Identifying Bacteria

Ask questions about projects relating to: biology, biochemistry, genomics, microbiology, molecular biology, pharmacology/toxicology, zoology, human behavior, archeology, anthropology, political science, sociology, geology, environmental science, oceanography, seismology, weather, or atmosphere.

Moderators: AmyCowen, kgudger, bfinio, MadelineB, Moderators

Locked
geesesandyoohoo
Posts: 2
Joined: Tue Jan 10, 2017 4:21 pm
Occupation: Student

Identifying Bacteria

Post by geesesandyoohoo »

We are doing a science fair project on bacteria in our school. We swabbed different surfaces around our school and grew the bacteria on agar plates. How can we successfully identify this bacteria? What type of materials and resources should we seek?
EAMills
Former Expert
Posts: 27
Joined: Tue Sep 27, 2016 11:46 am
Occupation: Other Adult

Re: Identifying Bacteria

Post by EAMills »

Hello geesesandyoohoo,

Trying to identify different types of bacteria in the school sounds like a very interesting science project! It will be fun to see if the types of bacteria are similar in different areas.

There are several types of staining and biochemical tests that can be done to help identify different species of bacteria.
The most straight-forward is to determine whether you have gram positive or gram negative bacteria based on staining.
Several other tests are available which also involve color reactions.
These links might be helpful, though they are probably more complicated than you need right now. If any of the tests seem like something you want to do, we can try to get more information about how exactly to perform them and everything you need.
http://vlab.amrita.edu/?sub=3&brch=76&sim=1109&cnt=1
http://www.ableweb.org/volumes/vol-24/8-christopher.pdf

Hope this helps,
Elizabeth
gandy
Posts: 5
Joined: Sun Jan 15, 2017 4:46 pm
Occupation: Student

Re: Identifying Bacteria

Post by gandy »

Hi! This is geesesandyoohoo's science fair partner. Thank you so much for replying.

We have another question. If one plate has more growth than a different plate, can we assume that the location that plate one was swabbed from has a higher concentration and a larger diversity of bacteria? For example:

Plate 1 contains bacteria swabbed from a bathroom.
Plate 2 contains bacteria swabbed from a water fountain.

Plate 1 has visibly more growth than Plate 2.
Does this mean that the bathroom has a higher concentration or a greater diversity of bacteria than the water fountain? Yes or no?

Another question. If the same bacteria is two different places on the same plate, will only one colony grow? Or will two grow?
gandy
Posts: 5
Joined: Sun Jan 15, 2017 4:46 pm
Occupation: Student

Re: Identifying Bacteria

Post by gandy »

We're also interested and looking into the gram-positive and gram-negative tests. We're doing research about it on our own but would you be able to give us some pointers and maybe even links to some sort of instructions, etc.? Are these tests expensive to do? (We've spent a lot on the agar plates and we don't want to spend much more.) How long does it take to do these tests? Our science fair is on Saturday, February 11th. We consider that deadline really close and incredibly fast-approaching. We want to get this done as soon as possible simply to avoid stress. Our bacteria is about a week and a few days old. Will this matter? A lot of sources mentioned "cultures" about 24 to 48 hours old, but they never specified. The idea of gram-staining is honestly our only hope and we really hope that we can still do it. :? :| :oops:

I've read at http://fermup.com/blog/what-does-gram-positive-mean/ that usually, gram-positive bacteria is usually good bacteria, and that usually, gram-negative bacteria is bad bacteria. Is that true?

"Usually, gram-positive bacteria are the helpful, probiotic bacteria we hear about in the news, like LAB. They are the happy ones that live in our gut and help us digests food (Behnes, et al; 2013). Gram-negative bacteria, by coincidence, are usually thought of as the nasty bugs that can make us sick and can be harmful. For example, several species of Escherichia coli (E. coli), are common causes of food-borne disease. Another example is Vibrio cholera, the bacteria responsible for cholera, is a waterborne pathogen (National Institute of Allergy and Infectious Diseases, 2012)."

That's a paragraph from the link I included above.

