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Exercise No.____
Instrumentation
Objectives:
1. To familiarize the dental student with the common glassware & apparatus used
in the microbiology laboratory.
2. To be able to differentiate the functions of some of the common instruments &
tools used in the performance of laboratory exercises in microbiology.
Illustration:
(1) Draw and label properly all functional parts of the compound microscope.
5. Colony counter:____________________
_________________________________
7. Inoculating loop:___________________
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8. Inoculating needle:___________________
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Review Questions:
1. Differentiate magnification , resolution & resolving power:
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2. Why is there a need for cedar wood oil & Xylol in the use of the Oil Immersion
Objective?_______________________________________________________________
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4. Differentiate an autoclave from a hot air sterilizer. Which is more commonly used in
the microbiology laboratory? Which is usually used in the dental clinic?
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Exercise No.____
Bacterial Cytology & Physiology
Objectives:
1. To study the basic structure of the bacterial cell.
2. To enable to student to distinguish the different morphological forms of the
bacterial cell.
3. To fully understand the functions of the fundamental parts and the specialized
structures contained in a bacterial cell.
Materials:
1. Compound microscope
2. Prepared microscopic slides of the following :
a. Staphylococcus aureus
b. Bacillus subtilis
c. Vibrio cholera
3. Cedar wood oil
4. Xylol
Illustrations:
(1) Draw a typical bacterial cell and label all its functional parts and structures:
(2) In the space provided , illustrate the different specialized structures which
could be present in a bacterial cell and give their classifications:
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(3) On the space provided , illustrate all pleomorphic forms of the cocci, bacilli &
spirilli:
a. Cocci - diplococci, staphylococci, streptococci, sarcinae, tetrads
b. Bacilli - single, diplococci, streptobacilli, palisade
c. Spirilli - spiral, comma-shaped
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Review Questions:
(1) Give a specific function for each of the basic structures of the bacterial cell:
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(2) Give the chemical composition of the cell wall and cell membrane of the
bacterial cell:
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(3) Name the specialized structures of the bacterial cell and give their functions:
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(4) Enumerate the different types of flagella based on number and location:
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(5) List down the different types of spores based on its location within the
bacterial cell:
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(6) Aside from the three morphological forms ( cocci, bacilli & spirilli) , are there
other special morphological forms?
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Exercise No._______
Microscopic Study of Protozoans & Observation of True Motility
Objectives:
(1) To observe the actual living microorganisms present in the samples obtained
from our present surroundings.
(2) To demonstrate and observe true motility as exhibited by the protozoans at the
same time also study the organs for locomotion.
Materials:
1. Specimen from any canal water
2. Vials
3. Hanging - drop slide or depression slide
4. cover slip
5. Vaseline
6. medicine dropper
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Procedure:
1. Clean the concave slide and coverslip thoroughly.
2. Take a drop of the specimen and carefully drop it on the center of the
coverslip. Ring the coverslip with a thin layer of Vaseline.
3. Carefully invert the prepared coverslip over the concavity of the depression
slide, making sure that the drop of specimen in the center is hanging into the
concavity without touching the slide.
4. Study the preparation under low power objective with the diaphragm partly
open and the condenser slightly lowered.
5. Switch the objective to high power and observe the specimen. The slide may
be moved to have a wider field of coverage in searching for motile organisms.
6. Make a note of all the different types of motile organisms observed.
Note: After the exercise, remember to immerse the dropper used and the
depression slide together with the coverslip into the disinfectant solution before
washing with soap & water.
Observations:
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Illustration : ( Set-up)
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Review Questions:
(1) What do you think is the purpose of ringing the coverslip with Vaseline before
inverting it onto the depression slide?
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(2) What are the other methods of testing for bacterial motility?
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(3) Could there be other types of movement aside from true motility? What do you
attribute this to?
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Exercise No._______
Staining Techniques
Objectives:
1. To introduce the different types of staining procedures.
2. To differentiate and distinguish the functions of the individual reagents
included in each staining procedure.
3. To have a realistic experience in the actual staining of microorganisms.
Materials:
1.
2.
3.
4.
5.
6.
7.
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A. Simple Staining:
Procedure:
Note : Inoculating loop must be flamed red hot before & after use.
1. Prepare 2 smears with the inoculating loop for each of the
microorganisms provided.
2. Heat fix and allow the specimen to cool.
3. Flood one set of the slides with Carbol fuchsin & the other set with
Methylene Blue.
