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208 MICROBIO LOGY

SECTION 1
LABORAT OR Y WEE K 3

- DIFFERENTIAL STAINING
- EX ER CIS E 5. GRAM STAIN
- CAPSULE STAIN
- FLAGELLA STAIN
- EX ER CIS E 6. ENDOSPORE STAIN
- E XE R CIS E 7. PURE CULTURE TECHNIQUE

DIFFERENTIAL STAINING

Definition: A process of staining that facilitates differentiation of various


structures in a specimen.

Exercise 5. GRA M STA IN

Hans Christian Gram was a Danish bacteriologist. He developed the Gram stain as a means to
differentiate pneumococci ( Labor atory exerc ise 19) from Klebsiella pneumonia
(Labora tory exerc ise 25) in 1884. It remains one of the most important staining techniques
in microbiology today. The Gram stain is often the first test performed in the identification of
bacteria.
Gram staining is partly based on the differences in the cell walls of Gram positive and Gram-
negative organisms. It is best to use younger cells (1 2-24 hours) because older Gram-positive
bacteria are subject to break down of the cell wall by enzymes that are produced with age which
may result in Gra m var iab le st aining . Gram-positive organisms have a cell wall composed
almost entirely of peptidoglycan whereas Gram-negative organisms have a lipid rich outer wall.
This fundamental difference allows the Gram stain to work. The crystal violet complex is an
insoluble complex that is difficult to remove. When alcohol is used as a decolorizer, it
dehydrates the Gram-positive cell wall and traps the complex inside the cell wall. However,
alcohol is able to remove the complex from the lipid rich wall of Gram-negative organisms. The
now clear Gram-negative organisms are stained with fuchsin or safranin so that the Gram
negative and Gram-positive organisms can be distinguished from one another.

CULTURE S

Micrococcus luteus Nutrient Agar Slant

Escherichia coli Nutrient Agar Slant

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PROC EDUR E

Step 1. PR EP ARE A FI X ED S ME AR

Prepare one fixed smear using both assigned cultures. (label the bottom of your slide)

Mix both cultures in


one smear together

Step 2. GR AM S TAI NI NG TH E SLID E

2a. Place the slide on your staining rack and flood the smear with Crystal Violet.

2b. After 1 minute remove the stain and wash gently with water.

2c. Flood the smear with Grams Iodine.

2d. After 1 minute remove the Iodine and wash gently with water.

2e. Flood the smear with 95% Ethanol.

2f. After 15 seconds remove the Ethanol and rinse gently with water.

2g. Flood the slide with Safranin.

2h. After 30 seconds remove the Safranin and gently wash with water

2i. Drain off excess water, blot dry with paper towel, and let the slide air dry.

Step 3. OBS ER VATI ON

Examine the stained smear with low-power and oil-immersion objectives. Record your
observations as sketches and descriptions.

You may save the slide in your slide box for further study or discard the stained smear in the
sharps container. Co lor p lat e 3 .1

CAPSULE STAIN

Capsules are structures that lay outside of an organism's cell wall and thus are in direct contact
with the environment. Many bacteria produce capsules under the right conditions. Bacterial
capsules are most often composed of long POLYMERS of sugar (or sugar derivatives) known as
POLYSACCHARIDES. Some capsules are composed of POLYALCOHOLS or AMINO ACID POLYMERS.
Capsules may be absent, thin, thick, well formed or loose. Capsule formation is dependent upon
the nutrient conditions. In general, media rich in sugars and low in nitrogen tend to induce
capsule formation. Capsules play a number of roles in the life of microbes. Both prokaryotes
and eukaryotes form capsules. Capsules can serve the following functions:

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1. Protect the cell from desiccation (drying).

2. Protect the cell from phagocytosis (being engulfed by white blood cells).

3. Provide a food reserve when certain organic compounds are in excess.

4. A virulence determinant of pathogenic microbes.

5. They serve as binding or adhesion agents for sticking cells together and/or to a surface such
as a rock in flowing stream or a tooth.

