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3-11 Practice staining

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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
Hay infusions and various other 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 described in the operating
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 such that it is oriented as illustrated above. Make sure
the clips on the stage hold the slide securely.
7. Begin your observations with the Bacillus cereus smear. (See figure below) 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)
Wet Mount
1. For observation of living microorganisms, various samples including a hay
infusion are available. To study the microorganisms in the aqueous materials
available, it is necessary to make wet mounts. The procedure is relatively simple:
2. Using a capillary pipette or inoculating loop, pick up some of the material from
around the surfaces of grass and leaves and from the bottom of the sample. Place
a drop of suspended material on a clean microscope slide.
3. With a toothpick, spread a very thin layer of vaseline over a small part of the palm
of your hand. Take a clean coverslip (always held by the edges) and gently scrape
all four edges along your palm, picking up a thin line of vaseline along each edge.
4. Place the coverslip directly onto the drop on the slide in such a manner that some
air bubbles are trapped. Place a small, multilayered piece of paper towel over the
coverslip and press down. Discard the piece of paper towel into the disinfectant.

5. Examine the wet mount with your light microscope or a phase CONTRAST
microscope set up by the instructor at a special station in the back or side of the
lab.
6. Without removing the coverslip, discard the slide into the disinfectant container
on the stage. (Refer to page viii for cleanup directions.)
If you haven't already, Figure 1-2 presents a movie of the types of life forms found in a
hay infusion.
Period 2
Materials
Bacterial cultures growing either in a liquid medium (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 if you need a refresher. Only when the smears have dried
completely should the slide be heat-fixed.
2. Perform the Gram 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,
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 gramvariability 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)
Period 3
Materials
This experiment will be done in class.
Young bacterial cultures growing on slants of Heart Infusion Agar:
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.
Period 4
Materials
For the capsule stain:
36-48 hour culture of Klebsiella pneumoniae growing on a slant of EMB Agar (a highsugar 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.
capsule stain
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

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 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 (page 148, observing the slide with the regular
light microscope) and record your observations below.
3. As with all stained smears, discard the slide in the appropriate container.
Spore stain:
1. Make a heat fixed smear of Bacillus as done previously.
2. Cover the smear with a small piece of paper towel, not hanging over the edges of the
slide.
3. Place the slide on top of a small beaker containing about 1-2 inches of boiling water,
being certain that the slide is horizontal. Instructor will demonstrate the ring stand set up.
Be careful of burning yourself or knocking down the ring stand.
4. Cover the smear and paper towel with malachite green, and steam. Don't let the slide
get dry--keep adding stain it is appears to approach dryness. Steam gently for about 5-10

minutes. REASON: You are cooking the malachite green into the endospore wall, as they
are very resistant to staining.
5. Remove the slide from the steaming beaker, turn off the flame, and dispose of the
green paper in the wastebasket, using your forceps. Wash the slide gently in running
water about 20 seconds, getting all the extra green off the slide.
6. Counterstain with safranin for one minute, at the staining sink. Gently rinse with water,
and shake off excess water into the sink. REASON: The running water washed the green
stain out of the vegetative cells and sporangia, and they became colorless. The
counterstain now dyes them red.
7. Gently blot the slide dry, no rubbing, and let it air dry. Examine with oil immersion
optics. Observe red vegetative cells and sporangia, and green endospores and free spores.

Spore stain of a overnight culture of Bacillus subtilis.


Note: The all red cells are vegitative cells. The free green "cells" are free spores. The
cells that are red with a green interior structure are the cells with endospores.

Capsule Stain:(Negative stain)

1. Place a drop of congo red on a clean slide. Mix a loop of Klebsiella culture into the
stain drop.
2. Using a second slide, spread the drop of stain/microbe across the first slide, making a
smear, and feathering the end.
3. Allow slide to air dry and then view under oil immersion.
Congo Red Capsule stain of Klebsiella pneumonia.
Note: The red interior is the bacterial cell and the clear zone around the cell is the area
where the capsule has excluded the congo red stain.

Questions:
1. What is the clinical value of the acid-fast stain? the capsule stain?
2. Why must the spore stain include a heating step? What would happen if this were
omitted?
3. Describe the different arrangements of flagella. What is their importance?
4. Why is staining bacterial components useful in strain identification?

Capsule Stain
Purpose: The capsule stain is a differential stain which selectively stains
external capsules surrounding bacterial cells.
How it works: Capsules are highly ordered polymers of sugars and proteins
that surround some bacterial cells, and can be easily dislodged by heat or
water. Accordingly, capsule stains are not heat-fixed, and water is never used
to rinse. The primary stain applied is crystal violet, which stains both the
bacterial cell and the surrounding capsule. A 20% copper sulfate solution is

then applied, which serves a dual function as both decolorizer and


counterstain. It removes and replaces the crystal violet in the capsule only. At
the end of the staining procedure, the capsule appears as a faint blue or white
halo around a purple cell.
Overview of capsule-staining process:
Crystal Violet
20% Copper Sulfate
No Capsule
Capsule

Results:

Simple Stain

Simple stains provide a quick and easy way to determine cell shape,
size, and arrangement.
1. Perform a bacterial smear, as discussed in Figure 3-35 on page 82 of
your lab manual.
2. Saturate the smear with basic dye for approximately 1 minute. You
may use crystal violet, safranin, or methylene blue.
3. Rinse the slide gently with water.
4. Carefully blot dry with bibulous paper.
5. Observe the slide under the microscope, using proper microscope
technique.

