Microscope Operating procedure

Below we describe detailed directions for the use of a microscope. This will give you
an appreciation of their operation. These directions have been written as generally as
possible, but it may be necessary for your instructor to make modifications for the
exact microscopes you are using. Light microscopes used in teaching laboratories are
designed for ease of use and with some practice should become automatic.
1. Raise the nosepiece using the course adjustment knob. This provides greater
access for positioning the slide on the stage.
2. Rotate the nosepiece so that the 10X objective lens is in operating position.
3. Open the iris diaphragm approximately half way.
4. Turn on the in-base illuminator by depressing the push-type switch.
5. Place a stained specimen slide on the stage and with the naked eye position the
specimen directly above the center of the condenser.
6. Use the thumbwheel below the eyepieces to adjust the interpupillary distance
between the two eyepieces. This is important to be able to view specimens with
both eyes, maximizing the quality of the image and preventing fatigue from
prolonged use of one eye.
7. Move the microscope condenser by means of the condenser adjustment knob
until the top of the condenser is almost at the highest position. There should be
enough room to slide a piece of paper between the stage and the condenser, but
no more. This will focus the light onto the slide.
8. Rotate the coarse adjustment knob in a clockwise direction to bring the 10X
objective closer to the slide. View through the eyepieces and, without
disturbing the coarse adjustment setting, slowly rotate the fine adjustment knob
until the specimen is in the sharpest possible focus.
9. The left eyepiece tube is focusable to compensate for refraction differences of
the eyes. The correct procedure is to bring the specimen into sharp focus
looking though the right eyepiece only. Then focus for the left eye by turning
the left eye tube collar fully counter-clockwise. Next, while viewing the
specimen with the left eye only turn the knurled collar clockwise until the
specimen is in sharp focus. Do not adjust the fine adjustment knob during this
procedure.
10. Remove an eyepiece to view the back aperture of the objective lens. Close the
condenser iris diaphragm, then re-open until the leaves of the diaphragm just
disappear from view. Replace the eyepiece and view the specimen. The iris
diaphragm may be closed slightly to enhance contrast, especially when viewing
unstained specimens.
Unstained specimens have only minimal contrast with their surrounding
environments. As a result they can usually be viewed more effectively by

setting the diaphragm at or near minimum opening. Reducing the diaphragm

setting increases definition, contrast, and depth of focus but introduces
diffraction problems and sacrifices resolution. Play with the diaphragm setting
and select the best compromise by trial and error.
11. Once the specimen is in sharp focus using the 10X objective lens, it is then
possible to rotate the nosepiece to the 40X objective lens without changing the
position of the coarse adjustment knob. Very little refocusing with the fine
adjustment knob is required since most light microscope objective lenses
are parfocal. Remember that the iris diaphragm setting must be changed to
allow more light to pass though the sample as the magnification increases.
12. If the specimen is to be viewed using the 100X oil immersion lens, immersion
oil must be applied to the slide.
13. Rotate the 40X objective lens slightly to the side so that a drop of immersion
oil may be placed on the specimen without getting it on the 40X lens.
14. Place a drop of immersion oil in the center of the circle of light formed on the
specimen slide.
15. Carefully turn the nosepiece until the 100X objective lens snaps into place. The
objective lens should be in the oil but must not touch the slide.
16. Increase the light intensity as required and rotate the fine adjustment knob to
obtain a sharp focus of the specimen. If necessary make further adjustments to
obtain optimal illumination.
If the microscope is not parfocal, it will be necessary to lower the objective lens as
close to the slide as possible without touching it. This is done only while looking at
the lens and slide from the side of the microscope. Bring the specimen into view by
slowly raising the objective lens with the coarse adjustment knob. Next, focus with
the fine adjustment knob and adjust the illumination as necessary. If this is not
successful the first time, repeat the entire procedure.
In many cases, a preparation needs to be observed only under the oil immersion lens.
In this case, first locate the specimen and center it in the field with the low power
objective lens. Then add oil and rotate the oil immersion objective lens into position.
Common Problems
Certain problems real or apparent, may be encountered while operating your
microscope. Here is a trouble shooting guide to help you if you are having difficulty
focusing a sample.
The sample can be focused at 10X, but it is difficult to find or blurry at 40X.

