Lab 2: Ubiquity Sampling, Aseptic Technique, Streak for Isolation, Smear Prep, and Gram Stain

Aseptic Technique and Lab Safety

• Aseptic: free of living pathogens; Disinfect: reduce microbial numbers to safe levels; Sterile: complete removal of all microbes, including spores; Contaminate: introduce unwanted microbes
• Core goal: work without contaminating environment, personnel, or cultures; protect patient safety in clinical contexts and ensure valid lab results
• Key practices to insure aseptic technique:
  • Work area disinfection before starting; disinfect after completing work
  • Use properly sterilized bacteriological loops and needles
  • Correct culture tube handling, flaming of the mouth, and inoculation technique
  • Proper handling of Petri plates during inoculation
  • Hand washing before and after procedures
  • Correct disposal and organization of materials
• Practical rationale: prevent introduction of exogenous microbes, maintain pure cultures, and reduce risk of contamination or exposure
• Ethical/safety note: avoid airborne or environmental contamination; note warning on breathing issues or allergies from moldy/furry colonies on plates

Work Area Disinfection and Contamination Prevention

• First disinfection occurs upon entering the lab; aims to destroy vegetative cells and viruses but generally will not destroy endospores; not a sterilization step
• Physical scrubbing of the bench removes microorganisms; helps reduce bioburden
• Disinfection should also occur after completing work
• Additional guidelines:
  • Disinfect the work area before starting experiments
  • Wash hands after bench cleaning
  • Properly sterilize inoculating loops and needles
• Minimize environmental contamination by:
  • Only bringing cultures out when ready to work
  • Being organized and taking time; avoid clutter and rushing
  • Labeling everything first to save time and limit contamination
  • Opening culture tubes only near the incinerator opening; hold cap and tube with one hand
  • Keeping the cap on when not in use
  • Opening Petri plates only when inoculating to limit airborne microbes
  • Avoid transferring bacteria over class items
  • Dispose materials in proper bins and disinfect bench and wash hands after storage

Bacteriological Loops & Needles: Sterilization and Handling

• Transfers of culture material use inoculating loops and needles; sterilize before and after contact with culture
• Incinerator used to sterilize loop/needle until they glow orange
• Loop/needle must be cooled prior to use (minimum ~5 seconds)
• Re-sterilize after transferring cultures and before returning to bench to prevent environmental contamination
• Do not store loop/needle in incinerator; can melt rubber holder
• Return sterilized tool to bench top location

Pure Culture Techniques and Streak Plate Overview

• Pure culture: culture containing only one bacterial species; Mixed culture contains more than one organism
• Purposes of pure culture: definitive results about identity, DNA, antibiotic sensitivity
• Pure culture vs mixed culture: observe colonies originating from a single cell (clones) vs multiple species with varying colony morphologies
• Streak plate technique (a common pure culture method): spreads a small inoculum across the plate surface to thin out cells and separate individual cells into colonies; each colony derives from a single original cell
• Goal: obtain colonies that are homogeneous (pure) for downstream identification
• Practical implication: you cannot reliably treat disease without knowing the infectious agent's identity

Preparing Streak Plates: Step-by-Step (Aseptic Procedure)

• Materials: TSA plates, a 2-species TSB broth culture, bacteriological loop
• Core steps (summarized):
  1. Obtain a TSA plate and a mixed culture; gently shake culture tube to disperse bacteria without spilling
  • ext{shake gently; avoid tilting too vigorously to prevent spills}
  1. Sterilize the loop in the incinerator for 5 ext{ s}; allow to cool ~5 ext{ s} before use
  2. Open culture tube with cap held in little finger; hold mouth near incinerator opening for ~5 ext{ s} to clean opening and create convection currents
  3. Insert loop and collect a loopful of culture
  4. Heat-opener the culture tube again and replace the cap
  5. Lift plate lid slightly and angle over the plate surface to protect from contamination
  6. Gently touch the agar surface with the loop at a shallow angle to begin streaking
  7. Sterilize loop again before returning to bench
  8. Label the plate; invert and incubate
• Quadrant-based streaking: Plate is divided into Quadrants 1–4; loop is flamed and cooled between quadrants; first set of streaks in Q1; subsequent sets in Q2, Q3, Q4 to progressively thin the inoculum
• Figure reference: Diagram shows the progression of streak sets and the need to flame/cool between quadrants

Culture Tube Handling, Flaming, and Inoculation (Best Practices)

