Lab 4271

2.1 OCULAR MICROMETER

Introduction:

An ocular micrometer is a glass disk that attaches to a microscope eyepiece. An ocular micrometer has a ruler that allows the user to measure the size of magnified objects. The distance between the marks on the ruler depends upon the degree of magnification. The ruler on a typical ocular micrometer has between 50 to 100 individual marks, is 2 mm long and has a distance of 0.01 mm between marks.

Microscope Eyepiece

Ocular Micrometer

We can used different type of the magnifications to observe the sample. Firstly, we need to calibrate ocular micrometer before used it to observe the cells. it is because the ocular micrometer is inserted inside the ocular lens, it will not change size when the objectives are changed. Therefore, each objective lens must be calibrated separately.

Ocular Micrometer as Seen Through a Microscope Eyepiece

Objectives:

To measure cells using a microscope.

Results:

Calibration Scale

Calibration:

400x:

5 x 0.01 = 0.05

0.05/ 20 = 0.0025 mm

1000x:

5 x 0.01 = 0.05mm

0.05/ 47 = 0.0011 mm

Lactobacillus sp :

400x magnification

2 division x 0.0025 mm = 0.005 mm

1000x magnification

5 division x 0.0011 mm = 0.0055 mm

Yeast :

400x magnification

5 division x 0.0025 mm = 0.0125 mm

1000x magnification

7 division x 0.0011 mm = 0.0077 mm

Discussion:

During this experiment we must take care any error of Calibration of the Ocular Micrometer on a Microscope. The determination of the objective lens magnification error:

The first step is to correctly line up the stage and ocular micrometer (Make sure two are line up completely paralleled) .Would calculate accurate magnification ration from error margin.
The image below shows a stage micrometer NOB1 (10μm pitch and 1mm/100 divisions) and an ocular micrometer S11 (100μm pitch and 10mm/100 divisions), with an objective lens magnification ratio at 20.
If magnification rate is correct, 10 pichs of stage micrometer should be equal to20 pichs (2000μm) of ocular micrometer (=[1 pitch of ocular micrometer is 10μm] x 10 pichs x [Objective lens x20] ). Instead, in below diagram, 10 pichs of stage micrometer shows equals to 21 divisions of ocular micrometer, therefore it can be calculated that magnification of objective lens is x21.
To achieve the calibrated measurement of the sample, multiply the measured value of the sample by the labeled magnification of the objective lens divided by the actual magnification ratio. (Ex. Calibrated measurement of sample = measurement of sample × labeled magnification ratio ÷ actual magnification ratio) (Calibration ratio = 20/21 = 0.95)

Conclusion:

This experiment has studied the correct way to calibrate ocular micrometer. The Ocular micrometer has a ruler those allows the user to measure the size of magnified objects but it has no units on them without numbers. A special slides which contains scales also used to place the objects being observed. This has to be done because the marks on the ocular micrometer are different distances apart depending on the magnification used on the microscope. It must be calibrated for each objective.This show how to calculate the scale using stage scale and ocular eyepiece. The small particles such as microorganisms or cell can be measure and the size can be compared.

References:

2.1 NEUBAUER CHAMBER

Introduction:

These 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.

We offer 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.

Objectives:

To count cells using microscope.

Results:

Yeast, Magnification: 400x

sum of the cell 10 box = 642

average = 642/10 = 64.2

volume box (16 box) = 0.2 mm x 0.2 mmx 0.1 mm

= 4 x 10^-3 mm

= 4 x 10^-6 cm^3

so that, 64.2 cell in 4 x 10 ^-6 ml

1 ml = 642/ 4 x 10 ^-6 = 1.61 x 10^8 cell/ml

Discussion:

During this experiment we must take consistency in filling the chamber with the cell. The consistency in filling technique is paramount to successful counts.

To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. 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. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power.

Conclusion:

For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types.
A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts. So that, the cells are counted by hemocytometer. The concentration of yeast is 161000000 cells per mL.

