Bacterial measurements using microscopic techniques

Bacterial measurements using microscopic techniques (Micrometery)


It is frequently necessary to accurately measure the size of the microorganism one is viewing. For example, size determinations are often indispensable in the identification of an unknown bacteria. The size of microorganisms is generally expressed in metric units and is determined by the use of a microscope equipped with an ocular micrometer. An ocular micrometer is a small glass disk on which uniformly spaced lines of unknown distance, ranging from 0 to 100, are etched. The ocular micrometer is inserted into the ocular of the microscope and then calibrated against a stage micrometer, which has uniformly spaced lines of known distance etched on it. The stage micrometer is usually divided into 0.01 millimeter and 0.1 millimeter graduations. The ocular micrometer is calibrated using the stage micrometer by aligning the images at the left edge of the scales. The dimensions of microorganisms in dried, fixed, or stained smears tend to be reduced as much as 10 to 20% from the dimensions of the living microorganisms. Consequently, if the actual dimensions of a microorganism are required, measurements should be made in a wet-mount.


  1. Bacterial culture
  2. Nutrient broth
  3. Reagents for Gram’s staining


  1. Ocular micrometer
  2. Stage micrometer
  3. Bright field microscope


Growth and stained preparation of bacteria

  1. Inoculate nutrient broth with the given bacterial culture and incubate the same at 37°C for overnight.
  2. After overnight incubation, take the culture and perform its Gram’s staining. Keep the slide in safe place its further use in bacterial measurement.

Calibration of ocular micrometer

  1. Fit the ocular micrometer in the eye piece of the microscope and fix the stage micrometer on the stage.
  2. Turn the ocular in the body tube until the lines of the ocular micrometer are parallel with those of the stage micrometer. Match the lines at the left edges of the two micrometers by moving the stage micrometer.
  3. Calculate the actual distance in millimetres between the lines of the ocular micrometer by observing how many spaces of the stage micrometer are included within a given number of spaces on the ocular micrometer. You will get the greatest accuracy in calibration if you use more ocular micrometer spaces to match with stage micrometer lines.
  4. Because the smallest space on the stage micrometer equals 0.01 millimeter or 10µm, you can calibrate the ocular micrometer using the following:
  5. 10 spaces on the ocular micrometer = Y spaces on the stage micrometer.
  6. Since the smallest space on a stage micrometer = 0.01 mm, then 10 spaces on the ocular micrometer Y spaces on the stage micrometer × 0.01 mm, and 1 space on the ocular micrometer = Y spaces on the stage micrometer × 0.01 mm/ 10 .
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