Benguet State University

COLLEGE OF VETERINARY MEDICINE

La Trinidad, Benguet

DEPARTMENT OF PARACLINICAL
SCIENCES

LABORATORY MANUAL OF Veterinary Medicine 111*

Laboratory Section A
VM111- 9:00-12:00 M
School Year 2018-2019
*Adopted from the Laboratory manual of VM111 prepared by Dr. Edlyn Mae N. Ciano., DVM,
MS, MVst, PGDip VPH
 INTRODUCTION
The primary purpose of this laboratory manual is to provide veterinary students with a tool that
would minimize their difficulties in the microbiology laboratory. Hopefully, the student will
explore the wonderful world of microbes by following the exercise carefully. These exercise
were selected and modifies as possible under our local conditions.
Microbiology deals with the study of very minute organisms that need a special instrument called
microscope. These organisms that we are going to study are so called microorganisms. These of
microscope is therefore an essential technic a microbiology student must learn properly in order
to study well this microorganisms. This basic course in microbiology intends to teach a
microbiology student the basics principles intend to teach a microbiology student that are
essential in the study of microbiology.
Each student is advised to read the exercises before coming to class. This will facilitate
understanding them before they will be performed in the class. The instructor will explain each
exercise at the start of the period. There two features of these exercises that were considered
essential in the learning process. Each exercise start with the objective(s) that will emphasize the
purpose of the procedure(s) and what are expected by the teacher that each student will
accomplish. At the end of the exercise, the student is expected to note down observation(s)
and/or result of each exercise. To make sure the student will understand the major features of the
exercise, questions are given for the student to answer.
Aside from learning the basic morphological techniques in this course, the students will be
expected to work with microorganism safely and properly. Each student must know the general
rules and regulations of laboratory safety.
Two to three students will comprise the group. Each group will work as a team and make sure
each member will be given a chance in performing the exercise. A list of the materials to be
provided by the students/group, laboratory operating procedures and laboratory safety
regulations are found in the next section.
After completion of this laboratory course, the student is expected to:
1. To develop proper microscopy technique
2. To perform proper aseptic bacteriological technique
3. To understand the basic technique used in the cultivation of bacteria and fungi
4. To know the different factors that influence microbial growth
5. To know how to properly described the cultural and morphological features of bacteria
and fungi
6. To know the proper staining techniques in demonstrating bacteria and fungi
7. To know the media and tests used to identify bacteria and fungi

LIST OF MATERIALS TO BE PROVIDED BY STUDENT

1. One laboratory gown
2. One laboratory manual
 3. One set of colored pencil
4. One hand towel

LIST OF MATERIALS TO BE PROVIDED BY THE GROUP

1. One box of glass slide (clear and brand new)
2. One box of cover slip
3. One staining can
4. One bar hand soap (with germicidal property preferred)
5. One bottle (500ml) of rubbing alcohol
6. One used face towel
7. One plastic can (one liter oil can)
8. One half gallon plastic container
9. One box of matches
10. 4 pieces of clothes pin
11. One pack of lens paper
12. One roll of tissue paper
13. One marking pen
14. One bar of laundry soap
15. One small plastic basin
16. One box tide (500g)
17. One roll cotton(200g)
18. One liter zonrox

LABORATORY OPERATING PROCEDURES

1. Read the exercise(s) assigned for the day before coming to the class
2. Wear your laboratory gown in the classroom, no lab gown, no exercise
3. Each group will be responsible for all materials
4. Note down the results and observations for each exercise
5. Leave your ID in the preparation room when borrowing microscope
6. Before conducting the exercise, place all books and other school materials in the assigned
places in the room
7. Each group will be responsible for keeping their of work neat and disinfected after each
session. Place discards and other materials in their proper places.
8. Be sure to stain only in the designed sink sing your staining can
9. Call the attention of the instructor in case of breakage, malfunction of equipment etc.
10. Avoid talking during the performance of each exercise. Keep only things needed on your
table top during the performance of the exercise(s).
 LABORATORY SAFETY REGULATIONS
1. Keep your bench top neat, clean and orderly at all times
2. Clean your bench top before performance an exercise
3. WEAR YOUR LABORATORY GOWN AT ALL TIMES IN THE LABORATORY.
4. NO EATING, DRINKING AND SMOKING DURING CLASS
5. Make it a habit to disinfect your table top with a used face towel previously soaked in a
disinfectant solution before and after performing the exercises for the day.
6. Place all waste materials in the waste can and not in the sink. AVOID LITTERING IN
CLASS
7. Place all discarded plates and tubes in designated baskets
8. Place all used slides, cover slips and others in can with disinfectant
9. Clean your microscope before and after using
10. BEFORE THE CLASS, MAKE SURE YOU HAVE
a. Cleaned and disinfect your table top
b. Put the stool in its proper place
c. Returned your microscope
d. Washed your hand and apply alcohol
11. In case of an accident like breaking of glass, spillage etc. Do not panic. Call the attention
of your instructor.
 LIST OF EXERCISES

Exercise No. 1 Microscopy and wet mount

Exercise No. 2 Proper handling of microorganism
Exercise No. 3 Gram Staining
Exercise No. 4 Acid Fast Staining
Exercise No. 5 Preparation of Culture Media
Exercise No. 6 Characterization of Bacterial Culture
Exercise No. 7 Pre Culture Technique
Exercise No. 8 Bacterial Morphology

