E. coli

[cddeguit_83]

hi i'm deo..

in my project, one of the methods concerned are of isolating E.coli...
i plan to isolate e.coli from different waste sources: confectionery wastes, feces, and sewage wastes.
here are some of my questions..

(1) could i ask for a link, pdf file, a manual that clearly explains the methods and principles concerned in isolating e.coli (if you have one that used LB agar plates in isolating the bacteria.. please do tell me)?
(2) how will i inoculate e.coli cultures with a glucose-rich medium?
(3) What does E.coli look like isolated in LB agar plates? what is the color, diameter, elevation, & type of colony Escherichia coli isolated in LB agar plates would usually appear?

[jmill9883]

these look like pretty easy things to google

you might also need to get this project approved by your school because e coli bacteria can be dangerous. This project might require a mentor or advisor.

[Emily Willingham]

Hi, Deo--

Welcome! It is true that some quick Googling will get you some answers to your questions. You can also go to our Microbiology Science Fair Projects page and find quite a lot of information about the questions you're asking. In addition, you can browse around among the "Related Links" on that page and learn more about microbes, safety, plating, and much more having to do with your questions. You can start here with your research: https://www.sciencebuddies.org/science- ... a=MicroBio

All of your questions are the right things to be asking and wondering about. I hope that the above link helps you discover some of the answers. Keep us posted about your progress, and please come back to this thread if you have further questions after your reading. Have fun!

Emily

[cddeguit_83]

Hi i'm deo,
part of my methods is isolating e.coli from different wastes in LB agar plates. My adviser says that i need to confirm first that the microbe that i have isolated is e.coli.
how will i confirm that e.coli was isolated in the LB agar plates?

[donnahardy2]

Hi Deo,

If you are working on the same project that you have posted about before, it would be helpful to continue with the same thread so that all of the experts can see the history of the inquiry. We will be able to help you better with all of the information.

LB agar is a general all purpose medium that will support the growth of a wide variety of microorganisms, including E. coli. You cannot identify E. coli directly on this medium. Generally E. coli is identified by growing samples on selective medium that allows this organism to grow, while inhibiting other microorganisms. Here is a website that describes various selective media for this purpose: http://www.m-techdiagnostics.ltd.uk/media.shtml

One key feature of E. coli that is useful for identification purposes is that it can ferment lactose. Here is a website that describes how to isolate and identify E. coli. http://www.foodsafety.gov/~ebam/bam-4.html. There are a number of steps required for positive identification, and you would need to do all of the steps.

At least one other expert and I have included safety precautions in our previous replies, and I would like to explain why we are doing this. E. coli is a microorganism that part of the normal flora found in the intestinal tract of warm-blooded animals, and in fact, it is essential to maintain a healthy physiology in the intestinal tract. There are also several strains of E. coli that are commonly used in the laboratory to make recombinant proteins, and the strains used for this purpose are relatively safe to work with. However, there are some strains of E. coli that are pathogenic and very hazardous to work with. The E. coli found in raw sewage could be normal flora or could be pathogenic, so this could be a hazardous project. We would not recommend that you do this experiment unless you have access to a BSL-2 laboratory and have someone who can show you the techniques you need to use.

If you are still working on the hydrogen gas project, you should know that wild type E. coli don't produce hydrogen gas as a rule. You will need to use a recombinant strain that has modified genes.

Please let us know if you need any additional information.


Donna Hardy

[ChrisG]

Hi Deo,
I merged your topics so that the experts can see the history of your project and the context of your question. Please keep future posts in this same topic.
Thanks!
Chris

[cddeguit_83]

ms. donna hardy,

It is true that the the info about natural e.coli capable of producing hydrogen isn't established yet but that is the reason why i'm pursuing the study. part of my plan is to verify if e.coli that haven't undergone metabolic engineering can still produce hydrogen even in small amounts. i just hope that my plan would be accomplished. and i have already done a lot of work and i think there is no turning back now. i'm actually doing a research paper about this study and starting all over would be really hard to do... could i ask for some tips/ advice that you could share with me...?
By the way, i asked the author, mr. wood, concerning the hydrogen low partial pressure assay... and he sent me the lab protocol about the assay.. problem is, i can't actually understand the protocol (seeing that there are terms and procedures that i have no idea about.) here's the protocol:

