Microbiology 3401-505

Lab Report 4
Exercises: 8,9, 32, 33, 35-38, 40, 41, 43, X
Jane Doe
T.A:

Exercise 8: Quantification of Bacterial Numbers in Milk 3/20/2023 -

3/22/2023
Objective: Students were to discover how the Standard Plate Count method is used to determine
the quality of milk by comparing the number of microbes found in the product.
Procedure: N/A
Activity 1: Quantitive determination of bacterial numbers in pasteurized refrigerated milk
- Our table was only responsible for testing the control (pasteurized refrigerated milk
Results:
As the milk continued to be diluted, it appeared clearer and less cloudy.

Refrigerated milk had the least number of bacteria, followed by unrefrigerated then Kifer milk
with the most bacteria.
Review Questions:
1. Describe how to perform serial dilutions.
A serial dilution is done by taking a small amount of a well-mixed solution (1ml) and
transferring it into a new container (9ml of H2O) to dilute the original solution.
2. How do microbes end up in our environmental samples.
Microbes end up in the samples when in contact with anything unsterilized in the environment
since microbes are ubiquitous, so it's bound for there to be decontamination.
3. How can we spread microbes from spreading?
By continuing to use sterilization and disinfection methods.

Exercise 9: Pour Plate 3/20/2023 - 3/22/2023

Objective: Students should observe how pour plates are used to obtain isolated colonies by
diluting a culture.
Procedure: N/A
Activity 1: Pouring the Plates
- Our table only tested the control, refrigerated milk.
Results:
Class results:
Refrigerated Milk:
Dilution Factor Replica #1 Replica #2 Average # of CFU/ml
10-3 0 6 - 6x10-3
10-4 0 2 - 2x10-4
Unrefrigerated Milk:
Dilution Factor Replica #1 Replica #2 Average (extra) # of CFU/ml
10-6 2 0 29,11,0 2x10-6
10-1 3 1 0, 11, 3 2x10-2
Keifer Milk:
Dilution Factor Replica #1 Replica #2 Average # of CFU/ml
10-6 TMTC - TMTC TMTC
10-7 TMTC - TMTC TMTC
Review Questions:
1. How do you calculate the CFU from a certain dilution?
You can find the dilution factor by taking the number of colonies found and
multiplying it by the positive dilution factor.
2. What is the difference between colonies and CFU
CFU is the number of live and active microorganisms in a specific amount of
food, while colonies are individual colonies of bacteria, yeast or mold growing
together despite the amount of food it's in.
3. What is pour plating? Why is it useful?
 Pour plating is a method of diluting bacterial cells in a mixed solution to count the
number of microorganisms in a mixed sample, which is added to a melted
agar before it solidifies. This is useful for counting viable colonies,
detecting very small amounts of bacterial counts, and does not require previously
solidified agar plates.

Exercise 32: Fungi 3/27/2023 - 3/29/2023

Objective: Students will view the diversity in structure, shape and reproductive methods of
different types of fungus.
Procedures: N/A
- Activity 2 was not performed
Activity 1: Drawing Fungal Structures
The features shown below were set up by the lab T.A
Activity 2: Spore Print
Results:
Activity 1:

Activity 2:
Review Questions:
1. Compare the two reproduction cycles of fungi, anamorphs and teleomorphs.
Teleomorphs have a sexual reproductive stage (morph), with a fruiting body. Anamorphs
have an asexual reproductive stage, more mold and spore like.
2. What are the similarities and differences between the five categories of fungi?
Item zygomycetes ascomycetes basidiomyce hyphomycet chytridiomy
tes es cota
Reproductio Sexually or Sexual and Sexually with Asexual with Motile spores
n method asexually asexual basidiospore conidiospores
spores from
asci sacks
Structure Tall Have four Mushroom/ Stalk and circular
structures toadstool spores
Living cond. Bread and Bread and Outdoor land Land areas Aquatic fungi
fruit molds food molds areas
other Called “sugar Best edible Common to Common Kills
fungi” fungus eat allergens amphibians
other Hallucinogen Morphology Most diverse Oldest form
ic (Salem is very type of fungi
Witch trials) distinct
3. The specimens located on the table had unique characteristics. Mention the common
name of the fungi, what group they belong to, and their unique features.
 - Zygomycetes: (Sugar Fungi) from the Zygomycota group, are known for their tall
filamentous structure.
- Ascomycetes: (Sac Fungi) from the ascomycetes group, are known for having LSD type
symptoms when ingested.
- Basidiomycetes: (Mushrooms) from the Basidiomycota group, are known for their
classic mushroom and toadstool structure.
- Hyphomycetes: (Molds) are from the Deuteromycota group and are best known to cause
most allergens.
- Chytridiomycota: (Chytrids) are from the Chytridiomycota group, commonly known as
the oldest type of fungi.

