History and Staining

• Microbiology

  The branch of biology that studies microorganisms including bacteria, viruses, fungi and protozoa
• Microbiology has a rich history that spans centuries with the contribution from numerous scientists who play pivotal roles in its development
• Anton Van Leeuwunhoek (1632-1723)

  • Dutch Scientist
  • Credited with the invention of the microscope
  • First to observe and describe micro-organisms (as Animacules), including bacteria, and protozoa using his simple microscope
• Louis Pasteur (1822-1895)

  • French Chemist and microbiologist
  • Disproved spontaneous generation through his experiment with swan-necked flasks
  • Developed germ theory of disease, establishing a connection between micro-organisms and infectious diseases
  • Credited with the development of vaccines against several diseases including rabies and anthrax
  • Development of aseptic techniques which prevent contamination during experiments
  • Developed pasteurization to prevent spoilage of food and beverages
  • Investigated the process of fermentation showing that micro-organisms are responsible for the conversion of sugars into alcohol
• Robert Koch (1843-1910)

  • German physician and microbiologist
  • Postulated the criteria for establishing a causative link between a micro-organism and a disease
  • Identifies the causative agents of tuberculosis (mycobacterium tuberculosis) and Cholera (vibro cholerae)
• Alexander Fleming (1881-1955)

  • Scottish bacteriologist
  • Discovered the first antibiotic, Penicillin, in 1928
  • Revolutionized medicine by introducing the era of antibiotics for treating bacterial infections
• Lazzaro Spallanzani (1729-1799)

  • Disproved Spontaneous generation
• Edward Jenner (1749-1823)

  • English Scientist
  • Smallpox vaccine pioneer
• Hans Christian Gram (1853-1938)

  • Danish Scientist
  • Developed the Gram Stain Technique
• Fanny Hesse (1850-1934)

  • German Scientist
  • Regarded as the mother of the Bacterial Culture
  • Developed Agar, a solid medium used in culturing
• Carl Woese (1928-2012)

  • American microbiologist
  • Revolutionized the classification of organisms into three domains
• Emil Adolf Von Behring (1854-1917)

  • German Scientist
  • Pioneered serum therapy, a method using antibodies from immunized animals to treat infectious diseases
  • Played a key role in the discovery of antitoxins, specifically for diphtheria and tetanus
  • His work significantly reduced childhood mortality from diphtheria, earning him the nickname "saviour of children"
  • Contributed to the foundation of the field of immunology
• Stains

  Colouring chemical substances that can interact with cells, making them easier to see under a microscope
• Microorganisms are tiny living creatures that are commonly extremely small and nearly transparent, making them very difficult to see directly using a microscope
• Benefits of Staining

  • Increases the feasibility of seeing microorganisms
  • Makes it much easier to clearly see the morphological (shape) features of a microorganism
  • Can accentuate (make prominent) specific morphological features (e.g., seeing flagella on some organisms)
  • Can preserve the stained specimen for future study
• Fixation

  The placement and preservation of a specimen on a glass slide for staining
• Heat Fixation

  Typically, a film/smear of the cell on the glass slide is gently heated
• Chemical Fixation

  Used when examining larger but more delicate micro-organisms such as protozoa, protects cellular structure as well as morphology, contains ethanol, Acetic acid, formaldehyde
• Basic Dyes

  Positively charged groups that bind to negatively charged cellular components such as nucleic acids, surfaces of bacteria, examples are crystal violet, methylene blue
• Acidic Dyes

  Negatively charged that bind to positively charged cell structures, example is carbolfuchsin
• Staining Techniques

  • Simple Staining
  • Negative Staining
  • Differential Staining
  • Acid-fast staining
  • Endospore staining
  • Capsule staining
  • Flagella staining
• Simple Staining

  1. Flooding/covering a shear with a stain for a short period and then the stain is washed off with water

  2. Uses basic dyes

  3. Used to determine size, shape and arrangement of bacterial cells
• Negative Staining

  The glass slide is stained and not the object, the unstained cells appear as bright against a dark background
• Differential Staining

  Employs the use of two different stains, the first stain applied is called the primary stain while the second stain applied is known as the secondary stain or counter stain
• Acid-Fast Staining

  Used to identify mycobacterium tuberculosis, and mycobacterium leprae which causes leprosy, they have a cell wall rich in mycolic acid which prevents dyes from easily binding, the primary Stain is Carbolfuchsin
• Endospore Staining (Schaeffer-Fulton Method)

  1. Flood the bacterial smear with malachite green and heat gently

  2. Rinse with water and counterstain with safranin

  3. Rinse, blot dry, and observe under the microscope
• Endospore Staining


  Endospores resist conventional staining methods due to their impermeable protein coat, malachite green is forced into the endospores by heat, while the safranin counterstain colours the vegetative cells, important for identifying organisms capable of forming endospores and assessing their environmental survival capabilities
• Capsule Staining (India Ink Method)

  1. Mix a small bacterial suspension with India ink on a clean slide

  2. Spread the mixture thinly over the slide using a second slide at a 45-degree angle

  3. Air-dry the smear without heat fixing

  4. Observe under the microscope using oil immersion
• Capsule Staining

  Capsules are non-ionic and do not readily take up standard stains, so they appear as clear halos against the dark background of the India ink, important for identifying encapsulated pathogens, such as Streptococcus pneumoniae and Klebsiella pneumoniae, Indian ink is commonly used due to its ease of use and ability to create a contrasting background
• Flagella Staining (Leifson's Method)

  1. Flood the bacterial smear with a mordant such as tannic acid or potassium alum

  2. Apply a stain such as basic fuchsin or carbolfuchsin

  3. Rinse, blot dry, and observe under the microscope
• Flagella Staining

  Enhances the visibility of flagella, which are thin and difficult to see with standard stains, mordants such as tannic acid or potassium alum increase the thickness of the flagella, making them more visible, important for studying bacterial motility and pathogenesis