Science Buddies: "Ask an Expert"

Hi Trader,

There are all kinds of obstacles in science, and this is just one more for you. The science teacher is concerned about your safety and liability for the school if anything happens to you. If there are a group of you who need to get your lab work done, perhaps you could recruit a parent or other adult to stay with you. It's only for a short time, so I hope you can work something out. After high school, you won't have this problem and you will be able to stay in the lab all night if you want to. I can still remember the late nights in the histology lab. Lab work is time consuming, but there’s no other way to get your answer in science except to do the experiment.

Your autoclave sounds like a large pressure cooker, and so it should automatically let the pressure out slowly unless it has a leaky gasket. If there's no choice for fast or slow cycle, then you don't have a choice about the exhaust times. Waiting 24 hours until you open the lid is more than enough time. Next time you autoclave something, look at the pressure guage, if there is one, and record the time it takes to go from 121 to 100 degrees Centigrade; if this is less than 30 minutes, then you probably have a leaky gasket.

The optimum temperature for minimum doubling time might be different than the temperature for maximum number of viable microorganisms. With the constraints on your experiment, I don’t think you are going to get the maximum number of viable microorganisms because this will occur briefly at the end of the log phase of growth. However, you might be able to measure an optimum temperature for minimum doubling time.

Since you have to do your growth curve experiment with samples taken between classes, go ahead and make the dilution. However, the microorganisms will continue growing if you leave them at ambient temperature. Can you keep the water dilution tubes on ice, and then make the dilution into cold water? You should store the tubes on ice until you can make the rest of the dilutions and plate the sample. You should try to keep the tubes as close to freezing as possible, without actually freezing the sample to minimize post-dilution multiplication. You should be able to plate everything between 3 and 6, but you are going to be very busy.

I was thinking about your experiment. If you are using the murky nutrient broth that had been contaminated before you autoclaved it, the broth will probably contain AHL’s when you do your growth curve. So you will be doing an experiment on your original topic, even if it is somewhat uncontrolled.

Good luck!


Donna Hardy

The optimum temperature for minimum doubling time might be different than the temperature for maximum number of viable microorganisms. With the constraints on your experiment, I don’t think you are going to get the maximum number of viable microorganisms because this will occur briefly at the end of the log phase of growth. However, you might be able to measure an optimum temperature for minimum doubling time.

Oh no! . Though, I thought that after the population reaches its maximum after the log phase, it stays constant in the stationary phase where the number of bacteria grown is equal to the death rate --> wouldn't this also be the highest amount of viable organisms?

Also, I want to let the ethanol airdry, but also want to make sure that the cover won't be contaminated, so I find that I am often closing the petri plate before all the ethanol is gone, and after about 24 hours the plate will be completely dry. Will this be OK? IF there are several drops of ethanol left, what are the chances that after pouring agar into the plate, that the plate won't be able to grow any bacteria?

Today I managed to get 3 hours! ... and "half" of 4 hours. I took the dilution, and put it in the fridge -- would putting it in the freezer be too much? I hope that I can get some good results from these.

I think I get what is meant by the autoclave. After the autoclaving is done, I waited approx 5 hours and then unscrewed the knobs that held the cover on... And there was a distinct leaking sound. This is probably gradual depressurization?

For the pressure cooker though, I only know that when the pointer is at 20 (i want to say the unit is psi?), that it's 121 C. I'm not sure what would be 100 C so I can't really tell .

Hi Trader,

Don't worry, your assumption is essentially accurate. If you look at growth curve numbers, you'll see a little peak at the end of stationary phase that is a little higher than the stationary phase number that remains constant for a long period of time. You should assume that the highest number you obtain is the highest number possible for the temperature you are using.

If the ethanol is evaporated, then the plate is good to use. Ethanol is very toxic to bacteria; it affects the cell membrane. This is why ethanol is used in hand sanitizers, for example. 20% ethanol is considered bacteriostatic (will prevent growth), but a trace of ethanol should not cause a problem. So one or two drops would be OK, but several drops would probably not be OK.

Freezing causes the formation of ice crystals inside the cell and damages the cell membrane, and can kill some cells so you should avoid freezing conditions.

Waiting 5 hours to open the autoclave should definitely be slow exhaust. Next time you autoclave, after you turn the heat off, watch and see how long it takes for the guage to go come down to 0 pressure. This will be 100 degrees Centigrade, the temperature for boiling water. This time should be at least 30 minutes and if the pressure comes down too quickly during this time, the tubes may be boiling over. After 5 hours, the water is ambient temperature, and the sound you hear to due to a vacuum inside the autoclave that is released when you open the lid.

Do you have any results yet?

Donna Hardy

First results...(I hope)... within a few hours. OK I saw the plates. And they weren't too good.

I've seen many plates with "half-lawns" of bacteria instead of actual bacteria...there were a few with speckles. Of course, I am using the nutrient broth that I thought was not contaminated --. Good news is that after the slow depressurization, I have nutrient broth (that is MUCH darker than the previous one), but CLEAR!. Perhaps it's clear because its dark?

Aii...if I am lucky, I will be able to run one test run per day. Why would the highest number of viable organisms occur briefly at the end of the stationary phase? (If you mean log phase, why is death rate suddenly larger than growth rate, and then equal?) --

Also, would it be possible to find out the doubling based on hourly measurements alone? I think I'll need at least one trial to pinpoint when the log phase comes out... approx. And then do measurements for 37, 35, 33, 31 or something, and then find the maximum number of viable organisms and the doubling time for both? Oh... but I can only run one test per day!

I'll have to do what I can do then <_<.

My 4th hour sample is still in the refrigerator -- if I plate them after the tubes are diluted and are 24 hours in the fridge, how inaccurate would they be in terms of representing the sample? If they aren't "that" inaccurate, I can fit in two test runs per day then...

Also, as I sometimes only have 4 hours to do platings, how different would a nutrient broth that has been incubated for 4 hours, then stored in the fridge for 24 hours, and then incubated for another 2 hours to conclude a 6 hour growth curve test, from a nutrient broth that has actually been incubated for 6 hours straight?

I'll do my best to avoid these inaccuracies though .

The bottles that I have say 95% ethanol -- does this mean that I can soak the plates for only 20 minutes, or should dilute it with water? Though something tells me that it won't really "mix" with water...

One last question regarding keeping the bacteria per mL constant. If the volume can be different, then that means that the amount of bacteria still needs to be in a certain proportion -- how can we control for this proportion? From my understanding, would 2.5 bacteria mean 2 colonies and half a colony of roughly the same size? Then does that mean that 500 mL with 1x bacteria and 500 mL with 2x bacteria would lead to largely different growth curves? -- but still result in same readings for optimum temperatures for the two factors?

I'll check out the times soon.

