Gram Staining and Bacterial Structure Quiz

Food Microbiology - FST 101

• Course: Food Microbiology (FST 101)
• Institution: Central Bicol State University of Agriculture
• Instructor: Alessandra M. Domanico, Assistant Professor IV
• Topics Covered: Gram Staining, Endospore Formation, Catalase Test, Motility, and Fermentation Test

Gram Staining

• Developed by: Hans Christian Gram (1853), a Danish physician
• Initial Work: Studied botany at the University of Copenhagen and discovered that certain stains are preferentially absorbed by bacterial cells.
• Early Process: His initial work focused on Streptococcus pneumoniae and Klebsiella pneumoniae.
• Refinement by: Carl Weigert (1845-1904), a German pathologist, added a final step using safranin as a counterstain.
• Importance: Gram staining remains the most crucial method to distinguish between major bacterial classes.

Gram Staining Procedure

• Preparation of Smear:
  • Spread the bacterial culture thinly on a glass slide.
  • Air dry the smear.
  • Fix the smear by passing the slide through a flame.
• Staining Procedure:
  • Flood the heat-fixed smear with crystal violet for 1 minute. All cells will be purple.
  • Add iodine solution for 3 minutes. All cells remain purple.
  • Decolorize with alcohol briefly (about 20 seconds).
  • Gram-positive cells remain purple, gram-negative cells become colorless.
  • Counterstain with safranin for 1-2 minutes. Gram-positive cells are purple, gram-negative cells are pink to red.

Gram Staining Results

• Gram-positive Bacteria: Appear purple under a microscope
• Gram-negative Bacteria: Appear pink to red under a microscope

Relationship of Cell Wall Structure to Gram Stain

• Gram-positive Bacteria: Possess a thick peptidoglycan mesh in their cell wall (50-90%). This thick layer prevents the decolorizing agent from extracting the crystal violet.
• Gram-negative Bacteria: Have thinner peptidoglycan layer (10%) and an additional outer membrane containing lipids. This outer membrane allows the decolorizing agent to easily wash away the crystal violet.

Endospore Formation

• Formation Trigger: Endospores form in response to unfavorable environmental conditions, commonly nutrient depletion.
• Spores vs. Vegetative Cells:
  • Vegetative Cells: Active, living cells that ingest nutrients and secrete waste materials.
  • Spores: Dormant cells (not actively metabolizing) highly resistant to heat, cold, chemicals, and radiation. This is a survival mechanism, allowing the bacterium to endure harsh conditions and resume its vegetative stage later.

Endospore Formation Diagram

• A series of detailed steps depict the formation process of endospores.
• The cycle includes steps like chromosome duplication, septation, spore coat formation, and the release of the mature endospore.

Endospore Formers

• Lists of various bacteria types that form endospores, including species and groups of bacteria (aerobic types, personnel contamination, and water/water sources).

Catalase Test

• Purpose: To detect the presence of the enzyme catalase, which is important to differentiate aerobic and anaerobic bacteria.
• Catalase Enzyme Function: A common enzyme in organisms surviving in oxygen, catalyzes the decomposition of hydrogen peroxide into water and oxygen.
• Principle of Catalase Test:
  • A small bacterial inoculum is introduced into hydrogen peroxide.
  • If catalase is present, rapid oxygen release causes bubbling.
  • Lack of catalase produces either no or a very weak bubble response.

Motility

• Defined: The ability of an organism to move by itself.
• Types: Propeller-like flagella in bacteria or gliding motility through special fibrils.
• Flagellar Structure: The flagella is rigid and helical and generates motility through rotation.
• Motility Testing: Demonstrated by observing the bacteria in a semi-solid agar medium.

Lactose Fermentation Tests

• Purpose: Identifying bacteria's ability to ferment lactose as a carbon source.
• Mechanism: If lactose is fermented, acid byproducts are generated, causing a pH drop in the medium, noticeable by a change in color using a pH indicator.

Requirements for Carbohydrate Tests

• Media: Carbohydrate broth, containing specific sugars (i.e., dextrose, sucrose, and lactose).
• Indicators: Include Andrade's indicator, Phenol Red, Bromocresol Purple, or Bromothymol Blue to determine the carbohydrate fermentation.
• Durham Tube: A small inverted tube used to detect gas production(CO2) during fermentation by observing the presence of bubbles.

Methyl Red and Voges-Proskauer (MR-VP) Test

• Purpose: Characterize bacterial metabolism through differentiation and classification.

Bacterial Identification and Classification

• Basis: Gram reaction, endospore formation, morphology, and other distinguishing features.
• Methodology: Often employs a dichotomous key to track similarities and differences among bacterial genera in a systematic manner.

Test your knowledge on Gram staining and bacterial structures with this quiz. Covering components of Gram-positive and Gram-negative bacteria, the quiz also delves into endospore formation and germination processes. Perfect for students of microbiology looking to reinforce their understanding.