Microscopy and staining

• Microscopy

  • is the most common method used both for the detection of microorganisms directly in the clinical specimens and for the characterization of organisms grown in culture
  • is defined as the use of a microscope to magnify (i.e. visually enlarge) objects too small to be visualized with the naked eye so that their characteristic is easily observable.
• TYPES OF MICROSCOPE

  • Bright-Field Light Microscopy
  • Phase Contrast Microscopy
  • Fluorescent Microscopy
  • Dark-Field Microscopy
  • Electron Microscopy
• BRIGHT-FIELD MICROSCOPY

  • Visible light is passed through the specimen and then through a series of lenses that reflect the light in a manner that results in magnification of the organisms present in the specimen
  • Maximum Magnification – 2000x
  • Resolution – 0.2 μm (200 nm)
• PHASE CONTRAST MICROSCOPY

  • Beams of light pass through the specimen and are partially deflected by the different densities or thickness of the microbial cells or cell structures in the specimen. This microscopy translates differences in phases within the specimen into differences in light intensities that result in contrast among objects within the specimen being observed.
  • Maximum Magnification – 2,000x
  • Resolution – 0.2 μm (200 nm)
• FLUORESCENCE MICROSCOPY

  • Microorganisms in a specimen are stained with a fluorescent dye. On exposure to excitation light, organisms are visually detected by the emission of fluorescent light by the dye with which they have been stained or tagged.
  • Maximum Magnification – 2,000x
  • Resolution – 0.2 μm (200 nm)
• DARK-FIELD MICROSCOPY

  • The condenser does not allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle. Only light that hits objects will be deflected upward into the lens for visualization.
  • Maximum Magnification -2000x
  • Resolution – 0.2 μm (200 nm)
• ELECTRON MICROSCOPY

  • Uses electrons to visualize small objects and the electrons are focused by electromagnetic fields and form an image on a fluorescent screen, like a television screen.
  • For bacterial morphology study
• TRANSMISSION ELECTRON MICROSCOPY (TEM)

  • Maximum Magnification – 100,000x
  • Resolution – 0.5 nm
  • Passes the electron beam through objects and allows visualization of internal structures.
• SCANNING ELECTRON MICROSCOPY (SEM)

  • Maximum Magnification – 650,000x
  • Resolution – 10 nm
  • Uses electron beams to scan the surface of objects and provides three-dimensional views of surface structures
• • Objective lens, which is closest to the specimen, magnifies object 100X.
  • Ocular lens, which is nearest the eye magnifies 10X.
  • The combination of the two lenses is called TOTAL MAGNIFICATION
  • Magnification of 1000X allows the visualization of fungi, most parasites and most bacteria.
  • Observing viruses requires magnification of 100,000X or more.
• RESOLUTION

  • is defined as the extent to which detail in the magnified object is maintained.
  • Without resolution, everything would be magnified as an indistinguishable blur.
  • RESOLVING POWER, which is closest distance between two objects that when magnified still allows the two objects to be distinguished from each other.
  • Oil Immersion is used to fill the space between the objective lens and the glass slide onto which the specimen has been affixed.
• CONTRAST

  • is needed to make objects standout from the background.
  • is most commonly achieved by STAINING TECHNIQUES, that highlight organisms and allow them to be differentiated from one another and from background material and debris.
  • In the absence of Staining, the simplest way to improve your contrast is to REDUCE THE DIAMETER OF THE MICROSCOPE APERTURE DIAPHRAGM, INCREASING CONTRAST AT THE EXPENSE OF THE RESOLUTION.
• STAINING TECHNIQUES

  • Staining techniques are used to produce color contrast.
  • Routine methods of staining of bacteria involve dying and fixing smears – procedures that kill them.
  • Bacteria have an affinity to basic dyes due to the acidic nature of their protoplasm.
• BASIC DYES

  • They are cationic dyes with positively charged groups (pentavalent nitrogen) that adhere to negatively charged molecules like nucleic acids and proteins
  • Examples are: Methylene Blue, Basic Fuchsin, Crystal Violet, Safranin, and Malachite Green
• ACIDIC DYES

  • They are anionic dyes with negatively charged groups (carboxyls and phenolic) that bind to positively charged cell structures
  • Examples are: Eosin, Rose Bengal, and Acid Fuchsin
• SIMPLE STAINING

  • A single stain is used
  • It is directed towards coloring the forms and shapes of the cells
  • The inoculum size is 105 CFU/mL
  • Example: Methylene Blue
• DIFFERENTIAL STAINING

  • Divides the bacteria into separate groups
  • It is directed towards coloring the components of the elements present
  • The use of more than one dye for contrast
  • Example: Gram Staining and AFB Staining
• Gram Staining:

  1. Crystal Violet
  2. Gram’s iodine
  3. Ethanol
  4. Safranin
• AFB Staining:

  • Carbolfuchsin
  • Methylene Blue
• was devised by histologist Christian Gram as a method of staining bacteria in tissues.


