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  Section: Biotechnology Methods » Microbiology
 
 
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Cell Size Measurements: Ocular and Stage Micrometers

 
     
 
Content
Microbiology
  Introduction
  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

Micrometery
Figure 11 Micrometery

Materials
  • Microscope
  • Ocular micrometer
  • Stage micrometer
  • Millimeter ruler
  • Prepared slide

Procedure
  1. Place a stage micrometer on the microscope stage, and using the lowest magnification (4X), focus on the grid of the stage micrometer.
  2. Rotate the ocular micrometer by turning the appropriate eyepiece. Move the stage until you superimpose the lines of the ocular micrometer upon those of the stage micrometer. With the lines of the 2 micrometers coinciding at one end of the field, count the spaces of each micrometer to a point at which the lines of the micrometers coincide again.
  3. Since each division of the stage micrometer measures 10 micrometers, and since you know how many ocular divisions are equivalent to 1 stage division, you can now calculate the number of micrometers in each space of the ocular scale.
  4. Repeat for 10X and 40X, and 100X. Record your calculations below:


    Using the stage micrometer, determine the smallest length (in microns) that can be resolved with each objective. This is the measured limit of resolution for each lens. Compare this value to the theoretical limit of resolution, calculated on the basis of the numerical aperture of the lens and a wavelength of 450 nm (blue light).

    Using the calculated values for your ocular micrometer, determine the dimensions of the letter “e” found on your microscope slide. Use a millimeter ruler to measure the letter “e” directly and compare it with the calculated values obtained through the microscope.
Notes
To measure an object seen in a microscope, an ocular micrometer serves as a scale or rule. This is simply a disc of glass upon which equally spaced divisions are etched. The rule may be divided into 50 subdivisions, or more rarely, 100 subdivisions. To use the ocular micrometer, calibrate it against a fixed and known ruler, the stage micrometer. Stage micrometers also come in varying lengths, but most are 2-mm long and subdivided into 0.01-mm (10-micrometer)

lengths. Each objective will need to be calibrated independently. To use, simply superimpose the ocular micrometer onto the stage micrometer and note the relationship of the length of the ocular to the stage micrometer. Note that at
different magnifications, the stage micrometer changes, but the ocular micrometer is fixed in dimension. In reality, the stage micrometer is also fixed, and what is changing is the power of the magnification of the objective.

 
     
 
 
     




     
 
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