"As a rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage" - the human body recognises a foreign body by its antigens; if they are hidden, it becomes harder for the body to detect the invader. Often the presence of a capsule will increase the virulence of a pathogen. Additionally, Gram-negative bacteria have lipopolysaccharide in their outer membrane. Lipopolysaccharide is an endotoxin which increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Gram-positive infections are generally less severe because the human body does not contain peptidoglycan, and in fact the human body produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also much more susceptible to beta-lactam antibiotics, such as penicillin."
This is from https://en.wikibooks.org/wiki/School_Sc ... m_staining.

If this idea is true, then it would help our project out a lot. Currently, we're really struggling with the idea of identifying the different bacteria. There's just so many different kinds! We feel like it's hopeless to attempt to identify the bacteria on our agar plates. Being able to categorize our bacteria into two different, easily distinguished, good-and-bad kind of ideas would be unspeakably helpful.

Also, what if we encounter fungi, molds, yeasts, etc.? What should we do then? From a student's point of view, do you think it would be best to ignore the fungi, etc. and only focus on the bacteria? Looking forward to your response. Thanks again.
EAMills
Former Expert
Posts: 27
Joined: Tue Sep 27, 2016 11:46 am
Occupation: Other Adult

Re: Identifying Bacteria

Post by EAMills »

Hi gandy,

You are correct that in general for the common bacteria more of the bacteria that are beneficial (good/helpful) tend to be gram positive, whereas those that are more likely to be pathogenic (harmful/cause sickness) tend to be gram negative, but of course there will be exceptions. I think for your purposes you are probably not going to be able to identify individual strains/species of bacteria so using generalities will be ok.

You can not really conclude that you have a higher density or greater diversity of bacteria on your plate with more growth. To look into diversity you might want to see if you can visibly identify colonies that have different shapes or colors which are likely different kinds of bacteria. In numbers it is possible that you have more bacteria from the source (bathroom) for the plate with more growth, however, it is also possible that the particular type of that bacteria grows at a faster rate - in both cases you would see more colonies.

If your plates are being stored someplace cool (say in the fridge) then the bacteria may be ok for the gram staining test. Ultimately, though it would be best to take fresh samples (you could swab the same locations- and rub the swab on a slide to do the gram staining. Here is another link that may be helpful.
http://www.wikihow.com/Gram-Stain

In terms of the reagents, these stains/dyes are not uncommon and it would be a good idea to check to see if the science department at your school has them, if not it is likely they would have them at your local high school science dept. Iodine should be easy to find at your local pharmacy (it is used in first aid to treat cuts), the violet stain may be more difficult to come by.

In terms of fungi, if you see fuzzy stuff or green stuff that is clearly mold/fungi you can report it, but sometimes the agar plates will start growing mold if you keep them too long, so it would depend on whether you say it right away or if it only developed after you had the plates for a while. If you have some that is easily identifiable (like the blue/green mold common on bread) you can report that, otherwise you might just want to focus on the bacteria.

Let us know if this helps and what other questions you have.

Good luck,
Elizabeth
gandy
Posts: 5
Joined: Sun Jan 15, 2017 4:46 pm
Occupation: Student

Re: Identifying Bacteria

Post by gandy »

Hi!
Thanks for replying.

About the gram-staining: we have some more questions.

There are some materials that we're concerned about. They are:

- Light Microscope
+ We have easy access to a light microscope. There are three objectives on the ones at our school: 4x mag., 10x mag., and 40x mag.. Of course, there's the 10x lens within the eyepiece, so everything is multiplied by 10. However, the WikiHow says: "Bacteria vary greatly in size, so the total magnification required will vary from 400x to 1000x." Our maximum available magnification is 400x. Will this dramatically impact our ability to look at the bacteria? After all, really, all we're looking for is if there are noticeable amounts of gram-neg bacteria, which (should) easily show up as pink.

- Methanol
+ Or, should we use a Bunsen burner? Would our school have a Bunsen burner? We thought it may be better to stray away from the burner, but after googling "methanol," we realized that it is toxic. How toxic is methanol, really? Would our school have it?

- Crystal violet
+ The WikiHow recommends "Hucker's" crystal violet. Will we need this particular brand of stain? Or any crystal violet?
+ You mentioned that our school may have it, but in the case that it doesn't, that we should seek a local high-school. Though this isn't really related to science, how would you recommend us to get in touch with a local high-school? Would we email a science teacher?

