Gram Staining and Using a Microscope
Reflection and Discussion Questions:
Virtual Labs:
• Gram Staining (testing yogurt)
• Using a Microscope
Objectives:
1. Students will apply knowledge gained regarding gram staining and using a
microscope.
2. Students will make interpretations from knowledge gained regarding gram
staining and using a microscope.
Introduction:
Studying bacteria is conducted through several methods. Gram staining to study and view
bacteria under the microscope is very common. This Discussion Guide is designed to
help students understand the concepts and applications involved with Gram Staining and
the use of an oil immersion microscope.
Below are two guided discussions to serve as a review of the module and to build upon
some basic concepts and applications that are related to food microbiology and food
safety.
1. Gram Staining Bacteria
2. Using an Oil Immersion Compound Microscope
Gram Staining Bacteria
(review after viewing Gram Staining Yogurt Lab)
Underlying Concept: The use of microbial analysis techniques such as growing
and culturing bacteria from food samples, Gram staining and using a microscope
are critical in investigating foodborne illness outbreaks and monitoring the safety
of the food supply.
1
Why is a gram stain helpful when monitoring bacterial contamination in food or
investigating a foodborne illness outbreak?
a. The Gram stain is usually one of the first tests conducted on a sample when
gathering data to identify the bacteria. The Gram stain classifies the bacteria
into one of two groups (gram + or −). From this reference point, additional
tests are conducted based on characteristics of gram + or − bacteria.
b. The gram stain reveals the shape of the bacterium cell, which is commonly
used to assist in decisions for further testing. More than one type of bacteria
are often visible in an initial sample.
c. Intestinal bacteria that are commonly associated with foodborne illness
outbreaks are gram negative bacteria. Therefore, gram stains are often
conducted on samples taken from foods or stool samples that are associated
with a food borne illness. This may direct the type of further testing and
investigating that should be conducted. Examples of bacteria that are gram
negative and have strains that cause illness include E. coli, Salmonella,
Campylobacter and Shigella.
d. Some gram + bacteria that are implicated in food borne illnesses include
Listeria monocytogenes, Clostridium botulinum, and Staphylococcus aureus.
Using an Oil Immersion Compound Microscope
(use after viewing oil immersion microscope lab)
Guiding Concept: “Micro” refers to the term small and “scope” means to view. Therefore the
origin of the term microscope is used to magnify and focus on very small objects that cannot
be seen with the naked eye. There are many types of microscopes that are used for specific
purposes – this virtual lab involves a compound microscope.
1. Why is this type of a microscope in the virtual lab referred to as a “compound
microscope”?
a. Has more than one lens
2. Do all compound microscopes have three objectives like the one in the virtual lab?
a. No, some may have just two objectives, usually 10X and 40X.
b. If only one lens it is referred to as a “simple microscope”
c. If a 100X objective then referred to as an Oil Immersion”
3. In order to see the bacteria using the microscope a specific type of staining procedure
was used – what was that procedure called, and are the bacteria alive when you are
looking at them?
a. Gram Staining
b. Dead - they are heat fixed and treated with several chemicals, so they are not
living
c. Most commonly used when studying bacteria associated with food
4. Do you think bacteria always have to be Gram stained in order for you to view them?
a. No – there are other methods, such as the Hanging Drop Preparation or wet
mount
b. This method uses a special hanging drop slide (thick glass with a circular
depression in the center - concavity) and a cover slip.
c. This method allows you to study live bacteria.
5. What is the magnification of the “eyepiece”?
a. 10 times
6. If the nosepiece of the microscope is located on the lowest objective (10X) what is
the total magnification of the object (sample) you are viewing through the
microscope? What is the total magnification at the 40X and 100X
a. 100 times
b. at 40X the magnification is 400 times and 100X it is 1000 times larger than it
would appear with the unaided eye.
7. Tell the students “I have a Gram stained slide of a bacteria specimen from a culture I
was growing in the lab. Can you tell me what I need to do to see this as good as
possible with my microscope?” Have a microscope on your demonstration table so
they can talk you through it.
a. Make sure the students start with the lowest objective lens and start with the
coarse focus. The stage needs to be positioned as close to the objective as
possible (if the nosepiece moves on the microscope you are using, then it will
need to be positioned as close to the stage as possible).
b. Bring the image into view with the coarse focus
c. Use the fine focus to make the image as sharp as possible
d. Move to the next objective lens (40x) and use the fine focus to make the
image as sharp as possible.
e. If you have an oil immersion lens – move the lens ½ between the 40X and
100X objectives – place a drop of oil on the lens. Move the 100X into position
and use the fine focus to make the image as sharp as possible.
8. If you can see the bacteria at 10X (100 times) and 40X (400 times) – why do you
need to have a stronger magnification?
a. Better view of their shape, color and other characteristics with greater
resolution and magnification.
9. Why does oil need to be used when using the 100X objective?
a. The oil prevents the light form distorting, which makes the image clearer
10. When done using the microscope how do you properly clean and store the
microscope?
a. Use lens paper to wipe the excess oil from the 100x objective – only use lens
paper that will keep from scratch the lens surface.
b. Microscopes are expensive, so we need to take good care of them.
c. Turn off the light and unplug the microscope and store it in the proper
location. Carry it using one hand supporting the bottom of the scope and other
holding onto the arm.