Lab 5:  Bacterial Enumeration

Introduction

In this laboratory, you will learn two different methods for determining the concentration of bacteria in a sample.  The concentration of bacteria in your sample is reported in units of bacteria/ml.  This fact will be important for the calculations that you will need to do in this laboratory.

You will need to know a few terms to describe the different methods that you will be performing.  I have defined these words below:

Viable counts – counting cells that can be cultured and/or are metabolically active.

Total counts – involves counting cells that are alive, dead or metabolically inactive.

Direct methods – involve counting actual cells or colonies.

Indirect methods – involve estimating the number of cells based on certain properties.  In this laboratory, you will estimate the number of cells based on the scattering of light through a culture (spectroscopy).

In this laboratory, you will count bacteria using a direct and viable method and will compare the results that you obtain to an indirect and total method that you will also perform. 

The direct and viable method is also known as the standard plate count method.  In this method, you will make various dilutions of your bacteria in an appropriate media.  You will then spread the bacteria on top of the agar in a petri dish.  After incubating your plates at 37°C overnight, you will count the number of colonies that grew on the plate.  Based on the number of colonies that grew and on the dilution that you previously performed, you will be able to calculate the number of colonies in your original culture.

You will need to make a number of dilutions to do this experiment.  After you incubate your plate, you do not want too many colonies but you do not want too few either.  The picture below, shows you plates where serial dilutions have been performed.

In the standard plate method, you want to end up with plates that have between 30 and 300 colonies in order to get the most accurate reading.  The plate on the right, with 87 colonies, would be a good plate to count the number of bacteria on.  It is important to realize that each colony that grew originated from a single cell.

In the document called sample calculations, you will learn how to do the dilutions and calculate the number of bacteria in your initial sample.  This will be very important to learn because IT WILL be on the lab practical.

In the indirect and total method, you will also perform dilutions but instead of putting the cultures on a plate and determining how many colonies grew, you will now put you sample into a spectrophotometer and determine the transmittance.  This will give you an estimate of the number of cells that you have.

It is important to understand that the more cells that you have in a culture, then the more turbid the solution will be.  This means that your culture will be more cloudy than a culture that has less cells.  When the turbidity increases, the OD or the optical density also increases.  In this laboratory, you will use a Spec 20 to determine the OD of your culture.  However, you will be measuring the %transmittance of your sample.  This value measures the amount of light that can pass through.  If you have more cells, then less light will pass through because it will be scattered by the cells.

The OD and the % transmission are inversely proportional to each other.

As OD increases, then % transmittance decreases.

As OD decreases, then % transmittance increases.

After you obtain the % transmittance of your samples, you will need to determine the OD of your sample.  You will need the following formula:

OD = 2-log%T

After you determine the OD, you will then multiply this number by 1.25 X 10⁶ cells/ml to determine the concentration of cells.