Microbio Lab Quiz #3

Coliform bacteria

A common contaminant in foods that may indicate greater risk of foodborne illness

• 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 & least 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

Stain

An organic compound containing benzene ring, chromophore, and auxochrome group

• Two types of stains: acidic & basic

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

• Ex: eosin and picric acid

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

• Bind effectively to negative charge of bacterial cell walls

• Ex: crystal violent & methylene blue

• Use BASIC DYES when staining bacteria

Heat fixation

When bacterial proteins are coagulated & adhere to glass slide

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)

• Darkens the color of the crystal violet stain

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

Outer membrane

Gram-negative cells have a second lipid bilayer outside of cell wall

Microorganisms need

• Water

• Carbon

• Energy

• Electrons

• Nitrogen

• Non-metallic elements

• Metals

• Vitamins

• Gaseous atmosphere

• Temperature

• pH

Water

Cytoplasm within cells contain 80% and 20% dissolved substances

• Need water outside of cells to facilitate movement

Carbon

Element most commonly found in living things; considered a macronutrient

• Autotroph: obtain carbon from inorganic compounds & fix it into organic compounds to use in cells

• Heterotroph: use organic carbon from their environment

• Mixotroph: capable of autotrophy and heterotrophy depending on environment

Energy

Most activities within the cell require energy

• Phototroph: obtain energy from solar radiation

• Chemotroph: obtain energy by oxidizing chemical compounds (organic/inorganic)

Electrons

All organisms during metabolism need electrons to complete reduction/oxidation (REDOX) reactions

• Organotrophs: organic chemicals used as electron donor source

• Lithotrophs: inorganic chemicals used as electron donor source

Nitrogen

Macronutrient that is the second most abundant element in microorganisms

Nitrogen fixers

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

Dentrifiers

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

Non-metallic elements

Mainly phosphorus and sulfur

• Phosphorus: key player in energy economy of cell and is an essential part of genomic structure

• Sulfur: common constituent of proteins, a component of some lipids, and is important in nitrogen metabolism

Metals

Includes elements like Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+. Fe3+, Cu2+, Zn2+

• Several of these function as essential cofactors in the operation of enzymes

• Others function as osmoregulators/in the transport of electrons during metabolism

• Most are considered micronutrients and are supplied via metal salts

Vitamins

Organic substances that function as metabolic coenzymes facilitating efficient metabolism

Gaseous atmosphere

Common for an organism to require atmospheric oxygen (obligate aerobe,
microaerophile), but it’s also common for organisms to thrive without it (obligate/ facultative anaerobes, aerotolerant)

Obligate aerobe

Requires oxygen to survive/grow (will die without O2)

Microaerophile

Require lower levels of atmospheric O2

• Too much will kill it but not enough will kill it

Obligate anaerobe

Unable to survive in normal atmospheric conditions (O2 is lethal)

• Exclude O2 to prevent oxygen radical

Facultative anaerobe

Able to survive in anaerobic conditions

• Grow best and fastest when O2 is present but will grow without it as well

Aerotolerant

Doesn’t use oxygen AT ALL for any metabolic processes but doesn’t die when exposed to oxygen

Every organism has an optimal ___________ where enzymes run most efficiently

temperature

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