Lab 1- Principles and Use of Microscope

1.1 Setting and using the microscope

Introduction

Microorganisms are organisms that cannot be seen by human naked eyes. They are too small and tiny. However, we can see these microorganisms by using the microscope. There have two types of microscope, electronic and light microscope, but the light microscope is still the instrument most frequently used for viewing microorganisms.

The parts of the light microscope

The figure below shows the typical light microscope:

              (Figure 1.1 Major Parts of a Light Microscope)

          A light microscope uses focused light and lenses to magnify a specimen. It send light through a path that first focuses the light into a tight beam and then passes that light through a sample, which creates an image. That image then passes through one or more lenses to magnify it until it reaches the user's eyes. Because light needs to pass through the sample, it must be either very small or very thin. Most cells (bacterial or otherwise) are both small and transparent, and so light can easily pass through them.

The light to illuminate the specimen comes from the light source. In many microscopes, this is a small bulb, but the ultimate light source may be from a lamp or even a candle - redirected by a mounted mirror. It also projects light upwards through the diaphragm, slide and lenses.

The diaphragm acts like the iris of the human eyes, it controls the intensity and size of the cone light projected on the specimen. As a rule of thumb, the less light is required when the more transparent the specimen which use for observe. The size of the opening at the center of diaphragm can be adjusted by turning the lever on the side of condenser until only the right amount of light enters the objective lenses.

The flat platform that can supports the slide beings analysed and is called stage. Above the stage, there got one or two stage clips to stable and hold the slide in place. This can make sure you to observe more easily and in the correct way.

A light microscope has three or four objective lens and attached to the revolving nosepiece. The magnification level will be changed due to this combination, by a simple process of turning the nosepiece which changes lenses. The light microscope has four objective lens which having the magnifications of 4 times, 10 times, 40 times and 100 times.

The light will be received by the eyepiece tube via the objective and be collected to the eyepiece. An additional magnification will be given by ocular lens in the eyepiece, usually 10 times.  You can see the image of the specimen via this ocular lens.

Last but not least, the coarse-focus and fine-focus adjustment knobs are used to adjust the height of the stage. You can adjust stage until you can focus up the figure of the specimen that you observe. The quality of the image also can be adjusted by both the coarse-focus and fine-focus adjustment knobs.

Magnification and resolution

        The total magnification can be calculated by multiplying the objective lens multiplication and the eyepiece lens multiplication. Therefore, four magnifications will be given by a microscope:

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 power means that the ability distinguish two close together images as being separate. The resolution power can be affected by adjusting the diaphragm, once the diaphragm is opened, the contrast will be decreased but the resolution power will be increased; the diaphragm is closed, the contrast will be increased but the resolution power will be decreased.

          Both magnification and resolution power are very important .The excellent magnification will give you a clear and magnified image, but the high resolution power will give you blurred image. Therefore, no any image will be seen without the magnification even you have an excellent resolution power.

Objective

Learn the proper way to use the simple light microscope.

Materials and reagents

Microscope slide and cover-slip

Results

Observation:     

Penicilium.sp under 40x magnification:   

                               

              

Penicilium.sp under 100x magnification:

             

Penicilium.sp under 400x magnification:

Penicilium.sp under 1000x magnification:

           

Discussion

          We were able to observe a Penicilium.sp clearly by using 40x, 100x and 400x magnification. The observation of the Penicilium.sp specimen can be seen clearly from the lowest magnification power which is 40x to the highest magnification power which is 1000x. The morphology of the Penicilium.sp can be seen very clearly under these magnification power. 

Scientific classification of Penicillium sp.:

Kingdom: Fungi

Class: Eurotiomycetes

Order: Trichocomaceae

Genus: Penicillium

Morphology:

1)    Red in colour

2)    Has highly branched network of multinucleate

3)    Has vacuole

4)    Has rough surface

                     

Reference

1.   Obtained from https://en.wikipedia.org/wiki/Penicillium#Characteristics on 29th September 2015

2.   Obtained from http://www.caltexmoldservices.com/section/mold_library/penicillium/ on 29th September 2015

3.   Obtained from http://elearning.usm.my/current/ on 22th September 2015

4.   Obtained from http://study.com/academy/lesson/light-microscope-definition-uses-parts.html on 22th September 2015

5.   Obtained from http://microbiologyonlineblog.blogspot.my/2009/11/microbiology-online-focus-on-parts-and.html on 22th September 2015

6.  Obtained from http://www.indepthinfo.com/microscopes/parts.htm on 22th September 2015

7. Obtained from http://www.yourdictionary.com/resolving-power on 22th September 2015

1.2 Examination of the cell

Introduction

         The bacteria sizes are very minute and can only be observed through a very high power lens in a microscope.  Bacteria can be motile or immobile and can exist in any shape or sizes. To enable the observation of bacteria under a microscope, we have to prepare them on a slide first. However, standard preparation method such as staining can distort or even destroy the cells. Thus, in order to study the bacteria in its natural state. The wet mount technique use water helps support the sample and it fills the space between the cover slip and the slide allowing light to pass easily through the slide, the sample, and the cover slip.

Objective

a) To provide an experience in use of microscope.
b) To illustrate the diversity of cells and microorganisms.
c) To learn the proper technique in preparing wet mount.
d) To practice the accurate aseptic technique.

Materials and Reagents

Lactusbacillus sp. culture
Immersion oil
Lens tissue
Inoculating loop
Bunsen burner
Slide and coverslip

Procedure

[Refer to Manual]

Result

The sample specimen is the bacteria Lactobacillus sp. which are the rod-shaped bacteria with transparent white colour.

       
Discussion

         Lactobaccillus. sp. is a genus of rod-shaped bacteria which is Gram-positive, fermentative, organotropism. These bacteria appeared to be translucent under microscopic view as it was not stained during this lab observation. They are often found in pairs or chains of varying length. Lactobacilli are classified as lactic acid bacteria, and derive almost all of their energy from the conversion of glucose to lactate during lactic fermentation. They generate ATP by non-oxidative substrate-level phosphorylation. Because of this characteristic, Lactobacillus sp. is majorly use in food industry. These bacteria also serve as beneficial flora in human digestive tract where they inhibit the growth of harmful bacteria by creating an acidic condition on the digestive membrane. Besides, Lactobacillus sp. is also used for therapeutic purpose in medical field , for instance treating diarrhoea in children. Some strains of lactobacilli also shown to have anti-tumour properties in clinical trial experiment.

Conclusion

            Based on the experiment conducted, with the aid of 100x power objective lens under oil immersion, we were able to observe the morphology of the microorganisms like Lactobacillus fermentum clearly under the magnification of 1000x achieved with high resolution with the aid of oil immersion method. The Lactobacillus fermentum is prepared with wet mount observer which enable the bacterium can be observe in its natural state without special treatment or staining the specimen. Besides, we managed to use the microscopes with proper techniques. To avoid contamination, we have learned to practise aseptic techniques while handling cultures.

References

1. Obtained from https://en.wikipedia.org/wiki/Lactobacillus on 28 September 2015

2. Obtained from https://www.nlm.nih.gov/medlineplus/druginfo/natural/790.html on 28 September 2015

3.Obtained from http://www.microbiologybytes.com/video/Bacillus.html on 28 September 2015

4. Obtained from http://www.connecticutvalleybiological.com/bacteria-typical-bacillus-c-1379_1384.html on 28 September 2015