Scientists use a variety of techniques to study microbes. One way scientists investigate bacteria in particular is by growing, or culturing them in the lab. In this activity, students will collect water samples and count how many bacteria are present in each sample by creating cultures in the lab.

Students will spread samples on agar media in petri plates, let the bacteria grow, and count the number of colonies.  They will then calculate the number of culturable bacteria in the original sample by dividing the number of colonies by the volume of sample spread on the plate. There is one challenge to using this method, however. If there are too many bacteria, then the colonies on the plate will overlap and we won’t be able to count them properly. So before spreading the samples on the plates students will prepare a ‘serial dilution’ of each sample so that they can spread several different volumes on the plates. Samples will be diluted by a factor of 10, 100, and 1,000.  Then, 100 microliters of each dilution will be spread on agar media in a petri plate.

Objectives

• Students will determine bacterial abundance in water samples
• Students will analyze and interpret results

Materials

Materials are listed in quantity per group; items 1-7 can probably be replaced with what your school has available; similar supplies can be purchased through Carolina Biological Supply as indicated)                                                      

• Sterile 15 ml Falcon tube for collecting sample
• 9 x 2ml sterile Eppendorf tubes, available at Fisher Scientific
• p1000 pipette (check out Carolina Biological Supply for pipettes that will fit your needs/budget)                                                                                                
• p1000 tip box
• p200 pipette                                                                                                                                                   
• p200 tip box                                                                                                                      
• 1 x 10ml sterile plastic pipette
• Pipette bulb
• Phosphate buffer saline (PBS) solution (if samples are salt water) available at Fisher Scientific
• 500 ml sterile water (if samples are fresh water)
• Sharpies for labeling tubes                                                                                         
• 9 petri plates with Luria Broth Agar media or Marine Agar 2216  
• 9 sterile spreaders          
• Parafilm for sealing plates
• Sharpies for labeling plates
• Worksheets                                                                                                                                      
• Pencils                                                                                                                                                                                                                                                                                                                        

Procedure

Preparing Dilutions

1. Divide students into teams. Each team will prepare three dilutions per sample.                                                 
2. Collect water sample from your local environment (pond, creek, river, lake, ocean, estuary, etc.) in the sterile 15ml falcon tube.
3. Label the small tubes with the dilutions (to keep track of which dilution is in each tube) 1/10A, 1/10B, 1/10C;1/100A, 1/100B, 1/100C; 1/1000A, 1/1000B, 1/1000
4. Add 0.9 ml sterile water to each of your 9 small tubes; if you collected salt water samples, add .9 ml PBS solution to each tube instead (to maintain constant pH).
5. Measure 100 microliters of your sample into each of the three Eppendorf tubes labeled 1/10A, 1/10B, and 1/10C (this is the 1/10 dilution).
6. Measure 100 microliters of your 1/10 dilutions into the three 0.9 ml sterile water tubes labeled 1/100A, 1/100B, and 1/100C (this is the 1/100 dilution).
7. Measure 100 microliters of the 1/100 dilutions into the last three tubes of 0.9 ml sterile water labeled 1/1000A, 1/1000B, 1/1000C (this is the 1/1000 dilution).
8. After completing these three steps of the procedure, your team will have (3) dilution series of one sample for a total of (9) tubes.  (See Figure 1).

Preparing Dilution Plates

1. Label plates with date, sample number, and dilution:1/10A, 1/10B, 1/10C; 1/100A, 1/100B, 1/100C; 1/1000A, 1/1000B, 1/1000C.
2. Pipette 100 microliters of each dilution onto a petri plate containing agar media.
3. Use a spreader to spread the sample. (See Figure 2).
4. Seal each plate with parafilm and allow to grow for 1-3 days.

Counting Bacterial Colonies

To calculate the abundance of culturable bacteria in a sample, count the number of bacteria colonies that appear on each plate after 1-3 days. To help keep track of which bacteria colonies you already counted, students can mark each counted colony with a sharpie on your petri plate. Divide the number of colonies by 0.1ml (the volume spread on the plate), and multiply by the dilution factor (e.g., times 100 for the 1/100 dilution). Students will not be able to count colonies on all the plates because there are none, or because there are too many. Calculate the mean bacterial abundance for each sample.  

 

Calculations

Dilution	Number of colonies counted	x  dilution factor	= # culturable bacteria/ml water (abundance)
1/10A	____________	x         10	= _______________
1/10B	____________	x         10	= _______________
1/10C	____________	x         10	= _______________
1/100 A	____________	x         100	= _______________
1/100 B	____________	x         100	= _______________
1/100 C	____________	x         100	= _______________
1/1000 A	____________	x         1000	= _______________
1/1000 B	____________	x         1000	= _______________
1/1000 C	____________	x         1000	= _______________

Average abundance (for the plates you could count) =_______________

Questions

• Which plates/dilutions had the most colonies growing on them? Which had the least?
• Were you able to calculate bacteria abundance for all of the plates? Why or why not?
• Why do scientists need to prepare different dilutions in order to calculate bacteria abundance?
• If you were able to sample from the ocean: bacteria abundance in the ocean is about 1,000,000/ml. How do your samples compare to this quantity? How can you account for differences?
• How many different types of bacterial colonies did you see (different colors, shapes, textures, etc)? 
• Describe each kind of colony you see and write down how many of each you can count.
• Were you able to positively identify the different species of bacteria growing on your plates?  What technique do scientists have to use to identify bacteria?  Why do they use this technique?

Lesson Resources

Student Instructions/Worksheet

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National Science Education Standards

K-12 Unifying Concepts and Processes

• Evidence, models, and explanation
• Constance, change, and measurement

9-12 A Science as Inquiry    

• Abilities necessary to do scientific inquiry
• Understandings about scientific inquiry

9-12 C Life Science

• The cell
• Behavior of organisms

9-12 E Science and Technology

• Abilities of technological design
• Understandings about science and technology