Easy way to measure the number protozoa on slides?

[Achiara]

Hi, I'm in the 4th grade and my project is about different microorganism habitats and the effect of sugar, bleach, fertilizer, etc on microorganism growth. I used a pond water and hay emulsion to create the different habitats, but now I want to measure the number of microorganisms I can see on samples of each habitat every day for a week, to see which grow or die out. Using my microscope, I can take pictures and count the number of creatures on each one, do you think that's a good enough counting method? Thanks for your help!

[deleted-71536]

Hi Achiara,

This is a great project! If you want to count the number of microorganisms you grew, you will probably want to take some samples, dilute them, and swab some of the dilution onto an agar plate for counting.

Here is a page with wonderful tips about working with microorganisms: https://www.sciencebuddies.org/science- ... ques.shtml
All About Agar: https://www.sciencebuddies.org/science- ... Agar.shtml
Here is a great thread that discusses diluting bacterial samples: https://www.sciencebuddies.org/science- ... hp?t=10820

I hope this helps! Please post again (in this same thread) if you have more questions.

Heather

[Achiara]

Thanks Heather!

I'm worried that doing the agar will be too much work for my family. Do you have any easier suggestions? Thanks so much for helping me with my project!

Nico

[SciB]

Hi Nico,

Since you are looking at ALL the living organisms in your pond water and hay infusion, I think your idea of taking photos and counting the organisms in each photos is a good one. Heather suggested using agar plates and that would be ok if you were only looking at bacteria which form colonies on agar; but protozoans and multicellular pond critters like rotifers wouldn't do well on agar. Plus your pond water will probably have green algae such as diatoms that also might not grow on agar.

Many protozoans and rotifers have hairlike flagellas that they use to move around as well as cilia to move tiny food particles into their 'mouths'. Treating the pond water with bleach, hydrogen peroxide, sugar, fertilizer, etc., may stop the movement of the little beasties even if it does not kill them outright.

You will need to test several different concentrations of your substances as well as different lengths of time of treatment in order to find a range that does not kill them instantly. Bleach is especially toxic to pond life and you will probably have to use quite a dilute solution so as not to kill everything immediately.

Were you planning to take a tube of pond water, add a tiny amount of the chemical to it, wait 30 minutes or so then take a picture? You would of course be comparing it to a similar tube that was not treated with anything. If you can take a short video--say 10 seconds--you might be able to do a rough count of how many protozoans are still moving after the treatment. This might be more useful than just counting dead bodies.

Let us know what you plan to do so we can advise you on the best way to approach it so you can get really good results.

Cheers,

Sybee

[Achiara]

Hi Sybee! Thank you so much. You're right about the bleach, we added a 1/4 cup to a cupful of the infusion and there was nothing moving around. I already have the colonies set up with different ingredients added and am trying to take pictures of all 7 every day but it's taking too long. So my mom and I really like your idea of just a half-hour treatment. Do you think that would be enough to show an effect? How do I calculate how much sugar, bleach etc to put in? I still have a huge bucket of the hay infusion that I can use for new samples. And yes, I have a control. I think I can take a video, that's an awesome idea!

Nico

[SciB]

Hi Nico,

Glad you like my ideas! Wish i could tell you how much of the bleach and other stuff to use, but i've never done this experiment. You'll just have to do a trial first. Maybe start with a quarter cup of hay infusion, put some on a slide and take a video of it to show what's in there and estimate how many critters are moving. I'm not sure what magnification would be best for a video. You could try 40X and 100X. You don't want to go too high on the mag because then the field of view is too small.

Add 1 drop of straight clorox to one-quarter cup of hay infusion, wait 10 minutes and look at it again. If nothing is moving, it's still too much and you will have to dilute the bleach to 10% [1 part bleach plus 9 parts water] and try that. If everybody is still rockin and rollin then try adding 2 drops. You get the idea.

You'll have to do the same sort of test for the other chemicals and find out which concentration gives you some effect on activity but not too much. Watch for changes in the speed and pattern of movement, but if you can see the cilia around the mouths, then look to see if they are beating at the same rate or if they stopped. The cilia are like little molecular motors and poisons can shut them down pretty quick.

If you want to see some super amazing video of protozoans, check out this: http://www.youtube.com/watch?v=TTbS7_vZLNg
The person uses a special optical technique called DIC which shows the insides of the bugs really well. He must have an expensive setup to get video like that, but it gives you an idea what can be done. There's a bunch more eye-boggling microscope videos on youtube so take a look at some of them while you are there.

I had a cheap microscope as a kid and I still remember how amazed i was to see all these teeny organisms racing around in a drop of water living their little lives invisible to our normal eyes. I never get tired of watching them!

Cheers!

Sybee

[Achiara]

Thanks a lot for all your tips, I won the Principal's Award and got another award for experimental technique. My family and I had a great time observing all these microorganisms on the computer. I used a chart to identify the different types I saw (8 different types of protozoa on some slides!). I also rated movement on a scale from 0 to 10. Then I added a drop of different contaminants and observed any changes in movement and number. I got interesting results. Adding sugar made the guys speed up, but a second drop of sugar solution made them slow down. The fertilizer killed 4 types right away but even a second drop didn't affect to he ones that survived, though they did slow down. Lemon juice and bleach basically had the same effect - everything died right away. The coolest thing about my experiment was that I could develop a habitat and then watch the protozoa in action. The toughest thing was the smell!

[SciB]

That's great news about the award! Thanks for letting us know. We always like to hear how your project turned out.


Protozoans are really cool and I hope you go on to study more about them and other critters. Biology is my favorite area of science. We look forward to hearing about your next science project.

Best wishes,

Sybee