Microbiology Lab Fundamentals: Growth, Metabolism, Genetics

Lab 8: Temperature and Microbial Growth

Key Concepts

• Psychrophiles grow best between 0–20°C. Often found in cold environments like glaciers.
• Mesophiles prefer 20–45°C. These include human pathogens like E. coli.
• Thermophiles thrive at 55°C or higher. They are often found in hot springs.

Materials and Their Functions

• Tryptic Soy Broth (TSB): A nutrient-rich medium that supports bacterial growth.
• Incubators set to different temperatures (4°C, 25°C, 37°C, 55°C): Used to simulate environmental conditions.
• Spectrophotometer (OD600): Measures turbidity (cloudiness) of a culture to estimate cell density.
• Classification by growth: “+++” indicates heavy growth; “0” means no growth.

In this lab, students investigated how temperature affects microbial growth. They were given cultures of Pseudomonas fluorescens, Bacillus stearothermophilus, and Saccharomyces cerevisiae, which they streaked onto nutrient agar plates. Each plate was incubated at a different temperature: 4°C, 25°C, 37°C, and 55°C.

After incubation, students observed which temperatures supported optimal growth for each organism. For example, B. stearothermophilus thrived at 55°C, identifying it as a thermophile. This lab demonstrated the classification of microbes by temperature range and the influence of temperature on enzyme activity and protein function.

• Pseudomonas fluorescens – mesophile
• Bacillus stearothermophilus – thermophile
• Serratia marcescens – mesophile
• Saccharomyces cerevisiae – eukaryotic yeast
• Temperature affects enzyme structure, protein expression, and membrane fluidity.

Lab 9: Fermentation Patterns of Gram-Negative Bacteria

Key Concepts

• Enterobacteriaceae are Gram-negative, rod-shaped, facultative anaerobes.
• Fermentation is the metabolic breakdown of sugar in the absence of oxygen.
• Mixed acid fermenters (MR+): Produce stable acidic end-products (e.g., E. coli).
• 2,3-Butanediol fermenters (VP+): Produce neutral end-products (e.g., Enterobacter).

Media and Indicators

• EMB Agar: Detects lactose fermentation; E. coli appears metallic green.
• MacConkey Agar: Lactose fermenters turn pink; non-fermenters remain colorless.
• Hektoen Agar (HEK): Detects H₂S production (black centers = positive).
• Phenol Red Broth Tubes: Turn yellow when acid is produced during fermentation.

This lab focused on fermentation behaviors in Enterobacteriaceae. Students used a range of media including glucose and lactose broths, citrate slants, and differential agars like EMB, MAC, and HEK. They inoculated these media with known and unknown bacteria.

Color changes and gas bubbles indicated fermentation types. For instance, glucose broth turning yellow signified acid production, while gas bubbles in Durham tubes showed gas production. On EMB plates, E. coli produced a green metallic sheen, confirming it as a strong lactose fermenter.

Temperature and Microbial Growth Summary

• Psychrophiles (0–20°C), Mesophiles (20–45°C), Thermophiles (55°C+)
• Temperature affects enzyme function, protein expression, and adaptation.
• Growth is tracked using OD600 over time in a spectrophotometer.

Fermentation Patterns Summary

• Enterobacteriaceae: Gram-negative rods, catalase-positive, facultative anaerobes.
• All ferment glucose to pyruvate; end products vary by species.
• Mixed Acid Fermentation (MR+): E. coli, Shigella, Salmonella.
• 2,3-Butanediol Fermentation (VP+): Enterobacter, Serratia.
• MacConkey agar: pink = lactose fermenter; colorless = non-fermenter.

Lac Operon and ONPG Test Summary

• Lac operon is inducible; active when lactose is present and glucose is low.
• ONPG test: Yellow = positive for β-galactosidase.
• Diauxic growth: Glucose is used first, then a lag occurs before lactose is utilized.
• Controlled by CAP (activator) and Lac repressor (inhibitor).

