Thursday, 4 April 2013

LAB 1: PRINCIPLES AND USE OF MICROSCOPE

1.1 Setting up and using the microscope

Introduction:
Microscope is an instrument use to see objects that are too small for the naked eyes such as microorganism. There are several types of microscopes. One of them is light microscope which uses light to image the sample.

The diagram below shows the parts of the light microscope :



The light switch functioning in turns on the current to the bulb whereas the brightness of the bulb is being controlled by the voltage control dial. The condenser collects the light into a column and directs it substage condenser. Then, it focuses the light onto the specimen.

The condenser diaphragm confines the column of light entering the substage so that the correct amount enters the objective lens.  

The function of the stage is holds the microscope slide. The stage may be moved backwards and forwards and from left to right by using the coaxial knobs.

Both the coarse-focus and fine-focus adjustment knob act to raise or lower the mechanical stage so that the objective lens is focused on the specimen. The stage can be moved quite rapidly by using the coarse-focus knob.

The light passing through the specimen to form a magnified primary image can be focused by using the objective lens. Several objectives are located on the revolving nosepiece, which rotates to bring the desired objective into the light path. The microscope usually has four objectives with magnification of 4x, 10x, 40x and 100x.

The eyepiece/ocular lenses functioning in receiving the light that coming through the objective and redirects it to the eyepiece. The eyepiece consists of several lenses that collect the light, focus it and transmit it to your eye.

Magnification and resolution
In order to know the total magnification of the image we se under the microscope, we have to multiply the objective lens multiplication (power) by the eyepiece lens multiplication (power). Thus, the microscope will give four magnifications:

·         4x objective X 10x eyepiece = 40x magnification
·         10x objective X 10x eyepiece =100x magnification
·         40x objective X 10x eyepiece =400x magnification
·         100x objective X 10x eyepiece =1000x magnification

The resolution of microscope is its ability to distinguish two very small and closely spaced objects as separate entities. One of the factors that affect resolution is the condenser diaphragm. Closing the diaphragm image contrast but decrease resolution while opening the diaphragm decreases contrast but increase resolution. Both magnification and resolution are equally important.

Objective: 
Learn to use a simple bright-field microscope correctly.

Materials and reagents:
Microscope slides, cover slip, and immersion oil

Methods:
1. The microscope is being setting up.
2. The light intensity is adjusted by the brightness control.
3. The 4X objective lens is brought into the light path by rotating the nosepiece.
4. A clean slide is marked with a marker pen and placed it on the stage.
5. The slide is focused and observed by us.

Low power (10X) objective viewing:
1. The marked slide is replaced with a specimen slide and placed it on the stage.
2. The view of specimen is obtained and focused by using fine adjustment knob and by moving the stage.
3. The condenser is focused by placing an object in the centre of the glass above the light.
4. The specimen is observed by us.

High power (40X) objective viewing:
1. The specimen is focused with the 10X objective and it changed to the 40X objective by watching from the side of our microscope.
2. The condenser is adjusted.
3. The specimen is focused by adjusting the fine control.

Oil immersion (100X) objective viewing:
1. A drop of immersion oil is put on the specimen slide.
2. The specimen is focused with the 40X objective and it changed to the 100X objective by watching from the side of our microscope.(  The objective is not allowed to touch the slide.)
3. The condenser is adjusted.
4. The specimen is focused by adjusting the fine control.

Results:

Salmonella enteritidis under 40x magnification:


Salmonella enteritidis under 100 x magnification:

Salmonella enteritidis under 400 x magnification:
                                               

Salmonella enteritidis under 1000 x magnification:


Penicillium under 400X magnification:
                                  
Discussion:
1)      Microscope is positioned so that it can be observed  through the eyepiece comfortably.
2)      Specimen is observed from the lowest power objective, 4x magnification followed with 10x  magnification and 40x magnification.
3)       Light intensity is adjusted to enable the correct amount of light enters the microscope and diaphragm is also adjusted to have a clearer view of the specimen.
4)      The fine adjustment knob is used first followed by the coarse adjustment knob to   ensure that the image is clear and focused at the centre while observing the images,.
5)   The microorganism that was observed and drawn is Salmonella enteritidis.
6)    Morphology of Salmonella enteritidis:
                 Shape: circular 
                 Size: tiny (punctiform)
                 Surface: shinny and smooth
                 Color: red
                 
         Morphology of Penicillium:
                  Shape: phialide
                  Size: colony
                  Surface:exudate
                  Colour: violet

Conclusion:
The higher the magnification of objective lens, the clearer the view of specimen that we can be observed. The view is narrower and more specific for higher magnification.

References:
http://en.wikipedia.org/wiki/Penicillium







1.2 Examination of cells

Introduction:
Generally, to study bacteria, the bacteria will be stained and observed with the oil immersion objective. They are not generally studied with the low-power or high power-power dry objectives. This is because they have extreme minuteness.
To study the sizes and shapes of the microorganism, we use the wet mount method. We also can know if the cells are motile by using this method. The wet mount method is quick and easy, and does not require special equipment.

Objectives:
·      To provide an experience in the use of microscope.
·      To illustrate the diversity of cells and microorganisms.

Materials and reagents:
Culture- a small bottle of microorganisms (Lactibacillus and E.coli respectively), Immersion oil, inoculating loop, bunsen burner, slide,and coverslip.

Methods:
1. One drop of culture is transferred aseptically to the centre of a glass slide by using a sterile inoculating loop.
2. The drop of culture is covered by cover slip at 45 degree.
3. The slide is placed on the microscope stage and using 4X objective focus on the culture.
4. The cell is observed by using 10X and 40X objectives and the image is taken down or drawn.
5. The cell is observed by using oil immersion lens. The condenser and diaphragm is adjusted to get clearer image.
6. The steps are repeated with other culture.

Result:

Lactobacillus  under 1000x magnification:

Eschericia coli under 1000X magnification:



Discussion:
1.)    A small drop of special immersion oil is placed on the slide and the front lens of the objective is dipped into it for viewing.
2.)    The oil immersion fills the space between the objective lens and specimen and matches the refractive index of the glass coverslip and glass objective lens. At a given focal length, this allows us to achieve a greater numerical aperture.
3.)    The microorganisms are observed in this experiment were Lactobacillus fermentum and Escherichia coli
4.)   Escherichia coli :
   -Shape    : rod
   -Size       : tiny
   -Surface : moist and smooth
   -Colour  : violet
5.)    Morphology of Lactobacillus :
   -Shape   : rod
   -Size      : tiny
   -Surface : moist and smooth
   -Colour  : violet


Conclusion:
100x objective lens immersed in oil could get a clearer view of the specimen. Hence, 1000x magnification with  100x oil immersion objective lens has a good resolution and allow us to observe clearer image.

References:

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