IBG 102 lab report

Sunday, 8 April 2012

Lab 2 Report Prepared by Teoh Leong Sin

Name: Teoh Leong Sin

Matric No: 111433

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 eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance superimposed may be calibrated using a stage micrometer on which parallel lines exactly 10 micrometer 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.

Results

Total magnification = objective lens power x eyepiece lens power(10x)

Yeast under 400x magnification:

2.5 micrometer x 2 = 5 micrometer

Yeast under 1000x magnification:

1.0 micrometer x 5 = 5 micrometer

Lactobacillus under 1000x magnification:

1.0 micrometer x 2.5 = 2.5 micrometer

Discussions

1) The ocular micrometer is a glass disc that attaches to a micrometer's eyepiece.It has a ruler that allow user to measure the size of magnified object.

2) The actual size of the letter on the microscope slide is measured using the millimeter ruler. This measurement will help us calibrate the ocular micrometer to determine if it is giving us accurate measurements.

3)The scale on the ocular micrometer changes with total magnification, and thus has no absolute value.A stage micrometer is essentially a ruler that is mounted on a microscope slide . One division of stage micrometer = 0.01 mm.

4) One ocular division = no. of divisions on stage micrometer

no. of divisions on ocular micrometer

5) For 400x magnification:

Stage scale = 0.01 mm x 5 divisions

= 0.05 mm

one ocular division = 0.05 mm/20 divisions on ocular micrometer

= 0.0025 mm

= 2.5 micrometer

measurement scale of sample yeast cells = 2.5 micrometer x 2 ocular divisions

= 5.0 micrometer

measurement scale of sample lactobacillus cells = 2.5 micrometer x 1 ocular division

= 2.5 micrometer

6) For 1000x magnification:

Stage scale = 0.01mm

one ocular division = 0.01 mm/10 divisions on ocular micrometer

= 0.001 mm

= 1.0 micrometer

measurement scale of sample yeast cells = 1.0 micrometer x 5 ocular divisions

= 5.0 micrometer

measurement scale of sample lactobacillus cells = 1.0micrometer x 3ocular division

= 2.5 micrometer

Conclusion

Ocular micrometer allows us to measure the size of microorganisms . Microorganisms or cells such as yeast and lactobacillus can be measured and the size can be compared. The result show that yeast cells is two times bigger than lactobacillus cells.

References

http://www.doctorfungus.org/thelabor/sec11.pdf

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

Results

Yeast under 400x magnification:

11	8	15	6
12	20		9
		14
	11	9

Discussions

1) Neubauer Chamber is device originally designed for the counting blood cells.It is now using to count other cells and microorganisms

2) The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

3) We randomly choose 10 of these 16 smaller squares and calculate the number of yeast cells in each of the squares,then we calculate the average of yeast cells to further determine the concentration by dividing the volume of a small square.

4) Volume of small square = 0.2mm x 0.2mm x 0.1 mm

= 0.004 mm^3

= 4 x 10^-6 mL

Sum of cells in 10 small box = 115 cells

Average cells = 115 cells/ 10

= 11.5

Concentration of yeast cell = 11.5 cells / (4 x 10^-6) mL

= 2875000 cells/mL

Conclusion

The number of cells in a population can measured by counting the number of cells in smaller squares and the volume of suspension is equal to area of smaller square times depth of film.With using Neubauer chamber, we can easily calculate the concentration of microorganisms or cells.The concentration of yeast cells in this experiment is 2875000cells/mL.

References

http://www.uvm.edu/~wschaeff/BasicCulture1.html
http://people.oregonstate.edu/~weisv/Protocols/Symbiodinium/Cell%20Counts.pdf
http://en.wikipedia.org/wiki/Hemocytometer

Saturday, 7 April 2012

Lab Report 2 by Ooi Sin Ying

Name : Ooi Sin Ying
No. Matrice : 111410

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 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 a eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance superimposed may be calibrated using a stage micrometer on which parallel lines exactly 10micrometer apart etched. By determining how many units of the ocular micrometer superimpoe 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 cells using a microscope.