Low partial pressure assay

- Streak for single colonies on LB agar plates.
- Make an aerobic overnight culture, 37oC, 250 rpm from a single colony in 25 mL of complex-formate or complex-glucose containing the correspondent antibiotic if plasmids are present. Do not add antibiotic for chromosomal mutations.
- Add 75 mL of fresh complex-formate medium or complex-glucose to a flask with tubes, and transfer the overnight culture. Close the specific flask with the rubber stopper that includes the tubes, then sparge for 5 min with nitrogen. Seal the flasks and incubate anaerobically at 37oC for 6 h.
- Sparge with nitrogen for 10 minutes complex-medium without formate or glucose (at least 35 mL per one sample) using a specific flask with tubes, and for 5 minutes 7 to 10 mL (dependent on sample number) of 1 M sodium formate or glucose using a sealed glass vial with arms.
- Seal glass vials (60 mL; also having stirrer) with a Teflon cap tightly and sparge for 2 min with nitrogen. The bottles should be checked for gas leaks as follows:
1. While sparging with nitrogen gas, one of two needles is connected with nitrogen gas (input of gas) and other needle is output of gas.
2. After sparging with nitrogen gas, the needle for output is closed using head cap. Then, wait 30 second. If the bottle will not leak gas, gas flow should stop (go to zero in the flow meter; from 100 of scale reading to 0).
- Open the glove box gate and introduce the flasks with cultures, and a 250 mL centrifuge tube per culture. The centrifuge tubes should be open to do not introduce air into the hood.
- Purge the glove box with nitrogen, first the entry compartment then the hood.
- Inside the glove box transfer the cultures to a 250 mL centrifuge tubes and close tightly; remove from the box and centrifuge at 7000 rpm, 4oC for 5 min.
- Return to the glove box decant the supernatant and resuspend the cell pellets in 30 mL of complex medium without formate or glucose (previously sparged).
- Remove the cap from one of the needles of the sealed vials and with a syringe add 18 mL of the cell suspension into the bottle, then close the needle with the cap.
- With a syringe add 2 mL of sparged 1 M formate or glucose to the vials, remove from the glove box, set up as indicated in Fig. 1, and incubate at 37oC by stirring.
- Analyze 50 μL of the head space by GC-TCD after different times, for example 15 min, 30 min, 1 h, 2 h and 16 h (16 h for hydrogen yield calculation) and measure gas volume in cylinder (see Fig. 1).
- Add the end of the experiment measure OD600.

Total proteins are calculated from 0.22 mg/OD/mL.

Total hydrogen amount = peak*0.181(nmol/peak area)* actual head space (head space volume in bottle + cylinder volume – background volume (5.1 mL)) (μL)/ 50(μL injection volume).

Hydrogen productivity = total hydrogen amount (μmol) / total protein (mg-protein)

i have a lot of questions concerning the procedure.
(1) how can i sparge the containers and flasks with nitrogen? what is sparging? what is it's purpose?

1. While sparging with nitrogen gas, one of two needles is connected with nitrogen gas (input of gas) and other needle is output of gas.
2. After sparging with nitrogen gas, the needle for output is closed using head cap. Then, wait 30 second. If the bottle will not leak gas, gas flow should stop (go to zero in the flow meter; from 100 of scale reading to 0).

(2)

Make an aerobic overnight culture, 37oC, 250 rpm from a single colony in 25 mL of complex-formate or complex-glucose

how can i do this, (making an aerobic culture)? what's with the rpm? does it mean "revolutions per minute"? do i have to centrifuge the cultures? what are the revolution's purpose in making the aerobic overnight culture?
(3)

Close the specific flask with the rubber stopper that includes the tubes, then sparge for 5 min with nitrogen. Seal the flasks and incubate anaerobically at 37oC for 6 h.

what does incubating anaerobically mean? how can i do this?
(4)what does the equations mean?
(5) could you please share to me what you understand about the set-up and how it is done.?

Thank you ma'am... Even the slightest information that you will share with me will contribute greatly for my research... I hope for your reply and i am truly grateful for spending some of your time for helping me in my past questions...
Thanks ma'am and more power!

[cddeguit_83]

another part of my methods is determining the bacteria concentration of the isolated e.coli....
i plan to use the McFarland standards in order to get the concentration...
how can i determine the bacteria concentration using the McFarland Standards?

[staryl13]

Hi,
Here's a great website discussing mcfarland standards and measuring bacteria concentration-
http://biology.fullerton.edu/biol302/30 ... quant.html
hope this helps, feel free to post back with more questions!

[donnahardy2]

Hi cddeguit,

I do understand that you are committed to this project, so I will definitely try to give you some suggestions. It’s great that you contacted the author of the original paper and that he sent you the protocols; this will really save a lot of time in figuring out what to do. One thing that would help is access to a microbiology laboratory and someone who could show you the basic techniques for working with microorganisms. Are there any laboratories in your local area? You should call around and ask if there is a local microbiologist that could help you out. The procedures that are mentioned here are very standard, and not technically difficult; you just need to know what to do, and this would be hard to do unless someone showed you.

