from mcbs*c

• Microscope


  An instrument used for observing organisms that are not readily observable using the naked eye
• Microscope


  • From the Greek word mikros (small) and skopein (to watch or see)
• Three Structural Parts of the Microscope

  • Head
  • Base
  • Arms
• Head


  Also known as the body, consists of the upper part of the microscope where optical parts are located
• Base


  Primarily supports the microscope and carries the illuminators
• Arms


  Acts to connect the head and base, and is usually where one holds when carrying the microscope
• Optical Parts of the Microscope

  • Eyepiece (or ocular lens)
  • Eyepiece tube
  • Objective lenses
  • Nose piece (or revolving turret)
  • Adjustment knobs
  • Stage
  • Aperture
  • Illuminator
  • Condenser
  • Diaphragm
• Eyepiece (or ocular lens)


  The part where one looks through for observation. It has a standard magnification of 10x and some ranging from 5x-30x
• Eyepiece tube


  The part holding the eyepiece above the objective lens
• Objective lenses

  Considered as the major lenses, usually 1-4 lenses of different magnification, utilized for viewing specimens. Their magnification can range from 10x to 100x
• Nose piece (or revolving turret)


  A movable part that can be rotated and holds the objective lenses
• Adjustment knobs

  • Coarse adjustment knob
  • Fine focus/adjustment knob
• Coarse adjustment knob


  Used for focusing the specimen approximately
• Fine focus/adjustment knob


  Used for maximally defining the specimen
• Stage


  The part where the specimen is placed
• Mechanical stage


  Have knobs that can used for moving the slides
• Aperture


  The hole on the stage where the light being transmitted from the illuminator passes through
• Illuminator


  Provides the light source for viewing. Sometimes, a mirror is in in place of the illuminator
• Condenser


  Lenses utilized to "condense" or focus the light coming from the illuminator to the specimen. They usually have higher magnification to ensure sharp images
• Diaphragm


  Also known as the iris, is the part found under the stage and primarily utilized for controlling the amount of light reaching the specimen
• Micrometers used in measuring microorganisms

  • Ocular micrometer
  • Stage micrometer
• Ocular micrometer


  Used to measure the microscopic object. It is usually a glass disc with a mounted scale and is inserted into the eyepiece. It is calibrated differently on different objectives
• Stage micrometer


  Used to calibrate the ocular micrometer. It has graduation of known intervals
• Steps in calibrating using the micrometers

  1. The eyepiece is first removed from the draw tube

  2. The lens cover of the eyepiece is then unscrewed

  3. The ocular micrometer is inserted

  4. The lens cover is replaced and the eyepiece is returned to the draw tube

  5. For checking, look into the eyepiece to check whether the disc is not inverted

  6. The stage micrometer is placed on the stage and the scale is focused

  7. The stage micrometer is focused under specific objective that will be utilized to measure the specimen size

  8. The SM is arranged so that a line on the far left should correspond to a line in the OM. It should be overlapping

  9. After coinciding the first line, another line to the right should coincide with that of another line in the OM

  10. The number of division is then counted on the OM that is transversed by the number of divisions on the SM, all within the area of proper line coincidence. This is done vice versa as well
• Calibration Factor (value of 1 OM division)


  CF = (SM division transversed by OM division) (value of 1 SM division in um)/OM divisions transversed by SM divisions
• Features of the Objectives

  • Focal Point (F)
  • Focal Length (FL)
  • Working Distance (WD)
  • Resolving Power (R) or Resolution
  • Numerical Aperture
  • Parfocal
• Focal Point (F)


  The point at which reflected or refracted rays meet
• Focal Length (FL)


  The distance between the lens and the focal point. It varies depending the objective lens being utilized; LPO (16 mm), HPO (4 mm) and OIO (1.8 mm)
• Working Distance (WD)


  The distance between the lens' surface and that of the focused specimen. It also varies depending on the lens
• Resolving Power (R) or Resolution


  The ability of the lens to dinstincly separate 2 close lines
• Limit of Resolution

  Differs depending on the microscope being used
• Numerical Aperture


  The measure of the objective's resolving power
• Parfocal


  This feature allows for focus viewing on one lens and can be observed with focus on other lenses without major adjustments
• Calculations


  Total M = (linear M of eyepiece) (linear of objective)

  Total M of HPO = (10X) (40X) = 400X

  Limit of resolution (d):

  d=0.5λ/nsin(Θ)

  where;

  d = limit of resolution

  λ = wavelength of the light used

  n = refractive index of the medium

  nair=1.0

  nglass = 1.5

  noil=1.5

  Θ = 1⁄2 of the angle of the cone of λ that enters the objective

  Numerical aperture (NA):

  n sin(Θ) also utilized in computing for d
• Advantages of observing live microorganisms

  • Observation of unaltered/undistorted characteristics of the organisms (e.g. size, shape)
  • Cellular processes like growth and reproduction can be studied
  • Motility can be observed
  • Simple to prepare
• Disadvantages of observing live microorganisms

  • Most of the time (except pigment microorganisms), the refractive index of the microbial cells is almost similar to that of water or commonly used mounting medium, thus, difficult to observe
• Methods for sample preparation for microscopic observation

  • Wet Mount Technique
  • Hanging Drop Technique
• Wet Mount Procedure


  Get sample

  Place a coverslip on top of the glass slide with a sample

  Observe the specimen using the microscope
• Hanging Drop Procedure


  Place droplets of water at the corners of the coverslip using a wire loop or a toothpick

  Get a loopful of the specimen and place it on the center of the coverslip

  Invert the depression slide and carefully press it onto the coverslip making sure that the sample will not be pressed, only the droplets

  Turn the slide right up quickly making sure that the specimen at the center of the coverslip does not spread

  Observe the specimen using the microscope
• General steps on staining


  Smear preparation

  Fixation

  Application of dye/stain