3.1.

5 Isolation & Gram Staining

Directions: As you go through Lesson 3.1 in CTLS, be sure to follow along and
complete the document. Be thorough, be complete, and be precise. Keep answer font
red or some color other than black or grey. This lesson should take you
approximately 120 minutes to complete. See directions in CTLS for where and how to
submit your work.

PART A: Isolation

How are bacterial species named?

By their genus and species. The first letter of the genus is capitalized and often abbreviated, and the species is written
out. Both names can either be underlined or italicized.

Microbiologist
What they do
They investigate the growth, structure,
development, and other characteristics of
microorganisms. They may also be concerned
with the isolation and identification of bacteria in
a specimen from a patient.
Read about microbiologist, Angela Jones, and her work
at the Mayo Clinic. In her profile, Angela notes that her
greatest challenge is dealing with antibiotic resistant
bacteria in patients at the hospital. Conduct some
research, if necessary, and record how bacteria develop
a resistance to antibiotics.
Antibiotic resistance occurs in bacteria when a few are eliminated
by the antibiotic, but some survive. Those that survive, the
resistant ones, reproduce and take over.

Isolation Notes:
Normal flora is the bacteria that normally lives in everyone. Scientists need pure cultures to accurately study a specific
bacterium. The bacteria that all the patients have in common may be the pathogen that causes them the illness

Reflection Question: Why is it important to use aseptic technique when working with bacteria in the lab?
It’s important to use aseptic technique to keep the area clean, keep the bacteria consistent, and keep yourself safe
from infections

If using a metal re-usable inoculating loop, what is the purpose of flaming it and letting the loop cool?
It’s important to sterilize the inoculating loop so that you clean off the bacteria from one specimen and keep it
separate when you dip the loop for new bacteria, so that your results aren’t mixed up. It is also done for the purpose of
cleaning off bacteria that was already there, like washing clothes you just bought from the store before wearing them.

Describe the process of producing an isolation streak on a petri dish.

You take an inoculation loop, sterilize it, put the patient’s sample over a Bunsen burner for a while, use the inoculation
loop and dip it into the patient’s sample, then use the streak technique; you zigzag the loop through half of the petri
dish, then rotate to zig zag ¼ of it, then finish of by zigzagging another ¼. You continue this method with the other
patients
Reflection Question: If isolated colonies do not grow on a plate, what may have happened?
If the colonies didn’t grow, that means you may have used the wrong technique

For each patient sample, record the following: 

a. How many different species of bacteria appear to be in each patient sample? How do you know?
Two species of bacteria; Patient two shows two greatly different colors

b. Complete the table:

Colony Characteristics
Patient: Color Form Elevation Margin Size (mm)
1 Lime-yellow Circular Raised Entire Large

2 Burgundy Circular Convex Entire Large

3 Lime-yellow Circular Convex Entire Large

c. Is there one organism that is common between the patients? Justify your answer.
Yes, the S. Aureus and S. melophilia share the same yellow color, circular form, convex elevation, and entire
margin.

Based on your observations, what are the possible identities of the organism causing the hospital outbreak?
How do you know?
The S. Aureus and S. melophilia could be causing this outbreak. I know this because they share many characteristics
with all the patient samples.

Reflection Question: Based on this experiment, has your team definitively identified the organism causing
the infection in these patients? If not, what else do you need to know? Is there other testing that needs to be
done?
No; I have two possible organisms with the exact same description. I need to know what differentiates
one bacterium from the one that are affecting my patients so that I can treat my patients properly.

Part A Conclusion Questions:

1. When identifying the agent responsible for causing a disease, why is evaluation of colony morphology
not enough?
It’s not enough due to the large amount of bacterial species and degree of variation between
them.

2. Why is it so important to determine the genus and species of an organism causing disease? What
could happen if the wrong organism is identified as the cause of disease?
It’s important so that you don’t identify the wrong organism and treat the patients ineffectively,
which will only worsen their condition.

