Science Buddies: "Ask an Expert"

Hi! I've been looking around a lot trying to figure out exactly how I would prepare my Helicobacter pylori and I would probably get it from ATCC.org. However, it would come frozen...
I looked in the FAQ of ATCC and it said that with frozen bacteria, I need to keep it frozen really really cold, like -80 C, and I need to "revive" it by rapidly heating it in a 37 C water bath. Then it said to dump everything in it into my medium, and incubate it all. But some of this confuses me a little:

The recommended medium is Trypticase Soy agar with difibrinated sheep blood. But I would be "autoclaving" all this so wouldn't that mean that if I poured the thawed bacteria in, they would all die from the high temperature? And if I do a low temperature, wouldn't the agar all just solidify anyway? And am I just supposed to keep taking out bacteria from the incubated sample now and forget about the freezed up thing? It's strange cause from what I can remember in the past, my science teacher was taking some kind of vial out from the fridge (we were doing E.Coli that time) and I always thought that was the bacteria, but now it seems like the bacteria is supposed to be incubated. Also, I remember my teacher using a sucking in pipet to transfer something to the agar plates. My memory and ATCC are conflicting! Help me guys please!

Hi bhh1988,

You are asking very good questions. I've already answered your other inquiry and suggested that you not use Helicobacter, but here's the answer to your question about growing it.

Many pathogenic bacteria will only grow on medium containing fresh blood. To make the agar for this type of organism, you first autoclave the trypticase soy agar and then cool it down to about 50 degrees C, then you add fresh, sterile sheep's blood to the agar and pour the agar into Petri dishes to solidify. Pathogenic organisms love this medium, but it's expensive because you have to buy sheep's blood and the agar. There are companies that sell the blood agar plates already prepared.

Stock cultures of bacteria are made by growing up a big batch of a single organism and then lyophilizing (freeze drying) it. The culture is frozen quickly and then the water from the sample is removed by pulling a vacuum on the vial overnight. The sealed vials are stored in the freezer until the organism is needed. Your teacher probably bought the bacteria in the lyophilized vial and revived the culture for the class experiment.

For your science fair project, you will have the keep the bacteria alive until you are through with the project. You can do this by preparing culture tubes of agar, which have been tipped while the agar is still liquid to form a slant. The bacteria are transferred to a new slant agar tube every few days to keep them alive and growing. If the cultures are left for too long, or the agar dries out, the bacteria will die off.

Do you have any other questions?

Donna Hardy

Hi bhh1988,

I agree with Donna. I work in a microbiology lab, and H. pylori is definitely a biosafety level 2 pathogen, meaning that it would require training and handling to deal with it. In addition, H. pylori requires growth under anaerobic (no oxygen) or microaerophilic (very little oxygen) conditions. This requires special incubators or special canisters to remove the oxygen from the atmosphere. This is incredibly expensive, so I think it would be better to work with a different bacteria.

I hope that we've discouraged you from trying to work with H. pylori. It's a rather difficult bacteria to maintain. For your reference (or for others who read this website), the agar slant method by Donna below will work for most bacteria, but NOT H. pylori. H. pylori is a rather finicky bacteria and will not grow well in an agar slant.

If you're planning to present your work at a science fair, be aware that many pathogens require very specific SRC approval (i.e. you need to fill out forms stating your experimental design, and exactly what precautions you'll take). Keep that in mind as you design your experiment.

I agree with Donna; testing a nonpathogenic Gram positive and Gram negative bacteria sounds like a good idea. I know that you do not need prior consent to work with E. coli (Gram negative) and Bacillus subtilis (Gram positive).

Please let us know if you need help choosing other types of bacteria to work with.

Best of luck,
Linh

thank you for your advice. I am thinking about switching because everyone seems to be telling me not to use H. pylori. But understand that I chose it because it seems much more applicable and realistic, since H. pylori is so prevalent and causes so many stomach problems, and because garlic is widely known to be very effective against it. I have looked online and some other alternatives seem to be Staphylococcus and Salmonella, but those bacteria don't seem as realistic and frankly, as impressive as H. pylori. I have heard a lot more about ulcers and stomach cancers than I have about getting infected with Salmonella or Staphylococcus. At the same time, Staphylococcus lives on the skin, which doesn't seem to make it that applicable since rarely do people use garlic to apply on their skin. Most people eat garlic the last time I checked...then again, I think garlic does come out through our pores after we've eaten it, so I suppose Staphyloccus might be ok. I wonder if there are any other bacteria susceptible to the Allicin on Garlic though. Do you guys know of any? Thanks!

Hi bhh1988,

It's funny that H. pylori seems so impressive to you. The researchers that discovered H. pylori infection is linked with ulcers just received the Nobel prize this year. However, when they made their discoveries, no one believed them. It took over 10 years for their research to gain acceptance.

And you probably have heard of Salmonella, Staphylococcus, and Streptococcus species and not realized it. Salmonella causes food poisoning and is commonly found in raw eggs. If you've ever had food poisoning, you'd realize that Salmonella is definitely "just as impressive" as H. pylori. Because Salmonella causes a foodborne disease, I definitely think it's relevant for the purposes of studying the effects of garlic consumption. Moreover, a very, very close relative of Salmonella is E. coli. The lab in which I work uses Salmonella routinely, and it's definitely a biosafety level 2 pathogen. However, as I mentioned before, E. coli is a biosfaety level 1 microbe, and you can definitely work with it in your school. Keep in mind that it's very closely related to Salmonella and just lacks some virulence genes that makes it cause severe disease in the host. In fact, E. coli can sometimes acquire virulence genes and causes very severe disease. Remember how Jack in the Box had that huge problem with contaminated meat? That was due to contamination with enteropathogenic E. coli 0111 and 0157. In fact, this strain of E. coli was *much* more pathogenic than Salmonella. The infectious dose of Salmonella requires that you eat millions of bacteria. In contrast, eating only 5 bacteria of enteropathogenic E. coli is sufficient to give you really bad poisoning and even kill some people (if you're very young or immunocompromised). So you see, even "boring old E. coli" can be very interesting. It'd be really cool to see if garlic consumption has an effect on E. coli, and I think that's a testable hypothesis that you could work on at your school.

As for Staphylococcus and Streptococcus species, the lab in which I work deals with these two microbes as well. Staphylococcus aureus is indeed found on your skin, but it is also a biosafety 2 level microbe. It does not cause "flesh eating disease" (that's Streptococcus pyogenes, and it's very, very dangerous. I definitely wouldn't work with it.). However, for immunocompromised inividuals (live the very young, the very old, and AIDS patients), Staph aureus causes problems with skin infections because it has developed various types of antibiotic resistance.

But back to the topic. I would not work with Staph aureus either because it is a biosafety level 2 microbe. However, Staphylococcus epidermidis is a biosafetly level 1 microbe that also lives on your skin, but normally does not cause any sort of disease. So I definitely think you could test this bacteria if you wanted to design some experiments.

By the way, E. coli is Gram negative and S. epidermidis is Gram positive.

I agree with you about eating garlic. I've seen pills of garlic extract that are sold to repel mosquitos. When you consume a lot of garlic, some of it does get excreted onto your skin (that's how it repels mosquitos). So I think working with S. epidermidis would be a really interesting thing to test as well.

I hope this was helpful and encourages you to keep working on this project. I think using a nonpathogenic bacteria is a perfectly viable option and will still provide very interesting results.

If you have any other questions, please don't hesitate to post.

Best of luck.

Linh