  Gram stain
• was discovered by Ehrlich, who found that after staining with aniline dyes, tubercle bacilli resist decolourization with acids. The method as modified by Ziehl and Neelsen, is in common use now.


  Acid Fast Stain
• NEGATIVE STAINING

  • Used to demonstrate the presence of diffuse capsule surrounding some bacteria
  • An excellent technique for studying bacterial gas vacuoles and viral morphology
  • Results in bacteria appearing as light-colored bodies against a dark background
  • Example: India Ink
• IMPREGNATION METHOD

  • Cells and structures too thin to be seen under ordinary microscope may be rendered visible if they are impregnated with silver on the surface.
  • Used for demonstration of spirochetes and bacterial flagella.
• FLUOROCHROMING


  • It is a nonselective staining of cells for fluorescence microscopy.
  • There is a direct interaction between the fluorochrome and bacterial cell component, specifically, the cell wall
  • Examples: Acridine Orange (for Mycoplasma ureaplasma) Auramine-Rhodamine (for Mycobacterium tuberculosis) Calcofluor White
• IMMUNOFLUORESCENCE


  A dye-antibody conjugate is used to “tag” specific cellular antigens

  Valuable in identifying difficult or slow-growing bacteria, as well as viruses and parasites
• Schaeffer-Fulton and Wirtz-Conklin Methods:

  Primary Stain – Malachite Green

  Counterstain – Safranin
• Dorner’s Method:

  Primary Stain – Carbolfuchsin

  Counterstain – Nigrosin
• Acetic Acid Method:

  Primary Stain – Carbolfuchsin

  Counterstain – Loeffler’s Alkaline Methylene Blue
• CAPSULE STAINING

  • A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining, bacterial cell wall takes up the dye. Capsules appear colorless with stained cells against dark background.
  • Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed.
• Anthony’s Method:

  Primary Stain – Crystal Violet

  Decolorizer and Counterstain – 20% Copper Sulfate solution

  /

  Graham and Evan’s Method:

  Primary Stain – Crystal Violet

  Counterstain – Safranin

  Background Stain – India Ink

  /

  Hiss’ Method:

  Primary Stain – Crystal Violet

  Decolorizer and Counterstain – 20% Copper Sulfate

  /

  Novelli’s Method:

  Primary Stain – Alcian Blue SCN 150

  Counterstain – Ziehl-Neelsen Cabolfuchsin

  /

  Maneval’s Method:

  Primary Stain – Congo Red

  Counterstain – Maneval solution
• FLAGELLA DEMONSTRATION

  • To be able to observe them under light microscopy, the thickness of the flagella is increased by coating them with mordant and staining them with several methods available.
  • Example: Gray’s, Leifson, Fischer and Conn, and West Method.
• unit of measurment

  A) one thousand of a millimeter

  B) one thousandth of a micron

  C) one millionth of a millimeter

  D) one tenth of a nanometer

  
• Microscopic dimentions

  A) algae

  B) rbc, wbc, ameoba

  C) rickettsia, E. coli

  D) Staphylococcus

  
• Microscopic dimentions

  A) Mycoplasma, Pox virus

  B) AIDS virus, Hepatitis B virus

  C) polio virus

  D) large protein DNA

  E) 200

  F) 100

  G) 10

  H) 1

  
• Microscopic dimentions

  A) Amino acids

  B) atom

  C) 0.1

  D) 1

  
• Shapes:

  1. Cocci are spherical or oval cells
  2. Bacilli are rod shaped cells
  3. Vibrios are comma shaped curved rods and derive their name from their characteristics vibratory motility
  4. Spirilla are rigid spiral forms
  5. Spirochetes (from speira meaning coil and chaite meaning hair) are flexuous spiral forms
  6. Actinomycetes are branching filamentous bacteria, so called because of a fancied resemblance to the radiating rays of the sun when seen in tissue lesions
  7. Mycoplasmas are cell-wall deficient and hence do not possess a stable morphology
• Shapes

  A) coccus

  B) rod/bacillus

  C) spirillum/spirochetes

  D) diplococci

  E) neisseriae

  F) coccobacilli

  G) vibrios

  H) spirilla

  I) corynebacteria

  J) mycobacterium

  K) sarcinae

  L) tetrads

  M) streptococci

  N) spore forming rods

  O) streptomycetes

  P) spirochetes

  Q) micrococci/staphylococci

  
• shapes

  A) coccobacillus

  B) bacilli

  C) palisades

  D) diplobacilli

  E) streptobacilli

  
• CELL WALL

  • prevent the cell from expanding & eventually bursting due to water uptake

  • is very rigid & gives shape to the cell

  • constitutes a significant portion of the dry weight of the cell

  • is essential for bacterial growth & division

  • is composed of peptidoglycan
• CAPSULE

  • is the outer most layer of the bacteria (extra cellular)

  • are highly impermeable

  • is antiphagocytic

  • helps in adherence of bacteria either to themselves forming colonial masses or to surfaces in their environment.