PART B: Gram Staining

Complete the table below by listing each bacterial cell morphology and draw or SNIP and paste what they
look like.

Cell Type DRAW what they look like

 Cocci

Bacilli

Spirilla

In the space below, list the different bacterial cell arrangements and DRAW what the arrangements look like.
For Bacilli, there’s individual bacilli and a chain of bacilli. For cocci, there are individual cocci,
diplococci, chain of cocci, tetrad, and staphylococci. For spirilla, there’s individual spirilla.

Draw and label the structures of Gram positive and Gram negative cell walls. Use a purple colored pencil to
draw the Gram positive peptidoglycan layer and use a pink or red pencil to draw the Gram negative
peptidoglycan layer.
Lipopolysaccharide
Gram Negative Outer membrane  Gram Positive

Peptidoglycan
Cell Membrane

What additional feature makes Gram negative more dangerous usually?

The extra layer on the Gram Negative bacteria, LPS, can have toxic effects. It can induce septic
shock.

Record your observations of the prepared slides (use colored pencils in your drawings):
Slide # Cell Cell Gram Drawing
Morphology Arrangement Results
(cocci, bacilli,
spirilla)
1 Cocci Individual cocci Negative
Diplococci
Chain of cocci
Tetrad
And
Staphylococci
2 Bacilli Individual bacilli Positive
 3 Spirilla Individual spirilla Positive

Diagram what happens in each step of the Gram stain.

Record your observations of your patient slide.

Slide Cell Cell Gram SNIP of Image
Morphology Arrangement Results

Cocci Individual cocci Positive

Diplococci
Chain of cocci
Patient Tetrad
And
Staphylococci

a. Record the identity of the organism causing the outbreak.

It was none other than Staphylococcus aureus

b. Conduct research into this organism and record the type of infections typically experienced by those
afflicted by it and its mode of transmission. 
i. How do the infections you read about compare to those the 11 affected hospital patients
experienced?
Staphylococcus aureus is the bacteria that is frequently found in the upper respiratory tract
and on the skin. My patients have been suffering from respiratory complications.

ii. Does the mode of transmission you read about fit the hypothesis your team developed with
regard to how this agent is spreading? 
Since my hypothesis about the spreading of the bacteria was through the Saline IV, I was
wrong because Staphylococcus aureus spreads through direct contact with an infected person by
using a contaminated objects, or by inhaling infected droplets from sneezes or coughs.

c. Revisit the treatment recommendation you previously made for the patients. Do you need to update
your recommendation? If so, how?
 Since my treatment was for the patients to wear masks, clean hands, socially distance, and
quarantine, the treatment should be the same. Anti-biotics are also useful to stop the spread
of the bacteria.

d. Compose a message to the staff at GNMH. You need to inform them of your conclusions: the cause
of the infections, the recommended treatment, and the steps to prevent further spread. Keep your
communication short and tactful.
Dear staff at GNMH,

I concluded that the infection is caused by a bacteria Staphylococcus aureus. I

recommend that you treat your patients with anti-biotics. And to prevent further spread, the
patients should thoroughly wash their hands, keep cuts and scrapes covered with bandages
until they heal, avoid contact with other people’s wounds, and to not share personal items.

Best Regards,
Isabel McLeod

Conclusion Questions:

1. List general measures GNMH could take to reduce the chance of nosocomial infections of any type.
Reflect on all the information you have learned in previous activities as well as the information you
have accessed through the Hospital Hub app.
Some general prevention methods include washing hands thoroughly, vaccinating, maintain
sanitary conditions, proper personal hygiene, covering coughs and sneezes, and keeping
hands away from your face.

2. If you and your teammates saw different Gram stain results, what might this mean? Why might this
have occurred? (Even if it didn’t happen to you – what would it have meant?)
If the results were negative, it means that the sample is not a bacteria; this may have
happened because my team didn’t use the proper technique to spread the sample on the petri
dish.