Hi Trader,

The plates that look like lawns rather than individual specks mean that you did not dilute the sample enough. This is a common problem when you are first starting to do growth curves. Just record the results as "TNTC, too numerous to count." The clear appearance of the nutrient broth is a good sign. How dark is the new batch of broth? If it's really dark brown, this indicates the medium may have been overheated, and really dark brown medium can inhibit the growth of bacteria compared to lighter brown media. It's so hard to keep all conditions constant when doing experiments. Make a note of the variation in appearance of your nutrient broth, and if you find there is a difference in the outcome of the experiment, the variation in color/cloudiness could be used to explain any discrepant results.

You can expect the highest possible number at the end of stationary phase to be between 10 to the 9th and 10 to the 10th. The number, for example, might go up to 6 x 10 to the 9th and then will slip back to 3 x 10 to the 9th for the remainder of stationary phase. The numbers will remain constant for a few days until death phase starts. I wonder if the number slips down slightly when the culture starts to form biofilms. A plate count will only measure bacteria in suspension, and will miss any biofilm bacteria. The number will be about 10 to the 7th just when the culture becomes turbid; if the broth is still clear, then you'll have less than 10 to the 7th. Hopefully, this information will help you plan your dilutions.


If you are lucky, you will be able to determine the generation time at different temperatures if you have 2-3 measurements in a row that are from the log phase of growth. This information will allow you to compare doubling times. Since you won't have enough time to plate an entire growth curve, you may miss the very highest number, but you can always take a sample the next day to find out what the top stationary phase number is.

I don't know what the inaccuracy of using stored samples is, but this is certainly a possible source for error in you experiment. If you have time and medium available, compare results of a sample plated immediate compared to the same sample plated 4 hours later. Or, just wait and see if your results make sense; you will know soon enough if storing samples causes a major problem. I would not put the entire culture in the refrigerator; just keep it incubating at the same temperature, and store the individual samples as cold as possible and plate them as soon as possible.

If you start one culture with 1000 organisms per ml and another at 2000 organisms per ml, both cultures will grow at the same doubling rate during log phase and will reach the same number at stationary phase. The culture starting at 1000 per ml, will reach stationary phase one generation time later than the 2000 per ml culture. Does this answer your question?

Donna Hardy

If it's really dark brown, this indicates the medium may have been overheated, and really dark brown medium can inhibit the growth of bacteria compared to lighter brown media.

Cool, I'll be sure to keep that in mind. I remember that at first it looked really dark brown, but after I tilted it at an angle it wasn't as dark brown. I'll keep a watch out though -- and I do think that this may have to do with it because I left the autoclave for 24 h than its supposed to... accidentally. I turned the dial to 1, but somehow the power was still on and it was at 10 psi for the entire time... that is not good . Next time I will plug the power off.

The number will be about 10 to the 7th just when the culture becomes turbid; if the broth is still clear, then you'll have less than 10 to the 7th. Hopefully, this information will help you plan your dilutions.

Thanks! I think this may be the reason why! For my platings, I did 10^-1 to 10^-4 for hour 1 to hour 3, and then did 10^-1 to 10^-6 for hour 4 because I really had no idea what the population would be... perhaps I should be diluting more to prevent the lawns from growing?

If you are lucky, you will be able to determine the generation time at different temperatures if you have 2-3 measurements in a row that are from the log phase of growth. This information will allow you to compare doubling times.

I hope I'm lucky ><. I'll need it . By plating 2-3 measurements in a row during the log phase that means that I'll have to go beyond hourly measurements right? Originally I planned to first do hourly measurements to find out approx when the log phase begins (is it correct to say that stationary phase starts approx. 5 h after first incubation, so log phase is in that short time?) -- I never knew that about the highest amount of viable organisms... I probably won't have time to get it, but I don't know, maybe I'll get lucky?

As for the inaccuracies regarding the stored samples, I'll plate tomorrow and conduct another mini-experiment .

As for starting one culture with a proportional amount of bacteria to mL, how would I be able to know the quantity of bacteria I'll be inoculating into the nutrient broth? Right now I am taking roughly the same size colonies into 500 mL nutrient broth for all, just to keep it simple. I get why both cultures will grow at the same rate and reach the same number at each phase, but I'm still not sure why (sorry!) the culture starting at 1000 per mL will reach stationary phase one generation time later than 2000 per mL...unless it's because the 2000 bacteria per mL had one generation more to start with? (OH...wait, 2000 bacteria is one generation after 1000!... I get it now). I think. Though, I'm still not sure how to control the amount of organisms that go into the broth?

As for the sterilization, would 20 minutes in 95% ethanol be sufficient? (I don't think diluting 95% to make it 75% or something will be helpful in this case) -- I'll need to try to sterilize as many petri plates, as fast as I can .

Thank you for your faith in me .

Hi Trader,

I'm glad the broth was not really dark brown. I remember autoclaving a very large volume of broth once in graduate school to grow a culture Streptococcus. The extra time required to autoclave the large volume overcooked the medium and turned it dark brown. The bacterium barely grew at all, and I had to start over from scratch. You always remember details like this when things go wrong. So I'm hoping your bacteria won't mind the slightly dark brown medium.

Here's a suggestion that should address your time limit problem and get your bacteria growing in log phase quickly. The colonies from the agar plate, which you are selecting, are in late stationary to death phase and are producing AHL's. It may take them 2-3 hours to get through lag time and start dividing in the nutrient broth. So, instead of using a colony from a plate, use a broth culture that has been growing for a few hours or overnight so you will start with cells that are still producing all of the enzymes needed for cell growth and division and not producing AHL's yet. Make a 1:10,000 dilution of the broth culture (1 ml culture into a "9" ml water tube, mix well, and then 0.5 ml into 500 ml of nutrient broth or 0.2 ml into 200 ml; dilution factor 1:10 x 1:100 = 1:10,000) and transfer into the prewarmed medium to start a culture that will have a mininum lag phase. Make sure the nutrient broth is pre-equilibrated to the temperature you want to use for the experiment. If you start with an overnight broth, you will diluting 10 to the 9th down to 10 to the 4th per ml. You could have as many as 3 generations per hour. If you then plate a zero time and a one hour time, lag phase will be over, and your 2 to 3 hour and 3 to 4 hour samples should be the linear part of the log phase. You should see the culture start to turn cloudy in about 3 hours. This should also minimize the number of TNTC and zero count plates, and you will have more plates in the ideal 30 to 300 range. You can plate a sample the next day to get the maximum number of organisms, since you won't be able to stay until the end of stationary phase.

Yes, I think you need to do higher dilutions. If the broth has just turned turbid, then your perfect dilution for counting should be 1: 10,000 and you should plate one dilution above and below your target. You would have gotten a lawn with the 1:100,000 (10^-6) only if the bacteria were at the end of stationary phase and the numbers over 10^9 per ml. Was the sample really cloudy for this result? Remember if you use 1 ml of sample and add it to 9 ml of water, the dilution is 1:10; if you use 0.1 ml of sample in 9 ml of water; this is a 1:100; if you use 0.1 ml at the end to transfer to the agar plate, and this is an extra 1:10 dilution. If you multiple all of the dilutions together, this is your final dilution. e.g. 1:10, 1:100, 1:10 = 1:10,000 or (10^-4).

