Microbiology Lab Essentials: Bacterial Growth, Metabolism & Identification

Microbiology Lab Practice Questions

What is the optimal temperature range for Bacillus stearothermophilus?

Answer: 55°C (a thermophile)

Which organism produces red pigment at 25°C?

Answer: Serratia marcescens

What does no pigment but visible turbidity in S. marcescens at 37°C suggest?

Answer: Growth occurred, but pigment production is temperature-sensitive.

What is the term for bacteria that grow best at cold temperatures but also at room temperature?

Answer: Psychrotroph (e.g., Pseudomonas fluorescens)

What color does E. coli appear on MacConkey agar?

Answer: Pink (lactose fermenter)

Which test detects mixed acid fermentation?

Answer: Methyl Red (MR) test

What does a black precipitate on HE agar indicate?

Answer: H₂S production (e.g., Salmonella)

Which organism is non-lactose fermenting and H₂S negative?

Answer: Shigella sonnei

What do cracks or bubbles in a TSI slant indicate?

Answer: Gas production during fermentation

What test result would you expect for Enterobacter aerogenes in a VP test?

Answer: Positive (red color)

Which bacteria swarm across the plate and produce H₂S?

Answer: Proteus mirabilis

What does a yellow ONPG result indicate?

Answer: β-galactosidase activity (indicating lac operon induction)

What sugar represses the lac operon?

Answer: Glucose

What conditions must be met for the lac operon to be active?

Answer: Lactose must be present, and glucose must be absent.

What does a negative ONPG test result look like?

Answer: Colorless (indicating no β-galactosidase enzyme activity)

What type of medium is used to isolate nitrogen-fixing bacteria?

Answer: Nitrogen-Free Mannitol Agar

Why do only nitrogen-fixing bacteria grow on this medium?

Answer: The medium lacks a nitrogen source; therefore, only bacteria capable of fixing atmospheric nitrogen can survive and grow.

Name a common free-living nitrogen fixer isolated from soil.

Answer: Azotobacter spp.

What is the role of mannitol in nitrogen-free medium?

Answer: Mannitol serves as a carbon source for bacterial growth.

How do you confirm growth is from nitrogen-fixing bacteria?

Answer: Confirmation involves Gram staining and additional biochemical testing of isolated colonies.

Key Microbiology Concepts & Definitions

Lab 8: Temperature and Microbial Growth

• Psychrophile: Microbe that grows best between 0–20°C.
• Mesophile: Grows best between 20–45°C (includes most human pathogens).
• Thermophile: Prefers high temperatures, 45–80°C.
• Hyperthermophile: Grows above 80°C.
• Psychrotroph: Can grow at cold temperatures but prefers 20–30°C.
• Optimal Temperature: Temperature where the growth rate is highest.
• Pigment Production: A temperature-dependent trait (e.g., red pigment from Serratia marcescens at 25°C).

Lab 9: Gram-Negative Bacterial Fermentation Patterns

• Enteric Bacteria (Enterobacteriaceae): Gram-negative, rod-shaped, facultative anaerobes that ferment glucose and reduce nitrate.
• Lactose Fermenter: Bacterium that breaks down lactose (e.g., E. coli), appearing pink on MacConkey agar.
• Non-Lactose Fermenter: Does not ferment lactose (e.g., Shigella), colonies remain colorless.
• MR (Methyl Red) Test: Detects mixed acid fermentation; a red result indicates positive.
• VP (Voges-Proskauer) Test: Detects neutral by-products (2,3-butanediol); a red result indicates positive.
• TSI (Triple Sugar Iron): Test for glucose, lactose/sucrose fermentation, gas, and H₂S production.
• H₂S Production: Results in a black precipitate; seen in Salmonella and Proteus.

Lab 10: Enzyme Induction in E. coli

• Lac Operon: Gene system in E. coli for lactose metabolism; induced when lactose is present and glucose is absent.
• Inducible Enzyme: Enzyme produced only in the presence of a specific substrate (e.g., β-galactosidase).
• β-galactosidase: Enzyme that hydrolyzes lactose into glucose and galactose.
• ONPG: A lactose analog that turns yellow if β-galactosidase is present (ONP = yellow product).
• Toluene: Chemical used to permeabilize E. coli membranes so ONPG can enter.
• Positive Induction: Lac operon is turned ON, leading to a yellow color from ONPG breakdown.

Lab 11: Nitrogen-Fixing Bacteria

• Nitrogen Fixation: Conversion of atmospheric nitrogen (N₂) into ammonia (NH₃).
• Nitrogenase: Enzyme complex that fixes nitrogen; sensitive to oxygen.
• Free-Living Nitrogen-Fixers: Bacteria that fix nitrogen without plant hosts (e.g., Azotobacter).
• Symbiotic Nitrogen-Fixers: Live in plant root nodules (e.g., Rhizobium in legumes).
• Cysts: Dormant, thick-walled form of Azotobacter seen in wet mounts.
• YEM Medium: Yeast Extract Mannitol medium; supports nitrogen-fixer growth without added nitrogen.
• Leghemoglobin: Oxygen-binding protein in root nodules that protects nitrogenase from oxygen.

Detailed Lab Procedures & Outcomes

Lab 8: Temperature and Microbial Growth

This lab tested how different temperatures affect bacterial growth. Broth tubes were inoculated and incubated at 4°C, 25°C, 37°C, and 55°C. Growth was observed by turbidity. Each organism exhibited a different optimal temperature range. Serratia marcescens produced red pigment only at 25°C, demonstrating that enzyme activity (not just growth) is temperature-sensitive.

