LABORATORY SAFETY RULES

The rules enumerated below shall be strictly enforced. The main objectives of
these sets of rules are to avoid dangers of infection that may arise from the neglect of
these necessary precautions. Each one must note that by neglecting any of these rules,
one not only put grave risk to himself but also exposes others to infection.
1. Read instructions carefully and thoroughly before coming to the laboratory. Know
what to expect to learn through each laboratory experiment and what you are
going to do in an experiment. This keep you informed and can prevent accidents
that occur when students are unprepared for laboratory. If you are in doubt about
correct procedures, double check the instructions and ask you laboratory
instructor.

2. Each student is obliged to wear a laboratory gown or coat while working in the
laboratory. This will be used in the Bacteriology Laboratory and properly kept in
the student’s locker. Gowns and coats should never be laid on the working tables.
With dirty, it should be properly wrapped before washing.

3. While working in the laboratory, avoid touching the mouth with the pencils, even
with your fingers and other materials used. DO NOT moistens labels with your lips or
tongue.

4. All accidents such as burns or abrasions and cuts as well as pillage of cultures and
breakages or loss of equipment should be immediately reported to the laboratory
instructor.

5. Eating and drinking are absolutely forbidden at all times in the laboratory. DO NOT
drink from the laboratory glassware. If one wishes to take a drink or snack, you are
at liberty to leave the laboratory for a short time, but before leaving, your hands
should be properly washed. Never go into an eating place wearing your coat or
laboratory gown.

6. Each group should provide themselves with a plastic cover/manila paper. At the
time beginning of the laboratory period, the plastic should be done on this plastic
cover. The contaminated surface should be immediately cleaned with
disinfectant solution.

7. All non-infectious solid wastes like paper, cotton, matchsticks, etc should be
placed in waste bags provided for that purpose. These are NOT to be discarded

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 on the table tops, sinks, much less on the floor. AT ALL TIMES, THE LABORATORY
SHOULD BE KEPT CLEAN AND NEAT.
8. Laboratory equipment which had been used in handling infective materials such
as test tubes, Petri dishes, beakers, flasks, pipettes should be discarded into discard
pans with disinfectant solutions.

9. Cultures either stock or those finished experiments should NOT be left on table tops
nor thrown into the sinks. They are to be returned immediately to the technician
for proper sterilization and disposal. Cultures are NOT to be taken out of the
laboratory without permission from the laboratory instructor.

10. LOITERING, MAKING UNNECESSARY NOISE AND BORROWING EQUIPMENT FROM

OTHER GROUPS WILL NOT BE TOLERATED.

11. At the end of the laboratory period, return all apparatus and microscopes to the
technician’s room, moisten your desktop with disinfectant and clean your
surroundings. Turn off any leaking gas or water outlets.

12. WASH YOUR HANDS THOROUGHLY WITH SOAP AND WATER.

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Experiment Microscopy: The Compound
1 Microscope

I. Objectives:
v The student must be able to recognized and know the function of the
different parts of the compound microscope and it’s usage in
microbiology laboratory.
v The student must be able to examine & visualize prepared slide under low
power objective, high power objective & oil immersion objective.

II. Introduction:
The microorganisms are miniscule organism which cannot be seen with
the naked eye. In order to visualize them, a tool called “the microscope” is
necessary.
The microscope is an essential tool for any microbiology laboratory and
one of the most important instruments in the study of microorganisms. It is
essential that the student should know the proper use of the microscope, its
different parts & functions. Bacterial identification and classification are based in
cell forms and structures visible only under high magnification and resolution.

III. Glossary of terms:

Magnification of the microscope is the product of the objective multiplied
by the magnification of the eye piece.
Resolving power refers to the ability to distinguish between two adjacent
points.
Low power objective (LPO) 10x objective power and 100x magnification.
High power objective (HPO) 40x objective power and 400x magnification.
Oil-immersion objective (OIO) 100x objective power and 1000x
magnification.
Parfocal is a property of the microscope where the objectives are so
adjustment and are fitted so that if even either objective is swung into
place, the image remains in focus or nearly focused as to require only fine
adjustment manipulation.

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IV. Materials:
1. Compound microscope
2. Prepared Slides
3. Xylene or xylol
4. Cedarwood oil

V. Procedures:

A. Use & Care of Microscope

The student can only really benefit from the use of microscope if he knows
very well the parts of the microscope, its capacity and limitations, its correct
operation and its proper use and care.

The following are important points to observe whenever using the microscope.

1. Carry the microscope with two hands—one hand grasping the handle and the
other hand supporting the from under. DO NOT carry the microscope with only
one hand, much less, swing it back and forth as you go to the table. The eye
piece and other parts may fall out or the microscope may hit a table or chair.
Avoid sudden jarring when you place the microscope on the table.

2. Always use the microscope with the tube in the perpendicular position. This is
to be strictly followed in the working with fresh mounts, hanging drop
preparations and smears being examined under the OIO. The oil and other
fluids of wet mounts tend to flow into the stage in the titled position, dirtying the
stage if NOT actually contaminating it.