I'm glad you mentioned the concentration of ethanol because I forgot to explain this to you. 70% ethanol is actually more bactericidal (kills faster) than 95% ethanol, so you should dilute the 95% in water to make it work better. The standard exposure time is 1 hour, but since you are not running a pharmaceutical lab, I think the 20 minutes will probably work. It would be better to go as long as possible, but you know what your time limitations are, so do the best you can.

It's actually a pleasure to work with someone who is interested in doing a science fair project, and realizes how intrinsically interesting and worthwhile science is. Just try to focus and concentrate on finishing your experiments; don't rush and reread the paragraph on making dilutions again, and I'm sure you'll be fine.

Donna Hardy

It seems like the nutrient broth may be what is called as "dark brown" because it doesn't seem to be growing...<_<. I'll still see if it inhibits growth after results come out.

Thank you for that explanation! It clears a lot of things up. From my understanding, that means that after keeping the nutrient broth for 2 h, I can do 10^-6 - 10^-8 for the 2 hour check, 10^-7 - 10^-9 for the 3 hour 20 minute check (I get a break every 80 minutes), and 10^-8 - 10^-10 for the 4 hour 40 and 6 hour intervals? (Where the "peak" of the viable population occurs roughly at around the 6 hour period?).

I've been doing too concentrated dilutions =P. I took 1 mL from a 500 mL nutrient broth and did 10^-1 - 10^-4 for hour 1, 2, 3, and 10^-1 to 10^-6 for hour 4. ... It doesn't really work that way does it? Perhaps thats why there are the lawns!

Ooh, the tip with the ethanol is interesting. A more dilute solution is more effective! . So given a lawn of bacteria that I do not want, bleach for 20 min, use bare hands (is this safe?) to remove agar, dip empty plates in ethanol, air dry, then pour fresh agar in = clean new plates? (Just to make sure).

As for some things with the autoclaving, I have a few quick questions: 1) it is OK to autoclave ziplock bags which is plastic right? Would the depressurization result in the bags filling up with air...? and 2) is it OK if I autoclave a 500 mL nutrient broth that is in a 600 mL Erlenmeyer flask? I think its a bit of a silly question, but I've noticed when I started out my teacher told me to fill up tubes of sterilized water half full, and only boil solutions with Erlenmeyer flasks half full. I think the latter is to prevent spilling, but given the extreme pressure and boiling that goes on in the autoclave, would a too full flask result in the solution pouring out?

I only have 500, 600 and very few 1 L flasks, so I'll also have to make 2 250 mL nutrient broth and combine them.

One last thing, just to make sure: inoculating a bacteria colony that is of roughly the same size as all the others would keep the "starting number of bacteria" roughly constant right?

Thanks

Hi Trader,

I think you may be planning to make dilutions that are too high. If the broth is not turbid, you will have less than 10^7 per ml and you would want to transfer between 30 and 300 bacteria to the agar plate to grow. So for a non-turbid sample, make a 1:100 (0.1 ml sample in 9 ml water) x 1:100, and use 0.1 ml sample for the Petri dish(1:10) (total 10^-5) dilution as your highest dilution. If your sample is just reaching 10^7 per ml at this sampling time, you will have 100 colonies growing on the plate. So you want to plate between 10^-2, 10^-3,10^-4, and 10^-5. Once the sample is turbid, then plate 10^-5,10^-6,10^-7.

Your bleach should be diluted 1:10 (one part bleach plus 9 parts water) for maximum effectiveness. It would be better not to use bare hands to remove the contaminated agar from the Petri dish. Do you have a spatula or something similar you could use? Your bacteria are not pathogenic, but they are very concentrated after growing in a dish of nutrient agar, and you never know when an unknown contaminant might be growing. Be sure to incubate one of your plates without inoculating it to make sure you have started with sterile plates. One or two stray colonies won't significantly affect your experiment, but gross contamination could cause a major problem.

Your teacher told you to fill containers only half full because the normal slow exhaust on the autoclave will result in a small amount of boiling over, so if you put 500 ml of broth in a 600 ml flask, you are going to lose some volume due to boiling over. Just put 250-300 ml in the 600 ml flask. If your water dilution tubes were more than half full when filled them, this is probably why the volume is not what you expected.

We discussed this a while ago, but I don't remember what you decided to do about aeration. Are you leaving a stir bar in the flask and did you put the stir plate in the incubator? If not, then you will be doing stationary incubation, and your bacteria will grow more slowly that I have suggested, and your doubling times will be longer. Your experiment will be controlled if you incubate all samples with or without aeration, but this is a consideration on making your dilutions during the 6 hour incubation time. If you are incubating stationary cultures, then swirl the flask to mix it well every time to take a sample to ensure that the bacteria will be evenly distributed, because there will be a higher concentration close to the surface where oxygen is more readily available.

Yes, if you take about the same amount of a colony and transfer it to the nutrient broth, you should be starting with approximately the same number of viable bacteria for each experiment. If would be best to use a freshly grown plate for this.

Donna Hardy

Thank you for your reply!

I've incubated some of the plates (that I've reused through the bleach/clear agar/ethanol/airdry technique) and it seems like there are colonies growing "under" the agar... the entire thing looks very contaminated at the bottom. Yet, there are actually colonies growing at the top (I've done a streak plate on it) -- an empty incubation appears to have colonies grow at the bottom too, though nothing at the top. Or at least I think its growing at the bottom.

Amazingly though, I checked one of the colonies that is pretty large (there is this one plate that had just 2 colonies) -- from the morphology, it had ridges and was a very "tall" colony, and was a lighter brown than all others; I thought it was some contamination. Yeeet when I observed it under the microscope (I'm proud to have done it in record time!!!! Straight away! Without adjusting the platform and up and down X100), it looks just like b. subtilis! Or at least bacillus. Even the gram stain shows purple/black! (That's gram positive right?) ... so perhaps it's b. subtilis after all. ... Is there a high chance of contamination of the culture by another bacillus species?

For aeration, I leave a stir bar in the flask (as the stirrer won't fit in the incubator). After realizing this I found out that plating the first 6 hours wouldn't be of much use either, but I'll find out tomorrow's results anyways and see how it goes. So many things learning last minute!

Sorry if I looked over this last question -- are there any precautions I should take when autoclaving "ziplock + pipettes"? I'm thinking that with the pressure, the ziplock might explode? (I'm basing this on how airtight bags are very full after a plane flight...) -- .

Here's everything I understand: from now on I should start plating at 6 hours, with 10^-2 - 10^-3 and when the nutrient broth is turbid (no matter what the volume is, assuming that appropriate reductions/additions have been made in proportion in terms of the starting colony size), a 10^-5 above plating is almost guaranteed. Log phase will continue for roughly 3-4 hours after this in standard incubation -- and as the experiment requires highest amount of viable organisms (if I'm lucky I'll get?) and lowest doubling time, it's those 3-4 hours that is critical. Anything before, after is not necessary.

I hope the above is (near?) right?

Thank you very much for all this..

