Week of 11/11/24-11/15/24

Intro

This week was largely dedicated to inoculations, with us also setting aside a flask to be measured for a growth curve. Media was made and plates were poured to ensure that we had enough for the rest of the semester. A lot of time was spent helping out Evan's group with inoculations, pouring plates, and gram staining for their various projects.

Methods

The process for gram staining is as follows:

1. clean station and gather materials

2.Clean slide with ethanol by applying ethanol to a kimwipe, then wiping the slide

3. Draw a circle with a wax pencil directly onto the slide, number the slide if multiple

4. Heat inoculation loop in flame until glowing then allow to cool To collect sample from beaker, wave beaker over flame to prevent contamination the use loop to collect and recap sample

5. Smear collected sample in the circle, then heat fix by passing the slide over the flame briefly.

6. Bring slide over to a sink, then flood plane with crystal violent and let sit for 1 minute before flushing with DI water.

7. Next flood the plane with Grams iodine and let sit for 1 minute, then flush with DI water, then waterfall with ethanol before flushing with DI water again.

8. fill the plane with safranin, let sit for 50 seconds and then flush with DI water.

9. Press slide dry with bibulous paper and then examine sample with microscope. If the sample is purple, it is gram positive. If it’s pink, it’s gram negative.

The procedure for inoculation is as follows:

1. clean the workbench with ethanol, then obtain plates for streaking.

2. Cure the inoculation loop in flame and then allow to cool before picking up a culture from the old plate and spread onto the new plate, going corner to corner before dragging into the center.

3. Repeat on a second plate in case of contamination.

4. Label the plate, then place into the correct incubator for best possible growth.

Results & Discussion

The results from the growth curve over 48 hours are shown here:


As seen above, the growth curve shows odd peaks and valleys as it progresses. This is because the D. desesrti in the flask clumped up as it grew, likely due to the speed at which the flask was being shaken. In order for a growth curve to actually be accurate we would need to slow down the speed at which the flask was being shaken dramatically in order to avoid clumping but still ensure plenty of cell growth.

Conclusion

Next week we will proceed with desiccation procedures as normal and hopefully be doing RNA extractions by the end of the week.

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