MIDTERMS | Gram Staining

• The following Gram staining method was developed empirically by the Danish bacteriologist Christian Gram in 1884. The differential ability of the Gram stain makes it useful in microbial taxonomy.
• MOST FUNDAMENTAL TEST IN MICROBIOLOGY is Gram Staining
• The Gram stain is used routinely and as requested in the clinical microbiology laboratory for the primary microscopic examination of specimens submitted for smear and culture.

  
  

  Always combo with C&S (acronym)

  
  

  It is ideally suited for specimen types in which bacterial infections are strongly suspected, but it may be used to characterize any specimen. Cerebrospinal fluid, sterile fluids, expectorated sputum or bronchoalveolar lavages, and wounds and exudates are routinely stained directly.

  
  

  Urine and stool may not be routinely stained directly.
• Both cell walls are made of peptidoglycans, cell wall for + is thick while – is thin, and top of – bacteria is an outer membrane made up of lipopolysaccharide which can easily be removed with ethanol
• Gram Stain:

  Primary stain - crystal violet

  Secondary stain - safranin

  mordant - iodine
• Stain specimen with crystal violet and leave for 1 minute, then wash

  
  

  Apply mordant (iodine – negatively charged, when added with violet, creates a strong bond) – strongly attaches primary stain to cell wall

  
  

  Leave for 1 minute, forming crystal violet-iodine complex

  
  

  Wash and apply decolorizer (95% alcohol, wash for 5 sec, over decolorization results to falsely negative)

  
  

  Apply safranin, gram negative retains color of safranin while gram positive remains purple as gram neg decolorizes
• COLORS

  • Gram negative = red/pink
  • Gram positive = violet/purple
• All cocci are GRAM POSITIVE except

  • Neisseria
  • Branhamella/ Moraxella
  • Veilonella
• All Bacilli are GRAM NEGATIVE except

  • Bacillus
  • Lactobacillus
  • Listeria
  • Actinomyces
  • Clostridium
  • Corynebacterium
  • Mycobacterium
  • Erysipelothrix
  • Nocardia
• AFB cell wall contains fatty acid known as mycolic acid (super thick) and cannot be stained easily
• Acid fast organisms are capable of retaining the primary stain even after treated with an acid alcohol decolorizer.
• Two methods for AFB staining:

  • Ziehl-Neelsen Method
  • Kinyoun Method
• Ziehl-Neelsen Method is hot method
• Kinyoun Method is cold method.
• AFB Stains:

  • primary stain - carbol fuchsin
  • secondary stain - methylene blue
  • mordant - tergitol [heat for ziehl-neelsen method]
• Ziehl-Neelsen Method decolorizer

  • 1% carbolfuchsin
  • 3% acid alcohol
  • 0.1% methylene blue
• If org is acid fast = red/pink

  If org is non-afb = blue
• Reporting AFB:

  • 0 - No AFB in 300 fields
  • n+ - 1-9 AFB in 100 fields
  • 1+ - 10-99 AFB in 100 fields
  • 2+ - 1-10 per field in 50 fields
  • 3+ - >10 per field in 20 fields
• Kinyoun Method Decolorizer

  • 1% acid alcohol