Hi Trader,

The colonies growing on the bottom of the plate are probably contaminants. Just count the top of the plate and then decide if the top data makes sense.

The plate with 2 colonies sounds like a contaminant because it looked a little different and had a different gram stain reaction compared to your original culture. Did you plate one lower dilution on the set that had about 200 colonies? Bacillus species can survive drying conditions so are common contaminants because they float around in the air in dust particles. Congratulations on perfecting your microscope skills; I am impressed that you can do the microscopic examination so much easier compared to just 2-3 weeks ago.

So your cultures are static, and your bacteria will be growing under essentially anaerobic conditions after a while. I am curious to know what the generation time is for this organism under the conditions you are using, because I have no idea what it will be.

You are just about right on your analysis of the growth curve. However, the samples that you take before the broth becomes turbid will also be in the log phase of growth and can give you information on doubling time. The bacteria will be in log phase all the way from 10^2 to 10^10 organisms/ml, and you just need 3 data points anywhere on the curve that are linear to determine the generation time at the temperature you are measuring. Knowing which dilutions to make will just help you get a countable plate.

I think that the ziplock bags might melt in the autoclave. However, you should never autoclave any tightly sealed container in the autoclave. There should always be an opening so the heat and pressure can get inside the container to sterilize it and a way for air to escape when the autoclave depressurizes. So try it, and leave a small opening in the seal.

I know you are approaching deadline, so remember to leave time to finish your board. You should have some results to include with all of the experiments you have done. Don’t try to do one last experiment too close to deadline; use your time on your board. When is your science fair? I have forgotten.

Donna Hardy

Thanks for your reply!

I've looked at the results for today and it seems like there are some colonies -- they are quite small (perhaps b/c they were just plated), so I left them out overnight -- hopefully they'll grow? What worries me is that they are not of the same size, and what I've noticed for all is that 1 mL is definitely too much for the plate (though 0.1 mL would be too little)--- in that case, if I drop 0.5 mL from a 1/10 solution ... that'd be a 1/20 dilution. Hmm, then I would be making 2 * 10^-2 to 2 * 10^-10 solution. I hope this is ok xD.

As for:

you just need 3 data points anywhere on the curve that are linear to determine the generation time at the temperature you are measuring. Knowing which dilutions to make will just help you get a countable plate.

I've noticed that after about 7 hours anaerobic incubation at 37 C the solution becomes turbid. Do you mean that if do a plate count as soon as it becomes turbid, and then two more samples later every 30 minutes I would be able to get the doubling time? (Is this what you mean? This would make things a lot easier)

Also, with this I'm a bit unsure about something. If I'm only focusing on the doubling time (if I want to ignore highest number of viable organisms for now) -- how would the initial amount of bacteria affect it? If I start one broth with twice the amount of bacteria, but I do the same as the above as soon as the nutrient broth becomes turbid, wouldn't I be finding out the same doubling time for the same species of bacteria? (It wouldn't matter how many bacteria it is -- it'll undergo the same doubling time?) -- Maybe there is something I'm missing here, sorry.

The ziplock bags didn't work . I'll try covering just the tip and the end (that connects to the pumper) with aluminum foil. What worries me is how easily the aluminum foil can just slip off. But I won't know until I try...

Based on what I know right now, tomorrow I'll incubate two 250 mL nutrient broth ... as soon as it becomes turbid (I still am not too sure about the log phase based on what I obtained above -- with the 5 hours = 10^-5 lawn, yet it was clear?) I'll get 3 points. That'll be it for 37. Turbid I'm now thinking is 10^-8 to 10^-10 (as the 20 mL 24 hour incubation resulted in countable number at 10^-7...).

My science fair is officially in ... 5 days. EEEK.

Thank you.

Hi Trader,

Yes, the 0.5 ml is OK as long as you can keep track of the dilution factor. If you do this project in the future, we will have to find a suitable spreader for you so you can make an even plate. If they are big enough to count, you can get a quantitative number for your count.

Yes, if you are there and notice when the culture becomes turbid, and then take a plate count at that time, and then at 30 and 60 minutes, you will obtain the generation time. If the culture is turning turbid at 7 hours, your generation time is probably about 40-50 minutes, but that is reasonable since the culture is anaerobic.

I think there is something about the growth that I'm not explaining clearly. I think if you can make of graph of the increasing numbers, you will see what your growth curve looks like. If you can plot the doubling time, you'll have the generation time; if your count the numbers at the end of the stationary phase, you will have the maximum number. These are two different measurements. Please send data so I can understand what your concern is.

Aluminum foil will protect the pipettes, but it is slippery. Maybe paper that is secured with tape would provide a better approach.

OK, this is your last experiment that you can possibly do. You need to start writing your results and conclusion. Remember that you are the expert on your project, and you need to communicate the reasoning behind the experiment and what you found very clearly. But, I'm sure you'll do fine.

Donna Hardy

Ok the plates are now in incubation.

The thing is, my plating of 1 hour - 5 hours after incubation from an individual colony all resulted in lawns. I took a sample to see if they were b. subtilis, and they were .. or were at least rod-shaped (perhaps that doesn't tell me much?)

Either way, I think the reason why there are all these lawns is because the dilution is too low?

But then whats contradicting this is that a 10^-6 sample of a 12 hour incubation of 20 mL broth actually resulted in countable colonies. This is making me think that perhaps its because the old plates (that are dry) are actually working while the new plates are too moist (which is supposed to be good), and are growing "too much" bacteria.

Please give me one more day to add everything I know together... is it possible for dilutions to exceed 10^-10? That would mean that 1 mL has ... 10,000,000,000 bacteria. With 0.5 mL, that means 20,000,000,000 bacteria...

Right now I just want to confirm several things:

1) No matter what the starting population is, if we take 3 points during the log phase of the bacteria with equal intervals, we can get the doubling time right? (This is what I did ... well they starting population was controlled, or attempted to be controlled, but if it isn't necessary it'll be nice to get rid of this uncertainty)

2) Different temperatures should result in different populations for most of the stationary phase right? I am taking a sample at 16 h incubation which I'm pretty sure is the stationary phase?

My hope is that I'll be able to get the results from today's doubling time and stationary phase count, tomorrow, and tomorrow I'll be plating two other temperatures, iF the plates plated today turns out good. If it doesn't, then I'm just completely lost.

Just one more day

Hi Trader,

If there was any free liquid on the agar plate, the growth of the lawn was probably due to diffision of the organisms in the free water. These bacteria are motile so they will swim all over if given the chance in a drop of water. I can't imagine that you missed all of the dilutions, so if all plates look the same, this is most likely reason.

1. You are correct, if you get 3 data points in a row that you can plot on a line, you will obtain the generation time, irregardless of the starting number.

2. Different temperatures may or may not give a different number at the 16 hour stationary time point. I don't know what to expect. Results are empirical, as they say, so we will find out when you get your counts.