This is often caused by immersion oil on the 40X lens. Wipe the 40X lens with lens
paper to remove the oil and refocus. This can be prevented by never viewing a
specimen with the 40X objective after adding immersion oil to a slide.
The sample can be focused at 10X but when the 40X lens is rotated in place it
contacts the slide.
In most cases this is caused by the slide being place on the stage upside down
with the smear facing the stage. Check your slide carefully to make sure it is placed
on the stage correctly.
The fine adjustment knob does not turn in the direction required for sharp
focusing.
This indicates that it has been turned to the limits of its threads, either upward or
downward, as the case may be. Screw it back to about one-half the thread distance
(About four turns), use the coarse adjustment to raise or lower the objective lens
sufficiently to bring the specimen into view; then refocus with the fine adjustment.
What I am viewing does not look like bacteria.
Check to make sure you are in the correct focal plane that you are focusing on the
smear and not dust on the lenses. To verify this, move the slide while looking at it.
Anything in the smear should move in the field of view.
What I am viewing does not look like bacteria. Part II
If you are in the correct focal plane, there may be problems with smear preparation.
Did you heat fix too much? Was the amount of culture applied sufficient? Did you
stain the slide correctly? Many apparent microscope problems can be attributed to
poor slide preparation.

Proper care of the microscope

The following rules, cautions and maintenance hints will help keep your microscope
in good operating condition.
1. Use both hands when carrying the microscope: one firmly grasping the arm of
the microscope; the other beneath the base. Avoid jarring your microscope.
To keep the microscope and lens systems clean:

2. Never touch the lenses. If the lenses become dirty, wipe them gently with lens
tissue.
3. If blurred specks appear in the field of view this may be due to lint or smears
on the eyepiece. If the specks move while rotating the eyepiece, the dust is on
the eyepiece and cleaning the outer lens of the eyepiece is in order. If the
quality of the image is improved by changing objective lenses, clean the
objective lens with lens paper.
4. Never leave a slide on the microscope when it is not in use.
5. Always remove oil from the oil-immersion objective lens after its use. If by
accident oil should get on either of the lower-power objective lenses, wipe it off
immediately with lens tissue.
6. Keep the stage of the microscope clean and dry. If any liquids are spilled, dry
the stage with a piece of cheesecloth. If oil should get on the stage moisten a
piece of cheesecloth with xylol and clean the stage, then wipe it dry.
7. When not in use, store your microscope in its cabinet. Put the low power
objective lens into position at its lowest point above the stage. Be sure that the
mechanical stage does not extend beyond the edge of the microscope stage.
Wrap the electrical cord around the base.
To avoid breaking the microscope:
8. Never force the adjustments. All adjustments should work freely and easily. If
anything does not work correctly, do not attempt to fix it yourself, immediately
notify your instructor.
9. Never allow an objective lens to jam into or even to touch the slide or coverslip.
10. Never focus downward with the coarse adjustment while you are looking
through the microscope. Always incline your head to the side with eyes parallel
to the slide and watch the objective as you move it closer to the slide. This will
prevent you from smashing the objective into the slide.
11. Never exchange the objective or eyepiece lenses of different microscopes, and
never under any circumstances remove the front lenses from objective lenses.
12. Never attempt to carry two microscopes at one time
If you follow these rules, you will never have trouble with your microscope.
Staining microorganisms
Preliminary identification of bacteria is usually based upon their cell morphology and
grouping and the manner in which they react to certain staining procedures. The
purpose of this section is to demonstrate some common staining reactions used to
categorize microorganisms.

An unstained bacterial smear

Unstained bacteria are mostly made of water and are nearly transparent when viewed
through a light microscope (pictured on the left). Note that most of the microbes are
not visible, but a dust spec in the center of the field of view is visible. Stains cling to
the positive and negative charges of bacteria, but do not bind as readily to the
background of a slide. They therefore differentiate microbes from their surroundings.
Stained bacteria are shown at 40X and 100X in the center and right panels.
Unstained bacteria are practically transparent when viewed using the light microscope
and thus are difficult to see as shown . The development of dyes to stain
microorganisms was a significant advance in microbiology. Stains serve several
purposes:

Stains differentiate microorganisms from their surrounding environment

They allow detailed observation of microbial structures at high magnification
Certain staining protocols can help to differentiate between different types of
microorganisms.

