BIO 461

MICROBIOLOGY

LABORATORY 1

THE USE AND CARE OF THE MICROSCOPE

NO. NAME GROUP ID NUMBER

1 PUTERI IMEIN SUFIA BINTI RAS2011A 2019253852
MOHD ADLY
2 QUTREN NADIA BINTI RAS2011A 2019683904
MURTADZA
3 NOR IWANI FARZANA BINTI RAS2011A 2019219726
MOHAMAD ZAMRI
4 FATIN NUR FATEHA BINTI S RAS2011A 2019883868
ABDUL JALIL

LECTURER NAME :

DATE PERFORMED : 25th SEPTEMBER 2019

DATE SUBMITTED : 2cd OCTOBER 2019

PRACTICAL 1 : THE USE AND CARE OF MICROSCOPE

OBJECTIVES :

1. To obtain accurate images.

2. To determine the depth of field.
3. To determine the field of view.
4. To calculate the actual magnification.
5. To apply the use of oil immersion with high magnification.

INTRODUCTION :

Microscope are precision instruments and therefore need to be handled carefully.

microscope can be used to magnify an object, determining the size of an object and
observing fine details of an object which are not discernible to our naked eyes. With the
advancement of technology in microscopy, many high-quality microscope has been
designed for many specific use. There are set of lens that constitutes the objectives lens
which supply the initial, real and magnified image. Another set of lens constitutes the
ocular lens which magnified further the image formed by other set of lens and convert the
real image into virtual image which is in turn viewed by the use’s eyes. In compound
microscope, the actual magnification is calculated as the magnification of object lens
multiplied by the magnification power of the ocular lens. The phase-contrast microscope
that allow user to view living cells or specimen without the use of stains to increase the
contrast. Contrast is the based on the differential absorption of light by parts of the
specimen. There are compound microscope, which employ ultra violet light as the source
of light, making it possible to view specimens that emit fluorescence. There are also
another comping microscope which use either dark field or light field. Another type of
microscope is compound microscope with inverted object, called inverted microscope,
which is used to observe living cell cultures. A microscope is not only capable of
producing the image of an object but also capable of distinguishing between two adjacent
points on the object. This capacity is terms as the resolving power of the lenses or the
resolving power of the microscope. The higher the resolution of the microscope, the
higher is the ability to distinguish details of the objects. The resolving power of a light
microscope depends upon the wavelength (colour) being used, and not on a value called
the numerical aperture (N.A) of the lens system used. The numerical aperture is derived
from a mathematical expression that relate the light transfer to the specimen by the
conduser to the light received by the objective lens.

MATERIALS : Microscope, glass slide, cover slip, lens paper, xylene, eye dropper,
newspaper, scissors and inoculating needle.

METHODS : The microscope that assigned was removed from the cabinet by taking the
arms with one hand and another hand support the instrument at the base.

1. The microscope approximately placed six inches from the edges of the laboratory
table with the microscope arm facing you.
2. The microscope’s component examined and their component listed.
a) Ocular g) Stage
b) Body tube h) Condenser
c) Revolving nosepiece i) Iris diaphragm
knob j) Course adjustment
d) Low power objective k) Fine adjustment
lens l) Mechanical stage
e) High-dry objective lens m) Pillar
f) Oil immersion
3. The exercise designed to familiarize with the certain properties of microscope
components were used.
a) A glass slide and cover slip were cleaned as indicated by instructor then a
drop of water placed on the slide.
b) Next, a series of letters from the newspaper provided are cut. One of the
small letter “e” curtained.
c) The string of the letters placed into the drop of water. The preparation
covered with cover slip. The following steps observed in order to limit the
number of air bubbles in the preparation.
i) The cover slip’s edge placed on the drop of water on the slide so
that the fluid completely wets the edge.
ii) The inoculating needle placed under the cover slip and lowered to
the slide.
 4. Letter “e” examined and made a sketch approximately in the same dimension as it
appeared in the slide.

RESULTS :

Low-power magnification High-power magnification

PROCEDURES FOR MICROSCOPE OPERATION :

1. All lenses wiped with lens paper.

2. The low-power objective moved into position under the body tube until it is
‘click’.
3. The light source connected and turned on.
4. The slide preparation examined over the central opening in the stage.
5. The portion of the preparation arranged to be examined over the central
opening in the stage.
6. The lower-power objective lowered with the aid of the course adjustment knob
to approximately one quarter of an inch above the cover slip of the
preparation.
7. The condenser raised as far as it will go with the aid of the condenser
adjustment knob.
8. The specimen looked through the ocular. Next, the iris diaphragm lever moved
so that the diaphragm opened to the maximum limit. Specimen determined by
looking through the eyepiece. Light intensity do not changed while the iris
lever is being manipulated. Necessary adjustment of light intensity admitted.
9. Next, fine adjustment knob focused upward until specimen comes clearly into
view. The respective adjustment knob controlled the stage because the
microscope having the body tube in the fixed position.
10. The objective simply swing desired into place and the body tube was not
raised. The microscope stay focused with the objective.
 RESULT :

OCULAR

BODY ARM
OCULAR
CONTROL
KNOB

100X
40X
OBJECTIVE
OBJECTIVE
LENS
LENS
SLIDE CLIPS
4X OBJECTIVE LENS

IRIS DIAPHRAGM 10X OBJECTIVE

LENS
FINE FOCUS
STAGE
KNOB
CONDENSER
ILLUMINATOR
BASE
CONCLUSION :

