Team:Baltimore BioCrew/Contribution

2020 Baltimore Biocrew

Contributions

Contributions

Overview

We made two main contributions to iGEM. The first was that we showed that iron is a limiting nutrient for cyanobacteria. We tested this by tracking the growth of cyanobacteria in various iron concentrations. The second was that we showed that Secchi sticks are a useful and accurate way to measure cell density for both cyanobacteria and yeast cells. Secchi sticks are a cheap and accessible alternative to spectrophotometers for anyone who may not access to a full lab setup — citizen scientists, scientists during a pandemic, or even people who want to run experiments at home.

Measuring Iron Limitation in Cyanobacteria - A First Attempt

Methods

For the first attempt, we filled 20 tubes with 20 ml each of SN-Media (excluding the iron). We then added different concentrations of iron to the tubes using serial dilutions. Four of the tubes had the normal SN-Media’s iron concentration, four had 1/10 of that, four had 1/100 of that, four had 10 times that amount, and the final four had no iron at all. Synechococcus CB0101 (the specific strain of cyanobacteria) was added to each of the tubes to bring the Optical Density 750 (OD750) up to 0.05.

The tubes were split up into four groups that contained one of each type. They were all kept in incubators, at a constant 30 ℃. They were also all kept under constant light. Measurements were taken of all four trials around twice a day with a Secchi stick

Secchi Stick
Figure 1: A Secchi stick, the tool used to take measurements of the growth of the cyanobacteria.

Due to the pandemic, we weren't able to run these experiments in the lab. However, Synechococcus CB0101 is classified as a biosafety level 1 organism and is safe to keep out of lab. Without access to the lab, we ran these experiments from our homes, growing the cyanobacteria in mini-incubators.

Mini-Incubator
Figure 2: An at-home Mini-incubator. Inside are the 5 tubes each containing cyanobacteria and differing iron concentrations.

Results

After three weeks, growth in ¾ of the trials was still undetectable to the Secchi stick, although it was clear to the naked eye. The fourth trial seemed to have been contaminated.

Measuring Iron Limitation in Cyanobacteria - A Second Attempt

Methods

Three changes were made to the original procedure for the second attempt. The first was decided after reaching out to Dan Fucich of the Institute of Marine and Environmental Technology, the graduate student who gave us the cyanobacteria. We were told that these particular cyanobacteria grew better at room temperature and with a day and night cycle. So the incubator and constant light were done away with and samples were kept at room temperature near a window.

The other two changes were done to increase noticeable growth.

1. The tubes were started at a higher OD750 (closer to 0.2)

2. The tubes themselves were taller and skinnier, this makes the Secchi measurements much more fine-tuned since we are starting with higher numbers and have a bigger range.

Initial states
Figure 3: A set of tubes from the second attempt. Iron concentrations from left to right: 60 mg/L (10x), 6 mg/L (1x), 0.6 mg/L (0.1x), 0.06 mg/L (0.01x), 0 mg/L (No Iron)

Results

This time, varied growth was clear. The higher the iron concentration, the faster the sample grew as demonstrated by the greater turbidity and green color in the tubes.

Final stats
Figure 4: (A) Estimated optical density of Synechococcus CB0101 at varying concentrations of Fe(II). Secchi depth was collected for 2 weeks of growth at room temperature and converted to OD using a correlation curve. Initial (t=0 hours) OD of 0.05. (B) Optical density of Synechococcus CB0101 growing with varying concentrations of Fe(II) after 1 week. Iron concentrations from left to right: 60 mg/L (10), 6 mg/L (1), 0.6 mg/L (0.1), 0.06 mg/L (0.01), 0 mg/L (0). All values are normalized to no Fe(II) control.

Opening an Avenue for Easier Citizen Science - Secchi Sticks

Reasoning

Not everyone has access to a spectrophotometer at home to do this kind of work. During a pandemic, it’s difficult to get into the lab to run tests on samples. We ourselves could only get into the lab to use a spectrophotometer to measure the ODs of the samples a couple of times. Using the Secchi Stick as a solution, we were able to accurately track the growth of the samples.

Most scientists, however, cannot imagine what a Secchi measurement of 11 mm means. So some conversion is needed to the more standard measurements of OD, cell count, or plate colonies.

Methods

Starting with a sample of cyanobacteria at an OD of 0.77 (Towards the upper end of the linear measurements of OD750) we measured the samples with a Secchi stick in both types of tubes that we used. That sample was then diluted to a ¾ concentration, its OD750 was measured, and the Secchi measurements were taken. This dilution process was repeated until they were too diluted to measure with the Secchi stick.

