for practicum

Differential Stains

  • Understanding of differential stains is crucial for identifying microbial cell structures.
  • Key types of stains include Gram stains and acid-fast stains.

Gram Stain Methodology

  • Primary Stain: Crystal violet
  • Mordant: Gram's iodine
    • Iodine reacts with crystal violet, forming a complex that crystallizes and retains the dye in Gram-positive cells.
  • Decolorizing Agent: Alcohol (specifically ethyl alcohol or reagent alcohol)
    • Removes the outer membrane of Gram-negative cells and allows the dye to wash out.
  • Counterstain: Safranin
Observations of Cell Wall Types

  • **Gram Positive Cell Wall: (Purple)

    • Composition:** Thick peptidoglycan layer
    • Retains the purple color after washing with alcohol due to crystallized dye.

  • **Gram Negative Cell Wall: (Pink/Red)

    • Composition:** Thin peptidoglycan layer sandwiched between an outer lipid membrane.
    • Alcohol washes away the purple crystal violet, and the counterstain imparts a pink/red color.

Acid-Fast Staining

  • Developed by: Robert Koch
  • Primary Stain: Carbolfuchsin
  • Decolorizing Agent: Acid alcohol
  • Counterstain: Methylene blue
Unique Characteristics of Acid-Fast Cells
  • Main challenge: Presence of mycolic acid in cell walls, making them waxy and resistant to staining.
  • Staining Characteristics: If a bacterium is acid-fast, it appears red/pink under the microscope.
  • Example: Mycobacterium tuberculosis can be visualized as pink “spaghetti” on a blue background.

Spore Staining

  • Organisms That Produce Spores:
    • Bacillus (aerobic) and Clostridium (anaerobic)
  • Primary Stain: Malachite green
  • Counterstain: Safranin
  • Observation: Vegetative cells appear pink, while spores appear green.

Capsule Staining

  • Not all bacteria produce capsules, which serve as protective layers.
  • Staining Method: Maneval’s method
  • Primary Stain: Congo red (stains background)
  • Secondary Stain: Maneval stain (changes Congo red to blue)
    • Allows visualization of the capsule, if present, by contrasting colors.

Motility Testing

  • Methods Used:
    • Soft agar method
    • Hanging drop method
  • Hanging Drop Explanation:
    • Drop of culture hangs from a cover-slip, examined for live organisms, requiring reduced light to visualize.
    • Non-stained organisms may require ambient light adjustments.

Characterizing Bacterial Growth

  • Observations include:
    • Growth rate, form on slants, and colony color (solid, opaque, diffusable pigments).
    • Methods of isolation include streak plate isolation and pour plate techniques, aimed at isolating pure cultures from mixed samples.

Anaerobic Cultivation

  • Equipment Used: BBL jar
    • Contains a gas pac to remove oxygen by producing water.
    • Indicator for Anaerobic Conditions: Methylene blue strip changes color based on oxygen presence.

Temperature and pH Implications

  • Key measurements: Thermal death point, thermal death time, decimal reduction time.

Quantitative Bacterial Enumeration

  • Counting CFUs and calculating original concentrations using formulas from lab manual.

Antimicrobial Testing

  • Media: Mueller-Hinton agar for susceptibility testing.
  • Tests Conducted: Kirby-Bauer test, E-test, broth dilution method for various antibiotics.
Definitions and Terminology
  • MIC (Minimum Inhibitory Concentration): Determined by observing the zone of inhibition around disks on agar plates.

Selective Media

  • Blood Agar: Tests for hemolysis (alpha, beta, gamma) relating to bacterial ability to lyse red blood cells.
  • McConkey Agar: Selects for Gram-negative bacteria, identifies lactose fermenters (pink growth) versus non-fermenters.
  • Phenylethyl Alcohol Agar: Selects for Gram-positive organisms.

Hydrolysis Tests

  • Starch Hydrolysis: Use Lugol's iodine to determine starch breakdown. Clear zones indicate breakdown by amylase.
  • Casein Hydrolysis: Protein breakdown visualized on plate, creating clear zones.
  • Tributyrin Plate: Tests for lipids, indicating breakdown by lipases.
  • DNase Plate: Hydrolysis tested by presence of pink color upon reaction with reagents.

Indol Test and Urea Hydrolysis

  • Indol Test: Checks for tryptophan breakdown producing significant metabolites; requires Kovac’s reagent for visualization.
  • Urea Hydrolysis: Positive test indicates pink coloration due to urease activity.

MRVP Test

  • Methyl Red and Voges-Proskauer tests for mixed acid fermentation and butanediol production.

Simmons Citrate Test

  • Observations for utilization of citrate as sole carbon source. Indicator changes from green to blue for positive results.

Gel Electrophoresis Practices

  • Procedures for loading and running gels with DNA samples, noting the roles of loading dye and electric current in separation.

Outbreak Investigation

  • Investigative methods involving media selection for isolating pathogens; examples include Salmonella from food samples.