• Hold culture tubes in the non-dominant hand
• Before inserting a sterile, cooled loop/needle, remove the cap; mouth near incinerator opening to create convection currents
• Maintain an upright angle to minimize airborne contamination
• After inoculation, flame the mouth again and replace the cap; place broth tubes in a rack (not on bench)

Expected Results: Mixed Broth Culture Streaks and Pure Cultures

• Mixed broth culture streak results: Serratia marcescens and Escherichia coli
• Pure culture success: Should see separate, single-color colonies (distinct colonies representing single clones)

Smear Preparation for Staining

• A smear is an application of bacteria to a slide for staining
• A good smear is essential to visualize cell shapes/arrangements and microstructure
• Requirements for a good smear:
  • Thin smears to avoid cell crowding; improves visualization of stain results
  • Cells must adhere to slide to resist washing or shrinkage during staining
• Drying/Fixation:
  • Air dry to prevent cell shrinkage
  • Heat fix to adhere cells to the slide and prevent wash-off during staining
• Expected appearance: a slide that retains cells evenly and does not show excessive clumping

Staining Pads and Staining Tray Protocols

• Staining pads are used to hold stain; helps reduce splash risk when moving trays
• A properly used staining pad (for Gram staining) should last the entire class period
• Do not rinse staining trays; this creates staining waste and splash hazards
• Replace pads when full or at class end
• Each pad has two sides: fluffy and smooth; place with fluffy side up for better absorption
• Used pads go to the designated disposal bucket (e.g., a 5-gallon bucket)

Microscopy Care and Handling

• Carry the microscope with both hands: one under the base and one on the arm
• Set the microscope gently on the bench; avoid damaging cords
• Do not let the cord hang over the edge of the table
• When finished:
  • Lower the stage and condenser; remove the slide
  • Turn the rheostat to the lowest setting and switch off light
  • Lock the 4x (scanner) objective in place
  • Wipe objectives and any oil with Kimwipe and cleaning solution
  • Wrap the cord around the microscope
  • Have the instructor check the microscope before putting it away

Staining: Types and Concepts

• Staining basics:
  • Positive stains use positively charged chromogens (usually basic) that adhere to bacterial cells
  • Simple stains use a single chromogen to color all cells the same color
  • Differential stains use 2+ chromogens to differentiate bacteria based on biochemical properties

Gram Stain: Theory and Importance

• Gram stain is a differential stain that separates bacteria by cell wall structure
• Gram-positive bacteria: thick peptidoglycan layer, teichoic acids, no outer membrane; typically stain purple
• Gram-negative bacteria: thin peptidoglycan layer, lipopolysaccharide and outer membrane; typically stain pink/red after counterstain
• Gram staining provides rapid clinical information and is a foundational tool for identification and treatment decisions

Gram Stain Procedure: Step-by-Step and Color Outcomes

• General steps (as described in lab materials and video reference):
  1. Prepare a smear of bacteria on a slide
  2. Apply Crystal Violet (CV) stain; leave on for 30 ext{ s}; rinse off
  3. Apply Gram's Iodine (mordant); leave on for 60 ext{ s} (1 minute)
  4. Decolorize with Gram's alcohol; apply for a brief period (commonly 2-5 ext{ s}; some protocols use 5-10 ext{ s})
  • Rationale: Alcohol shrinks peptidoglycan pores in Gram-positive cells, trapping CV-I complexes; in Gram-negative cells, CV-I is washed out, rendering cells colorless
  1. Counterstain with Safranin; leave on for 30 ext{ s} to 60 ext{ s}; rinse
  • Gram-positive cells remain purple; Gram-negative cells appear pink/red after counterstain
  1. Blot and air-dry
• Note: The critical step is the decolorization; errors here lead to false positives/negatives
• Color results after each step:
  • After CV: Both Gram-positive and Gram-negative cells stained (purple/blue)
  • After iodine: CV-I complex forms and remains in both cell types
  • After decolorization: Gram-positive remain purple; Gram-negative become colorless
  • After counterstain: Gram-positive stay purple; Gram-negative become pink/red
• Reference: A video animation and step-by-step notes are provided in the lab manual and linked video

Gram Stain: Color Results and Data Recording

• Data recording examples (Table 2 reference):
  • Columns typically include: Name, Gram reaction (positive/negative), Shape, Arrangement, and a Sketch
  • Examples cited in lab materials include organisms such as Staphylococcus aureus, Neisseria sicca, Corynebacterium pseudodiphtheriticum, Enterococcus faecalis, Escherichia coli, Bacillus cereus
• Practical use: correlates Gram reaction with morphological characteristics to narrow down identity

