Name: KNEDRICK KEVIN C.

MACALMA Date: FEBRUARY 2, 2017

Group No.5
Rating:_________

Laboratory Exercise No .1
The Preparation of Bacterial and Oral Smears , the
Use of Simple Stains and Study of Bacterial Morphology

Objectives: After completing this exercise, you should be able to:

1.effectively perform the appropriate aseptic techniques required in the handling of bacterial
cultures.
2.prepare and stain bacterial and oral smears.
3. locate ,examine and interpret stained bacterial and oral smears.
4. distinguish among basic bacterial shapes
5. give the steps and perform the following staining procedures:
a. Simple stain
b. Gram stain
c. Acid Fast Stain (Kinyouns Method)

Bacteria are microscopic organisms that cannot be seen with the naked eye and as such must
be viewed using the microscope. Microscopic examination of clinical specimen in stained and
unstained states is one of the steps usually done in the identification of bacteria. Staining
reactions, morphology,size, arrangement ,motility ,and presence of special structures can be
visualized by microscopic examination.

The first step in the preparation of smears of microorganisms involves the removal of a
small amount of microbial growth from a culture, which is then spread on the surface of a clean
glass slide .This is done with the aid of an inoculating loop .The wire portion of the instrument
,either in the form of loop or needle ,is sterilized by holding it in a flame or alcohol lamp until it
is entirely heated to redness. Such flaming incinerates any microorganism on the wire.

After a few seconds of cooling , the needle or loop can be used to lift the bacteria from
surfaces or remove them from broth cultures and transfer them to a slide or to other media. The
inoculating instrument then is flamed (resterilized) once more to kill any remaining bacteria.
Flaming the wire portion from where it is connected to the handle to its tip is the accepted
practice. The use of the inoculating loop or needle is one of several that belong to the group
referred to as aseptic (sterile) techniques. Aseptic techniques are procedures used to prevent
contamination of cultures and to protect individuals and the environment from microbial
exposure.

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 Source: http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab3.html

Figure 1. The correct way to use an inoculating loop/needle. Take up the inoculating loop by the
handle and hold it as you would a pencil, loop down. Hold the wire in the flame of the Bunsen burner or
over an alcohol lamp until it glows red. ). Remove loop and hold it steady a few moments until cool. Do
not wave it around, put it down, or touch it to anything.

After the small amount of bacterial growth is spread on a clean glass slide, the resulting thin
film is allowed to air-dry and is then usually heat fix by passing over the alcohol lamp 3 to 5x.
The resulting heat-fixed smear is then ready for the application of staining solutions.

There are three basic categories of staining methods utilized in diagnostic bacteriology. The
most basic is the simple stain which makes use of aniline dyes. However, very little information
can be obtained from this method and as such, this method is used only if there are no other
stains are available. The most widely used staining technique is the differential stain which
consists of the Gram stain and the Acid Fast staining technique. Gram stain differentiates
gram-positive organisms from the gram-negative organisms. Organisms that are gram-positive
will appear purple while gram-negative organisms will appear red.

In 1884, Hans Christian Gram, a Danish physician, devised a staining procedure that can
divide all the true bacteria into 2 physiological groups. This is the most important staining
method done in bacteriology to facilitate the identification of the bacterial species. The cells are
exposed to more than 1 dye or staining procedures.

The Gram stain is one of the oldest and most useful methods of staining bacteria. By this
method, bacteria can be classified as Gram(+) positive or Gram (-) negative. They are gram(+) if
they retain the initial stain (crystal violet) after treatment with mordant (iodine) and
decolorizer(alcohol) and thus resist staining with the counter-stain (safranin). They are gram(-) if
they pick up the counter-stain (safranin) after the initial stain is washed out by the mordant and
decolorizing agents.

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 Research involved in determining the composition of bacterial cell walls indicated that there
was close correlation between the gram staining results and the presence or absence of lipid cell
wall constituents. This basic chemical difference between gram(+) and gram (-) cells provides a
reasonable explanation for the differential mechanism associated with the gram stain. Some of
the information used to support the Gram staining mechanism is provided below:

The following rules will help the student in remembering the Gram staining reaction of the
more important bacteria:

1.All bacilli are gram negative EXCEPT Corynebacterium, Mycobacterium, aerobic spore
formers (Bacillus) and anaerobic spore formers (Clostridium).
2. All cocci are gram positive with the exception of Neisseria and Veillonella.
3.Gram stain is usually not applied to spirochetes.