Results

The following graph allows the Secchi measurements to be converted into OD750.

OD to Secchi
Figure 5: Graph that compares OD750 to Secchi depth measurements. It also provides an equation to accomplish a conversion.

Using our graphs, we show that measurements can be taken with a Secchi stick and be converted into OD750 by creating a standard curve. This not only allows for easier sampling but also for greater access to science across the board. Seeing as spectrophotometers cost many times what Secchi sticks cost ($4), this method makes this process much more accessible while still being accurate.

Counting Cells

Now that we had OD750 measurements of Cyanobacterial growth, we wanted to see if Secchi depth was also proportional to cell count on plates.

Methods

To make an agar gel of the SN Media, all the ingredients for the SN Media were mixed in ½ a liter of water and then combined with 15 grams of agar mixed into ½ a liter of water. 18 petri dishes were made. In 6 of them, 100 µL of 2⋅10⁷ OD750 was spread around using beads. In 6 of them, 100 µL of 2⋅10⁶ OD750 was spread around using beads. In the final 6, 100 µL of 2⋅10⁵ OD750 was spread around using beads.

After a week, we would have counted the number of groups on each dish, but there was no growth. We repeated the plating experiment but instead diluted the liquid cultures by 10-4 before plating and counting colonies after 5 days.

Also, we counted with a hemocytometer, revealing the number of cells from liquid culture. We diluted the culture by 10-4 and then measured one 4x4 square of the 25 squares of the hemocytometer. So the expression to get the estimated total cell count is (cell count from hemocytometer) × 25 × 104 (Protocol)

Results

Log(Cell Count) to Secchi Depth
Figure 6: Graph that compares the log of the cell cyanobacterial count (obtained from counting cells on plates) to Secchi depth using data from the table below. It also provides an equation to accomplish a conversion.
  • Cell Counts from Plates arrow_downward
    Cyanobacteria Dilution Colonies counted on plate Calculated total colonies per mL Secchi Depth OD750
    1x 108 1.08 × 106 15 mm 0.400
    0.75x 66 6.6 × 105 28 mm 0.268
    0.375x 69 6.9 × 105 49 mm 0.136
    0.25x 36 3.6 × 105 56 mm 0.082
    0.1875x 16 1.6 × 105 >75 mm 0.066
    0.125x 11 1.1 × 105 >75 mm 0.021
Log(Cell Count) to Secchi Depth
Figure 7: Graph that compares the log of the cyanobacterial cell count (obtained from the hemocytometer) to Secchi depth using data from the table below. It also provides an equation to accomplish a conversion.
  • Cell Counts from Hemocytometer arrow_downward
    Cyanobacteria Dilution Counted by hemocytometer Calculated total cell count per mL Secchi Depth OD750
    1x 621 1.55 × 108 15 mm 0.400
    0.75x 675 1.69 × 108 28 mm 0.268
    0.375x 572 1.43 × 108 49 mm 0.136
    0.25x 299 7.48 × 107 56 mm 0.082
    0.1875x 52 1.30 × 107 >75 mm 0.066
    0.125x 39 9.75 × 106 >75 mm 0.021

Yeast Experiments

To further validate the Secchi depth measurement and whether it is proportional to cell count, plate count and OD, we repeated the above experiments with yeast.

Methods

We grew a liquid culture of yeast. Then made dilutions of which we measured the Secchi depth and optical density (OD750). Then we used the dilutions to grow yeast on plates.

Results

Log(Cell Count) and Log(OD600) to Secchi Depth
Figure 8: Graph that compares the log of the yeast colony count and the log of the OD (600) to Secchi depth using data from the table below. It also provides an equation to accomplish a conversion.
  • Yeast Dilutions: Secchi Depth to OD to Colony Count arrow_downward
    Yeast Dilution Average Secchi Depth (mm) OD (600) Number of colonies (10-5 dilution)
    1x 10 2.153 1020
    0.5x 14.333 1.724 695
    0.25x 21 1.217 395
    0.125x 24 0.795 149
    0.0625x 34 0.401 190
    0.0313x 46.667 0.211 112
    0.0157x 65.333 0.119 55

Importance

Our results show that cyanobacteria are limited by iron concentration. They also show that Secchi sticks are an effective and cheap way to get accurate measurements of cell density. Other teams can use our standard curves or make their own to convert between Secchi depth, OD, cell count, and plate count. (Check out our calculator for converting Secchi depth to other units on our Software page)