Dr. Lu lab

1. 0

2. 1
Exercise No.____
Instrumentation
Microbiology as a science includes the study of bacteria (bacteriology),
viruses (virology), fungi (Mycology), and animal parasites (Parasitology). After
the discovery of microscopic life forms by Leeuwenhoek, other fields such as
Immunology & Genetics are also classified as branches of Microbiology.
In the study of Microbiology, the single most important apparatus used in
routine examinations is the compound microscope. The microscope is not only
essential , it is also a must to master the proper usage and handling of this. Aside
from the microscope comes many other laboratory equipment and glassware
which are also needed in performing the daily tasks in a microbiology laboratory.
This is the main goal of the initial chapter: To introduce to the dental students the
basic instrumentation in the microbiology laboratory.
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.
Part 1. The Compound Microscope:
Objectives:
a. To train the dental student in the use of the compound microscope
b. To enforce proper operation of the microscope specially with the Oil
Immersion objective
Materials:
a. Compound microscope
b. Prepared slide specimen
c. Cedar wood oil
d. Xylol

3. 2
Part 2. Apparatus & Glassware
Objectives:
a. To acquaint the student with the various apparatus used in microbiology
b. To know the different functions & uses of the different glassware in
microbiology.
Materials:
a. All apparatus & glassware checked & distributed to the students
b. Apparatus in the Micro Lab ( GDLSC Stockroom )

4. 3
Illustration:
(1) Draw and label properly all functional parts of the compound microscope.

5. 4
(2) Draw & give a use for each of the following :
1. Plain slide: _______________________ 2. Depression slide: _____________________
_________________________________ ____________________________________
3. Candle jar:________________________ 4. Gas Pak: _____________________________
_________________________________ _____________________________________
5. Colony counter:____________________ 6. Wire basket: _______________________
_________________________________ __________________________________
7. Inoculating loop:___________________ 8. Inoculating needle:___________________
_________________________________ _________________________________

6. 5
9 Petridish : _______________________ 10. Coplin jar : _________________________
_________________________________ ____________________________________
11. Bunsen burner:___________________ 12. Waterbath: _________________________
__________________________________ _____________________________________
13.Dilution bottle :____________________ 14. Dropping bottle: _____________________
__________________________________ ____________________________________
15.Petridish can: ___________________ 16. Pipette can:_________________________
_________________________________ ____________________________________

7. 6
Review Questions:
1. Differentiate magnification , resolution & resolving power:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. Why is there a need for cedar wood oil & Xylol in the use of the Oil Immersion
Objective?_______________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
3. Briefly discuss the following:
a.Electron microscope: __________________________________________________
___________________________________________________________________
__________________________________________________________________
b. Fluorescence Microscope: ______________________________________________
___________________________________________________________________
___________________________________________________________________
c. Phase Contrast Microscope: ____________________________________________
___________________________________________________________________
___________________________________________________________________
d. Inverted Microscope: _________________________________________________
___________________________________________________________________
___________________________________________________________________
e. Darkfield Microscope: _______________________________________________
___________________________________________________________________
___________________________________________________________________

8. 7
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?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
5. Why do you think is it necessary to take precautionary measures in handling your
specimen and for yourselves prior to coming in and out the laboratory as dental students?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

9. 8
Exercise No.____
Bacterial Cytology & Physiology
Bacteria are characteristically smaller than the more advanced microscopic
eukaryotes and range in size from 0.2 um, in width , which is barely visible with
the compound microscope, to some spiral-shaped species that reach 400-500 um
in length. Generally speaking, most bacteria are 1 to 6 um in length with
diameters between 0.2 to 1.5 um.
A typical bacterial cell taken apart could consist of a capsule, cell wall ,
cytoplasmic membrane, some organelles, nucleus, fimbriae & flagella plus the
presence of spores. They could be classified according to shape into: spherical,
rod shaped, curved or spiral and square, with variations in most types which is
known as pleomorphism. They also have the ability to exhibit reproduction and in
some cases motility and cellular interactions.
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