4. Describe how to do a spore print and prepare a tape slide.

To do a spore print, press the interrupted gills of a fungus onto a clean piece of
paper for 24 hours with a cup covering it until the residue of spores creates a print
on the paper. To make a tape slide take the spores from the spore print, add some
blue dye to the slide, and a cover slide on top.

Exercise 33: Microbial Analysis of Food Products 3/20/2023 -

3/22/2023
Objective: Students will become familiar with the importance of food microbiology and how to
create safe food without being able to completely sterilize it.
Procedure: N/A
- The times under the UV light were changed to 2, 5 and 10 minutes
Activity 1: Microbes in different foods
Activity 2: Microbes in different types of tea
- Raising Canes sweet tea was used in this activity
Results:
Activity 1:
We did not have E. coli in the food item
The spinach had too many bacteria to count
Activity 2:
There were E. Coli traces found in the tea samples.
Review Questions:
1. What is the importance of Food Microbiology?
Food Microbiology is important since it detects and determines the germ content,
minimizing the risks of contamination and preventing outbreaks of foodborne
diseases.
2. Mention the methods used to control microbe growth in food.
Methods to control microbe growth in food are by keeping sanitation requirements up,
pasteurization, keeping continuous high or low temperatures, radiation, filtration, and
desiccation (drying).
3. What do EMB and BGA select for and how?
EMB selects for E. coli with a lactose medium, and BGA selects E. coli and Salmonella
with a lactose and phenol red dye medium depending on what gets fermented and the end
products of that fermentation
4. What are some bacteria commonly found in beef, chicken, pork and fish.
Some bacteria found in beef, chicken and pork are Y. enterocolitica, E. coli, Salmonella,
Campylobacter, C. perfringens, and L. monocytogenes. Some bacteria and parasites
found in fish are V. parahaemolyticus and C. botulinum.
5. How do you perform serial dilutions and plate samples with spread plating techniques?
The spread plate technique involves using a sterile rod (Spreader) with a smooth surface
to apply a small number of bacteria suspended in a solution over a Petri dish so that the
bacteria are easy to count and isolate.
 6. How do you filter tea or water samples?
They are filtered by using a vacuum pump.

Exercise 35: Epidemiology 4/17/2023

Objective: Students will learn the difference between epidemics and pandemics and how
pathogens spread to start an epidemic.
Procedure: N/A
Activity 1: Demonstration of disease Transmission via Handshaking
- I shook the hands of five people, with numbers: 1, 25, 23, and 3
Results:
I ended up having bacteria in the culture yet negative for S. marcescens.
- The infected numbers turned out to be #9 and #7

Review Questions:
1. What is Epidemiology?
Epidemiology is a branch of study which deals with the incidence, distribution, and
possible control of diseases and other factors relating to health.
2. What are the three things it focuses on?
It focuses on the spread, control, and prevention of diseases.
3. What did we do in class to demonstrate this concept?
We shook hands with strangers who had the possibility of spreading S. marcescens.
 4. Were you able to trace back a data set to the person that was the index case? Explain
how.
Yes, since we asked who was infected by the bacteria and we traced back who each
infected shook hands with until it was back to the index case.
5. Briefly describe the terms.
Index case: The first record of a person with a disease that spreads widespread
Pandemic: When a disease is widespread worldwide
Epidemic: When a disease is widespread in an area
Fomite: Inanimate objects which are likely to carry infections, such as clothes, utensils,
and furniture.
Mechanical: Like flies, carriers that can pick up infectious agents on the outside of their
bodies and transmit them through physical contact
Biological: Like mosquitoes and ticks, carriers that may carry pathogens that can
multiply within their bodies and be delivered to new hosts, usually by biting
Carrier: Someone who has a disease, but no symptoms shown yet is able to spread it to
others
6. What are the agencies that focus on epidemiology and what do they do?
- Centers for Disease Control and Prevention (CDC) - control and prevention of
diseases
- Federal Drug and Administration (FDA)- approve safety of food and drugs
- World Health organization (WHO) - control and prevent diseases worldwide