FIRST PRACTICAL EXAM

Exercise No. 9 Bacterial Anatomy

Exercise No. 10 Physiological Characteristics of Bacteria
Exercise No. 11 Enumeration of Bacteria
Exercise No. 12 Effects of Environment on Microorganism
Exercise No. 13 Microbial Control
Exercise No. 14 Introduction to Mycology

SECOND PRACTICAL EXAM

 THE MICROSCOPE

Each student of microbiology must learn good and proper microscopic techniques. Microbiology
entails the detailed study of minute organism that are not visible to naked eye. These minute
organism otherwise known as microorganism need to be observed with the use of apprecision
instrument called the microscope. Unlike other fields of microbiological sciences microbiology
rely heavily on the use of the oil immersion objectives. The use of this objective properly is
essential to good visualization of bacteria. The student must know how to adjust properly the
component of a light microscope as an effective tool in the study of microorganisms. The first
exercise in the laboratory course will acquaint the student on how to use properly a light
microscope when studying microorganism.

COMPONENTS OF A LIGHT MICROSCOPE

Familiarize yourself with the different components of a light microscope, their function, and
their proper adjustments. First, know how to handle properly the microscope. You will be
expected to know how to handle and care for the microscope. So, whenever you have to move
the microscope from the preparation room to your bench top, grasp the microscope arm firmly
with one hand steady the scope with the other hand under the base support. While walking, keep
the microscope as close as possible to the front part of your body. Make sure you have properly
rolled the cord before transporting the scope.
The different component of the microscope will be discussed by the laboratory instructor but
each student is expected to refer to the recommended references for the detail and theory of
microscopy. A diagram of the microscope will be shown and orient yourself with the different
parts by comparing it with the diagram. Before manipulating the microscope make sure you have
placed it at least two inches away from the edge of the bench top.
The condenser is the first component of the microscope to which the light is directed by the
mirror (use the concave side of the mirror in focusing the light). The condenser concentrates (or
condenses) the pencils of the light into a cone, the apex of which is the focused on the object of
the study. The size of this cone can be adjusted by using the an iris diaphragm. This can be found
underneath the stage. Above the condenser is found the stage, a platform on which the object is
located in the light path so that stage is present to secure the glass slide containing the specimen.
There are two lens system in the compound light microscope: objectives and ocular. The
objective lens is found near the object. The light microscope you will use in this class has three
objectives: (1) low power objective which has a 16 mm distance from the object to the lens of
16mm. ; (2) high power objective which has 4 mm 40x dry lens with greater magnification but
shorter working distance, and (3) oil-immersion objective which has 1 mm 100x oil immersion
lens which is very small, admit only very little light and provides the highest magnification but
with very limited working distance.
When using the oil-immersion objective, the light must be concentrated by the condenser and
must not be allowed to scatter as it passes through the space between the objective and the
object. To prevent this light lose the space between the object and the objective is filled with
immersion oil has nearly the same refractive index as glass. The ability of the condenser to
concentrate the light is accomplish by putting the condenser as high as possible.
The lens system into which one looks is the ocular. The image formed and magnified in the
objective lens system is further magnified by the ocular lens, usually ten-fold. The magnifying
power of the microscope is the product of the objective magnification and the ocular
magnification.
PROPER CARE AND USE OF THE MICROSCOPE
The microscope is a delicate precision piece of instrument that needs proper care and handling.
Here are some recommended tips to follow in handling and care of the microscope:
1. When holding the microscope, grasp it by the pillar arm. Carry it with one hand and the
other hand beneath the base to steady fit in an upright position. Hold the microscope
firmly close to your front body.

2. Place the microscope carefully on top of the laboratory bench. If you want to move it
from place to another, make sure to lift it up and do not just pull it. AVOID
SUBJECTING THE microscope TO SUDDEN JARS.

3. Clean the microscope’s body parts (metal) with an ordinary tissue paper. However, use
only lens paper for cleaning lenses. It is ideally recommended to use a camel brush to
clean the microscope. Remember that the lenses are soft and easily scratched.

4. Avoid using too much oil immersion. After using the oil objective, remove the oil
immersion as soon as possible by wiping it off with lens paper. Then gently wipe off
surface of the lens. Clean it with a piece of lens paper. SLIGHTLY MOISTENED WITH
XYLOL AND WIPE IT OFF AT ONCE WITH DRY LENS PAPER TO REMOVE THE
DISSOLVED OIL AND XYLOL. Too much xylol must be avoided since it may loosen
the lens attachment.

5. Before returning the microscope, always make it a point swing back the low power
objective into the line with ocular and lower the tube carefully to the bottom stop. Also as
an added precaution, lower the sub stage condenser.

6. Report any malfunction to the instructor. NEVER DISMANTLE OR PLAY WITH ANY
PART OF THE MICROSCOPE.

7. Never look down through the microscope while rapidly closing the distance between the
objective and the slide. Always observe this movement from the side of this instrument.
8. Before you return the microscope, be sure to adjust the mechanical stage so that it does
not project too far on either side. Also, make sure that you have lowered down the low
power objective as low as possible.