10. 9
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:

11. 10
(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

12. 11
Review Questions:
(1) Give a specific function for each of the basic structures of the bacterial cell:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
(2) Give the chemical composition of the cell wall and cell membrane of the
bacterial cell:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
(3) Name the specialized structures of the bacterial cell and give their functions:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
(4) Enumerate the different types of flagella based on number and location:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________

13. 12
(5) List down the different types of spores based on its location within the
bacterial cell:
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
(6) Aside from the three morphological forms ( cocci, bacilli & spirilli) , are there
other special morphological forms?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________

14. 13
Exercise No._______
Microscopic Study of Protozoans & Observation of True Motility
Protozoans are unicellular, free living, in some cases parasitic
microorganisms. They belong to the Kingdom Protista. Protozoa are excellent
tools for research because they are easily cultivated in the laboratory. They
reproduce asexually and clones are easily generated with the same genotype of the
desired “parent”. The effects of environmental factors, such as radiation are easily
analyzed in these organisms.
Protozoans are classified into the Sarcomastigophora, Sporozoa,
Myxospora and the Ciliophora. Specific examples of protozoans include:
Amoeba, Mastigamoeba, Euglena, Paramecia, Volvox, Bicoeca, Ochromonas,
Opalina, Stentor, Hymenostomina & Pleuromona. Some of the morphological
structures present on them are the pellicle, shell, mucocyst, cytosome, oral groove,
cytopharynx, contractile & food vacuole, cytopyge, extrusomes & Uroid.
One of the most common characteristic of protozoans is the ability of
movement or locomotion. This is brought about by organs of locomotion which
are flagella and pili or fimbriae. True motility could easily be observed with a
sample from any canal water
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

15. 14
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:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
Illustration : ( Set-up)

16. 15
Review Questions:
(1) What do you think is the purpose of ringing the coverslip with Vaseline before
inverting it onto the depression slide?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(2) What are the other methods of testing for bacterial motility?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

17. 16
(3) Could there be other types of movement aside from true motility? What do you
attribute this to?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

18. 17
Exercise No._______
Staining Techniques
Staining is a technique used in the microbiology laboratory with the help of
coloring dyes, indicator and reagents to enhance or distinguish a specific organism
or a morphological structure to facilitate their study under the microscope. The
most commonly used dyes in microbiological work are known as cationic dyes.
The color containing portion of the molecule is known as chromatophore while the
nonpigment containing part is known as auxochrome.
Before staining, one must first prepare a smear of the specimen by
spreading a thin film of the bacterial suspension on a clean slide with the use of
an inoculating loop. The smear is then air dried, heat fixed r immersed in
chemical solutions. Stains could also be used in combination to produce
contrasting colors and effects to distinguish 2 separate group of organisms in one
sample.
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. Specimen of microorganisms to be provided in the lab.
2. Different staining solutions
3. Clean glass slides
4. Inoculating loop
5. Bunsen burner
6. Dropping bottle
7. Staining Rack

19. 18
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 Gram’s Iodine.

20. 19
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:
II. Acid Fast Staining:
Procedure:
1. Prepare a thin smear of the specimen provided.
2. Heat fix and cool.
3. Flood the slide with aqueous Carbol Fuchsin solution. Steam the slide
for 5 minutes over a flame. Do not allow the preparation to dry up by
adding Carbol Fuchsin from time to time.
4. Wash off the excess stain with tap water.
5. Decolorize the smear with acid alcohol until no excess stain comes off.
6. Counterstain with Methylene Blue for 30 seconds to one minute.
7. Wash off excess stain with tap water.
8. Blot dry and examine under Oil Immersion objective.