The polysaccharide or polypeptide composition of the capsules makes staining difficult. The
capsule stain technique stains around the capsule with a negative acidic stain. Then a basic
stain will colorize the cell itself. The capsule appears as a white halo between the cell and the
darker background. The smear is prepared in the same manner as the negative stain, and the
slide is not heat-fixed, since application of heat may destroy or distort the capsule. The negative
stain is air-dried before the basic stain is applied. Co lor p la te 3.2

FLAGELLA STAIN

The flagella stain allows for the direct observation of flagella. Presence and arrangement of the
flagella can be used in identification of the bacteria.

Since the flagella are too thin to be seen in ordinary stains, special stains and techniques need
to be used to coat the flagella with enough stain to obtain a visible thickness. There are several
staining techniques and some commercially available flagella stains. This technique takes more
time and specialized stains than are usually found in introductory microbiology labs. Co lor
plat e 3 .3

Exercise 6. E ND OSP ORE STAIN


Bacillus and Clostridium are bacterial genera that sporulate (i.e. make endospores) to protect
cells through times of poor nutrient availability, low humidity, and high temperature. The
cell’s DNA is tightly packaged in small basic proteins which protect it from damage by oxygen,
heat as high as boiling for up to an hour and drying. Although it is not completely clear why
the spore is so resistant to environmental insults, its properties are probably related to the
exclusion of water from the spore. Sporulation is induced – turned on – under starvation
conditions, particularly starvation for nitrogen. During the sporulation process, a whole set of
vegetative genes are turned off and a new set of sporulation genes is turned on. These genes
are responsible for the creation of the spore, initially part of the mother cell, the packaging of
chromosomal DNA, ribosomes, and proteins into the spore, and subsequently the destruction of
the mother cell. While spores can last many thousands of years, they can be reactivated to
generate
vegetative cells in response to moisture and proper nutrients.
Sporulation has important implications for human health as you will see in laboratory exercise
34 and 35; spores of pathogenic bacteria introduced into a human body can germinate and lead
to disease.
Endospores may be located in the middle of the cells (central), at the end (terminal), or
between the end and the middle of the cells (subterminal). Endospores themselves, once
stained, may be viewed under oil immersion as round or oval.

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FIGURE 3.1 TH E BACTERIA L ENDOSPORE

Exosporium

Spore coat
(cross-linked
Keratin)
Spore cortex region
(peptidoglycan)

Endospore

Bacterial endospores are resistant to antibiotics, most disinfectants, and


physical agents such as radiation, boiling, and drying. The impermeability of
the spore coat is thought to be responsible for the endospore's resistance to
chemicals. The heat resistance of endospores is due to a variety of factors:

1. The cortex is a prominent region composed of loosely cros s- lin ked p ept id iglyca n .

2. Calciu m-d ip icolinat e is abundant within the endospore. This salt of Dipicolinic acid
stabilizes the endospore's DNA along with Specialized DNA-binding proteins that protect it from
heat, drying, chemicals, and radiation.

3. De sicca t ion . The cortex may osmotically remove water from the interior of the endospore
and the dehydration that results is thought to be very important in the endospore's resistance
to heat and radiation.

4. Finally, DNA repair enzymes contained within the endospore are able to repair damaged DNA
during germination.

CULTURE S

Bacillus subtilis Nutrient agar slant


Micrococcus luteus Nutrient agar slant

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PROC EDUR E

Step 1. PR EP ARE A FI X ED S ME AR

Prepare two fixed smear on one slide using your assigned species. (label the bottom of your
slide)

Step 2. E ND OSP ORE S TAI NI NG TH E S LIDE

2a. Place the slide on your staining rack and flood the smear with Malachite Green.

2b. Stea m s lide for 3-5 minu tes (t he in struc tor will de mon s trat e), Cool, remove the
stain and wash gently with water.

2c. Flood the slide with Safranin.

2d. After 30 seconds remove the Safranin and gently wash with water

2e. Drain off excess water, blot dry with paper towel, and let the slide air dry.