Nitrogen Fixation Summary

• Azotobacter fixes atmospheric N₂ to NH₃ via nitrogenase.
• Maintains anaerobic conditions through a high respiration rate.
• Rhizobium forms root nodules in legumes; uses leghemoglobin to reduce O₂.
• Identified using Gram stain and YEM plates.

Bacteriophage Plaque Assay Summary

• Soft agar overlay method used to count plaques on bacterial lawn.
• Each plaque = infection by one phage particle.
• pfu/mL = (Number of plaques) × (1/mL plated) × (1/dilution).

Microbial Growth Curve Summary

• Phases: Lag → Log (Exponential) → Stationary → Death.
• Log phase is used to calculate generation time.
• Tracked using OD600; a standard curve is built to estimate cell numbers.

Lactic Acid Bacteria Summary

• Ferment sugars to lactic acid (homofermentative or heterofermentative).
• Includes species like Lactobacillus.
• Found in yogurt, cheese, and the gut microbiome.

Photosynthetic Microbes Summary

• Algae: Eukaryotic, contain chlorophyll a, perform oxygenic photosynthesis.
• Cyanobacteria: Prokaryotic, also photosynthesize and can fix nitrogen.
• Both play key roles in oxygen production and the carbon cycle.

Enumeration Calculations Summary

• Used in standard plate count and plaque assay.
• Only count plates with 30–300 colonies or plaques.
• CFU/mL = Colonies × (1/dilution) × (1/mL plated).

Lab 10: Lac Operon and Nitrogen Fixation

Lac Operon Key Concepts

• Operon: A gene cluster regulated as a unit.
• Inducible system: Activated only when needed.
• β-galactosidase: Breaks lactose into glucose + galactose.
• ONPG: A lactose analog; when cleaved by β-galactosidase, turns yellow (positive test).
• CAP/cAMP system: Needed when glucose is low; helps activate the operon.
• Diauxic Growth: Glucose is used first, followed by a lag, then lactose is utilized.

Nitrogen Fixation

• Azotobacter: Free-living, fixes N₂ to NH₃. Protects nitrogenase through high oxygen consumption.
• Rhizobium: Symbiotic, forms root nodules on legumes. Uses leghemoglobin to reduce oxygen exposure.
• YEM agar (Yeast Extract Mannitol): Used to grow nitrogen fixers.
• Gram stain: Shows pleomorphic shapes inside nodules (varied morphology).

This lab introduced gene regulation and nitrogen-fixing bacteria. For the lac operon, students performed the ONPG assay. Tubes containing E. coli and ONPG were observed for a color change—yellow indicated β-galactosidase activity, meaning the lac operon was active.

In the second part, students examined Azotobacter on nitrogen-free media and stained Rhizobium from pea root nodules. Azotobacter protected its nitrogenase enzyme through rapid oxygen consumption, while Rhizobium used leghemoglobin in root nodules to limit oxygen exposure.

Lab 11: Bacteriophage T3 and Microbial Growth Curve

Bacteriophage Concepts

• T3 Phage: Lytic phage that infects E. coli and causes visible plaques.
• Plaques: Clear zones on agar where phage lysed bacterial cells.
• Soft Agar Overlay: Traps bacteria in semi-solid media to allow local infection by phages.
• Titer Formula: pfu/mL = plaques × (1/dilution) × (1/mL plated).
• Serial Dilutions: Reduce concentration for accurate counting (10⁻¹, 10⁻², etc.).

Growth Curve Concepts

• OD600 (Optical Density at 600nm): Indicates cell turbidity.
• Lag Phase: Cells adapt, no division.
• Log Phase: Cells divide exponentially.
• Stationary Phase: Growth rate = death rate.
• Death Phase: More dying than dividing.
• Hemocytometer + Crystal Violet: Used for direct cell counts.
• Generation Time (G): G = t / n (time divided by number of generations).

