USING BRIGHT FIELD MICROSCOPE TO VIEW
BACTERIA
INTRODUCTION:
This manual gives you detailed instructions on how to prepare a bacterial slide and view
it under the microscope, assuming that you have some very basic knowledge about the bacteria
and microscope. The process is divided into three parts: Preparing a bacterial smear, heat-fixing
and staining and finally viewing it under the microscope.
Bacteria are small sized living organisms that cannot be seen by naked eye. Thus, to view
bacteria, we always have to use a microscope. Anything viewed under the microscope is first put
onto a clear glass slide. Bacteria are transparent and in order to view them, we use a simple stain
to color them. (There are different stains that can dye the bacteria to different colors.)
Magnification of lenses differs in various microscopes. This manual gives detailed procedure for
using Bright field light microscope that can magnify the bacteria up to 100 times its initial size.
Below is the labeled figure of microscope showing all its major parts.
The figure shows a Bright field light Microscope. It has four objective lenses of different
magnifications attached to the rotatable nosepiece. The magnifications of the lenses are given
below:
Name of lens
Magnification
1 Scanning lens
4X
2 Low dry lens
10X
3 High dry lens
40X
4 Oil immersion lens
100X
When viewing a specimen, it is always good to start from the least magnifying lens. Once you
get the specimen in focus, you can change the lenses and view under other magnifications.
CARE AND HANDLING OF MICROSCOPE
Transporting: When you take the microscope from one place to another, use both hands to hold
it. Grab the arm with one hand and place the second hand on the bottom of the base.
Cleaning: Never touch the lenses with your fingers. Your skin produces oil that smudges the
lens. Use only lens paper to clean the lenses and no other kind of tissue like Kleenex or
paper towels. For the oil lens, you can use 95% ethanol to clean it once you have blotted
the excess oil from the lens. But never let any excess ethanol stay on the lens.
Storage: When you are finished using microscope, rotate the nosepiece so that low power
scanning objective is in position. Cover the microscope with a dust cover.
1) Be very careful when handling the microscope as it has delicate lenses and is very costly.
2) Always wear gloves to avoid direct contact with bacteria and the time for which Perti
dish is opened should be minimum.
3) Immersion oil should not go on any lenses other than the oil immersion lens because oil
can permanently smudge the lens.
MATERIALS REQUIRED:
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Two clean glass slides
Methylene Blue stain
Bright-field microscope
Culture of Staphylococcus bacteria
Inoculation loop
Bibulous paper
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Permanent marker
Bunsen burner
A pair of gloves
Lens paper
95% ethanol
Slide holder
PROCEDURE:
PREPARING A SMEAR
1) Wear on the gloves before you start. Place a drop
of water on one of the glass slides.
2) Take the second clean glass slide and draw a
circle in its center with the help of a permanent
marker to specify the area your smear would
occupy.
3)
Turn on the Bunsen burner and flame the
inoculation loop holding it at an angle and
keeping it in the flame until it gets red hot. Allow
it to cool down for few seconds.
4) With the help of inoculating loop, take a loopful
of water from the first slide and spread it within
the circle you marked for your area.
5) Open the Petri plate and very gently scrap a single bacterial
colony with the flame sterilized inoculation loop.
6) Mix in the bacteria and spread the drop over the circle you
had drawn. Avoid spattering the emulsion as you mix. Flame
your loop when done.
7) Allow the smear to air dry. The will appear a little cloudy
when it dries. Flame the loop again.
HEAT FIXING TECHNIQUE
The purpose of heat-fixing is to kill the bacteria, to adhere them to the slide and modify their
proteins to take-up more stain. The slide is passed through the flame several times. But keep
in mind that it should only be passed through the flame and not kept over it for longer time
because that might lead to distortion of their shape. Thus, the technique is carefully performed
to adhere the bacteria to the slide but still keeping their shape intact.
8) Hold the slide with forceps and pass the smear through the
upper part of a flame two or three time. This heat-fixes the
preparation. Avoid overheating the slide as aerosols may be
produced.