The second method of differential staining is the Acid Fast Staining Method. This can be done
by the Ziehl-Neelsen or the Kinyoun Method.The staining method will differentiate acid fast
from the non-acid fast organisms. As a general rule: all Mycobacterium species are acid-fast.
Nocardia is partially acid fast.

The third category of staining method is the special stain. Special stains are used to help
visualize certain bacterial structural components which ordinarily cannot be seen using any of
the preceding staining methods. Examples are the special stains used to demonstrate the capsule,
flagella or spores.

Reagents Used in Differential and Selective Staining

1.Initial Stain also known as the primary stain , it is the first stain that is applied on the
specimen wherein the cell wall will appear colored.

2. Mordant - any substance which will form a bridge between the cell and the initial stain, so
that the cell being studied will better retain the stain. There are two types of mordant:
a. Physical mordant such as heat or cold
b. Chemical mordant such as iodine, ferrous sulfate, tannic acid

3. Decolorizer any substance that may be used to remove the initial stain. This is especially
important when you want to contrast the staining affinity of some parts of the cell to the initial
stain. The decolorized stain will then be replaced by the secondary stain.

4. Secondary Stain also known as the counterstain. It is the stain that is applied to the
decolorized cell or cell parts. This will help in differentiating the physiological characteristic or
some special structures that are present in the cell.

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 Study of the Morphological and Physiological
Characteristics of Microorganisms

A.Morphology of the Bacteria

Morphology deals with the study of the structure and form of living organisms. There are
three (3) forms generally recognized on the basis of the shape of the bacteria.

1.Cocci- spherical or round in shape; means berry in Greek.

Examples:

Genus Arrangement
Staphylococcus Irregular or grape-like clusters
Diplococcus In pairs
Streptococus In chains
Gaffyka In groups of four
Sarcinae In cubical pockets of eight
Micrococcus singly

2. rods straight, sausage, cigarette-shaped, cylindrical or elongated; sometimes known as

bacilli which means little sticks. The name bacilli with the small letter b should indicate and
describe the shape of the bacterium while the word Bacillus with a capital letter B indicates a
genus of a group of bacteria.

Examples;
Genus Arrangement
Streptobacillus In chains
Clostridium singly
Corynebacterium Palisade/Chinese letter-
arrangement

3. spiral- curved rods having a helicoidal or cork-screw shape. The spiral bacteria are divided as
follows:

a.Comma-shaped- curved rods resembling a comma. It is an incomplete spiral and forms an S-

shaped strand or serpentine formation when grown together. Ex. Vibrio cholerae the causative
agent of cholera

b. Spirilla actual or complete spirals, helices or may resemble the appearance of a cork-screw,
Its body is relatively rigid. Ex. Campylobacter

c. Spirochetes- they look like spirilla but their bodies are flexible and they wiggle while moving
about. The movement results from the contraction of an axial filament or flagellum which spirals
around the organism between the cell membrane and cell wall. Ex, Treponema pallidum-
causative agent of syphilis

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Materials:
1.broth culture of Escherichia coli, Bacillus subtilis, Staphylococcus aereus
2. Teeth and cheek scrapings
3.cotton swabs, toothpicks
3. Methylene blue dye,carbol fuschin
4. distilled water
5. glass slides and cover slip
6. wire loop, alcohol lamp, microscope

Procedure:

1.Preparation of Smears:

Fixed dried, and stained smears are often used in light microscopy to provide for greater
resolution of bacteria and of internal structures in larger organisms. Such treatment results in the
whole organism, or part of it, achieving a dramatic contrast to the unstained background.

1.Wash slide thoroughly with a detergent, rinse with water and dry with a clean cloth. A drop of
water placed on a clean slide will spread out evenly. If the drop rounds up, clean the slide again.
The slide would be thoroughly cleaned and freed of grease. Slide should always be held along
the edges to prevent recontamination with grease from the finger tips.

2. If the specimen comes from liquid culture, remove a loopful using sterilized inoculating/wire
loop. Spread the loopful evenly and thinly on a clean glass slide.There is no need to mix such
material with distilled water unless the culture or material is thoick , in which case dilution of the
preparation is preferable

3. If the source of your specimen is growing on solid media, place a loopful of sterile distilled
water in the center of your slide. With your sterilized wire loop, pick a small colony and
emulsify in the drop of distilled water on the slide.