Students explored bacteriophage activity and microbial growth patterns. In the plaque assay, phage T3 was mixed with E. coli and poured onto nutrient plates using a soft agar overlay. After incubation, plaques (clear zones) were counted to calculate viral concentration using the formula:

pfu/mL = plaques × (1/dilution) × (1 /volume plated)

For the growth curve, students inoculated E. coli into broth and incubated it in a 37°C shaker. They measured cell density at regular intervals using a spectrophotometer (OD600). Cell counts were also taken using a hemocytometer after staining.

Lab 12: Lactic Acid Bacteria, Photosynthesis, and Enumeration

Lactic Acid Bacteria (LAB)

• Gram-positive rods (e.g., Lactobacillus), catalase-negative, acid-producing.
• YDC Agar (Yeast Dextrose Calcium carbonate): Clearing zones = acid produced (positive test).
• Fermentation Types: Homolactic: Only lactic acid.
• Heterolactic: Lactic acid + gas + ethanol.
• Durham Tubes: Catch gas bubbles from fermentation.

Photosynthetic Microbes

• Cyanobacteria: Prokaryotic, fix nitrogen, oxygenic photosynthesis.
• Algae: Eukaryotic, contain chloroplasts and chlorophyll a.
• Important for oxygen production, symbiosis, aquatic food chains.

Enumeration: Serial Dilution and Spread Plate

• Common method to count microbes.
• CFU/mL formula: CFU/mL = colonies × (1/dilution) × (1/mL plated).
• Only count 30–300 colonies per plate for accuracy.

This lab combined food microbiology, photosynthesis, and microbial quantification. Students streaked yogurt, sauerkraut, and buttermilk onto YDC agar. Clear halos around colonies indicated acid production. Glucose fermentation tubes with Durham tubes helped classify the isolates as homofermentative (acid only) or heterofermentative (acid + gas).

Students then examined photosynthetic microbes under a microscope, comparing cyanobacteria (prokaryotic) and algae (eukaryotic). These organisms play essential roles in oxygen and nitrogen cycles. Finally, students performed serial dilutions and plate counts to calculate colony-forming units (CFU/mL). Only plates with 30–300 colonies were considered valid.

Quiz Questions

Which condition turns the lac operon ON?
A. High glucose, no lactose
B. Low glucose, no lactose
C. Low glucose, lactose present ✅
D. High glucose, lactose present

ONPG is a lactose analog that turns yellow when cleaved by β-galactosidase.

Which of the following is a characteristic of lactic acid bacteria?
A. Gram-negative, catalase positive
B. Gram-positive, catalase negative ✅
C. Spore-forming rods
D. Alkaline fermentation

Clear zones on YDC agar indicate acid production.

Which of the following organisms is a thermophile?
A. E. coli
B. Saccharomyces cerevisiae
C. Pseudomonas fluorescens
D. Bacillus stearothermophilus ✅

Mesophiles grow best at temperatures between 20 and 45 °C.

Key Organisms Studied

1. Pseudomonas fluorescens

• Gram-negative, rod-shaped, motile bacterium.
• Mesophile – optimal growth around 25°C.
• Found in soil and water.
• Used to show mesophilic growth behavior and test temperature effects on metabolism.

2. Bacillus stearothermophilus

• Gram-positive, endospore-forming rod.
• Thermophile – thrives at high temperatures like 55°C.
• Common in hot springs and compost.
• Used to show heat resistance and thermophilic growth.

3. Serratia marcescens

• Gram-negative, rod-shaped, produces a red pigment at room temperature.
• Mesophile – grows best at 25–37°C.
• Used to illustrate how pigment production can depend on temperature.

4. Saccharomyces cerevisiae

• Eukaryotic yeast used in baking and brewing.
• Grows best at moderate temperatures.
• Demonstrates temperature response in a eukaryotic microbe.

1. Escherichia coli

• Gram-negative, rod-shaped, lactose fermenter (produces acid and gas).
• MR-positive, VP-negative.
• Used to show mixed acid fermentation.

2. Enterobacter aerogenes (now Klebsiella aerogenes)

• Gram-negative, rod-shaped, VP-positive, MR-negative.
• Lactose fermenter.
• Demonstrates 2,3-butanediol fermentation.

