Micro Lab Exam 1

Canvas Form Requirement: A Lab Make-up Request Form

must be completed on Canvas within 48 hours of the missed absence

Make-up Arrangements

Once Lacy receives the request, she will email you to coordinate a physical makeup lab or issue a written makeup assignment.

Email Protocol:

Do not send a separate email to Lacy alongside your form submission. Only follow up if it has been one full WORKING day without a response

Advance Notice Requirement:

The Canvas Lab Make-up Request Form must be submitted at least 72 hours in advance of the lab to be missed. Failure to do so results in an unexcused absence.

Gloves go in what

biohazard bin

Glass Microscope Slides go in what

glass disposal box

Glass tubes go in what

Biohazard bin

Pipette tips go in what

biohazard waste bag

Paper towels go in what

regular trash can

Ubiquity of Microorganisms

Microorganisms are "ubiquitous in nature," meaning they are found everywhere other lifeforms exist. They have a rich history on Earth and have adapted to diverse environments.

Isolation Sources

Soil (all types), water (spanning wide salinities), plants, animals, and humans.

Extreme Habitats:

Can be found in seemingly uninhabitable locations, such as the hot acid pools in Yellowstone National Park (roughly 85°C with a pH near 1).

Caveat:

Ubiquity applies to microorganisms as a collective group, not to every individual species.

saprophytes

acting as essential decomposers in ecosystems

Pathogens:

Microbes that cause damage or disease to their host

Mutualism:

A relationship where both the host and the microbe benefit

Commensals

Microbes that benefit from the host without affecting it.

Opportunistic Pathogens:

Many commensal or mutualistic strains living in/on our bodies can cause a disease state if introduced into a suitable, vulnerable area

Reservoir

Any site or area where a microbe resides that harbors the potential to cause an infection.

Blood Agar (Theory) Purpose

A differential medium used to distinguish bacteria based on their hemolytic characteristics.

Blood Agar (Theory) Composition

Composed of 5% sheep blood inside a Tryptic Soy Agar (TSA) base.

Blood Agar (Theory) Mechanism

Certain Gram-positive cocci produce exotoxins called hemolysins (Streptococci produce streptolysins) that destroy red blood cells (RBCs) and hemoglobin.

beta-hemolysis (Beta)

Complete destruction of RBCs, producing a clear zone around the colony.

alpha-hemolysis (Alpha)

Partial destruction of RBCs, producing a greenish discoloration on the agar

gamma-hemolysis (Gamma):

No hemolysis occurs; the agar remains unchanged

Colony Formation

Single cells multiply exponentially (1—> 2 —>4—>8) until they form a visible mass called a colony

Determinants

A colony's color, size, shape, and texture are dictates of its genetic makeup

Environmental Variables

Growth properties are heavily influenced by nutrient availability, temperature, and incubation time

Shape

Irregular, or Punctiform (tiny pinpoint dots)

Margin

(smooth, regular), Undulate (wavy), Lobate (lobed), Filamentous (unbranched strands), or Rhizoid (branched root-like structure)

Elevation

Flat, Raised, Convex, Pulvinate, or Umbonate

Texture

Moist, Mucoid, Butyrous, Dry, Shiny, or Dull

Optical/Color

Pigment production (color) and optical properties (Opaque vs. Translucent)

Purpose of Slants

Chiefly utilized for the cultivation, maintenance, and long-term storage of stock cultures

Filiform

Dense, opaque growth exhibiting a completely smooth edge

Friable

Crusty, dry growth

Spreading Edge

Produced by motile organisms spreading outwards

Optical traits

Can be highly pigmented or translucent/transparent.

Pellicle

Microbial growth floats on the top surface of the liquid broth

Sediment

Growth settles entirely at the bottom of the tube.

Uniform Fine Turbidity

Even, cloudy distribution of cells throughout

Flocculent

Clumped, floating chunks of growth throughout the broth.

Germicides

Physical or chemical substances/systems designed to disrupt and prevent the spread of pathogens. Most target a broad spectrum

Levels of Control (Low to High)

Decontamination —> Disinfection —> Sterilization.

Decontamination

The lowest tier of microbial control

Decontamination definition

Reduction of pathogenic microorganisms to a safe handling threshold without needing protective attire.

Decontamination methods

Involves physical cleaning utilizing soaps or detergents, and the rigorous removal of organic and inorganic matter.

Disinfection

Intermediate control tier. Kills most targeted pathogens but does not typically destroy large numbers of bacterial spores. Separated into low, medium, and high sublevels.

Disinfectionn delivery

Usually liquid chemical agents, but can be solid, gaseous, dry heat, moist heat, or UV light

Chemical Sterilants

High-level disinfectants capable of destroying all vegetative cells and some spores.

Antiseptics

Disinfectants specifically formulated to safely eliminate or reduce pathogens on living tissues.

Sterilization

The highest level of pathogen control.

