ExerciseⅤ Microscopic Measurement of Organisms
Materials per Student
compound microscope；lens paper and lens cleaner；immersion oil；prepared slides of yeast；coverslips；dropper with bulb；tweezers；ocular micrometer；stage micrometer；cellular dilution to measure；hemocytometer，or counting chamber
Learning Objectives
Each student should be able to
1.Understand how microorganisms can be measured under the light microscope.
2.Calibrate an ocular micrometer.
3.Perform some measurements on different microorganisms.
1.The measurement of bacterial size
Principles
It frequently is necessary to accurately measure the size of the microorganism one is viewing.For example，size determinations are often indispensable in the identification of a bacterial unknown.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.
Procedure
Calibrating an Ocular Micrometer
1.If you were to observe the ocular micrometer without the stage micrometer in place，it would appear as shown in[Figure 1（a）].In like manner，the stage micrometer would appear as illustrated in[Figure 1（b）].
2.When in place，the two micrometers appear as shown in[Figure 1（c）].Turn the ocular in the body tube until the lines of the ocular micrometer are with those of the stage micrometer[Figure 1（d）].Match the lines at the left edges of the two micrometers by moving the stage micrometer.
Figure 1 Calibrating an Ocular Micrometer
3.Calculate the actual distance in millimeters 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.Because the smallest space on the stage micrometer equals 0.01 millimeter or 10 mm[Figure 1（b）]，you can calibrate the ocular micrometer using the following：10 spaces on the ocular micrometer=Y spaces on the stage micrometer.
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.
For example，if 10 spaces on the ocular micrometer=6 spaces on the stage micrometer，then 1 ocular space=6×0.01 mm/10，1 ocular space=0.006 mm or 6.0μm.
This numerical value holds only for the specific objective-ocular lens combination used and may vary with different microscopes.
Calibrate for each of the objectives on your microscope and record below.Show all calculations in the space following the Table 1；also show your calculations to your instructor.
Table 1 Calibrating an Ocular Micrometer
2.The measurement of of bacterial numbers
Principles
Cell counting is any of various methods for the counting or similar quantification of cells in the life sciences，including medical diagnosis and treatment.It is an important subset of cytometry，with applications in research and clinical practice.For example，the complete blood count can help a physician to determine why a patient feels unwell and what to do to help.Cell counts within liquid media（such as blood，plasma，lymph，or laboratory rinsate）are usually expressed as a number of cells per unit of volume，thus expressing a concentration（for example，5，000 cells per milliliter）.It's also used for bacteria and yeast counting.
A counting chamber（also known as hemocytometer），is a microscope slide that is especially designed to enable cell counting.The hemocytometer has two gridded chambers in its middle，which are covered with a special glass slide when counting.A drop of cell culture is placed in the space between the chamber and the glass cover，filling it by capillarity.Looking at the sample under the microscope，the researcher uses the grid to manually count the number of cells in a certain area of known size.The separating distance between the chamber and the cover is predefined（The distance between the bottom of the chamber and the cover is 0，1 mm），thus the volume of the counted culture can be calculated and with it the concentration of cells.Cell viability can also be determined if viability dyes are added to the fluid（Figure 2）.
Figure 2 The counting chamber（hemocytometer）
Their advantage is being cheap and fast；this makes them the preferred counting method in fast biological experiments in which it needs to be merely determined whether a cell culture has grown as expected.Usually the culture examined needs to be diluted，otherwise the high density of cells would make counting impossible.The need for dilution is a disadvantage，as every dilution adds inaccuracy to the measurement.
Counting chamber's counting grid is 3 mm x 3mm in size.The grid has 9 square subdivisions of width 1mm.（Figure 3）.The central square is split in 25 squares of width 0.2 mm（200μm）.Each one of the 25 central squares is subdivided in 16 small squares.Therefore，the central square is made of 400 small squares（Figure 4）.
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Figure 3 Hemocytometer chamber counting grid detail
Figure 4 The squares of hemocytometer
1.Sample preparation（250.000 cells/ml and 2，5 million cells/ml.）.
2.Introducing the sample into the counting chamber.
1）Put the glass cover on the counting chamber central area.Use a flat surface to place the chamber，like a table or a workbench.
2）Introduce the dropper with bulb on the dilution previously prepared
3）Push the dropper plunger slowly until you feel it has arrived to the end of its travel.
4）Remove the dropper tip from the dilution，and bring it to the counting chamber.When the pipette is loaded，it must always be held in vertical position.
5）Place tip close to the glass cover edge，right at the centre of the counting chamber.
6）Release the plunger slowly watching how the liquid enters the chamber uniformly，being absorbed by capillarity.See Figure.5
7）In case of the appearance of bubbles，or that the glass cover has moved，repeat the operation.
3.Microscope set up and focus on.
1）Place the counting chamber on the microscope stage，fix the counting chamber with fixing clamp.
2）Turn on the microscope light.Focus the microscope until you can see a sharp image of the cells looking through the eyepiece and adjusting the stage.
3）Look for the first counting grid square where the cell count will start.In this example，5 big squares from a hemocytometer will be counted.See figure 6.
4）Start counting the cells in the first square.
Different laboratories have different counting protocols，but there is a popular unwritten rule that states，“Cells touching the upper and left limits should be counted，unlike cells touching the lower and right limits which should not be taken into account.”
Figure 5 sample filling a Neubauer chamber Figure 6 Count in a Neubauer chamber big square
4.Concentration calculation.We apply the formula for the calculation of the concentration.
The number of cells will be the sum of all the counted cells in all squares counted.
The volume will be the total volume of all the squares counted.
Since the volume of 1 big square is，
0.1 cm x 0.1 cm=0.01 cm2 of area counted.
Since the depth of the chamber is 0.1mm，
0.1 mm=0.01 cm，
0.01 cm2*0.01 cm=0.0001 cm3=0.0001ml=0.1μL.
So，for the counting chamber，the formula used when counting in the big squares.
In case a dilution was applied，the concentration obtained should be converted to the original concentration before the dilution.In this case，the concentration should be divided by the dilution applied.