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Microscopy of bacteria
Related Topics
Bacteria, sterility, microscope, and staining.
Principle
The microscopic examination of microorganisms can be performed based on live specimens or in
fixed and stained preparations. Live specimens of microorganisms are prepared in a drop of water (or
culture liquid). The aim of staining bacteria preparations is to increase the contrast between the
bacteria and their environment from which they hardly stand out when unstained.
Equipment
11Culture vessel
64834-00
1 Scissors, straight, blunt, l = 140 mm
64625-00
1 Glass beaker, DURAN®, tall, 100 ml
36002-00
1 Microscope slides, 50 pieces
Cover glasses, 18 mm x 18 mm, 50
1
pcs.
Inoculation loop with holder, l = 230
1
mm
1 Water, distilled, 5 l
64691-00
1 Immersion oil, 50 ml
Glass rod, boro 3.3, l = 300 mm, d =
10
7 mm
Safety gas tubing, DVGW, sold by
1
metre
Bunsen burner with stop cock, for
natural gas
SWIFT student microscope
1
M3602C-3
1 Staining tweezers, after Kühne
1
32167-05
63021-99
64726-00
64796-00
31381-05
1 Petri dish, d = 200 mm, glass
Graduated cylinder, 100 ml, BORO
1
3.3
Filter paper, 580 mm x 580 mm, 10
1
sheets
1 Carbol-fuchsin solution, 100 ml
40485-05
1 Entellan for microscopy, 100 ml
31294-10
1 Ethyl alcohol, absolute, 500 ml
Methylene blue B, for microscopy 25
1
g
30008-50
39281-10
64685-00
64936-00
31246-81
36629-00
32976-03
31463-10
31567-04
Fig. 1: Staining of bacteria preparations.
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TEB
Microscopy of bacteria
Tasks
Examine a bacteria preparation under a
microscope and apply the standard methods that
are described herein.
Procedure
Procurement of microorganisms for microscopic
examinations
A quick way to obtain microorganisms for
microscopic examinations, e.g. in order to
identify and observe different forms of bacteria,
the existence or non-existence of proper motion,
the formation of spores, etc. in living or fixed and
stained preparations, is an infusion of plant
parts.
Infuse a hand full of grass, hay, or lettuce leaves
in tap water in a culture vessel and let this
infusion stand uncovered at room temperature.
The water level should be several centimetres
above the plant parts and approximately three
quarters of the culture vessel should be filled Fig. 2: Hay infusion in a glass.
(Fig. 2). Large, cumbersome plant parts can be
broken up beforehand.
Within three to five days, a biofilm forms on the liquid surface. At first, it consists solely of bacteria. Later,
after one to two weeks, it includes also various types of microscopic fungi next to protozoa (mainly
ciliates).
The reason for the formation of the biofilm is that the plant parts that are filled into the culture vessel are
infected with microorganisms and their spores. The addition of water leads to excellent culture
conditions, which is why they multiply rapidly. They grow in the form of a biofilm since they are mainly
aerobic microorganisms, which means that they can develop only under sufficient oxygen partial
pressure. At the surface of the liquid, the oxygen level is the highest.
Observation of microorganisms with the aid of a live specimen
Transfer a drop of water onto a microscope slide. Flame an inoculation loop in the flame of a gas burner
and let it cool. Then, use it to withdraw some of the
microorganisms to be studied, e.g. from the biofilm
on an infusion of plant parts or from a colony that
has developed in a Petri dish, and stir them carefully
into the drop of water (Fig. 3). Do not use an
excessive amount of material, since otherwise a
compact mass of close-packed microorganisms
would result. In such a mass, details are difficult to Fig. 3: Transfer of bacteria onto a microscope slide.
discern. Only a very slight, milky turbidity should
form in the drop of water. This can be checked
rather easily by placing the microscope slide on a dark surface. Flame the inoculation loop again in the
flame of a gas burner until it is red hot in order to clean and sterilise it.