4. Allow the stain to remain for one minute.
5. Wash with tap water and blot dry.
6. Examine all specimen under oil immersion objective.
7. Note down all observations.
Observations:
B. Differential Staining:
I. Gram Stain:
Procedure:
1. Prepare a thin smear for all specimen provided.
2. Fix & allow to cool.
3. Flood the smear/s with a few drops of Crystal or Gentian Violet.
4. Let the slide stand for 60 seconds then wash with tap water.
5. Add a few drops of Grams Iodine.
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6. Let the slide stand for 60 seconds then rinse with tap water.
7. Add 95% alcohol to the smear until no more color comes off from the
alcohol (this may take 30 seconds to 2 minutes)
8. Wash again with tap water.
9. Add a few drops of Safranin
10. Allow to stand for 30 seconds
11. Wash off the excess stain.
12. Blot dry and examine the specimen under Oil Immersion Objective.
Observations:
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Observations:
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Review Questions:
(1) Enumerate and give the functions of the reagents used in Grams & Acid Fast
Stain:
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(2) What is the purpose of heating the bacterial smear before staining?
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(3) What is the factor behind an organism being gram (+) or gram (-)?
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(4) What is the purpose of steaming the smear in acid fast staining? ____________
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(6) Give the (+) and (-) color results of both the Grams stain & Acid fast stain:
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(7) Explain the reason why sometimes the color of the background of the
specimen is in contrast to that of the microorganism?
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(8) Give the importance and the circumstances when Negative staining is used:
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(9) What could be the possible explanation for you obtaining a result of having
both Gram positive and Gram negative results on one slide:
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Exercise No._____
Media Preparation
In media preparation, the reagents and materials required may vary from
one to the other but there a standard set of procedure to follow:
1. Gathering all the materials needed.
2. Dissolving the ingredients in the proper solvents
3. Adjusting the pH of the media
4. Filtering the media
5. Dispensing the media in their appropriate containers
6. Cotton plugging the tubes
7. Sterilizing the media
8. Testing the sterility
9. Storing the media.
Objectives:
1. To develop the ability to prepare the different types of culture media based on
the standard procedures and the requirements of the individual microorganisms.
2. To know the various nutrient requirements of various individual
microorganisms
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Materials:
1. Beef Extract
0.3 gm
2. Peptone
2.0 gm
3. Sodium Chloride
0.5 gm
4. Distilled water
100 ml
5. Erlenmeyer flask
6. Pipettes, funnel, test tube
7. Graduated cylinder
8. Indicators
9. Comparator block & pH standards
A. Nutrient Broth:
Procedure:
1. Mix the ingredients in a 100 ml flask. Add aliquot portion of distilled
water.
2. Dissolve by heating over flame.
3. Adjust the pH to 7.4 - 7.5 using comparator block method.
4. Filter the media if necessary.
5. Dispense into 10 ml amount in test tubes, Plug with cotton plugs.
6. Autoclave at 25 psi for 15 to 20 minutes
7. Store in refrigerator for future use.
B. Nutrient Agar:
Procedure:
1. Same as the above preparation but add 2 gram agar.
2. Boil until all agar is completely dissolved.
3. Dispense 10 cc into each test tube.
4. Plug the test tube with non-absorbent cotton.
5. Autoclave the tubes at 15 psi for 15 to 20 minutes.
6. Slant 5 tubes and allow the other tubes to solidify in the upward position.
7. Store the media in the refrigerator for future use.
Review Questions:
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(1) What constituent in nutrient broth satisfy the essential requirements for energy,
cellular growth & repair?
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(5) Is there danger or harmful effects in oversterilizing the media? _____ Explain:
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(7) Can all microorganisms grow in nutrient broth and media ? ________ Why or
why not?
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(8) Name the other types of media and give examples of organisms which grow in
them :
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(9) Give the differences between a butt culture and the slant culture aside from
their physical appearance:
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(10) Differentiate pure, stock & mixed culture. Give their uses and examples for
each:
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Objectives:
1. To develop the skill of proper inoculation of different kinds of culture media.
2. To be able to practice asepsis in the laboratory which will serve as a training
ground for the dental students future practice in the clinic & actual practice.
3. To study bacterial growth in different types of culture media.
Materials:
1. Culture media
a. plain broth tube
b. plain agar butt
c. plain agar slant
d. agar plates
2. Inoculating loop and needle
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3. Bunsen burner
4. Organism/specimen
Procedure:
A. Inoculation of Liquid media:
1. Hold the tube gently on the left hand and the loop or needle ( which
must be previously heated ) with the right hand.
2. With the pinkie ( little finger) of the right hand, pull out the cotton plug
of the tube.
3. Heat the mouth of the test tube and proceed with the actual inoculation
by slightly rubbing the loop with the inoculum on the side of the tube or
simple just by twirling the loop to dislodge the inoculum.