- Decoloriser
+ A 1:1 mix of acetone and ethanol is what it recommends. Do we have to mix this on our own? Or are decolorisers already mixed? Would our science dept. or a local high-school dept. have some? If they don't have the decoloriser itself, would they possibly have acetone and/or ethanol?
+ The WikiHow says that it "typically takes under 10 seconds" for it to work. How will we know when to stop? "Stop immediately, or the decoloriser will remove the crystal violet stain from both gram-positive and negative cells, and the stain will have to be repeated," it says, and we're a little scared that we're going to over-do it. :| Should we simply eyeball it? Is there any obvious, visible change as we look at it?

- Counterstain
+ The WikiHow talks about safranin or fuchsin as the counterstain.

Do you think that our school will have these materials? We're in 7th grade and our school has students in grades 6-8 only.

We don't want to spend much. The agar plates were a decent expense.

If we were to swab a surface with a sterilized swab and proceed to complete the gram staining test within, say, an hour of swabbing, would the test results be accurate? I know that we can't have anything over 48 hours, but can we have anything less? Or does it have to be at least 24 hours old? How would we grow it? Does it even have to be grown? Would it grow directly on the microscope slide? Would they somehow multiply on the swab itself? So many questions. :|

You've been ENORMOUSLY helpful and we can't express our gratitude enough. We're most definitely going to give you acknowledgements on our poster. :D

Can't wait to hear back. We're genuinely excited to see your next reply. Thanks so much. :mrgreen: :mrgreen: :mrgreen:
EAMills
Former Expert
Posts: 27
Joined: Tue Sep 27, 2016 11:46 am
Occupation: Other Adult

Re: Identifying Bacteria

Post by EAMills »

Hi gandy,

I think for your purposes, the microscope you have at school will be sufficient, since you are just looking at shape and color of the bacteria.

A Bunsen burner is a great tool for sterilizing and common in most science classrooms, however there is a risk of burns, so protective gloves should always be worn if you choose to use it. Methanol is toxic if it is consumed, but will not harm otherwise, though it is always good practice to wear gloves to avoid getting it on your skin.
Ethanol and acetone are easy to come by and common in science classrooms, you might even have some at home as acetone is used in types of nail polish remover.

Emailing the science teacher would be a good idea, or if you have friends with an older sibling at the HS, maybe you can ask them to connect you with the teacher.
I found online a company that sells a kit for gram staining, I am not sure what your budget is for this project, but it may be an option for you. I do not know anything about it, so I can not say how well it works.
http://www.hometrainingtools.com/gram-stain-kit

In terms of getting the timing right, doing a cheek swab will be a really good test, since we all have (healthy) bacteria in our bodies. If you have bacteria on a surface you dont have to worry about trying to grow it on a slide, you are just looking at the bacteria that is already present.
Another potential option if you have more plates, is to try to grow up (ideally someplace warm and shaking) some of the bacteria (taking a tiny bit from bacterial colonies that look different) from the plates you already have in media (typically Luria broth- which may be found in a science classroom- more likely in HS than MS) and then streak a little of that inoculated media onto new plates (you can divide up the plates into sections with a marker) and grow for a day or so and use that 'fresh bacteria' for staining.

I will look into further about how critical the timing is and whether the counterstains are necessary and get back to you soon.

Elizabeth
gandy
Posts: 5
Joined: Sun Jan 15, 2017 4:46 pm
Occupation: Student

Re: Identifying Bacteria

Post by gandy »

Hi Elizabeth!

The gas to our school was shut off, so we're doing the methanol method. We asked our science teacher -- we'll call him Mr. Frog, because there's frogs all over his classroom :lol: -- and he said that the school has all of the materials we need. We're going to email Mr. Frog today and ask him about staying after school to do the gram-staining some time next week.

Reading the WikiHow that you provided a link to, it says: "If you have a swab sample, roll the swab lightly across the slide."
We're thinking that we can simply:
- Swab the surface
- Roll the swab lightly across the slide

and then the bacteria would be on the slide.

Next, we would just follow the WikiHow's instructions:
- "Alternatively, the smear may be fixed by methanol instead, by adding 1-2 drops of methanol onto the dried smear, draining off the excess methanol, and allowing it to air dry. This method minimises damage to host cells, giving a cleaner background."
- "Position the slide on a staining tray."
- "Flood the smear with crystal violet."

and so on.