Most dyes consist of two functional chemical groups as shown in Figure 3-3. The
chromophore group, which give dyes their characteristic color; and the auxochrome
group, containing an ionizable chemical structure, which helps to solubilize the dye
and facilitates binding to different parts of microorganisms. Previously, dyes were
classified as acidic or basic, depending upon whether the pigment was negatively or
positively charged at neutral pH. More accurately, dyes can be referred to as anionic
(-) or cationic (+) and this is the convention that will be used in this manual. Cationic
dyes (crystal violet, methylene blue) will react with groups on bacteria that have a
negative charge. Anionic dyes (eosin, nigrosine) will react with groups that have a
positive charge. Since most bacteria have many positive and negative groups in their
cell walls and other surfaces, they will react with both cationic and anionic dyes.

Figure 3-3 The structure of crystal violet

The auxochrome groups of crystal violet is the charged carbon in the center of the
molecule. This is typically neutralized by a Cl- ion. The chromophore group consists
of the three benzene rings and the central carbon. These structures readily absorb
light.
Staining protocols can be divided into 3 basic types, simple, differential, and
specialized. Simple stains react uniformly with all microorganisms and only
distinguish the organisms from their surroundings. Differential stains discriminate
between various bacteria, depending upon the chemical or physical composition of the
microorganism. The Gram stain is an example of a differential stain. Specialized
stains detect specific structures of cells such as flagella and endospores.
Preparation of a Bacterial Smear for Staining
Before staining and observing a microbe under a microscope, a smear must be
prepared. The goal of smear preparation is to place an appropriate concentration of
cells on a slide and then cement them there so that they do not wash off during the
subsequent staining procedure. Figure 3-4 demonstrates smear preparation.
The best smears are made from bacteria that have grown on a solid surface such as an
agar slant or plate. A bit of growth from a culture is mixed with distilled or tap water
to form a slightly turbid solution and this is spread on a clean grease free slide. When
staining broth cultures, a drop of broth is transferred directly to a slide, using no extra
water. The procedure for making a smear is as follows:
1. If more than one culture is to be examined using the same stain, it is possible to
prepare up to 6 smears on the same slide. Before preparing the slide, divide it
into the appropriate number of sections and clearly label each section on the
underside of the slide.
2. If your culture has been grown on a agar slant or agar plate. Place a small drop
of water on a clean, grease-free slide. Next, using a sterile loop or straight wire

3.

4.

5.

6.

7.

needle, transfer a bit of the growth to the drop of water and rub the needle
around until the material is evenly emulsified. Spread the drop over a portion of
the slide to make a thin film. The suspension should be only slightly turbid.
If you are using a broth culture, the broth culture must have clearly
visible turbidity. Transfer a loopful of culture from the broth onto a clean
grease free slide. Spread the drop over a portion of the slide to make a thin film.
Allow the film to air-dry. To get a good stain, it is important to let the smear
dry completely. Excess water left on the slide will boil during the fixing stage,
causing most microbe present to rupture. Rushing this step will result in a poor
final stain.
Once dry, "fix" the smear to the slide by passing the bottom of the slide through
the tip of the burner flame several times for a one second. After heat fixing,
touch the heated portion of the slide to your hand. It should be comfortably
warm, but not burning hot.
Take care not to under-fix (the smear will wash off) or over-heat (the cells will
be ruptured or distorted) the slide. The correct amount of heat fixing is learned
by experience.
Allow the smear to cool and apply the stain.

The Simple Stain

In a simple stain, the smear is stained with a solution of a single dye which stains all
cells the same color. Differentiation of cell types or structures is not the objective of
the simple stain. However, certain structures which are not stained by this method
may be easily seen, for example, endospores and lipid inclusions.
Simple stains are, well simple. One makes a smear and the applies a single stain to the
slide. Below is a procedure for a simple stain.
1. Prepare and heat-fix a smear of the organism to be studied.
2. Cover the smear with the staining solution. If crystal violet or safranin is used,
allow one minute for staining. The use of methylene blue requires 3-5 minutes
to achieve good staining.
3. Carefully wash off the dye with tap water and blot the slide dry with blotting
paper, an absorbent paper pad or a paper towel.
Three steps, now wasn't that easy? Figure 3-5 contains a movie demonstrating the
simple stain. Figure 3-10 shows a light micrograph of what a simple stain should look
like.

