CSN BIO 251 LAB 7-11 Practical #2

Non-ionizing, short, high energy wavelength radiation (from 4 - 400 nm).

UV radiation

UV-A (longest wavelength)

UV-B (280-315 nm)

UV-C (100-280 nm, most damaging, kills 99.9% of all pathogens)

3 types of UV radiation

Caused by prolonged exposure to UV radiation at wavelengths of 254-260 nm.

DNA damage

Molecular lesions formed by UV radiation that kink the DNA strand, preventing replication (thymine-thymine most common).

Pyrimidine Dimers

A repair system activated by major DNA damage that removes pyrimidine dimers and restores DNA. If system is overwhelmed, the bacteria dies.

SOS system

Time length of exposure

Wavelength (260 nm is most damaging)

Distance from the source

Barriers

Factors affecting UV radiation lethality

Acid-soluble proteins that protect DNA and enzymes.

Can withstand UV radiation. Biofilms also provide protection.

Bacterial Endospore

They repair damaged DNA within endospores.

What is the role of enzymes in bacterial endospores regarding UV damage?

Chemical metabolites produced by fungi and bacteria to treat infections.

Natural antibiotics

The first commercial antibiotic discovered.

Penicillin

Alexander Fleming

Who discovered penicillin?

Entirely produced inside laboratories (ex. Sulfonamide).

Synthetic antibiotics

Antibiotics produced chemically in the lab by modifying the R side chain of natural antibiotics; Used if parent (original) antibiotic is unsuccessful (ex. Penicillin).

Semi-synthetic antibiotics

Bactericidal antibiotics kill bacteria, while bacteriostatic antibiotics inhibit their growth.

Bactericidal vs. Bacteriostatic antibiotics

Narrow spectrum antibiotics target specific bacteria.

Broad spectrum antibiotics target multiple types.

Narrow spectrum vs. Broad spectrum antibiotics

Target a specific Gram class of bacteria.

Vancomycin - active against Gram-positive bacteria.

Polymyxin - active against Gram-negative bacteria.

Narrow spectrum antibiotic

Target both Gram types of bacteria (positive and negative).

Tetracycline - target both Gram-positive and Gram-negative bacteria but remove normal microbiota (found in colon), allowing superinfections.

Broad spectrum antibiotic

1. Inhibit cell wall synthesis

2. Inhibit protein translation

3. Disrupt cell membrane

4. Inhibit DNA/RNA synthesis

Antibiotics Mechanisms of Action

The antibiotic targets bacteria and not humans (very specific, ex. Penicillin)

High Selective Toxicity

The antibiotic can cause more serious side effects in humans like polymyxin B (not very specific for bacterial cell membranes). Used externally only.

Low Selective Toxicity

Bacteria swabbed over entire agar to form a uniform growth.

Bacteria lawn

A method used to determine the in vitro sensitivity of antibiotics or the antibiotic resistance of bacteria.

Incubation 37°C for 16-18 hours.

Kirby Bauer Method

Mueller-Hinton II agar

pH 7.2 - 7.4

Agar thickness of 4 mm

Standardized variables for Kirby-Bauer test

The diameter of the zone of inhibition in millimeters is compared to a standardized chart.

Susceptibility rating determination in Kirby-Bauer test

A quantitative technique for determining antimicrobial susceptibility using a predefined gradient of antibiotic concentrations on a plastic strip.

E-TEST

Indicates the lowest concentration of an antimicrobial agent that prevents visible growth of bacteria.

Minimum Inhibitory Concentration (MIC)

Code for Antibiotic

What is A?

Inhibition Ellipse

What is B?

Bacterial Growth

What is C?

MIC (ug/ml)

What is D?

Etest MIC predefined gradient

What is E?

MIC Interpretation

Study this chart

The lowest concentration of an antibacterial agent required to kill a bacterium over a fixed period.

Minimum Bactericidal Concentration (MBC)

The process of transferring genetic information from one donor bacterial cell to a recipient cell through a sex pilus.

Bacterial conjugation

Small, circular, double-stranded DNA molecules that exist outside of the bacterial chromosome and can carry antibiotic resistance genes.

Plasmids

Genes providing bacteria resistance to antibiotics.

Antibiotic resistance genes

The transfer of genetic information from a parent cell to a daughter cell as a result of binary fission.

Vertical transmission

Zone of inhibition

The area around the antibiotic disk in which no bacteria grow, indicating susceptibility to the antibiotic.

What is this?

Sensitive - when a bacterium is killed or inhibited in growth.

Resistant - when a bacterium is not susceptible to an antibiotic.

Intermediate - when a bacterium gains resistance and another concentration may be necessary.

Susceptibility Classification

Results for Antibiotic Sentitivity

A form of horizontal transfer that cells use to incorporate novel DNA into their genome.

