microbiology labs

• Types of microscopes


  • Light microscope
  • Electron microscope
  • Dark – ground microscope
  • Fluorescent microscope

  
• Light microscope


  • Magnification
  • Resolution

  
• Electron microscope


  Provides higher magnification and resolution than light microscope
• Dark-ground microscope


  The organism appears brightly illuminated in a dark background, used for delicate organisms like Spirochetes of Syphilis
• Fluorescent microscope


  Uses fluorescent stains and ultraviolet radiation to illuminate specimens
• Bacterial staining properties


  • Size (0.2-1.2 um, 0.4-14 um)
  • Shape
  • Arrangement
• Bacterial structure


  • Cell wall (Gram positive, Gram negative)
  • Cytoplasmic membrane
  • Nucleoid
  • Ribosomes
  • Intracytoplasmic inclusions
  • Plasmids
  • Capsule
  • Flagella
  • Pili
• Methods of bacteriological diagnosis

  • Specimen collection
  • Smear preparation
  • Cultivation (Film preparation, Biochemical reactions, Colony morphology)

  
• Microscopic examination value


  • TB
  • Leprosy
  • Syphilis
  • Acute male gonorrhea
  • Second case of cholera
  • Vincent's angina
  • Relapsing fever
• Staining methods


  • Gram's stain
  • Ziehl-Neelsen stain
  • Wet mount preparation

  
• Gram's stain


  Crystal violet, Iodine, Decolorizer (alcohol), Safranin
• Ziehl-Neelsen stain


  Strong carbol fuchsin, Decolorizer (Acid alcohol), Methylene blue
• Wet mount preparation


  Unstained bacterial suspensions, Living bacteria, Used to study motility
• Bacterial cultivation


  A method of multiplying the organisms by letting them reproduce in predetermined culture media under controlled lab conditions
• Why to cultivate bacteria

  • 1. Study the properties of bacteria isolated from pathological specimens
  • 2. Diagnosis of infectious diseases
  • 3. Prepare antigens, toxins and vaccines
  • 4. Further antibiotic susceptibility testing
• Culture media


  An artificial culture medium must provide similar nutritive & environmental conditions that exist in the natural habitat of a bacterium
• Culture media


  • Liquid or solid
  • Mostly, Temp: 35 – 37 °C
  • Time: 24 – 48 hrs
• Types of media


  • Simple
  • Enriched
  • Selective
  • Differential
• Simple media


  Basic, highly nutritive, enrich number of bacteria
• Enriched media


  Enrich growth of fastidious organisms and detect hemolytic activity
• Selective media


  Select growth of certain bacteria & suppress others (contain bile or chemicals or antibiotics)
• Differential (indicator) media


  Differentiate among different kinds (contain indicator)
• Simple media


  • Peptone water
  • Nutrient broth
  • Nutrient agar plate & slope
• Peptone water


  • Composition: 1% peptone + 0.5% NaCl
  • Used for: Base for preparation of other media, Alkaline peptone water for Vibrio cholera
• Nutrient broth


  • Composition: 1% peptone + 0.5% NaCl + meat extract
  • Used for: Growth medium for cultivation of nonfastidious microorganisms, General maintenance of cultures and routine work, Basic for other media
• Nutrient agar


  • Composition: nutrient broth + agar agar
  • Used for: Growth medium for cultivation of nonfastidious microorganisms
• Enriched media


  • Blood agar plate
  • Chocolate agar plate
• Blood agar plate


  • Composition: nutrient agar + 10% blood
  • Used for: Isolate fastidious organisms and detect hemolytic activity: identify bacteria by their hemolytic action on the red cells
• Chocolate agar plate


  • Composition: heated blood agar
  • Used for: Growing fastidious bacteria as Neisseria & Haemophilus
• Selective media


  • Lowenstein Jensen (L-J) slope
  • Blood tellurite agar plate
• Lowenstein Jensen (L-J) slope


  • Composition: beaten egg + mineral salts + malachite green (solidified by heating)
  • Used for: M. tuberculosis
  • Growth: 2-8 weeks
• Blood tellurite agar plate

  • Composition: blood agar + potassium tellurite
  • Used for: Identification of types of Corynebacterium diphtheria
• Differential (indicator) media


  • MacConkey agar plate
  • Triple sugar iron (TSI) agar slope
  • Sugar fermentation
• MacConkey plate


  • Composition: (differential, selective & indicator) nutritive: peptone, indicator: lactose, neutral red, selective: bile salts, agar
  • Used for: Cultivation of enteric bacteria, Differentiate between lactose fermenters & non lactose fermenters
• Triple sugar iron (TSI) slope

  • Composition: nutritive: meat extract, indicator: glucose, lactose & sucrose, ferrous sulphate, phenol red
  • Prepared in slopes: slant & butt
  • Used for: Differentiate bacteria according to sugar fermentation and H2S production
• Sugar fermentation test


  • Used for: Differentiate bacteria according to fermentation of a specific carbohydrate
  • Composition: single carbohydrate: glucose, lactose, sucrose, mannite, maltose, Indicator: Andrade's solution
• Anaerobic cultivation


  Anaerobic bacteria such as Clostridia, PeptoStreptococci, Actinomyces, Lactobacilli need special media for growth (low oxygen content, reducing substances)
• Types of bacteria

  • Obligate aerobes
  • Facultative anaerobes
  • Obligate anaerobes
  • Micro-aerophilic
  • Aerotolerant anaerobes
• Anaerobic culture methods


  1. Anaerobic culture media (Robertson cooked meat)

  2. Anaerobic GasPak system
• Robertson cooked meat


  Cooked minced meat + broth + reducing substances (haematin & glutathione)