Lab 9: Standard Plate Count
The colonies on the plate can then be counted and the concentration of bacteria in the original culture can be calculated. In the pour plate method a diluted bacterial sample is mixed with melted agar and then that mixture is poured into a petri dish. Again the colonies would be counted and the viable cell count calculated. The primary trick in counting colonies is to count each colony dot once. One approach is to set the Petri dish on a grid background and count the colonies in each grid cell, moving in a methodical pattern through all of the cells. Marking counted colonies on the back of the Petri dish can also be a .
We use serial dilutions to create decreasing concentrations of the original sample that are then plated so that a plate will be created with a low enough number of bacteria that we can count individual colonies.
From that number, we can calculate the original cell density in the broth. A turbid broth tube contains millions of bacteria. If we transferred directly numbrr that bcateria, we would ot a plate that had a lawn of bacteria. This plate would be uncountable and we could not use it to estimate bacterial cell numbers.
Therefore, we need to dilute our original sample before plating. Therefore, we do a dilution series to create a number of plates, with a range of dilutions, in the hope of producing a countable plate. We then divide this by how much we diluted the original sample to get the estimated concentration of bacteria in the original tube:.
Fill in the chart below before proceeding you do not need to turn this in. Using the magic of fire to bend a glass Pasteur pipette into a spreader. Place it in your beaker of alcohol. Flame your glass spreader, wait for ALL the alcohol to evaporate and repeat this 3 times.
Turn the plate at least 5 times as you move the nummber spreader to distribute the bacteria over the bacterai. At this point, the liquid has been absorbed by the plate. How do we know how many bacteria are in a liquid? Why do we do a dilution series? Exercise Fill what makes a function a function the chart below before proceeding you do not need to turn this in.
Complete t Following Lab Individually: 1. Obtain 7 microcentrifuge tubes and label them Obtain 4 TSA plates and label them. Place the glass spreader back into your beaker of alcohol. Volume Pipetted: Total Volume Ratio. Sample Volume: Total Volume Ratio.
Individual Dilution Factor. Total Dilution Factor.
Mar 27, · Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to liquefied agar. number of colonies (CFUs) = # of bacteria/ ml dilution X amount plated. Jun 25, · The number of colonies we count on a plate gives us the CFU or colony forming units, when we divide the CFU, by the volume we plated we get CFU/volume a CFU/mL. For example, if we counted 32 bacteria on a plate that had received ?l of . Thus each colony is a clone of cells. We can now determine the number of live bacteria (or Colony Forming Units [CFU]) per ml of original culture be using the formula: CFU/ml = number of colonies per ml plated Total dilution factor As plate E has colonies, in the original culture: The CFU/ml = colonies/ml plated = x = x
Many studies require the quantitative determination of bacterial populations. The two most widely used methods for determining bacterial numbers are the standard , or viable , plate count method and spectrophotometric turbidimetric analysis. Although the two methods are somewhat similar in the results they yield, there are distinct differences. For example, the standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria.
The spectrophotometric analysis is based on turbidity and indirectly measures all bacteria cell biomass , dead and alive. The standard plate count method consists of diluting a sample with sterile saline or phosphate buffer diluent until the bacteria are dilute enough to count accurately. That is, the final plates in the series should have between 30 and colonies. Fewer than 30 colonies are not acceptable for statistical reasons too few may not be representative of the sample , and more than colonies on a plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units CFUs.
The assumption is that each viable bacterial cell is separate from all others and will develop into a single discrete colony CFU.
Thus, the number of colonies should give the number of bacteria that can grow under the incubation conditions employed.
A wide series of dilutions e. Greater accuracy is achieved by plating duplicates or triplicates of each dilution, although we will not be doing that in this exercise. Increased turbidity in a culture is another index of bacterial growth and cell numbers biomass. By using a spectrophotometer, the amount of transmitted light decreases as the cell population increases. The transmitted light is converted to electrical energy, and this is indicated on a galvanometer.
The reading, called absorbance or optical density , indirectly reflects the number of bacteria. This method is faster than the standard plate count but is limited because sensitivity is restricted to bacterial suspensions of 10 7 cells or greater. The procedure for the spectrophotometer use is at the end of this exercise. Why Is E. When working with large numbers and a short time frame, one of the most reliable microorganisms is one that has been used in previous experiments, namely, Escherichia coli.
Thus, it reproduces very rapidly and is easy to quantify i. Your agar deeps should already be liquefied and sitting in a water bath. If not, you will have to boil them. When the deeps are liquefied, they can be placed into the water bath to cool so that the pours can be made without killing the bacteria. Coolingtakes about 20 minutes.
This section does not have to be done aseptically! Your data will appear already plotted on an x-y axis system. Now you will learn how to set-up the axes, write the title of the graph and write the legend. You will also command the computer to draw best-fit lines and calculate the slope of each plot. You can also choose to make some cosmetic touches changing the size of fonts, colors of the plot etc.
Click on space below Value X Axis and type: Absorbance. Your graph will appear on the screen as shown below. You can alter its size by clicking the mouse button on the black squares around the graph and dragging the mouse with the mouse button pressed down. As you do this, information about this data point will appear on the screen. A box with choices appears. Another box will appear. Select Linear should already be highlighted. The graph will show a best-fit line.
On the graph you should see an equation which gives you the value of the slope for the line. Learning Objectives Correlate absorbance value for a bacterial suspension with an accurate bacterial count. Become proficient at dilutions. Become proficient at performing a standard plate count and determining bacterial counts in a sample. Calculate the number of bacteria in the original tube of E. Here is an example of a graph. If you have a graph for E. How is transmission different from absorbance?
Give the formula for determining bacterial counts. Give the bacterial count per milliliter of E. Double click on the Logger Pro icon on the computer desktop.
When you open the Logger Pro 3. Insert the cuvette in the sample chamber, making sure that the clear side of the cuvette is in the path of the light. On the top menu, click on Experiment. Scroll down to Calibrate and select Spectrometer 1. Wait 60 seconds for the lamp to warm up. Click on Finish Calibration and then OK. The spectrophotometer is now calibrated.
Select Absorbance vs. Concentration as the Collection Mode. Below the concentration menu, select Individual Wavelengths. Under the column showing a list of wavelengths, click on Clear Selections and the select the wavelength closest to nm as the desired wavelength.
A graph will appear with concentration on the X-axis and absorbance at on the Y-axis. Wait until a red dot appears on the screen and click on Keep on the top bar. A menu appears, asking for the concentration of the sample. Use 1 for this dilution. Click Stop on the top bar menu. Numbers data Open the Microsoft Excel program.
You will see a grid. Each box in the grid is called a cell. Double click on cell A1 to start entering information. Using the diagram on this next page as a reference, enter the data of bacterial numbers and absorbance readings of your table. Click on cell A2 with the left mouse button LMB and drag the mouse to highlight the area containing all the data points go down to cell B8.
The screen should look like this:. A box with graph options will appear. Leave the chart sub-type alone: the top chart should be highlighted. Another box will appear with options to modify titles on the graph.
Click on space below Value X Axis and type: Absorbance c. Now you are going to add the line that goes through the data points trendline. Click on the graph to make sure that the black squares around the graph are there. Cosmetic changes for graphs. You can change font size, color, etc. However, I will not get into those directions here.
Go to the top toolbar. Before proceeding any further, you want to be sure that all your work is saved.