Result :

1. With 40x objective len, each division of ocular micrometer :
(1x0.01) / 4 = 0.0025mm

(a) Saccharomyces cerevisiae (Yeast)

Length of Saccharomyces cerevisiae (Yeast) :
2 x (0.0025) = 0.005mm

(b) Lactobacillus fermentum

Length of Lactobacillus fermentum :
1 x 0.0025 = 0.0025mm

2.With 100x objective len, each division of ocular micrometer :
(1x0.01) / 10 = 0.001mm

(a) Saccharomyces cerevisiae (Yeast)

Length of Saccharomyces cerevisiae (Yeast) :
5 x (0.001) = 0.005mm

(b)Lactobacillus fermentum

Length of Lactobacillus fermentum :
2.5 x 0.001 = 0.0025mm

Discussion :
1.An ocular micrometer has a ruler that allows the user to measure the size of magnified objects.
2.Ocular micrometer can be used to measure any planar dimension in a microscope field since the
   ocular can be turned in any direction and the object of interest can be repositioned with the stage
   manipulators.
3.With difference magnification, the division of ocular micrometer will has difference value. Thus, it
   is necessary to calibrate the scale by focusing on a fix and known scale micrometer (a stage
   micrometer) placed directly on the stage for every magnification.
4.After calibrated, the ocular micrometer can be use to measure the dimension of cells.

Conclusion :
Ocular micrometer with a known scale stage micrometer , the cell can be measure easily and accurately especially with oil immersion method.

Reference :
1.How to Use an Ocular Micrometer | eHow.com
2.http://www.ehow.com/how_5019336_use-ocular-micrometer.html#ixzz1rQeXORkR
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 heavy glass slide with two counting areas separated by a H-shaped trough. A special coverslip is placed over the counting areas and sits precise distance above them.

Objective :
To count cells using a microscope

Result :

Observation of yeast on Neubauer chamber under 40x objective magnification.

1. Volume of one small box :
    Height   = 0.2mm
    Length  = 0.2mm
    Breadth = 0.1mm
    - 0.2mm x 0.2 mm x 0.1mm = 0.004mm^3
                                                   = 4x10^-6 cm^3
                                                   = 4x10^-6 mL
2. Total no. of cells calculated from 10 small box :
    9+14+8+13+22+11+20+15+5+7 = 124
    - average = 124 / 10
                     = 12.4
3. Concentration of cell (no. of cells / each mL) :
    - 12.4 / (4.0x10^-6)mL = 3100000 cells / mL

Discussion :
1.The Neubauer chamber is a device used for determining the number of cells per unit volume of a
   suspension.
2.Coverslips for counting chambers are specially made and are thicker than those for conventional
   microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid.
3.Suspensions should be dilute enough so that the cells or other particles do not overlap each other on
   the grid, and should be uniformly distributed.
4.When counting the cells, cells that overlap a ruling, count a cell as "in" if it overlaps the top or right
   ruling, and "out" if it overlaps the bottom or left ruling.
5.Counting :
   (a) The chamber contains many grids, producing nine major large squares.
   (b) For calculation purposes, only the middle large square which has 16 small squares with area
         (0.2mm x 0.2mm) is used.
   (c) Choose randomly 10 small boxes and count the total number of cells and its average per square.
   (d) Assuming the average number of cells = Z, and volume of a square = 4x10^-6mL.
        Then the concentration of cells = Z / (4x10^-6) cells/mL.

Conclusion :
Neubauer Chamber is a useful advice use to count concentration of the cell by calculate the average of cells in a sample on the small boxes of chamber which chosed randomly and divided to the volume of each box.

Reference :
1. http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html
2.http://www.uni-greifswald.de/~immuteach/methods/counting_chamber/counting_chamber.html#

Lab 2 Report written by Tan Hwee Feng

Name: Tan Hwee Feng
Matric No: 111427

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 eyepiece, the line superimposed certain distance markers on the microscope field. The actual distance superimposed may be calibrated using a stage micrometer on which parallel lines exactly 10micrometer 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.

