CMMB 343 Lab Practical Exam 2 (Labs 5-8)

LAB 5

Oxygen Tolerance & Aerotolerance (4)

• Molecular oxygen (O₂) is important in aerobic respiration because it acts as the final electron accepter in the ETC

• Reducing O₂ to H₂O can generate toxic oxygen by-products (O₂^-, H₂O₂, OH·)

• If not removed, organic cellular components are oxidized and the cell dies

• Microorganisms differ in their ability to detoxify reactive oxygen species, so they differ in the level of oxygen used for metabolism; some cannot tolerate oxygen at all

Testing Oxygen Tolerance: Deep Shake Culture (3)

• Molten tryptose agar is inoculated and allowed to solidify

• As oxygen diffuses from the top, an oxygen gradient forms

• The area where bacteria grow indicates oxygen preference

Testing Oxygen Tolerance: Thioglycolate Medium (3)

• Thioglycolate removes oxygen from the medium, creating a gradient (top of the tube = oxygen presence; bottom = anaerobic)

• A pink layer at the top indicates the oxygenated region

• Growth patterns reveal oxygen tolerance

Oxygen Tolerance: Interpreting Results - 4 Things to Look For

1. Location of Growth

2. Amount of Turbidity

3. Presence or Absence of Gas

4. Relationship of Growth to Oxygen Gradient

Oxygen Tolerance Classes

*Reference Midterm 1 Flashcards?

Interpreting Deep Shake Culture Results: What to Record (3)

1. Where is the turbidity?

2. Is growth only at the top, throughout the tube, or deeper in the agar?

3. Is there cracking in the agar?

• If gas was produced in the anaerobic portion of the tube, cracking of the agar may be observed

Interpreting Thioglycolate Medium Results: Pink Layer (2)

• The pink layer at the top marks the oxygenated region (Resazurin redox indicator)

• This layer is pink when oxidized and colourless when reduced

Interpreting Thioglycolate Medium Results: Growth Relative to Pink Layer (2)

• If growth is below the pink layer, the organism is growing away from the oxygen-rich region

• If the pink layer disappears, oxygen may have been consumed and the indicator was reduced

Interpreting Thioglycolate Medium Results: What to Record (3)

1. Location of growth

2. Relationship of growth relative to the pink layer

3. Turbidity pattern

Metabolic Diversity: Testing Glucose (3)

• Glucose is a central substrate in microbial metabolism; many microbes use glucose to generate energy, but not all use it in the same way

• Depending on the organism and oxygen availability, glucose may be used in: aerobic respiration, anaerobic respiration, fermentation

• The key idea is that different organisms can process the same substrate using different metabolic pathways

Testing Glucose: Hugh & Leifson (H&L) Glucose Medium (3)

• Two H&L glucose tubes are stab inoculated with the same organism: one tube remains open to air, and the other is overlaid with mineral oil

• Mineral oil limits oxygen exposure to create more anaerobic conditions

• The purpose of the test is to compare glucose metabolism in the presence vs. absence of oxygen

Hugh & Leifson (H&L) Glucose Medium: What This Test Asks (3)

Does The Organism Use Glucose:

1. Only when oxygen is available

2. In both aerobic and anaerobic conditions

3. Or not at all

*Distinguishes oxidative, fermentative, and non-utilizing organisms

Hugh & Leifson (H&L) Glucose Medium: Interpreting The Indicator (3)

• H&L glucose medium contains bromothymol blue, a pH indicator

• If the organism metabolizes glucose, acidic end products are released into the medium (lowers pH)

• If the organism does not utilize glucose, it may instead use peptones in the medium which produces alkaline end products (raises pH)

Hugh & Leifson (H&L) Glucose Medium: Interpreting The Indicator - Colour Change (3)