Figure 3-10 The Simple Stain

A photomicrograph of a simple stain at 1000X magnification. Note that all cells,
regardless of species or cell wall construction, stain the same color.
The Gram Stain
The Gram stain, performed properly, differentiates nearly all bacteria into two major
groups. For example, one group, the gram-positive bacteria, include the causative
agents of the diseases diphtheria, anthrax, tetanus, scarlet fever, and certain forms of
pneumonia and tonsillitis. A second group, the gram-negative bacteria, includes
organisms which cause typhoid fever, dysentery, gonorrhea and whooping cough. In
Bacteria the reaction to Gram stain reagents is explained by different cell wall
structures. Gram-positive microbes have a much thicker cell wall, while that found in
Gram-negative microbes is thinner. Microbes from the Archaea domain contain
different cell wall structures than that seen in microbes commonly found in the lab
(Bacteria domain). However, they will still have a species specific Gram stain
reaction, even though the underlying macromolecular structures are different.
The Gram stain is one of the most useful differential stains in bacteriology, including
diagnostic medical bacteriology. The differential staining effect correlates to
differences in the cell wall structure of microorganisms (at least Bacteria, but not
Archaea as mentioned above). In order to obtain reliable results it is important to take
the following precautions:

The cultures to be stained should be young - incubated in broth or on a

solid medium until growth is just visible (no more than 12 to 18 hours old if
possible). Old cultures of some gram-positive bacteria will appear Gram
negative. This is especially true for endospore-forming bacteria, such as species
from the genus Bacillus. In this class, many of the cultures will have grown for
more than 2 days. For most bacteria this is not a problem, but be aware that
some cultures staining characteristics may change!
When feasible, the cultures to be stained should be grown on a sugar-free
medium. Many organisms produce substantial amounts of capsular or slime
material in the presence of certain carbohydrates. This may interfere with
decolorization, and certain Gram-negative organisms such as Klebsiella may
appear as a mixture of pink and purple cells.

Gram stain procedure

Below is a procedure that works well in the teaching laboratories.
1. Cover the slide with crystal violet stain and wait one minute.
2. After one minute wash the stain off (gently!) with a minimum amount of tap
water. Drain off most of the water and proceed to the next step. It may help to
hold the slide vertically and touch a bottom corner to paper toweling or blotting
paper.
3. Cover the slide with iodine solution for one minute. The iodine acts as a
mordant (fixer) and will form a complex with the crystal violet, fixing it into
the cell.
4. Rinse briefly with tap water.
5. Tilt the slide lengthwise over the sink and apply the alcohol-acetone
decolorizing solution (dropwise) such that the solution washes over the entire
slide from one end to the other. All smears on the slide are to be treated
thoroughly and equally in this procedure. Process the sample in this manner for
about 2-5 seconds and immediately rinse with tap water. This procedure will
decolorize cells with a Gram negative type of cell wall but not those with a
gram-positive type of cell wall, as a general rule. Drain off most of the water
and proceed.
6. As the decolorized gram-negative cells need to be stained in order to be visible,
cover the slide with the safranin counterstain for 30 seconds to one minute.
7. Rinse briefly and blot the slide dry. Record each culture as Gram positive
(purple cells) or Gram negative (pink cells).
Figure 3-11 shows the results of a Gram stain for gram-positive and gram-negative
negative bacteria.