Transformation

1% of cells in a natural population that have expressed a protein system on their surface which allows them to move intact ssDNA into the cells.

Competent

A family of Gram Negative rods that ferment glucose.

Can be normal gastrointestinal microbiota, opportunists (E. coli), and pathogens(Salmonella, Shigella).

These bacterial pathogens can cause diarrhea, dysentery, hemolytic uremic syndrome,UTI, pneumonia.

Pathogens can release toxin that cause illness and can be fatal.

Many normal flora can cause nosocomial (hospital acquired) infections and are often resistant to multiple antibiotics.

Enterobacteriaceae

Allows us to determine the presence of enzymatic activity because some enzymes cause changes in the pH of the media.

Differential Media

1. Sulfide-Indole-Motility (SIM) medium

2. Triple Sugar Iron (TSI) agar

3. Simmon's Citrate Agar (SCA)

4. Urea agar

Differential Tests

Sulfide, Indole, Motility

Differential Media: SIM

Some bacterial reduce sulfur directly or can remove sulfur from the amino acid cysteine to produce H2S.

Media contains ferrous ammonium sulfate that combines with H2S gas to form ferric sulfide, a black precipitate.

Sulfide

Some bacteria have the enzyme tryptophanase that hydrolyzes the amino acid tryptophan to produce Indole and pyruvate.

Detected with Kovac's reagent (red).

Indole

Semi-solid media

Some bacteria move via flagella

Can be seen moving away from stab line.

Motility

Enzyme that breaks down the amino acid tryptophan to indole, pyruvic acid, and ammonia.

Tryptophanase

Differentiates bacteria based on sugar fermentation and sulfur reduction.

Triple sugars: Glucose, Sucrose, Lactose

Ferrous sulfate (Iron sulfate)

Differential Media: Triple Sugar Iron (TSI) Agar

SCA tests for citric acid utilization

Media contains sodium citrate as the sole carbon source

Also has ammonium phosphate as a single nitrogen source and bromthymol blue (pH indicator)

Organisms with the enzyme citrase are able to utilize the sodium citrate for energy to convert the ammonium phosphate to ammonia

Differential Media: Simmon's Citrate Agar (SCA)

Differentiates bacteria based on the presence of the enzyme urease.

Bacteria are small and typically can not perform endocytosis.

Urease hydrolyzes certain amino acids and urea to form ammonia.

Differential Media: Urea Agar

Enzyme that changes urea into ammonia and carbon dioxide

Urease

Helps differentiate many members of the Enterobacteriaceae based on color.

Chromagar

It facilitates the transfer of genetic material from the donor to the recipient bacterial cell.

Sex pilus

The transfer of genetic information between bacteria through methods like conjugation, transformation, transduction; Also uses transposons.

Horizontal gene transfer

To ensure that the water is safe to drink (potable) and free from microbial contamination.

What is the primary purpose of testing public drinking water?

Parasites (e.g., Cryptosporidium, Giardia)

Viruses (e.g., Norovirus)

Bacteria (e.g., Salmonella, Shigella, E. coli, Vibrio cholera, Campylobacter, Leptospira)

Pathogens found in contaminated drinking water

Pathogens in water, some of which are resistant to chlorine and can cause gastrointestinal outbreaks.

Fecal Contamination

Bacillus that indicate fecal contamination and the possible presence of gastrointestinal pathogens; Ferments lactose.

Gram-Negative

Rod shaped

Facultative Anaerobes

Coliform Bacteria

Rapid'E.coli 2 Agar

Eosin Methylene Blue (EMB)

Hektoen Enteric Agar (HEA)

MacConkey Agar (MCA)

Selective-Differential Media used for screening water

Detects E. coli and other coliforms by identifying two enzymatic activities:

β-D-Galactosidase (GAL; pink)

β-D-Glucuronidase (GLUC; blue).

Rapid'E.coli 2 Agar

Rapid'E.coli 2 Agar

Coliform (GAL+/GLUC-) = blue-green colonies

E. coli (GAL+/GLUC+) = violet-pink colonies

What type of media is this?

Contains two dyes: eosin Y and methylene blue

Methylene blue inhibits Gram-positive bacterial growth.

Eosin Y is an indicator that reacts with the acid produced by lactose fermenters.

Eosin Methylene Blue (EMB)

Eosin Methylene Blue (EMB)

Coliforms present

E. coli produce a striking metallic green sheen due to excessive acid production in addition to turning the media to a pink-dark purple color.

What type of media is this?

Eosin Methylene Blue (EMB)

Salmonella; Non-coliforms

If there is no lactose fermentation, the agar remains red in color. This means that no coliforms are present, but there are non-coliforms growing in the media.

What type of media is this?

Differentiates Salmonella and Shigella from other enteric bacteria.

