Microbiology Lab Techniques

• These notes cover various microbiology lab techniques and concepts.

Streak-for Isolation Technique

• Uses an inoculating loop to dilute an organism onto a nutrient agar plate.
• This technique leads to macroscopic colonies on the plates.

Types of Medium

• Include solid, semi-solid, and liquid (broth) mediums.

Turbidity

• Cloudiness or growth observed throughout a liquid medium.

Sterilization

• Achieved through methods like electric incineration.

Subculturing

• The process of transferring a microorganism from one medium type to another.
• Soy agar is used to promote the healthy growth of bacteria.

Procedure Guidelines

• Sterile swabs must be moistened before collecting dry samples.
• Agar plates should be streaked inverted and placed on top of the cap, or streaked and immediately closed with the cap.
• When opening broth, always pass the top through the flame, dip, reflame, and cap.
• Plates are incubated upside down to prevent drips from causing colonies to merge.
• When performing a streak for isolation, make sure to overlap the streaks.

Broth Growth Characteristics

• Pellicle: A thin layer forms at the top of a broth.
• Membrane: A thick layer forms at the top of a broth.
• Flocculent: Characterized by large clusters of organisms.
• Turbid: Indicates cloudy growth.
• Flaky: Shows the presence of flakes.
• Granular: Characterized by small flakes.
• Sediment: Accumulation at the bottom.

Growth Results

• Growth at the surface of the test tube indicates a preference for oxygen.
• Growth at the bottom indicates that the bacteria do not require oxygen.
• Bacteria can stick together, making it hard to confirm a single microorganism culture.

Compound Microscope

• Uses two lenses: ocular and objective.
• Condenser: Located below the stage, it aligns and focuses the light.
• Aperture iris diaphragm: Controls the amount of light passing through the condenser.
• Kohler Illumination: Should be adjusted at the beginning of every microscope use.

Microscope Results

• Maximum magnification: 100x.
• Higher magnifications can result in blurry images.
• Immersion Oil is used to improve clarity.
• Field iris diaphragm regulates light.
• Resolution defines the resolving power of the microscope.

Bacterial Motility

• Brownian Movement: Erratic movement of organisms in an aqueous environment due to molecular interactions.
• True Motility: Requires a functioning flagella, but some flagellated organisms might be non-motile.
• Hanging Drop Method: Uses vaseline on the cover slip to create a hanging drop on a depression slide.
• Organisms are visible near the edge of the drop.

Factors Affecting Motility

• Temperature, light, food availability, and moisture.
• Adjusting the aperture iris helps to focus the light on the organism.

The Winogradsky Column

• Illustrates the interactions between environmental conditions and microbial activity.
• Shredded paper: provides carbon source in the form of cellulose (electron donor).
• Calcium carbonate: provides CO2.
• Calcium sulfate: acts as a source of sulfur, which is an electron acceptor.
• Columns are placed in indirect sunlight.

Oxygen Gradient

• Cellulose depletion of O2 in the mud creates an O2 gradient, with low O2 at the bottom and high O2 at the top.
• Aerobes & microaerophiles grow at the top, and anaerobes grow in the remainder of the column.

Sulfide Levels

• High H2S at the bottom and low H2S at the top due to sulfate-reducing bacteria.
• Black Area: Indicates where H2S reacted with iron in the mud.
• Green Sulfur Bacteria: Grow immediately above the dark layer.
• Purple sulfur Bacteria: Grow above the green layer due to low H2S tolerance.
• Purple Non-sulfur bacteria: Photoheterotrophs that grow in anaerobic low sulfide conditions.
• Photoheterotroph: Obtain energy from light but use organic acids from anaerobic bacteria as their carbon source.
• Sulfur oxidizing bacteria: Chemoautotrophs that oxidize H2S to sulfate, allowing them to produce their own materials (CO2).

Water Layer

• Contains cyanobacteria and algae, which use light for energy, release O2, and fix CO2.

Simple Staining

• Uses a single dye (crystal violet or methylene blue) to view microorganisms.
• Chromophores (colored cations) are positively charged and attracted to the negative bacterial surface.

Gram Staining

• Gram Positive: Retain the purple stain due to a thick peptidoglycan layer.
• Gram Negative: Easily decolorized by alcohol due to thinner and more lipid-containing layers, thus taking up the counterstain (pink).

Gram Staining Process

• Primary Stain: Crystal violet (purple).
• Mordant: Iodine stain (intensifies reactions, helping trap the color).
• Decolorizing Agent: 95% ethanol or isopropyl alcohol.
• Counterstain: Safranin (pink).
• Organisms are better seen when grown in broth.
• Gram Variable: Some stain purple, others stain red due to repeated staining.

Gram Staining Procedure

• Flood smear with crystal violet for 1 minute, then rinse.
• Flood with iodine for 1 minute, then rinse.
• Apply decolorizer until it runs clear, then rinse.
• Counterstain with safranin for 1 minute, then rinse.

Gram Stain Results

• E. Coli: Gram-negative.
• Staphylococcus epidermidis: Gram-positive.
• Bacillus Subtilis: Gram-positive.
• Mycobacterium Smegmatis: Gram-positive.
• Heat-fixation helps microorganisms stick to the slide during staining.
• The type of water used does not affect the staining process.

Endospore Staining

• Vegetative Cell: An actively growing bacterial cell.
• Spore Formation: Vegetative cells form spores under harsh conditions, splitting into a mother cell and a forespore.
• Spore Coat: The mother cell wraps around the forespore to form a protective coat.
• Endospore: A fully protected spore released when the mother cell breaks down.

