BIO_114 LAB 2

Lab Overview

  • Biology 114 Lab

    • Focus: The Diversity of Life: Kingdom Protista

Exercise A: Observing Colonies

  • Observe the growth of your colonies on the plates.

  • Answer the corresponding questions in your lab packet.

Exercise B: Bacterial Staining & Aseptic Technique

  • Aseptic Technique Procedures:

    • Start with hand washing and cleaning lab surfaces with disinfectant.

    • Turn off HVAC systems that create air currents.

    • Clean and sterilize all equipment before use.

    • Wash hands again after cleaning; wear sterile gloves.

    • Avoid contamination of the sterile work environment with non-sterile items.

Slide Preparation

  • Materials Needed:

    • Clean slide, label it.

    • Prepare smear with broth or solid cultures + water.

    • Air dry the slide before heat fixing to kill organisms and ensure adherence.

    • Common problems: Smear may be too thick, leading to washing off of the specimen.

Gram Staining Procedure Timing

  • Timing for Steps:

    1. Crystal violet: 1 minute

    2. Iodine: 2 minutes

    3. Alcohol wash (decolorization): 15 seconds

    4. Safranin (counterstain): 1 minute

  • Rinse with water between each step.

Gram Staining Steps

  • Procedure:

    • Step 1: Apply crystal violet and rinse after 1 minute.

    • Step 2: Apply iodine and rinse after 2 minutes.

    • Step 3: Alcohol wash for 15 seconds and rinse.

    • Step 4: Apply safranin for 1 minute.

  • Critical Timing:

    • Particularly during the alcohol wash to differentiate cell types.

Gram Stain General Theory

  • Mechanism:

    • Based on cell wall structure's ability to retain crystal violet during solvent treatment.

    • Gram-positive: Higher peptidoglycan, lower lipid content.

    • Gram-negative: Structure allows easier removal of the dye.

    • Iodine: Acts as a mordant forming the crystal violet-iodine complex aiding stain retention.

Decolorization Process

  • Impact of Decolorizer:

    • Ethanol and acetone dissolve lipid layer of Gram-negative bacteria, allowing stain leaching.

    • Thicker Gram-positive walls dehydrate, shrink, and close pores, preventing stain loss.

    • Critical timing to differentiate between bacterial types; prolonged exposure may lead to misclassification (Gram-variable).

Results Documentation

  • Record the following for each staining:

    • Type of staining

    • Name of stain

    • Shape of cells

    • Arrangement of cells

    • Color observed

Gram Stain Experiment

  • Conduct Gram staining on the cultured bacteria.

  • Document results through sketches and responses in the lab packet.

Kingdom Protista Overview

  • Eukaryotic Origins:

    • Protists are the first eukaryotic organisms; ancestors of all other eukaryotes.

    • Divided into four supergroups:

      • Excavates

      • Clade 'SAR'

      • Archaeplastids

      • Unikonts

Excavates

  • Features: An 'excavated' feeding groove along the cell body.

  • Groups Include:

    • Diplomonads

    • Parabasilids

    • Euglenozoans

  • These groups are often anaerobic and found in animal guts.

Clade 'SAR'

  • Largest Supergroup:

    • Divided into three groups:

      • Stramenopiles

      • Alveolates

      • Rhizarians (not covered)

    • Formerly known as Chromalveolata; now more refined and excludes some lineages.

Stramenopiles

  • Meaning 'made of straw'; includes many photosynthetic organisms and decomposers.

  • Subgroups:

    • Diatoms

    • Brown Algae

    • Golden Algae

    • Oomycetes (Water Molds)

Alveolates

  • Defined by the presence of alveoli (air sacs) beneath the membrane.

  • Subgroups:

    • Dinoflagellates

    • Apicomplexans

    • Ciliates

Archaeplastids

  • Photosynthetic ancestors of modern land plants.

  • Composed of:

    • Red Algae

    • Green Algae

Alternation of Generations

  • Unique life cycle in Archaeplastids, involving both multicellular haploid and diploid forms.

  • Sporophyte: Multicellular diploid undergoing meiosis for haploid spores.

  • Gametophyte: Multicellular haploid produces haploid gametes by mitosis which fertilize to form diploid zygotes.

Exercise C: Archaeplastids

  • Practice using textbooks to explore the alternation of generations.

  • Obtain slides and specimens for observation.

Unikonts

  • Most are heterotrophic organisms; ancestors to Fungi and Animals.

  • Groups:

    • Amoebozoans (including amoebas and slime molds)

    • Opisthokonts

  • Notable for one or no flagella presence.

Exercise F: Unikonts

  • Conduct slide observations of amoebozoans and slime mold life cycles.

  • Sketch and observe protists from pond water samples.

  • Complete Kingdom Protista outline in lab manual.

Exercise G – Brassica Rapa

  • Half exposed to Gibberellic Acid (GA) for observation over time.