LAB 2: MEASUREMENT AND COUNTING OF CELLS USING MICROSCOPE

2.1 OCULAR MICROMETER

INTRODUCTION

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.

OBJECTIVE

To measure and count cells using a microscope

MATERIALS AND REAGENT

Microscope fitted with an ocular micrometer

Slide micrometer

Stained preparation of yeast and bacteria

PROCEDURE

(refer to lab manual)

RESULT

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

The ratio of magnification was calculated by:

the observed  diameter of yeast is 1mm ocular

Using 40x magnification,

1mm on stage represent 8.9mm on eyepiece under 40x magnification view

Magnification	Ocular(mm)	Stage(mm)
10x	0.89	0.1
40x	8.9	1.0

CONCLUSION

The size of the microorganism can be measured by using ocular micrometer and stage micrometer.

REFERENCES

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

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

2.2 NEUBAUER CHAMBER

INTRODUCTION

The hemocytometer is a device used to count cells. It was originally designed for the counting of blood cells.

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 cellsor particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

MATERIALS AND REAGENTS

Serial dilutions of bacteria cultures
Neubauer and coverslip
70% ethanol
Sterile Pastuer pipettes

PROCEDURE

(refer to lab manual)

RESULT

Number of cell = 1550

Boxes = 5

1550 = 310 (average for 1 box)

   5

Total 256 small box = 310 x 256

                               = 79360

Volume       = 1 x 1 x 0.1

                   = 0.1mm³

1mm³              = 0.001cm³

0.1  mm³         = 1 x 10^-4mL

1 x 10^-4 cm³ = 1 x 10^-4mL

79360

1 x 10-4mL

 Concentration =

                        

                         = 793 600 000

CONCLUSION

By using the neubauer chamber, we can estimate and hence calculate the number of microorganisms

REFERENCES

http://www.microbehunter.com/the-hemocytometer-counting-chamber/
http://groups.molbiosci.northwestern.edu/morimoto/research/Protocols/II.%20Eukaryotes/A.%20Cell%20Culture/3b.%20Hemacytometer.pdf

LAB 1 : PRINCIPLES AND USE OF MICROSCOPE

INTRODUCTION

 "Micro" refers to tiny, "scope" refers to view or look at. Microscopes are tools used to enlarge images of small objects that can not be seen by naked eye so as they can be studied.
There are many type of microscopes such as electron microscope and ultramicroscope but the most widely used are the optical microscope or the light microscope. The light microscope is an instrument which uses two lenses which magnifies and helps to focus the image of the small object.

                                                    Light Compound Microscope

Part of microscope	Function
Eyepiece Lens	the lens at the top that you look through.  They are usually 10X or 15X power.
Tube	Connects the eyepiece to the objective lenses
Arm	Supports the tube and connects it to the base
Base	The bottom of the microscope, used for support
Illuminator	A steady light source (110 volts) used in place of a mirror. If the microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage.
Stage	The flat platform where the slides is placed. Stage clips hold the slides in place. Mechanical stage  able to move the slide around by turning two knobs. One moves it left and right, the other moves it up and down.
Revolving Nosepiece or Turret	This is the part that holds two or more objective lenses and can be rotated to easily change power.
Objective Lenses	There are 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X.  To have good resolution at 1000X, you will need a relatively sophisticated microscope with an Abbe condenser.  The shortest lens is the lowest power, the longest one is the lens with the greatest power.  Lenses are color coded and if built to DIN standards are interchangeable between microscopes.  The high power objective lenses are retractable (i.e. 40XR).  This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide.  All quality microscopes have achromatic, parcentered, parfocal lenses.
Rack Stop	This is an adjustment that determines how close the objective lens can get to the slide.  It is set at the factory and keeps students from cranking the high power objective lens down into the slide and breaking things. Adjust this if very thin slides been using and the specimen unable to focus on high power.
Condenser Lens	The purpose of the condenser lens is to focus the light onto the specimen.  Condenser lenses are most useful at the highest powers (400X and above).  Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X).
Diaphragm or Iris	Many microscopes have a rotating disk under the stage.  This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide.  There is no set rule regarding which setting to use for a particular power.   Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use.

