Microscopy is the act of using Microscope to view thinly things that cannot be seen with unaided eye.

1. CHAPTER 2
BASIC CONCEPTS OF MICROSCOPY AND
CULTURE TECHNIQUES

2. 2.1. MICROSCOPE
 Microscopy is the act of using a microscope to view tiny
things that cannot be seen with the unaided eye.
 A microscope is a laboratory instrument used to examine
objects that are too small to be seen by the naked eye.
 It enables us to see microorganisms and their structures
which are invisible to the naked eye.

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 Depending upon the principle on which magnification is
based, microscopes are of two categories
 Light or optical microscope
 Electron microscope

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A) Light microscopy: magnification is obtained by a system of
optical lenses using light waves.
 Two types namely Simple and Compound Microscope
1. Simple Microscope consists of a single lens.
 A hand lens is an example of a simple Microscope.
2. Compound Microscope consists of two or more lenses in
series.
The image formed by the first lens is further magnified by
another lens.

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 Simple microscope: consists of only a single lens.
 it is similar to a magnifying glass

6. Cont.…
• Parts of a Compound Microscope

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 Light microscopy, in which magnification is obtained by a system
of optical lenses using light waves.
 Types of light microscopes
1. Bright field microscope
2. Dark- field microscope
3. Phase-contrast microscope
4. Fluorescence microscope
 Is the ordinary light microscope
 It is called a bright field because it forms a dark image against a
brighter background

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2. Dark- field microscope:
 The effect produce by the dark- field technique is that of a
dark back ground against which objects are brilliantly
illuminated
3. Phase-contrast microscope:
 Is valuable for studying living unstained cells and is widely
used in applied and theoretical biological studies.
 It uses a conventional light microscope fitted with a phase
contrast objective and a phase contrast condenser.

9. Cont.…
4. Fluorescence microscope:
 Exposes a specimen to ultraviolet, violet or blue light and
forms an image of the object with the resulting fluorescent
light.
 Usually the specimens have been stained with dye
molecules, called fluorochromes
 To understand how a light microscope operates one must
know the three principles of a light microscope;
 Magnification, Resolving power, Illumination

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 Magnification:
 The magnification of a compound microscope depends on
ocular and objective lens systems.
 The total magnification of a microscope is equal to the
product of the ocular lens and the objective lens
magnifications.

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 Resolving power:
 The ability of a microscope to distinguish two adjacent
points (particles) as distinct and separate
 Resolving power can be increased either
o By reducing the wave length of the light or
o By increasing the numerical aperture.

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 Illumination:
 The easily available source of illumination is ordinary
day light but usually artificial light is used.
 The light from illumination source is refracted in to the
sub stage condenser via the mirror located just below the
condenser.

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B) Electron microscope
 Uses a beam of electrons in place of light waves to
produce the image.
 Specimens can be examined by either transmission or
scanning EM
 EM has a practical resolution roughly 1,000 times better
than the light microscope

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 electron illuminates the
specimen, the microscope’s
resolution is enormously
increased because the wave
length of the radiation is a
round0.005nm,
approximately100,000 times
shorter than that of visible
light.

15. 2.2. Preparation and staining of specimen
 There are several types of microscope slides preparations. These include:
 Dry mount: A specimen is placed on a slide with or without a coverslip
over it. This method is useful for viewing dry specimens such as hair or
pollen.
 Wet mount: A specimen is placed on a drop of water on the slide, with a
coverslip placed over it. This is often used for viewing cells.
 Prepared slides: This type of slide has already been professionally
prepared, and can last long-term. Many types of prepared microscope
slides can be purchased.

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 Smears: A smear is a sample that is spread across a slide
and allowed to dry. A coverslip may be used in this type
of preparation. This technique is helpful for viewing
blood samples.
This slide has been prepared with a smear, visible on the right side.

