Microbio Lab Quiz #2

Lab 2 Content

Spread plate

Using a glass spreader to spread the liquid culture over the surface of the TSA plate

Mutualistic relationship

Both the host and symbiont organism benefit

Commensal relationship

Host organism is not affected; symbiont organism benefits

Parasitic relationship

Host organism is harmed; symbiont organism benefits

Cultural characteristics

Used to describe colony morphology

Colony morphology

Visual appearance/overall shape of bacterial colony on plate

• Size, pigmentation, form, margin, elevation

Size

Relative size of bacterial colony

• Pinpoint, small, medium, large

Pigmentation

The color (if any) that the bacterial colony produces while growing on plate

• Chromogenic bacteria: produce pigments that result in colored colonies

• Most are not chromogenic (appear white, gray, tan)

Form

The overall shape of the whole colony

• Circular: smooth, unbroken periphery

• Irregular: non-uniform edge

• Rhizoid: root-like

• Spindle: ovoid

Margin

Outer edge of the colony

• Entire: even

• Lobate: irregular

• Undulate: wavy

• Erose: sawtooth-like

• Filamentous: fuzzy, thread-like

Elevation

The height of the colony when compared to agar surface

• Flat: no difference

• Raised: slightly above

• Convex: dome-like

• Pulvinate: convex

• Umbonate: convex portion on top

Your skin and natural microbiota are the ______ defense against pathogens

Best

Your skin and natural microbiota are…

Typically commensal/mutualistic and can outcompete invading pathogens

How can something be ocassionally pathogenic?

1) The organism changes physical location

• Ex: Staphylococcus on skin vs. bloodstream

2) The organism contains virulence factors

Virulence factors

Strain-level differences (different genes) within a bacterial species that aid an organism in pathogenesis

• Some are non-pathogenic (avirulent or harmless)

• Some are pathogenic (virulent or disease-causing)

Cellular product (“tools”) used/secreted by pathogens

Virulence factors fall into one of three categories:

1) Colonize host

• Ex: flagella/cilia, adhesive properties for attachment of host cells

2) Evade host defenses

• Ex: capsules - thick outer covering/shell that helps intestinal pathogens survive stomach acid

• Antibiotic-resistant genes

3) Cause damage to host cells

• Ex: toxins, hemolysins, proteases

Nosocomial infections (HAIs)

Infection acquired in a hospital or other healthcare facility

• Also known as healthcare-acquired infections (HAIs)

• Results from breakdown in proper sanitation protocols (improper handwashing, contaminated equipment, poor air filtration, contaminated bed linen)

TSA

General growth medium (grows everything that is culturable)

MSA (mannitol salt agar)

Selective and differential growth medium

• Selective: ingredients in agar prevent some bacteria from growing

• Differential: bacteria/microbes that visually appear in a certain color

  • Indicates whether or not it ferments mannitol

Selective function of MSA

Only grows halophilic (salt-loving) bacteria

• 7.5% NaCl concentration only allows for growth of organisms that can tolerate salinity

  • Bacterial growth = halophilic organism

  • No bacterial growth or inhibited/unhealthy growth = organism is not halophilic

MSA differential function

For the bacteria that grows, we can determine if it ferments mannitol

• Mannitol: sugar that some microbes can use for energy

• Phenol red: pH indicator that starts red but turns yellow when conditions are acidic

  • Bacterial growth & NO color change = halophile cannot ferment mannitol

  • Bacterial growth & color change = halophile can ferment mannitol

Coliform bacteria

Gram-negative bacilli capable of fermenting lactose

Sickness from foodborne illness can be achieved through…

• Infection from actual bacterial infection

  • Slower onset of illness — bacteria need time to multiply and colonize host

  • Usually requires medical intervention

  • Ex: salmonella & Clostridium botulinum

• Illness from exotoxin production

  • Symptoms tend to come quicker

  • Self-limiting (need to run its course)

  • Ex: Staphylococcus aureus & Bacillus cereus

MacConkey Agar

Selective and differential culture medium for bacteria

• Designed to selectively isolate Gram-negative & enteric (found in intestinal tract) bacteria and differentiate them based on lactose fermentation

MacConkey Agar selective function

Grows Gram-negative bacteria (inhibit Gram-positive)

• Use bile salts and dye crystal violet (inhibit Gram-positive growth)

  • Growth = Gram-negative bacteria

  • No growth = Gram-positive bacteria

MacConkey Agar differential function

Separates Gram-negative enteric bacteria into two groups based on ability to ferment lactose

• Coliform bacilli (Escherichia’s, Klebsiella’s)

  • Colonies will be pink: Gram-negative organism, positive for lactose fermentation

• Dysentery, typhoid, or paratyphoid (Salmonella’s, Proteus’s, Shigella’s)

  • Colonies will be tan: Gram-negative organism, negative for lactose fermentation

Eosin Methylene Blue Agar (EMB)

Selective for Gram-positive bacteria against Gram-positive bacteria

• Used to identify Escherichia coli (E. coli) → used as the Fecal Contaminate Indicator Organism

E.coli is used as the fecal contaminate indicator organism because…

It’s a commonly occurring commensal bacteria found in the gut of most mammal species (including humans)

• Can commonly be isolated from fecal material

• Heavy contaminated water = increase numbers of E.coli = high chance other coliform gut microbes are present

EMB selective function

Grows up Gram-negative; partially inhibits Gram-positive (not as strong as MacConkey)

• Use eosin Y and methylene blue (pH indicator dye) to form dark purple precipitate at low pH → inhibit Gram-positive