21. 20
Observations:
III. Negative Staining:
Procedure:
1. Obtain gingival scrapings from crevices by means of a toothpick or a
periodontal scaler.
2. Spread the scrapings on a glass slide and apply a drop of distilled water.
3. Mix Nigrosin with the smear and allow to dry.
4. Wash the excess dye off , dry and examine under Oil Immersion
objective. Use lead pencil in illustrating results.
Observations:

22. 21
Review Questions:
(1) Enumerate and give the functions of the reagents used in Gram’s & Acid Fast
Stain:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
___________________________________________________________________
(2) What is the purpose of heating the bacterial smear before staining?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(3) What is the factor behind an organism being gram (+) or gram (-)?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(4) What is the purpose of steaming the smear in acid fast staining? ____________
________________________________________________________________________
________________________________________________________________________
_______________________________________________________________________

23. 22
(5) Give examples of Acid fast bacteria:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(6) Give the (+) and (-) color results of both the Gram’s stain & Acid fast stain:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(7) Explain the reason why sometimes the color of the background of the
specimen is in contrast to that of the microorganism?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(8) Give the importance and the circumstances when Negative staining is used:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

24. 23
(9) What could be the possible explanation for you obtaining a result of having
both Gram positive and Gram negative results on one slide:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

25. 24
Exercise No._____
Media Preparation
Culture media refers to any artificial or natural substance or nutrients
provided to the microorganism for it to obtain nourisment in order to sustain
growth and reproduction.
An optimum balance of nutrients and a suitable growth environment are
needed to culture a microbe in the laboratory. The culture media should contain
all the necessary materials for the microbe to move quickly into the log phase of
growth. Water, Carbon, Nitrogen, minerals and growth factors must be available
in a usable form. Many growth media can be prepared by the dental student to
isolate , grow and identify a particular specie of microbe in the laboratory.
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

26. 25
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:

27. 26
(1) What constituent in nutrient broth satisfy the essential requirements for energy,
cellular growth & repair?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(2) Why is agar used in solid media preparation?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(3) How would you adjust the pH of solid media ?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(4) What are the different sterilizers available in the laboratory?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(5) Is there danger or harmful effects in oversterilizing the media? _____ Explain:
________________________________________________________________________
________________________________________________________________________

28. 27
________________________________________________________________________
________________________________________________________________________
(6) What types of media are sterilized in the autoclave ?
_______________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(7) Can all microorganisms grow in nutrient broth and media ? ________ Why or
why not?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(8) Name the other types of media and give examples of organisms which grow in
them :
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(9) Give the differences between a butt culture and the slant culture aside from
their physical appearance:
___________________________________________________________________

29. 28
________________________________________________________________________
________________________________________________________________________
______________________________________________________________________
(10) Differentiate pure, stock & mixed culture. Give their uses and examples for
each:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

30. 29
Exercise No. ______
Aseptic Technique & Methods of Inoculation
One of the most important laboratory procedures in microbiology is to
obtain pure cultures for the identification of organisms. When the samples from
soil, water, food, or body are collected into nutrient containing petridishes, it is
clear that each sample contains a variety of microbes. This is particularly true of
specimens collected from the skin or the mucous membranes of patients with
infection. The clinical specimens contain both the harmless microbes ( normal
flora ) of the body and the microbes causing the disease ( pathogens ) . In order to
accurately identify which microorganism is responsible for the illness, the many
different types of microbes must be separated. After separation the individual
microbes must be placed on the medium that will allow them to reproduce into a
clearly visible population or colony containing only one specie. Samples from this
colony can then be picked out and transferred to a separate culture for further
growth and identification. The microbiologist will then be able to study such
characteristics such as morphology, metabolism and antibiotic sensitivity.
Special wires known as the inoculating loop and needles are used in the
transfer of specimen. Aseptic technique is followed to prevent both the mixing of
cultures and contamination of the student or working area with pathogenic
microbes or their toxins.
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

31. 30
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.
B.Inoculation of Butt culture:
1. Repeat steps 1 & 2 from the previous instructions but this time use the
inoculating needle.
2. Heat the mouth of the test tube and plunge the needle into the agar in one
swift downward motion stabbing about 2/3 of the media. Pull out the
needle, reheat the mouth of the tube and replace the cotton plug onto
the test tube.
3. Flame the inoculating needle until red hot and cool before finally setting
it down on the table.
C. Inoculation of Slant culture:

32. 31
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.
D. Inoculation of Plated Media:
I Interrupted Streak:
1. Flame the loop and pick up the specimen from the test tube after flaming
the mouth of the tube. Reflame the mouth and replace the cotton plug.
Set down the test tube.
2. Hold the petridish with your left hand, with the cover side up. With the
thumb and little finger, raise the lid up and proceed with the
inoculation.
3. Start inoculation at the farther side of the plate tracing a zigzag course
from side to side until you reach the middle of the plate or the widest
part of the petridish.
4. Close the lid and rotate the plate 180 degrees. Repeat the procedure from
the other end of the plate in the same manner.
II. Multiple Inoculation:
-This is done with the same steps as Procedure A except that this is a
procedure done when the available media is insufficient for the number of

33. 32
specimen. The plate is then divided into about 5 to 8 quadrants as that of
a pizza pie, and each organism is streaked into each quadrant.
III. Overlap streak Method:
-This procedure is carried out in the same manner as above except that in
the overlap method, we depend on the overlap zones to provide the
isolated colonies in the second & the third areas of inoculation.
IV. Radial Streak Method:
1. Follow all the preliminary steps as in procedure A.
2. Place a loopful of the broth culture near the edge of the agar plate.
3. From here make radial straight lines streaking towards the other side.
Start from one side of the plate until the whole plate has been inoculated.
This method is usually used in culturing fungi.
V. Pour plate method:
1. Prepare 3 sterile test tubes & petridishes .
2. Put 9 cc of sterile saline to each of the 3 test tubes.
3. To the first tube add 1 ml of the broth culture of Specimen A and mix.
4. Transfer 1 ml from the contents of the first tube into the second tube and
mix. You now have a dilution of 1:10
5. Transfer 1 ml from the contents of the 2nd tube into the 3rd tube and
mix. You now have a dilution of 1:100
6. From each of the test tubes pipette out 1 ml of the contents separately
and transfer them into a corresponding petridish.
7. Add about 10 - 12 cc of melted agar to each of the petridish.
8. Allow the agar to solidify then incubate.
Illustrations:
(1) Graphically present the different types of culture media used in inoculation:

34. 33

35. 34
(2) On the space provided , graphically illustrate and label the 7 different methods
of inoculation

36. 35

37. 36
Review Questions:
(1) Why are the inoculated plates incubated in an inverted position ?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(2) Differentiate a pure culture, mixed culture and a stock culture : _____________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(3) What are the other ways of identifying microorganisms? ___________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(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.
______________________

38. 37
Show complete solution in the space provided:

39. 38
Exercise No. _____
Biochemical Activities of Bacteria
Bacteria are capable of performing several biochemical activities, one of
which is fermentation of many molecules including carbohydrates, amino acids &
organic acids. These properties could serve as tools of identification since they
demonstrates some metabolic capability of the microbe.
The set-up used in the laboratory to detect biochemical activity is with the
use of Durham tubes inverted in test tubes containing different carbohydrates. The
Durham tube would collect the gases such as Carbon Dioxide, Methane or
Hydrogen that might be produced in the fermentation process. If gas is produced ,
a visible bubble will form clearly in the Durham tube. Some bacteria produce acid
& gas as fermentation products and in some instances only one of the two is
produced.
Differential media is also used to differentiate microbes. One of the most
important sets of tests is the IMViC Test. It is a series of biochemical test used to
identify members of the Enterobacter family. Many members of this family
produce diseases such as :
E. coli - Infantile diarrhea
Enterobacter - Urinary tract infection
Klebsiella - Pneumonia
Salmonella - Typhoid fever & Gastroenteritis
Shigella - Bacillary Dysentery
An example would be two of the abovementioned organisms exhibits the
same properties. E.coli & Enterobacter are both found in the colon of humans,
they are both Gram negative rods, and also exhibit the same symptoms in parasitic
relationships. The IMViC test would be the easiest way to distinguish them apart
.
Objective :
1. To understand the biochemical activity & metabolic capabilities of some
closely related microbes.

