LAB 1

1.1 Setting up and using the microscope

Introduction

Since microorganisms are invisible to the unaided eyes, the essential tool in microbiology is the microscope. Microscope is an instrument used to magnify objects that are too small to be observed through our naked eyes such as microorganisms. The microscope must be able to accomplish three tasks: produce a magnified image of the specimen (magnification), able to separate the details in the image (resolution) and to render the details visible to the naked eyes, camera or other imaging device (contrast). There are two types of microscopes, light microscope and electron microscope. Light microscope is used to conduct this experiment.

Parts of the microscope

The description below is to highlight the main parts of the light microscope (Diagram 1.1)

Diagram 1.1

The light is the main source of illumination. Eyepiece tube is used to connect the eyepiece to the objective lenses. Eyepiece is another component located at the top of the microscope which contains magnifying lens that focus the image from the objective lens into our naked eyes. Typically, standard eyepieces have a magnification power of 10x.

Objective lenses consist of 4x, 10x, 40x and 100x magnification powers which will focus the light passing through the specimen to form a magnified image. The shortest lens have the lowest power while the longest ones have the highest power.

Coarse adjustment knob make major adjustments to the focusing of an object by adjusting the stage upward or downward. Fine adjustment knob focus the object in order to produce low resolution and clearer image. A coarse adjustment knob is usually larger than fine adjustment knob. Both are located on the side of the microscope. The intensity of light can be adjusted by illumination intensity knob.

The mechanical stage is another part of the microscope which acts as a  platform to place the microscope slide. Iris diaphragm is located above the condenser and below the mechanical stage. It is helpful in controlling the amount of light passing through the mechanical stage. A condenser is lens that serves to concentrate light from the illumination source which in turn is focused through the object and magnified by the objective lens. It is located under the mechanical stage often in conjunction with an iris diaphragm. Base provides basal support for the microscope.

Magnification and resolution

Magnification is a change in the apparent size of an object so that the object can be seen more easily. The total magnification of the image can be calculated by using the formula below:

Objective lens multiplication X Eyepiece lens multiplication

Therefore, four magnifications will be produced by the 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 resolving power is defined as the ability of an optical device to produce separate images of closely packed or smaller objects. Both magnification and resolution are important in viewing the image. It is possible to have high magnification with low resolution or low magnification with high resolution causing a blurred image. 

Microscope resolution is affected by several elements. An optical microscope set on a high magnification may produce a more blur image and yet at the maximum resolution of the objective lens. The numerical aperture of the objective lens affects the resolution. 

These numbers indicate the ability of the lens to gather light and resolve a point at a fixed distance from the lens.  Closing or opening the condenser diaphragm controls the angle of the light rays emerging from the condenser. This enables the rays to reach the specimen from all angles. As the condenser diaphragm is opened, the working numerical aperture of the microscope increases, resulting in greater resolving power and light transmittance.

Objective

·        Learn to operate the simple light microscope properly with the correct way.

·        Identify and understand the principle components of a light microscope.

Material and reagents

·        Microscope slide

·        Specimen slide

·        Cover slip

·        Immersion oil

·        Marker pen

·        Lens tissue

Procedure

Setting up:

1. We made sure that we are sitting comfortably at the proper position.
2. The power lead was plugged in and the light source was switched on using the on-off switch.
3. The binocular tubes were grasped with both hands while looking through the eyepieces of the binocular observation tubes. The tubes were brought closer together or further apart so that the two circles of light were fused into one circle.
4. The  objective lens with 4x magnification was rotated into the light path.
5. The slide was taken and marked with a small “X” in the middle of the slide by using a marker pen. The slide was then placed properly onto the mechanical stage and was held by a spring clip.
6. The slide was adjusted until a clear image comes into focus with the use of coarse adjustment knob and fine adjustment knob.
7. The slide was removed from the mechanical stage of the microscope after the observation was made.

Low power (10x) objective viewing:

1. A marked slide was replaced with a specimen slide and was held by a spring clip.
2. The specimen slide was adjusted until a clear image was obtained with the use of coarse adjustment knob and fine adjustment knob.
3. The objective lens was changed with the 10x objective.
4. The specimen slide was then refocused with the adjustment of coaxial stage control knobs.
5. Observation was made.

High power( 40x) objective viewing:

1. The nosepiece of 10x objective was rotated for 40x magnification.
2. The mechanical stage was raised up within 1 cm of the objective lens.
3. The specimen slide was refocused using the fine focus and coarse focus.
4. The light intensity was adjusted using condenser diaphragm and illumination intensity knob to obtain the optimum brightness. 
5. The specimen slide was removed for the oil immersion procedure.