Figure 3-11 The Gram Stain - A photomicrograph of gram-positive and gramnegative bacteria. Note that Gram reaction is dependent upon cell wall structure. A) E.
coli a common gram-negative rod found in the colon. B) Staphylococcus
epidermidis a gram-positive cocci found on the skin. C) Bacillus cereus a grampositive rod found in the soil.
The Endospore Stain
Cells of Bacillus, Desulfotomaculum and Clostridium (and several other, lesserknown genera) may, as a response to nutrient limitations, develop endospores that
possess remarkable resistance to heat, dryness, irradiation and many chemical agents.
Each cell can produce only one endospore. It is therefore not a reproductive spore as
seen for some organisms such as Streptomyces and most molds. The endospore is
essentially a specialized cell, containing a full complement of DNA and many
proteins, but little water. This dehydration contributes to the spores resistance and
makes it metabolically inert. The endospore develops in a characteristic position (for
its species) in the vegetative cell. Eventually the cell lyses, releasing a free endospore.
For more information on endospores, read the Figure 3-7.
Endospore Stain Procedure
Endospore stains require heat to drive the stain into the cells. For a endospore stain to
be successful, the temperature of the stain must be near boiling and the stain cannot
dry out. Most failed endospore stains occur because the stain was allowed to
completely evaporate during the procedure.

1. Place the heat-fixed slide over a steaming water bath and place a piece of
blotting paper over the area of the smear. The blotting paper should completely
cover the smear, but should not stick out past the edges of the slide. If it sticks
out over the edges stain will flow over the edge of the slide by capillary action
and make a mess.
2. Saturate the blotting paper with the 5-6% solution of malachite green. Allow
the steam to heat the slide for five minutes, and replenish the stain if it appears
to be drying out.
3. Cool the slide to room temperature. Rinse thoroughly and carefully with tap
water.
4. Apply safranin for one minute. Rinse thoroughly but briefly with tap water, blot
dry and examine. Mature endospores stain green whether free or in the
vegetative cell. Vegetative cells stain pink to red.
Figure 3-12 shows a photomicrograph of an endospore stain.

Figure 3-12 The Endospore Stain - A photomicrograph of an enodspore stain.

Spores present in the picture stain green, while the vegetative cells stain red.
A) Staphylococcus epdiermidis which does not form endospores. B) The endosporeforming rod, Bacillus cereus.

The acid-fast stain

Because of the waxy substance (mycolic acids) present on the cell walls, cells of
species of Mycobacterium do not stain readily with ordinary dyes. However, treatment
with cold carbol fuchsin for several hours or at high temperatures for five minutes will
dye the cells. Once the cells have been stained, subsequent treatment with a dilute
hydrochloric acid solution or ethyl alcohol containing 3% HCl (acid-alcohol) will not
decolorize them. Such cells are thus termed acid-fast in that the cell will hold the
stain fast in the presence of the acidic decolorizing agent. This property is possessed
by few bacteria other than Mycobacterium.
Microscopic examination of tissues or of sputum stained by the acid-fast staining
procedure is an aid in the diagnosis of tuberculosis. If an individual has pulmonary
tuberculosis, and if the tubercles in the lungs are open, the bacteria (Mycobacterium
tuberculosis) will be present in the sputum. The bacteria which cause leprosy
(Hansen's disease; caused by M. leprae) can also be detected with this staining
procedure. The finding of acid-fast cells in milk, on the skin, or in feces is of no great
signifi-cance, because these bacteria may be commonly-found saprophytic species
of Mycobacterium.
1. After preparation of the heat-fixed smear, place the slide over a steaming water
bath.
2. Place a piece of paper towel or blotting paper over the smear. The paper should
be about as wide as the slide and cover an area just slightly greater than the
smear itself. Saturate the paper with carbol fuchsin and let the slide remain
above the steaming water bath for five minutes. Add more carbol fuchsin to the
paper if it appears the stain is drying out.
3. Allow the slide to cool to room temperature. Remove the paper and wash off
the excess stain with water.
4. Decolorize the smear with acid-alcohol for 10-15 seconds. Wash gently with
tap water.
5. Counterstain with methylene blue for 3 minutes. Rinse the slide gently and dry.
6. Examine the smear first with the 10X and then the 100X (oil-immersion)
objective. Those cells which retained the primary stain (carbol fuchsin) through
the acid-alcohol treatment are stained red; these are the acid-fast organisms.
Mycobacterium cells characteristically appear as clusters of long, red rods. All
other cells are blue.
Below is pictured an example of an acid-fast stain