                 Magnification and Resolution

When viewing a slide through the microscope make sure that the stage is all the way down and the 4X scanning objective is locked into place. Place the slide that you want to view over the aperture and gently move the stage clips over top of the slide to hold it into place. Beginning with the 4X objective, looking through the eyepiece making sure to keep both eyes open (if you have trouble cover one eye with your hand) slowly move the stage upward using the coarses adjustment knob until the image becomes clear. This is the only time in the process that you will need to use the coarse adjustment knob. The microscopes that you will be using are parfocal, meaning that the image does not need to be radically focused when changing the magnification. To magnify the image to the next level rotate the nosepiece to the 10X objective. While looking through the eyepiece focus the image into view using only the fine adjustment knob, this should only take a slight turn of the fine adjustment knob to complete this task. To magnify the image to the next level rotate the nosepiece to the 40X objective. While looking through the eyepiece focus the image into view using only the fine adjustment knob, this should only take a slight turn of the fine adjustment knob to complete this task.

Total Magnification:

To figure the total magnification of an image that you are viewing through the microscope is really quite simple. To get the total magnification take the power of the objective (4X, 10X, 40x) and multiply by the power of the eyepiece, usually 10X.Total magnification = Objective lens power x eyepiece lens power

OBJECTIVE -To learn the importance of magnification and resolution of microscope. -To learn the ways to take care of the microscope. MATERIALS AND REAGENT Microscope slide and cover-slip. PROCEDURE (refer to manual) RESULT 10x4 10x10 CONCLUSION -We observed that magnification enebles us to see speciment in details, while resolution enebles us to see clearer object. We find that both magnification and resolution is important for us to get a quality image of speciment. -Thus, microscope is an expensive instrument. When we walk with it, we must pick up the microscope and grab the arm with one hand and place your other hand on the bottom of the base. DON'T SWING THE MICROSCOPE ! Never touch the lenses with our fingers. Body produces an oil that smudges the glass. This oil can even etch the glass if left on too long. Use only LENS PAPER to clean the glass. When we finished with our "scope" assignment, rotate the nosepiece so that it's on the low power objective, roll the nosepiece so that it's all the way down to the stage, then replace the dust cover. All slides must be cleaned, materials, and work area when you're done. Please, be careful with the slides and cover slips. They are made of glass and if broken, we will get cut and will bleed. REFERENCES http://www.microscope-microscope.org/basic/microscope-parts.htm http://en.wikipedia.org/wiki/Microscope 1.2 EXAMINATION OF CELL INTRODUCTION Historically, the study of cell biology could not have happened without the invention of microscopes because cells were not known to exist before Antonin van Leeuwenhoek and Robert Hooke saw them in their primitive microscopes Today, much cell biology research still requires careful microscopic examination of cells and their internal structures. It is not too strong a statement to say that microscopy is the single most important tool for the cell biologist. OBJECTIVE To provide an experience in the use of microscope To illustrate the diversity of cell and microorganisms. MATERIAL AND REAGENTS  -Culture  -Immersion Oil -Lens Tissue -A Microscope slide containing stained microorganism -Inoculating loop -Bunsen Burner -Slide and coverslip PROCEDURE  (refer to lab manual ) RESULT Lactobacillus Lactobacillus DISCUSION Lactobacillus Lactobacillus are rod-shaped, Gram-positive, fermentative, organotrophs. They are usually straight, although they can form spiral or coccobacillary forms under certain conditions. They are often found in pairs or chains of varying length. Lactobacillus are classified as lactic acid bacteria, and derive almost all of their energy from the conversion of glucose to lactate during homolactic fermentation. In this process 85-90% of the sugar utilized is converted to lactic acid. They generate ATP by nonoxidative substrate-level phosphorylation. Gram positive bacteria In general, the following characteristics are present in .Gram-positive bacteria has the following characteristic, that is it they have cytoplasmic lipid membrane, thick peptidoglycan layer, and they are also a class of bacteria that take up the crystal violet stain. used in the Gram staining method of bacterial differentiation. The thick peptidoglycan layer in the cell wall that encases their cell membrane retains the stain, making definitive identification possible. Only some species have a capsule usually consisting of polysaccharides. Also only some species are flagellated, and when they are, they only have two rings to support them. CONCLUSION Based on the experiment that we observe, The Lactobacillus is a rod shape-bacteria. We found that some of them are straight while others are spiral. REFERENCE https://microbewiki.kenyon.edu/index.php/Lactobacillus http://water.me.vccs.edu/courses/env108/lesson5_2.htm https://answers.yahoo.com/question/index?qid=20080718220806AABkm2Z http://en.wikipedia.org/wiki/Gram-positive_bacteria