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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.

1. Draw T on bottom of plate.

2. Flame sterilize inoculating loop, let cool 10 seconds

3. Pick up culture with loop.

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

5. Flame sterilize and let cool

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

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

8. Flame sterilize, let cool

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

10. 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 due to condensation

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.

How to do a Gram Stain (continued)

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

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

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

4. Look under microscope

5. Blue stain: Gram positive

6. 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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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)

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How Do We 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

LB Agar

YMA

Potato Dextrose

MacConkey Agar

Sheep’s Blood

Identification

Narrowing down growth allows microbiologists to identity their cultures

Types of Agar
LB Agar

Common

Not very selective

Potato Dextrose

Grows both bacteria and fungi

Yeast Mannitol

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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Chain of Infection
Organism → Reservoir →

Portal of Exit → Transmission → Portal of Entry → Susceptible Host

Spread of Infection

Airborne

Long distance, remain suspended in air up to 6ft - Measles, TB, Chickenpox

Vector-borne

Transfer from host (carrier) → victim - Malaria (mosquitoes)

Common Vehicle

Transfer by contaminated source - Salmonella

Droplet

Close proximity, coughing, sneezing, talking - Pertussis, Flu, TB

Direct Contact

Person/Person, Animal/Person, Pregnancy -     Rabies, HIV, Ebola, Lice, STDs

Virulence Factors

Invade the host

Cause disease

Evade host defenses

What Medication Would We Use?
Antibiotics

Used for a bacterial infection

Common Bacterial Infections: Bronchitis, ear infections, UTIs

Common Antibiotics: Doxycycline, Penicillin, Amoxicillin

Antivirals

Used for a VIRAL infection

Common Viral Infections: Influenza, HIV, Herpes

Common Antivirals: Tamiflu, Retrovir, Zovirax

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Viruses - Structure

Consists of a nucleic acid

DNA or RNA

This is its genetic material

Single or double stranded

Outer layer is the...

Cospid: rigid structure to withstand harsh environments.

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

Viruses - Shape

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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Gram Staining

1. Add crystal violet stain

2. Add Iodine

3. Rinse gently with ethyl alcohol

4. Counterstain with safranin

5. Gram + will be violet/blue (+ Thick peptidoglycan layer)

6. Gram - will be pink/red (- Thin/no peptidoglycan layer)

Identifying Aerobic Bacteria - Catalase Test

Catalase: Enzyme produced by bacteria that uses OXYGEN

A positive catalase test will have bubbles present

A negative catalase test will have no reaction

Identifying Aerobic Bacteria - Thioglycolate 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

6. Aerobic: Love O2

7. Obligate anaerobic: Avoid O2

8. Facultative: Prefer O2, but not hurt by the lack of O2

9. Microaerophiles: Low O2 levels

10. Aerotolerant: They do not care

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Microbial Taxonomy - The Prokaryotes
Bacteria

Unicellular

May possess a flagella for movement

Aerobic (live in O2)

Anaerobic (can’t live in O2)

Facultative (An)aerobe (lives in either environment)

Gram + or Gram - (depends on peptidoglycan in cell wall)

Archaea

Unicellular

Live in extreme environments

Thermophiles (extreme hot temps)

Halophiles (salty environment)

Methanogens (methane-producing)

Psychrophiles (extreme cold)

Microbial Taxonomy - The Eukaryotes
Protozoa

Aerobic eukaryotes

Cellulose in cell wall

Many ways of locomotion (movement)

Cilia (hair-like)

Flagellates (whip-like)

Pseudopodia (fake feet)

Algae

Unicellular or multicellular

Cyanobacteria or blue-green algae

Photosynthetic

Live in damp soil, water, and on rocks

Microbial Taxonomy - Fungi
Fungi

Mushroom, molds, yeast

Cell wall contains chitin

Reproduce by releasing spores

Decomposers

Microbial Taxonomy - Viruses
Viruses

Non-living, non-cellular entities

Consist of:

DNA or RNA (nucleic acid)

Surrounded by a protein coat

Require a host to reproduce

Microbial Taxonomy - The Helminths
Helminths

Not technically microorganisms, but part of their life cycle is microscopic

Flatworms

Lung Fluke Disease (Paragonimiasis)

Roundworms

Ascariasis (infection of small intestine)

Their parasitic tendencies are important for clinical reference

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Review of Cells

Cells are the simplest unit of living matter

Cell theory
1) All living organisms are composed of cells. They may be unicellular or multicellular
2) The cell is the basic unit of life
3) Cells arise from pre-existing cells

Prokaryotes vs. Eukaryotes
Prokaryotes

Simple, no nucleus or membrane-bound organelles

Bacteria and Archaebacteria

Grow and reproduce commonly through asexual reproduction - binary fission

Typically rod, spherical, or corkscrew shape

Eukaryotes

Complex, membrane-bound organelles

Protists, plants, fungi, animals

Grow and reproduce via mitosis or meiosis

Prokaryotic Metabolism
Autotroph

Carbon is obtained directly from CO2

Photoautotroph- Light

Chemoautotroph - inorganic oxidation (of water, minerals, nitrogen gas)

Heterotroph

Carbon is obtained from other things (not directly CO2)

Photoheterotroph - Energy from light

Chemoheterotroph - depend on organisms for carbon

Prokaryotes Reproduction

Genetic Transfer/Bacterial Conjugation

Prokaryotes can grab stray DNA from environment

Plasmid transfer***

This is how prokaryotes get variation!

Antibiotic resistance, new virulence factors, diversity

Binary Fission

Rapid reproduction of 2 identical daughter cells

Eukaryotes Reproduction
Cell Cycle

Mitosis

Somatic/Body cells

Meiosis

Gametes

Egg/Sperm

Variations & Diversity come from mutations

Fermentation

Fermentation: A natural metabolic process in which microorganisms convert carbohydrates into byproducts such as alcohol, Carbon Dioxide, or acid.

It is an anaerobic process (aka - without oxygen)

Can be prokaryotic or eukaryotic cells!

Lactobacillus - breaks down lactose into lactic acid

Saccharomyces (a fermentative yeast) - helpful for bread and alcohol

Penicillium (a fermentative mold) - preserves shelf life of dairy and meat products