1. Green → Yellow: Positive for carbohydrate utilization

2. Green → Blue: Negative for glucose utilization

3. Green: Neutral/No Major Change

Four Common H&L Result Patterns

1. Both Tubes Green

• Negative/No glucose utilization

2. Only Open Tube Yellow

• Oxidative metabolism/aerobic glucose use

• Positive for respiration

3. Both Tubes Yellow

• Fermentation/Facultative Glucose Use

• Positive for fermentation

4. Blue Colour Develops

• Peptone utilization/alkaline end products

• Positive for peptone utilization

*Blue colour developing is still considered a negative result

Fermentation

• Fermentation is a way for heterotrophic microorganisms to obtain energy from carbohydrates without using an ETC

4 Key Features of Fermentation

1. Occurs in the absence of oxygen

2. No energy-producing electron transport

3. Less energy-efficient than respiration

4. Metabolic intermediates or fermentation end products are produced

Fermentation: Two Stages

1. Oxidation of Glucose

• Glucose is split and oxidized; pyruvate is produced; reducing power is generated

2. Reduction of Pyruvate-Derived Compounds

• Pyruvate or its derivatives are reduced; regenerates oxidized cofactors needed to continue glycolysis; characteristic products are formed

Distinguishing Bacteria Via Fermentation

• Different bacteria produce different end products, and these products help distinguish species

Homolactic Fermentation (3)

• In homolactic fermentation, carbohydrate (glucose, lactose, etc.,) is first converted to pyruvate via the EMP pathway

• Pyruvate is then reduced almost entirely to lactic acid

• Lactic acid production changes the chemistry of the medium and can be used to detect fermentation activity

Homolactic Fermentation: Examples (2)

1. Streptococcus

2. Lactobacillus

Testing Homolactic Fermentation: Litmus Milk (3)

• Litmus milk is used to detect lactose fermentation and protein metabolism produced by microorganisms growing in milk-based medium

• Litmus milk is a pH indicator; several different metabolic reactions can occur in litmus milk

• The medium contains: skim milk (source of lactose and casein protein) and litmus dye (acts as pH indicator and redox indicator)

The initial colour of the litmus milk medium is ___

• Light purple (lilac)

Litmus Milk: Interpreting Results - 5 Potential Metabolic Reactions

1. Acid Production

• Litmus turns pink

• Indicates lactose fermentation (acidic end products are produced)

2. Alkaline Reaction

• Litmus turns purple or blue

• Indicates casein digestion (ammonia is produced)

3. Reduction

• Litmus turns white

• Occurs when litmus dye is chemically reduced during fermentation

4. Coagulation

• Medium solidifies

• Acid causes casein precipitation

• Gas production may create cracks or fissures

5. Peptonization

• Casein is digested

• Medium becomes translucent or watery

Anaerobic Respiration

• Respiration does not always require oxygen; some microorganisms can use alternative terminal electron acceptors (e.g. NO₃^-, SO₄^2-, CO₂, Fe³⁺, fumarate)

Anaerobic (Nitrate) Respiration (2)

Two Different Ways Nitrate Can Be Reduced:

1. Assimilative Nitrate Reduction

2. Dissimilative Nitrate Reduction

Anaerobic (Nitrate) Respiration: Assimilative Nitrate Reduction - Biosynthesis (3)

• Nitrate is reduced for use as a nutrient source (providing nitrogen for amino acids and proteins - typically ammonia)

• Can occur under aerobic or anaerobic conditions

• Assimilative nitrate reductases are soluble proteins that are ammonia repressed

Anaerobic (Nitrate) Respiration: Dissimilative Nitrate Reduction - Energy Metabolism (3)

• Nitrate is used as a terminal electron acceptor in energy metabolism (anaerobic respiration)

• Restricted to bacteria

• Dissimilative nitrate reductases are membrane-bound proteins that are repressed by O₂ and synthesized under anaerobic conditions only

Nitrate Reduction (2)

• Ability to reduce nitrate to nitrite in the absence of oxygen (anaerobic respiration)

• This process is present in many bacteria

Denitrification (3)