Results

Total magnification = objective lens power x eyepiece lens power(10x)

Yeast under 400x magnification:

2.5 micrometer x 2 = 5 micrometer

Yeast under 1000x magnification:

1.0 micrometer x 5 = 5 micrometer

Lactobacillus under 1000x magnification:

1.0 micrometer x 2.5 = 2.5 micrometer

Discussions

1) The ocular micrometer is simply a glass disc with etched lines on its surface.The distance between

graduations of an ocular micrometer does not have any standard value.

2) The distance is worked out by calibrating it with a known scale, stage micrometer.

3) Stage micrometer is a special glass slide which have a known distance. One division of stage micrometer = 0.01 mm.

4) One ocular division = no. of divisions on stage micrometer

no. of divisions on ocular micrometer

5) For 400x magnification:

Stage scale = 0.01 mm x 5 divisions

= 0.05 mm

one ocular division = 0.05 mm/20 divisions on ocular micrometer

= 0.0025 mm

= 2.5 micrometer

Average dimensions of sample yeast cells = 2.5 micrometer x 2 ocular divisions

= 5.0 micrometer

6) For 1000x magnification:

Stage scale = 0.01mm

one ocular division = 0.01 mm/10 divisions on ocular micrometer

= 0.001 mm

= 1.0 micrometer

Average dimensions of sample yeast cells = 1.0 micrometer x 5 ocular divisions

= 5.0 micrometer

Average dimensions of sample lactobacillus cells = 1.0 micrometer x 3 ocular divisions

= 3.0 micrometer

Conclusion

Ocular micrometer has a measurement scale that allows the us to measure the size of magnified objects. A special slides which contains scales also used to place the objects being observed. Besides that, this experiment also allow us to learn how to calculate the dimensions of cells using stage micrometer and ocular micrometer. By learning this method, microorganisms or cells such as yeast and lactobacillus can be measured and the size can be compared.

References

http://www.ehow.com/how_5019336_use-ocular-micrometer.html

http://www.doctorfungus.org/thelabor/sec11.pdf

2.2 Neubauer Chamber

Introduction

Objective

To measure and count cells using a microscope

Results

Yeast under 400x magnification:

11	8
12			9
		14	8
8	11	9	5

Discussions

1) A device used for determining the number of cells per unit volume of a suspension is called a counting chamber (Neubauer Chamber).
2) Inside the middle large squares, there are 16 smaller squares, each with the size of 0.2mm x
0.2mm.
3) We randomly choose 10 of these 16 smaller squares and calculate the number of yeast cells in each of the squares.
4) Volume of small square = 0.2mm x 0.2mm x 0.1 mm
                                           = 0.004 mm^3
                                           = 4 x 10^-6 mL
     Sum of cells in 10 small box = 95 cells
     Average cells = 95 cells/ 10
                            = 9.5
     Concentration of yeast cell = 9.5 cells / (4 x 10^-6) mL
                                                 = 2.375 x 10^6 cells/mL

Conclusion

The number of cells in a population can measured by counting the number of cells in smaller squares and the volume of suspension is equal to area of smaller square times depth of film.With using Neubauer chamber, we can easily calculate the concentration of microorganisms or cells which is average number of cells in 10 box divide by volume of suspension.

References

http://b110-wiki.dkfz.de/signaling/wiki/display/rnaiwiki/Counting+cells+with+haemocytometer+%28Neubauer+chamber%29
http://www.emsdiasum.com/microscopy/products/magnifier/counting.aspx
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html

Lab 2 by Tang Su Xian

Name:Tang Su Xian
Matric card no.:111431

Lab 2: Measurement and Counting of Cells using Microscope

2.1 Ocular Micrometer

Introduction:

Ocular micrometer is used 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 eyepieces. The micrometer, which serves as a scale or ruler, 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 micrometer field. The actual distance superimposed may be 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 the cells by using a microscope.