Schaeffer-Fulton Method

• Stain with malachite green while heating for 3-5 minutes.
• Rinse with distilled water.
• Counterstain with safranin for 1 minute.
• Rinse and dry.

Endospore Stain Results

• Bacterial endospores protect the cell from harsh environments.
• Cells may appear gram-positive or negative, with the endospores appearing clear.

Acid-Fast Staining

• Used for mycobacterium due to high levels of waxy lipids in the cell wall.
• Acid-fast organisms have high percentages of waxy lipids in their cell walls.
• Simple staining is ineffective because dyes cannot penetrate the waxy cell wall.
• Utilizes Ziehls-Neelsen (uses heat) and Kinyoun Method (cold stain).
• Carbolfuchsin (red stain) stains the waxy cell wall and is not decolorized by acid-alcohol.
• Counterstain: Brilliant green (bluish-green).

Bacterial Nutrition and Growth

• Heterotroph: Uses an organic carbon source.
• Autotroph: Uses CO2 as a carbon source.
• Chemotroph: Energy source is chemical.
• Phototroph: Energy source is light.

Measurement Techniques

• Direct Measurement: Counting cells under a microscope using a counting grid.
• Indirect Measurement: Measuring cell mass (dry, wet, or turbidity) using a colorimeter or spectrophotometer.
• Absorbance is measured at 600nm.

Media Types

• Medium A: Davis minimal broth + 1% glucose.
• Medium B: Medium A + 1% nutrient broth.
• Always use a blank for calibration.
• Microorganisms used: E. coli and Staph epidermis.
• Direct measurement: Dilutions were made by diluting original bacteria broth into saline, then plate dilutions were made (beads to spread).
• Turbid cultures should be further diluted.
• Selective Media: Contains chemicals that inhibit the growth of some microbes.
• Differential Media: Distinguishes between different microbes.

Growth Inhibition

• Staph epidermidis and Salmonella enteritidis do not grow in methylene blue agar.
• E. coli, Klebiella aerogenes, and Shigella Sonnei do not grow in salt agar.
• Staph areus, staph epidermidis, and salmonella enteritidis do not grow in enteric agar.

Anaerobic Culture Techniques

• Anaerobes: Grow in the absence of oxygen.
• Facultative Anaerobes: Grow in both the presence and absence of oxygen.
• Strict Anaerobes: Only grow in the absence of oxygen.
• Thioglycollate Broth: Contains reducing agents like cysteine and sodium sulfhydryl groups that donate hydrogen to organic molecules.
• Mineral oil layer can be used to prevent oxygen from entering.

Brewer Anaerobic Jar

• Contains a gas generation package that produces hydrogen and carbon dioxide when water is added.
• Palladium pellets (catalyst): Combine hydrogen with oxygen to produce water.
• Blue methylene strip indicates the presence of oxygen; white indicates no oxygen.

Anaerobic Growth

• Peptu Mangus and Bacteroides are anaerobic.
• Oxygen can diffuse into tubes, creating an O2 gradient.
• Anaerobes grow at the bottom, and aerobes grow at the top.

Metabolic Activities of Bacteria

• Exoenzymes: Start the extracellular breakdown of microorganisms.
• Hydrolysis: Water breaks down large molecules.
• pH changes indicate metabolic activity.

Starch Hydrolysis

• Requires enzymes like amylase to break down starch into smaller molecules.
• Starch -> Dextrins -> Maltose -> Maltase -> Glucose.

Lipid Hydrolysis

• Lipases break down lipids to produce glycerol and fatty acids.
• Spirit Blue Agar: Contains vegetable oil; bacteria with lipase break down the oil, causing spirit blue to migrate to the cleared region.
• Some microorganisms metabolize carbohydrates in the presence or absence of oxygen.
• Durham Tube: Detects the production of gases caused by fermentation

Starch Hydrolysis Results

• Bacillus Subtilis: Produced amylase enzymes that break down starch, so iodine stayed clear.
• E. Coli: Did not break down the starch, causing the plate to darken.

Fat Hydrolysis Results

• Staphylococcus aureus: Produced lipases that broke down fats, resulting in growth with a halo due to the blue agar plate.

Wine Production

• Wine is produced by enzymatically degrading fruit sugars (fructose and glucose) into acetaldehyde and then alcohol by the yeast cells.
• Must: Crushed grapes used in wine making.
• Acidity: Liveliness to the taste; too acidic is sharp, and less acidic is bland.
• Higher amount of sugar dictates higher alcoholic content.
• Specific Gravity: Used to measure the amount of sugar in a solution.
• Fermentation has stopped when no carbon dioxide bubbles form.
• Wine is pasteurized to kill bacteria while preserving its flavor.

Coliform Analysis of Environmental Water

• Coliforms: Rod-shaped, gram-negative, facultative anaerobes found in poop and sewage.
• Large amounts of coliforms indicate the presence of other bacteria in the water.
• ECA Check Plust Easy Gel: Used to mix the sample with liquid gel, plate, and incubate.
• Results:
  • E. coli will appear dark blue.
  • Other coliforms will appear pinkish-purple green.
  • Salmonella will appear greenish.
  • Aeromonas will appear pink/red or uncolored.

Questions about common microbiology lab techniques. Covers streak plating, broth cultures, inoculation, and bacterial growth patterns. Aimed at understanding sterile techniques and culture analysis.