  • Growth = Gram-negative organism

  • No growth or inhibited/unhealthy growth = probable Gram-positive organism

EMB differential function

Separates Gram-negative enteric bacteria into two groups based on ability to ferment lactose and specifically identifies E.coli

• Gram-negative lactose fermenter: colonies will be pink

• Gram-negative non-lactose fermenter: colonies will be tan

• Escherichia coli (Gram-negative vigorous lactose fermenter): colonies will be black with metallic green sheen

MacConkey vs. EMB

MacConkey

• MUCH better inhibiter of Gram-positive (inhibits almost ALL)

EMB

• Selective function not as strong - some Gram-positives can grow on EMB

• Able to DIRECTLY identify E.coli

All modern microscopes are…

compound → use more than one lens

• Will be talking about brightfield microscopes (simplest & leasy expensive)

To get total magnification power, you need to…

take the fixed eyepiece lens and multiply it by objective lens you’re using

• Ex: 40x objective lens → 40 × 10 = 400x total magnification power

100x oil immersion lens

The strongest objective lens in a standard brightfield microscope

Bacterial smear

Use bacterial culture and spread/smear the culture on glass slide

• Let it air dry and perform heat fixation → kill cells and adhere & anchor cells to glass slide so they stick

Benzene ring

Scaffold that the stain is built from

Chromophore

Portion of the molecule that is the color-bearing group of the stain

Auxochrome

Functional group in the compound that helps chromophore bind to substance being stained

Acidic vs. basic stains

Acidic stain: negative charge (anionic) → same charge as cell walls & membrane

• Stain not taken inside cell

Basic stain: positive charge (cationic) → strong affinity for negatively charged cellular components (cell walls & membrane)

• Bind effectively to negative charge of bacterial cell walls

Cellular morphology & arrangement

Cellular morphology: cell shape

Arrangement: how multiple cells are organized with one another

Bacterial names

• Diplo: pairs

• Staphylo: clusters/groups

• Strepto: chains

• Tetrad: cluster of 4 cells

• Sarcina: cluster of 8 cells

• Palisades: rod-shaped cells stacked side-by-side

Gram staining process

1) Primary stain:

• Use crystal violet— has affinity for negatively charged peptidoglycan & stains all bacterial cells dark purple

2) Mordant

• Use Gram’s iodine — combines & binds with primary stain to form large complex molecule that is water-soluble (dark purple)

3) Decolorizer

• Use ethyl alcohol

  • Washes away CV-I (crystal violet-iodine) in Gram-negative → colorless

  • Dehydrates peptidoglycan layer of Gram-positives → purple

  • Differential step of Gram stain procedure

4) Counterstain

• Use safranin — re-stain cells that were colorless from decolorizer

  • Color is different from primary stain (Gram-negative cells pink)

Peptidoglycan

Compound that serves as the cell wall in BACTERIA ONLY

N-acetyl glucosamine and N-acetyl muramic acid

Bind to one another in an alternating pattern to create long glycan chains that surround entire bacterial cell

• Layered chains bind together between peptide side chains

Microorganisms need

• Water

• Carbon

• Energy

• Electrons

• Nitrogen

• Non-metallic elements

• Metals

• Vitamins

• Gaseous atmosphere

• Temperature

• pH

Organotrophs vs. lithotrophs

Organotrophs: organic chemicals used as electron donor source

Lithotrophs: inorganic chemicals used as electron donor source

Nitrogen fixers

Take N2 gas in the air and convert it to a biologically usable form of nitrogen to ammonium ion (NH4+)

Nitrifiers

Take ammonium ion and convert it to an even more biologically usable form for more microbes in the form of Nitrate (NO3-)

Denitrifiers

Use nitrate and reduce it to other forms like nitrite (NO2-), nitrous oxide (N2O), nitric oxide (NO), etc. all the way back into a non-biologically available form of N2 gas

Psychrophiles

“Cold-loving” organisms that grow between -5°C to 20°C

• Optimal is usually around 0-5°C

Mesophiles

Grow between 20°C to 45°C

• All mesophiles grow at 37°C (body temp)

• Most human pathogens are mesophiles

Thermophiles

“Heat-loving” organisms that grow above 35°C

• Facultative: will grow at 35°C and range between 45°C-60°C

• Obligate: will grow only above 50°C (optimum 60°C)

Hyperthermophiles

“Extreme heat-loving” organisms that grow as high as 121°C

Acidophiles

Organisms that thrive under highly acidic conditions

• Optimum usually around 2-3 pH; <5.5 pH

Neutrophiles

Organisms that prefer an environment around a pH of 7 (neutral)

• Cannot withstand major shifts in either direction (5.5-7.9 pH)

Alkalophiles

Organisms that thrive under highly basic conditions

• Optimum >8pH, usually around about 10pH

Bacterial growth curve

1) Lag phase: bacteria prepare their cell machinery for growth

• No increase in cell number

• Cells adjust to new environment, activate needed genes, and prepare for growth

2) Log phase: growth approximates an exponential curve

• Cells increase at exponential rate, nutrients are plentiful, space is abundant, and cells replicate at its fastest rate

• Phase we want to keep our bacterial cells in while running experiments

3) Stationary phase: cells stop growing and shut down their growth machinery while turning on stress responses to help retain viability

• Waste is building up, space and nutrients become limited, environment is becoming unhealthy for cells

4) Death phase: cells begin to die at exponential rate

• No nutrients, inhospitable environment