Common Mistakes and Problems in Gram Staining

• Smear prep too thick:
  • Difficult to visualize cell shapes/arrangements
  • May hinder alcohol penetration causing false positive
  • Thick layers can trap decolorizing solution, leading to false negative
• Over-decolorization:
  • Leaving alcohol too long can remove color from Gram-positive cells (false negative)
  • Not decolorizing long enough can cause false positives or mixed results
• Culture age:
  • Gram-positive culture over 16–18 hours old can shrink peptidoglycan layers, causing false negatives

Morphology and Arrangement of Bacteria (General Observations)

• Gram-positive cocci in irregular clusters
• Diplococci (pairs) sometimes Gram-negative
• Gram-negative cocci singly or in tetrads
• Gram-positive: V formation or palisades patterns
• Gram-positive rods: singly or in short chains
• Gram-negative rods: singly, in short chains, or other arrangements
• (These patterns help with initial identification alongside Gram staining results)

Hemolysis and Blood Agar (BAP) Patterns

• Hemolysis terminology (Hemolysis on Sheep Blood Agar)
• A = beta-hemolysis (β-hemolysis): complete lysis of red blood cells; clear zone around colony
• B = alpha-hemolysis (α-hemolysis): incomplete lysis; greenish discoloration around colony
• C = gamma-hemolysis (γ-hemolysis): no red cell lysis; no color change around colony
• Beta-hemolysis (1): complete clearing, colorless zone around colony
• Alpha-hemolysis (2): zone of partial lysis with greenish discoloration
• Gamma-reaction (3): no hemolysis or discoloration around colony
• Practical significance: helps differentiate species (e.g., certain streptococci vs staphylococci) based on their effect on RBCs

Notes on Safety and Lab Conduct

• Moldy/furry colonies should not be opened due to allergy risks; use caution and avoid exposure to students with allergies
• Maintain cleanliness and proper disposal to reduce risk of contamination and exposure
• Instructors may check equipment (microscopes for oil, cleanliness of lenses) to ensure safe use
• Do not erase lab manuals; proper labeling and organization help track experiments

Additional Resources and References

• YouTube: Gram stain procedure (video animation): https://www.youtube.com/watch?v=_k3uuOpbQI8
• Lab manuals: Step-by-step Gram stain procedures, figure references, and table formats for data recording
• Diagnostic Microbiology context: Gram stain as a foundational diagnostic tool with implications for clinical decision-making and treatment choices

Summary of Key Concepts and Their Significance

• Aseptic technique and clean workspaces are essential to obtain reliable results and ensure safety; contamination can invalidate experiments and pose health risks
• Pure culture techniques, especially streak plating, enable isolation of single colonies from mixed cultures, which is critical for accurate organism identification and downstream testing
• The Gram stain differentiates bacteria based on cell wall structure and provides rapid, clinically actionable information; it is considered the gold standard for rapid bacterial identification
• Proper staining technique requires precise timing and handling; the decolorization step is the most critical and most often yields erroneous results if performed incorrectly
• Microscopy care and accurate data recording are essential for reproducibility and accurate interpretation of results
• Understanding colony morphology on Blood Agar (hemolysis patterns) aids in preliminary identification and can guide further testing

Quick Reference Timings and Numerical Details (LaTeX)

• Loop sterilization: 5 ext{ s}; loop cooling: 5 ext{ s}
• CV stain contact time: 30 ext{ s}
• Iodine mordant contact time: 60 ext{ s} (1 minute)
• Decolorization time: typically 2 ext{--}5 ext{ s} (some protocols 5 ext{--}10 ext{ s})
• Safranin counterstain: 30 ext{ s} to 60 ext{ s}
• Dehydration and drying: air-dry after staining; blot to remove excess water
• In Gram staining, decolorization is critical because improper timing can lead to incorrect results by either over- or under-decolorizing the cells

Connections to Foundational Principles

• Sterile technique reflects the fundamental microbiology principle of avoiding contamination to preserve sample integrity and ensure accurate interpretation
• The concept of a pure culture aligns with the idea that observed characteristics (morphology, staining, genetic features) come from a single lineage, enabling consistent identification
• Differential staining (Gram stain) uses chemical interactions with cellular components (cell wall structure) to distinguish organisms, illustrating the link between chemistry and biology in diagnostic microbiology
• Ethical and safety considerations emphasize responsibility in handling infectious agents and protecting people with allergies or sensitivities to environmental microbes