Bacteriology

• Taxonomy is the science of classifying living things, while nomenclature assigns scientific names to taxonomic categories and individual organisms
• Classification involves arranging organisms into a hierarchy of taxa, and identification is the process of discovering and recording the traits of organisms for recognition and placement in a taxonomic scheme
• Carl von Linné (Linnaeus) laid down basic rules for classification and established taxonomic categories or taxa
• Robert Whittaker's classification system is based on structural similarities and differences, including prokaryotic and eukaryotic cellular organization and nutrition methods, dividing organisms into Monera, Protists, Plants, Fungi, and Animals
• Carl R. Woese devised the Three-Domain System of Classification, which includes two domains of prokaryotes (Archaea and Bacteria) and one domain (Eucarya or Eukarya) for all eukaryotic organisms
• The International Code of Nomenclature of Bacteria provides accepted labels for universally recognizing organisms, using a binomial system where each organism is assigned a genus and species of Latin or Greek derivation
• In bacterial morphology, cocci are round or spherical shaped bacteria, bacilli are rod-shaped, and spirals are helical or twisted bacteria
• Staining in bacteriology imparts artificial coloration to bacteria and other materials on clinical specimen smears for better visualization under a microscope, with categories like Direct/Simple Stain, Differential Stain, Selective/Special Stain, and Indirect/Negative/Relief Stain
• Direct/Simple Stain enhances the appreciation of bacterial size, shape, and arrangement using stains like Crystal Violet, Gentian Violet, Methylene Blue, and Malachite Green
• Differential Stain contains two or more chromogens to differentiate specific components within bacterial cells, including the critical decolorization step, with examples like Gram Stain, Acid Fast Stain, and Fluorochrome Stain
• Selective/Special Stains highlight specific bacterial cell structures or components aiding in presumptive identification, such as stains for cell wall, capsule, and metachromatic granules
• Loeffler’s Alkaline Methylene Blue (LAMB) stain:

  • LAMB granules stain RED
  • Albert granules appear BLUE BLACK
  • Neisser granules appear DARK BLUE
  • Lindegran granules appear REDDISH BROWN
  • Burke’s Technique is a modified gram’s staining technique
  • Ljubinsky granules stain DARK VIOLET
• Stains for Bacterial Spores/Endospores:

  • Fulton-Schaeffer: spores are GREEN
  • Dorner: spores are RED
  • Wirtz-Conklin: spores are GREEN
• Selective/Special Stains for Flagella:

  • Leifson: Gray
  • Tannic Acid is an important component in flagellar stain
  • Silver coats, swells, and precipitates the flagella enhancing its visualization
• Stains for Rickettsia:

  • Castaneda stains BLUE
  • Machiavelo stains RED
  • Giemsa stains BLUE
• Stains for Chlamydia:

  • Gimenez: elementary bodies stain RED
  • Machiavelo stains RED
  • Giemsa stains PURPLE
• Selective/Special Stains for Spirochetes:

  • Fontana-Tribondeau: spirochetes stain DARK BROWN or BLACK
  • Levaditi Silver Impregnation: spirochetes stain BLACK
  • India Ink Negative Stain: spirochetes are UNSTAINED; background is BLACK
• Stains for Mycoplasma:

  • Dienes stains BLUE
• Stain for Bipolar Bodies (Yersinia pestis):

  • Wayson: bipolar bodies stain RED
• Indirect/Negative/Relief Staining:

  • Provides coloration to the background of the smear while rendering the bacteria and covering structures such as capsules unstained
  • Useful in the identification of medically important capsulated bacteria and capsulated strains of Cryptococcus spp., especially in cerebrospinal fluid samples in cases of meningitis
  • Background is COLORED/STAINED
• Methods of Studying Bacteria:

  • After standard incubation, examine colonial or cultural characteristics of bacterial colonies grown in each culture media for each specimen, known as PLATE READING
  • Characteristics include MARGIN (appearance of the edge of the colony) and ELEVATION (height of the colony)
• Antigenic Determination by Serological Typing:

  • O Antigen is associated with the cell wall
  • H Antigen is associated with the flagella
  • K Antigen is associated with the capsule
  • Vi Antigen is the specific capsular antigen of Salmonella typhi
• Microbial Control:

  • Phenol Coefficient Test evaluates the effectiveness of disinfectants, also known as the Disinfectant test
  • Utilizes Staphylococcus aureus or Salmonella typhi for the test
  • Based on the dilution of phenol and experimental disinfectant inoculated with bacteria and incubated at 20-37 deg C for 2-3 days
  • Calculated by dividing the dilution of the test disinfectant by the dilution of phenol
  • Methods include Rideal Walker Test and Chick Martin Test
• Bartlett’s Grading System:

  • Scores of 0 or less indicate a lack of inflammation or the presence of saliva
• Biologic Indicators:

  • Bacillus subtilis var. niger: used to check the effectiveness of sterilizers, especially dry heat sterilizers
  • Bacillus subtilis var. globigii: biologic indicator for gas sterilizers
  • Bacillus pumilus: biologic indicator for ionizing radiation sterilizers
  • Bacillus stearothermophilus: used for moist heat autoclave
• Specimen Collection:

  • Specimen Transport, Preservation, Labelling, Requisition
  • Specimen Processing includes Gross Examination, Direct Microscopic Examination, Selection of Culture Media, Specimen Preparation, Inoculation on Solid Media, and Incubation Conditions
  • Specimen Workup involves the extent of identification and communication of laboratory findings
• Collection Techniques:

  • Samples collected during the acute phase of illness before antibiotics are administered
  • Swabs are inferior if tissue or needle aspirates can be obtained
  • Provide clinicians with a collection manual and follow safety considerations
• Specimen Transport:

  • Specimens should be transported to the laboratory within 2 hours of collection in sterile, leak-proof containers marked with a biohazard symbol
• Specimen Storage:

  • Specimens should not be stored in the refrigerator and should remain at room temperature
• Specimen Labelling:

  • Labels must contain patient's name, hospital number or birthdate, date and time of collection, and source
• Rejection of Unacceptable Specimens:

  • Reasons for rejection include improper transportation, preservation, or storage of specimens
• Laboratory Grouping Requirements:

  • Individual requirements include complete PPE, pencils, colored pencils, short bond paper
  • Group requirements include manila paper with plastic cover, scotch tape, permanent marker, yellow sando bags, glass slides, small disinfectant spray