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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
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.
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.
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.
V. Procedures:
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.
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.
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.
i. Place the specimen or slide under the clips. Swing the LPO into place.
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.
LPO HPO
I. Objectives:
II. Introduction:
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.
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.
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?
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:
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:
Slide 1 Slide 2
Describe the complete morphology of the organism on the basis of its simple staining
reaction.
VII. Questions:
1. Give the advantages of simple-stained preparations.
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:
IV. Materials:
V. Procedures:
Slide 1 Slide 2
Describe the complete morphology of the organism on the basis of its simple staining
reaction.
VII. Questions:
b. Gram’s iodine
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?
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.
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.
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)
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:
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:
Describe the complete morphology of the organism on the basis of its simple
staining reaction.
Method: ______________________________
Result: ________________________________
Name of Organism: ___________________
5. What cellular structure is responsible for the difference in the Gram staining
reaction of microorganisms?
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.
IV. Materials
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:
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:
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:
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
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:
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?
II. Introduction:
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
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:
VI. Computations.
A. Solid Medium
B. Liquid Medium
C. Semi-Solid Medium
VII. Questions.
b. Manner of formation:
c. Composition:
3. Give an example of differential and selective culture media and state the
purpose of each incorporated substance.
I. Objectives:
II. Introduction:
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.
IV. Materials
1. Autoclave
2. Bunsen burner
3. Hot air oven
4. Wire loop
5. Forceps
V. Procedures:
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
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.
1. Define sterilization.
3. Explain the mechanism of action of dry heat and moist heat on bacteria.
5
B. Chemical Methods: Disinfectants &
Antiseptics
I. Objectives:
II. Introduction:
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.
IV. Materials
V. Procedures:
A. Lysol
B. Alcohol
b. Disinfectant
c. Bactericidal
d. Bacteriostatic
I. Objectives:
II. Introduction:
II. Materials
1. Antimicrobial soap
2. Paper/hand towel
VI. Questions.