40. 39
2. To differentiate two groups of microorganisms based on their biochemical
properties. (Escherichia coli & Enterobacter aerogenes)
3. To have an actual demonstration of the most common biochemical tests used in
the microbiology laboratory.
Materials:
1. Broth culture of both E. coli & E. aerogenes
2. Erlich’s reagent
3. Clark & Lub’s 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 Ehrlich’s 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.
B. Methyl Red Test
- a pH indicator ( Methyl Red ) is used to determine whether the microbe
has produced acid or not.
Procedure:

41. 40
1. Prepare 2 tubes containing Clark & Lub’s broth
2. Inoculate specimen A & B into the tubes
3. Incubate the tubes for 72 hours at 37 degrees
4. After incubation, add 2 to 3 drops of Methyl Red.
5. Red Color would be an indication for the production of acid and yellow
color would be indicative of alkaline or negative results
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 acetyl-
methylcarbinol 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.
Observations & Results:
In the space provided, draw a 3 column table and tabulate your results:

42. 41
Review Questions:
1. Give the positive results & at least 2 organisms identified by the following tests:
a. Catalase test: ____________________________________________________
_________________________________________________________________
__________________________________________________________________
b. Coagulase Test: __________________________________________________
__________________________________________________________________
__________________________________________________________________
c. Oxidase Test ____________________________________________________
_________________________________________________________________
_________________________________________________________________
d. Urease Test _____________________________________________________
_________________________________________________________________
e. Hemolysis Test __________________________________________________
_________________________________________________________________
_________________________________________________________________

43. 42
f. Hydrogen Sulfide Test _____________________________________________
_________________________________________________________________
_________________________________________________________________
g. Greig’s Test _____________________________________________________
_________________________________________________________________
_________________________________________________________________
2. What is the API 20 system?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

44. 43
Exercise No. _____
Factors Influencing Bacterial Growth
Aside from the nutritional requirements of the microorganisms which are
provided for in the culture media, different microbes require different
environmental conditions for them to exist, grow and reproduce. Environmental
factors such as temperature, pH, Osmotic pressure, salt concentration, Oxygen &
Carbon Dioxide concentration and presence of moisture are all essential & should
be taken into consideration as part of the whole living environment. This set of
exercise is designed at altering and adjusting some of the variables and observing
the effects of these variants on the behavior and growth of specific microbes.
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.

45. 44
2. Incubate all tube A’s at 37 degrees.
3. Store all tube B’s in the refrigerator ( 4 - 12 degrees )
4. Incubate all tube C’s in the incubator at 50 degrees.
5. Store all tube D’s in the locker ( 25 degrees )
6. After 24 hours check and tabulate results.
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:

46. 45
1. To each of the specimen provided, inoculate each into three tubes.
2. To the first set of tubes , add 1 ml of mild acid.
3. To the second set of tubes, add 1 ml of mild base
4. Use the third set of tubes as control.
5. Incubate the tubes for 24 hours.
6. Check and tabulate your results.
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:

47. 46
1. Differentiate the following : Optimum growth temperature, minimum growth
temperature & maximum growth temperature:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. Name the different types of microorganisms based on their oxygen requirement:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
3. Different psychrophiles, mesophiles & thermophiles: _______________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. Aside from the factors discussed in the laboratory exercise, what other factors
do you think influence bacterial growth? ____________________________________
________________________________________________________________________
________________________________________________________________________
_______________________________________________________________________
(5) Is there an actual theoretical value for optimum growth temperature? Why or
why not? Justify your answer:

48. 47
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________

49. 48
Exercise No. _____
Microbial Control
Microbes present in the environment which are harmful to us need to be
controlled or eliminated. This is carried out by inhibiting their growth and mode
of actions, destroying the microbes thereby eliminating them from the
environment. This is carried out by elements known as antimicrobial agents.
These agents could be classified into physical, chemical and through the process
of filtration. The choice of a particular agent depends on the type of material to
be treated, specific kind of microbe to be controlled and the environmental
conditions present at the time of use.
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

