Lab training

• Microbiological Tests


  Laboratory methods used to identify specific etiologic agents or pathogens that cause disease
• The cause of disease may be bacteria, virus, fungi and parasite
• The diagnosis is based on the selection of suitable specimens for microbiological examination
• Most common samples that reach the microbiology lab


  • Swab from wound, vagina, abscess, nasal and ear secretion, skin and mucous membranes
  • Urine
  • Sputum
  • Blood
  • Stool
  • Fluids like CSF, Ascetic
  • Biopsy
• The physician should supply important information about the patient, such as age and sex, details of the clinical syndrome, date of onset, significant exposures, prior antibiotic therapy, immunologic status
• The type and timing of specimens to be collected, the conditions for their transportation and storage are very important for the microbiologist to give decisions during the diagnostic studies
• Specimens should be collected before the administration of antibiotics
• Close communication between the clinician and the microbiologist is essential to ensure appropriate specimens are selected and collected and they are appropriately examined
• Endogenous infections


  The microorganism is a component of the patient's indigenous flora (opportunistic), e.g. aspirated from the upper to the lower respiratory tract
• Exogenous infections


  The microorganism is acquired from the environment (e.g., from soil, water, droplets) or from another person or animal
• The differential diagnosis is based on a careful history, physical examination, and appropriate radiographic and laboratory studies including the selection of appropriate specimens
• Causes of Infection


  • Bacteria (including many kinds of Gram-positive and Gram-negative bacteria)
  • Viruses
  • Fungi
  • Parasites
• Microbiological Examination


  • Direct Examination & Staining
  • Culture
  • Biochemical tests
  • Sensitivity test
• Wet mount


  Little amount of specimen on the slide examined under microscope (10x,40x) to detect microorganism morphology, pus cells, RBCs
• Types of Staining


  • Acidic dyes (e.g. Eosin, Nigrosine, picric acid)
  • Basic dyes (e.g. Crystal Violet, Methylene blue, Safranin)
  • Neutral dyes (e.g. Leishman's stain, Giemsa stain)
• Types of Staining Procedures


  • Simple Staining
  • Differential Staining (Gram stain, Ziehl-Neelsen stain)
  • Special Staining (Capsule, flagella, spores)
• Biochemical tests


  Important in the identification of bacterial species
• Biochemical tests


  • Depend on the presence of certain enzymes
  • Different bacteria produce different enzymes such as catalase, oxidase, urease, gelatinase, etc.
• Catalase test


  1. Determine the ability of bacteria that produces Catalase enzyme

  2. Catalase converts hydrogen peroxide into oxygen and water

  
• Catalase positive


  Numerous bubbles produced, active bubbling
• Catalase negative


  No bubbles produced
• Catalase positive


  • Staphylococcus sp.

  
• Catalase negative


  • Streptococcus sp.

  
• Oxidase test


  1. Differentiates organism into oxidase-positive and oxidase-negative microorganisms

  2. Based on the existence of cytochrome oxidase enzyme in their electron transport chain system

  3. Oxidase positive organisms can oxidize the TMPD reagent into a blue coloured complex

  4. Oxidase negative organisms cannot oxidize the reagent and remains in a reduced form (appears colourless)

  
• Oxidase positive


  Development of dark purple color (indophenols) within 10 seconds
• Oxidase negative


  Absence of color
• Coagulase test


  1. Differentiates Staphylococcus aureus from other coagulase-negative staphylococci

  2. S.aureus produces two types of coagulase: free coagulase and bound coagulase

  3. Free coagulase is detected in tube coagulase test

  4. Bound coagulase is detected in slide coagulase test

  
• Optochin sensitivity test


  Distinguishes Streptococcus pneumoniae (optochin sensitive) from other a-hemolytic streptococci (optochin resistant)

  
• Bacitracin sensitivity test


  Distinguishes between the b-hemolytic streptococci: Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin sensitive)

  
• Gelatin hydrolysis test


  1. Determines the ability of an organism to produce extracellular proteolytic enzymes, gelatinases that hydrolyze gelatin

  2. Gelatinases hydrolyze gelatin into polypeptides and then polypeptides are further converted into amino acids
• Gelatin hydrolysis positive


  Liquefaction of the inoculated tube even after exposure to cold temperature
• Gelatin hydrolysis negative


  Complete solidification of the inoculated tube
• Indole test


  1. Demonstrates the ability of certain bacteria to decompose the amino acid tryptophane to indole

  2. Indole production is catalyzed by the enzyme tryptophanase

  
• Indole positive


  Formation of a pink to red color ("cherry-red ring") in the reagent layer
• Indole positive


  • Escherichia coli, Salmonella sp., Klebsiella sp., Neisseria sp.

  
• Indole negative


  • Proteus mirabilis, Enterococcus faecalis, Vibrio sp., Proteus vulgaris

  
• Methyl Red (MR) test

  1. Detects the production of sufficient acid during the fermentation of glucose

  2. Bacteria that produce a stable acid from glucose metabolism will turn the methyl red indicator from yellow to red
• Methyl Red positive


  A distinct red color
• Methyl Red positive


  • E. coli
• Methyl Red negative


  A yellow color