3. Keep the microscope free from the dust at all times. One must acquire the
habit of cleaning the microscope before and after using it. The body if the
microscope is wiped cleans with a piece of clean dry cloth while the lenses are
wiped clean with the use of lens paper. If any of the objectives or the stage is
smeared with oil, use lens paper with xylene. An excess of xylol may, however,
dissolve cement and loosen other parts of the microscope.

4. Carry the microscope with two hands – one hand grasping the handle and the
other hand supporting the from under. DO NOT carry the microscope with only
one hand, much less swing it back and forth as you go to the table. The eye
piece and other parts may fall out or the microscope may hit a table or a chair.
Avoid sudden jarring when you place the microscope on the table.

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 5. Always use the microscope with the tube in the perpendicular position. This is
to be strictly followed in the working with fresh mounts, hanging drop
preparations and smears being examined under the OIO. The oil and other
fluids of wet mounts tend to flow into the stage in the titled position, dirtying the
stage if NOT actually contaminating it.

6. Keep the microscope free from dust at all times. One must acquire the habit
of cleaning the microscope before and after using it. The body of the
microscope is wiped clean with a piece of clean dry cloth while the lenses are
wiped clean with the use of lens paper. If any of the objectives or the stage is
smeared with oil, use lens paper with xylene. An excess of xylol may, however,
dissolve cement and loosen other parts of the microscope.

7. Illumination- successful microscopic work depends a great deal on the

adjustment and control of the illumination. Adjustment of light consist of
reflecting light form the best source with the reflecting mirror, lowering or raising
the condenser and opening or closing the diaphragm to control the amount
of light required for different kinds of objects for visualization under the different
objective.

Unstained or fresh preparations and visualization with the lower objectives does
NOT require the minimum amount of light unlike visualization under the HPO
and OIO. If close to a window anti utilizing daylight as source of illumination,
the use of the concave mirror becomes imperative using artificial light as the
source of illumination, the plain mirror may be used for work for the higher
magnification.

8. The course adjustment screw is only used to obtain an approximate focus and
the fine adjustment screw fails to function. DO not force it for it may have been
screwed so far and reached its limit. To read just use the course adjustment to
get an approximate focus and then turn the fine adjustment screw until it is
midway within its range. Use the fine adjustment for fine focus.

9. Focusing the objectives:

a) LOW POWER OBJECTIVE (LPO)- intended to have a general view of an object.

i. Place the specimen or slide under the clips. Swing the LPO into place.

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 ii. Using the coarse screw, lower the objective until it is about ¼ inch from
the top surface of the specimen on the slide. Looking through the eye
piece, slowly raise the objective using the coarse adjustment screw until
the object becomes more or less distinct. For better focus, use the fine
adjustment.
iii. It is a safe part to use LPO when one begins a microscopic work showing
a larger portion of the specimen thus facilitating the choice of parts to
be examined under the HPO.

b) HIGH POWER OBJECTIVE (HPO)- intended to have specific object.

B. Microscopy
1. Adjust the amount of light received by the microscope using the correct
mirror (plane mirror for bright light and concave mirror for poor or artificial
light) opening or closing the iris diaphragm and/ or raising or lowering the
condenser.
2. Place the slide to be examined on the stage and apply the clip on the
end.
3. Focus the object using the LPO. Raise the LPO slowly, using the coarse
adjustment know until the object is brought into the focus. Study and draw
the object.
4. Shift to the HPO. Making a few turns with the fine adjustment knob will
focus the object. This is Possible because the microscope is par focal.
Study and draw the object.
5. The object may then be examined using the OIO. Place a drop of Cedar
wood oil over the object. Shift to the oil immersion lens and focus the
object using the fine adjustment knob.

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VI. Observations:
1. Draw and label the parts and function of the microscope.

2. Draw the image of the specimen in the prepared bacterial slide.

LPO HPO

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Experiment Preparation of Bacterial
2 Smear

I. Objectives:

v The student must be able to prepare a slide with a bacterial

inoculums/colony.
v The student must focus the prepared smear using oil-immersion
objective.
v The student must be able to describe & visualize accurately the
morphology of the microorganism as viewed under the microscope.

II. Introduction:

In their natural state bacteria, yeast, molds, protozoa, ricketsiae, and

PPLO (Pleuro-pneumonia-like organism) except viruses, appear under the
microscope as tiny, colorless, translucent spheres, rods or spirals that are difficult
to see clearly. In order to see them distinctly and study them closely, some
pretreatment is done to the cells. These include preparing, fixing, & staining
bacterial smear before viewing under a compound microscope.

III. Glossary of terms:

Bacterial smear: a thin film of bacteria spread in the surface of the slide.
Heat fixation: manner of permanently placing the microorganism to a slide in
order to be viewed under the microscope.
PPLO: any group of bacteria that lacks cell wall can survive without the
presence of oxygen. It usually associated with pneumonia & urinary tract
infection.
Staining: an auxiliary technique employed in microscopy to enhance contrast
in the microscopic image which aid in the identification of the unknown
organism.