4. Heat the mouth of the tube , put back the cotton plug onto the test tube
before setting it down and reflame the loop or needle before setting it
down on the table.
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1. Repeat steps 1 & 2 of procedure A, but this time heat the inoculating
loop.
2. With the little finger remove the cotton plug and heat the mouth of the
test tube.
3. Starting from the butt end of the slant draw the loop over the surface in a
straight line towards the end of the slant.
4. Starting again from the butt end, trace a zigzag course from side to side,
at the same time slowly drawing the loop towards the end of the slant.
5. Heat the mouth of the test tube , replace the plug, heat the loop, cool and
set it down on the table.
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Illustrations:
(1) Graphically present the different types of culture media used in inoculation:
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(2) On the space provided , graphically illustrate and label the 7 different methods
of inoculation
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Review Questions:
(2) Differentiate a pure culture, mixed culture and a stock culture : _____________
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(4) Assuming that the same serial dilution was carried out in a laboratory
experiment in 10 tubes with 4 ml of saline in each . Pour plate method of
inoculation was performed. It was later found out that on the 7th petridish
cultured there was a total of 16 individual microorganisms found. Deduce a way
based on the given and compute the total number of microorganisms present in
tube one neglecting all chances of human error in the transferring process.
______________________
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Infantile diarrhea
Urinary tract infection
Pneumonia
Typhoid fever & Gastroenteritis
Bacillary Dysentery
Objective :
1. To understand the biochemical activity & metabolic capabilities of some
closely related microbes.
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Materials:
1. Broth culture of both E. coli & E. aerogenes
2. Erlichs reagent
3. Clark & Lubs media
4. Ether
5. Methyl red indicator
6. Simon Citrate media
7. 40 % KOH
8. Test tubes
A. Indole Test:
- demonstrates the production of indole based on the presence or absence of
tryptophan
Procedure :
1. Prepare 2 test tubes containing Specimen A ( E. coli) & Specimen B ( E.
aerogenes ) in broth culture.
2. Add 1 ml of ether ( note: do not aspirate by mouth) to 48 hour broth
culture of both specimen A & B
3. Shake well and allow to stand until ether rises to the surface.
4. Gently add about 0.5 ml of Ehrlichs reagent drop by drop down the side
of the test tube so that it forms a ring between the medium and the ether
layer. The presence of indole is indicated by a cherry red or deep red
color in the reagent layer at the top.
Procedure:
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C. Citrate Test
- indicates whether or not the organism can utilize citrate as a sole source of
Carbon.
Procedure:
1. Prepare two tubes with Simon Citrate media.
2. Inoculate by streaking on the slant portion of the media.
3. Incubate the tubes at 37 degrees for 24 hours.
4. Blue color would be indicative of positive results and green color would
be negative.
D. Voges-Proskauer Test
- indicates the production of an organic molecule called acetylmethylcarbinol or acetoin from glucose in microorganisms.
Procedure:
1. Prepare 2 tubes of Clark & Lubs media
2. Inoculate specimen A & B into the tubes
3. Incubate the tubes at 37 degrees for 72 hours.
4. After incubation add an equal amount of 40% KOH and shake
vigorously
for 2-5 min.
5. Allow the tubes to stand and note the color reaction.
6. Production of an Eosin like color up to 2 hours would be positive. Color
would depend on the oxidation of acetoin.
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Review Questions:
1. Give the positive results & at least 2 organisms identified by the following tests:
a. Catalase test: ____________________________________________________
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b. Coagulase Test: __________________________________________________
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c. Oxidase Test ____________________________________________________
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d. Urease Test _____________________________________________________
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e. Hemolysis Test __________________________________________________
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Objectives:
1. To know the different factors behind the growth of microorganisms.
2. To differentiate microorganisms based on their ability to exist under different
environmental conditions.
Materials:
1. Broth culture of different microorganisms
2. 20 test tubes
3. Candle jar or anaerobic jar
4. Dessication chamber
5. Mild acid & base
6. Water bath
Procedure:
A. Temperature Requirement:
1. For every specimen provided , inoculate each microorganism into 4 tubes
and label them A, B, C, D.
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B. Oxygen Requirement :
1. For every specimen provided, inoculate each into 3 sets of tubes
2. Store the first set of tubes in the incubator.
3. Store the second set of tubes in a candle jar or anaerobic jar
4. Store the third set of tubes in the cabinet.
5. Incubate for 24 hours. Check and tabulate the results
C. pH Requirement:
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D. Moisture Requirement:
1. Inoculate the specimens provided in a set of tubes and store them in a
desiccated chamber.
