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MICROSCOPY

MICROORGANISMS
➢ Are miniscule organisms that cannot be seen
with the naked eye.
➢ The discovery of the microscope has led to their
close observation, allowing microbiologists and
other scientists to study them further.

MICROSCOPE
➢ Is an optical instrument that can magnify
organisms a hundredfold or even a
thousandfold. From the time of its initial
Figure 1.1 Two compound light microscopes which differ in their light source.
development in the 1600s, the microscope has
undergone great revolutionary changes, making
it more advanced and complex throughout
time.

DIFFERENT TYPES OF MICROSCOPES

That have evolved from van Leeuwenhoek’s simple prototype.

1. Compound Microscope
• The compound microscope is a type of
microscope that contains more than one
magnifying lens.
• It can magnify objects approximately a
thousand times their original size.
• Visible light is its main source of
illumination.
• As such, it is also known as the compound
light microscope.
• The compound microscope utilized today
consists of two magnifying lens systems.
• The eyepiece (or ocular) contains what is
called the ocular lens that has a magnifying
power of 10x.
• The second lens system is located in the
objective that is positioned directly above
the organism to be viewed.
2. Brightfield Microscope
• Made up of a series of lenses and utilizing
visible light as its source of illumination, the
brightfield microscope can magnify an
object 1,000 to 1,500 times.
• This is used to visualize bacteria and fungi.
Objects less than or thinner than 0.2 μm
cannot be visualized by this type of
microscope.
• The term “brightfield” is derived from the
fact that the specimen appears dark against
the surrounding bright viewer field of this
Figure 1.1
microscope.

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• However, it has very low contrast and most
of the cells need to be stained to be
properly viewed.
3. Darkfield Microscope
• This microscope utilizes reflected light
instead of transmitted light, with a special
condenser that has an opaque disc that
blocks the light, such that only the specimen
is illuminated.
• The specimen to be studied appears bright
against a dark background.
• This type of microscope is ideal for studying
specimens that are unstained or transparent
and absorb little or no light.
• It is also useful in examining the external
details of the specimen such as its outline or
surface.
• This type of microscope is used to view
spirochetes.
4. Phase-contrast Microscope
• Phase contrast microscopy is based on the
principle that differences in refractive
indices and light waves passing through
transparent objects assume different
phases.
• This type of microscopy was first introduced
by Frits Zernike, a Dutch physicist, in 1934.
• The phase contrast microscope has a
contrast enhancing optical technique in
order to produce high contrast images of
specimens that are transparent which
include thin tissue slices, living cells in
culture, and subcellular particles (such as
nuclei and organelles).
Figure 1.2 Phase-contrast Microscope and its parts.
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5. Differential Interference Contrast Microscope
• The differential interference contrast
microscope is similar to the phase contrast
microscope except that it utilizes two beams
of light instead of one and therefore has
higher resolution.
• The resulting contrasting colors of the
specimen being studied are due to the
prisms that split the light beam.
• It was developed by Georges Nomarski in
1952 as an improvement to the phase
contrast microscope.
• It is useful in examining living specimens
when normal biological processes might be
inhibited by standard staining procedures.
• However, the three-dimensional image of
the specimen produced may not be accurate
since the enhanced areas of light and
shadow may distort the appearance of the
image.
6. Fluorescence Microscope
• The fluorescence microscope makes use of
ultraviolet light and fluorescent dyes called
fluorochromes.
• The specimen understudy fluoresces or
appears to shine against a dark background.
• Fluorescence microscopy is based on the
principle that certain materials emit energy
that is detectable as visible light when they
are irradiated with the light of a given
wavelength.
• It uses a higher intensity of light source and
this in turn excites a fluorescent species.
• The fluorescent species then emits a lower
energy light of a longer wavelength which
produces the magnified image instead of
the original light source.
• Fluorescence microscopy can be used to
visualize structural components of small
specimens such as cells and to detect the
viability of cell populations.
• It may also be used to visualize the genetic
material of the cell (DNA and RNA).
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7. Confocal Microscope
• Also known as the confocal laser scanning
microscope (CLSM) or laser confocal
scanning microscope (LCSM), the confocal
microscope uses an optical imaging
technique that increases optical resolution
and contrast of the micrograph by using a
spatial pin hole to block out of focus light in
image formation.
• The specimen is stained with a fluorescent
dye to make it emit or return light.
• The object is scanned with a laser into
planes and regions.
• This is used, together with computers, to
produce a three-dimensional image.
• It is also useful in the study of cell
physiology.
9. Scanning Probe Microscope
• The scanning probe microscope was
developed in the 1980s by the Swiss
scientists Dr. Gerd Binnig and Dr.
HeinrichRohrer.
• It is used to study the molecular and atomic
shapes of organisms on a nanoscale.
• A physical probe is used to scan back and
forth over the surface of a sample.
• A computer then gathers data that are used
to generate an image of the surface.
• It can also be used to determine the
variations in temperature inside the cell as
well as its chemical properties.