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 IV. Materials:

1. Bacterial suspension or colony 4. Alcohol lamp

2. Slide 5. Gum label
3. Wire loop 6. Used newspapers/manila papers

V. Procedures:

1) Get a clean glass slide and gently heat one side to remove any grease.
Slide should be held along the edges to prevent recontamination with
the grease from the fingertips. Lay the slide on the table with the
flamed side up.
2) Sterilized wire loop until it is red hot and allow it to cool.
3) Place a drop of sterile distilled water or 0.85% NSS on the center of the
slide.
4) Pick a small colony of organisms from a solid media with the use of
sterilized wire loop and emulsify in the drop of distilled water or 0.85%
NSS of the slide.
Note: Smears made from liquid media are directly spread on the slide.
To do this, take one or two loopfuls of the culture and spread over an
area of 1 inch by ½ inch. If otherwise, the culture of material is thick,
there is a need to dilute the preparation prior to smearing.
5) Air-dry by laying the prepared smear on the table.
6) Heat fix the smear by passing on the slide (with the smear upside up)
over the flame 5x or 6x, then allow it to cool. Fixation of the smear causes
the preparation to adhere to the slide and will not be easily washed off
during staining process.
7) Stain the preparation with the desired staining method.
8) Wash off the excess stain with top water.
9) Air or blot dry with filter paper.
10) Place a drop of immersion oil on the smear and examine under the OIO.

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 VI. Observations:

1. Draw a proper labeled smear.

2. Draw the appearance of the microorganism under the Oil-immersion

objective.

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VII. Questions:
1. Give some precautions in the preparation of bacterial smear.

2. Give the reasons why you have to flame sterilize the inoculating loop before picking
up a colony from the stock culture.

3. What are the reasons for flaming the mouth of the culture tube after the cotton plug
has been removed and before it is reinserted?

4. Why do you have to pass the bacterial smear four to five times over the flame before
staining?

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 Experiment Microscopy: Staining Methods
3 A. Simple Staining

I. Objectives:

v The student must be able to know and perform the method and techniques
of bacterial isolation & identification.
v The student must be able to know the basic concepts behind staining
procedures.
v The student must be able to prepare bacterial smears from a bacterial
suspension or stock cultures without error.
v The students must be able to describe the microorganism’s morphology after
staining.

II. Introduction:

Bacteria and other microorganisms are usually transparent which makes

the study of its morphologic details difficult when they are examined in the
natural state. The pretreatment of fixing and following staining allows the
microbiologist to distinguish many structural features of microorganisms that is not
formerly seen. In the staining process, it uses staining dye of different colors, thus
the color of the organism will depend upon the stain used. Basic staining dyes
are the simplest stain used for the bacteria due to its high affinity.

III. Glossary of terms:

Staining: it is the process of artificially coloring the microorganisms with dyes in

order to visualize accurately their morphological characteristics under the
microscope.
Culture: it contains microorganism that is artificially grown in the culture
media.
Culture media: it is an artificial environment that is suitable for keeping the
microorganism in a viable condition.
Aniline dyes: chemical reagents which bind to cellular components of the
microorganism rendering them visible under the microscope.

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 IV. Materials:

1. Bacterial suspension or colony 5. Alcohol lamp

2. Methylene blue (aq) 6. Wire loop
3. Carbolfuchsin (aq) 7. Gum label
4. Glass slides 8. Used newspapers/manila papers

V. Procedures:

1. Prepare two smears from the bacterial suspension or colony and label it as 1
& 2.
2. Heat-fix the prepared smear and allow it to cool.
3. Take the first smear and cover the smear with aqueous Carbol fuchsin but use
only sufficient amount of the stain to cover the smear, not the entire glass
slide.
4. Allow the stain for 1 minute.
5. Wash the slide with tap water and air to blot dry.
6. Repeat the above procedure using the aqueous methylene blue stain for the
second smear.
7. Place the drop of cedarwood oil on the smears and examine under oil
immersion objective (OIO).
8. Draw and label your observations.

VI. Observations:

Illustrate and label your results and observations.

Slide 1 Slide 2

Describe the complete morphology of the organism on the basis of its simple staining
reaction.

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Slide #1: Slide #2:

Result: ________________________________ Result: ________________________________

Name of Organism: ___________________ Name of Organism: ___________________

VII. Questions:
1. Give the advantages of simple-stained preparations.

2. Give the causes of error in simple staining procedure.

 Experiment Microscopy: Staining Methods
3 B. Gram Staining

I. Objectives:

v The students must able to know and perform the methods and techniques
bacterial isolation & identification.
v The students must able to know the principle involved in Gram staining.
v The students must able to enumerate the reagents in Gram stain and give
the purpose of each.
v The students must able to classify the bacteria as to Gram positive or
Gram negative.

II. Introduction:

In 1884, Hans Christian Gram, a Danish physician devised as staining procedure

that can divide all the true bacteria into two physiological groups and it is called as
GRAM STAIN.
Gram stain is valuable diagnostic tool in differentiating the microorganism into
two groups, gram positive and gram negative based on its cell wall composition. Gram
positive bacteria retain the primary stain, crystal violet resulting to purple color due to its
high content of peptidoglycan and the presence of techoic acid on the cell wall while
gram negative bacteria lose primary stain when decolorized, thus taking up the color of
the counter stain, safranin and appear pink to red in color.