3. Shigella spp.

• Gram-negative, non-motile rods.
• Does not ferment lactose.
• Important human pathogen; shows lactose-negative fermentation.

4. Salmonella spp.

• Gram-negative, motile rods.
• H₂S producer (black precipitate on HEK agar).
• Does not ferment lactose.

1. Escherichia coli (again)

• Used in ONPG test to demonstrate inducible enzyme activity (β-galactosidase).
• Shows gene regulation (lac operon ON/OFF) based on sugar availability.

2. Azotobacter vinelandii

• Gram-negative, free-living nitrogen fixer.
• Aerobic but uses high respiration to protect nitrogenase from oxygen.
• Grows on nitrogen-free media.

3. Rhizobium leguminosarum

• Gram-negative, symbiotic nitrogen fixer.
• Forms root nodules in legumes (e.g., peas, clover).
• Inside nodules, cells become pleomorphic and fix nitrogen under low-O₂ conditions with leghemoglobin.

1. Escherichia coli (host)

• Acts as the bacterial host for the T3 bacteriophage.
• Easily infected by lytic phages; produces clear plaques.

2. T3 Bacteriophage

• Lytic virus that infects E. coli.
• Causes host lysis and plaque formation.
• Used to quantify viral titer via soft agar overlay method.

1. Lactobacillus spp.

• Gram-positive, rod-shaped, catalase-negative.
• Produces lactic acid (acid dissolves CaCO₃ on YDC agar → clear zone).
• Present in yogurt, pickles, vaginal flora.

2. Leuconostoc, Lactococcus, Streptococcus, Enterococcus

• All part of the lactic acid bacteria group.
• Differ in fermentation: homolactic vs. heterolactic.
• Found in dairy, fermented vegetables.

3. Cyanobacteria (e.g., Anabaena, Oscillatoria)

• Prokaryotic, photosynthetic, some fix nitrogen.
• Found in aquatic ecosystems.

4. Algae (e.g., Chlamydomonas, Spirogyra)

• Eukaryotic, contain chloroplasts, conduct oxygenic photosynthesis.

Common Lab Questions and Explanations

How temperature affects microbial enzymes:

Cold temperatures slow metabolism; hot temperatures may denature proteins.

Why incubate at different temps:

To determine optimal growth temperature and microbial classification (e.g., mesophile).

Why multiple media used (EMB, MAC, HEK):

Different bacteria grow and appear differently; each medium is selective and differential.

Why pH indicators used:

Color change (e.g., red to yellow) signals acid from fermentation.

Durham tubes purpose:

Collect gas from fermenting microbes — shows gas production.

What MR/VP tells you:

Metabolic pathway used for glucose fermentation.

Why ONPG used instead of lactose:

ONPG is cleaved by β-galactosidase but doesn’t require permease — gives faster, clearer results.

Why Azotobacter and Rhizobium are grown on nitrogen-free media:

To force bacteria to fix nitrogen for survival — confirms their metabolic ability.

Function of leghemoglobin:

Oxygen carrier that keeps nodules anaerobic so nitrogenase isn’t inactivated.

Why Gram stain shows different morphology in nodules vs. free-living cells:

Symbiotic state changes cell shape and size.

Why soft agar used in plaque assay:

Allows diffusion of phages and local infection of bacteria, creating visible plaques.

How to interpret plaque counts:

Only plates with 30–300 plaques are accurate for titer calculation.

Why OD600 readings matter for the growth curve:

Help generate the actual curve and identify phases of growth.

Importance of generation time:

Measures how fast a microbe replicates — critical for understanding its behavior in infection or production.

Why YDC plates contain calcium carbonate:

Acid dissolves the CaCO₃ → clear halo = positive acid production.

Homolactic vs. Heterolactic fermentation:

Homo = lactic acid only; Hetero = lactic acid + gas/alcohol.

What Durham tube gas bubble tells you:

Heterolactic fermentation.

Why serial dilutions done:

To reduce cell density for accurate CFU counts.