LAB 2 MAYA EMIRA BT MAD SAAD 111381

2.1 OCULAR MICROMETER

INTRODUCTION

Ocular means of or connected with eyes or vision, and a lens is a piece of glass with curved sides that concentrates light rays and magnifies images. Thus the ocular lens quite simply is the one you look through. It causes the contents of the slide to appear magnified. The curvature of the lens determines the magnification level. On the microscope the ocular lens is found at the top of the tube which connects it to the objectives lens or lenses.Most ocular lenses have either a 10X (magnified ten times) or 15X magnification level. When a person looks through the ocular lens, light reflects from the objective lens, and the slide is magnified by the multiplied magnification levels of both the ocular and the objective lenses. So if a person is using a microscope with an ocular lens of 10X and they set the objective lens to 4X, then the slide will appear 40X when viewed through the ocular lens. Most microscopes have several settings for objective lenses.

OBJECTIVE

To measure and count cells using ocular microscope.

 

RESULT

	400x magnification		1000x magnification
calibrate	Stage micrometer	Ocular division	Stage micrometer	Ocular divison
	0.03 mm	39 division	0.09 mm	96 division
	7.69 x10⁻⁴ mm	1 division	9.38x10⁻⁴ mm	1 division
	0.769 µm	1 division	0.94 µm	1 division

a ) Yeast

 

400x magnification

       4 division x 0.769 µm

       =3.076 µm

1000x magnification

       3 division x 0.94 µm

       = 2.82 µm

b ) Lactobacillus

400x magnification

       4 division x 0.769 µm

       =3.076 µm

1000x magnification

      5 division x 0.94 µm

      =4.7 µm

 

DISCUSSION

   

An ocular micrometer scale, or reticule, is a scale etched on a glass disk and placed within an eyepiece. The scale is superimposed over any image seen in the microscope, allowing the user to measure any object in the field of view. Such measurement does require the reticule to be calibrated.

To calibrate and/or use an eyepiece reticule, start by focusing the eyepiece itself on the reticule. Looking through that eyepiece only, focus the microscope, then make the adjustment for the other eye as described previously. Then place and center a stage micrometer in the light path. To line up the ocular reticule with the stage micrometer, the eyepiece can be rotated in its tube (without changing focus) and the mechanical stage controls used so that the two images are superimposed. Then measure the distance over which the eyepiece reticule extends and divide by the number of divisions to determine distance per division. 

For example, suppose that at 100x an ocular micrometer scale with 50 evenly spaced divisions is superimposed over a portion of stage micrometer that is 1.0 mm (1000 µm) long. In this case, each division of the reticule represents 1000 ÷ 50 = 20 µm per division. The calibration for other magnifications is inversely proportional to objective magnification. For example, if you have 20 µm per division at 100x, you have 5 µm per division at 400x. 

(field of view 1) X (magnification 1) = (field of view 2) X (magnification 2)

 

 

CONCLUSION 

 

Basically, calibration is the "zero" setting on a measuring object. For example a weighing scale that is incorrectly calibrated would, for example, read 50 pounds as 49 pounds. This means that whatever is actually on the scale would be one pound heavier than what it actually is. This is not a problem when comparing two things of different weight (the difference will be the same) but if we measuring something as a single unit, it could be troublesome.

An ocular micrometer is what's on microscopes and basically tells  how big an object is. An incorrectly calibrated micrometer would be catastrophic since a small difference in the reading could have a massive effect on what we're trying to do. For example, a 2mm difference could mistakenly read a 300% increase in bacteria growth.

Bottom line is that calibration is important in measuring results and an incorrect calibration on an instrument would yeild incorrect results.

 

 

REFERENCES

 

Lodish, H., et al. (2000). Molecular Cell Biology (4^th ed.). New York: W.H. Freeman and Co.

Wolfe, S.L. (1993). Molecular and Cellular Biology. Belmont, CA: Wadsworth Publishing Company.

aprette, D. (1995). Light Microscopy. Retrieved 8-22-2006 from http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy.html

http://www.mecanusa.com/microscope/micrometer/micrometer-use_en.html

2.2 NEUBAUER CHAMBER

INTRODUCTION

Counting chamber: This one is called the Neubauer improved. There are other standards with different grids available as well.