Bile salts = Gram-positive

Hektoen Enteric Agar (HEA)

Hektoen Enteric Agar (HEA)

Yellow-pinkish orange = presence of coliform

Blue-green = no coliforms but non-coliforms growing

Ferric ammonium citrate > H2S gas > Ferrous sulfide = Black (Salmonella)

What type of media is this?

Differentiates Salmonella/Shigella from other enterics. MCA contains bile salts and crystal violet which inhibits Gram-positive growth.

MacConkey Agar (MCA)

MacConkey Agar (MCA)

Pink = E. coli (coliforms present)

Fermentation occurs

What type of media is this (left)?

MacConkey Agar (MCA)

Colorless = Proteus Vulgaris (no coliforms present but non-coliform growing)

What type of media is this (right)?

A process where bacteriophages mistakenly package host DNA and transfer it to another bacterial cell during infection.

Transduction

Viruses that infect and replicate inside bacteria.

Bacteriophages

The phage injects its DNA into the bacterial cell, uses the host's machinery to replicate, and then lyses the cell to release new phages.

Bacteriophage - Lytic Infection

Dormant viral DNA that is incorporated into the bacterial chromosome during lysogenic infections.

Prophage

Simmon’s Citrate Agar

Sodium citrate, which bacteria with citrase enzyme can use to produce ammonia.The agar changes from green > blue due to an increase in pH; Ammonia produced (bromothymol blue).

Blue = positive citrate

Green = negative citrate

What is this?

Urea Agar

Hydrolyzes urea, releasing ammonia and carbon dioxide, which raises the pH.

Pink = Positive (Alkaline)

Orange = Uninoculated or Negative

Yellow = Negative

(NH2) 2CO + 2 H2O (Urea) > Urease > CO2 + H2O + 2 NH3 (Carbon Dioxide, Water, Ammonia)

What is this?

Sulfide-Indole-Motility (SIM) Medium

Tests for sulfur reduction, tryptophan metabolism, and motility.

Yellow = Negative

Black = Positive (H2S gas present)

What is this?

It indicates that the bacteria are motile.

What does cloudiness extending from the stab line in SIM Medium indicate?

SIM Medium

Pink = Indole positive

Yellow = Indole negative

What is this?

Added to SIM to detect indole; contains DMABA; reacts with indole to form rosindole (cherry red compound).

Kovac's Reagent

Some bacteria have the enzyme, tryptophanase, which hydrolyzes the amino acid, tryptophan. This hydrolysis produces indole and pyruvate.

Tryptophan Metabolism (Indole)

Triple Sugar Iron (TSI) Agar

The bacteria fermented glucose and either sucrose or lactose, producing acid.

Yellow = Acidic pH (A)

Pink-red = Alkaline pH (K)

What are these?

Yellow slant and butt in TSI Agar (A/A)

The bacteria fermented the glucose and either the sucrose or lactose (or both), resulting in a yellow slant and butt. Pockets = gas.

What is this?

Pink-red slant and yellow butt in TSI Agar (K/A)

The bacteria fermented glucose but could not use sucrose or lactose, leading to peptonization.

What is this?

Red slant and butt in TSI Agar (K/K)

The bacteria did not ferment any sugars and only used peptones; alkaline reversion/conversion

What is this?

Indicates that bacteria reduced sulfate during anaerobic respiration, producing H2S gas.

Black medium in TSI Agar

pH indicator in TSI Agar that changes color with acidity.

Phenol Red

Occurs when the bacteria initially rapidly ferment all the sugars, turning the media yellow. After depleting the sugars, the bacteria then use the amino acids for food and the media turns red.

Alkaline reversion/Conversion

Small, non-resistant colonies that grow around a central colony due to beta-lactamase diffusion.

Satellite colonies

Toxic metabolic product of cellular respiration (ex. hydrogen peroxide).

Reactive Oxygen Species (ROS)

Is pyogenic (pus-forming) and can cause abscesses and systemic diseases.

Staphylococcus Aureus

MRSA causes thousands of deaths annually in the U.S. and is resistant to common antibiotics.

Methicillin-Resistant Staphylococcus Aureus (MRSA)

Is typically not hemolytic on blood agar, it is halotolerant and generally ferments mannitol. It is coagulase negative.

Staphylococcus Saprophyticus

Is non-hemolytic on blood agar, halotolerant, does not ferment mannitol, and is coagulase-negative.

Staphylococcus Epidermidis

May be found in gastrointestinal tract. It's an opportunistic and is associated with nosocomial infections.

Enterococcus Fecalis

Coagulase, which coagulates blood plasma.

What enzyme is produced by Staphylococcus aureus that aids in its pathogenicity?

Is an enzyme that breaks down hydrogen peroxide (H2O2) into water and oxygen (H2O + O2).

Staphylococci species = catalase-positive

Catalase

Bubbles = Catalase-positive

No bubbles = Catalase-negative

Catalase test