III. Glossary of terms:

Gram positive microorganisms: organism which retain the color of the

primary dye even after decolorization.
Gram negative organism: Organisms which do not retains the color of the
primary dye after decolorization and take the color of the secondary dye.
Cocci: spherically-shaped microorganisms.
Bacilli: rod-shaped microorganisms.

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 Mordant: auxiliary regent added during the staining process which serves
as a bridge to crosslink to the cell wall of the bacteria resulting to intensify
color of the primary dye.
Primary dye: first coloring reagent added to the smear to impart
violet/purple color to the microorganism.
Secondary dye: second agent added to the smear to impart red to pink
color to the microorganism.
Decolorizer: reagent added to the smear during the staining process
which removes the color rendered by the primary dye.

IV. Materials:

1. Bacterial suspension or colony 6. Glass slides

2. Primary stain - Crystal violet 7. Alcohol lamp
3. Mordant - Gram’s iodine 8. Used manila papers/newspapers
4. Decolorizer - 95% alcohol 9. Wireloop
5. Secondary stain – Safranin 10. Gumlabel

V. Procedures:

1. From the bacterial suspension given, prepare two bacterial smears.

2. Cover ach entire smear with a few drops of the primary stain, crystal violet
for one minute
3. Wash off excess stain gently with running water until no more stain comes off
4. Cover the smear with the mordant, Gram’s iodine for one minute
5. Wash off the iodine with tap water
6. Decolorize the smear by flooding with 95% alcohol. Allow it to stand for 15-
30 seconds. Repeat this procedure until no more color comes off with the
alcohol.
7. Wash again with tap water.
8. Counter-stain with Safranin for 30 seconds.
9. Wash off the excess stain with tap water. Air or blot dry.
10. Place a drop of immersion on the smear and examine under the oil
immersion objective (OIO).
11. Draw and label your observations

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 VI. Observations:

Illustrate and label your results and observations.

Slide 1 Slide 2

Describe the complete morphology of the organism on the basis of its simple staining
reaction.

Slide #1: Slide #2:

Result: ________________________________ Result: ________________________________

Name of Organism: ___________________ Name of Organism: ___________________

VII. Questions:

1. Give the purpose of the following reagents:

a. Crystal violet

b. Gram’s iodine

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 c. 95% Ethyl Alcohol

d. Safranin

2. What is the step in Gram stain which can be omitted and still allow
differentiation of Gram negative from Gram positive organism?

3. List three genera that are Gram negative cocci.

4. List three genera that are Gram negative bacilli.

5. What cellular structure is responsible for the difference in the Gram

staining reaction of microorganisms?

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Experiment Microscopy: Staining Methods
3 C. Acid Fast Staining

I. Objectives:

v The student must be able to know the principle involved in acid-fast staining.
v The student must enumerate the reagents used in acid-fast staining and give
the purpose of each.
v The student must be able to classify bacteria as acid-fast or non-acid fast
organisms.

II. Introduction:

Some bacteria are not readily stained by the Gram staining procedure,
therefore a more rigorous staining procedure may be required using more
concentrated biological dyes and longer staining time which is the ACID FAST
STAIN.
Acid fast staining is differential staining procedures commonly use to stain
organism which have a mycolic content in their cell wall. The acid-fast organisms
resist depolarization with acid alcohol due to its mycolic content in the cell wall
thus retaining the primary stain, carbol fuchsin resulting to red color while non acid
fast organisms on the other hand, are easily decolorized with acid alcohol hereby
taking up the color of the counterstain, methylene blue or malachite green.
Heat and/or solvents acts as mordant are needed to drive the stain into the
cell wall of the acid fast organism. Acid fast organisms are hard to stain but one
stained, difficult to decolorize.

III. Glossary of terms:

Acid-fast: organisms which are not decolorized by acid alcohol once they
have been stained resulting to red color.
Non-acid fast: organisms which are decolorized by acid alcohol after they
have been stained resulting to blue or green color.

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 IV. Materials

1. Sputum sample 6. Applicator sticks

2. Aqeuous carbol fuchsin 7. Glass slides
3. Acid alcohol 8. Gumlabel
4. Methyline blue 9. Used newspapers/manila papers
5. Alcohol lamp

V. Procedures:

1. SMEAR PREPARATION

Procedure:
1. Using an applicator sticks, spread a small amount of the specimen on
the slide, making a thin oval film about 1-2cm x 2-3 cm in size. Air dry and
heat fix the smear.
2. Perform either Ziehl-Neelsen or Kinyoun method (whichever staining
protocol is available)

2. ZIEHL-NEELSEN’S METHOD (HOT METHOD)

Procedure:
1. Prepare a smear using sputum sample.
2. Flood the entire slide with carbol fuchsin and pass over low flame. Steam
gently. Do this for 3-5 minutes.
3. Rinse the smear with tap water.
4. Decolorize the smear by adding with acid alcohol until pink/red color
disappears
5. Gently rinse with tap water.
6. Flood the entire slide with methylene blue for 60 seconds
7. Rinse and air dry.
8. Examine stained slide under OIO
9. Draw and label your observations.