9. Please be reminded that you and your group are responsible for the microscope that was
assigned to you. You will account for any lost or damage to be unit during the course
period. The instructor or assistant will check your microscope if it was properly handled
and cleaned.
EXERCISE 1. MICROSCOPE AND WET MOUNT PREPARATION
After completing this exercise, the student is expected to:
1. Know the parts of a light microscope
2. Know the function of each part of the microscope,
3. Be able to manipulate properly the different parts of a microscope to effectively study
microorganism.
Materials
Yeast suspension of Saccharomyces sp.
Glass slide Inoculating loop
Cover slip Microscope
Methylene Blue stain Immersion oil, xylene, lens paper
Procedure
1. The microscope should have been returned and stored with the low power objective in
position. If such is the case, turn the nosepiece until the low power objective clicks into
position and make it a habit to return it in that position before returning the microscope.
Look through the ocular lens while adjusting the mirror to the desired light intensity. Do
this while the objective is about 1cm ABOVE THE STAGE WITH THE DIAPHRAGM
OPEN.
Observe the effects of adjusting the condenser and changing its iris diaphragm.
2. With a dropper, place a drop of water near the center of a clean glass slide and stir into a
small amount of yeast suspension with an inoculating loop. Prepare a faintly turbid
suspension on the slide. Place a coverslip over a suspension. Put the slide on the stage and
secure with the clip. Examine the slide with scanner (4X) or low power objective (10X).
3. By looking at the slide on the stage, lower the scanner or lower power objective as low as
possible to the surface of the slide and focus upward with the course adjustment until the
yeast suspension comes into focus. Then use the fine adjustment wheel for sharp focus.
Record your observation and prepare sketches of the yeast cells.
4. Alter the light, condenser position and slide position, making note of the effect of this
manipulations. To help you manipulate better the adjustments of the microscope, try to
use left fingers for manipulating the fine adjustment wheel using your right fingers for
adjusting the position of the slide by rotating the knob on the stage.
5. Our microscope is of parfocal design so what we can assume that the specimen under the
high-dry objective will focus if it was in focus under low power. When changing from
low power to high-dry, keep in mind the following:
a. Once the objective is in focus under the low power, the high dry can be safely
rotated into place.
b. To insure that the high dry is directly over the specimen, make sure that the
nosepiece is locked in place.
c. For high power and oil immersion objectives, the condenser must be elevated
to the full stop position.
 Try to bring the specimen into focus by adjusting the iris diaphragm for the right
illumination and at the same time try to adjust the fin adjustment knob. Try to move up
and down the condenser to see the difference between having it far and close to the stage.
Record your observations at this magnification.
6. Although it is not necessary to start with low power objective before using the oil
immersion lens, it is recommended. Once the image has been focused using the high dry
lens put a drop of oil immersion objective. Focus with the fine adjustment. NEVER
FOCUS WITH THE COARSE ADJUSTMENT WHEN USING THE HIGH AND LOW
POWER OBJECTIVE! For better focusing try to adjust the iris diaphragm. Note the yeast
cells under the oil immersion objective. Record your observations and sketches under this
magnification.
7. After you have noted down your observation and make your sketches of the cells under
varying magnification, make sure to:
a. Wipe the immersion oil from the lens with your lens paper, wipe it off gently
until it is clean.
b. Switch the nosepiece until the low power or scanner is in position and the
barrel tube is at its lowest position.
c. Adjust the stage and wipe the stage with ordinary tissue paper to remove
whatever dust or oil/liquid.
d. Return the microscope to its proper place.
8. Remember to answer all the questions at the end of your exercise. Try to do them on your
own by referring to the recommend references or lecture given by the instructor.
9. CLEAN UP YOUR LABORATORY TABLE TOP AND DISINFECT IT WITH
ALCOHOL AFTER EVERY USE. KEEP EVERYRTHING IN PLACE AND MAKE
SURE TO WASH YOUR HANDS AND APPLY ALCOHOL BEFORE LEAVING THE
CLASSROOM. MAKE THIS A HABIT.
NOTES AND OBSERVATIONS
Use of scanner/low power objective:
It must be the common position of the objective lens when the microscope is carried and stored.
Saccharomyces appeared plane blue color in scanner and under LPO it looks like dots and in
cluster.
Use of high power objective:
Its use reveals the shape, size and arrangement that is very important in determining
morphological characteristics of minute
organisms.

Use of oil immersion objective:

To see the microorganisms more closely and to
observe some movements.
QUESTIONS:
1. What is the total magnification obtained with
the:
a. Low power objective: 10X10=100X
b. High power objective: 10X40=400X
c. Oil immersion objective: 10X100X=1000X

2. Distinguish between resolving power and parfocal

The resolving power is the quality of the sharpness of the image and gives the ability to
separate two closely spaced objects through lens so that we are able to see two distinct
images instead of one whereas Parfocal means that when one objective lens is in focus,
then the other objectives will also be in focus.

3. Differentiate between bright field from dark field microscopy

In bright field microscopy, the specimen is dark while its background is bright, hence the
cells are dead, whereas Dark field microscopy, the specimen is bright while the
background is dark and the cells are alive.
4. Give the function(s) of the following parts:
Ocular:
It is used to see the objects under study with a magnification from 10X to 15X.
Objectives:
It magnifies the specimen in the slide and it can be adjusted from low magnification to
highest magnification.
Condenser:
It collects and focuses the light from the illuminator onto the specimen.
Iris diaphragm:
It helps in controlling the amount of light that is passing through the opening of the
stage.
In base illuminator:
It is the main source of light that release light into the specimen.
Stage:
A flat platform that holds the slide in place, it also have stage clips to secure the slide
and you will be able to move the slide around by turning two knobs under it.
Coarse adjustment knob:
 It moves up and down to bring the specimen into focus and most commonly during
scanning.
Fine adjustment knob:
It is used to bring the specimen into its sharp and precise focus for fine details of
microorganisms.