Step 3. OBS ER VATI ON

Examine the stained smear with low-power and oil-immersion objectives. Record your
observations as sketches and descriptions. Co lor pla te 3.4

You may save the slide in your slide box for further study or discard the stained smear in the
sharps container.

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RESULTS TABLE 3.1

AS SI G NE D MI CR OOR G ANIS M DI AG R AM: MORP H OLOG Y, CE LL GR OUPI NG


GR AM S TAI NI NG D ATA AND S TAI NI NG RE ACTI ON 10 00X

Escherichia coli

Micrococcus luteus

AS SI G NE D MI CR OOR G ANIS M DI AG R AM: MORP H OLOG Y, CE LL GR OUPI NG


E ND OS P OR E STAI NI NG D ATA AND S TAI NI NG RE ACTI ON 10 00X

Bacillus subtilis

Micrococcus luteus

Exercise 7. PURE CULTUR E TECHNIQUE

The skin and many mucosal surfaces of the human body support large numbers of
microorganisms that comprise the normal, or indigenous, flora. When clinical specimens are
collected from these surfaces and cultured, any pathogenic microorganisms being sought must
be recognized among, and isolated from, other harmless organisms. Colonies of the pathogenic
species must be picked out of the mixed culture and grown in isolated pure culture. The
microbiologist can then proceed to identify the isolated organism by examining its biochemical
and immunological properties ( la boratory w eek 6 a nd 13) . Pure culture technique is
critical to successful, accurate identification of microorganisms.

CULTURE S
Each desk is provided with a mixed broth culture labeled “Mixed Bacteria”. The mixture
contains the following microorganisms:

E. coli White colonies, flat, irregular


M. luteus Yellow colonies, small round, smooth, convex
M. roseus Red colonies, small round, smooth, convex, (slow growth)
Streak Mixed Broth for Isolation (the instructor will demonstrate this procedure)

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PROC EDUR E

The streak plate technique is essentially a method to dilute the number of organisms,
decreasing the density. This allows for individual colonies to be isolated from other colonies.
Each colony is considered "pure," since theoretically, the colony began with an individual cell.

Step 1. Begin with inoculating the first, or primary, quadrant of the agar plate (See f igur e
3.2 b elo w) . Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid.

Step 2. Flame the loop, cool by touching an uninoculated portion of the surface.

Step 3. Now rotate the plate. Open lid and streak again, following the diagram Remember:
you are picking up growth from quadrant one, and using this as your inoculum for quadrant
two.

Step 4. Flame loop; rotate plate, and repeat procedure for quadrants three and four.
The proper wrist action and light touch takes practice.

Step 5. Incubate plate inverted at room temperature until the next laboratory meeting.

FIGURE 3.2 STREAK FOR ISOLATI ON

Culture flame loop flame loop flame loop

Quadrant 1 Quadrant 2 Quadrant 3 Quadrant 4

RESULTS
Draw your results showing the approximate number of isolated colonies and record any
suggestions the instructor may have for improvement.

Comments and suggestions:

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TERMS AND QUEST IONS F OR STUDY

1. What is the function of the Iodine solution in the Gram stain?

2. What is the function of the 95% ethanol in the Gram stain?

3. What counter stain is used in the Gram stain and why is it necessary.

4. What is the advantage of the Gram stain over the simple stain?

5. Describe two conditions in which an organism might stain gram variable?

6. What is the accepted theory regarding the mechanism of the Gram stain?

7. What is the importance of the capsule stain, the flagella stain and the endospore stain?

8. Describe the process of endosporulation, is this a reproductive mechanism? Explain.

9. Why is the location of an endospore important?

10. What is the value of a capsule to a microorganism?

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11. When an agar plate is inoculated, why is the loop flamed between quadrants?

12. What is a mixed culture? What is a pure culture?

13. What is a bacterial colony?

14. How would you differentiate colonies of E. coli from M. luteus ?

15. Why is it necessary to isolate individual colonies from a mixture in the clinical lab?

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