4. Dry the smear by air. Fix smear by heat or by alcohol. In heat fixing, the slide is passed film-
side up quickly over the flame of the alcohol lamp 5 or 6 times. Do not scorch. The slide should
feel hot to the fingers, but not burn them. The alcohol treatment consists of covering the smear
with 95% ethyl alcohol for 1 minute, then draining off. Heat fixing is never used with
preparations made from milk.
The purpose of fixation is to kill the microorganisms, coagulate the protoplasm of the cell
and cause it to adhere to the slide.

5. If it is desired to preserve and file the smear, place a drop of Canada balsam or paraffin on the
cover slip.

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2.Simple Bacterial Stain

Prepare the following smears:

a.Teeth scraping
Remove some of the film from your teeth with a toothpick and spread on a clean slide as
thin,even film without water, Air dry. Fix by heat. Stain for 3-5 minutes with methylene blue.
Wash with water and air dry. The bacteria will be stained blue.

b. Cheek scraping
With a toothpick, scrape the inner surface of your cheek and make a smear. Air dry and fix by
heat. Stain with methylene blue for 5 minutes. Wash with water. Counterstain with carbol
fuchsin for 3 minutes. Wash with water and air dry.
The epithelial cells appear pink, while the bacterium Diplococcus salivarius should appear
dark blue

c. Culture of Bacteria
1.. Place a clean slide on the table and label each slide with the test organism.
2. Sterilize the wire loop by heating to redness the entire wire portion over the flame. Allow it to
cool before use.
3. Pick up a tube of bacterial culture with your free hand. Remove the cotton plug with the free
fingers of the hand holding the wire loop.
4. Heat the lip of the tube by passing it through the upper cone of the flame.
5. For organisms in liquid medium, insert the sterilized loop into the culture and withdraw a
loopful of broth. Place the loopful of culture onto the surface of the clean glass slide, spread it
over. Flame the lip of the tube and reseal with the cotton plug. Resterilize the loop, by passing
again over the flame.
6. For organisms on an agar plate, place a loopful of distilled water to the center of a glass slide.
Flame the loop . Lift the lid of the Petri dish just enough for the wire loop to enter. Transfer a
portion of bacterial colony to the water on the glass slide and close the Petri dish. Use the loop
to mix the bacteria and water together and spread them into the area. Sterilize the loop and return
to holder.
7. Allow the film to dry. To hasten drying, you may hold the slide about one foot above the
flame.
8. Fix the dried smear by passing the slide rapidly just over the flame. This prevents the smear
from being washed away and also kills the bacteria. Avoid excessive heating since it will alter
the composition of the organisms
9.. Flood the smear with methylene blue for 1 minute
10. Wash with tap water
11. Blot dry with filter paper
12. Examine under the microscope

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Results/Illustrations

Record the appearance of your stained bacteria indicating cellular morphology and cellular
arrangement for each organism observed.(5 pts each)

Specimen: Teeth Scraping (1000x magnification)

Arrangement: clostridium
Shape: Bacillus
Color: Pinkish Blue

bacteri
a

cheek
epithelium

Specimen: Cheek Scraping (1000x magnification)

Arrangement: Micrococcus, diplococcus and streptococcus
Shape: Coccus
Color: Blue

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Specimen: Specimen:
Arrangement: Arrangement:
Shape: Shape:
Color: Color:

Answer the following:

1.Why is methylene blue an appropriate dye for staining bacterial cells?(2 pts)
Methylene blue is a basic dye. It will readily give up a hydroxide ion or accept a hydrogen ion,
which leaves the stain positively charged. Since the surface of most bacterial cells and cytoplasm
is negatively charged, these positively charged stains adhere readily to the cell surface. After
staining, bacterial cell morphology (shape and arrangements) can be appreciated.

2. What is staining affinity?(2pts)

Staining affinity refers to the molecular attraction (or affinity) of a specific stain for a particular
type of cell component. an example would be the gram stain. the crystal violet has an affinity for
the molecules found in the cell wall of gram positive organisms, while safranin has an affinity for
the cell wall of gram negative organisms.