EXPECTED RESULT:

ACID FAST = red bacilli against a blue background

NON-ACID FAST = blue bacilli against a blue background

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 3. KINYOUN’S METHOD (COLD METHOD)

Procedure:
1. Overlay the smear with Kinyoun’s carbolfuchsin reagent for 5 minutes
2. Rinse the smear with tap water
3. Decolorize the smear with acid-alcohol for 3 minutes or until the red
color is washed away. Rinsed the smear immediately.
4. Over the smear with methylene blue for 30 to 60 seconds
5. Washed the smear with distilled water, blot, and allow to air dry.
6. Examine it under OIO.
7. Draw and label your observations.

RESULT:
ACID FAST = red bacilli against a blue background
NON-ACID FAST = blue bacilli against a blue background

VI. Observations:

Illustrate and label your results and observations.

Describe the complete morphology of the organism on the basis of its simple
staining reaction.

Method: ______________________________
Result: ________________________________
Name of Organism: ___________________

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 VII. Questions:

1. What substance is responsible for the acid-fastness of an organism?

2. Give two genera of microorganisms which are acid-fast.

3. Differentiate Ziehl-Neelsen and Kinyoun method.

4. Why is carbol fuchsin not allowed to dry out or boil?

5. What cellular structure is responsible for the difference in the Gram staining
reaction of microorganisms?

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Experiment Microscopy: Staining Methods
3 D. Special Staining

I. Objectives:

v The student must be able to know and perform the methods and techniques
of isolation and identification of bacteria.
v The student must able to know the principle involved in the special staining
techniques.
v The student must be able to perform and familiarized with special staining
techniques and procedures.
v The student must be able to demonstrate the bacterial structures using
special stains.

II. Introduction:

Different species of bacteria have different features that are often helpful
in identifying them under the microscope. The demonstration of the structures
however, depends on the type of stain used. Different stains can be used to
look for the presence of such structures.
These special bacterial structures include spores, capsule, flagella, and
granules.

III. Glossary of terms:

Special staining: this technique demonstrates the different structures within

or outside the bacterial cell wall.
Endospore: spore formed within the cell by some species of bacteria.
Flagellum: thin and fragile hair-like appendages that protrude through the
cell wall responsible for motility.
Capsule: slimy layer or gelatinous substance forming a layer around the cell.
Negative staining: staining method which stains the background rather than
the bacterial cell.

IV. Materials

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 1. Bacterial suspension or colony of S. pneumoniae, K. pneumoniae, C.
diphtheriae, B. subtilis, E. coli
2. Crystal Violet
3. 20% Copper sulfate solution
4. Loeffler’s alkaline methylene blue stain
5. Albert’s stain
6. Gram’s iodine
7. 5% acetic acid
8. Carbolfuchsin
9. 5% aqueous malachite green
10. 0.5% aqueous safranin
11. 10% aqueous solution of nigrosin
12. 5% Alcoholic iodine solution

V. Procedures:

CAPSULAR STAIN

A. HISS METHOD
Procedure:
1. From your bacterial suspension or colony, prepare a bacterial smear.
2. Air dry and don’t heat fix.
3. Cover the entire smear with crystal violet for 4-5 minutes.
4. Wash off the excess stain with a 20% aqueous solution of Copper sulfate.
5. Air or blot dry examine under OIO.

NOTE:
The crystal violet is used here as a contrast stain.

EXPECTED RESULT:

CAPSULE = clear or halo around the bacterial cell;

BACTERIA = violet (like the background)

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 METACHROMATIC GRANULES STAIN

This method of staining is significantly used to demonstrate the

metachromatic granules of Diptheria bacillus.

A. L.A.M.B METHOD
Procedure:
1. Make a thin smear from a saline suspension C. diptheriae
2. Cover the smear with a few drops of Loeffler’s alkaline methylene blue for 5
minutes. Wash off the excess stain with tap water.
3. Air dry and blot dry and examine under OIO.

RESULT:
Bacteria will appear blue with both its polar ends as darker blue.

B. ALBERT METHOD
Procedure:
1. Prepare the smear.
2. Heat-fix and allow to cool.
3. Flood the smear with Albert’s stain for 2-15 minutes.
4. Wash with tap water.
5. Flood the smear with Gram’s iodine for 1 minute.
6. Wash with tap water.
7. Air dry and examine

EXPECTED RESULT:

GRANULES = blue to black

BANDS = blue to blue
CYTOPLASM = green

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 SPORE STAINS

A. ACETIC ACID METHOD

Spores are generally hard to stain but once stained, they are likewise
difficult to decolorize. Hence, special stains have to be made to show these
structures.

Procedure:
1. Make a thin smear. Air dry and heat fixed.
2. Steam with carbol fuchsin for 5 minutes.
3. Decolorize with 5% Acetic acid until the firm assumes a light pink color.
4. Stain with Loefflers alkaline methylene blue for 3 minutes.
5. Wash off the excess stain with tap water.
6. Air dry and examine under the oil immersion objectives.

