MICROSCOPY
Biotechnology Protocols
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Microscopy
Microscopic examination of micro-organisms
Source: NQ Curriculum Support Intermediate 2 Biotechnology,
(Unit 2 Student Materials)
Micro-organisms are so small that you cannot see them without the aid of a
microscope. Microscopes are used to produce an enlarged image of objects
too small to be seen with the naked eye.
Although microscopes produced by different manufacturers may look quite
unlike each other, they all work on the same principle and consist essentially
of similar working parts.
To obtain the clearest image of micro-organisms using a microscope, you
must learn how to set it up properly. You must first know the principles on
which the microscope works.
Structure of the microscope
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Microscopy
Part
Description
Eyepiece lens
It fits into the body tube. It contains lenses which
magnify the image produced by the objective lens and
produces the image the operator sees. Eyepiece lenses
usually have a magnification of x10. You may use x15 to
observe bacteria.
Objectives
Contain lenses which magnify the specimen. Objective
lenses bear a coloured ring which indicates their order of
magnification. Microscopes usually have objective
lenses of power x10 and x40. They may also have an oil
immersion objective (x90 or x100).
Stage
A flat platform on which the specimen is placed. It has a
hole in the centre which allows light to pass up through
the specimen. There are usually two side clips which
hold the slide in position.
Condenser
Contains lenses which focus the light into a cone. A
condenser focus control allows the condenser to be
moved up and down so that light is focused at the
specimen. The condenser may not be present in some
microscopes.
Iris diaphragm
The iris diaphragm can be opened or closed to control
the angle of the cone of light passing through the
condenser. It makes the specimen brighter and the
image clearer.
Mirror
Can be adjusted to reflect light up on to the specimen.
One side of the mirror is flat, the other is concave. The
flat side is used if a condenser is present. The mirror
may be replaced with an electrical light source.
Principles of the light microscope
1 Visible light passes through a substage condenser which focuses the light
into a sharp cone.
2 The light passes through the opening in the stage into the slide illuminating
the specimen.
3 The light passes through the objective lens and forms a magnified image
of the specimen which is usually darker than the background.
4 The eyepiece lens magnifies this image further and creates the image that
the user sees.
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Microscopy
Oil immersion
To observe bacteria clearly with a microscope, it is usually necessary to use
an objective lens of x100. The lens must be immersed in a drop of oil which is
placed on the slide. This helps direct more light into the objective lens.
Care of the microscope
Dust from the air, grease from fingers, liquids and chemicals from slides all
damage microscopes. Students should develop a routine of cleaning all the
glass surfaces of the microscope with lens tissue before, during and after use.
It should be stressed that if immersion oil is left on lenses, it hardens and blurs
the image. Ordinary tissue should not be used as it may scratch the glass
surfaces of the lenses and leave fibres behind which obscure the image.
Magnification
The magnifying power is the degree of enlargement; that is, the number of
times the image appears greater than the original specimen.
To calculate this, multiply together the separate magnifying powers of the
objective and eyepiece lenses.
For example, the total magnification of a microscope fitted with:
a x10 eyepiece lens and a x10 objective lens
is
10 x 10 = x100.
Similarly, if a x40 objective lens was being used on the same microscope,
the total magnification would be:
eyepiece lens x objective lens
that is
10 x 40 = x400.
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Microscopy
Common problems and their solutions
Problem
Cause
Solution
Image is not sharp
Dirty objective
Slide upside down
Clean with cleansing fluid
Invert slide
