems to be, a paranoid last line of defense) but you will want to protect your cotton bungs from getting wet. The cotton bungs should be sterile to start with and thus full autoclaving shouldn't be necessary*. You really don't want them getting sodden. But when you wrap the aluminium foil around the bung don't do it very tight, just wrap it round and secure with a squeeze of your hand.

But aye, I wasn't very exact in what I said. klinmed's description is far better than mine.

*Actually, now that I've typed this I hate it. It's standard practice in a few places I've been but it does seem not to be best practice.

Its good that we brought up this discussion.. so now we kow the standard practice in most of our labs are unnecessary.. so the next time when i autoclave i wont use aluminium foil to cover unneccessary items like medium and pipette tips...its just wasting resources...

### #22058Centrifuge

Posted by on 16 April 2009 - 09:46 PM in General Lab Techniques

Most benchtop centrifuges can't reach 30,000g. Few protocols require it that wouldn't work better at ultracentrifuge speeds.

Im not sure about this cuz I am an amateur researcher but in articles concerning isolation of compounds from bacteria usually they use around 30 000g. Maybe they use different kind of centrifuge. Anyone done this kind of research before? Can you confirm about this or is it just the paper that i read is an exception??

But i wonder how they come up with the time and rcf to be used..

### #22049WHen you autoclave

Posted by on 16 April 2009 - 06:33 PM in General Lab Techniques

I dont know if it is the norm in other labs but in my lab when we autoclave we will use aluminium foil to cover our materials. A lot of people I ask just say that it is the norm but I think there should be a reason to it.
When autoclave:

1) Medium: We use aluminium foil to cover the cap.. Why?
2) Pipette tips: WHen autoclaving pipette tips, we use aluminium foil to cover the box.. Why?

ALso when we put culture in the shaker, in the conical flask we use cotton wool to stuff the mouth.. Then some people use aluminium foilt to cover the cootn wool.. WHy?

There seems to be some miss-understandings of the principles behind steam-sterilization.

Non heat-conductive items like cotton wool, plastic pipette tip boxes (and the tips!!) etc MUST be in contact with steam throughout the sterilization process. If aluminium foil keeps the items "dry" (prevents access of the steam) they will not be sterilized with the times/temperatures used for autoclaving.

Autoclaving is usually undertaken at 121 oC for ca 30 mins or at higher temps for shorter periods. In contrast, dry heat (oven) sterilization requires much higher temperatures/times ca 160 oC for 2-3 hours. Thus, in the absence of steam contact, autoclaved pipette boxes etc will not get hot enough, for long enough, to assure sterility.

The principle behind this is simple: Steam is many times more effective at conveying thermal energy to the item than hot (dry) air. For example, in the kitchen, potatoes can be cooked in a few minutes in a steam pressure cooker (115 - 121 oC) while cooking at THE SAME TEMPERATURE would take many hours in a hot-air oven.
In addition, steam "wets" the bugs and thus facilitates lethal heat-induced protein coagulation.

Small items like forceps can be autoclaved when wrapped tightly but only because they conduct heat rapidly.

In critical applications (hospitals for example) special indicators are placed in the center of dry loads to check for adequate steam penetration (eg Bowie-Dick test).

Thus, if you must wrap dry items with foil before autoclaving, do it loosely. However, it is better (and required for GMP, ISO..) to use steam-permeable bags specially made for the purpose.

Remember that standard autoclave tape ONLY shows that the article has been exposed to steam. Not that it has been treated long enough for sterility. Appropriate autoclave test strips that integrate time and temperature can be purchased from a number of sources.

Sorry to go on, but think this is important.

I like your explanation. So does it mean that usng the aluminium foil to cover our non heat condusive items are wrong that we should not use the foil?

### #22048Centrifuge

Posted by on 16 April 2009 - 06:29 PM in General Lab Techniques

My quick summary (not applicable to ultracentrifuges!):

It doesn't much matter except for breaking things. 2000-3000g for 96 well plates. 6000g or so for thin wall pcr tubes (you will be unhappy when your precious sample goes into the rotor). 8000g for most conical 15 and 50 ml tubes. 15000g for eppendorfs, including column minipreps. Final elution of miniprep columns into capped tubes can be done at 6000g if you want to keep the loose caps on the tubes.

How about 30 000g?? It was used in isolation of unnown growth factor from bacteria.. the g u mentioned are all small compared to it

### #21952WHen you autoclave

Posted by on 16 April 2009 - 12:16 AM in General Lab Techniques

I dont know if it is the norm in other labs but in my lab when we autoclave we will use aluminium foil to cover our materials. A lot of people I ask just say that it is the norm but I think there should be a reason to it.
When autoclave:

1) Medium: We use aluminium foil to cover the cap.. Why?
2) Pipette tips: WHen autoclaving pipette tips, we use aluminium foil to cover the box.. Why?

ALso when we put culture in the shaker, in the conical flask we use cotton wool to stuff the mouth.. Then some people use aluminium foilt to cover the cootn wool.. WHy?

### #21951Centrifuge

Posted by on 16 April 2009 - 12:11 AM in General Lab Techniques

Thanks dude,, A lot of information there... I think it should help...

### #21844Centrifuge

Posted by on 14 April 2009 - 06:59 PM in General Lab Techniques

I dunno where I should post this so i think that this is the best place..

