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