If you find that you don't have any usable data, please don't be concerned. You still have a lot of information to present on your science board, and you will have lots to discuss to explain what happened, even if the results are not what you were expecting. You can describe and even do a graph of what you think the results would have been, and you can use most of your conclusion and discussion section to explain what when wrong and what you would do differently next time. The science behind your project and your rationale are intrinsically very interesting and based on recent scientific findings. A science fair judge will evaluate the project based on the scientific content and explanation of your results. Your bibliography on this project is exceptional. A science project with no results cannot score a high as one that has results, but it can do surprisingly well in competition. Don't forget your microscope work; you can at least present the microscope and colony morphology results confirming that you are working with B. subtilis.

Do let me know if you have some results. Don't forget, the lawns are "TNTC," and that's a somewhat measurable result.

Donna Hardy

Thanks.

I have 4 plates at 37 C, stationary phase (24 h). One of them I'm sure is good plate, and it has around 200 at 10^-6 ... From my understanding, this is ... 200,000,000 per mL? The original was 20 mL, so that was interesting. I have 3 other plates at 10^-6, 10^-7 and 10^-8 b/c I wanted to see further dilutions, and originally I couldn't find any colonies so I incubated them for quite a long time. The 10^-6 I'm still not sure what to make of it, but I can clearly see that there are 3 "big" colonies in the 10^-8, and by the looks of it 25 "big" colonies in the 10^-7. Do these results make sense? I'm thinking that some of them seem like they're off... a lot. I'm not sure.

I do have a lot of TNTC...they might come in handy.

Sadly I have yet to get the results back on my doubling time for 37 C.

I've also gathered samples of 3 points of 30 C. I'll be able to get those results very soon. As for other possible "results", I have a few other "mini-experiments" that I conducted during the big experiment, such as time it took for the nutrient broth to actually get turbid (perhaps I'll find some other support that shows when the "normal" lag phase would be?) -- I've also jotted down notes of the differences in duration of the lag phase w/ colony that is originally from agar, and another with a colony that was inoculated in nutrient broth, and then put in the fridge... would this be relevant at all?

If tomorrow works out well, I'll be able to get the max number in 24 h incubation w/ 30 C as well as the doubling time for 37 and 30 C.

If only I had one more week .

Aii.

I'm writing up my paper, and I'm a bit stuck on what the significance for finding the lag phases/doubling time or stationary number for 30 and 37 C (where support says b. subtilis' optimum temperature is from 30 - 37 C)...

I know that I originally set out to do growth curves, and didn't manage to do this. Perhaps if I find a difference in 30 and 37 C doubling time (chances are I will... IF the plates turn out OK), ... well, doubling time will help me increase the efficiency for my hope of a future experiment w/ accurately predicting bac growth as a substitute for a sensor strain for AHLs. ... That seems a bit insignificant =P.

IF I find a difference in 24 h, I think that the use of b. subtilis as industrial enzymes is generally an anaerobic process? If more of the b. subtilis is required to increase efficiency, in the given conditions (of that nutrient broth and temperature), this difference in temperature would help. But then, I have no idea whether the creation of those industrial enzymes would be under 'nutrient-broth' like conditions .

I wouldn't really have any chance at anything at the fair, but I'll go for the experience and see what others in microbiology are doing and how they're successful =P.

Thank you for cheering me up and encouraging me through this whole way -- I'll do better next year.

Hi Trader,

Good job! You have results. And the numbers make perfect sense. If you have 200 colonies on the 10^-6, then you would expect to have 20 colonies on the 10^-7 and 2 colonies on the10^-8 dilution. The numbers between 30 and 300 colonies are considered the most accurate, so the 25 and 2 colonies are actually very close to the expected numbers. If these numbers are accurate, then it means that the bacteria only can get to about 10^8 when grown under non-aerated conditions. This is a very reasonable result. The TNTC's will be good to use because you know that the numbers are above a certain number. You should be able to see lots of dots on the TNTC, not a continuous lawn, which would indicate smearing due to excess moisture.

You can include all of your results and observations about the difference in lag time between agar plate and broth-started cultures. Even if you didn't record the exact times, you should be able estimate the times and make a reasonable conclusion about these results.

I would be very nice to have 30 and 37 degree C results, but don't worry if you don't. Just present whatever real data you have. If you had another week, you could have a perfect project. It's always this way with science projects.

Donna Hardy

It looks I'll be able to have a doubling time for the 37 C growth, as well as the 30 C growth. I still have to finish up counting b/c it seems like many of the colonies are quite small, though it looks like its below 300.

There are some plates that have only 3 colonies or something -- I've made a slide from one of them and they look like b. subtilis -- should I accept these results?

As I'm writing up the research paper I was also wondering -- would a varying nutrient broth size be something that would be an uncontrolled variable? Its a different worry from the previous "wouldn't it result in less bacteria per mL" question. Since I'm partly investigating how much longer lag time and doubling time is under anaerobic conditions, ... wouldn't a 500 mL broth in an 1 L Erlenmeyer flask be exposed to more air than a 250 mL broth in a 500 mL Erlenmeyer flask, and a 10 mL nutrient broth test tube w/ much less exposure to air? Or is the entire process going to be the same "degree" of anaerobic-ness because there is a cotton plug and it is pretty much air tight?

I've seen some pertinent literature (a long time ago) that had growth curves and doubling times for b. subtilis and I can see that their lag time and doubling time is much shorter. Is it "common sense" that the lag time and doubling time will be longer that I shouldn't state that in my conclusion, or is it worth mentioning?

It's also a bit confusing because in these articles, it seems to say that b. subtilis anaerobic growth requires this and that to be possible. I didn't add any of the below, yet it still worked...or at least, there was growth, but very little.

http://aem.asm.org/cgi/content/abstract/70/9/5252
http://arjournals.annualreviews.org/doi ... o.52.1.165

Does this mean that my incubation was semi-anaerobic? (Is this possible?) ... it'd be interesting to see if doubling time increased among the results I got from the 500 mL (in the very beginning) and the results I got from the 250 mL. I obtained lawns from the 500 mL first 5 hours, with up to 10^-6 dilutions, while I obtained countable colonies in 10^-6 250, for 37 C at least. Didn't have the time to do 30 C. If I wanted to include this possible difference b/c of anaerobic/semi-anaerobic conditions, is it possible to incorporate it without adding "another independent variable"? It's more like two separate projects though ...

The big day is on Tuesday! I hope I'll do OK. I guess I somewhat found that anaerobic growth increased the lag time and doubling time by X amount in these temperatures ... what would be the significance?

I think the use of b. subtilis in industrial enzymes is largely anaerobic. Perhaps if they can make the process aerobic they'll be much more efficient? (Then again, I don't think this is a practical suggestion)...

One last thing! For the research paper, what would be "common sense"? I find myself writing out the procedure for pouring plates and making streak plates -- is this necessary?

And another technicality issue -- is it necessary to write out a materials list if I've already listed out all that is needed in the procedures section?

Ahhh!