50. 49
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:
B. Effect of Chemical Agents on Microorganisms :

51. 50
1. Make dilutions of the following disinfectants:
a. Phenol
b. Hydrogen Peroxide
Prepare dilutions of 1:10, 1:100, 1:1000
2. Place all the tubes in a water bath at 20 degrees for 5 minutes
3. Transfer 0.5 ml of the culture in each of the tubes
4. Incubate the nutrient broth for 24 hours.
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 :
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

52. 51
2. What is phenol coefficient? ____________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
3. If results obtained were all ( ++ ) in all dilutions, what would be the most
sensible alternative or approach?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. Another method of microbial control is filtration. List down at least 5 kinds of
filters which could be used for this method? _________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
5. Define the following terms:
a. Antiseptic: ______________________________________________________
__________________________________________________________________
__________________________________________________________________
b. Asepsis:_________________________________________________________
_________________________________________________________________
__________________________________________________________________
c. Disinfectant : ____________________________________________________
_________________________________________________________________
d. Sterilization : ____________________________________________________

53. 52
__________________________________________________________________
__________________________________________________________________
e. Bactericidal :_____________________________________________________
__________________________________________________________________
__________________________________________________________________
f. Bacteriostatic : ___________________________________________________
_________________________________________________________________
_________________________________________________________________

54. 53
Exercise No. _____
Bacterial Growth Curve
Large population of bacterial cells are routinely used in the laboratory
experiments in which bacterial growth and related processes are measured. A
small inoculum of cells is usually introduced into the culture media and after a
short period of time, when the cells have reached confluency, harvested. It is
therefore of utmost importance to determine the time it takes for the organism to
reach confluency and the stages of growth that the culture is in.
Cultures after being inoculated undergo several stages:
A. Lag phase is a phase wherein cells are given ample time to react to their
new environment and there is only increase in size,
B. Log phase or the exponential phase is a stage of rapid growth and cell
multiplication,
C. Stationary phase is achieved after a period of time and the number of
organisms remains constant due to the depletion of nutrients, pH change
from waste accumulation or simply overcrowding,
D. Death or decline phase occurs when the organism starts to die off due to
the increasingly irreversible effects from the stationary phase.
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

55. 54
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. ___________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2.. What are the factors influencing the decline of a bacterial culture? ___________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

56. 55
3. Would all microorganisms exhibit the same bacterial growth curve? ______
Why?__________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. Why is there an existence of a stationary phase? Is it really because the
microorganisms “stop” growing ?
_____________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
5. What are the other methods of determining cell population? _________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

57. 56
Exercise No. _____
Antibiotic Susceptibility Test
The single most important clinical test performed by many microbiologists
is the culture and susceptibility test because the results would provide the
physician, dentist or any medical personnel with significant information regarding
the pathogen and a possible means of controlling it.
A culture and susceptibility test is the growth of a known pathogen in pure
culture and its exposure to known antibiotics of different concentration to
determine which drug will kill or inhibit the growth of the microorganism.
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.

58. 57
Observations:
Review Questions:
1. What is the Kirby-Bauer method in susceptibility testing? ___________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. What are the three ranges of inhibition zones? How are they interpreted? _____
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

59. 58
3. How do you differentiate a broad from a narrow spectrum antibiotic?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. What is MIC in relation to antibiotic susceptibility? How does it affect the
choice of antibiotic prescription? __________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
5. Would the same procedure done above be applicable in determining allergies in
man? _______________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
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:
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

60. 59
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
7. What is the basis of determining a microorganism in being susceptible or
resistant to a specific antibiotic? _______________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________

61. 60
Exercise No. _____
Normal Flora of the Oral Cavity
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. Gram’s Stain
5. Nigrosin Dye
6. Inoculating loop and needle
7. Bunsen burner
8. Glass slides