Figure 3-17 The acid fast stain- A photomicrograph of Mycobacterium

smegmatis (pink) and Micrococcus luteus (blue) at 1000x magnification. M.
smegmatis is acid-fast, retaining the carbol fuchsin dye, thus appearing pink. M.
luteus is not acid-fast, loses the carbol fuchsin during decolorizaiton, and is counterstained with methylene blue.
Practice staining
In the study and identification of bacteria, the microscope is indispensable! The series
of micro-scopic observations in this exercise is designed to illustrate how bacteria
may be viewed individually in their basic form, the cell . The second and third periods
herein coincide with those of Experiment 1 where organisms isolated by the student
are examined microscopically (and could be found to be more interesting than those
provided in this exercise!).
Period 1
Materials
Items from nature
Slide with smears of Bacillus cereus and Staphylococcus epidermidis

Simple stain.
1. You are provided with a microscope slide with two smears. Following the
directions for microscopy and staining, heat-fix the slide, making sure the slide
goes through the flame smear-side up.
2. Gloves are available for the staining procedure. Placing the slide on the staining
rack in the sink, cover the slide with crystal violet for one minute. For a review,
look at the directions for the simple stain
3. Carefully rinse off the dye with tap water and blot the slide dry with paper
towel or blotting paper.
4. With both hands, obtain the light microscope from the cabinet (corresponding
to your desk number). This is the type of microscope which we will always use
to observe stained smears.
5. Unless the instructor has other directions more directly applicable to the
microscope you are using, use the simple procedure. Refer to this procedure as
you study the cells in the two smears in the following steps.
6. Place the slide on the stage. Make sure the clips on the stage hold the slide
securely.
7. Begin your observations with the Bacillus cereus smear. When observing this
organism with the oil-immersion objective, you will notice that the cells are
relatively large and rod-shaped (bacilli) and are usually in chains. Record your
observations on the next page.
8. Repeat this procedure with the Staphylococcus epidermidis smear. Cells of this
organism are spheres (cocci) which are usually arranged in clusters
(staphylococci) and pairs.
9. When you are through, be sure the microscope is put away properly (i.e., all oil
wiped off, 10X objective lens in place, stage centered). It is recommended that
you keep the slide. (To remove immersion oil from smears, place a few pieces
of lens paper on the slide to absorb the oil. Then, add several drops of xylol to
the lens paper. Peel the paper, now soaked with xylol, off the slide. Xylol is
flammable! Keep it away from flames!)

Figure 3-13 Simple stain - A simple stain of S. epidermidis and B. cereus. S.

epidermidis (A), B. cereus old (B), B. cereus young (C)
Period 2
Materials
Bacterial cultures growing either in a liquid medium [40] (Heart Infusion Broth) or on
a slant of an all-purpose medium followed by suspension in saline:
Escherichia coli - young culture, incubated 12-15 hours
Bacillus cereus - young culture, incubated 12-15 hours
Bacillus cereus - old culture, incubated 2-3 days
Figure 3-14 Gram stains

Gram stains of demonstration species. Below are shown typical Gram stain reactions
of two species. E. coli (A), B. cereus old (B), B. cereus young (C). The images are
slightly larger than what would be visible in a light microscope to improve clarity.
1. On one clean glass slide, prepare smears of the three cultures. Go to smear
prepapration [41] if you need a refresher. Only when the smears have dried
completely should the slide be heat-fixed.
2. Perform the Gram [42] stain procedure as described.
3. As with any stained smear, definitive observations are made with the 100X, oilimmersion objective. Refer to the microscope directions already given [43],
remembering to focus the slide initially with the 10X objective, moving then to
the oil immersion objective.
4. Keep in mind that the young cultures of B. cereus and E. coli are your positive
and negative control cultures, respectively, for the observation of probable
gram-variability of the older B. cereus culture.
5. Using the figures below, record your observations in your notebook, noting the
Gram reaction (positive if purple, negative if red) and the cellular shape. Is
there any difference seen between the two cultures of Bacillus cereus? Is gramvariability evident for the older culture? Recall from the introduction to
Experiment 1 that we can refer to old and young cultures but should not do so
for individual cells. (Remember to discard the tubes and slides properly)
Materials
This experiment will be done in class.
Staphylococcus epidermidis
Pseudomonas fluorescens
An unknown
Record the number of your unknown!

