MICRO LAB EXAM Review questions

Week 1

Exercise 1: Ubiquity of Microbes

• Ubiquity:

  • found everywhere

• Reason for Ubiquity:

  • Microorganisms are able to thrive in diverse environments due to their high adaptability due to rapid reproduction and genetic variation

  • small size

  • ability to form endospores or cysts: withstand harsh conditions

  • various metabolic pathways.

• Growing Microorganisms from Air:

  • Techniques to isolate and grow microbes from the air include using Petri dishes with culture media and incubating them to allow microbial growth.

  • TSA: allow any microbe

  • PDA: target fungi

  • exposing nutrient agar plates

Exercise 3: Preparing a Wet Mount

• Definition of a Wet Mount:

  • laboratory technique where a specimen is placed in a liquid suspension (usually water) and covered with a glass slide for microscopic examination.

• Observations Possible with a Wet Mount:

  • It allows for the observation of living microorganisms

  • observe if motile, size shape, and arrangement

  • cellular structures like nuclei in eukaryotic cells.

Week 2

Exercise 7: Streak Plate

• Objective/Aim of Streaking a Plate:

  • The primary goal is to isolate pure/specific colonies from a mixed culture, allowing for further study of individual species.

  • Done by spreading a culture of mixed organisms so that it becomes diluted over the agar plate then it is incubated

  • petri plate containing type of sterile nutrient agar (TSA, BHI,etc.)

• Definition of a Colony:

  • A colony refers to a visible mass of microorganisms that arise from a single mother cell (asexually reproducing) and contain genetically identical cells.

Exercise 8: Quantification of Bacterial Number in Milk

• Serial Dilution:

  • Standard plate count method: involves diluting a sample in a stepwise manner to reduce the concentration of LIVE bacteria to countable numbers.

  • important for food e.g. milk

• CFU Calculation:

  • CFU (Colony Forming Units) is calculated by counting colonies on an agar plate and factoring in the dilution to determine the concentration of bacteria in the original sample.

  • CFU/mL = (# of colonies x Dilution factor) / volume plated

Week 3

Exercise 11: Simple Smear Preparation

• Smear Preparation:

  • This process involves spreading a aqueous suspension of microbial culture on a slide to prepare it for staining and microscopic viewing.

• Performing Simple Smear Preparation:

  • Key steps include spreading the sample, allowing it to air dry, and then heat fixing the slide.

• Steps

  • drop of water on slide

  • add bacteria via inoculating loop

  • spread and air dry

  • heat-fix for adherence

  • add 2-3 drops methyl blue then rinse after 1-2 min

Exercise 12: Gram Stain

• Gram Stain Observations:

  • Cells will appear either purple (Gram-positive) or pink (Gram-negative) under the microscope based on the characteristics of their cell walls following staining.

  • Gram (+): purple due to thick peptidoglycan retaining crystal violet

    • resist decolorizer

  • Gram (-): Pink/red due to thin peptidoglycan, absorbing safranin

    • less resistant to decolorizer (alcohol)

    • take up counterstain (safranin)

• Precautions for Accurate Results:

  • over-decolorization or improper staining.

  • ensure proper smear and heat fixing

  • use fresh reagents

  • dont spray directly when rinsing

Week 4

Exercise 13: Capsule Stain

• Function of the Capsule:

  • The capsule provides protection against phagocytosis

  • osmotic barrier

  • helps in adherence to surfaces

  • aid in nutrient retention.

  • prevent desiccation

• Negative Stain Definition:

  • staining technique that colors the background but leaves the cells transparent, allowing visualization of structures like capsules (unstained).

  • colors everything except structures wished to visualize

Exercise 22: Streptococci in the Mouth

• Differentiation of Hemolysins:

• Alpha hemolysin:

  • partially lyses red blood cells, producing a greenish discoloration around the colonies.

• Beta hemolysin:

  • completely lyses red blood cells, resulting in a clear zone around the colonies.

• Gamma hemolysin:

  • no hemolysis of red blood cells, maintaining the original color of the blood agar.

Week 5

Exercise 14: Bacterial Endospore Stain

• Endospore Production:

  • Bacteria produce endospores as a survival mechanism when environmental conditions become unfavorable. e.g nutrient depletion

• Role of Endospores:

  • Endospores protect the bacterial DNA from extreme conditions like heat, desiccation, chemical damage, and radiation

• Endospore Appearance under Microscope:

  • Endospores may appear as refractive bodies within the bacterial cells during microscopic examination.

  • spores: oval/spherical, green due to malachite green staining, still within red-stained mother

  • vegetative cells: ord-shaped, red due to safranin counterstain

  • Free form: stain green

Exercise 17: Selective and Differential Media

• Definitions:

  • Selective Media:

    • Contains agents that inhibit the growth of certain microbes while allowing others to grow.

  • Differential Media:

    • Contains indicators that allow for the differentiation of closely related organisms based on biochemical reactions. differentiates 2 groups. Bacteria appear differently.

• Examples - MSA and MAC:

  • MSA (Mannitol Salt Agar):

    • Selective for Gram-positive bacteria. Staphylococcus species (high salt tolerance) and differential for S. aureus (mannitol fermentation turning agar yellow).

  • MAC (MacConkey Agar):

    • Selective for Gram-negative bacteria and differential for lactose fermentation (pink colonies) from non-fermentors (colorless colonies).

    • inhibits growth of Gram +

Week 6

Exercise 20: IMViC Test

identify enteric bacteria (Gram-negative)

• Tests Included:

  • Indole Test:

    • detects tryptophan breakdown in indole

  • Methyl Red Test

    • identifies strong acid production from glucose fermentation

    • red: positive

  • Voges-Proskauer Test

    • detects acetoin production from glucose fermentation

    • rose color: positive

  • Citrate Utilization Test:

    • determines if bacteria can use citrate as a sole carbon source

    • green → royal blue = positive

• What They Test For:

  • Each test examines different biochemical properties like indole production, acid production, and utilization of citrate as a carbon source.

Exercise 21: Urea Hydrolysis

• Definition and Explanation:

  • Urea hydrolysis tests the ability of organisms to hydrolyze urea, using urease, into ammonia and carbon dioxide, which increases the pH of the medium.

  • important decomposers

• Media and Positive Species:

  • Media used:

    • stuarts urea broth containing phenol red as pH indicator or agar

  • Positive Test:

    • bright pink color due to alkaline pH (ammonia production)

  • Common positive organisms:

    • Proteus and Helicobacter

Exercise 24: Carbohydrate Fermentation

• Interpreting Results: Results are interpreted based on color changes in the media and gas production, which indicates whether the organism can ferment specific carbohydrates.

• Yellow broth:

  • acid production (+) fermentation

• Pink Broth:

  • peptone utilization, negative carbohydrate

• Red broth

  • no fermentation

• Gas bubble in durham tube

  • gas production (fermentation with gas)

• no color change

  • no fermentation = negative