Hi Trader,

You are amazing. I think you are going to actually have a complete project with data and excellent analysis for your project. And your questions and concerns about the final presentation are very perceptive. Your observation that the lag time and doubling time are slower than published results in the literature, and the analysis and explanation of this may just carry your project to prize-winning status. But that, of course, will depend on the quality of the other projects that are entered in the science fair. However, I think that your knowledge and understanding of the science behind your results will give you a definite advantage in the competition.

Here are some answers.

If you have 3 colonies on the lowest dilution you plated, and the result seems to make sense based on your expectation of numbers, you can use this result, but with a note that you know 3 colonies is not an ideal number for a plate count. 3 colonies could easily be a contaminant, so you can disregard the results if it seems appropriate. If this were a continuing research project, you would of course be planning to repeat the experiment and verify your results. So a plate with 3 colonies could be considered a tentative result.

Varying the volume of nutrient broth in the flask would be a minor uncontrolled variable because the surface area exposed to oxygen and the depth of the nutrient broth would be different with 250 and 500 ml in a 1 L Erlenmeyer flask. The conditions you used for your growth curves with either volume of broth would be considered microaerophilic in either container, so the availability of oxygen was a limiting factor in the growth rate of the B. subtilis. (Anaerobic is no oxygen, microaerophilic is about 5-7% oxygen, and aerobic would be 20% oxygen). You know from the literature that B. subtilis is an aerobic organism, so would grow better with unlimited oxygen. I wouldn’t spend too much time discussing this in your paper because you didn’t measure the oxygen levels. You might just want to point out that it is a possible source of error.

You should definitely state that the reason that the log phase and doubling times are longer with a microaerophilic environment. Your literature references cite the difference in metabolism and nutrient requirements for growth in anaerobic conditions compared to aerobic conditions. B. subtilis grows well with just about any carbon and nitrogen source in an aerobic environment, but requires a sugar source and extra nucleosides with anaerobic conditions. When you take biochemistry in college, you will learn about all of the biochemical pathways that organisms use for metabolism. B. subtilis gets a lot more energy per sugar molecule with aerobic metabolism. The organism can grow in microaerophilic and anaerobic conditions, but metabolism is much slower. The growth curves from the literature were all done on a shaker flask with aerobic conditions; you used static conditions so growth was slower. The references you have cited provide documentation to support this statement.

The significance of your results is in the basic understanding of how microorganisms grow in different conditions. You have quantitated the doubling time of B. subtilis under microaerophilic conditions. I don’t this information has been published in the scientific literature, so this may indeed be a new fact to add to our scientific knowledge. This information could be important, for example, if you were planning to do additional studies on the nutrient requirements of this organism under microaerophilic conditions, or if you were designing a fermenter to produce industrial enzymes with microaerophilic conditions. Basic facts about microorganisms, like doubling times, are always useful and highly significant.

With science fair projects, teachers and judges expect students to write out all of the materials used and all of the procedures. So you would need to include a list of all materials, and the procedure you used for pouring plates and doing streak cultures. It would be acceptable to use a copy of a published method if you had followed the exact method. You don’t need to include every tiny detail like how you autoclaved the nutrient broth and disposed the used agar, so use your judgement on what to include. What you want to do is describe your method with enough detail that someone would be able to follow your method and obtain exactly the same results that you did. Making streak plates is a standard, common technique, so you don’t have to describe this if you are out of time. In the future, you can refer to earlier publications that describe the technique you are using, so you don’t have to rewrite everything every time you publish new results.

Since you have your research paper with all of the details, make sure your board just has the basic information, and is very clear and easily readable. You have so much information to present that it would be easy to make it too complicated. You will want the judges to be able to understand the basic project within about 30 seconds after glancing at the board.

You are doing great! Don’t forget to get some sleep this week-end.

Donna Hardy

Thank you for this.

Wow it's so exciting typing my first real research paper . I find that the procedures section takes up so much, but there WAS a lot of preparation work ...

I am a bit worried about the control group. In the experiment I've noticed that lag times are longer (just by measuring the time from inoculation to turbidity), the doubling time is longer, though the stationary phase population appears to be the same.

But how can I claim that the lag phase and doubling time is longer than the same conditions, only w/ aerobic growth? . I have various sources displaying growth curves of b. subtilis and it shows that their lag time is around 2, but I have no idea whether that is from agar to broth, or broth to broth, and I don't know if whatever their setup is (their addition of chemicals/use of different broth) would have impacted the results enough to make their lag time shorter). I did record that there was approx. 7 hour lag phase (or at least before turbidity) for agar to broth, and around a 5 hour phase to first sign of turbidity, from broth to broth, and I'm pretty sure b. subtilis really takes less than that under aerobic growth -- but I can't make a comparison if the experimental setup is different right?

Something interesting I found: I was previously worried about the different colonies that I found that all turned out to be b. subtilis. It seems that they were all b. subtilis (probably) for a reason!

http://www3.interscience.wiley.com/jour ... 6/abstract

It mentions the ridges that I found! It looks like that it was b. subtilis after all, and was because of the variations in nutrient in the agar itself. Cooool.

If I'm not mistaken, it seems like b. subtilis can survive anaerobically (with the aid of so and so), microaerphilicly and aerobically. Would this mean that its an facultative anaerobe?

I love the history of the oxygen requirements of b. subtilis growth. It seems like until 2004, b. subtilis was also thought of as an obligate aerobe. And then research for its anaerobic conditions were investigated and it was found that they could survive anaerobic conditions, given the addition of nitrite, nitrate, etc. I can't find any characterizing the growth of b. subtilis and its microaerophilic growth on places such as Google, so it seems like additional nitrite, nitrate, etc is not required in the presence of oxygen.

Thanks for all this!

Hi Trader,

Yes, I can imagine that your procedures section was a lot of work. But it’s very important for anyone reading your report to know exactly what you did, so I’m glad you took the time and effort to include the details.

You should not be worried about the control group results. Your results are empirical, or in other words, they are what they are. This is why we do controlled experiments. Results can’t be wrong because they are what actually happened. If your hypothesis doesn’t match the results, then it’s the hypothesis that is incorrect or something was wrong with the experimental design, and you need to revise the hypothesis or the experimental design and try again. This is how progress in science is made. (Does this help?)

So it’s definitely possible that you will obtain the same maximum number of organisms at different temperatures. I’m sure there is a significance to this. Maybe it’s the AHL’s that are produced at a certain population density that inhibit doubling. Whatever your result is, I’m sure it’s significant and it would be possible to think of an experiment to explain why.

I’m not sure what data you have, but you should be able to make a general comparison between the generation time reported in the literature and your results, and I was trying to encourage you to compare your results with previously published results. The time between inoculation and the first sign of turbidity includes the lag time (no doubling) and early log phase. You don’t know exactly what conditions were used in the literature, but the standard method for this type of study is to put the flask of broth on the shaker and incubate under aerobic conditions. I believe that the literature sources show a doubling time of about 20-25 minutes for Bacillus species at 30-37 degrees Centigrade (but you could verify that with a specific reference). If you have 3 data points in a row from log phase, you can estimate the doubling time for your experiment, and I am guessing from your description that the doubling time is about twice as long as the published data. You probably don’t have enough data points to know how long lag time was in your experiment, so it would be difficult to make a comparison about the length of the log times between published studies and your data. So, if you have data from your experiment, you can compare results to the published references and offer reasonable explanations for the similarities and differences. .

