Micro Lab Midterm Exam

Aseptic technique

Preventing contamination

Culture

Medium that contains living microbes

Neutrophiles

Organisms that prefer pH levels between 5.5 and 8.5

Dichotomous

Divided into two parts

What are the 3 basic stains?

• Methylene blue

• crystal violet

• safranin

Active Movement

Independent movement over greater distances

Brownian Movement

Random collisions between cells and water

Nutritional components

Types of carbon source (citrate) or sugar (glucose, Lactose, maltose, etc)

Christmas Tree Streaking

IT IS NOT AN ACTUAL CHRISTMAS TREE!!!

Inhibitors

High NaCl concentration, bile, salts, selective dyes

Differential Media

Uses biochemical and physiological properties of bacteria to identify them

Key

Consist of a series of choices that lead the user to the correct name of a given item

Flagella

• - Structures used by cells to move in aqueous environments

• - Act like propellers in bacteria

• - Basal body “run” — Hook “tumble” — Filament “run”

Endospores

• - Lack ATP

• - Environmentally stable, dormant

• - Found intracellularly

• - Spore coat is resistant to staining

• - Malachite Green

Inoculation

Transferring microorganisms into or onto a medium

Aerotolerance

The ability or inability to live in the presence of oxygen

Aerobic

Living in the presence of oxygen

Anaerobic

Living without air

What are the 2 types of media to test Aerotolerance?

• - Fluid thioglycolate broth

• - Trypticase soy agar deep stain

Fluid Thioglycolate Broth

Used red dye to show O₂ presence

Trypticase Soy Agar Deep Stab

Uses depth of agar to provide O₂ - free environment

Pure culture

One type of microorganism

Mixed culture

More than one type of microorganism

Magnification

Making an image appear bigger

Resolution

Ability to distinguish two close objects clearly

Contrast

Difference between an object and its background

Immersion oil

Because the glass microscope slide and immersion oil have the same refractive index, the oil keeps the light rays from refracting

USED ONLY IN 100X!!!

Inoculating loop

Transfer microorganisms, especially when working with liquid cultures or streaking plates

Inoculating needle

Transfers microorganisms and it is useful for inoculating agar deeps

Compound light microscope

Uses lenses and visible light to magnify specimens

Total magnification

eyepiece magnification × objective magnification

4x (red) = 40x

10x (yellow) = 100x

40x (blue) = 400x

100x (black) = 1000x

What are the 5 culture methods?

Streak plate

Spread plate

Broth

Agar slant

Agar deep

Streak plate

Separates bacteria to obtain isolated colonies

Spread plate

Spreads a sample across the agar surface

Broth

Liquid growth medium

Parts of the Microscope

Microorganisms

Are free living, they do not reside on or in a specific plant or animal host and are not known to cause diseases.

• - They are nonpathogenic

• - They are saprophytes and perform the important ecosystem role of decomposing organic matter

Ubiquity of Microorganisms

Liquid media

A liquid nutrient solution used to grow microorganisms. It is also called broth and does not contain solidifying agar.

Agar slant

Solid medium in a tube, with a slanted surface

Agar deep tube

Solid medium in a tube, useful for growth patterns below the surface

Agar Plate (solid)

Semi-solid

can be either solid or liquid

What are the 8 Colony Morphology Characteristics?

1. Pigmentation

2. Size

3. Surface

4. Appearance

5. Opacity

6. Shape/Form

7. Elevation

8. Margin

Colony morphology

The visible appearance of bacterial colonies growing on solid agar

Form

Overall shape

Margin

Edge of the colony

Appearance

Elevation

Height or profile

Size

How large it is

Pigmentation

Color

Surface/texture

Smooth, rough, wrinkled, etc

Opacity

Transparent, translucent, or opaque

Obligate aerobe

Requires oxygen, growth occurs at the top

Obligate anaerobe

Oxygen is harmful; growth occurs at the bottom

Facultative anaerobe

Can grow with or without oxygen; growth occurs throughout, usually heavier at the top

Aerotolerant anaerobe

Does not use oxygen for respiration but tolerates it'; growth is fairly uniform

Microaerophile

Requires oxygen at a lower concentration than atmospheric oxygen; growth forms a band below the surface

Why do we stain bacteria?

Most bacteria are nearly colorless, so they are difficult to see clearly under a microscope. Staining increases contrast and helps you observe their shape and arrangement.

Simple stain

• - Uses a positively charged basic dye.

• - Colors the bacterial cells.