Negative Stain

Negative staining is an excellent way to determine an organisms


cellular morphology. Since the cells themselves are not stained, their
morphology is not distorted in any way. The nigrosine provides a dark
background against which the shapes of the unstained cells are clearly
visible. This method provides a high degree of contrast not available in
most other staining procedures.
1. Place a single drop of nigrosin on the left-hand end of a clean
microscope slide.
2. Using a flamed loop and sterile technique, remove some organism
from your slant and mix
it into the drop of nigrosin. Be sure there are no large clumps of
organism, but try to avoid
spreading the drop.
3. Place the end of another, clean microscope slide at an angle to the
end of the slide
containing the organism and spread the drop out into a film. This is
done by contacting the
drop of nigrosin with the clean microscope slide and using the capillary
action of the
dye/microscope slide to spread the nigrosin across the smear:
4. Allow the film to air dry.
5. Observe the slide under the microscope, using proper microscope
technique.

Gram Stain

1. Perform a bacterial smear, as discussed in Figure 3-35 on page 82 of


your lab manual.
2. Saturate the smear with crystal violet for 1 minute.
3. Rinse the slide gently with water.
4. Saturate the smear with iodine for 1 minute.
5. Rinse the slide gently with water.
6. Decolorize with Gram decolorizer (acetone/alcohol) for 3-5 seconds
ONLY; if you leave the
decolorizer on too long, it will bleach the crystal violet out of a gram
positive cell!

7. Rinse the slide gently with water.


8. Counterstain with safranin for 1 minute.
9. Rinse the slide gently with water.
10. Carefully blot the slide dry with bibulous paper.
11. Observe the slide under the microscope, using proper microscope
technique.
With proper staining technique
Gram positive bacteria will stain purple.
Gram negative bacteria will stain red/pink.
TIP: When making a smear of an unknown organism to gram stain,
place three
organisms on your slide: a known gram positive, a known gram
negative, and your
unknown (and LABEL which is which!). That way, if theres a problem
with your
staining technique, it will be reflected in the known organisms, and
youll know you
need to do the stain again to get accurate results.

Capsule Stain

1. Place a single drop of India ink on the left-hand end of a clean


microscope slide.
2. Using a flamed loop and sterile technique, remove some K.
pneumoniae (or the organism you
want to stain) from your slant and mix it into the drop of India ink. Be
sure there are no large
clumps of organism, but try to avoid spreading the drop.
3. Place the end of another clean microscope slide at an angle to the
end of the slide containing the
organism. Spread out the drop out into a film. This is done by
contacting the drop of India ink
with the clean microscope slide and using the capillary action of the
dye/ slide to spread the
India ink across the smear. Refer to the Negative Stain portion of this
handout for a diagram.
4. Allow the film to air dry. DO NOT heat or blot dry!!!! Heat will melt
the capsule!
5. Saturate the slide with crystal violet for 1 minute.
6. Rinse the slide gently with water.
7. Allow the slide to air dry. DO NOT heat or blot dry!!!! Heat will melt
the capsule!
8. Observe the slide under the microscope, using proper microscope
technique.
The background will be dark.
The bacterial cells will be stained purple.
The capsule (if present) will appear clear against the dark
background.

background
capsule
bacterium
TIP: If you are staining an unknown organism, be sure your
culture is several days
old. Young, fresh cultures wont have developed capsules yet.

Acid Fast Stain

1. Perform a bacterial smear, as discussed in Figure 3-35 on page 82 of


your lab manual.
2. Place a small piece of bibulous paper over the smear and saturate
the paper with carbolfuchsin.
3. Heat the slide gently over the bunsen burner for 5 minutes. Be sure
to keep the bibulous paper
saturated with carbolfuchsin during heating; if the slide is steaming,
youre okay; if it stops
steaming, add more carbolfuchsin!
4. Rinse the slide gently with water and dispose of the used bibulous
paper in the trash. DO NOT
leave the bibulous paper in the sink or drain!
5. Decolorize the slide with acid-alcohol until the rinsate runs clear.
6. Rinse the slide gently with water.
7. Counterstain with methylene blue for 2 minutes.
8. Rinse the slide gently with water.
9. Carefully blot the slide dry with bibulous paper.
10. Observe the slide under the microscope, using proper microscope
technique.
Acid-fast cells will stain fuchsia. Non-acid-fast cells will stain
blue.

Endospore Stain

1. Perform a bacterial smear of Bacillus or your unknown, as discussed


in Figure 3-35 on page 82
of your lab manual.
2. Place a small piece of bibulous paper over the smear. Saturate the
paper with malachite green.
3. Heat the slide gently over the bunsen burner for 5 minutes. Be sure
to keep the bibulous paper
saturated with malachite green during heating; if the slide is steaming,
youre okay; if it stops
steaming, add more malachite green!
4. Rinse the slide gently with water and dispose of the used bibulous
paper in the trash. DO NOT
leave the bibulous paper in the sink or drain!
5. Counterstain with safranin for 2 minutes.
6. Rinse the slide gently with water.
7. Carefully blot the slide dry with bibulous paper.

8. Observe the slide under the microscope, using proper microscope


technique.
Endospores will stain green. Parent cells will stain red.