What is "titering"?
Titering is the laboratory measurement of a concentration of a substance.
In this course, titering will be done to measure the concentration (or density) of microbes. The first problem lies, as many of you may already have guessed, in the fact that microbes such as the bacterium Escherichia coli are rather small. It is quite impractical to count all of them in a flask or tube, so we must be a little craftier.
Although an individual bacterium can't be counted by the naked eye, a colony of bacteria can. For instance, if a single E. coli cell landed on a suitable solid growth medium, it would divide through enough generations to form a visible colony. Therefore, by counting colonies, one can measure the amount of individual bacteria present in a sample (1 cell --> 1 colony).
A second problem is that the concentration of microbes we will be using will be quite high. For instance, a grown culture of bacteria can easily have 100,000,000 cells per mL of fluid. If we were to simply spread such a bacterial culture on a Petri dish with solid growth medium, it would form an uncountable (and smelly) lawn. Therefore we dilute our samples before plating.
For this and the other labs, we will be using 96 well microtiter (MT) plates for performing dilutions. Each well in the MT plate is filled with 270 µL of saline. Saline (instead of say, water) is used so that the cells are not osmotically stressed in the course of a dilution. In order to achieve a ten-fold dilution, 30µL of the fluid to be sampled will be drawn (aspirated) into a pipette tip and dispensed into a well (30µL into 270µL is a 1/10 dilution). This process is repeated several times; 30µL from the previous well into a fresh well.
After the sample has been sufficiently diluted, it needs to be plated (introduced into a Petri dish where colonies can later be counted). To plate bacteria, sterile glass beads (5-8 or thereabouts) will be placed in Petri dish directly on top of the solid growth medium. Then you will aspirate 100µL from the well of the appropriate dilution and dispense it onto the Petri dish. Shaking the Petri dish (so that the beads roll across its surface) ensures an even spreading of the sample. After shaking, the beads are removed and placed in a beaker containing ethanol. After being placed in a 37°C incubator overnight, the Petri dishes will sport white, circular colonies.
Remember to look over the Example Math Problems before class and make sure you understand how to solve them.
In this lab, you will receive a tube containing a culture of Escherichia coli, grown up the night before the lab. You will titer the sample to determine the density of bacterial cells. You will also do a "thumbprint exercise" to demonstrate the importance of sterility when working with microorganisms.
Lab Logistics:
This lab will take only one lab session. (You will count the colonies on your Petri dishes the following week.)
You will each titer a sample of bacteria.
Background:
Microorganisms are everywhere! Good sterile technique is the first and most important step in ensuring consistent results when working with microbes. Wear gloves not only to protect yourself but also to prevent contaminating cultures.
Lab Overview:
In this lab you will place your thumb on a Petri dish with your hands unwashed, washed, wearing gloves, and after using ethanol on your gloves.
Materials:
Protocol: