Tests in Microbiology Lab - Microbiology for Lab Technicians
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Tests in Microbiology Lab - Microbiology for Lab Technicians
- 1. Microbiology by, Miss Areesha Ahmad Senior Lecturer B.Sc (H), M.Sc, M.Phil and Ph.D scholar (Microbiology)
- 2. Prokaryotes Bacteria: Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis Archaea: Methanobrevibacter smithii, Halobacterium salinarum, Thermoplasma acidophilum Name of some Microorganisms
- 5. Pseudomonas
- 6. Acid-fast bacillus (AFB) • Mycobacterium tuberculosis • Mycobacterium leprae
- 7. Eukaryotes • Fungi: Candida albicans, Aspergillus niger, Saccharomyces cerevisiae • Protozoa: Plasmodium falciparum (causes malaria), Amoeba proteus, Giardia lamblia • Algae: Chlorella, Spirogyra, Diatoms
- 8. Fungi Aspergillus niger Penicillium crustosum Candida albicans
- 9. Viruses DNA Viruses: Herpes simplex virus, Varicella-zoster virus (chickenpox) RNA Viruses: Influenza virus, HIV, SARS-CoV-2 (causes COVID-19)
- 10. Virus SARS-CoV-2
- 28. Tests in Microbiology Lab For lab technician students, microbiology tests are essential to develop practical skills in diagnosing infections, understanding microbial cultures, and conducting various diagnostic procedures. Here are some common Microbiology tests that should be covered:
- 29. Tests in Microbiology Lab 1. Gram Staining 2. Acid-Fast Staining (Ziehl-Neelsen Staining) 3. Culture Media Tests 4. Antibiotic Sensitivity Testing (Kirby-Bauer Disc Diffusion) 5. Biochemical Tests 6. Urine Culture 7. Blood Culture 8. Stool Culture 9. Microscopy of Wet Mount Preparation 10. ELISA (Enzyme-Linked Immunosorbent Assay) 11. DNA Testing (PCR - Polymerase Chain Reaction) 12. Hemolysis Test on Blood Agar
- 31. Preparation of Microscopic slide Preparing a slide for viewing microorganisms, especially for techniques like acid-fast staining, involves a few careful steps to ensure clear observation. Here’s a general step-by-step guide for preparing a slide for microscopic examination:
- 32. Procedure • 1. Clean the Slide: • Start with a clean, dry glass slide. • If necessary, wipe it with ethanol and a lint-free tissue to remove any dust, oils, or fingerprints. • 2. Prepare the Specimen Smear: • Bacterial Specimen: Use an inoculating loop to pick a small sample of bacteria (from a culture plate or liquid culture) and spread it thinly on the slide. If using a liquid sample like sputum, place a drop on the slide and spread it evenly. • Tissue or Sample: For solid tissues or larger samples, use a scalpel or needle to obtain a very thin section. Place a small piece on the slide and press it down gently.
- 33. 3. Air Dry: • Allow the smear to air dry completely. Avoid using heat at this stage, as it can cause artifacts in the sample. 4. Heat Fixation: • Once dry, pass the slide quickly through the flame of a Bunsen burner (3–5 times) with the smear side facing up. This step: • Kills the organisms • Fixes them to the slide to prevent washing off during staining • Preserves the shape of cells Note: Be careful not to overheat, as this can distort the cells. 5. Staining (Optional): • After fixation, apply the desired stain to the slide based on the type of observation you need: • Simple Stain: For a basic view, use stains like methylene blue. • Gram Stain: For bacterial classification, use crystal violet, iodine, alcohol (decolorizer), and safranin. • Acid-Fast Stain (Ziehl-Neelsen): Use carbol fuchsin, acid-alcohol as a decolorizer, and methylene blue as a counterstain to view acid-fast bacilli. • Fluorescent Stain: For more sensitive detection, fluorochrome stains like auramine-rhodamine can be used.
- 34. 6. Rinse and Dry: • Gently rinse the slide with distilled water to remove excess stain. Blot dry with absorbent paper, avoiding smudging or disturbing the smear. 7. Microscopic Examination: • Place the slide on the microscope stage, start with a low magnification to locate the specimen, and switch to higher magnifications as needed. • For oil immersion (usually at 1000x), add a drop of immersion oil to the slide and carefully adjust the focus.
- 36. 1. Gram staining • Gram staining is a differential staining technique that is used for microscopic examination of bacteria. • In differential staining, specimen is subjected to a series of stains (dyes) in which different organisms are stained differently so that they can be distinguished from each other. • Gram staining differentiates bacteria into two groups; Gram positive and Gram negative • It was developed by Hans Christian Gram in 1884 and modified by Hucker in 1921.