Check by;
EXERCISE NO.2 PROPER HANDLING OF MICROORGANISM
Microorganisms are extremely minute entities that metabolize rapidly and readily multiplying
into big numbers within a short period of time. This is very important concept a microbiology
student must take into consideration whenever working with these microorganisms. These
microorganism must be handled properly in order to cultivate only what is desired by worker and
avoid contamination and also prevent possible human infection with these microbes. Therefore,
after learning how to handle the microscope, the next basic knowledge the microbiology student
must learn is how to handle microorganisms properly. This exercise today will expect the student
to:
1. Be able to handle bacterial cultures aseptically
2. Be able to prepare and stain bacterial smears
3. Be proficient in properly focusing and examining bacterial smears under the oil
immersion objective.
4. Be able to distinguish the different morphological features of bacteria using stained
smears.
PART A. ASEPTICE TRANSFER OF CULTURES
The study of one particular microorganism like a bacterial species can only be successful in one
has pure culture. If other bacterial species are present in the culture, it would be very difficult to
study them with regards to their biological activities. One can only identify the bacterial species
by considering its particular reactions. These reactions will be altered if more than one bacterial
species are present in the culture. Therefore, the technique of properly obtaining pure cultures is
an essential technique a microbiology student must master. This technique can only be learned
and developed proficiently by following closely the instructions and taking real effort in
practicing the steps in class.
Materials;
1 nutrient agar slant of Serratia marcescens
1 nutrient broth of Serratia marcescens
1 nutrient agar slant of Staphylococcus aureus
1 nutrient broth of Staphylococcus aureus
2 nutrient agar culture
2 nutrient agar broth

Procedure
1. Learn how to handle the inoculating loop. Handle the inoculating loop with your right
hand by grasping it firmly like a pencil. Sterile the loop by heating the entire wire portion
to redness over the hottest part of the flame.
2. While allowing the loop to cool down, remove the cotton plug from the test tube as
follows: first grasp the end of the lower tube cotton plug and hold it in that position.
Flame the mouth of the tube by passing it over the flame once or twice.
3. After you practice unplugging and plugging the test tube containing culture, you are
ready to do different technique of culture transfer. You can transfer inoculum from an
agar slant into another agar slant, agar slant in broth, broth into an agar slant or slant to an
agar plate, etc.
4. Whenever performing aseptic transfer of culture observe the following:
a. Always flame the mouth of the tube whenever you unplug and plug it with cotton
b. Avoid touching the lip of the culture as you unplug and plug it with cotton
c. Never put the cotton plug down on the top of laboratory table
d. Always remember to label first properly the tubes before inoculating them
e. Be careful not to flame the cotton plug
f. Avoid tilting the culture tube during the process of inoculating specially when
handling broth cultures. The liquid will wet the cotton plug.
g. Do not talk while performing the transfer technique.
h. Remember to close the front part of your laboratory gown whenever doing any
transfer of culture.
i. Avoid spattering the inoculating and sterilization of the loop. Hold the loop with a
steady hand when doing the transfer. If you sterilize the loop full of liquid do not
plunge it directly into the flame. Put the end of the loop close to the flame first for a
few seconds to allow the liquid to evaporate first and then sterilize the loop in the
hottest part of the flame.
j. Never touch the loop after sterilizing to test if it is cool enough to transfer.
k. Incubate all culture tubes/slant/plates as soon as possible using the recommended
temperature and gaseous requirement.
5. All the transfer techniques will be demonstrated by your laboratory instructor. Observe
them carefully.
6. Incubate all inoculated tubes at 37 °C. Place the tubes of the group your plastic can
(properly label it). REMEMBER NOT TO ALLOW OVERCROWDING OF THE
PLASTIC CAN. If you have a lot of tube to incubate, use another can to hold them in the
incubator.
REMINDER; PLEASE OPEN TAND CLOSE GENTLY AND PROPERLY OUR CLASS