Exercise 36: Bacteriophage Titer 3/20/2023

Objective: Students will learn how viruses infect bacteria and complete their lifecycle.
Procedure: N/A
Activity 1: Preparation of lysate dilutions
Activity 2: Inoculating host bacterial suspensions with the lysate dilutions
Results:
On each plate, the 10-8 had a count of 88 colonies and they 10-7 had 29 colonies.
The PFU for 10-8 is 2.7x10-9
The PFU for 10-7 is 8.8x10-8
Review Questions:
1. What is a bacteriophage?
A bacteriophage is a virus that has the ability to infect bacteria.
2. What are the differences between lytic and lysogenic phages? What are the differences in
their plaques?
In a lytic cycle, the phage replicates and lyses (kills) the host cell. In the lysogenic cycle,
phage DNA is placed into the host genome, where it is passed on to later generations
until activation. Lytic phages produce clear plaques and lysogenic phages produce bulls-
eyed plaques
3. How do you determine phage concentrations using serial dilution? What is the difference
between using these variations in making your dilutions 1/10, 10/90 or .1/.9.9?
Serial dilutions allow us to count the number of bacteriophages in each area by diluting it
to a countable ratio. 1/10 shows a solution with 1/10 of the original concentration, 10/90
is a solution with 1/9 of the original concentration and 0.1/9.9 represents a solution with
1/100 of the original concentration.
4. What is the definition of plaque, lysate, prophage, and titer?
Plaque: Clear area of no growth due to bacteriophage
Lysate: When a bacteria lyses to release new viral particles, its suspension of
bacteriophages
Prophage: The integration phase of when viral and bacterial DNA combine
Titer: A test that measures a number of bacteriophages or other microbes
5. How do you calculate titer?
The titer is obtained by dividing the measured concentration through the nominal
concentration.

Exercise 37: Transformation 4/17/2023 - 4/19/2023

Objective: Students will learn the basic biology of bacterial cells and plasmid DNA goes through
transformation and selects acquired genes.
Procedure: N/A
Activity 1: Make E. coli competent cells (done by T.A)
Activity 2: Transformation of competent cells
Activity 3: Make LB Amp (100 ug/mL) plates
Results:
In the experiment, only the control TSA and the

Review Questions:
1. What is transformation? What are other modes for DNA to enter bacterial cells?
Transformation is the process of inserting new DNA into a bacterial cell that results in a
new genetic trait. Other modes for DNA transfer are transduction and conjugation.
2. What are the steps to a transformation and what does each step mean?
The steps are competent cell preparation, transformation of cells, cell recovery, and cell
plating.
3. What do the positive and negative control tell us?
The positive and negative controls tell us what to expect the results to look like when
given one of these results.
4. What is special about the GFP/AMPr genes on our plasmid?
These genes cause bioluminescence which makes it easier to see if transformation was
successful in the experiment.
5. How do you calculate the transformation efficiency?
The transformation efficiency is calculated by having the Number of transformants/
micrograms of plasmid multiplied by the final volume of recovery/volume plated equal to
the number of transformants/ micrograms of plasmid DNA.
6. How do you make AMP plates?
 They are made by adding 500ul of Ampicillin to each 250ml of TSA and allowing it to
solidify.
7. How do you look at plasmids?
You can look at plasmids under a strong microscope, by seeing if the gene is altered or
looking at a plasmid map.

Exercise 38: Biofilms 3/28/2023 - 3/30/2023

Objective: Students will learn about the steps to create a biofilm and what they consist of.
Procedure: The biofilm observed was prepared by the T.A
Activity 1: Biofilm formation on material set up
Results:
From the biofilm slide, there was mainly algae and other immobile miscellaneous items in the
slide, yet our table were able to observe a live microorganism.