B. WIRTZ-CONKLIN METHOD

Procedure:
1. Flood the entire smear with 5% aqueous malachite green.
2. Steam for 3-6 minutes
3. Rinse under running tap water
4. Counterstain with 0.5% aqueous safranin for 30 seconds.

EXPECTED RESULT:

SPORE = green spherules

BACTERIA = red

C. DORNER’S METHOD

Procedure:
1. Make a heavy suspension of the organism in a test tube.
2. Add an equal amount of freshly filtered carbol fuchsin.
3. Place the tube in boiling water for 5-10 minutes.
4. Mix a loopful of the above with one loopful of boiled and filtered 10%
aqueous solution of Nigrosin on a clean side.
5. Spread mixture and dry quickly gentle heat.
6. Examine under OIO.

EXPECTED RESULT:

SPORE = red
BACTERIA = colorless against a dark gray background

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 POLYSACCHARIDE STAIN

Procedure:
1. Make a smear, air dry and fix by heat.
2. Apply iodine solution for 1 minute
3. Wash with water and blot dry.
4. Other than yellow color stained material in the bacteria is indicative
of polysaccharide, Glycogen stains reddish brown.

VI. Observations:

Illustrate your results and observations.

A. Capsular Stain (Hiss method)

B. Metachromatic Granules Stain

B.1 LAMB B.2 ALBERT

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C. Spore Stains

C.1 Acetic Acid Method C.2 Wirtz-Conklin Method C.3 Dorner’s Method

VII. Questions:

1. What are the different types of spores as to their location on the bacterial
cell?

2. Classify bacterial cells according to the location number of flagella.

Illustrate each type.

3. Give the capsule’s chemical composition and its significance.

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 Experiment
Preparation of Culture Media
4
I. Objectives:

v The students must be able to accurately prepare the different types of

culture media according to consistency and manner of formation
v The students must correctly dispense the culture media into test tubes and
petri dishes.
v The students must identify the characteristics of the different types of
culture media

II. Introduction:

The study of microorganisms requires techniques for isolating cells from

natural sources and growing them in the laboratory on artificial media. Thus,
developments of culture media and culture techniques had played important roles
in the diagnosis of disease and advancement of the microbiology. Microbiologists use
bacterial culture media for many purposes and applications.
Media are used to isolate and identify bacteria, reveal their colonial
morphology and allow long-term storage of pure cultures. Taxonomic descriptions of
bacteria commonly include information about their cultural requirements; species
that are poorly characterized are frequently those most difficult to culture under
laboratory conditions.
Indeed, Koch’s second postulate requires culturing of a suspected pathogen
in pure form. Knowledge about the composition and types of culture media and how
different types of media can be use is essential as part of studying the properties of
bacteria.

III. Glossary of terms:

Culture media: serve as an artificial environment in which the bacteria are

planted for purposes of laboratory study such as demonstrating particularly
biochemical activities and/or physiological properties of microorganisms.

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CLASSIFICATION OF CULTURE MEDIA ACCORDING TO:
1. PHYSICAL STATE
A. Solid Media – with 2-3 solid agar
B. Semisolid media – with 1% solid agar motility determination
C. Liquid media – with NO solidified gelatin or agar

2. COMPOSITION
A. Synthetic media – exact chemical composition of the ingredient is KNOWN
examples: Ringers solution Loeke’s solution
B. Non-synthetic media – precise chemical composition of some or all of nutritive
supplement is NOT known.
examples: Meat extract broth, vitamin agar
C. Living Tissue Media – with living tissue cells used for cultivation or Rickettsiae and
Viruses
examples: Embryonated egg, Maitland’s tissue culture, tissue plasma roller tube.

3. USE
A. Simple media – an ordinary media with NO enrichment materials for growing non-
fastidious organisms.
- For general laboratory purposes
examples: Nutrients aga/broth, plain agar
B. Enriched media – solid media with nutritive supplements for growing fastidious
organisms.
C. Enrichment media – liquid media with nutritive supplements for growing fastidious
organism.
examples: Selenite broth, Alkaline peptone water
D. Differential media – differentiate and identify microorganisms.
example: MacConkey
E. Selective – growth of particular microorganism while inhibiting the growth of
undesired.
-specific and/or special media such as this follows:
a) Loeffler’s blood serum – Diptheria bacillus
b) Cooked meat – anaerobes like clostridia
c) Lowenstein Medium – tubercle bacilli

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 IV. Materials

1. Erlenmeyer flasks
2. Analytical balance
3. Spatula
4. Wire gauze
5. Electric Stove
6. Autoclave
7. Graduated cylinder
8. Stirring rod
9. Test tubes
10. Petri dish
11. Pipette
12. Aspirator
13. Dehydrated media: Mueller Hinton Agar, Brain heart infusion broth, SIM
medium