Basically, what we're going to do is swab the surface, then, about a minute later, we'll roll it on the slide, and then, about another minute later, we'll use methanol the fix the bacteria to the slide, and so on. We're planning on completing the entire process within about an hour after school. Depending on how long everything takes, we may or may not look at the slides under the microscope on the same day of swabbing it. If this happens and we have to look at the slides, say, the next day, where would we store the slides? In what kind of conditions?

Thank you so much. Looking forward to your reply.
EAMills
Former Expert
Posts: 27
Joined: Tue Sep 27, 2016 11:46 am
Occupation: Other Adult

Re: Identifying Bacteria

Post by EAMills »

Hi gandy,

I am happy to hear that you are able to get all the supplies you need from your science teacher.
Your plan sounds really good. As I mentioned earlier, I would try doing a cheek swab first as a positive control to make sure you have everything working properly.
If you have to look at the slides later I would recommend adding a glass coverslip to the slide. You will need some type of mounting media to attach the coverslip and keep the dye from fading. Permount is a very common form of mounting media that you may have in your school (though it is quite smelly), but your teacher may have something else, so it would be a good idea to ask him. Glycerol can also sometimes be used as a mounting media, but I am not sure how well it works for gram staining.
The slides can then be stored at room temperature, to avoid fading it is probably a good idea to keep them in the dark (in a drawer or slide folder).

Good luck with the experiment!

Elizabeth
gandy
Posts: 5
Joined: Sun Jan 15, 2017 4:46 pm
Occupation: Student

Re: Identifying Bacteria

Post by gandy »

Hi Elizabeth,

We're definitely going to do the cheek swab first as a test. Good idea. Thanks.

We're staying after school this Wednesday, February 1st with our science teacher to do the gram-staining. We're going to ask him about the Permount and Glycerol, just in case we're going to have to look at the bacteria over a period of more than one day.

Question: So, for our science fair, we have to come up with a problem -- "The question that you are trying to answer or the design challenge that you are addressing by doing your project."

Currently, our problem is:
"Do [school name] students encounter any harmful bacteria throughout their school day?"
but we changed it to
"What type of germs do students at Southampton encounter on a daily basis and are any of them harmful?"

We came up with this problem long before we had known what gram-staining was, let alone deciding to do it. I was thinking that we should change it to something more catered towards gram-staining, for example:
"Do [school name] students encounter any gram-negative bacteria throughout their school day?"

Do you think this is a good idea? What would you say our problem should be?

Thanks!
EAMills
Former Expert
Posts: 27
Joined: Tue Sep 27, 2016 11:46 am
Occupation: Other Adult

Re: Identifying Bacteria

Post by EAMills »

Hi gandy,

Sorry for the delayed response, I hope the experiment went well.
In terms of your question, from the initial post it sounded like you were trying to do a comparison study about the types of bacteria found in different places in the school. Therefore you might want to try to frame your question in those terms. I think it is still ok to use gram staining as a correlate for 'good/bad' bacteria, and just say in your methods that we chose to look for the presence of bad bacteria using the gram staining method.
Maybe something along the lines of "Are students at (school) exposed to potentially negative bacteria? Which places in the school harbor potentially negative bacteria?"

Good luck,
Elizabeth
geesesandyoohoo
Posts: 2
Joined: Tue Jan 10, 2017 4:21 pm
Occupation: Student

Re: Identifying Bacteria

Post by geesesandyoohoo »

Hi Elizabeth,

Thank you for all your help! We ended up naming our project "Got Germs?" and we not only won first place for our grade level, we also won the overall fair award! Our adventure isn't quite over, as we're planning to participate in our city science fair. We cited you in our "Acknowledgments" section, too. If you hadn't helped us, we doubt that our project would have been as successful as it is. We can't thank you enough.

With lots of gratitude, geesesandyoohoo
EAMills
Former Expert
Posts: 27
Joined: Tue Sep 27, 2016 11:46 am
Occupation: Other Adult

Re: Identifying Bacteria

Post by EAMills »

Hi geesesandyoohoo,

Congratulations! I am so happy to hear that all of your hard work paid off! Best of luck as you continue onto the next science fair.

Elizabeth
Locked

Return to “Grades 6-8: Life, Earth, and Social Sciences”