Review Questions:
1. What is a biofilm?
A biofilm is a layer of microorganisms stuck to each other on a surface.
2. What is EPS? What are the components of EPS?
EPS are extracellular polymeric substances the biofilm id composed of. They are made of
DNA, proteins and polysaccharides.
3. Where can biofilms be found? What are the benefits and dangers of biofilms?
Biofilms can be found on a wide variety of surfaces, including living tissues, indwelling
medical devices, water piping, or natural aquatic areas. They are beneficial since they can
be useful in water purification and nutrient cycling. Biofilms can also be harmful due to
causing persistent infections or buildup on mechanical equipment.
4. How does the crystal violet stain work?
The stain works by being able to see the biofilm form with a crystal violet assay by
staining the biomass attached to the surface.
Exercise 41: Immunology 4/20/2023
Objective: Students will learn about basic immunology concepts and how to perform a rapid
strep test.
Procedure: N/A
Activity 1: Positive and Negative Controls
Activity 2: Student Throat Swabs for Streptococcus pyogenes
Results:
Activity 1: The positive control ended up being positive.

Activity 2: My throat swab and the entire tables tests turned out with a negative result

Review Questions:
1. What kit did you use for our rapid strep test and how is it performed? How do you
interpret the results?
The kit used was an Areta rapid strep test. It is performed by swabbing one's throat and
tonsils, adding saliva to a tube, adding the strep test to the tube for 30 seconds and
waiting 10 minutes for the results. If both lines are bold its positive, the top line is bold its
negative, and if the lines are dull or only the bottom line is bolded then the test is false.
2. Define the following terms.
Antibody: A protein created by the body's immune system to help fight infections
Antigen: Something, usually a microorganism, that causes an immune response
GAS: Streptococcus pyogenes Group A, a gram-positive bacterium
 3. What defines group A: Streptococcus pyogenes?
Bio
4. What type of infections does Streptococcus pyogenes cause?
The bacteria cause Strep 90% of the time but can also cause impetigo, pneumonia,
necrotizing fasciitis, and streptococcal toxic shock syndrome.

Exercise 43: Contamination of Common Items 4/18/2023 - 4/20/2023

Objective: Students will observe how microbes are ubiquitous in the environment, even around
our daily routines and items we use.
Procedure: N/A
Activity 1: Swab your sample
- My sample was swabbed in the bathroom sink handles of my apartment
Results:
There were multiple filamentous microbes (fungus) on the TSA plate and one circular colony on
the MAC plate. This shows how my faucet is contaminated with some type of gram-negative
bacteria and fungi.
TSA: 11 colonies
EMB: 0 colonies
MAC: 1 colony
MSA: 0 colonies

Review Questions:
1. What was the purpose of using each of the
following media?
NA: General-purpose, nutrient medium
MSA: Genus Staphylococcus, to determine if the
bacteria are salt loving and if the bacteria can ferment
mannitol.
MAC: Selective and differentiating agar that only
grows gram-negative
EMB: Isolation of gram-negative rods
2. How do you swab a nonliving surface?
 Take a sterile cotton swab, moisten it in saline, and thoroughly swab the surface. Take the
swab and streak the plate on all sides.

Experiment X: Microbial Competition: Natural Products 4/4/2023 -

4/6/2023
Objective: Students will observe how microbial competition between species often leads to the
production of natural products with antimicrobial effects as microbes compete for survival in
harsh environments or to be used as antibiotics and medicine.
Procedure: N/A
Activity 2: Observing competition between bacterial species
Results:
S. marcescens and E. coli had the highest result while B. subtilis had the lowest. This shows how
S. marcescens and E. coli had the most natural products produced to grow the best in the
experiment.

Review Questions:
1. What is a natural product and why is its discovery important?
Natural products are compounds produced “naturally” by any living organism. They are
important since they can be used to treat disease and often have antimicrobial effects and
75% of our current antibiotics were originally isolated as natural products.
2. Where are some places where natural products are produced?
Natural products could be found by “mining” for them by harvesting the natural products
from microbes or other living organisms that create the specific one. Scientists look to
places untouched by man with great biodiversity, such as coral reefs and rainforests or
now even everyday microbes can produce natural products.
3. How does microbial competition lead to the production of natural products?
Natural products would originally be used to fuel microbial competition and to survive
harsh environments.
4. Are all identified natural products safe for human use? Why or why not?
No, since some natural products would be beneficial in some ways yet still toxic in
others.
5. How can genome sequencing help us identify potential natural products?
Genome sequencing can be used by having microorganisms get their genomes screened
for genes encoding for potential natural products and further tested to see if it can be used
for human use.