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Materials:
1.pure cultures of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas
aeruginosa; Streptococcus pyogenes ; oral smear/swab
2. staining solutions: crystal violet,Grams iodine, 95% ethyl alcohol; safranin
3. glass slides
4. wire loop
5. alcohol lamp
6. microscope
7. distilled water

Procedure:
1.Label the slide.
2. Make a thin smear of each of the pure cultures of bacteria
3.. Fix the smears over heat and stain as follows:
a. flood the slide with crystal violet for 3 min
b. wash with tap water for not more than 5 seconds or until no more stain comes off
c. flood the slide with Grams iodine and let stand for 2 minutes
d. decolorize by flooding with 95% ethyl alcohol until free stain has been washed off.
Allow it to stand for 30 seconds to 1 minute. Repeat this procedure until no more color
comes off with the alcohol . Decolorization may take place from 30 seconds to 2 minutes.
e.Wash again with tap water
f. counter-stain with safranin (carbol fuchsin or Bismark brown) for 30 seconds to 1
minute
f. wash with tap water for 5 seconds
g. blot dry with tissue
h. examine under the microscope
i. in the Results /Report Sheet section, sketch the morphology of and indicate the Gram
reaction with the color for each culture of bacteria used.

Note:The smear may seem to disappear after the process of decolorization.It is best to mark off
the smear by drawing a circle around it with marker on the side of the slide where the smear was
made. Bacteria retaining the original (violet) stain are Gram positive while those that are
decolorized by the alcohol and take up the counterstain (red ,pink or brown) are Gram negative.

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 (a) Crystal violet ; 30 se<:onds (b) Rinse for 5 se<:onds

(c) Cover with Gram'siodine; (d) Rinse with water for

60 seconds 5 se<:onds

(e) Decotorlze for 1-5 (f) Rinse with water for

se<:onds 5 seconds

(g) Counterstain with safranln; (h) Rinse for 5 seconds

30 se<:onds

( ) Blot dry with a paper towel

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Results:

Staphylococcus aureus (1000x) Pseudomonas aeruginosa (1000x)

(contaminated)

Escherichia coli Bacillus subtilis (1000x)

Oral smear (1000x) Streptococcus pyogenes (1000x)

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B.Acid-Fast Stain

The acid-fast stain is a differential staining procedure commonly used to stain Mycobacterium
and Nocardia organisms. This procedure divides bacteria into 2 major groups: the acid-fast
organisms (which retain the primary dye throughout the staining procedure) and the non-acid-
fast organisms (which are decolorized and then counterstained).

The Acid-Fast staining procedure was developed by Paul Ehrlich in 1882. He found that
tubercle bacilli (Mycobacterium tuberculosis) retained a dye reagent composed of crystal violet
and aniline in water even after a wash treatment with acidified ethanol solution.After the
development of this initial technique,changes in methodology resulted in the formation of the
Ziehl-Neelsen procedure.

The bacterial cell wall of acid-fast organisms contains a substantial amount of lipid material,
called mycolic acids.The lipid material is not only difficult to stain, but it appears to resist the
penetration of the primary dye to the underlying cellular cytoplasm. Because of this factor, long
staining time is required to stain the acid-fast organisms. However, once stained they are very
resistant to decolorization. Acid-fast organisms are also very resistant to drying. They remain
viable and infectious in a room environment for as long as 6 weeks, following contamination by
a patient having a disease like tuberculosis. Again, this characteristic appears to be related to the
wax-like lipid composition of the cell wall associated with acid-fast bacteria.

The 2 important acid-fast pathogenic organisms are Mycobacterium tuberculosis, the

causative agent of human tuberculosis; and Mycobacterium leprae, the causative agent of
leprosy or Hansens disease. Acid-fast staining of sputum from patients suspected to have
tuberculosis is routinely carried out and the presence of acid-fast bacteria is diagnostic.

These bacteria once they are stained, however, they retain the stain so that even acid alcohol
could not decolorize it. Hence, they are referred to as acid-fast. Non acid-fast organisms do not
resist acid alcohol decolorization and readily pick up the color of the counter-stain. In the
commonly used acid-fast staining method of Ziehl Neelsen, the primary stain used is carbol
fuchsin and the counterstain is methylene blue. Thus, acid-fast organisms stain red and non-
acid fast organisms stain blue.

Materials:
1. unstained sputum smears containing Mycobacterium sp.
2. staining solutions: Ziehl-Neelsen carbol fuchsin;acid alcohol (3% HCl +95% ethanol);
methylene blue

A.Ziehl-Neelsen Method

Procedure:
1.Place a square piece of filter paper approximately 2x2 cm over the bacterial smear
2. Saturate the filter paper with aqueous carbol fuschin solution
3. Steam the slide for 5 minutes over the low flame of a burner. Do not allow the stain to dry or
boil. Add stain if necessary or from time to time.