8. Electron Microscope
• The electron microscope utilizes a beam of
electrons to create an image of the
specimen.
• The electron beams serve as the source of
illumination and magnets are used to focus
the beam.
• The first prototype of this microscope was
built by the German Engineer and physicist
Ernst Ruska in 1933, which had a resolution
power of up to 50 nm.
• Modern electron microscopes are capable
of magnifying objects up to 2 million times.
• It is used to visualize viruses and subcellular
structures of the cell.
• There are two types of electron microscopes
• The transmission electron microscope
(TEM)
➢ is the original form of the electron
microscope.
➢ It produces two dimensional, black and
white images, and magnifies objects up
to 200,000 times.
• The scanning electron microscope (SEM)
➢ relies on interactions at the surface
rather than transmission.
➢ It can magnify bulk samples with greater
depth of view so that the image
produced represents the 3D structure of
the sample, but the image is still only
black and white. Generally, it can
magnify the object 10,000 times.

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Parts of Compound Microscope

Eyepiece

Body Tube

Revolving
Nosepiece
Arm
Medium Power
Objective Lens
Low Power
Objective Lens
High Power Stage
Objective Lens

Stage Clip Fine Adjustment

Iris Diaphragm Course

Adjustment

Lamp /
Light Source
Base

15. Power Button

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Basic Laboratory Equipment
Name Function Appearance
1. Beaker Glass container, most are pyrex; common sizes are
100 ml, 25 ml, 400 ml; it can be used as a container,
shows approximate volume, and may be heated

2. Erlenmeyer flask Container; common sizes are 125 ml, 250 ml, 500 ml;
may be heated; it has a thin neck and a wide base;
used to hold liquids when carrying out reactions and
preparing solutions

3. Test tube Glassware that comes in many sizes; it has many

uses and can be heated

4. Graduated cylinder Marked with milliliter (ml) scale and is used to

measure volume

5. Pipette Used to measure and dispense small amounts of

liquids (ex. 1 mL)

6. Burette It is marked with a milliliter scale and fitted with a

stopcock; can be used to withdraw and measure
accurate volumes of solutions in titrations

7. Test tube brush Brush with wire handle; used to scrub thin glassware

8. Cotton swabs Cotton swabs also known as cotton buds or Q-tips

consist of a small wad of cotton attached to one end
of a stick or handle. They are commonly used for
various purposes, including cleaning wounds,
applying or removing ointments, taking samples for
laboratory testing, and precise application of
medications.

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9. Microscope slide A microscope slide is a thin piece of glass used to
support a sample or specimen which needs to be
examined under a microscope.

10. Cover slip Cover slips are small squares of glass that cover the
specimen placed on the microscope slide.

11. Oil immersion Oil immersion is essential for viewing individual

bacteria or details of the striations of skeletal
muscle. It is nearly impossible to view living, motile
protists at a magnification of 1000x, except for the
very smallest and slowest. A disadvantage of oil
immersion viewing is that the oil must stay in
contact, and oil is viscous.

12. Lens paper Lens paper is primarily used for cleaning microscope
lenses and delicate optical equipment in
laboratories.

13. Hot plate Used for consistent heat; used to heat substances
that may be flammable.

14. Centrifuge Centrifuge is a laboratory device that is used for the

separation of fluids, gas or liquid, based on density

15. Tripod stand A laboratory tripod is a portable, three-legged

platform equipment made of lightweight metal such
as stainless steel or aluminum.

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16. Wire gauze Wire screen with ceramic fibered center; used to
spread the heat of a flame

17. Bunsen burner A metal heating device connected to a gas outlet

with rubber tubing; used to heat chemicals in
beakers or test tubes; has adjustable air-hole
allowing some control of temperature

18. Microscope Microscopes are used in laboratories to view

specimens that are too small to be seen clearly with
the naked eye123. They are used to view the cellular
structures of organs, germs, and bacteria.

19. Beaker tongs Beaker tongs are a type of lab tongs that are used to
grasp and lift beakers of heat-resistant material.

20. Forceps A handheld, hinged instrument used for grasping and

holding objects,

21. Inoculating needle An inoculation needle is a laboratory equipment

used in the field of microbiology to transfer and
inoculate living microorganisms. It is one of the most
commonly implicated biological laboratory tools and
can be disposable or re-usable.

22. Volumetric flask A volumetric flask is a piece of laboratory apparatus

that is used to measure and mix precise amounts of
liquids.

