Untitled Quiz

Lecture 9: Laboratory Diagnosis of Bacterial Infections (I)

• This lecture covers the laboratory diagnosis of bacterial infections, focusing on initial steps.
• The process involves three main stages: specimen collection and transport, bacterial identification, and result interpretation and release.
• Specimens must be collected before antimicrobial therapy begins.
• Specimens must represent the infection.
  • Examples of specimens based on infection location include CSF for meningitis, throat/nasopharyngeal swab and sputum for respiratory tract infections (RTIs), vomitus/stool for intestinal infections, and mid-stream urine for urinary tract infections (UTIs).
• Specimens should be an adequate volume.
• Specimens should be collected aseptically in sterile containers.
• A complete request form, including date, type of sample, and relevant department details, is essential.
• Specimens should be transported rapidly to the lab; transport media should be used if delay is anticipated.

Bacterial Identification

• Direct detection methods seek the causative bacteria or their components.
  • Microscopic examination is used.
  • Culture and antibiotic sensitivity testing is done.
  • Antigen detection is performed.
  • Molecular tests (e.g., PCR) identify genetic material.
• Indirect detection methods identify antibodies produced in response to bacteria.
  • Serology is used to detect specific antibodies.

Microscopic Examination

• Different types of microscopes are used for bacterial identification:
  • Ordinary light microscopes use visible light for standard observation.
  • Dark-field microscopes illuminate the specimen from the side and are used for bacteria like Treponema pallidum.
  • Fluorescence microscopes use UV light and are used to detect bacteria with fluorescent dyes (e.g., Mycobacteria and intracellular pathogens like Chlamydia).
  • Electron microscopes are used for viewing extremely small structures like viruses.
• Unstained wet mount (fresh specimens) allows examination of bacterial motility.
• Stained specimens, such as Gram stains, Ziehl-Neelsen stains (acid-fast stain), flagellar stains, and capsular stains, provide more detail about shape, arrangement, and staining properties of bacteria.

Gram Stain

• A crucial method for classifying bacteria into Gram-positive and Gram-negative groups.
• Gram-positive bacteria appear purple after staining.
• Gram-negative bacteria appear red after staining.
• The difference lies in the cell wall's structure: Gram-positive have thicker peptidoglycan layers that retain the crystal violet stain.
• Steps in the Gram stain procedure include fixing the specimen, applying a primary stain (crystal violet), mordant (iodine), decolorization (alcohol), and counterstain (safranin).

Ziehl-Neelsen Stain

• Identifies acid-fast bacteria, specifically Mycobacterium species, which have waxy layers in their cell walls.
• Procedure is similar to the Gram stain; uses Carbol fuchsin stain, decolorization with sulfuric acid, and methylene blue counter-stain to visualize acid-fast organisms.

Bacterial Culture

• Clinical samples are grown (cultured) on various media under specific temperature and atmospheric conditions.
• Media types are classified by consistency: liquid (broth), solid, or semi-solid (agar).
• Media types are also classified by ingredients: transport media, ordinary/simple media (nutrient agar/broth), enriched media (Loffler's serum agar, Blood agar, Chocolate agar), selective media, or indicator media.

Transport Media

• Preserves viability of microorganisms during transport to the laboratory.
• Examples include Stuart, Amies, and Cary-Blair media.

Culture Media

• Simple (ordinary/basic) media provides basic nutritional needs.
  • Examples include nutrient agar and nutrient broth.
• Enriched media supports the growth of fastidious bacteria.
  • Addings like serum, whole blood, or specific growth factors to simple media result in enriched media.
  • Examples are Loffler's serum agar, blood agar, and chocolate agar.
• Blood agar can be differential, based on hemolysis (destruction of red blood cells) produced:
  • Beta (complete) hemolysis.
  • Alpha (partial) hemolysis.
  • Gamma (no) hemolysis.

Case Study

• A 45-year-old man with a six-month history of night sweats, fever, cough, and weight loss (30 kg) is suspected of Mycobacterium tuberculosis infection.
• Ziehl-Neelsen stain would be necessary to identify the bacteria.