Dirt or grease on slide
Clean both sides with lens
tissue
Clean top lens with
cleansing fluid & lens
tissue
Clean front lens with lens
tissue and cleansing fluid
Dirty marks
Marks move when
slide is moved
Marks move when
eye-piece is rotated
in draw-tube
Marks move when
objective is rotated
in nosepiece
Marks appear when
lamp is focused
Too bright
Bright specimen on
dark background
‘Worms’
Dark edged circles
Uneven illumination
Too dark
Image goes slowly
out of focus
Jagged lines
Dirt or grease on eyepiece
Dirt on objective
Dirt on lamp surface
Iris too far open
Lamp too bright or too
close
Light shining on top of
specimen
Fibres on slide, eyepiece
or objective
Air bubbles
Mirror and/or lamp askew
Objective not properly
clicked into position
Filter tray blocking light
into condenser
Iris closed down too far
Lamp too weak or too
distant
Filter tray obstructing light
into condenser
Condenser not focused
properly
Insufficient friction in focus
control mechanism
Coverslip edge or broken
part of coverslip
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Clean lamp or defocus
condenser slightly
Close down iris slightly
Change bulb or move
lamp further away
Adjust position of lamp
Clean with fibre free lens
tissue
Move slide and examine
area without air bubbles
or make fresh slide
Adjust mirror and/or lamp
Rotate nosepiece
Swing in or out
Open iris
Change bulb or move
lamp closer
Swing in or out
Focus condenser
Adjust friction focus
control (likely to require
trained operator)
Move slide to examine
different region. If c/slip
broken, make new slide
Microscopy
SMEAR PREPARATION
Source: James Watt College
The first step in many staining procedures, a bacterial smear is a dried
preparation of cells on a glass slide. In preparing the smear a number of
points should be considered:
1. Bacteria should be evenly spread out on slide so they are adequately
separated from one another.
2. Bacteria shouldn’t be washed off slide during staining
3. Bacterial form should not be distorted
*Remember: Sterile techniques throughout*
The initial part of this procedure differs depending on whether bugs are from
(a) liquid culture or (b) solid medium (e.g. plate).
a) From liquid media: degrease a microscope slide.
Swirl culture to resuspend.
Put two loopfuls of liquid culture in centre of slide
Use the loop to disperse the liquid over a circle
Allow the slide to “air dry” (by normal evaporation; no applied heat).
b) From solid media: degrease a microscope slide
Put two loopfuls of water in centre
Then disperse (very thoroughly) in the water a very small amount of
colony.
Allow the slide to air dry.
Then: to heat kill and fix the organisms to the slide it is passed (after air
drying) through a Bunsen flame several times (i.e. 4 to 6 times).
N.B. Bugs uppermost (i.e. away from direct flame).
Unless fixed on the glass slide, the bacterial smear will wash away during the
staining procedure. During heat fixation the bacterial proteins are
“coagulated” (heat denatured) and “stick” to the glass surface (a bit like
cooking an egg on a dry, sticky frying pan).
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Microscopy
SIMPLE STAINING
Source: James Watt College
This uses a single stain / dye to create contrast between the bacteria and the
background.
Often used to obtain information about cell shape, size and arrangement.
Basic dyes such as crystal violet, carbol-fuchsin or methylene blue are often
used.
Method
Prepare a fixed smear
Place staining rack over sink
Stain slide as follows (cover sample on slide with a ‘puddle’ of dye in
each case):
Methylene blue
1 – 1½ minutes
or
carbolfuchsin
5 – 10 seconds
or
crystal violet
20 – 30 seconds
Wash stain off slide with water for few seconds
Blot slide dry. Do not rub
Examine under oil immersion (i.e. x 100 lens)
Record the SHAPE of the micro-organism you see
Bacilli (rods)
Cocci (round)
Spirilla (spirals)
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Microscopy
Source: NQ Curriculum Support Intermediate 2 Biotechnology
(Unit 2 Student Materials)
Staining a smear preparation with a simple stain
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Microscopy
GRAM STAINING
Source: James Watt College
This is a differential stain.
Method