Figure 3-15 Typical reactions of example strains for test - The classic Gram
reactions for Staphylococcus epidermidis (A) and Pseudomonas fluorescens (B). From
this, determine whether they are Gram (+) or Gram (-). Note we do not show an
unknown as this must be done in class. The images are slightly larger than what would
be visible in a light microscope to improve clarity.
1. On a clean glass slide, prepare heat-fixed smears of the three cultures, noting
that these cultures are growing on a solid medium. Therefore the cells must be
dispersed in a drop of water when preparing the smears, as a smear is always a
dried suspension of cells. Take care not to make the smears too thick! S.
epidermidis and P. fluorescens are your positive and negative control cultures
(respectively) for your unknown.
2. Perform the Gram stain procedure and note the Gram reaction and cellular
shape. Record your results. Fill out and turn in your description of your
unkonwn. Save your slide until your graded unknown is returned.
Materials
For the capsule stain:
36-48 hour culture of Klebsiella pneumoniae growing on a slant of EMB Agar (a
high-sugar medium)
Dropper bottle of filtered India ink
For the acid-fast stain:
3-day culture of Mycobacterium smegmatis growing on a slant of Trypticase Soy Agar
plus 1% glycerol

18-24 hour culture of Micrococcus luteus (the negative control culture) growing in
Nutrient Broth
Dropper bottles of carbol fuchsin (freshly-made), acid alcohol and methylene blue
Capsule stain

Figure 3-16 The capsule stain - A capsule stain using India ink at 1000x
magnification. The cells of Klebsiella pneumoniaeare surrounded by a dark
background. The capsule is the clear area surrounding the cells. The
photomicrographs is slightly enlarged for clarity.
1. Using the culture of Klebsiella pneumoniae, Place one loopful of water on a
slide and emulsify in it a bit of growth from the slant or plate culture of the
designated organism. Add a drop of filtered India ink to the cell suspension. It
often works out well to place the drop of India ink adjacent to the cell
suspension on the glass slide.
2. Obtain a clean coverslip (no fingerprints, smudges, dirt, etc.) and rim it lightly
with vaseline; the vaseline can be gently scraped from a thin layer applied to
the palm of the hand. Place a small, multi-layered piece (about 1-2 cm2) of
paper towel over the coverslip and press down firmly; discard the paper towel
into the disinfectant.
3. Using the regular light microscope, focus initially with the 10X objective,
switching to the 45X objective and then - if needed - the 100X, oil-immersion

objective. Adjust the light intensity as required with the iris diaphragm. The
outline of the cell can be seen within the area of the clear capsule.
4. Alternatively, the phase microscope can be used. Heed the precautions
regarding use of this microscope. Excellent observations can be made with just
the 40X objective lens (which takes no immersion oil).
5. When finished, without removing the coverslip, discard the slide directly into
the disinfectant. Never discard capsule stains and other wet mounts with the
stained smears, as viable cells are still present and the slides must be
disinfected!. Record your observations below.
Acid-fast stain

The acid fast stain A photomicrograph of Mycobacterium smegmatis (pink)

and Micrococcus luteus (blue) at 1000x magnification. M. smegmatis is acid-fast,
retaining the carbol fuchsin dye, thus appearing pink. M. luteus is not acid-fast, loses
the carbol fuchsin during decolorizaiton, and is counter-stained with methylene blue.
Acid fast stain.
1. Prepare a mixed smear of two organisms as follows: Place a drop of
the Micrococcus luteus broth culture on a slide. Into this drop, add cells from
the Mycobacterium smegmatis culture. Disperse the cells as much as you can

(the Mycobacterium cells tend to clump), and prepare a smear about the size of
a nickel. Let it air-dry completely, and then heat-fix it well, passing the slide
through the flame an extra one or two times.
2. Perform the acid-fast procedure and record your observations below.
3. As with all stained smears, discard the slide in the appropriate container.
Summary of Microscopy and Staining
Staining and viewing microbes under the microscope is often necessary in for their
identification and classification. The identity of a microbe can help in determining the
cause of a disease or the source of food spoilage. Microscopes also have important
roles in genetics, cell structure, biochemistry and many other scientific disiplines.
Hopefully, this short introduction has helped you to understand the visualization of
microorganisms.