Quantification of Bacterial Colonies on an Agar

Plate

Report made by:

Radhika Jayant Nibandhe (1803020)
Pranali Prasad Suryawanshi (1803023)
Tejashree Kondiba Dabade (1803027)
Shwetali Anandrao Patil (1803029)

Objective:
To understand the growth of a bacterial samples on an agar plate using image
analysis software.

Theory:
Bacteria are a group of microscopic prokaryotic organisms belonging to the
kingdom Monera. They grow in large numbers everywhere and live in almost
every environment on the surface of earth. Most of the bacteria reproduce by
binary fission, which is an asexual mode of reproduction. The growth of
bacteria can be defined as the gradual increase in the quantity of cell
components, as well as the number of bacterial cells.

In laboratories bacterial cells are cultured in a predetermined media under

controlled conditions. Microbial cultures are used for the determination of
unknown microorganisms and their abundance in the sample. It is one of the
primary diagnostic methods in microbiology for the identification of causative
agents of infectious disease.
Bacteria and other microorganisms grow in specially prepared Petri plates
containing nutrient rich media. Solid medium used for the cultivation of bacteria
cells is known as bacterial agar. They form colonies in the agar which can be
recovered for further work or analysis.

The qualitative analysis of the bacterial culture can reveal many characteristics
of cell culture. It is necessary to quantify those characteristics of culture media.
There are several methods for quantitative enumeration of bacterial colonies.
Quantitative analysis of these samples can be done by obtaining the snapshots
of the Petri dishes at different time points and analyzing the images through
image analysis software like ImageJ.

ImageJ is a Java based image analysis software by the National Institute of

Health (NIH, USA). If bacterial cells were cultured on an agar plate, the number
of cells would increase with time.

Procedure:

Step 1: Follow the instructions from simulation tab to download and install
ImageJ software.

Step 2: ImageJ will be open in a new window as shown below.

Step 3: To analyse an Image into ImageJ, we must import the image into
ImageJ. To perform that, Go to files -> Open. A popup window will appear. Use
Choose button to choose your respective images into ImageJ software.
Step 4: The imported picture will show in a new window.

Step 5: Choose the line symbol from the shape buttons and draw a line that was
placed in the image for scaling.
Step 6: Go to Analyse ->set scale and set the known value to the box and click
“OK”
Step 7: The image will be in the RGB format. Convert the RGB image into a
gray-scale image: Image -> Type -> 8-bit.
Step 8: To invert the image, Go to Edit ---> invert. This step is not necessary if
the bacteria are already darker than the agar.
Step 9: Click on Image -> Adjust -> Threshold. The "thresholding" of the
image is done by setting all pixels above certain intensity value to black and
leaving background as white. This is called image segmentation.

Step 10: Adjust the threshold by using the sliders to highlight only the bacteria.
Step 11: Select the circle image from the tool bar. Draw a circle around the
colonies where the imageJ will measure from.

Step 12: To count the objects, Open: Analyze -> Analyze particles.
Step 13: Adjust the size: “0- infinity”, show: “Outlines” and tick on all boxes
except “Record Starts”. Click "OK"
Result interpretation:
 
The number of colonies present per selected area is 4. The area covered by
colonies are 1487 which is 14.431% of the total area. From these 4 colonies,
forth colony is large and third colony is small compared to others.

Conclusion:

Track plating can be used in quantitative assessment of the faecal microbiota

with an accuracy comparable to spread plating. It makes quantitative assessment
of the faecal microbiota in multiple samples feasible by greatly reducing the
time and number of agar plates required, and can be performed using
inexpensive standard laboratory equipment.