This hydrogen production experiment is done under anaerobic (without oxygen) conditions. To duplicate this experiment, you need a tank of nitrogen gas. The nitrogen gas is bubbled (sparged) through the culture medium to keep the oxygen out. The flasks are also incubated in a special glove box, which is an enclosed container that is kept free of oxygen. If the E. coli have access to oxygen, they will use the oxygen to ferment the sugar to form carbon dioxide, so it’s important to keep the oxygen away.

E. coli grow best when they have access to oxygen, so the culture for the experiment is first grown at 37 degrees Centigrade on a shaking incubator (250 revolutions per minute) that will provide the conditions for optimum growth. The hydrogen-production experiment will work best if you transfer rapidly growing young cells to the anaerobic culture flasks. If you start with old, dying cells, the hydrogen gas production will be greatly reduced.

In the experiment, after the cells are grown aerobically overnight, they are centrifuged to pellet the cells, and a portion of the cell pellet is transferred to the anaerobic flask for the hydrogen production experiment. If you do not have access to a centrifuge, you would have to transfer a portion of the overnight culture to the anaerobic medium. However, this would add some oxygen to your culture, and this would affect the hydrogen production.


The total protein formula (0.22 mg/OD/mL) assumes that you measure the optical density of the culture at a wavelength of 280 nanometers in a cuvette that has a 1 cm pathlength. It assumes that there is 0.22 mg of protein for every 1 OD unit measured on the spectrophotometer. Do you have access to a spectrophotometer to make this measurement? If not, you could do the experiment without measuring the amount of protein.

In this experiment, the hydrogen gas is measured by an instrument called a gas chromatograph with thermal conductivity detector (GC-TCD), which is a very specialized laboratory instrument. The fomula, peak*0.181(nmol/peak area)* actual head space (head space volume in bottle + cylinder volume – background volume (5.1 mL)) (μL)/ 50(μL injection volume is used to calculate the total hydrogen produced. Without a GD-TCD, you would have to record the volume of gas and the temperature and do the flammability test with a match.

Please do talk to your teacher and discuss the possibility of limiting the scope of your project. You could try to do one finite part of this project and try to obtain results.

Please le me know if you have any other questions.


Donna Hardy

[cddeguit_83]

ms. donna hardy
thank you for answering some of the questions that needed to be answered in my research..and i hope that you would still answer some more of my questions... like this time...
do you remember the hydrogen low partial assay? remember that it's parts (flasks, and flasks with the e. coli culture) was supposed to be sparged with nitrogen gas in a glovebox so as to keep the E.coli in an anaerobic environment. well i just want to ask:
(1) is it okay that i don't sparge the parts with nitrogen and keep the assay in an aerobic environment? it seems that a glovebox is not quite available in our school..
(2) is there any other acquirable equipment that work like a glovebox?
(3) will the e. coli still produce hydrogen even though it's in an aerobic environment?
(4) do you think the assay will work? do you think, theoretically and in reality, that it can measure the volume of hydrogen produced by e. coli

thank you! and i am hoping for your reply.

[cddeguit_83]

one last thing,

my research is on the hydrogen production of e. coli isolated from diff. waste sources: fecal matter, confectionery waste and wastewater....
(5) seeing that i will isolate e. coli from different sources, will it also follow that the e coli that i'll be isolating will be different strains... i mean, the e.coli from the feces will be one strain and on the confectionery waste will be another and on the waste water will also be another... will this be possible?? cuz my research is maximizing the possibility of having diff. strains so as to have a good comparison of which waste source contains the best hydrogen producing e.coli...

(6) if e.coli are of different strains... will their hydrogen producing ability differ from each other?
and why?

(7) if yes, what aspect about their hydrogen metabolism is unique from each other??

[donnahardy2]

Hi,

It sounds like you are making good progress on your project. I remember this project because it is a completely unique project. Here are some answers that I hope will help you:

1. Since you don't have access to a nitrogen tank or glove box, you will need to keep your culture medium static (still) to minimize the amount of oxygen that is dissolved in the medium. The E. coli will growth and rapidly use the oxygen, and hopefully the conditions will be anaerobic enough for hydrogen production, if it occurs.

2. A glovebox is a completely unique item and only labs that do experiments that require no oxygen have them. Most microbiology labs don't have glove boxes. I can't think of anything that would be comparable. I think it's best to go ahead and try your experiment without the glove box.

3. The E. coli in the reference paper were genetically modified so they would produce hydrogen gas. I don't know if native E. coli produce hydrogen gas. You will have to find out when you do your experiment.