Results:

Photo that shows the size of yeast cell under 400x magnification.

Photo that shows size of yeast cell under 1000x magnification.

Photo that shows the size of Lactobacillus cells under 1000x magnification.

1.) For 400x magnification:

Calibration= stage micrometer ( mm ) / eyepiece division ( no. of intervals )

= 0.01mm / 4

= 0.0025mm

=2.5μm

2.) For 1000x magnification:

Calibration= stage micrometer ( mm ) / eyepiece division ( no. of intervals )

= 0.001mm / 10

= 0.001mm

= 1μm

1.) Therefore,
a.) The size of Yeast under 400x magnification= Intervals on the eyepiece graticule X

calibration under 400x magnification

= 2 X 2.5μm = 5μm

b.)The size of Yeast under 1000x magnification = Interval on the eyepiece graticule X

calibration under 1000x magnification

=5 X 1= 5μm

c.) The size of Lactobacillus under 1000x obj.magnification = Interval on the eyepiece graticule X

calibration under 1000x

magnification
= 3 X 1μm= 3μm

Discussions:

1.) An ocular micrometer is a glass disk that attaches to a microscope’s eyepiece. I t has a ruler that allows the user to measure the size of magnified objects.

2.) The distance between the marks on the ruler depends upon the degree of magnification. When we change magnifications, it appears as though as the size oat the stage micrometer is changing while the ocular micrometer remains fixed. Both micrometers actually stay fixed, but the view of the stage microscope becomes distorted as the magnification changes.

3.) Before we started to measure the cells, we took some time to move the stage until the lines of the ocular micrometer superimposed on the stage micrometer. This step is taken due to we could only able to count the spaces of each micrometer to a point at which the lines of the micrometers coincided.

Conclusion:

As a conclusion, the size of microorganisms can be measured by using ocular micrometer. Besides, i found that the size of Yeast is bigger than the Lactobacillus.

Reference:

http://wiki.answers.com/Q/Why_it_is_necessary_to_calibrate_ocular_micrometer

2.2 Neubauer Chamber

Introduction

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

Objective:

To count cells by using microscope.

Results:

Photo that shows the no. of cells (Yeast) under 40x objective magnification.

1.) Volume of the 16 small boxes = 0.2mm x 0.2mm x 0.1mm

= 0.004mm³

= 0.000004 cm³

= 0.000004 mL

	12	11	12
9			16
15	15		17
8		8

2.) Total no. of cells counted in the 10 small boxes = 123cells

3.) Average no. of cells in 10 small boxes = 123cells/ 10

= 12.3cells

4.) The cell concentration = 12.3cells / 0.000004 mL

= 3075000 cells / mL

Discussions:

1.) There are several precaution steps in this experiment:

a.) Clean chamber and coverslip with lens paper with a little ethanol to remove any grease.

b.) Hold the pipette with one hand and use the upper surface of the index finger of the other hand to guide the tip; the pipette should be held at 45 ̊ angle to the chamber. The chamber should fill very quickly with liquid by capillary action. Not much pipette bulb pressure is required. If there is any slow filling or if the liquid front is not even, for example when a bubble is formed, we need to start over.

c.) Place the cover slip over the counting platform, pressing on the elevated ridges of the hemocytometer, but not the center.

2.) During calculation of cells, we do not need to count the whole chamber. We just count in a section of the chamber and use the grid to determine what proportion of the chamber that is. Then we continue our calculation by estimating how many cells are in the chamber.

3.) Scan square subdivisions from left to right, up to down and count the cells. Count the cells if they are touching the left or top line of each square (doesn’t matter which ones- it can be bottom and right line but be consistent!) too, make sure that the cells that touching the lines are not counted twice.Do not

count the cell in the bottom row either.

Conclusion

From this experiment, I found that the cell concentration for yeast is 3075000cells per mL. Besides, I have learned the method to calculate the cells by using the Neubauer chamber.

References:

http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html#top

http://www.immunocytometry.com/hematocytometer.pdf