LAB 2: Measurement and Counting of Cells Using Microscope

2.1 Ocular Micrometer

            Introduction :

            Ocular micrometer is use in order to measure and compare the size of prokaryotic and eukaryotic microorganisms. Microorganisms are measured with an ocular micrometer which is inserted into the one of the microscope eyepieces. The micrometer, which serves as a scale or rule, is a flat circle of glass upon which are etched equally spaced divisions.This is not calibrated, and may be used at several magnifications. When placed in the   eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance superimposed maybe calibrated using a stage micrometer on which parallel lines exactly 10μm apart etched. By determining how many units of the ocular micrometer superimpose a known distance on the stage micrometer, you can calculate the exact distance each ocular division measures on the microscopic field. When you change objectives you must recalibrate the system. After calibration of the ocular micrometer, the    stage micrometer is replaced with a slide containing microorganisms. The dimensions of the cells may then be determined.

            Objective :

            To measure and count cells using a microscope.

            Materials :

            Light microscope

            Ocular micrometer

            Stage scale

            E.coli slide

                Methods :

1. One of the eyepiece on the light microscope is changed with the eyepiece that contains the ocular    micrometer.
2. The stage scale is then put on the stage of the microscope.
3. Start from the lowest power objective (4X) , focus the microscope until the image on the stage scale is superimposed with the eyepiece scale.
4. The number of divisions on the ocular micrometer is determined and calculated.
5. The procedure is repeated when using high power magnification.

Results :

4.0 ocular micrometer = 0.01 mm stage micrometer ( 40 x magnification )

9.6 ocular micrometer = 0.01 mm stage micrometer ( 100 x magnification )

4.1 ocular micrometer = 0.001 mm stage micrometer ( 400 x magnification )

Lactobacillus

Discussion :

1. Ocular micrometer has no units on it.
2. We have to make sure that the first line of the ocular micrometer is in line with the first line of the stage micrometer to avoid errors when reading the measurements.
3. The primary challenge is to measure the size of one Lactobacillus cell from our sample.
4. For our experiment, we are able to calibrate a scale at 400x magnification which is already enough to be used to convert into other magnifications.
5. The actual size of the Lactobacillus sample through our observation is 1.708x10^-4mm.

Reference :

1. http://en.wikipedia.org/wiki/Ocular_micrometer
2. http://wiki.answers.com/Q/Why_is_it_necesary_to_calibrate_the_ocular_micrometer_with_each_objective
3. http://www.ruf.rice.edu/~bioslabs/methods/microscopy/measuring.html

2.2 Neubauer Chamber

Introduction :

                Neubauer chambers are more convenient for counting microbes. The neubauer is a       heavy glass slide with two counting areas separated by a H-shaped trough. A special            coverslip is placed over the counting areas and sits a precise distance above them.

Objective :
To measure and count cells using a microscope.

Materials :
Light microscope
Neubauer chamber       

Coverslip

Bunsen burner
Sterile Pasteur pipettes

Lactose bacillus culture

Methods :

1. Lactose bacillus culture is added to the space between the coverslip and the counting chamber with the use of sterile Pasteur pipettes.
2. The cells is allowed to settle about one minutes.
3. The number of cells on the counting chamber is then observed and counted under the microscope.
4. The procedure is repeated when using high power magnification.
5. After finish the experiment, the Neubauer and coverslip is cleaned with 70% alcohol.

Results :

Figure 1 . Neubauer Chamber 

Figure 2

Figure 2 and 3 . Neubauer Chamber 's Grids

           Discussions :

           The neubauer  chamber`s grip common was 24 x 16. Mostly use to observe the blood     
           cell and now also being use to calculate concentration or density of others cell.
 

1. The depth of the chamber was 0.1 mm, Therefore the volume = 0.04mm² x 0.1mm     = 0.004mm³ = 4x10^-3mm³ = 4x10^-6cm³
2. The most important things is use the correct cover (neubauer counting chamber cover glass). If not you will not able to see the grip.
3. If lose count when counting the cell, has to recount one more time.
4. Count the cell immediately after loading, because some cell may aggregate and this will affect the result.

References :

1. http://shop.gabsystem.com/data/descargas/Camara%20thoma%20neubauer_Eng.pdf
2. http://people.oregonstate.edu/~weisv/Protocols/Symbiodinium/Cell%20Counts.pdf
3. http://wiki.answers.com/Q/Why_is_70_percent_alcohol_a_better_disinfectant_than_100_percent_alcohol