9) Allow the slide to cool and perform the staining protocol.
STAINING TECHNIQUE
10) Add a few drops of Methylene Blue stain such that
it completely covers the smear. Keep the slide
undisturbed for 2 minutes.
11) Grasp the slide with a slide holder. Rinse it with
water.
12) Gently blot the slide dry with bibulous paper. Do
not rub the slide dry. Only place it between the
pages and press so that the paper takes up all the
water on the slide.
VEIWING UNDER MICROSCOPE
13) Plug in the microscope and turn on the lamp such
that the light intensity is highest.
The best image is visible under maximum light
intensity when we stain the bacteria. But if we do
not use any staining technique, we image would
be more clear under little light intensity.
14) Revolve the nose piece ring and move 4X lens
into position. The magnification of the lens is
written on the lens itself.
4X lens is actually the scanning objective lens
and has the lowest magnification of the entire
four lenses on the nose piece of the microscope
we are using.
15) Place the slide on the stage in the mechanical
slide holder and center it on to the source of lightaperture in the stage. You can set the slide using
the stage adjustment knobs located close to the
mechanical stage.
16) Rotate the coarse focus knob to view as clear an
image as is possible by rotating the knob. Adjust
the glass slide if you do not see much to move an
area having more bacteria under the objective.
17) Rotate the fine focus knob to focus the image
further. Rotate the knobs both clockwise and
counter clockwise until you get the sharpest
image.
18) To view the specimen with a higher
magnification, scan the specimen (slide) to locate
an area on the slide where there is a higher
population of the bacteria.
19) Keeping the slide and the mechanical stage
unchanged, rotate the nosepiece to bring 10X lens
into position.
20) Generally the image is in focus or can be brought
into focus by slight rotation of the fine focus
knob. But if the fine focus knobs cannot do it,
very carefully move the coarse focus knob to
focus the image.
21) To view the specimen under 40X magnification,
rotate the nosepiece until high dry objective
(40X) clicks into position. Do not move the slide
while you do this otherwise you will lose your
specimen from the focus.
VEIWING UNDER OIL IMMERSION LENS
22) Rotate the 40X lens out of position and place a
drop of immersion oil on the slide at the spot
where the subject is illuminated by the condenser.
23) Lower the oil immersion objective into position
so that the lens is immersed in the oil when it
clicks into position.
24) Usually the image is in focus when you move the
lens. If is not the case, slightly move the fine
focus knob and then coarse focus knob.
Cleaning the apparatus: After you are finished viewing the specimen, clean the glass slide with
glass slide powder. Lower the stage, blot dry oil from the oil immersion lens, clean it
with ethanol if needed, and move the lowest magnification lens in position. Replace the
microscope carefully to its place and cover it with dust cover.
TROUBLE SHOOTING
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If you do not get a clear image under the oil immersion lens, try to rotate the fine focus
knob to get a focused image.
If the image still remains blurred, very slightly move the coarse focus knob.
If the problem still persists, clean off all the oil from the oil immersion lens. Then, start
by viewing the specimen under lowest magnification lens and repeat the process. Be
careful to not to move the slide when you move different lenses into position by rotating
the nose piece.
If you are unable to get a clear image under the scanning objective, make sure that the
lens are clean. Clean them with lens paper.
If you are still unable to get a clear image under 4X lens, rotate the coarse focus knob
over its whole range. It the problem still persists, you may have washed the bacteria off
the slide while rinsing the stain. In that case, you will have to prepare a bacterial smear
on a fresh slide. (This could also be the result of poor heat-fixing technique.)
REFERENCES
1) Lea, C. (2002) Microscope [Figure]. University of New Mexico. Retrieved on September
29, 2009 from
<http://biology.unm.edu/ccouncil/Biology_203/Images/Microscopes/microscope6.jpeg>
2) Leboffe, M.J. & Pierce, B. E. (2006). Microbiology Laboratory Theory and Applications.
Englewood, CO: Morton Publishing Company.