Lab 9: Fermentative Patterns of Gram-Negative Bacteria

Students identified Gram-negative enteric bacteria using selective media and fermentation tests. MacConkey agar distinguished lactose fermenters (pink colonies) from non-fermenters (colorless). Hektoen Enteric agar showed H₂S production (black colonies). Biochemical tests like MR, VP, and TSI were used to determine sugar fermentation, gas, and sulfur production patterns. These results helped differentiate bacteria such as E. coli, Salmonella, and Shigella.

Lab 10: Enzyme Induction in E. coli

This lab demonstrated how the lac operon in E. coli controls enzyme production. Cells only produced β-galactosidase (an inducible enzyme) when lactose or ONPG was present and glucose was absent. Toluene was used to permeabilize the cells. If ONPG was cleaved, a yellow color appeared, indicating enzyme induction. This experiment illustrated how gene expression can be regulated by environmental conditions.

Lab 11: Isolation of Nitrogen-Fixing Bacteria

Soil samples were used to isolate nitrogen-fixing bacteria using nitrogen-free mannitol agar. Only bacteria capable of fixing atmospheric nitrogen (like Azotobacter) could grow on this medium. Colonies were Gram stained and viewed by wet mount or phase contrast microscopy. Students also learned about Rhizobium, which fixes nitrogen in legume root nodules through a mutualistic symbiosis.

Characteristics of Key Microorganisms

Lab 8: Temperature and Growth Organisms

Pseudomonas fluorescens

• Psychrotroph
• Grows well at 4°C and 25°C
• Gram-negative rod
• Common in soil and water

Serratia marcescens

• Mesophile
• Produces red pigment (prodigiosin) at 25°C only
• Gram-negative rod
• Opportunistic pathogen

Bacillus stearothermophilus

• Thermophile
• Grows best at 55°C
• Gram-positive rod, spore-former
• Common in hot springs and soil

Saccharomyces cerevisiae

• Yeast (eukaryote)
• Grows at 25–37°C
• Used in baking and alcohol fermentation

Lab 9: Gram-Negative Enteric Organisms

Escherichia coli

• Lactose fermenter (pink on MacConkey agar)
• MR+ / VP−
• No H₂S production
• Gram-negative rod
• Common gut bacterium, indicator of fecal contamination

Enterobacter aerogenes

• Lactose fermenter
• MR− / VP+
• Gram-negative rod
• Found in soil, water, and the gut

Salmonella typhimurium

• Non-lactose fermenter
• H₂S producer (black on HE agar)
• MR+ / VP−
• Pathogen causing foodborne illness

Shigella sonnei

• Non-lactose fermenter
• No H₂S production
• MR+
• Gram-negative rod
• Causes dysentery

Proteus mirabilis

• Non-lactose fermenter
• Strong H₂S production
• Swarming motility
• Urease positive
• Opportunistic pathogen

Lab 10: Enzyme Induction Organism

Escherichia coli

• Model organism for studying gene regulation (lac operon)
• Produces β-galactosidase when induced
• Used with ONPG to test enzyme induction
• Lac operon turned off in the presence of glucose

Lab 11: Nitrogen-Fixing Organisms

Azotobacter spp.

• Free-living nitrogen-fixing bacterium
• Gram-negative
• Forms cysts (phase-bright under microscope)
• Found in soil
• Grows on nitrogen-free medium

Rhizobium spp.

• Symbiotic nitrogen fixer (in legumes)
• Lives in root nodules
• Gram-negative, pleomorphic
• Requires a plant host to fix nitrogen efficiently

Microbiology Culture Media Types

General-Purpose Media

Nutrient Agar (NA)

• Used for general growth of non-fastidious organisms.
• Example: Human microbiome sampling (Staphylococcus spp.).

Nutrient Broth

• Liquid form of general-purpose media.
• Used in Lab 8 to test microbial growth at different temperatures.

Selective & Differential Media

MacConkey Agar

• Selective for Gram-negative bacteria (inhibits Gram-positives).
• Differential for lactose fermentation:
  • Pink = lactose fermenter (E. coli)
  • Colorless = non-fermenter (Shigella, Salmonella)

Hektoen Enteric (HE) Agar

• Selective for Gram-negative enterics.
• Differential for H₂S production (black colonies) and lactose fermentation (orange/salmon colonies).

Mannitol Salt Agar (MSA)

• Selective for Staphylococcus spp. (high salt concentration).
• Differential for mannitol fermentation:
  • Yellow = S. aureus
  • Red = S. epidermidis

Biochemical Test Media

MR-VP Broth

• Used for Methyl Red and Voges-Proskauer tests.
• Detects types of glucose fermentation (acidic or neutral by-products).

TSI (Triple Sugar Iron) Agar

• Tests for fermentation of glucose, lactose/sucrose, gas production, and H₂S production.
• Yellow butt = glucose fermentation; black = H₂S.

Enzyme/Induction Assay Media

ONPG Test Broth

• Used in E. coli enzyme induction experiments.
• Turns yellow if β-galactosidase is present.

Lactose and Glucose Broths

• Used to test lac operon induction versus repression.
• Glucose represses enzyme production; lactose induces it.

Nitrogen Fixation Media

Nitrogen-Free Mannitol Agar

• Used to isolate nitrogen-fixing bacteria (like Azotobacter).
• No nitrogen source; only organisms that fix N₂ can grow.

YEM (Yeast Extract Mannitol) Medium

• May be used to grow Rhizobium in symbiosis studies.
• Contains yeast extract for general nutrition, mannitol as a carbon source.

Food Science / Fermentation Media

YDC (Yeast Dextrose Calcium Carbonate) Agar

• Used to culture lactic acid bacteria.
• Clearing around colonies = acid production (dissolves CaCO₃).