2. Incubate for 24 hours.
3. Observe and record the results.
Review Questions:
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4. Aside from the factors discussed in the laboratory exercise, what other factors
(5) Is there an actual theoretical value for optimum growth temperature? Why or
why not? Justify your answer:
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Objective:
1. To have a general overview on the different methods of inhibiting & controlling
microbes.
2. To distinguish the different physical & chemical agents used in microbial
control
3. To be able to understand the bacteriostatic & bactericidal effects of some
common agent used in microbial control
Materials:
1. Inoculating loops ( 3 )
2. Nutrient broth
3. Water bath
4. Tripod
5. Bunsen burner
6. Broth culture of Staphylococcus aureus
7. Hydrogen peroxide
8. Phenol
9. Clean test tubes
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Procedure:
A. Effect of heat on Microorganisms:
1. Boil water in a water bath
2. Label three test tubes nutrient broth as:
A - control
B - dry heat
C - moist heat
3. Expose the three tubes to the same environment ( to be given)
4. Dip one inoculating loop directly into tube A
5. Flame another inoculating loop directly for 10 minutes then dip into tube
B.
6. Place the 3rd inoculating loop in boiling water for 10 minutes then dip in
tube C.
7. Incubate the three tubes for 24 hours.
Tabulate your results using the following symbols:
( - ) no growth
( +- ) slight growth or trace
( + ) moderate growth
( ++ ) heavy growth
Observations:
Using the symbols provided from the previous page draw a table & tabulate your
results in the space provided below:
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Observations:
Using the same symbols given in part A, tabulate your results in the space
provided below:
Review questions:
1. Name the dental uses of phenol :
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3. If results obtained were all ( ++ ) in all dilutions, what would be the most
sensible alternative or approach?
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d. Sterilization : ____________________________________________________
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e. Bactericidal :_____________________________________________________
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f. Bacteriostatic : ___________________________________________________
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Objective:
1. To study the development of a bacterial culture from inoculation to its decline.
2. To be able to quantitatively measure the bacterial population at given points
during culture.
3. To graphically illustrate the different stages of the bacterial growth curve based
on the results obtained in the exercise.
Materials:
1. Broth culture of Staphylococcus aureus & Escherichia coli
2. Spectrophotometer
3. Cuvette
4. Graphing paper
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Procedure:
1. Prepare the broth culture of the different specimen provided in serial
dilutions of 1:10, 1:100 and 1:1000 labeled as tube A, B, & C.
2. Prepare a control by storing broth in a tube without any specimen.
3. Incubate the tubes at 37 degrees.
4. Take the readings of the tubes with a spectrophotometer at the given
intervals:
0 minutes, 30 minutes, 2 hours, 4 hours , 8 hours, 12 hours, 24 hours ,
48 hours
5. Take the reading of the control and subtract the value from the values
obtained in all tubes at all times.
6. Tabulate all results and plot with a graphing paper.
Review Questions:
1. What are the stages of the bacterial growth curve? Discuss Briefly. ___________
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2.. What are the factors influencing the decline of a bacterial culture? ___________
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3. Would all microorganisms exhibit the same bacterial growth curve? ______
Why?__________________________________________________________________
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Objectives:
1. To understand the principle behind a culture and sensitivity test.
2. To illustrate the effects of different antibiotics on a given microorganism
3. To quantitatively measure the delimiting power of a specific antibiotic to a
given microbe.
Materials:
1. Broth cultures of specimen provided
2. Agar plates
3. Antibiotic paper discs
4. Sterile swabs
5. Sterile forceps
6. Transparent ruler
Procedure:
1. Prepare one agar plate for each specimen provided. Label properly.
2. Inoculate the agar plates.
3. Using flamed forceps, place the antibiotic discs on the agar surface .
Gently press down the discs to ensure contact with the agar.
4. Incubate the plates in an inverted manner at 37 degrees for 48 hours.
5. Measure the clearing zone or zone of inhibition with your rulers.
6. Record and tabulate your results in millimeters.
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Observations:
Review Questions:
1. What is the Kirby-Bauer method in susceptibility testing? ___________________
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2. What are the three ranges of inhibition zones? How are they interpreted? _____
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6. Pick out any one microorganism which you have studied and try to classify it in
terms of susceptibility to any 10 common antibiotics used in Microbiology or
Pharmacology:
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There are more than 30 species of microorganisms that have been isolated
from the oral cavity. From shortly after birth till death, the individual supports a
relatively stable oral microbiota. Its composition depends upon the introduction of
various microorganisms from the external environment and more importantly
upon the various inherent and acquired factors that control the environment within
the oral cavity.