I use centrifuge very often and I would say that many other researchers also used it in their experiments. However, I only use it based on the protocols that have been written. I would like to understand the principles of centrifugation so that I can use it without following the protocols blindly and determine my own parameters when carrying out my own research.

My questions are:

1) I understand that the in centrifugation, the heavier product will be sedimented as the pellet while the lighter product will stay as supernatant. There will be a certain minimum threshold to the size/density of the particle to become the pellet. Insufficient time or centrifugal force will result in more components to stay in the supernatant while excessive time or centrifugal force will make you lose your desired product to the pellet. How do you determine the time and centrifugal force (g) to be used to obtain your desired product in the supernatant/pellet?

For example in a paper, one of the step is

"Samples, 10 ml, were centrifuged for 5 min at
5000 g. The biomass pellets were washed twice and
then dried in an oven at 110 ◦C to constantweight. The
culture supernatant was analysed for glucose using a
commercial kit (Sigma)."

How did you come to the parameter 5 mins as the time and centrifugal force as 5000g?

2) The second question, is somewhat similar to the first question. In an experimental design requiring centrifugation as one of the methods, what is the best way to determine the parameters ( time, centrifugal force) to get the an uncharacterized product in the supernatant so that the minimum size in the supernatant is my product where bigger molecule will be sedimented as pellet?

Hopefully someone understand the principles and share wif us. thanks

### #217011.5% agar plate

Posted by on 13 April 2009 - 02:38 PM in Microbiology

This is strictly a matter of taste and convenience. 1% agar is a bit soft and is easy to tear with a loop. 1.5% agar has a harder surface. The choice may slightly change the morphology of colonies, so if this is an important phenotypic characteristic, it might matter. 1.5% is the "normal" choice.

so there are no published paper on the different percentage of agar used?
Then it means that the hardness of agar varies from 1 - 1.5% are not so important right.. thanks..

you could find some papers on the subject.
Like:
1 or2 or 3 but how they got the 1.5% ? No idea, I guess trail and error? Someone found out that it was good to use 1.5% and well since then... I have no idea of an article is written on why 1.5%.. but maybe you could find some references in the texts I gave that refer to some older literature that speak about the 1.5% agar.

ALright thanks.. Ill look into it... Maybe its not so important after all but I just like to know my work in detail...

### #216541.5% agar plate

Posted by on 13 April 2009 - 04:44 AM in Microbiology

This is strictly a matter of taste and convenience. 1% agar is a bit soft and is easy to tear with a loop. 1.5% agar has a harder surface. The choice may slightly change the morphology of colonies, so if this is an important phenotypic characteristic, it might matter. 1.5% is the "normal" choice.

so there are no published paper on the different percentage of agar used?
Then it means that the hardness of agar varies from 1 - 1.5% are not so important right.. thanks..

### #21652NPB medium

Posted by on 13 April 2009 - 04:42 AM in Microbiology

Hi again,

I guess I have found the function of most of the components. The remaining components that I am not sure of is MgSO4. I guess MgSO4 provides Mg++ and S to the culture but doesnt the yeast extract already provide the Mg++ ions and peptone provide the S?

Also why is the Mg++ so important that it needs to add extra MgSO4 for it? I think that Mg act as a cofactor for enzymes but does it really need a high amount?

tryptone = amino acid source ( N and C )
glucose = carbon source
yeast = growth factor mainly.
the key is actually Mg . mgcl2 probably would work fine. anyway, Mg2+ serves as co factor for alot of enzyme . even on ribosome and used to stabilize dNTP.

the phosphate is there to help resist the change in pH.

thats what I found out.. But thanks anyway.. U help to confirm my findings.. I still have a lot more to learn

### #21633NPB medium

Posted by on 12 April 2009 - 11:46 PM in Microbiology

Hi again,

I guess I have found the function of most of the components. The remaining components that I am not sure of is MgSO4. I guess MgSO4 provides Mg++ and S to the culture but doesnt the yeast extract already provide the Mg++ ions and peptone provide the S?

Also why is the Mg++ so important that it needs to add extra MgSO4 for it? I think that Mg act as a cofactor for enzymes but does it really need a high amount?

### #216321.5% agar plate

Posted by on 12 April 2009 - 11:08 PM in Microbiology

Im an amateur researcher and I just got into the mucrobiology field. Before this I was doing animal science. I hope to learn more about the basics and principles of microbiology. I have been trying to figure this out but couldnt get any information on the net. They didnt explain why..

I would like to know what is the reason behind researchers using 1.5% agar plate? What is the significance between 1.5% and other percentages? If we use 1% is there any difference?

### #21631NPB medium

Posted by on 12 April 2009 - 11:05 PM in Microbiology

Any of you heard of NPB medium before? IM not sure if it is a standard medium or a special medium created by the researcher. I am an amateur in research and would like your help. The composition of the medium is:

10g tryptone peptone
2g yeast extract
1g mgso4.7h2o
1g k2hpo4
0.5g kh2po4
5g glucose

I cannot find the information on the medium online. I would like to know the function of each component of the medium and why they add the component into the medium.

The literature where i encounter this medium is

Tanaka Y, Hanada S, Manome A, Tsuchida T, Kurane R, Nakamura K, Kamagata Y (2004). "Catellibacterium nectariphilum gen. nov., sp. nov., which requires a diffusible compound from a strain related to the genus Sphingomonas for vigorous growth". Int. J. Syst. Evol. Microbiol. 54: 955–959

Thanks