Sterilization Definition

The absolute, complete elimination of all viable organisms, including highly resilient bacterial spores

Sterilization Methods

Attainable via select chemicals/gases, incineration, dry heat, moist heat, ethylene oxide gas, ionizing radiation, low-temperature plasma, or low-temperature ozone.

Autoclave

The most common and reliable method of laboratory sterilization, using superheated steam under high pressure to destroy heat-resistant structures

Autocave standard parameters

• Sterilization temperature: Set between 121°C and 127°C.

• Must hold this optimum temperature for at least 15 minutes.

• Note: Total run time varies depending on the overall size and consistency of the load.

Autoclave monitoring tools

Color-coded autoclave indicator tape and biological indicators

Yellow Broth (Acidic)

Autoclave failure. Spores survived, germinated, and fermented the sugars, generating acid

Purple Broth (Negative/Control)

: Successful sterilization. Spores were killed; no fermentation occurred

Rules of Nomenclature when typing

Use italics. Full genus name first, then species (e.g., Escherichia coli). Abbreviated form uses the first letter of the genus followed by the species name (e.g., E. coli).

Rules of Nomenclature when hand-writing

Underline the genus and species names separately (e.g., Escherichia coli or E. coli)

Aseptic Transfer

Moving living microbes from one vessel to another without contaminating the culture, the sterile medium, or the external lab environment.

Pure culture

Contains only a single species

Mixed culture

Contains multiple species

Broths

Liquid media used to produce large volumes of cells or fresh cultures

Agar Slants

Solid media in tubes used to cultivate and maintain stock cultures; can be refrigerated for weeks

Plated Media

Solid media in Petri dishes used to isolate individual species, run differential assays, and quantify bacterial concentration.

Methods of Isolation Core Principle

A bacterial sample collected from a source is ALWAYS assumed to be a mixed culture until proven otherwise

Three Isolation Techniques:

spread plate

streak plate

pour plate

Incinerator Safety

Incinerators become extremely hot. Do not leave the loop resting inside the incinerator core, as the handle will conduct intense heat and cause burns.

Labeling Rule:

Always label the bottom of agar plates and glass tubes, never the removable lids or caps.

Louis Pasteur (1850s)

Established the concept of a pure culture to study individual species, but his isolation method (diluting cultures down to a single cell) was highly inefficient

Robert Koch (1880s)

Developed the practical "streaking for isolation" technique on solid media

he main goal of streaking for isolation is to produce distinct

individual colonies

Colony Definition

A collection of millions of identical cells that arose from a single original cell or group of cells

Pure Culture Generation

Cells from a single isolated colony can be sampled and transferred to a new sterile medium to build a confirmed pure culture

Quadrant Streak Method Mechanism

A microbial sample is spread across the surface of an agar plate[cite: 2]. As the streak pattern progresses through successive quadrants, the cell density decreases[cite: 2]. This mechanically dilutes the sample until individual cells are deposited far enough apart to grow into isolated colonies

Quadrant Streak Method Application

This is the standard method used for samples suspected of having a high cell density

Plunger

Top button pressed down to draw or dispense liquid

Tip Ejector Button

Side button that drops the contaminated tip

Volume Adjustment Dial

Dial turned to set the target volume

Volume Readout

Digital display window showing the set volume

Tip Ejector Shaft & Tip Attachment Cone

Lower sections that hold and release the plastic pipette tips

Large Clear Tips

Exclusively for the P1000

Small Yellow Tips:

Shared by both the P200 and P20

Spread Plate Method

An isolation technique where a pre-diluted liquid sample is pipetted onto an agar plate and spread evenly across the surface using a sterile tool

Spread Plate Method Mechanism

Proper dilution ensures cells (colony-forming units, or CFUs) are deposited far enough apart to grow into isolated colonies

Spread Plate Method Application

Used to pick clean colonies to start a pure culture, or to back-calculate and quantify the cell density of a liquid culture

Tube A dilution

(10^-2)

Tube B dilution

(10^-3)

Tube C dilution

(10^-4)

Tube D dilution

(10^-5)

Serial Dilution Calculations

Formula 1 (First Tube)

$$\text{Dilution} = \frac{\text{Volume Transferred}}{\text{Total Volume of Tube}}$$

Serial Dilution Calculations Subsequeent Tubes

$$\text{Dilution} = \text{Previous Concentration} \times \left(\frac{\text{Volume Transferred}}{\text{Total Volume}}\right)$$

Antimicrobials

A broad umbrella term for any physical or chemical agent used to kill or inhibit the growth of microorganisms (including bacteria, fungi, viruses, or protozoa)

Antibiotics

A specific subset of antimicrobial agents that target bacterial cells exclusively. They can be naturally occurring or synthetic

Bactericidal:

Directly kills the target bacterial cells

Bacteriostatic

Arrests metabolic growth and replication without directly killing the cells

Kirby-Bauer / Disk Diffusion Test:

A standardized diagnostic test used to measure how effectively various antimicrobials control specific pathogens

Zone of Inhibition

If the organism is susceptible, a clear zone with no bacterial growth forms around the disk. The size of this zone depends on the bacteria's sensitivity to that specific agent