INCUBATOR

PART B. PREPARATION OF BACTERIAL SMEARS

Morphological studies of bacteria can be done by examining either live or killed (fixed cells).
Preparation of live bacterial cell can only demonstrate the outline and structural arrangement of
the cells. A more detailed study of the internal structures of the cell can be done on stained,
however, they must be properly attached or fixed to the glass slide and killed. The application of
stain to unfixed organisms removes them completely from the slide. This exercise will guide you
in the preparation of bacterial smears for staining. Procedure from liquid media.
Broth culture of Serratia marcescens and Staphylococcus aureus prepared from part A will be
used in this exercise.
1. Wash a glass slide thoroughly with soap and water to remove the dirt and grease from the
surface, after rinsing the slide thoroughly, be sure to handle the slide by edge only.
2. Dry the slide thoroughly with a lens paper preferably or by careful blotting on folded
tissue.
3. On the dry slide, write the initials of the microorganism with a marking pen on the revers
e side of the glass slide.
4. Get the fluid medium (nutrient broth) and shake the culture sidewise with care and
transfer two loopful of the organism on the center of the glass slide.
5. Spread the organism over an area that is about the size of a ten centavo coin. In order to
remember where the smear is, you can encircle it with a marking pen on the reverse side.
6. Allow the slide to dry by normal evaporation of the liquid.
 7. After the smear has completely dried, pass the slide over the flame a few times to kill the
organism to the slide.
Procedure form solid media
Agar slant cultures of Serratia marcescens and Staphylococcus aureus prepared from part A will
be used in this exercise.
1. Clean the glass slide following the same procedure from liquid media. Dry it and label
properly.
2. Place a loopful of water on the slide (you can use a dropper of get a loopful of water from
the faucet with an inoculating loop).
3. With an inoculating loop pick up a very small amount of organism from the surface of the
agar slant culture and mix them with the water on the slide. Spread them in an area
similar to a ten centavo coin. Prepare a good emulsion.
4. Allow the slide to dry by normal evaporation of water.
5. After the smear has completely died, pass the slide over the flame to heat- kill and fix the
organism to the slide.
Part C. INTRODUCTION TO STAINING
The bacterial smears prepared in Part B are now ready for staining. The chemical substance
commonly used to stain bacteria are called dyes. These dyes can either be acidic or basic.
Cationic or basic dyes, in which the color group is associated with the cationic portion of the dye
molecule, are the most useful for general staining of bacterial cells. Basic dyes such as crystal
violet, methylene blue and safranin. Combine with molecular elements that are acidic in nature.
On the other hand, anionic or cationic stains are on those in which the color group is anionic
portion of the dissociated compound. Examples of the acidic dyes are acid fuschin and eosin.
These dyes combine with the cytoplasmic components of the cells that are alkaline in nature.
Simple staining
1. Place the slide containing the bacterial smears you prepared in Part B on the staining can.
REMEMBER TO STAIN IONLY IN THE DESIGNATED SINK IN THE
CLASSROOM.
2. Avoid contaminating your finger with dye, clip the edge of the slide with clothes pin.
3. Put enough methylene blue stain on the surface of the smear. Let the stain stay for 2-3
minutes and wash it gently with tap water.
4. Drain the slide of excess water and allow it to air dry.
5. Examine the methylene blue stained bacterial smears under the microscope. Practice
manipulating the microscope so that you will gain proficiency in focusing the
microorganism under the oil immersion objective. Compare the bacterial cells of S.
marcescens and S. aureus with respect to size, shape and uniformity of staining. Note
down your observation and draw sketches of the representative fields of each bacterial
smear.
6. Once you have focused and examined each bacterial smear, call your instructor to
evaluate your proficiency in preparing the stained smear. Feel free to ask question
relevant to the exercise(s) in class.
7. Repeat as often as necessary to obtain smear of proper density.

NOTES AND OBSERVATION

Staphylococcus Serratia marcescens
aureus Gram stain: Gram
Gram stain: Gram positive
positive Shape: Bacilli
Shape: Cocci Arrangement: Singly
Questions
1. What is the preparation of wet mount of bacterial cells recommended?
a. Clean the slide with distilled water.
b. The slide is dried with lens paper preferably and can be subsequently moving over
flame.
c. On the dry slide, label(Microorganism, date)
d. If bacteria is grown on a solid medium, a drop of distilled water is put at the center of
the slide. A loop is sterilized by heating it over and is cooled to room temperature,
then aseptically small portion of bacteria culture is scraped by the loop and
transferred to the portion of bacteria culture is scraped by the loop and transferred to
the drop of water.
e. If grown in a liquid medium, aseptically a drop of bacteria suspension is placed at the
center of the slide directly, using a flames-sterilized loop.
f. Petroleum jelly or similar material is applied surrounding the drop so that when the
coverslip covers the drop, evaporation and effect of air current is minimized.
g. A cover slip is kept in a scanning position near the drop of bacteria suspension with
one edge touching the slide.
h. Slowly cover slip is lowered, till it touches the surface of the drop.
i. The cover slip is released so that the drop of bacteria suspension spread in all
direction under the cover slip. Care should be taken so that no air bubble is formed.
j. Ready for observation under microscope.
2. What are the three advantages of examining stained bacterial smears?
a. It provides a better visualization of the examined bacteria. It gives the clarity of the
observed specimen.
b. It help for the identification of cellular structure.
c. It helps to identify the classification and specification of bacteria.
3. What is one disadvantage of stained bacterial smear?
a. The cell die and lose their natural shape and size due to heat- fixation as well as due
to exposure to chemicals.

Checked by:
EXERCISE NO.3 GRAM STAINING

The previous exercise on simple staining demonstrated to you the technique of demonstrating
bacterial cells using methylene blue. Although this technique of using simple stain like
methylene blue enabled you to observe the cells, it cannot categorize the cells by their reaction to
stain. It was Christian Gram, a Danish bacteriologist, who discovered in 1884 a staining method
that can divide the bacteria into two groups based on the ability of bacteria to retain the crystal
violet dye during decolorization with alcohol. The Gram stain referred to as differential stain
because of bacterium may be categorized by its reaction to this stain. THE STUDENT IN
MICROBIOLOGY MUST MASTER THIS STAINING TECHNIQUE AND BE ABLE TO
NOTE THE CORRELATION IN SUBSEQUENT APPLICATIONS.

Gram negative bacteria are decolorized by the alcohol, losing the purple color of crystal violet.
The crystal violet stain is also referred to as the primary stain. On the other hand, bacteria that
are decolorized by the alcohol and so retain the primary stain are described as gram positive. In
the case of the Gram negative bacteria, they are decolorized by alcohol and take up a second
stain (counterstain), safranin. The Gram negative bacteria stain pink to red. However, there are
bacteria that are Gram variable.