The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name “hemo-”) but also the concentration of sperm cells in a sample. The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm). Additionally, a grid is etched into the glass of the hemocytometer. This grid, an arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to determine the number of cells in a specified volume.

 

OBJECTIVE

To find the number of cells in 1ml of original solution.

 

RESULT

 

CALCULATION :

 Cell in 10 boxes = 39

Average of cell: 39 ÷ 10 = 3.9

Volume = 0.02 x 0.02 x 0.01 mm
        = 4 x 10⁻³ mm

       = 4 x 10⁻⁶ cm 
      = 4 x 10^-6 ml

1ml = 3.9 ÷ (4 x 10^-6 )

              = 9.75 x 10^-5 cells/ml

 

 

DISCUSSION

 

Neubauer chambers are the finest quality, optically ground, and polished milled glass chambers available. The chamber is diamond etched and has a double improved Neubauer Ruling, which has a worldwide reputation in hospitals and laboratories for unmatched reliability, meeting the most demanding of standards. The standard Hausser blood counting chambers are one piece construction (measuring 75mmx32mmx4.5mm) ensuring long term durability and absolute accuracy in measurement and count.
Both the standard (incline) and the new “V-Load” counting hambers for different charging methods. The tolerances are ( 2% of the volume). Cell Depth: 0.100 mm ( +/-2%); Volume: 0.1 Microliter Ruling; Pattern: Improved Neubauer, 1/400 Square mm Rulings cover 9 square millimeters. Boundary lines of the Neubauer rulings are the center lines of the groups of three. (These are indicated in the illustration ). The central square millimeter is ruled into 25 groups of 16 small squares, each group separated by triple lines, the middle one of which is the boundary. The ruled surface is 0.10 mm below the cover glass, so that the volume over each of 16.

To get an accurate result ,the fluid containing the cells must be appropriately prepared before applying it to the hemocytometer.

• Proper mixing: The fluid should be a homogenous suspension. Cells that stick together in clumps are difficult to count and they are not evenly distributed.
• Appropriate concentration: The concentration of the cells should neither be too high or too low. If the concentration is too high, then the cells overlap and are difficult to count. A low concentration of only a few cells per square results in a higher statistical error and it is then necessary to count more squares (which takes time). Suspensions that have a too high concentration should be diluted 1:10, 1:100 and 1:1000. A 1:10 dilution can be made by taking 1 part of the sample and mixing it with 9 parts water (or better saline of correct concentration to prevent bursting of the cells). The dilution must later be considered when calculating the final concentration.

 The most important part in this experiment is the calculation for the counting cells.

• Counting cells that are on a line: Cells that are on the line of a grid require special attention. Cells that touch the top and right lines of a square should not be counted, cells on the bottom and left side should be counted.
• Number of squares to count: The lower the concentration, the more squares should be counted. Otherwise one introduces statistical errors. How many squares? To find out one could calculate the cell concentration per ml based on the numbers obtained from 2 different squares. If the final result is very different, then this can be an indication of sampling error.

 It is necessary to do some simple math. The following numbers are needed: number of cells counted in a square, area of the square, height of the sample, dilution factor.

• Averaging: If one did not count all of the cells in a large square (1mmx1mm) then it is necessary to average the results first before proceeding.
• Computing the volume: It is necessary to determine the volume represented by the square. The width and height of the square (e.g. 0.2mm x 0.2mm) must be multiplied by the height of the sample : v = 0.2mm x 0.2mm x 0.1mm = 0.004mm³ =0.000004 mL 
• Calculating the number of cells in 1 ml
• Correcting for dilution: If the sample was diluted before counting, then this must be taking into consideration as well. We assume that the sample was diluted 1:10.  

 

CONCLUSION

There are different types of counting chambers available, with different grid sizes. One counting chamber also has grids of different sizes. Take care that  the grid size and height (read the instruction manual) otherwise we'll make calculation errors.We need to use cover glasses so that the height of the fluid is standardized.They are thicker than the standard 0.15mm cover glasses. They are therefore less flexible and the surface tension of the fluid will not deform them. For moving cells (such as sperm cells) are difficult to count. These cells must first be immobilized.

 

REFERENCES

http://www.emsdiasum.com

http://www.microbehunter.com

http://www.brand.de/index.php?id=497&L=0