References:

lab 2: MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

2.1 Ocular Micrometer

Introduction
Ocular micrometer is use in order to measure and compare size of prokaryotic and eukaryotic microorganisms. Suitable scale for their measurements should be somewhere in the microscope itself.An ocular micrometer is a glass disk that fits in a microscope eyepiece that has a ruled scale, which is used to measure the size of magnified objects. The physical length of the marks on the scale depends on the degree of magnification. the ocular micrometer is inserted in the right eyepiece of the microscope. the calibration factor for the ocular micrometer specific for each ocular objective combination because the objectives have different values of magnification. we can used different type of the magnifications to observe the sample. firstly, we need to calibrate ocular micrometer before used it to observe the cells. it is because the ocular micrometer is inserted inside the ocular lens, it will not change size when the objectives are changed. Therefore, each objective lens must be calibrated separately.

ocular micrometer

Objective

To measure and cells using a microscope.

Results

1. Lactobacillus sp.

400x magnification

1000x magnification

2. Yeast

400x magnifications

1000x magnifications

calibration:

400x:

5 x 0.01 = 0.05

0.05/ 20 = 0.0025 mm

1000x:

5 x 0.01 = 0.05mm

0.05/ 47 = 0.0011 mm

1. Lactobacillus sp.

400x magnification :

2 division x 0.0025 mm = 0.005 mm

1000x magnification :

5 division x 0.0011 mm = 0.0055 mm

2. Yeast

400x magnfication :

5 division x 0.0025 mm = 0.0125 mm

1000x magnification :

7 division x 0.0011 mm = 0.0077 mm

Discussion

from our experiment, we need to measure the cells that we observe which are Lactobacillus sp. and Yeast using the ocular micrometer. An ocular micrometer is a small glass disk with thin lines and numbering etched in the glass.

the ocular micrometer

we can used the ocular micrometer to observe the eukaryotes and the prokaryotes cell and measure their length by using this device. first we need to calibrate both the ocular micrometer at the eyepiece and the stage micrometer at the stage so that both of them superimpose to each other. then we can get the actual number for one devision of the micometer. The vertical distance of an object that is in focus. When magnification is increased, less of the object is in focus (depth of field decreases) – but greater detail of the area in focus can be seen. in this experiment we used Lactobacillus sp. the prokaryotes and the yeast as for the eukaryotes.

Prokaryotes are organisms made up of cells that lack a cell nucleus or any membrane-encased organelles. Eukaryotes are organisms made up of cells that possess a membrane-bound nucleus (that holds genetic material) as well as membrane-bound organelles. Genetic material in eukaryotes is contained within a nucleus within the cell and DNA is organized into chromosomes. we used 400x magnification and 1000x magnification to observe both of the cell. then when the measurement is recorded, we calculated the result obtained to get the actual length of the cells.

Conclusion

as the conclusion, we can determined the sizes and the measurement of the cells by using the ocular micrometer. An ocular micrometer is a glass disk that fits in a microscope eyepiece that has a ruled scale, which is used to measure the size of magnified objects. by using the devices, it can help us to estimate the cells in our experiment. we need to calibrate the ocular micometer placed in our microscop so that we can know the measurement for one division.

Reference

http://en.wikipedia.org/wiki/Ocular_micrometer

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

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

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

http://animals.about.com/od/animalswildlife101/a/diffprokareukar.htm

2.2 NEUBAUER CHAMBER

Introduction

The hemocytometer or haemocytometer is a device originally designed for the counting of blood cells. It is now also used to count other types of cells as well as other microscopic particles. The hemocytometer was invented by Louis-Charles Malassez and consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. 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. For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types. A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts.

Objective

1. To count cells using microscope.

Results

400 x magnification

sum of the cell 10 box = 642

average = 642/10 = 64.2

volume box (16 box) = 0.2 mm x 0.2 mmx 0.1 mm

= 4 x 10^-3 mm

= 4 x 10^-6 cm^3

64.2 cell in 4 x 10 ^-6 ml

1 ml = 642/ 4 x 10 ^-6 = 1.61 x 10^8 cell/ml

Discussion

the method for using Neubauer chamber are we must ensure that the special coverslip provided with the counting chamber (thicker than standard coverslips and with a certified flattness) is properly positioned on the surface of the counting chamber. the cell suspension is applied to the edge of the coverslip to be sucked into the void by capillary action which completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture the sample comes from. It is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start). some of the aseptic technique is applied to avoid contamination to occur.

Conclusion

Neubauer chamber is a tecnique to observe and calculate the cells observed in a specific way. at the last of the experiment, we get the result and achieved our objective. some of the safety measures must be taken to avoid contamination and error in our result.

Reference

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

http://en.wikipedia.org/wiki/Hemocytometer

http://en.wikipedia.org/wiki/File:Neubauer_improved_counting_chamber.jpg