Microbiology Day 11 - Microbiology Day 11 We need to know...

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Microbiology Day 11 We need to know what we want to get to. So if we used 10^9 in the original culture. We want to get to 200 cells on plate. We have to plate about .1 or .2 uL. In reality, you never plate 1 mL since if you had to use that, you’d need to dilute agar, and centrifuge it out. If you keep diluting agar, it will break up easily and plates stay wet. If you put 1 mL on, they’ll never dry out. So if you wait 30 min, and turn it around, it would drip and ruin the experiment. So in this case, if we want 10^2 plated, we need some number times 10^3. So if you want to 200 cells, you need to plate 2000 cells. It might be hard to do it in your mind, it’s hard. If I had 200, but don’t know concentration, instead of diluting, you go backwards, so multiply by 10, by 100 and keep doing it until you get your number. Difficulty is devising the original serial dilution Haemocytometer needs to be an even dilution and if it isn’t we can use the numbers. We have different squares and different numbers Slide 6 The cover slip is designed so that theres a .1mm depth. So you square the volume of a p- square, which is 1 mm x 1 mm x 0.1 mm (it will tell you how big p-square is). Then you put in bacteria in a broth, and through capillary action, it brings the bacteria up. SO if you don’t flood it properly, you run into problems. This is how they use p-square, and the little square would be an s-square. In this case, you cut number in p-square since it’d be too big. You have to determine (on your own), how to calculate or count the cells on a grid. Sometimes, they give you rule, but there is no rule Slide 7 So which square, would cells on the line count? So we can say that they would count in the upper square (if counting p-square). This determination is subjective. The point is not to count it twice. It looks evenly distributed and in counting, you count several of whatever squares you use in different regions. If the cells counts are different per square significantly, then you have an issue. A significant difference would vary, but a range from 180-220 is ok. If you got back to original haemocytometer, then you’d count as far away as possible to make sure you have an even distribution of cells/mL Slide 8 So when doing count of p-square, if the volume is in mm squared, is cc (cubic centimeter). If we look at s-sqaure, there’s 25 squares, then that’s 5 x 5, it would be 0.2 x 0.1 (always the same). If you were doing a fraction Slide 9
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If we didn’t dilute, it would be too many cells to count. Then when we get the number, we multiply it by how much we dilute it. Generally, we use s-sqaure for bacteria. Slide 11 We have realistic picture. There’s a lot that follow the lines, we again need to make the same rule and if a cell touches the lines at all, then we follow it.
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