LABORATORY LESSON

Activity No. 1
INTRODUCTION TO THE LIGHT MICROSCOPE

Guide Questions

VARIETY OF LIGHT MICROSCOPE

1. Differentiate between bright-field and dark-field microscopy. When would you use
the dark-field microscope?

Bright-field microscopy is most widely used by microbiologists. It

is where the specimen is seen darker in the surrounding field, thus
suitable for observing specimens with natural color or stained
samples. On the contrary, dark-field microscopy shows bright
specimens on a dark background. Dark-field microscopes are useful
for unstained specimens and cells that are difficult to stain such
as spirochetes, which are difficult to see in bright-field
microscopes.

2. When would you use the phase-contrast microscope? What advantage does the phase-
contrast microscope have over the ordinary bright-field microscope?

Phase contrast microscope is used for viewing certain transparent

microorganisms that are difficult to see using bright-field
microscope. It contains a condenser with an annular diaphragm, as
well as objectives with phase plate, that throw the light “out of
phase” which causes to pass through the object at different speeds
(phase change). This allows the microscope to view the internal
structure of live, unstained organisms, which cannot be seen over
the brightfield microscope.

3. What is the difference between a bright-phase contrast and a dark-phase

contrast microscope?

The optics of the phase contrast microscope would make objects

appear darker (using a positive phase plate that delays the
diffracted light, producing a dark-phase contrast) or brighter
(using a negative phase plate that advances the undiffracted light,
producing a bright-phase contrast), thereby increasing their color
contrast with the surrounding mounting medium.
 4. When is fluorescence microscope used in the laboratory? List two fluorochromes that
are used in staining bacteria.

Fluorescence microscope is used to identify the microorganisms

present in the smear, stains and fluids, and also for viability
counts for cell population. Auramine O and auramine-rhodamine are
commonly used fluorochromes in staining acid-fast bacilli (AFB) such
as Mycobacterium tuberculosis.

5. Differentiate between phosphorescence and fluorescence. Which one usually uses short
wavelength, high frequency light and which usually uses ultraviolet light?

Fluorescence is an absorption of energy followed by immediate emission

of absorbed electromagnetic radiations. Meanwhile, some photons may be
emitted following a delay after the absorption, which is a process
called phosphorescence. Fluorescence tends to use UV light often, has
shorter wavelength and has higher frequency light than phosphorescence.

MICROSCOPE MEASUREMENT AND MAGNIFICATION

1. Calculate the total magnification of the scanning and three common objective lenses
partnered with an ocular that magnifies tenfold (10x).

Total
Objective Type Magnification Ocular
Magnification
Scanning Objective 4x 10x 40x
LPO
(Low Power Objective) 10x 10x 100x

HPO 40x 10x

(High Power Objective) 400x
OIO
(Oil Immersion Objective) 100X 10x 1000x

2. What is an ocular micrometer and a stage micrometer?

3. Why is it necessary to calibrate the ocular micrometer with each objective?

4. Differentiate magnification from resolution.

5. What is the difference of a light microscope from electronic microscope in terms of their
resolving power?
PARTS OF A MICROSCOPE

1. In your experiment notebook, write the function of each of the following?

a. Ocular b. High-power objective

c. Substage d. Oil Immersion objective

e. condenser f. Coarse adjustment knob

g. Irish diaphragm h. Fine adjustment

knob
i. Condenser knob
j. Exciter filter
k. Revolving nosepiece
l. Barrier filter
m. Low-power objective
n. Heat filter

2. Describe the position of your hands when carrying the microscope to and from your
laboratory bench.

3. (a) What two adjustments can be made to the condenser? (b) What effect do these
adjustments have on the image?

4. A phase-contrast microscope differs from a brightfield microscope by having a

a. blue filter in the ocular lens.

b. diaphragm with an annular stop.
c. phase plate in the objective lens.
d. Both (b) and (c) are correct.
e. All are correct
 5. Identify the parts of the compound microscope. (Paste the image in your laboratory
notebook)

References:
Brown, A, and Smith, H (2015). Benson’s Microbiological Applications, Laboratory Manual in
General Microbiology, Short Version 13th Edition. New York, U.S.A.: McGraw-Hill Education.

Harley, J. and Prescott, L. (2002). Laboratory Exercises in Microbiology 5th Edition. New
York, U.S.A.: McGraw-Hill Education.