4. If you can follow the experimental set-up, you will be able to collect gas from the growing culture if it is produced. I know that native E. coli will produce carbon dioxide, so you will need to distinguish between carbon dioxide and hydrogen gas.

5/6/7 There is variation in the characteristics of E. coli, and you will probably isolate different strains from different sources. As I recall, the authors evaluated several strains of E. coli, but I believe that all had been genetically modified. Why don't you write back to the authors who sent you the protocol, and ask them these questions? Otherwise, you will have to wait and answer them with the results of your experiment.

I recommend that you contact your local Society for Microbiology and see if there is a local microbiologist who could help you with the techniques you will be using, and possibly with equipment. In the US, the American Society for Microbiology has volunteers in every state that are available to help high school students. I don't know what country you are in, but try searching for "microbiology society." You should find a name and phone number of someone to contact to ask. A local mentor would be a big help with this project.

This is exciting that you are finally starting your research. Do write back to let us know if you have any other questions, and to let us know about your results. Good luck!


Donna Hardy

[cddeguit_83]

uhm.... i really need help concerning this assay... unfortunately, i don't have a very goof background on microbiology procedures.

Low partial pressure assay

- Streak for single colonies on LB agar plates.
- Make an aerobic overnight culture, 37oC, 250 rpm from a single colony in 25 mL of complex-formate or complex-glucose containing the correspondent antibiotic if plasmids are present. Do not add antibiotic for chromosomal mutations.
- Add 75 mL of fresh complex-formate medium or complex-glucose to a flask with tubes, and transfer the overnight culture. Close the specific flask with the rubber stopper that includes the tubes, then sparge for 5 min with nitrogen. Seal the flasks and incubate anaerobically at 37oC for 6 h.
- Sparge with nitrogen for 10 minutes complex-medium without formate or glucose (at least 35 mL per one sample) using a specific flask with tubes, and for 5 minutes 7 to 10 mL (dependent on sample number) of 1 M sodium formate or glucose using a sealed glass vial with arms.
- Seal glass vials (60 mL; also having stirrer) with a Teflon cap tightly and sparge for 2 min with nitrogen. The bottles should be checked for gas leaks as follows:
1. While sparging with nitrogen gas, one of two needles is connected with nitrogen gas (input of gas) and other needle is output of gas.
2. After sparging with nitrogen gas, the needle for output is closed using head cap. Then, wait 30 second. If the bottle will not leak gas, gas flow should stop (go to zero in the flow meter; from 100 of scale reading to 0).
- Open the glove box gate and introduce the flasks with cultures, and a 250 mL centrifuge tube per culture. The centrifuge tubes should be open to do not introduce air into the hood.
- Purge the glove box with nitrogen, first the entry compartment then the hood.
- Inside the glove box transfer the cultures to a 250 mL centrifuge tubes and close tightly; remove from the box and centrifuge at 7000 rpm, 4oC for 5 min.
- Return to the glove box decant the supernatant and resuspend the cell pellets in 30 mL of complex medium without formate or glucose (previously sparged).
- Remove the cap from one of the needles of the sealed vials and with a syringe add 18 mL of the cell suspension into the bottle, then close the needle with the cap.
- With a syringe add 2 mL of sparged 1 M formate or glucose to the vials, remove from the glove box, set up as indicated in Fig. 1, and incubate at 37oC by stirring.
- Analyze 50 μL of the head space by GC-TCD after different times, for example 15 min, 30 min, 1 h, 2 h and 16 h (16 h for hydrogen yield calculation) and measure gas volume in cylinder (see Fig. 1).
- Add the end of the experiment measure OD600.

Total proteins are calculated from 0.22 mg/OD/mL.

Total hydrogen amount = peak*0.181(nmol/peak area)* actual head space (head space volume in bottle + cylinder volume – background volume (5.1 mL)) (μL)/ 50(μL injection volume).

Hydrogen productivity = total hydrogen amount (μmol) / total protein (mg-protein)

i really have a lot of questions concerning the procedure.
(1) how can i make a complex-glucose media?
(2) are LB plates solid?
(3) what are cell pellets?
(4) Could anyone explain to me all the laboratory procedures concerned in doing the assay?
(5) what is the chronological order of all the methods in the assay??

Thank you... your help will be truly appreciated...

[barretttomlinson]

Hi,

I just noticed this thread and your problem with a lack of glove box. Here are some options for building one inexpensively:

http://www.omnisterra.com/botany/cp/slides/tc/hood.htm

http://virtualastronaut.tietronix.com/t ... ovebox.pdf

Hope this helps explore the options.

Good luck with what sounds like a fantastic project!

Barrett Tomlinson