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4. Wash the slide with tap water.
5. Decolorize with acid alcohol until color no longer runs off the slide. This should take from 25
to 30 seconds. Repeat this until no more stain comes off with the alcohol.
6. Wash well with water.
7. Counterstain with methylene blue for 30 to 1 minute.
8. Wash with water, blot or air dry and examine under the microscope.

Note:Acid-fast organisms resist decolorization by dilute acids and retain the original stain.They
appear red against a blue background. Non-acid fast organisms are decolorized by dilute acids
and take up the counterstain. They appear blue like the rest of the smear.

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Acid fast: Bright red to intensive purple (B), Red, straight or slightly
curved rods, occurring singly or in small groups, may appear beaded
Non-acid fast: Blue color (A)

Mycobacterium tuberculosis visualization using the ZiehlNeelsen stain.

Source: Wikipedia
Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.
Non-Mycobacterial bacteria: Nocardia

Source:
www.microbiologyinfo.com/acid-fast-stain-principle-procedure-interpretation-and-examples/

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B.Kinyouns (Cold) Method

1.Prepare a bacterial smear.

2.Fix with gentle heat.
3.Stain with kinyouns carbol fuschin for 3 minutes. Do not heat.
4.Wash gently in running water.
5.counterstain with malachite green for 30 seconds
6. wash gently in running water
7. air or blot dry then examine under the microscope

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Results:
Draw the field of vision where you have observed the acid-fast organisms. Describe according to
their shapes, arrangements, color of the stained cells (10 pts)

ZEIHL-NEELSEN METHOD KINYOUNS METHOD

Specimen: Sputum Smear (x1000) Specimen: Sputum Smear (x1000)

Arrangement: Streptococcus Arrangement: Streptococcus
Shape: coccus Shape: coccus
Color: blue violet Color: violet (non-acid fast)

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Answer the following:
1.What is the significance of the presence of acid-fast bacteria in the sputum of a
patient? 5 pts.

The presence of acid-fast bacteria indicates the presence of mycobacterium.Acid-fast

bacteria in the sputum is most likely to be mycobacterium tuberculosis, indicating active
TB of the lungs. There is a possibility that it might be some other form of mycobacterium
causing lung infection, especially if the patient has a defective immune system because
of medication or chronic disease. Culture of the bacterium is necessary to distinguish
between mycobacterium tuberculosis and non-tuberculous mycobacteria.

2. List 2 human diseases caused by acid-fast bacteria. Give their pathogenecity.(10 pts)

A. Tuberculosis

Pathogenicity of Mycobacterium tuberculosis (Mtb) has been attributed to the

plasticity of its central carbon metabolism (CCM) machinery, which facilitates ready
adaptation of pathogen to the intracellular milieu of the macrophage. Emerging
evidence, however, suggests that Mtb pathogenicity is also supported by engagement
with metabolic pathways of the host cell. Thus while Mtb adaption to host requires the
switch in bacterial CCM towards catabolism of host lipid substrates, optimal exploitation
of this switch involves pathogen-induced promotion of lipid body (LB) accumulation by
the host macrophage. This ensures an abundant supply of the lipid substrates.

B. Leprosy

Mycobacterium leprae replicates intracellularly inside histiocytes and nerve cells and has
two forms. One form is tuberculoid, which induces a cell-mediated response that limits
its growth. Through this form M. leprae multiplies at the site of entry, usually the skin,
invading and colonizing Schwann cells. The microbe then induces T-helper
lymphocytes, epitheloid cells, and giant cell infiltration of the skin, causing infected
individuals to exhibit large flattened patches with raised and elevated red edges on their
skin. These patches have dry, pale, hairless centers, accompanied by a loss of
sensation on the skin. The loss of sensation may develop as a result of invasion of the
peripheral sensory nerves. The macule at the cutaneous site of entry and the loss of
pain sensation are key clinical indications that an individual has a tuberculoid form of
leprosy.

The second form of leprosy is the lepromatous form. This form of the microbe
proliferates within the macrophages at the site of entry. It also grows within the epithelial
tissues of the face and ear lobes. The suppressor T-cells that are induced are
numerous, however the epithelioid and giant cells are rare or absent. With cell-mediated
immunity impaired, large numbers of M. leprae appear in the macrophages and the
infected patients develop papules at the entry site, marked by a folding of the skin.
Gradual destruction of cutaneous nerves lead to what is referred to as "classic lion face".
Extensive penetration of this microbe may lead to severe body damage; for example the
loss of bones, fingers, and toes.

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