At the end of this experiment, obtain accurate image that can see through the eyepiece by
the objective lenses of 4x,10x, 40x, 100x. Next, we able to determine the depth field of
the image that can be seen through for a clearer and focused image. Also, the total
magnification for each image were calculated. The high magnification used by applying
the oil immersion.
PRACTICAL 2 : EXAMINATION ON STAINED CELLS

OBJECTIVES :

1. To observe stains cell under oil immersion .

2. To observe the size, shape and structure of organisms

INTRODUCTION :

Medical science depends on the staining of cell in tissues to make accurate

diagnoses of a wide range of disease from cholera to sexually transmitted disease,
to parasitic disease and skin infection. Staining techniques performed routinely in
microbiological laboratories include gram’s stain, acid-fast stains, acridine orange,
calcofluor white, toluidine blue, methylene blue, silver stain and fluorescent stain.

Stain are classified broadly as basic, or neutral stains. The chemical natural of the
cells under examination determine which stain is selected for use. Cell staining is
important in the diagnosis of microorganism because bacteria can identified by the
colour differentiation of stain. Microscopic examination of stained cell samples
allowed examination of the size, shape and arrangement of organelles, as well as
external appendages such as the whip-like flagella, which are the cell’s organ of
motion. When sample cell are stained to show their chemical composition it is
called differential staining

MATERIALS : Plant cell, animal cell, Staphylococcus aureus, Bacillus subtilis,

Salmonella typhimurium.

PROCEDURE :

1. All the slides examined under the oil immersion objectives.

2. The size, shape and any visible structures of these organisms observed.
3. Illustrations/drawings of all observation made.
RESULT :
 Salmonella typhimurium under 100x magnification

CONCLUSION

At the end of this experiment we can observe stains cell under oil immersion. Next we
also can observe the size, shape and structure of organisms in the microscope.
PRACTICAL 3 : EXAMINATION OF LIVING BACTERIA

OBJECTIVE:

1. To apply the technique of hanging drop

2. To apply the technique of temporary wet mount

INTRODUCTION :

Many bacteria can move using a variety of mechanism like flagella are used for
swimming through water, bacteria gliding and twitching motility move bacteria across
surface and change of buoyancy allow vertical motion. Flagellum of gram-negative
bacteria. The base drives the rotation of the hook and filament. Swimming bacteria
frequently move near 10d lengths per second and a few as a fast as 100. This make them
at least as fast as fish, on a relative scale.

Bacteria species differ in the number an arrangement of flagella on their surface. Some
have a single flagellum (monotrichous), a flagellum and each end (amphitrichous),
clusters pf flagella at the poles of the cell (lophotrichous), while others have flagella
distributed over the entire surface of cell (peritrichous).

Many bacteria such as E.coli have tool distinct modes of movement, forward movement
(swimming) and tumbling. The tumbling allows them to reorient and makes their
movement a three-dimension random walk. The flagella of a unique growth of bacteria,
the spirochaetes, are found between two membrane in the periplasmic space.they have a
distinctive helical body that twist as about as it move.

MATERIALS:

1. 24-hour nutrient broth cultures of Staphylococcus aureus and E.coli, hollow gound
depression slides, vaseline, inoculating loops, glass slides, cover slips and applicator
sticks.

PROCEDURE :

A. HANGING DROP TECHNIQUE

 This technique used to determine whether or not living bacteria are flagellated and
hence motile. This can be of diagnostic importance when followed by the flagellum
stain. The care taken when handling the living bacteria. All microbial cultures treated
as they were pathogenic.

1. Vaseline used to apply to the edges on one surface of cover slip with the aid of a
wooden applicator stick. The treated surface faces placed on the table upward.
2. A drop of bacterial broth culture placed on the centre of the cover slip using a sterile
inoculating loop.
3. A depression slide inverted and lowered it onto the prepared cover slip. The slide
down pressed gently so that Vaseline forms a seal between the slide and the cover
slip.
4. The slide turned over carefully for the drop does not run sideways.
5. The drop focused using the 10x objective. Then, changed to 40x objective and the
light intensity reduced.
6. The type of movement observed:
a) The bacteria drifting across the field of view caused by evaporation of the
drop through an incomplete Vaseline seal.
b) Brownian motion.
c) True motility-a characteristic of flagellated bacteria. The bacteria observed
darting about and changing direction.
7. The findings recorded.

B. TEMPORARY WET MOUNT TECHNIQUE

PROCEDURE :

1. The inoculating loop flamed to redness.

2. The plug from the bacterial culture removed to flame the mouth of the tube.
3. A loopful of the culture removed and placed it in the centre of a clean glass slide.
4. The mouth of the tube removed and the plug returned.
5. The inoculating loop sterilized.
6. A cover slip put over the bacterial suspension and pressed gently.
7. The preparation examined under the low power and the under the high power
objectives.
RESULTS :

E.coli under 1000x magnification

Staphylococcus aureus under 1000x magnication

 CONCLUSION

At the end of this experiment, we can apply the technique of hanging drop. Next, we also
can apply the technique of temporary wet mount.

REFERENCES :

1) Reece, J. B., & Campbell, N. A. (2011). Campbell biology. Boston: Benjamin

Cummings / Pearson.
2) https://www.microscopeworld.com/t-dissecting_microscopes.aspx
3) Kementerian Pendidikan Malaysia. (2015). Laboratory manual (2nd Ed.),
Matriculation Division Kementerian Pelajaran Malaysia.