Materials
1 nutrient agar slant culture of Staphylococcus aureus
1 nutrient agar slant culture of Bacillus subtilis
1 nutrient agar slant of Psuedomonas aeruginosa
1 set of Gram staining reagents
Glass slides

Procedure
1. Each member of the group must perform this exercise alone to gain proficiency in
performing the Gram staining technique
2. Prepare smears of each culture on a glass slide ( a slide can accommodate 2-3 smears at a
time)
3. Heat fix each bacterial smear.
4. Add enough crystal violet to the smear; allow the dye to remain on the slide for two
minutes.
5. Rinse the slide with tap water gently. Add enough lugol’s iodine on the smear and let it
stand for one minute.
6. Rinse the slide with tap water gently. Add enough acetone-alcohol on the smear for 10
seconds. Thick smear will need more than 10 seconds. Immediately rinse with tap water.
 7. Add enough safranin to the smear for 30 seconds and rinse with tap water.
8. Air dry slide in a tilted position on the top of the folded tissue paper. Examine under OIL.
9. Sketch representative fields of each smear and indicate Gram staining reaction. Call your
instructor to show your smear under oil.

NOTES AND SKETCHES

100x 100x 100x

Staphylococcus aureus Bacillus subtilis Pseudonomonas aeruginosa
Gram positive Gram positive Gram negative
Cocci Bacilli Bacilli
Clusters Short chains Singly or pairs
Unknown: each student will be given an unknown culture. Prepare a gram stained smear of the
culture and record the Gram stain reaction and morphological features of the culture.
Unknown: Escherichia coli
Gram reaction: -
Morphologic feacture
Shape: Bacilli
Occurrence: individually and large clumps
Arrangement: single cell arrangement
Special feature if any:
Sketch of unknown culture:

100x
Escherichia coli
QUESTIONS
1. Give the principle behind the Gram stain
-The basic principle behind the Gram stain is most of the bacteria can be differentiated by
the gram reaction to the difference of the content peptidoglycan in their cell wall.

2. Are there any chemical differences between the cell walls of Gram positive and Gram
negative bacteria that might account for the differences in the rate of decolorization?
-Yes, due to the thickness of a peptidoglycan layer in the cell membrane, gram positive
bacteria has thicker peptidoglycan so it will retain the crystal violet stain during the
decolorization process, while the gram negative has thinner peptidoglycan and its easy to
decolorized.

3. List three factors that may affect the Gram stain reaction
-Age of bacteria (old cells loses to hold the ability to hold stain)
-Overcrowding of the smear
-Reaction time (important factor effect gram staining because of time of dye applied)

4. Define the following

a) Mordant
- It is a substance that intensifies the primary stain color.
b) Counterstain
-An additional dye to produce a contrasting background or to make clearer
distinction between different kinds of microorganism.
c) Gram variable
- A bacterium that is stained irregularly with Gram stain, it can be positive stain
or negative stain.

5. Suppose a smear prepared from a Gram positive control gave a Gram negative result.
Give three possible reasons for this incorrect reaction.
-The three reasons for this incorrect reaction are over decolorization, aged bacteria and
the properties of the organism.

Checked by:
EXERCISE NO. 4. ACID FAST STAINING
Another differential staining technic a microbiology student must learn to perform is the Ziehl-
Neelsen method. This method demonstrates the ability of bacteria to resist decolorization with
acid-alcohol. These bacteria are called acid-fast and members of the Mycobacterium group
exhibit this reactor. Ordinary bacteria or those not members of the Mycobacterium group are
considered non-acid fast bacteria they cannot resist the decolorizing action of acid-alcohol.
This technic was develop by Paul Ehrlich in 1882. He found that tubercle bacilli retained a dye
reagent composed of crystal violet and aniline in water ever after a wash treatment with a
acidified ethanol solution. Changes in the original methodology of Ehrlich was developed giving
rise to the Ziehl-Neelsen technic. One of the advantage of Ziehl-Neelsen method involves the use
of reagent with better preserving quality than those use by Paul Ehrlich. Basic fuchsin in aqueous
5% phenol is the primary staining reagent. Methylene blue is the counterstain, replacing
Bismarck brown, as used by Ehrlich.
Acid fast bacteria contain large amounts of lipid material in their cell wall which combines
tenaciously with the primary stain, carbol fuchsin. After decolorization, the counterstain
(methylene blue) is added to the bacteria to stain any material that is not acid-fast. In this staining
technic, the primary dye, acid fuchsin, is formulated with formalin to allow permeation of the
primary dye. Ethyl alcohol is prepared with hydrochloric acid to aid in decolorization of the non-
acid fast bacteria. The most important step in this method is to prevent complete drying of the
smear while staining with steaming carbol fuchsin.
Material
Nutrient agar slant of Bacillus subtilis
Agar slant of Mycobacterium spp.
Unknown culture
1 set of acid fast staining reagent

Procedures

This exercise will be performed by students individually.

1. Prepare half-fixed smears of B. subtilis and Mycobacterium spp.

2. Place the slides on the staining can and cover the slide with enough carbol fuchsin. Allow
the stain to stand for 1 minute.
3. Heat the preparation gently by passing the flame under the slides on the staining rack, or
you can hold the slide with a clothes pin and pass over a flame. Heat the slide until a
steam is created and maintain the steam for at least 5 minutes by passing the flame as
 needed. DO NOT ALLOW THE SURFACE OF THE SLIDE TO DRY BY ADDINGH
ENOUGH CARBOL FUCHSIN AS NEEDED. BE CAREFUL NOT TO OVERHEAT
THE SLIDE IN ORDER TO AVOID ITS BREAKING.
4. Allow the slide to cool on the staining rack for at least a minute. Rinse in tap water.