Oil immersion (100x) objective viewing:

1. One or two drops of immersion oil was put over the specimen slide cover slip for oil immersion (100x) objective viewing.
2. The specimen slide was put on the mechanical stage and was secured by a spring clip.
3. The 100x objective lens was rotated into the light path. The immersion oil was brought into contact with the 100x objective lens by the adjustment of coaxial stage control knob to produce a clearer image.
4. The light intensity was adjusted using condenser diaphragm and illumination intensity knob to obtain the optimum brightness. 
5. The specimen slide was then removed.
6. The light source was switched off and the power lead was plugged off.
7. The specimen slide and the four objective lens was cleaned with lens tissues.
8. The specimen slide was then returned.

After use:

1. The specimen slide was removed and put into the special beaker.
2. The light brightness control was turned to the lowest setting.
3. The objective power was turned to the lowest working position.
4. The oil was cleaned from the 100x objective by using lens tissue.
5. The switch of the microscope was turned off and the plug was removed.

Care of the microscope: 

1. The base and the metal support arm was held when carrying the microscope, the microscope should not be lifted up using the middle part of microscope or stage.
2. The microscope cannot be placed near the lower edge of the bench.
3. The plug (not the cable) was held when unplugging the illuminator.
4. The stage and lenses were clean before putting the microscope away.
5. The lenses should not be handled with your fingers. The specified lens tissues were used for cleaning lenses.
6. The oil immersion lens were cleaned with chemicals provided in the lab.
7. The stage was lowered before placing or removing a slide.
8. The lowest power objective was put into a working position and the cover was replaced before the microscope was put away.

Introduction

Some samples can be placed directly under the microscope. However, many samples look better when a drop of immersion oil is placed on the microscope slide. This is known as “wet mount” . The immersion oil helps support the sample and allow light to pass easily through the slide and the cover slip. The wet mount method enables us to study the shapes and sizes of living microorganisms. It also enables us to determine whether the cells are motile or not.

Objective

·        To provide an experience in the usage of microscope and aseptic technique.

·        To learn wet mount method of microorganisms.

Materials and reagents

- Culture (lactobacillus)
- Immersion oil
- Lens tissue
- A microscope slide containing stained microorganisms
- Inoculating loop
- Bunsen burner
- Slide and cover slip 
 



Procedure

1.  The inoculating wire was held by its handle and the wire was heated with the flame of a Bunsen burner.
2.  The vessel was held with the left hand at 45°C and the closure was removed gently.
3. The opening of the vessel was passed near the Bunsen burner flame to burn off any contaminating matter.
4.   The sterile inoculating wire was used to transfer a few drops of culture to the center of a glass slide aseptically. Then, one edge of the cover slip was placed onto the slide and lowered gently.
5.   The slide was placed on the microscope stage and the 4x objective focus was used to view the culture. The step was repeated by using the 10x objective focus.
6.   The cells was then observed by using oil immersion lens (100x objective focus). The condenser and diaphragm was adjusted.

Result

1.     Vibrio cholerae

Colour: red

Shape: comma

10x objective viewing

40x objective viewing

 

2.     Salmonella entritidis

Colour: colourless

Shape: rod

40x objective viewing

3.     Clostridium  perfringens
 

Colour: violet

Shape: rod

40x objective viewing

DISCUSSION

Vibrio cholerae is a type of gram-negative bacteria which is red in colour due to counterstain (usually safranin). It is a comma-shaped bacteria and has a singular polar flagellum used for motility. Vibrio cholerae is a facultative anaerobic organism. Some strains of Vibrio cholerae will cause pandemic disease cholera by infecting the intestine and increase mucous production resulting in diarrhea, vomiting and dehydration. This is because Vibrio cholerae will be transmitted through faeces, contaminated water and even food.

The other type of bacteria studied in this experiment is Clostridium perfringes. It is a gram-positive rod-shaped bacteria which grows under anaerobic condition and forms spore. It is found as a normal component of decaying vegetation, marine sediment, intestinal tract of human, vertebrates and soil. It is also the most common bacterial agent for gas gangrene which will causes myonecrosis (muscle tissue death). 

Salmonella entritidis is a rod-shaped gram-negative bacteria. This bacteria have flagella which has tail like projections made of protein aiding bacteria movement. Salmonella entritidis live in the intestine of both cold and warm blooded animals. This kind of bacteria also causes food poisoning in humans known as salmonellosis. Salmonella entritidis produces enterotoxins which acts on small intestine. Other than that, infection will occur when a number of these bacteria are ingested through contaminated food such as meat, milk and eggs.

CONCLUSION

From the experiment, we can draw a conclusion that the higher the magnification power with lower resolution which uses oil immersion method (100x), the clearer the image produced. The image with higher magnification power is able render the separated details visible to our unaided eyes.

REFERENCE

1.     Obtained from www.wikipedia.org  on 28 Oktober 2014

2.     Obtained from www.biologyjunction.com on 29 Oktober 2014

3.     Obtained from www.cliffsnotes.com on 29 Oktober 2014

4.  Michael L. Schuler, Fikret Kargi (1992). Bioprocess Engineering- Basic concepts, Prentice Hall International Series, France.