Although there are many factors which make it difficult to obtain valid
information concerning the kinds and number of microorganisms in the oral cavity
at a given time, this exercise is designed to only identify indigenous organisms
present. Some examples of these organisms are lactobacilli, streptococci,
veillonella, spirochetes, filamentous forms, fusiform bacilli & vibrios. They are
constantly present in large numbers.
Objectives:
1. To confirm the presence of microorganisms in the normal flora of the oral
cavity
2. To identify the organisms present in the oral cavity by studying the saliva.
3. To practically apply the different laboratory procedures introduced in the
laboratory to be used in the identification of the microorganisms.
Materials:
1. Broth culture of salivary secretions.
2. Agar plates
3. Methyl violet
4. Grams Stain
5. Nigrosin Dye
6. Inoculating loop and needle
7. Bunsen burner
8. Glass slides
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Procedure:
1. Using clean glass slides perform the following staining procedures:
a. Direct methyl violet staining
b. Grams Stain
c. Negative staining
2. Inoculate the agar plate with the broth culture of saliva
3. Invert the plate and incubate for 48 hours.
4. Observe and record your results for all procedures performed.
Observations:
Review Questions:
1. What are the microorganisms most commonly found in the healthy oral cavity?
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2. What is the nature of the oral environment of the infant at birth ? _____________
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Objectives:
1. To confirm the presence of specific microorganism present in dental caries
2. To compare the microbiota of patients with rampant dental caries from those
without..
Materials:
1. Saliva from a patient with rampant dental caries
2. Saliva from a patient without caries
3. Rogosas agar slant
4. Sterile test tubes
5. Clean slides
6. Reagents for gram and negative staining
Procedure:
1. Collect a small amount of saliva from both types of patients and store in
sterile tubes
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Review Questions:
1. What are the microorganism which are predominant in the occurrence of dental
caries?
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Objectives:
1. To identify the presence of microorganisms in dental plaque
2. To know the different types of plaque.
Materials:
1. Clean glass slides
2. Reagents for Grams and Negative staining procedure
3. Toothpick
Procedure:
1. Collect plaque from the enamel and dentin with the use of a toothpick.
2. Prepare a bacterial smear from the collected sample.
3. Perform Grams and negative staining procedure.
4. Wash and dry stained smears.
5. Examine specimen under OIO.
6. Record your results and observations.
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Observations:
Review Questions:
1. What organisms make up the considerable bulk of the microbial flora of the
dental plaque?
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________________________________________________________________________
________________________________________________________________________
3. Name the bacterial species which are predominant in superficial enamel decay.
________________________________________________________________________
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The tissues that surround and support the teeth like the gingival tissues,
alveolar bone, periodontal membrane are subject to various diseases, collectively
known as periodontal diseases. In these pathological processes, the oral microbial
flora is greatly involved.
Bacteria do not produce disease simply by their physical presence, although
their adequate multiplication is essential to infection. To produce a disease, some
constituents or products of bacteria like toxins and enzymes must react with the
tissues or cells to destroy them or interfere with their normal functions.
Objectives:
1. To distinguish the microorganisms present in the periodontal pocket and the
root canal.
2. To identify the predominating organisms generally causing periodontal
diseases.
Materials:
1. Sample from a periodontal pocket & infected root canal
2. Brain Heart Infusion broth ( BHI )
3. Reagents for grams and negative staining .
4. Clean glass slides
Procedure:
1. Inoculate the collected specimen in BHI
2. Incubate at 37 degrees for 48 hours
3. Perform Grams and negative staining procedures.
4. Examine under OIO.
5. Record your results and observations.
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Observations:
Review Questions:
1. What factors influence the initiation & progression of periodontal diseases? __
________________________________________________________________________
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3. Name the enzymes produced by the members of the oral microbial flora? ______
________________________________________________________________________
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Objectives:
1. To study the microscopic appearance of a common fungi.
2. To enumerate the cultural characteristics and mode of reproduction of fungi
Materials:
1. An old piece of bread
2. Hand lens
3. Clean glass slide
Procedure:
1. Moisten a little piece of old bread and incubate
2. Study the bread from the incubator.
3. With the hand lens, examine the bread and identify the colonies.
4. Prepare a wet mount using the fresh state, and focus under the
microscope.
5. Draw and label all parts observed
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Observations:
Review Questions:
(1) Why are fungi considered plant-like organism and yet studied under
microbiology?
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(2) Give the different types of mycoses and examples of each: _________________
________________________________________________________________________
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(3) Name some of the members of the fungi family which are beneficial to
mankind & give their uses: _______________________________________________
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