Excellent! You have carefully documented observations about the variation in colony morphology and a literature reference that explains why this organism can have ridged colonies. Be sure and include this information in your report.

Yes! Bacillus subtilis is a facultative anaerobe because it normally grows aerobically, but does have the ability to grow anaerobically. Because you did not aerate the broth cultures in your experiment, it is likely that your cultures were growing under microaerophilic conditions (it would be reasonable to say this even though you didn’t measure oxygen levels because you know from the literature that an aerobic organism would use up the oxygen, and you weren't adding it back into the culture). The statement that your cultures were growing under partially anaerobic conditions is a reasonable explanation for your results, which showed a slower than expected generation times. Your use of literature references to show that the organism can grow anaerobically helps support your statement. Another possibility would be that the bacteria were still growing aerobically, but growth was just slower due to limited oxygen availability.

http://www.sciencedirect.com/science?_o ... a2ee1b3d3d

You are going to love biochemistry when you get to it, because it helps explain everything. Bacillus species need a place to park electrons after they metabolize nutrients and they prefer to use oxygen because, metabolically, this is more efficient. However, if oxygen isn’t available, they can use nitrate, and probably other electron acceptors. Nitrite is already reduced, so it can’t be used. You are correct, if oxygen is available, nitrate is not needed.

I agree that it’s very interesting that Bacillus species are no longer obligate aerobes, as I had learned when I studied microbiology. And, we had to wait until 2004 to find out about this. I wonder what other “facts” will change in the future.

Donna Hardy

Attached is how I organized the data I had. Would this be the most accurate way to display the data that I have?

I'm using a formula that can find doubling time with just two points (is this the wrong approach?) -- I've based my discussion on this, and instead found the average of the two doubling times obtained from the three points for more accuracy. ...

Eeek! As for graphs, I only am graphing the growth curves as well as a linear relationship between growth rate and time (unless I got my axes wrong?) --

I don't think I'll be able to graph my observations of lag time and highest amount of viable organisms in stationary phase?

(Also, I originally hypothesized that the number of viable organisms in stationary phase would be lower b/c of lowered oxygen levels -- it turns out that the number is approximately the same as that in literature, so oxygen levels do not affect the amount of viable organisms -- I'm not sure why though ...?)

I wish I learned biochem right now -- I would be able to know the answer.

Also, from my understanding microaerophilic is 2-10% oxygen -- how am I sure that the conditions I've tested the b. subtilis is actually microaerophilic? Or is it a, if its not aerobic and if its not anaerobic, its everything else in between = microaerophilic?

Thank you so much! Second last day ...before fair. Travel tomorrow...

Hi Trader,

Data! I love data because it’s always so interesting. Your data is very good, but is going to require some interpretation. You did a very nice job of putting the data into the tables.

If you graph your results using a semi-log scale during log phase, you would expect a linear curve as shown in this example:

http://www.splammo.net/bact102/102gcurve.htm

So you could also include a simple line plot of time (x-axis, linear) vs number of organisms (logarithmic scale) with each table of results, so the judges can “see” what your data shows.

Please check your calculator formula because the average doubling time seems off. For example, on the 37 degree date, if the population increased from 2575 to 2,900,000 from hour 1 to hour 2, this would represent 10 generations in one hour, or a doubling time of 6 minutes. The increase of 2,100,000 to 140,000,000 from 1-2 hours on the 30 degree data represents 6 generations or 10 minutes doubling time. Try the formula from the above website and see if you don’t get a different answer. I wish we had one more day to work on the data, but you are at deadline, so do the best you can.

Now, we know that data is data, so how do you explain this? The fastest generation time published anywhere in the literature is about 20 minutes, so generation times significantly less than this are impossible. One likely cause of the unexpected results is that the bacteria kept multiplying after you took the samples and before you could plate them. Another possibility is that the reuse of the pipettes and Petri dishes allowed contamination and affected your results. Your trend in your numbers is from low to high, so I doubt you made a major mistake in making dilutions or mixing up samples. Since you did the experiments, perhaps you could offer another possibility.

So, recheck your calculations, print the tables (highlight or make bold the “average population” and the “average doubling time” values so your viewers will know to focus on those values and won’t have to read through the whole table trying to figure it out. And add a graph if possible, and offer an offer a logical explanation of the results. Since the results appear to be a little unexpected, also offer a description of what you would do differently next time. One item that I am regretting now, is that I didn’t suggest that you include OD readings at 660 taken at the same time as the plate counts. That information would have helped clarify the plate count results, so that’s something that you could plan to do next time.

One advantage you have with this data is that the judges are not going to think you made it up. Sometimes, when judging projects, data is too good to be true, so one wonders if the experiment was actually performed. No one will have any doubt that your data is real.

Microaerophilic can be 2 to 10%; I don’t think there is an absolute number. It would be conditions that are less than atmospheric conditions, but more than zero% oxygen.

If you are planning to take pictures of your project, I would love to see your board, if you can post it. If you can do this, be sure to follow internet safety rules, and make sure there is no way to identify you or your school from the pictures.

Good luck at the judging! This is an excellent project, and I’m sure you will do well in the fair, depending on the competition, of course. One thing that all judges are expected to do is ask a challenging question that is difficult to answer. If this happens to you, just relax, and think. You know more about this subject than anyone else, so you will have a good response available in your vast knowledge of this topic. I’m anxiously waiting for a report. Be sure to take some time and look at all of the prize-winning projects to get ideas for next year for presentation and analysis of results.

Donna Hardy

Sorry for the abrupt reply: Can you give me an alternate website? I can't seem to be able to load it ><.

I've got pictures of my semi-finished board...big day is tomorrow

As for finishing up the graphs, would there only be a linear relationship b/w population (units: Log N?) and time, as well as an exponential growth using x = N0 * 2^(t/d) formula? with x axis population and y axis population? Oh no, this is a bit confusing. Does this mean that are two possible graphs to be obtained form the same axes?

According to the formula that I've used (I found out my calculation error) -- yes, I do know that I left out the bacteria in the fridge overnight b/c I didn't have time. They must have multiplied then.

Attached is what I'm putting on for data...as well as pictures Oh no! I forgot the camera cord! I really wanted you to see this because I thought the layout was very cute ><.

Right now its one day before the fair and I'm still typing out the graphs and discussion and conclusion.

This reminds me: for the conclusion, my teacher tested me before asking me exactly how "oxygen" is substituted under anaerobic conditions (making b. subtilis a facultative aerobe) ... I didn't know how to answer (I wasn't too familiar with the literature) -- as for how b. subtilis uses oxygen under microaerophilic conditions, all I know is that it is crucial in metabolism to get more energy per glucose... I hope this is OK?