17. Cont.…
• Staining of Specimen
Sometimes a stain is necessary to help view cells
under a microscope.
By staining certain cells, it may be easier to view
features such as shape for classification and to see
specific cell structures like the nucleus.
There are a few different techniques for staining cells.

18. Cont.…
1) Permeabilization is a technique that is used to allow
larger dye molecules into a cell.
 Since the cell membrane normally blocks larger
molecules from entering the cell, a surfactant, or soap,
is used to dissolve the cell membrane.
 This allows the larger dye molecules into a cell to
stain internal structures.

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2) Mordant application is used to help set stains in a
specimen.
 This helps maintain the dye application during viewing.
 A mordant is usually a chemical.
3) Heat-fixed means that heat was applied to help preserve
a specimen and attach it to the slide.
 Heat fixation is often used for bacterial samples and
maintains the structure of the cells.

20. 2.2.Development of Culture Medium
Culture media
 Media (Medium sing.): is a substance used to provide nutrients
for the growth and multiplication of microorganisms.
 It gives artificial environment, simulating natural condition
necessary for growth of bacteria
 Used for:
 Isolation
 Identification
 antibiotic sensitivity test

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 Culture media should contain
 Energy source
 Carbon source
 Nitrogen source
 Salts
 Satisfactory PH
 Adequate oxidation-reduction potential and
 Growth factor

22. 2.2.1. Theory and Practices of Sterilization
 Bacteria are present on the surface of all laboratory
apparatus, in the dust, upon the hands and are generally
found every where.
 They are the source of contamination, infection, and
decay.
 Hence it is necessary to remove them from materials and
areas.

23. Sterilization
• Sterilization:- is a total destruction of all microbes
including the more resistant forms such as bacterial
spores, mycobacterium, non-enveloped viruses and fungi
• Sterilization means “destruction of all forms of
microbial life”.
• Sterilization can be achieved by
 Heat
 Chemical
 Filtration methods

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• Methods of sterilization

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 Heat sterilization
 Most common method of sterilization.
 The heat kills the microbes in the substance.
 The amount of heat and duration of heating are the factors
that affect extent of sterilization.
 two types based on the type of heat used
Moist heat method
Dry heat method

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Moist heat methods of sterilization:
 Boiling
 Pasteurization
 Use of pressurized steam (Autoclaving)

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A) Boiling is preferred for metallic devices like surgical
scissors, scalpels, needles etc.

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B. Pasteurization is a process of heating the milk at a
temperature of 63 ⁰C or 72 ⁰C for 30 min and 15 sec, respectively
C. Steam (autoclaving): Substances are subjected to
sterilization in an autoclave.
 Three major factors required for effective autoclave
 Pressure
 Temperature
 Time

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Dry-heat sterilization
 Requires a higher temperature than moist heat and a longer
exposure time.
 More convenient for heat-stable, non-aqueous materials that
cannot be sterilized by steam (strong glasses like Petri dish and
tubes)
Temperature ⁰C time in min
• 160 180
• 170 60
• 180 30

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• This method includes techniques like
 Red heat
 Flaming
 Incineration
 Hot air oven
 Radiation sterilization

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 Chemical methods of sterilization
 Articles are subjected to sterilization by using toxic Gases
 Gas penetrates quickly into the material
 But the chances of explosion and cost factors are to be
considered
 The commonly used gas is ethylene oxide with
combination of CO2.
 CO2 is added to minimize the chances of an explosion

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 Filtration method of sterilization:
 Liquids are filtered though bacterial filters to remove any
microbes present
 Useful to sterilize heat sensitive objects.

34. Disinfection
• Disinfection: is a process, which involves use of physical
procedures or chemical agents (disinfectants) to destroy
most microbial forms
• A disinfectant is a chemical substance that kills
microorganisms on inanimate objects, such as exam tables
and surgical instruments.