5. Add acid-alcohol on the slide until the thinnest areas are colorless. This usually takes
place 15 - 20 seconds or more depending upon the thickness of the smear.
6. Wash with tap water and counterstain with methylene blue for 1 minute.
7. Rinse the slide with tap water, air dry and examine under oil immersion. Acid fast
bacteria retain the primary dye pink to red in color; non-acid fast cells are blue in color.
8. Write down your observations and sketch representative fields of each culture.

Observation and sketches

Unknown culture: perform the same exercise using the unknown culture:
Acid Fast Non-Acid Fast

Sketch of unknown culture

QUESTIONS
1. Give the principle behind the acid-fast staining technique.
 Acid fast stain is used to distinguish Mycobacterium spp. and some Nocardia
species. In the cell wall of acid-fast bacteria, a lipid and waxy substance called
mycolic acid is present. The mycolic acid layer weakens and becomes porous when
carbol fuschsin enters the cell wall turning the bacteria into a red color. A non-acid
fast bacterium when decolorized by acid alcohol and counterstained by methylene
blue appears blue in color.

2. Distinguish between the old tuberculin (OT) and purified protein derivative (PPD).
 Old tuberculin is a solution of heart concentrated filtrate of tubercle Bacillus culture
grown on a special medium used for tuberculin test
  Purified protein derivative is a sterile solution of purified protein fraction precipitate
from a filtrate of tubercle Bacillus grown on a special medium used in tuberculin
test.

Checked by:

EXERCISE NO. 5 PREPARATION OF CULTURE MEDIA

Cultivation of bacteria and fungi requires creation of an artificial environment where all the
requirement for growth are met. This environment is otherwise called culture medium. The
components of the culture medium vary according to the growth requirements of bacteria. There
are those that can grow on a simple preparation composed of peptones and extracts of beef. A
culture medium that can support the basic needs of ordinary bacteria is called a basic medium.
The nutrient medium (either liquid or solid) that is commonly used in laboratory is called the
nutrient broth or agar. Frequently the components of nutrient broth serve as a culture medium
base, to which other components maybe added to encourage the growth of more fastidious
organism or allow the identification of certain microorganisms, or inhibitors may be introduced
to provide selective growth condition for forms not affected by their inhibitors.

The basic requirements of all microorganisms include the following: water, carbon, nitrogen,
minerals and growth factors.

Water. Like other living organisms or components, water is very major component, 70-85%. It is
essential component which is responsible for transport of nutrients, secretions, excretions and all
biochemical reactions rely on water. Because of this vital role of water it is essential to
incorporate it in culture medium.

The quality of water used in preparing media is very important. Hard tap water, high calcium and
magnesium ions should not be used. Insoluble phosphates of calcium and magnesium may
precipitate in the presence of peptone and beef extract. DISTILE WATER IS ALWAYS USED
TO PREPARE CULTURE MEDIA IN THE LABORATORY.

Carbon. Bacteria can be divided into two groups according to their source of carbon. Those that
can use the carbon in carbon dioxide for the synthesis of all cell components are classified as
autotrophs. If they must have one or more organic compounds for their carbon source they are
called heterotrophs. In addition to organic sources of carbon, heterotrophs are also dependent on
carbon dioxide. If this gas is completely excluded from their environment their growth is greatly
retarded, particularly in the early stages of starting a culture.

Energy. Chemosynthetic autotrophs oxidize simple inorganic substances for energy. This
substance maybe incorporated in the medium in the form of nitrate or sulfide. The
chemosynthetic heterotrophs need an organic source of energy and the usual substance that is
incorporated not the medium to provide this glucose. The amount of energy yielding ingredients
in media for chemosynthetic type is on the order of 0.5%.

Nitrogen. Heterotrophs get their nitrogen from amino acids intermediate protein compound such
as peptides, peptones and proteoses. Beef extract and peptone, as used in nutrient broth, provide
the nitrogen need for the heterotrophs grown on this medium.

Minerals. All microorganism require several metallic elements such as Na, K, MG, Fe, Zn, Cu, P
and Co for normal growth. The amount required for bacteria are very small.

Growth Factors. Any essential components of cell material that an organism is unable to
synthesize from its basic carbon and nitrogen sources is classified as being growth factor. These
may include amino acids and vitamins. Many of the heterotrophs are satisfied y the growth
factors present in beef extract. More fastidious organisms require enrichment media such as
blood agar for ample growth factors.

Hydrogen ion concentrations (pH). The growth of organism in a particular medium may be
completely inhibited if the pH of the medium is not within the limits required by the organisms.
The enzymes of microorganism are greatly affected by this factor. Since most bacteria grow best
at around 7 or slightly lower, the pH of the medium is usually adjusted to pH 6, 8 before
autoclaving.

Year ago, culture media were prepared from various raw materials. The preparation of these
media involved the use of many ingredients and so the process was tedious. Fortunately, at
present there are dehydrated media that are available that only need basic reconstruction with
water, pH adjustment, dispensing and sterilization.

PREPARATION OF NUTRIENT AGAR/BROTH (A DEMONSTRATION)

The preparation of nutrient broth or agar will be demonstrated in class. All the materials needed
in the preparation of the media will be available in class for your study. Take note of availability
of the media, source, etc. Observe carefully the essential steps followed in the preparation of
basal media.

