Untitled
Microbiology 351 - Week 9 Notes
Biochemical Tests
Function:
Differentiate and sometimes identify microorganisms based on specific biochemical characteristics.
Selective Media
Definition:
Designed to enhance isolation by inhibiting the growth of some organisms while encouraging others.
Process:
A mixed culture must be isolated and cultivated as pure cultures before biochemical tests can be conducted.
Differential Media
Definition:
Comprises multiple types of media that may also contain indicators to expose differences between organisms.
Coliforms
Definition:
Subgroup of Enterobacteriaceae that produce acid and gas from lactose fermentation; E.coli is the most prominent member.
Ecological Role:
Most coliforms are normal inhabitants of the human intestinal tract, indicating potential fecal contamination in the environment if detected.
Features of Media for Biochemical Tests
Categorization:
Media may be selective, differential, defined, or undefined, or any combination thereof.
Nutritional Components:
Selected to achieve optimal growth for the microorganisms under study.
Inhibitors:
Serve to make media selective by preventing the growth of specific microorganism groups.
Substrates:
Essential for differentiating organisms based on their ability to perform certain chemical reactions.
Indicators:
Make expected reactions visible; often dyes that change color depending on pH variations or reactions with produced chemicals.
Results Interpretation:
Results can be recorded as positive or negative based on the presence or absence of expected biochemical characteristics.
Basics of Fermentation
Definition:
A metabolic process where energy is extracted from the oxidation of organic compounds (e.g., carbohydrates).
Preferred source of energy, where glucose is first consumed before sucrose or lactose.
Byproducts:
Fermentation typically produces acid byproducts that lower the pH and may produce hydrogen gas.
Alternate Metabolism:
In cases where carbohydrates are depleted or fermentation is not possible, microbes utilize peptones or amino acids, resulting in ammonia production, which raises pH.
Microbial Metabolic Capability:
Microbes can be categorized as:
Only capable of fermenting glucose.
Capable of fermenting glucose and either sucrose or lactose.
Incapable of carbohydrate fermentation.
Triple Sugar Iron Agar (TSIA)
Purpose:
Differentiates bacteria based on sugar fermentation (glucose, sucrose/lactose), sulfur reduction, and gas production.
TSIA Medium Composition
Slant Structure:
Contains a shallow agar slant with a deep butt to provide both aerobic and anaerobic growth conditions.
Components:
Contains three carbohydrates (glucose, sucrose, lactose), ferrous sulfate, and sodium thiosulfate.
Indicators:
pH indicator: Phenol red for fermentation; ferrous sulfate for sulfur reduction.
Interpretation of TSIA Results
When inoculated with a glucose-only fermenter:
Produces acid, lowers pH, leading to a yellow medium initially.
As glucose is depleted, aerobic breakdown of amino acids occurs, converting the medium to pink in the slant region due to ammonia production.
When inoculated with an organism fermenting glucose AND lactose and/or sucrose:
Similar initial lowering of pH and coloration to yellow since lactose and sucrose concentrations are 10X higher—leading to greater acid production.
When capable of no carbohydrate fermentation:
Utilizes peptones and amino acids resulting in a red medium appearance (alkalinization).
Gas Production in TSIA
Observation:
Gas appears as fissures within the agar or by lifting the agar off the tube's base.
Evidence of gas can be seen as cracks or bubbles.
Hydrogen Sulfide Production in TSIA
Mechanism:
H2S can be produced by the reduction of thiosulfate or breakdown of cysteine in the peptone.
The ferrous sulfate indicator reacts with H2S resulting in a black precipitate, indicating sulfur reduction occurs only in acidic conditions.
Recording Results for TSIA
Use the format of
Slant/Butt/Gas/H2Sfor result documentation.Color Codes:
Yellow indicates acidic (A).
Pink/red indicates alkaline (K).
Results for gas or H2S production indicated as positive or negative (+ or -).
Lysine Iron Agar (LIA)
Purpose:
To differentiate enteric bacteria based on lysine decarboxylation or deamination ability.
Medium Composition:
A combination medium (slant and deep butt) to detect lysine activity and sulfur reduction with bromocresol purple as the pH indicator.
Decarboxylation and Deamination of Lysine
Decarboxylation Reaction:
Glucose fermentation lowers pH, turning the medium yellow, promoting decarboxylase enzyme production.
Decarboxylation yields an alkaline reaction, turning it back to purple.
Deamination Reaction:
If lysine deaminase is produced, it leads to a burgundy/red coloration at the slant.
Observed with only specific genera (Proteus, Providencia, Morganella).
Result Interpretation for LIA
Symbols used in results recording:
Purple indicates alkaline (K), yellow for acidic (A), and red for deamination (R).
Interpret specific results based on color changes in the slant and butt of the tube.
Oxidase Test
Purpose:
To identify bacteria containing cytochrome c oxidase through a color change reaction.
Mechanism:
The electron transport chain (ETC) uses oxidized/reduced coenzymes, requiring cytochrome c for electron transfer to O2.
Performing the Oxidase Test
A bacterial colony is transferred to filter paper and saturated with oxidase reagent.
A color change within 20 seconds indicates presence or absence of oxidase enzyme (+ or -).
Catalase Test
Purpose:
Identifies organisms producing the enzyme catalase; differentiates between catalase-positive Staphylococceae and Micrococcaceae and catalytic-negative Streptococcaceae.
Mechanism:
Involves the electron transport chain functioning under aerobic or anaerobic conditions, producing toxic byproducts.
Performing the Catalase Test
A bacterial colony is exposed to hydrogen peroxide; bubble formation indicates the presence of catalase (+) while lack of bubbles indicates absence (-).
Phenol Red Broth – Carbohydrate Fermentation
Purpose:
To differentiate members of Enterobacteriaceae from other Gram-negative rods using carbohydrate substrates like glucose, sucrose, or lactose.
Components:
Contains carbohydrate, Phenol Red as a pH indicator with distinct color changes based on acid production from fermentation.
Result Interpretation for Phenol Red Broth
Observations for fermentation status (yellow = positive, red = negative) and gas production indicated by bubbles in Durham tubes.
Urease Hydrolysis
Purpose:
Differentiate rapid urease-positive bacteria from slower urease-positive and urease-negative bacteria.
Mechanism:
Urease hydrolyzes urea to ammonia and carbon dioxide, which can alter the medium’s pH indicator, Phenol Red.
Result Interpretation for Urease Hydrolysis
Observed as color changes:
Pink indicates rapid urea hydrolysis (+); orange/yellow indicates lack of hydrolysis (-).
Laboratory Procedures
Oxidase Test (E. coli and P. flu):
Transfer bacteria to filter paper, apply oxidase reagent, and observe color change.
Catalase Test (S. lac and E. coli):
Transfer bacteria to slide, add hydrogen peroxide, and check for bubble formation.
TSIA and LIA Slants:
Stab and streak each organism, incubate at 37°C.
Urease Hydrolysis and Phenol Red Broths:
Perform transfers into respective broths, incubate, observe changes, and record findings.
Effects of UV Radiation on Microbial Growth
Experiment Details:
Compare bacterial growth on plates exposed to UV light for varying durations (30 seconds to 8 minutes).
Analyze differences in susceptibility between E. coli and B. sub strains.
Bacterial Mutation Examination:
Evaluate effects on plates with varied concentrations of Kanamycin and observe resulting changes as concentrations increase.
Disposal Protocol:
Cultures, plates, gloves should only be discarded in designated biohazard containers.
Safety Measures:
Return writing utensils, disinfect benchtops, and wash hands before leaving the laboratory.