- 37. • Gram-positive bacteria retain the primary dye, crystal violet (CV), following the application of the mordant, iodine. • Mordant is a substance that increases the cells’ affinity for a stain. • The iodine and crystal violet form a complex (CV-I) within the peptidoglycan. When a decolorizer is applied to the cells, the CV-I complex remains within the cell, making it appear dark purple to blue. • In Gram-negative cells, following the application of the crystal violet and iodine, the CV-I complexes are not trapped within the peptidoglycan. Principal
- 38. • Application of the acid-alcohol decolorizer dehydrates the outer cellular membrane, and also dissolves the lipids leaving holes in the membrane and effectively washing or removing the CV-I complex from the cells. • The cells appear colorless. Therefore, a counter stain, safranin, is applied, to make the cells distinctly visible (either red or pink).
- 39. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram- negatives. Threading through these layers of peptidoglycan are long anionic polymers, called teichoic acids.
- 46. Common Shapes of Bacteria • Coccus - spherical or round • Bacillus – rod shaped • Spiral - curve, spiral, or twisted
- 49. Bacteria arrangements • Bacteria can also have different arrangements of cells. So the common bacterial Cell Arrangements are: • Diplo - cells remain in pairs after dividing. • Strepto - cells remain in chains after dividing. • Tetrad - cells remain in groups of four and divide in two planes. • Sarcinae - cells remain in groups of eight and divide in three planes. • Staphylo - cells remain in clusters and divide in multiple planes
- 52. 2. Acid-fast bacilli (AFB) • Acid-fast bacilli (AFB) refer to bacteria that are resistant to decolorization by acids during staining procedures, particularly the Ziehl-Neelsen or Kinyoun acid-fast stain. • This property is mainly due to the high lipid content (particularly mycolic acid) in their cell walls. • The term "acid-fast" means these bacteria can retain the primary stain (carbolfuchsin) even after being treated with an acid-alcohol solution, which removes the stain from non-acid-fast cells.
- 57. 3. Culture Media Tests Types of Media: •Nutrient Agar: General-purpose media for bacterial growth. •MacConkey Agar: Selective for Gram-negative bacteria and differentiates lactose fermenters. •Blood Agar: Used for detecting hemolysis (alpha, beta, gamma). •Procedure: Inoculate specimens on media and incubate at optimal temperatures.
- 58. 3. Culture Media Tests
- 59. 4. Antibiotic Sensitivity Testing (Kirby- Bauer Disc Diffusion) Purpose: To determine bacterial susceptibility to antibiotics. Procedure: Discs containing antibiotics are placed on bacterial lawns on agar plates. Result Interpretation: Zones of inhibition around discs indicate sensitivity or resistance.
- 60. Introduction
- 73. 5. Biochemical Tests 1) Catalase Test: Purpose: To detect the presence of the catalase enzyme. Procedure: Adding hydrogen peroxide to bacteria. Bubbling indicates a positive result.
- 74. 5. Biochemical Tests 2) Coagulase Test: Purpose: Differentiates Staphylococcus aureus (coagulase-positive) from other staphylococci. Procedure: Mixing bacteria with plasma to check for clot formation.
- 75. 5. Biochemical Tests 3) Oxidase Test: Purpose: To identify bacteria producing cytochrome c oxidase (e.g., Pseudomonas). Procedure: Oxidase reagent turns purple when positive.
- 76. 6. Urine Culture Purpose: Diagnose urinary tract infections (UTIs). Procedure: Inoculate urine on agar plates and check for colony growth. Result Interpretation: Significant growth indicates infection; colony count is important.
- 77. 7. Blood Culture Purpose: Detect bacteremia (presence of bacteria in blood). Procedure: Blood samples are inoculated into bottles containing media and incubated. Result Interpretation: Growth of organisms indicates a bloodstream infection.
- 79. 8. Stool Culture Purpose: Identify enteric pathogens (e.g., Salmonella, Shigella). Procedure: Stool is plated on selective media like MacConkey and XLD agar. Result Interpretation: Pathogens show specific colony characteristics (color, shape).
- 81. 9. Microscopy of Wet Mount Preparations Purpose: Observe motility or structure of organisms, such as protozoa or fungi. Procedure: Place a drop of liquid specimen on a slide and observe under a microscope.
- 83. 10. ELISA (Enzyme-Linked Immunosorbent Assay) Purpose: Detect antigens or antibodies (e.g., in viral infections like hepatitis or HIV). Procedure: Sample is tested in microplate wells, and color change indicates a positive result.
- 84. 11. DNA Testing (PCR - Polymerase Chain Reaction) Purpose: Detect specific genetic material of microorganisms. Procedure: Amplifies DNA sequences to diagnose infections like tuberculosis or COVID-19.
- 85. 12. Hemolysis Test on Blood Agar Purpose: To identify types of hemolysis produced by bacteria, especially Streptococcus species. Result Interpretation: •Alpha hemolysis: Greenish zone (partial hemolysis). •Beta hemolysis: Clear zone (complete hemolysis). •Gamma hemolysis: No hemolysis.