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• Examples of disinfectants include:
 Glutaraldehyde
 Hydrogen peroxide
 Per-acetic acid
 Chlorine dioxide and other chlorine compounds

36. Antisepsis
Antisepsis:- Is the use of chemical agents on skin or other
living tissues to inhibit or eliminate microbes; no sporicidal
action is implied.
 Antiseptics include
• Alcohols
• Iodophors
• Chlorhexidine
• Triclosan etc

37. Cont.…
 An antiseptic is a chemical that is applied to a living body
to inhibit the growth of microorganisms.
 Hand sanitizers are antiseptics.
 Asepsis is the absence of harmful microorganisms in
living tissue.
 Skin can never be completely sterile.
 The inside of the body contains no bacteria and is
referred to as aseptic.

38. 2.2.2. Culture Media and Its Preparation

39. 2.2.3.Pure Culture Techniques

40. 2.2.4. Preparation of Smear and Staining Techniques
 Preparation of Smear
 The first step in most bacterial staining procedures is the
preparation of a smear.
 In a smear preparation, cells from a culture are spread in a
thin film over a small area of a microscope slide, dried,
and then fixed to the slide by heating or other chemical
fixatives.

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 Staining Techniques
 Because microbial cytoplasm is usually transparent, it is
necessary to stain microorganisms before they can be viewed
with the light microscope.
 In some cases, staining is unnecessary, for example when
microorganisms are very large or when motility is to be studied,
and a drop of the microorganisms can be placed directly on the
slide and observed.
 A preparation such as this is called a wet mount.

42. Cont.…
 In preparation for staining, a small sample of
microorganisms is placed on a slide and permitted to air
dry.
 The smear is heat fixed by quickly passing it over a
flame.
 Heat fixing kills the organisms, makes them adhere to the
slide, and permits them to accept the stain.

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 Simple stain techniques: Can be performed with basic dyes
such as crystal violet or methylene blue, positively charged dyes
that are attracted to the negatively charged materials of the
microbial cytoplasm.
 Such a procedure is the simple stain procedure.
 Negative stain technique: use a dye such as nigrosine or Congo
red, acidic, negatively charged dyes.
 They are repelled by the negatively charged cytoplasm and
gather around the cells, leaving the cells clear and unstained.

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 Differential stain techniques: distinguishes two kinds of
organisms.
 An example is the Gram stain technique.
 This differential technique separates bacteria into two groups,
Gram‐positive bacteria and Gram‐negative bacteria.
 Crystal violet is first applied, followed by the mordant iodine,
which fixes the stain
 Then the slide is washed with alcohol, and the Gram‐positive
bacteria retain the crystal‐violet iodine stain; however, the
Gram‐negative bacteria lose the stain.

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 The Gram‐negative bacteria subsequently stain with the
safranin dye, the counterstain, used next.
 These bacteria appear red under the oil‐immersion lens,
while Gram‐positive bacteria appear blue or purple,
reflecting the crystal violet retained during the washing
step.

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 Acid‐fast technique: differentiates species
of Mycobacterium from other bacteria.
 Heat or a lipid solvent is used to carry the first stain,
carbolfuchsin, into the cells.
 Then the cells are washed with a dilute acid‐alcohol
solution.
 Mycobacterium species resist the effect of the acid‐alcohol
and retain the carbolfuchsin stain (bright red).

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 Other bacteria lose the stain and take on the subsequent methylene
blue stain (blue).
 Thus, the acid‐fast bacteria appear bright red, while the nonacid‐fast
bacteria appear blue.

49. Cont.…
• Other stain techniques seek to identify various
bacterial structures of importance. For instance, a
special stain technique highlights the flagella of
bacteria by coating the flagella with dyes or
metals to increase their width.
• Flagella so stained can then be observed.

50. Cont.…
• A special stain technique is used to examine
bacterial spores.
• Malachite green is used with heat to force the
stain into the cells and give them color.
• A counterstain, safranin, is then used to give color
to the non-sporeforming bacteria.
• At the end of the procedure, spores stain green
and other cells stain red.

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