62. 61
Procedure:
1. Using clean glass slides perform the following staining procedures:
a. Direct methyl violet staining
b. Gram’s 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?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. What is the nature of the oral environment of the infant at birth ? _____________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

63. 62
3. Name the factors that regulate the oral microbiota: _________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. What is commonly known as the indicator microorganism in the oral cavity?
Why?__________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

64. 63
Exercise No. _____
Microbiota of Dental Caries
The teeth, because of their composition, inertness and environment are
particularly susceptible to destruction. Caries is a destructive disease of the teeth
of man and all other forms of life that possess calcified dentition, the etiology of
which is intimately associated with the oral microbial flora.
Dental caries begins most often in areas of coronal enamel where saliva
may stagnate, food debris may impact, and the oral microbial flora may find a
suitable environment for growth such as enamel pits & fissures, and near the point
of interproximal areas. Microbial activity initiates the destruction of enamel by
converting carbohydrates to organic acids which demineralize the enamel or by
chelation of enamel calcium by a variety of bacterial metabolites.
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. Rogosa’s 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

65. 64
2. Inoculate the Rogosa;s agar slant. Incubate for 48 hours at 37 degrees.
3. Perform Gram and negative staining procedures.
4. Examine under oil immersion objective.
Observations::
Review Questions:
1. What are the microorganism which are predominant in the occurrence of dental
caries?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. Discuss the factors that mediate dental caries: _____________________________

66. 65
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
3. What is Snyder’s colorimetric test? ______________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. What is the DMF index ? _______________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

67. 66
Exercise No. _____
Microbiota of Dental Plaque
Dental plaque is the mass of bacteria in a matrix of organic material which
adheres tenaciously to the tooth surface. It is made up largely of protein and
polysaccharide which is partly derived from saliva and partly produced by the
organism . Plaque does not include food debris or exfoliated epithelial cells which
are only transient features.
Dental plaques are composed of about 20% precipitated salivary mucoid or
mucin and about 80% microorganisms.
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 Gram’s 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 Gram’s and negative staining procedure.
4. Wash and dry stained smears.
5. Examine specimen under OIO.
6. Record your results and observations.

68. 67
Observations:
Review Questions:
1. What organisms make up the considerable bulk of the microbial flora of the
dental plaque?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. .How are plaque formed? ______________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

69. 68
________________________________________________________________________
________________________________________________________________________
3. Name the bacterial species which are predominant in superficial enamel decay.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. What are the three types of dental plaque? ________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

70. 69
Exercise No. _____
Microbiota of the Periodontal Pocket & Root Canal
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 gram’s 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 Gram’s and negative staining procedures.
4. Examine under OIO.
5. Record your results and observations.

71. 70
Observations:
Review Questions:
1. What factors influence the initiation & progression of periodontal diseases? __
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. What is Vincent’s angina? Ludwig’s angina? ______________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

72. 71
3. Name the enzymes produced by the members of the oral microbial flora? ______
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. Why is BHI commonly used in culturing root canal specimen? _______________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
5. What is the microbiota of an infected root canal? ___________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

73. 72
Exercise No. ______
The Study of Fungi
Mycology is the study of fungi which are non-photosynthetic
microorganisms that grow typically in filaments and reproduce by sporulation.
Example of this group of organisms would include the lichens, mushrooms,
toadstools, puffballs and the microscopic plants known as molds and yeast. Some
of them have evolved into true pathogens and are able to actively infect, cause
harm and be transmitted from one suitable host to another.
Infections caused by fungi is known as mycoses. The types of clinical
mycoses could be divided into cutaneous, subcutaneous & systemic. These
different types of fungi that are clinically significant could be acquired from soil
or are opportunistic normal flora of the body.
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

74. 73
Observations:
Review Questions:
(1) Why are fungi considered plant-like organism and yet studied under
microbiology?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
(2) Give the different types of mycoses and examples of each: _________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

75. 74
(3) Name some of the members of the fungi family which are beneficial to
mankind & give their uses: _______________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________