V. Procedures:

1) Weigh the desired amount of dehydrated culture media and transfer

them to suitable containers. Use the Erlenmeyer flask for the plated
media and the beaker for the tubed media.
2) Add the appropriate volume of distilled water to the medium (If paper
was used in weighing the culture medium, let the water run down the
sides of the paper to remove the particles that cling to the paper.
3) Dissolve the medium-containing agar by heating and constabtly stirring
(discontinue as soon as the solution becomes clear). This process is not
necessary when preparing a liquid medium.
4) After dissolving, place the exact amount of solution in an appropriate
container. For tubed media, use test tubes and plug the mouth of the
tubes with cotton.
5) After plugging the test tubes with cotton, the culture media are
generally sterilized by autoclaving at 121C for 15-20 minutes/15psi
6) After autoclaving, prepare the following forms of culture media.
a. Slant: Solidy the agar in the test tube in a slightly raised position
b. Butt-slant: Solidify the agar in the test tube in a slightly raised position
at an acute angle.
c. Butt: Allow the agar in the test tube to solidify in a vertical position

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 d. Liquid: Leave the test tubes containing the liquid medium in a vertical
position.
e. Plated medium: Pour the agar from the Erlenmeyer flask into several
sterile Petri dishes and allow the agar to solidify on a flat surface.

Note: All materials to be sterilized such as the tubesd media as well as

the prepared culture must be placed in a metal basket or bucket and
put in the chamber.

VI. Computations.

A. Solid Medium

B. Liquid Medium

C. Semi-Solid Medium

VII. Questions.

1. Illustrate the types of culture media according to manner of formation.

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 2. Give examples of culture media classified as follows:
a. Physical state:

b. Manner of formation:

c. Composition:

3. Give an example of differential and selective culture media and state the
purpose of each incorporated substance.

4. Give a classification of culture media according to function and state their

corresponding use.

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Experiment Control of Microbial Growth
5 A. Physical Methods: Heat

I. Objectives:

v The students must be able to perform control of microbial growth by

physical means
v The students must have the knowledge about the mechanism of action of
moist heat, dry heat & direct flaming
v The students must identify the different means of sterilization by physical
methods

II. Introduction:

Sterilization is done by either physical or chemical means. Physical means

of sterilization includes Incineration, moist heat, dry heat, filtration & ionizing radiation.
During this process hazardous organism including their spores are killed. Incineration is
performed by literally burning the hazardous material into ashes at temperature of 80
to 980C while moist heat was performed in the form of saturated steam under pressure
which causes irreversible denaturation of enzymes and structural proteins. Dry heat
ovens are used to sterilize item such as glasswares, oil, or powders and Filtration is a
method of choice for antibiotic solutions, toxic chemicals, radioisotopes, vaccines
and carbohydrates. Last is the ionizing radiation which is used for sterilizing disposables
such as plastics syringes, catheters or gloves before use.

Culture media should be sterilized prior use to ensure that proper isolation and
subsequent identification of microorganisms is possible in the laboratory without any
resulting error. Media are BEST prepared frequently in small amounts so that the period
of storage is kept at a minimum. When large amounts are to be kept on hand, it should
be stored so as to prevent evaporation and usually kept in an icebox or cold room.

Finished media should be checked for sterility by incubation. A representative

sample is incubated overnight and examined for growth. This is especially important
for mixtures containing ingredients NOT autoclaved such as blood agar plates.
III. Glossary of terms:

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 Sterilization: the process whereby all forms of microbial life, including
bacterial spores are killed. It can be accomplished by physical or chemical
methods
Disinfection: process whereby pathogenic organisms, but not necessarily all
microorganism or spores are destroyed.
Moist Heat: also known as “steam under pressure”. Eg. Autoclave
Incineration: is the most common method of treating infectious waste
wherein the hazardous material is literally burned to ashes at temperate of
870 to 980C
Dry heat: serve as an artificial environment in which the bacteria are planted
for purposes of laboratory study such as demonstrating particularly
biochemical activities and/or physiological properties of microorganisms.

IV. Materials

1. Autoclave
2. Bunsen burner
3. Hot air oven
4. Wire loop
5. Forceps

V. Procedures:

A. Moist Heat: Autoclave

1. Close the drain valve behind the drain mouth located beside the drain tank
2. Turn the exhaust known (exhaust valve) clockwise until it is closed
3. Open the lid and fill the chamber with water until the pupe heater is
submerge up to the level of the drain board.
4. Place the object to be sterilized in a basket or bucket. Put it in the chamber,
close the lid, and turn the hand wheel clockwise to tighten it.
5. Switch on the breaker
6. Set the timer to the appropriate sterilization time
7. Press the “start” button. The pilot light (“sterilize”- green button) goes on. The
heater is energized by the power and the temperature in the chamber rises.
Until the temperature of the automatic air exhaust valve shut (about 101C)
is reached, the exhaust valve is kept open and air with steam moves out into
the exhaust drain tank

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 8. After the air exhaust valve is close, the temperature and pressure rises
further, and the timber begins to move when the set temperature (pressure)
– 121C for 15 minutes at 15lbs (per square inch) – is reached. The small round
red mark moves down to show the remaining sterilization time. To maintain
the temperature/pressure at the set figures (red zone) during sterilization, the
heater automatically repeats switching ON-OFF. To decrease the
temperature/pressure, turn the pressure control known to the right (high) or
to the left (low). After controlling this knob, it is unnecessary to touch it during
sterilization at the same temperature/pressure.
9. When the set sterilization time is completed, note that the buzzer sounds,
and the pilot light goes off.
10. Turn the exhaust knob (exhaust valve) to the left, Steam inside the chamber
will be released. If the exhaust known is left closed after the operation is
completed, the temperature/pressure in the chamber gradually decreases
due to natural cooling. This is recommended especially when sterilizing
liquids in bottles.
11. Wait until pressure goes to the “0”. Open the lid and remove the object with
care. Watch out for plumes of steam.
12. If the water level in the drain tank reaches the high level mark, drain excess
water until it reaches the love (minimum) level mark.

