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*Cells are the simplest unit of living matter
*All living organisms are composed of cells and can be unicellular or multicellular
*Cells are a basic unit of life
*Cells arise from preexisting cells
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Prokaryotes
Simple, no nucleus or membrane-bound organelles
Bacteria and archaebacteria
Grow and reproduce through asexual reproduction and binary fission
Typically rod and spherical and corkscrew shaped
Eukaryotes
Complex, Membrane-Bound organelles
Protists, Fungi, plants, animals.
Grow and reproduce via Mitosis and meiosis
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Autotroph
Carbon directly obtained via CO2
Photoautotroph - light
Chemoautotroph - inorganic oxiditation (of water, minerals, gas)
Heterotroph
Carbon is obtained from other sources that aren't direct CO2
Photoheterotroph - energy from light
Chemoheterotroph - depend on organisms for carbon
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Prokaryotes Reproduction
Genetic Transfer/ Bacterial Conjugation
-Prokaryotes can grab stray DNA from the environment
-Plasmid Transfer
This is how prokaryotes get variation
-Antibiotic, New virulence factors, diversity
Binary Fission
-Rapid reproduction of 2 identical daughter cells

Eukaryote Reproduction
Cell Cycle
*Mitosis
- Somatic cells
*Meiosis
- Gametes
-Egg/sperm
-Variations/diversity come from mutation
-Mutations that happen in the gametes are passed down to the offspring.
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Fermentation: A natural metabolic process that is anaerobic and that which converts carbs into byproducts such as alcohol, carbon dioxide, or acid.
- can be prokaryotic or eukaryotic cells
-Lactobaccilus - breaks down lactose into lactic acid

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Microbial Taxonomy
Prokaryotes
*Bacteria
-Unicellular
-may possess a flagella or movement
-Aerobic(able to live in O2)
-Anaerobic(Unable to live in O2)
-Facultative (an)aerobe (lives in either environment)
- Gram- and gram+ - (depends on peptidoglycan in cell wall)
*Archaea
-Unicellular
-Living in Extreme enviroments
-Thermophiles(lives in extreme temps)
-Halophiles(Salty environments)
-Methanogens(methane-producing)
-Psychrophiles(Extreme-cold)
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Eukaryotes
*Protozoa
-Aerobic Eukaryotes
-Cellulose in the cell wall
-Many ways of locomotion
-Cilia(hair-like)
-Flagellates(whip-like)
-Pseudopedia(fake feet)
*Algae
-Unicellular or multicellular
-Cyanobacteria or blue-green algae
- Photosynthetic
-Live in damp soil, water, and on rocks
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Fungi
-Mushroom, molds, yeast
-Cell wall contains chitin
-Reproduce by releasing spores
-Decomposers
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Viruses
-Non-living, non-cellular entities
-Consists of DNA, RNA(genetic material)
-Protein Coat
-Requires a host to reproduce
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Helminths
-Not technically microorganisms, but part of their life cycle is microscopic
-Flatworms
-Lung fluke disease(Paragonimiasis)
-Roundworms
-Their parasitic tendencies are important for clinical reference

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Structure

Consists of a nucleic acid

DNA or RNA

This is its genetic material

Single or double stranded

Outer layer is the...

Capsid: rigid structure to withstand harsh environments.

Envelope: can only survive in aqueous solutions (usually transmitted via fluids)

Shapes

Complex,

Bacteriophage: A virus that infects bacteria

Helical/Filamentous

Tobacco mosaic virus, Ebola

Spherical

COVID-19, Influenza, Hepatitis B and C

Enveloped

Polyhedral

Poliovirus, adenovirus, rhinovirus

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*Bacilli
rod-shaped

Important Examples:
Streptobacillus,

Bacillus
*Cocci
round, spherical

Important Examples:

Staphylococci,

Streptococci

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Bacteria Tests - Gram Staining & Catalase Test

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Bacteria Tests - Gram Staining & Catalase Test

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Gram +: Thick peptidoglycan layer
Gram -: Thin/no peptidoglycan layer
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*Gram Staining
- Add Crystal Violet stain
- Add Iodine
- Rinse gently with ethyl alcohol
- Counterstain with safranin
- Gram - will be pink/red
- Gram + will be violet/blue

Catalase: Enzyme produced by bacteria that uses OXYGEN

Catalase Test
- A positive catalase test will have bubbles present
- A negative catalase test will present no bubbles

Thioglycollate Test

1. Obligate Aerobic: Love O2
2. Obligate Anaerobic: Avoid O2
3. Facultative: Prefer O2, but not hurt by the lack of O2
4. Microaerophiles: Low O2 levels
5. Aerotolerant: They do not care

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*Chain of Infection
- Organism → Reservoir → Portal of Exit → Transmission → Portal of Entry → Susceptible Host

*Spread of Infection

1. Airborne
Long distance, remain suspended in air up to 6ft - Measles, TB, Chickenpox
2. Vector-Borne
Transfer from carrier (carrier) → victim - Malaria (mosquitoes)
3. Common Vehicle
Transfer by contaminated source - ex: Salmonella
4.Droplet
Close proximity, coughing, sneezing, talking - Pertussis, Flu, TB
5. Direct Contact
Person/Person, Animal/Person, Pregnancy - Rabies, HIV, Ebola, Lice, STDs

Virulence Factors

Invade the Host

Cause Disease

Evade Host defenses

Endotoxins

On Gram Negative bacteria - released when cell dies

Exotoxins(Live cells)

Secreted - neurotoxins, cytotoxins, enterotoxins

Siderophores

Iron binding factor - competes host for iron
Hemoglobin uses iron to carry O2

Virulence Factors

Antibiotics

Used for a Bacterial infection.

