3. Smear Preparation and Staining Techniques

Smear Preparation and Staining Techniques

• Course: MLT 1303 Lab Techniques Microbiology

• Learning Outcomes: 1.1, 1.4-1.6, 4.1, 4.7-4.11, 5.1

Identification of Bacteria

• Traditionally classified based on morphological characteristics

  • Macroscopic Appearance: Shape, color, texture

  • Microscopic Appearance: Cellular arrangement, shape, Gram stain characteristics

  • Biochemical Reactions: Metabolic characteristics

• Recent classification includes genotypic characteristics

  • Identification of bacteria, viruses, parasites, fungi

Routine Identification Procedures

• Conducted prior to microorganism growth:

  • Specimen Collection

  • Media Choice and Inoculation

  • Staining and Microscopic Examination

• Further identification post-growth:

  • Biochemical testing

  • Antibody-antigen reactions

  • Molecular techniques (e.g. PCR)

  • Automation

Knowledge for Identifying Etiologic Agents

• Understanding growth and nutritional requirements critical for recovery

  • Varying requirements (air, temperature, specific agar) for different bacteria

  • Importance of identifying normal flora and pathogens

Smear Preparation and Staining Basics

• Identifying bacteria through microscopic examination

  • Shape, arrangement, staining characteristics are preliminary indicators

• Unstained bacteria appear transparent; staining aids visualization

  • Common stain: Gram stain

Types of Smear Preparation

• Wet Prep: Specimen added to saline, cover slipped

• Gram Stain: Demonstrates specific bacteria (e.g., Listeria monocytogenes)

• Bacterial Forms:

  • Listeria: Gram-positive bacilli with tumbling motility

  • Lactobacillus: Normal vaginal flora

Steps for Smear Preparation

• Thinly Spread Specimen on glass slide

  • Sputum specimens using cotton swabs

  • Liquid specimens pipetted or rubbed onto slide

  • Smears made from agar colonies with sterile saline

• Film Thickness: Should be thin for visible cells

  • Avoid overlapping of cells

• Heat Fixing: Used after air-drying to attach bacteria; heat drying distorts cell shape

Essential Points of Slide Preparation

• Proper slide labeling:

  • Include specimen #, last name, and specimen source

• Fixation Post-Drying: Prevents cell wash-off during staining

• Fixation methods: Heat or methanol immersion

Types of Stains

• Simple Stains: Not differentiated; one color for all organisms

• Differential Stains: Most common is Gram stain

  • Distinguishes organisms based on cell wall structure (gram-positive vs. gram-negative)

Gram Stain Basis

• Key reagents include:

  • Primary Stain: Crystal violet

  • Mordant: Gram iodine forms complex with dye

  • Decolorizer: Alcohol-acetone solution

  • Counterstain: Safranin or carbol fuchsin

• Outcomes of Staining:

  • Gram-positive: Stain purple/blue

  • Gram-negative: Stain red/pink

Gram Stain Procedure Summary

1. Label smear with details

2. Add organism/specimen

3. Dry and fix the smear

4. Stain with crystal violet (1 min)

5. Apply Gram's iodine (1 min)

6. Decolorize until no purple runs off

7. Counterstain with safranin

8. Air dry or blot, then microscope examination

Acid-Fast Stain Overview

• Mainly detects Mycobacterium tuberculosis

  • A different staining method for acid-fast organisms

• Difficult to decolorize due to mycolic acid in cell walls

• Common methods include Ziehl-Neelsen and Kinyoun

  • Ziehl-Neelsen uses heat for penetration

  • Kinyoun uses phenol, avoiding heat

Microscopy Methods

• Brightfield Microscopy: Routine use, dark specimens against a bright field

• Fluorescent Microscopy: Uses UV light and specific antibodies for enhanced detection

• Fluorescent microscopes utilized for various bacteria like Legionella

Quantitating Organisms in Gram Stains

• Guidelines vary by institution; key formats include:

  • Many or 4+ (>25 organisms per field)

  • Moderate or 3+ (10-25)

  • Few or 2+ (1-10)

  • Occasional or 1+ (<1)

Lecture Summary Highlights

• Gram Staining: Powerful identification tool in microbiology

• Three basic bacterial shapes: cocci, bacilli, spirochetes

• Color reactions: purple/blue (gram-positive) or pink/red (gram-negative)

• Importance of precise gram stain procedures in identifying and classifying bacteria

Learning Check Questions

1. Organism morphology outlined in blue: spirochete

2. Organism morphology in orange: Gram-negative bacilli

3. Organism morphology in green: Gram-negative cocci in chains

4. Over decolorization effect: Gram-positive organisms will stain negative

5. Under decolorization effect: Gram-negative organisms will stain positive

6. Best communication for Gram-positive cocci in chains: Gram-positive cocci in chains

7. Description for long, thin, purple-staining bacteria: Gram-positive bacilli

8. Likely cause of dark stained gram-positive organisms: Crystal violet not filtered before use

9. Interpretation of purple gram-positive and pink gram-negative: Quality control acceptable, patient testing may occur

10. Preferred method for Acid-Fast Bacteria: Kinyoun or Ziehl-Neelsen staining

References

• Textbooks:

  • Clinical Laboratory Science by Mary Louise Turgeon

  • Bailey & Scott’s DIAGNOSTIC MICROBIOLOGY (15th ed.)

• Online Resources:

  • Various educational links regarding Gram staining and microbiology practices.