• - Examples: methylene blue, crystal violet, safranin.

• - Commonly uses heat fixation.

Hans Christian Gram (1884)

Negative stain

• - Colors the background rather than the cells.

• - Cells appear clear or lightly colored against a dark background.

• - Usually air-dried, not heat-fixed.

• - Helps preserve the natural appearance of cells.

Cocci

Round cells

Bacilli

Rod-shaped cells

Spirilla/spirochetes

Spiral-shaped cells

Diplo-

Pairs

Strepto-

Chains

Staphylo-

Clusters

Tetrads

Groups of four

Streptobacilli

Chains of rods

Monotrichous

Lophotrichous

Amphitrichious

Peritrichous

What are the 6 steps to Heat Fixation?

1. Prepare the smear.

2. Allow it to air-dry.

3. Heat-fix the smear.

4. Apply the stain.

5. Rinse and dry as directed.

6. Observe under the microscope.

Crystal violet

Stains all peptidoglycan cell walls

Gram’s iodine

• - Modorant

• - Fixes crystal violet to cell walls

Ethanol (decolorizer)

Removes the purple stain from Gram-negative cells when used correctly.

Safranin

• - Counterstain

• - Stains unstained cell walls

Phenylethyl Alcohol Agar (gram positive)

inhibits growth of gram negative and some gram positive

Gram-positive

• - PURPLE

• - Thick peptidoglycan layer retains the crystal violet–iodine complex.

Gram-negative

• - PINK

• - Loses the purple complex during decolorization and takes up safranin.

• 
  

Mobility

An organism's ability to move independently.

True motility

Cells move actively in a direction and may change direction.

Brownian motion

Random, jiggling movement caused by collisions with surrounding molecules. It is not true motility.

Capsule stain

Capsule surrounding a cell

Remember: Clear halo around the cell

Flagella stain

Flagella used for movement

Remember: Makes thin flagella visible

Endospore stain

Resistant endospores

Remember: In the common Schaeffer–Fulton method, spores appear green and vegetative cells pink/red

Selective medium

Allows certain organisms to grow while inhibiting others.

Differential medium

Shows visible differences between organisms based on their reactions.

PEA agar

• - Type: Selective.

• - Inhibits many Gram-negative bacteria.

• - Helps select for Gram-positive bacteria.

• - It is not presented as a differential medium in your slides.

Mannitol Salt Agar (gram positive)

• - Type: Selective and differential.

• - High salt selects for salt-tolerant organisms.

• - Mannitol fermentation produces acid, turning the medium yellow.

• - Yellow = mannitol fermentation.

• - Staphylococcus aureus is a classic positive example.

Bile Esculin Agar (gram positive)

• - Bile helps select for bile-tolerant organisms.

• - Tests for esculin hydrolysis.

• - Dark brown or black = positive.

• - Commonly used to help identify Group D streptococci and enterococci.

Blood agar

• - Alpha hemolysis: Partial breakdown; greenish zone.

• - Beta hemolysis: Complete breakdown; clear zone.

• - Gamma hemolysis: No hemolysis; no clearing.

Starch agar

• - Tests whether bacteria hydrolyze starch.

• - Iodine is added after incubation.

• - Clear halo around growth = positive starch hydrolysis.

Staphylococcus epidermidis (Gram-positive)

Gram-positive cocci in clusters; typically mannitol-negative; gamma hemolysis

Staphylococcus aureus (Gram-positive)

Gram-positive cocci in clusters; mannitol fermentation; beta hemolysis

Catalase test

2H₂O₂ —— 2H₂0+O₂

_{Positive: Bubbles appear}

_{Negative: No bubbles appear}

MacConkey Agar

Type: Selective and differential.

• - Bile salts and crystal violet inhibit most Gram-positive bacteria.

• - Differentiates bacteria based on lactose fermentation.

• - Pink/red colonies = lactose-positive.

• - Colorless or pale colonies = lactose-negative.

• - Example: E. coli typically forms pink colonies.

EMB Agar (Eosin Methylene Blue)

Type: Selective and differential.

• - Eosin Y and methylene blue inhibit most Gram-positive bacteria.

• - Differentiates lactose fermenters.

• - Metallic green sheen: Strong acid production from lactose fermentation, classically E. coli.

• - Other lactose fermenters may appear pink or purple.

• - Non-lactose fermenters are usually pale or colorless.

Triple Sugar Iron Agar (TSI)

TSI differentiates bacteria by glucose, lactose, and sucrose fermentation, sulfur reduction, and gas production.