B. Dry Heat: Hot Air Oven

1. Wrap all clean, empty and dry petri dishes in aluminum foil before placing
them in the oven.
2. Raise the temperature to 160C to 180C
3. Maintain the temperature for on and a half to three hours
4. Allow the temperature to go down before opening the oven.

C. Incineration

1. Inoculating loops, needles, and forceps are sterilized by flaming the entire
length of the nichrome wire or platinum wire and the top of the forceps
until they are red-hot.

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 VI. Questions.

1. Define sterilization.

2. Compare the effectiveness of autoclaving and boiling.

3. Explain the mechanism of action of dry heat and moist heat on bacteria.

4. What is the danger in using direct flaming as a method of microbial control?

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Experiment Control of Microbial Growth

5
B. Chemical Methods: Disinfectants &
Antiseptics

I. Objectives:

v The students must be able to perform control of microbial growth by

chemical means
v The students must have knowledge regarding the mechanism of action of
the several disinfectants and antiseptics
v The students must identify the difference between disinfectants from
antiseptics and bactericidal from bacteriostatic agents

II. Introduction:

Disinfection is a process whereby pathogenic organisms, but not

necessarily al microorganisms or spores are destroyed. It can be accomplished by
physical & chemical means. Physical means includes Boiling at 100C for 15 minutes,
Pasteurizing at 63C for 30 minutes or 72C for 15 seconds and using non-ionizing
radiation. Chemical means includes Alcohols, aldehydes, halogens, heavy metals,
quarternary ammonium compounds and phenolics.

Working table must be disinfect before and after use but several factors
influence the activity of disinfectants such as type of organism present, temperature
and pH, microbial load or the number of organisms present, concentration of
disinfectant, amount of organics present, length of contact time and etc.

Chemical disinfectants that are used on living tissue like skin are called
antiseptics. Antiseptics are similar to disinfect but differ in the manner of action for it
mainly inhibit the growth of microorganism in living tissue while disinfects inhibit
organism in inanimate objects. Antiseptic includes alcohols, anilines, iodine and
iodophors, quaternary ammonium compounds and etc.

III. Glossary of terms:

Sterilization: the process whereby all forms of microbial life, including

bacterial spores are killed. It can be accomplished by physical or chemical
methods

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 Disinfection: process whereby pathogenic organisms, but not necessarily all
microorganism or spores are destroyed.
Antiseptics: disinfectants used on living tissue as way of inhibiting
microorganisms
Biocides: are chemicals used to destroy all microbial life in the process
.

IV. Materials

1. Lysol – liquid concentrate and spray

2. Alcohol – 70% isopropyl or ethyl alcohol
3. Gloves
4. Rags

V. Procedures:

A. Lysol

Lysol is used as a disinfectant in killing microorganism in cultures or other

inanimate objects or environmental surfaces. Liquid Lysol Is placed on the
surface of bacterial culture to be discarded and allow to stand for 10 minutes
prior discarding. It may be applied or sprayed on pre-cleaned surfaces of
warking tables until the entire surface are misted. Air-dry surfaces for around 10
minutes.

B. Alcohol

Isopropanol or seventy percent ethyl alcohol is used as skin antiseptic in killing

microorganism on the skin surfaces. Rub skin briskly with 70% alcohol and air dry for
1-2 minutes.

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 VI. Questions.

1. Define the following terms:

a. Antiseptic

b. Disinfectant

c. Bactericidal

d. Bacteriostatic

2. Enumerate chemical agents that are bactericidal or bacteriostatic

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Experiment Control of Microbial Growth
5 C. Hand washing

I. Objectives:

v The students must be able to perform proper hand washing

v The students must have knowledge regarding the standard precaution to
observe during hand washing.

II. Introduction:

Handwashing is like a "do-it-yourself" routine—it involves five simple and

effective steps (Wet, Lather, Scrub, Rinse, Dry). It can reduce the spread of common
illness so you can stay healthy. Regular handwashing, particularly before and after
certain activities, is one of the best ways to remove germs, avoid getting sick, and
prevent the spread of germs to others. It's quick, it's simple, and it can keep us all from
getting sick. Handwashing is a single and most important thing to stop the spread of
the infection or germs.

II. Materials

1. Antimicrobial soap
2. Paper/hand towel

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 III. Procedures:

VI. Questions.

1. What is the important of handwashing?

2. Enumerate the 8 steps of proper handwashing.

LABORATORY MANUAL IN PHARMACEUTICAL MICROBIOLOGY AND PARASITOLOGY