Common Bacterial Infections: Bronchitis, ear infections, UTIs.

Common Antibiotics: Doxycycline, Penicillin, Amoxicillin

Antiviral

Used for a Viral infection

Common Viral Infections:
Influenza, HIV, Herpes

Common Antivirals:
Tamiflu, Retrovir, Zovirax

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Preparing the Petri Dish

Always read directions on how to prepare the agar!

On the petri dish, write this on the agar side:

Your Name

Date

Time

Sample (Example: Ms. Fusco’s door handle)

DO NOT WRITE ON THE LID

Plating Techniques

METHOD 1: Triple-Z - This should give you isolated colonies.

Draw T on bottom of plate.

Flame sterilize inoculating loop, let cool 10 seconds

Pick up culture with loop.

Streak like a z-method back and forth in quadrant 1. DO NOT CROSS your T.

Flame sterilize and let cool

Drag inoculating loop through the end of quadrant 1 into quadrant 2.

Keep streaking in quadrant 2 with a z pattern. DO NOT CROSS ANY T again.

Flame sterilize, let cool

Drag inoculating loop through the end of quadrant 2 into quadrant 3.

Keep streaking in quadrant 3 with a z pattern. DO NOT CROSS the T again.

Method 3: 4-Quadrant Streak

1. Sterilize loop, let cool for 10 seconds

2. Pick up desired colony and spread horizontally in Quadrant 1.

3. Sterilize loop, let cool for 10 seconds.

4. Overlap Quadrant 1 and spread horizontally in Quadrant 2.

5. Repeat steps 3&4 for Quadrants 3 and 4.

Incubation

1. Wrap parafilm around the edges of the petri dish.

2. Place dish into incubator (can stack dishes to save time).

3. Make sure plates are turned upside down!

Why??? It doesn’t allow for condensation to build up.

Plating Viruses

Can be cultured, but it looks a little different

Viruses can be grown in vivo (inside a whole living organism) or in vitro (in a test tube, agar plate, cell culture flask)

On agar plates: Bacteriophages need a bacterial lawn first! They will not grow without any cells to infect.

How to do a Gram Stain

1. Add one drop of distilled water onto a slide.

2. Put a loopful of the desired bacteria colony into that drop (you do not need the entire colony - too much will crowd your stain).

3. Fix culture onto slide using a flame (wave it over flame, never HOLD it over).

4. Add a drop of crystal violet onto sample and let sit for 20 seconds.

5. GENTLY rinse slide with distilled water or you’ll lose your sample.

6. Add a drop of iodine and let sit for 20 seconds.

7. GENTLY rinse slide with distilled water or you’ll lose your sample.

8. Add a few drops of decolorizer (acetone/ethanol) and let it run off the slide. Rinse with water after 5 seconds.

9. Counterstain with fuchsin for 40-60 seconds then rinse off with water.

10. Gently shake of excess water and let air dry.

11. Look under microscope

12. Blue stain: Gram positive

13. Red stain: Gram negative

How to do a Catalase Test

1. Put a sample colony onto the slide using an inoculating loop.

2. Add one drop of hydrogen peroxide and look for bubbles.

3. Bubbles = catalase positive (catalase is present).

4. No bubbles = catalase negative (no catalase is present).

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How does one grow Bacteria?
"

1. First, we need to make sure we practice aseptic technique.

2. Second, we need a media to grow them on.

3. Third, we’ll need to wipe surfaces with a sterile applicator (looks like a really long Q-Tip or cotton swab).

4. Fourth, we would inoculate the agar plate using a spreading technique.

5. Fifth, wrap plates in parafilm and incubate 48-72 hours!"

What's a Media

Nutrients for Microbes

Different media can have different nutrients that grow specific bacteria

Different Kinds of Medias

LB Agar

YMA

Potato Dextrose

MacConkey Agar

Sheep’s Blood

Identification of Media

Narrowing down growth allows microbiologists to identify their cultures

Types of Agar

LB Agar

Common not very selective

Potato Dextrose

Grows both bacteria and fungi

Yeast-Mennitol

used for soil microbiology

Sheep's Blood

Determines type of hemolysis (breaking down blood)

MacConkey

Selects for Gram (-) bacteria and lactose fermentation

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Hemocytometer

Used to count number of cells in a sample under the microscope

Trypan Blue: A dye that exclusively labels dead cells to differentiate them from live cells

Cells that are white (not filled in with blue) are LIVE cells!

Solid blue cells are dead cells.

Equation for Total Viable Cells:

(Average Viable Cells) x 10,000 x DF

(DF = Dilution Factor)