23. Dropper Used to obtain small amounts of liquids (not precise)

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24. Petri dish Petri dish, also known as a Petri plate or cell-culture
dish, is a shallow, transparent container used by
biologists to hold growth medium for cultivating
cells. These dishes are commonly employed for
culturing microorganisms such as bacteria, fungi, and
small mosses.
25. Pipette Used to measure and dispense small amounts of
liquids (ex. 1 mL)

26. Test tube holder A test tube holder is a device that is used to hold test
tubes in place. It is useful for holding a test tube
when the tube is hot or should not be touched, such
as when it is being heated. A test tube holder can
also help to organize test tubes and prevent them
from falling and breaking. It can also provide a safe
way to store test tubes when they are being used for
ongoing experiments.
27. Rubber tubing Rubber tubing is used to circulate and transport
liquids and gases.

28. Crucible tongs Crucible tongs are mostly used to handle intense
materials at high temperatures.

29. Evaporating dish Porcelain dish; used to hold a solution whose solvent
is being separated from the solvent by evaporation
(often using heat)

30. Wash bottle Squeezable plastic bottle; used to dispense distilled

water

31. Filter paper Filter paper is a semi-permeable paper barrier placed

perpendicular to a liquid or air flow. It is used to
separate fine solid particles from liquids or gases.

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32. Clay triangle Used to hold a crucible while crucible is heated.

33. Mortar and pestle Heavy porcelain dish with grinder; used to grind
chemicals to a powder

34. Stirring rod Stirring rods are a piece of laboratory equipment

used to mix chemicals and liquids. Stirring rods are
usually made of solid or hollow glass materials being
borosilicate (Pyrex) or polypropylene.

35. Thermometer Used to measure temperature.

36. Litmus paper Litmus paper is a tool used to test whether a

substance is an acid or base.

37. Gloves Gloves protect the hands and skin from harmful
substances, provide a safe work environment and
minimize the risk of contamination.

38. Safety goggles Safety goggles used for protection against chemical
splash hazards, use of concentrated corrosive
material, and bulk chemical transfer.

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39. Lab coat Lab coats provide protection of skin and clothing
from incidental contact and small splashes of
hazardous substances, chemicals, or biological
agents present in the lab.

40. Test tube rack May be made of wood, metal, or plastic; used to
hold test tubes in an upright position

41. C CLAMP A device (shaped like a “C”) is used to hold or secure

objects to a retort stand.

42. Iron stand A metal rod attached to a heavy metal base. The
heavy base jeeps the stand stable, and the vertical
metal rod allows for easy height adjustment for the
iron ring/clamp.

43. Rubber stopper Used to cap the openings of glassware such as test
tubes or Erlenmeyer flasks

44. Glass tubing Glass tubing is a hollow piece of glass that is used to
connect other pieces of lab equipment or glassware.
It can be cut, bent and stretched for a variety of uses.
It is used to convey or deliver chemicals, solvents,
liquids, gases and other products.

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45. Crucible Made of porcelain; used to heat small amounts of
solid substances that are being heated strongly at
high temperatures

46. Wire brush Wire brushes are typically designed with a wire
handle and nylon bristles. These brushes are used for
cleaning carboys, bottles, vials, pipettes, burettes,
beakers, graduated cylinders, flasks, centrifuge
tubes, plastic test tubes, and glass test tubes.

47. Tongs Tongs are large pincers for grasping and lifting
vessels of heat-resistant material used in high
temperature chemical reactions.

48. Weighing scale

*Double beam balance A double beam balance is primarily used to measure
the mass of an object by comparing it to known
masses. It's commonly used in laboratories and
educational settings for precise measurements of
mass. The double beam design allows for greater
accuracy and versatility in measuring various masses.
*Triple beam balance The triple beam balance is also used to measure the
mass of an object, but it offers more precision and
flexibility compared to a double beam balance.

49. Autoclave An autoclave is a machine that provides a physical

method of sterilization by killing bacteria, viruses,
and even spores present in the material put inside of
the vessel using steam under pressure.

50. Funnel Used to pour liquids into containers with small

openings; also used to hold filter paper

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51. Watch glass Watch glass is a circular concave piece of glass used
as a surface to evaporate a liquid, to hold solids
while being weighed, for heating a small amount of
substance and as a cover for a beaker.

52. Retort stand A piece of scientific equipment, to which can be

attached to hold test tubes and other equipment
(such as burettes)

53. Test tube holder A gripping device used to carry a test tube after it
has been subjected to heat and/or cold
temperatures

54. Scoopula A utensil used to transfer solids (usually in powdered

form)

55. Spot plate Used to perform reactions on a very small amount of

materials

Additional
Staining solutions:
Crystal violet It is the primary stain in the Gram staining process. It
stains all bacterial cells purple.

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Gram’s iodine This is a mordant in the Gram staining process. It
forms a crystal violet-iodine complex within the cell,
aiding in the retention of the crystal violet stain.

Acid alcohol This is the decolorizing agent. It dehydrates the

peptidoglycan layer of Gram-negative bacteria,
making them more porous and allowing the crystal
violet-iodine complex to be washed out.

Safranin It is the counterstain used in the Gram staining

process. It stains the decolorized Gram-negative
bacteria pink or red, allowing them to be visualized
after the primary stain has been removed.

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