• Ability to further reduce nitrate beyond nitrite to gaseous products

• This process is limited to a smaller number of genera

• Denitrification is considered a detrimental process as gases can escape into the atmosphere, removing biologically available nitrogen from the environment (N₂ less readily available)

Testing Nitrate Respiration: Nitrate Reduction Broth Test - Interpreting Results (3)

• Turbidity in the tube indicates organism growth, but turbidity by itself does not prove nitrate reduction

• A positive result is the presence of a bubble in the Durham tube (indicates formation of nitrogenous gas - denitrification)

• A negative result is no bubble in Durham vial; no evidence of gas production; organism does not fully reduce nitrate (unknown result)

Effect of Chemical Agents on Bacterial Growth: 3 Mechanisms

1. Cidal

• Concerned with the killing of microorganisms (bactericidal, fungicidal)

2. Static

• Inhibits growth (bacteriostatic)

3. Lytic

• Causes cell lysis

Antiseptics (2)

• Antimicrobial agents safe for living tissue (used on skin or other tissues)

• May be cidal, static, or lytic

Disinfectants (2)

• More potent antimicrobial agents used on non-living surfaces (unsafe for living tissues due to toxicity)

• Usually cidal or lytic

Evaluating Claims of Commercially Prepared Antibacterial Agents: Experiment (3)

• Many commercial preparations claim to be antibacterial; experiment will evaluate these claims and compare effectiveness of competitive products

• The effectiveness of products is tested against both Gram-positive bacterium (Staphylococcus), and Gram-negative bacterium (Enterobacteriaceae)

• Each test solution is applied to a paper disc on a bacterial lawn; after incubation the inhibition of growth around the disc can be compared

Evaluating Claims of Commercially Prepared Antibacterial Agents: Products Tested (4)

1. Tea Tree Oil

2. BandAid

3. Pine Sol

4. Mr. Clean

Effect of Chemical Agents on Bacterial Growth: Kirby-Bauer Disc Diffusion (2)

• Chemotherapeutic agents are antimicrobial compounds that can be administered internally (important in both human and veterinary medicine)

• The Kirby-Bauer test is used to evaluate how sensitive a bacterium is to different antibiotics (standardized method to compare antibiotic susceptibility)

Kirby-Bauer Disc Diffusion: Experiment (4)

• Each antibiotic disc contains a specific amount of antibiotic and is placed on a bacterial lawn

• The antibiotic diffuses outward into the agar to create a concentration gradient; antibiotic concentration is highest near the disc and decreases with distance

• If the bacterium is inhibited, a zone of inhibition forms around the disc

• The zone diameter can be compared to a standard chart to classify the organism as: susceptible, intermediate, resistant

Kirby-Bauer Disc Diffusion: Antibiotics Tested (3)

1. Erythromycin

2. Vancomycin

3. Penicillin

Effect of Chemical Agents on Bacterial Growth: Phenol (2)

• Phenol was one of the first disinfectants widely used in Western medicine

• Because phenol became an early standard disinfectant, other chemical agents are compared against it

Phenol Coefficient (2)

• The phenol coefficient is the ratio of the test disinfectant’s effectiveness to phenol’s effectiveness (reciprocal effective dilution of test disinfectant/reciprocal effective dilution of phenol)

• Both agents are tested against the same organism in the same conditions

Phenol Coefficient: Interpretation (3)

Coefficient > 1: Test disinfectant is more effective than phenol

Coefficient < 1: Test disinfectant is less effective than phenol

Coefficient = 1: Equally effective

Phenol Coefficient: Effective Dilution (2)

• An effective dilution is the dilution of an agent that completely inhibits growth after 10 minutes, but does not completely inhibit growth after 5 minutes

• This gives a standard way to compare disinfectants fairly

Phenol Coefficient: Effective Dilution - Why 5 & 10 Minutes (2)

• Because effectiveness depends on both concentration and exposure time

• A disinfectant may not kill quickly at 5 minutes, but becomes effective by 10 minutes

LAB 6