Make and fix bacterial smear (as per page 14)

Allow to cool

Stain with crystal violet for 30 seconds

Drain off crystal violet

Rinse with iodine solution

Cover with fresh iodine and leave for one minute

Drain off the iodine

Rinse alternately with water then alcohol until no more colour comes off
(i.e. not until the slide is decolourised). Make sure you don’t leave the
alcohol on for too long.

Cover with 1% saffrannin and leave for 30 seconds

Rinse with water

Blot dry (do not rub)

Examine by oil immersion (i.e. x 100 lens)
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Microscopy
GRAM STAINING (continued)
Observations and Results
Following your observation of all slides under oil immersion, record your
results in the space provided here.

Make a drawing of a representative microscopic field.

Describe the cells according to their shape and arrangement.

Describe the colour of the stained cells.

Classify the organism as to the Gram reaction: Gram-positive or Gramnegative.
Organisms
name 
Drawing of a
representative
field of view
Individual
shape
Arrangement
Cell colour
Gram reaction
Test your understanding of Gram staining by answering these
questions:
-
What are the advantages of differential staining procedures over simple
staining techniques such as the use of methylene blue?
-
Identify the reagents used as the primary and counter stains:
a) Primary stain:
b) Counter stain:
-
State the purpose of each of the following reagents in the Gram staining
procedure:
a) Primary stain:
b) Counter stain:
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Microscopy
CAPSULE STAINING (Differential Stain)
Source: James Watt College
a) Aseptically prepare a smear of culture on a labelled slide (seep14 for
details)
b) Air dry
c) Do not heat fix
d) Place slide on staining rack
e) Flood with crystal violet (Primary stain)
f) Leave for 4 – 7 minutes
g) Rinse thoroughly with 20% copper sulphate solution not water as the
capsule is water soluble
h) Blot dry
i) Examine under oil immersion
j) Record results
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Microscopy
NEGATIVE STAINING
Source: Adapted from NQ Curriculum Support Intermediate 2 Biotechnology,
(Unit 2 Student Materials)
This is a type of simple staining which determines the overall morphology
without harsh-staining or heat-fixing techniques that change cell shape.
These stains do not penetrate the cells due to repulsion between the negative
charge of the stains and the negatively charged cell wall, so it doesn’t actually
stain the bacteria but their background. The result is that bugs appear as light
areas in a dark background.
Place a drop or nigrosin toward one end of
the slide
Place a loopful of the inoculum into the drop
of stain and use the loop to mix thoroughly.
Place a slide in the drop of suspended organism
and pause to allow the drop to spread along the
edge of the applied slide.
Pushing down to maintain contact with the glass,
push the slide away from the previously spread
droplet, forming a thin smear.

Allow to air dry

Do not heat fix

Examine by oil immersion (i.e. x 100 lens)
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Microscopy
SPORE STAINING
Source: James Watt College
i.e. stain to show up a specific feature in a micro-organism, namely a
bacterial endospore.
1 Make individual smears in usual manner
2 Air dry
3 Heat fix
4 Flood with malachite green
5 Place over boiling bath to steam for at least 3 minutes (up to 10 minutes)
Do not allow stain to evaporate; replenish as needed
(Stain should steam not boil)
6 Cool slide
7 Wash under running water for 30 seconds
8 Counterstain with safranin for 30 seconds
9 Wash under running water for 30 seconds
10 Blot and examine under oil immersion (i.e. x 100 lens)
Note:
Heating drives malachite green into all areas of cell including the spore.
Rinsing in water easily removes this stain again from all structures except the
spore i.e. it remains green. The safranin counterstain is then required to
colour the rest of the cell.
Result is that spores appear as green structures whilst all else is red.
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Microscopy
MOULD STAINING
Source: NQ Curriculum Support Intermediate 2 Biotechnology.
(Unit 2 Student Materials)
To show the hyphal structure of moulds the following method is used (see
figure on p23 also):

Add small drop of lactophenol cotton blue to a clean slide

Aseptically transfer a small portion of a typical mould to the stain and tease
it out with a pair of needles

Lower coverslip avoiding air bubbles
Examine final slide to observe hyphae arrangements:
 You will NOT need oil immersion here (i.e. use x 40 objective
magnification)
 You may continually adjust focus to see detail of different areas
of the slide, as not all the hyphae will be in the same plane
 Lactophenol blue stains the fungal cytoplasm. The fungal walls
should therefore appear colourless against a light blue
background.
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Microscopy
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Microscopy
VITAL STAIN
Source: HSDU Biology and Biotechnology Microbiological Techniques
(Intermediate 1-Advance Higher) Folder
This is a staining technique used to show if cells are VIABLE i.e. alive
Materials
Lab coat
Eye protection
Benchkote if necessary
Disinfectant and paper towels
Discard jar with disinfectant
Bunsen burner
Microscope
Lens tissues
Glass slide and coverslip
Wire loop
Sterile water
Plate culture of yeast
Neutral red
Fibre free blotting paper
Instructions
1
Wear a lab coat and use eye protection.
2
Clean slide and coverslip.
3
Using aseptic technique, transfer two loopfuls of sterile water to the centre
of the slide.
4
Using aseptic technique, transfer a small amount of yeast from a single
colony into the water on the slide and mix.
5
Carefully lower the coverslip.
6
Using the Pasteur pipette, draw up a little neutral red.
7
Slowly release the stain along one edge of the coverslip.
8
Place the edge of the blotting paper against the opposite edge of the
coverslip to draw through the stain.
9
Observe under high power (x40) of the microscope.
10 Record the colour of the background and the colour of the cells at five
minute intervals for a period of twenty minutes.
N.B. This method can be used with other vital stains and micro-organisms
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Microscopy
SPACE FOR EXTRA NOTES & OBSERVATIONS
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Microscopy
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Microscopy