One last thing! ... Iiiif I can't find any specific articles that state the doubling time of b. subtilis, would it be a problem? It seems like one of those "assumed and true" things ...

I've calculated the times for 30, the trial that I did right before leaving, with what I know. I think the results can be called "good"?

Now to graphing! And I hope to leave in 2 hours to sleep...

Hi Trader,

I'm sorry I didn't see your request yesterday. Here it is.

Donna

Hi Trader,

You can see from the plots how to plot bacterial growth curves. It's possible to plot the data on semi-log graph paper, but not linear graph paper. If you plot both the 30 and the 37 degree data the same way, you can compare results. But if you are out of time, don't worry about it. Your tables look great, and you have lots of data to present. And, since there was a delay in sample collection and plating, you can explain your results. That's all you need! I'm sure you'll do fine! I can hardly wait to hear your post science-fair report.

Donna Hardy

Thank you for the link! From what I understand now, for a linear relationship it is

y axis = "bacteria population" and increments are 10^1, 10^-2, 10^-3?

and x axis = time.

For an exponential relationship y axis is still "bacteria population" and increments are "normal".

Is there anything else I can graph? Though I find that a lot of my centerpiece board has results taking up the space. There is so much to talk about in the discussion!

I included a formula to calculate doubling time, but not generation time ... on the board. I guess I'll add it verbally. I like formulas

ONE LAST worry: for the data tables, the final population that I obtain -- is that the population in the 1 mL that I took from the broth? In that case it would be "OK" if I base all "bacteria population" as not the bacteria that resulted from that one individual colony inoculated, but just as how the bacteria population in 1 mL changes over the course of time?

I have also seen CFU used a lot -- is there any way I can apply this term to my graphs? (I'm not too sure what it is).

Sorry for all these questions!!

Hi Trader,

I like your teacher; he asks very good questions, but does not supply answers, even if you haven't had biochemistry. When bacteria use sugars for metablism and there is no oxygen available, there are some extra electrons left at the end of the electron transport chain. If oxygen is not available to accept the electrons, then the electrons can be given to another molecule like nitrate (N03) which is thereby reduced to nitrite (NO2). Bacillus subtilis cannot use nitrite as an electron acceptor, but some organisms can use it and reduce it to NO. An obligate aerobe would not be able to use an alternate molecule, so would only be able to grow if oxygen was available.

http://en.wikipedia.org/wiki/Fermentati ... chemistry)

Here's a reference that shows generation time, or doubling time of Bacillus subtilis in a defined growth medium with ammonium sulfate as the nitrogen source. The authors found a doubling time of 40 minutes; you were using a medium contained tryptone, which contains preformed amino acids and other biomolecules and your medium contained glucose as an easily usable energy source. You would expect a faster doubling time with your medium because the bacteria would not have as much work to do to synthesize proteins.

http://jb.asm.org/cgi/reprint/104/2/762.pdf

Generation time and doubling time are two ways of saying the same thing; it's the time required for all of the bacteria in the medium to divide in half and thereby double in number.

I'm not sure I understand you concern about the 1 ml sample. The bacteria growing in the nutrient broth are a like homogenous suspension so a 1 ml sample represents the population density of the entire sample. Hopefully your two hours of sleep will help this worry disappear.

CFU, or colony forming unit, is the same thing as organisms or bacteria per ml. It's a standard term for this application and your judges would understand it if you put in on your graphs. But, I would not change your graphs or tables at this point; it would not add anything to the presentation.

Good luck!!!

Donna

The brief version, or the fancy version?

Brief version: (Highlight text below)

I got 1st place for the regional science fair and qualified for ISEF in Reno Nevada! That one...

Fancy version:

Night before I was hyperventilating xD because I kind of didn't have my results and discussion typed out yet. Today morning (of the fair) I printed out 1/3 of my board (pictures soon...sponsor said all pictures would be uploaded soon: http://www.tiseagles.com/extracurricula ... .php?id=18)

And approx 3 minute to deadline I was still pasting my board ... . Sorry for that.

1st round of judging. They were somewhat intimidating, but nice at the same time. Just asked w/ "tell us about your project", and I went onto talking ... and talking . They asked about the practical value of my research ... and more about the project itself, but amazingly not one of those "so if you had more resources..." or other questions.

They just left... I thought I didn't do so good, so I just said 'thanks' and sat down awkwardly. Buuut then, the fair manager came to me and said "pssst, the judges that just interviewed you -- they said you did an exceptional job...they were the only judges I've had mention this about anyone"... and then he started talking about "if you get to qualify as an ISEF Finalist..." -- I stopped listening cuz I didn't want to get my hopes up =P.

2nd round of judging. I loved this group! I thought I did same as previously, but this pair of judges nodded and listened, and said "really good job" in the end . I somewhat stopped hyperventilating. They asked quite a lot of questions regarding how I controlled the variables, and where I would see the biggest problem be in duplicating this research.

Then there was break, and they were to announce the first place winners of the 5 categories. We were told that of these 5 individuals, 2 to go to ISEF?

One final round of judging. 4 JUDGES! Stuttering quite a bit...even though I'm much better in debate <_<. Don't know why. I felt that this round was the worst... and the judges were relatively non-responsive. One nodded though. I really liked that .
They asked for how I made sure the environment was microaerophilic, and I said that the nutrient broth w/o aeration would be microaerophilic, but I felt that something was missing there.

Then final judging was over! (It turned out that the final judgings were only made by the judges who didn't see our project yet). We waited a whole 30 minutes ... in anxiety.

Right! Our school had 3 people and all 3 were part of the 5 first place winners. But by then it was a bit awkward b/c we know that best case scenario, one of us wouldn't make it.

I was already "okay, you guys tell me how it is in the states..." because I was very grateful about getting a prize already ><

Then there was the awards ceremony...and when they came to announce the two "best of show" winners...THEY SAID MY NAME O=.

I can't believe this <_<.

Anxious and very excited about being able to go... but I'm worried that I'll not do so well there. There'll be so many finalists competing!!

But first I must MUST thank you for sticking with me this whole time. I can't believe I made it here ... definitely wouldn't have done it without you!!!!

Now... I'll want to recheck those doubling times... and get my board "non-last-minute" looking.

WILL YOU GO TO THE FAIR IN MAY? I may see you there!!!!

Hi Trader,

Congratulations!! You won!! I'm so happy for you. Your knowledge and understanding of your topic more than compensated for the data and your hard work was worth it. I'm really glad you are going to the next step. It will be a really good experience for you. Thanks for sending all of the details of the judging; it sounds like it was a well organized fair, with decisions coming from a consensus of several judges. And, it sounds like you handled the questions very well.

It's also good that you can look at the data again, and see if there's something else to interpret. And it will be nice to have the time to polish the appearance of the board. Did the judges write out any comments about your board?

Before this, I was not planning to go to ISEF, but I will see if it possible, now that there would be an excellent reason to do so. I've worked with so many students in the past, but I've never provided advice for an ISEF final project before, so this is very exciting for me as well.

I will look at the data again as well, and see what can be done about that.

Donna Hardy