Cover the preparation with a cover glass and place it under the microscope with a 50 times microscopic
magnification (objective 10x, eyepiece 5x). Depending on the size of the objects, examine it with a 400
and 1000 times magnification (objective 40x or 100x, eyepiece 10x). In many cases, however, the
bacterial cells are so large that it is not necessary to use an immersion objective.
Microorganisms that are already distributed in a nutrient solution are usually transferred to a microscope
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Microscopy of bacteria
TEB
slide, which is then covered with a cover glass,
without using a drop of water.
Unstained living preparations of microorganisms
have a relatively low contrast. The cells or union of
cells hardly set themselves apart from their
environment. The contrast of the preparation can
be increased by closing the iris diaphragm of the
illuminator of the microscope by half or two thirds.
Fixing of bacteria preparations
The internal structures of most types of bacteria
are so small that they cannot be resolved by a light
microscope, which means that they are not visible.
This is why the conservation of the internal
structures of the cells is usually refrained from and
the bacteria preparations are fixed by heat.
Flame an inoculation loop in the flame of a gas
burner and let it cool. Then, use it to withdraw
some of the bacteria mass, e.g. from the biofilm on
an infusion of plant parts and stir it as evenly as
possible into a drop of water on a microscope slide.
The quantity of the bacteria mass is correct if the
Fig. 4: Fixing of bacteria preparations.
drop of water shows a slight milky turbidity when
viewed on a dark surface. Flame the inoculation
loop again in the flame of a gas burner until it is red hot in order to clean and sterilise it.
After the preparation has dried completely in the air, pass the microscope slide, with the coated side up,
three times for approximately one second for each pass through the non-bright and non-crackling flame
of a gas burner (Fig. 4).
During this process, the microscope slide reaches a temperature of approximately 120°C. As a result,
the bacterial cells are killed while preserving their outer shape and they are fixed on the microscope
slide.
Staining of bacteria preparations
Nearly all stains are based on the electro-adsorption of dye ions to free valences of the molecules that
are involved in the formation of the bacterial cell. To a lower degree, stains are also based on the affinity
of a dye for certain parts of the bacterial cell or on chemical reactions inside the cell if the reaction
product is a dye.
Prior to staining, however, the bacteria must be fixed.
For staining, the fixed preparation is placed on a staining bench above a staining tray that can be
assembled rather easily.
Use half a Petri dish with a diameter of 200 mm. Attach four medium-sized paper clips to the rim of the
dish approximately in the middle so that two pairs of paper clips face each other from opposite ends of
the dish. Then, push two glass rods (300 mm long, diameter 5 mm) through the eyelets that are formed
by the paper clips and push the paper clips down in order to fix the glass rods in place.
Drop some staining solution onto the microscope slide so that the fixed bacteria mass is completely
covered. After the staining time, let the staining solution flow into the staining tray by tilting the
microscope slide. Then, rinse the preparation under tap water until no more dye flows off. Dry it in the air
by placing it on some filter paper and by tilting it against the staining tray.
The stained preparations can be viewed with 400x and 1000x magnification (objective 40x or 100x,
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Microscopy of bacteria
eyepiece 10x). If an immersion objective is used, they can also be examined without a cover glass. If,
however, they are to be stored as permanent preparations, add a drop of embedding agent (Entellan) to
the microscope slide and cover it with a cover glass.
For bacterial preparations, the following standard dyes can be used:
Diluted carbol-fuchsin solution
Prepare a 1:5 dilution of a carbol-fuchsin solution according to ZIEHL-NEELSEN in distilled water. This
means that four parts of distilled water are added to one part of staining solution.
The staining time is 5 minutes.
The bacteria are stained bright red.
Diluted methylene-blue solution
Prepare a 1:5 dilution of a saturated, alcoholic methylene-blue solution in distilled water. This means that
four parts of distilled water are added to one part of staining solution.
The staining time is 5 minutes.
The bacteria are stained in a deep blue.
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