1. Formula for nutrient broth:

a. Beef extract 3g
b. Peptone 5g
c. Distilled water 1000 ml
To prepare nutrient agar, add 15 grams of agar.

2. The beef extract and peptone will be weighed and placed in a flask and the required
distilled water is then added.
 3. The ingredients are brought into solution either through stirring or gentle heating

4. The pH is then determined in a known aliquot of the media by a pH meter or by one of

the colometric methods, and adjusted to a desired range using 1.0 N NaOH. The total
amount of 1.0 N NaOH required to adjust the entire amount is calculated, added, and
again the pH is re-checked using a pH meter. This final preparation is called nutrient
broth/agar.

5. To prepare agar, 16g is added (1.5 % concentration) to the above solution.

6. The nutrient broth/agar are brought into solution completely by heating (agar dissolves at
97⁰C and remains in a liquid state until it is cooled to approximately 40⁰C at which
temperature it will solidify.

7. The hot nutrient broth/agar is then dispensed into flask or test tubes, plugged with cotton
(or screw cap loosely), put into test tube baskets, sterilized in an autoclave.

Here are some recommended tips in preparing media

1. Prepare media under aseptic conditions as much as possible to prevent contamination

2. Follow the recommendation of the manufacturer of the dehydrated medium whenever

preparing the medium

3. As soon as the tubes of media have been filled with enough broth/agar they must be
plugged as soon as possible. At present there are many types of plugs that are available in
the market like caps of plastics (color coded) and aluminum. They are available in the
market like caps that are easy to use because they are simple put on top of the tubes.
There are tubes with screw caps. However, the use of cotton plug is still widely used
especially in the Philippines. In our laboratory class, you will be handling most of the
time cotton-plugged tubes. It will be demonstrated to you how to construct a good cotton
plug. A well-constructed cotton plug will extend about 1 to 1 ¼ into the tube; protrude
about ¾ inch out of the tube, and hold firmly in the tube so that it does not get
accidentally pulled out during normal handling.

4. As soon as the tubes of media have been stoppered they must be sterilized. The
organisms present on the walls of the test tubes, distilled water and dehydrated medium
will begin to grow within a short time at room temperature and so destroy the medium.

5. Before autoclaving the media, be sure to have labeled them properly (using special
masking tape and permanent marking pen) with the name of the media prepared, and date
prepared.
 6. When using the autoclave, do not overload it. Check if the temperature reaches 250⁰F
(121.6⁰C). Small loads may take only 10-15 minutes while a full load may require 30
minutes for complete sterilization.

7. If the nutrient agar will be in the form of slant, lay the tubes in a slant (1/2 inch diameter)
under the plugged ends. This should be done immediately after removing from the
autoclaving.
8. Let all sterilization media cool down at room temperature and keep them (if they will not
be used) inside a refrigerator (4⁰C). It is recommended to prepare media that will be used
within 1-2 weeks’ time.

9. Note down all your observation during the demonstration.

NOTE AND OBSERVATION
Continuous stirring

Photo source:
https://www.ebay.com/itm/PROFESSI
ONAL-18L-STEAM-AUTOCLAVE-
STERILIZER-TATTOO-DENTAL-LAB-
EQUIPMENT-/262788374164
The test tubes that contain the
nutrient agar are autoclaved for 10 -
15 min. at 121.6⁰C

Let it cool
down
QUESTIONS

1. What is the agar concentration (%) of :

a. Agar slant – 1.5% to 2%
b. Semi solid medium – 0.5% or less

2. What are the advantages of agar-agar in comparison with the sue of gelatin as a
solidifying agent in the media :
a. Provides a solidified medium to observe growth patterns.
b. Allow for surface area for isolating bacteria
c. It doesn’t melt at normal incubation.

3. Compare the following type of media

Types of media Description/indication(s)

Basal medium This media supports the growth of non-
fastidious bacteria.
Enriched Contains the nutrients required to support
the growth of wide variety of organisms,
usually by adding blood, serum or egg.
Enrichment This type of media enhances the growth of a
specific microorganism that grows in a small
population. This amount of bacteria can
easily be unnoticed that is why enrichment
culture are necessary.
Selective They are designed to suppress the growth of
unwanted bacteria and encourage the growth
of the designed microbes.
Differential It eases in distinguishing different colonies
of microorganisms.
Maintenance Satisfactory maintenance of the viability and
physiological characteristic of culture
overtime.
Synthetic or defined Energy sources are provided to support the
 microbial growth of the bacteria. These
energy sources are carbon, nitrogen, sulfur,
phosphorus, and any organic growth factors
the organism is unable to synthesize

Checked by :

EXERCISE NO. 6 CHARACTERIZATIONS OF BACTERIAL CULTURES

After completing this exercise, you are expected to:

1. Be able to distinguish basic features of bacterial culture on agar plates, agar slants and in
broth.
2. Be able to recognize the advantages and limitations of culture characteristics in the
identification of bacterial species.

Materials
The class will be provided with 3 sets of the following: bacterial grown in agar plates, agar slant
and broth
Staphylococcus aureus
Bacillus subtilis
Pseudomonas aeruginosa
Serratia marcencens
Escherichia coli
Mycobacterium spp.
Proteus vulgaris

Procedure

Each student will perform this exercise individually.

1. Examine the different cultures and note down the selected characteristics of bacterial
growth on the plate, agar slant and broth of each organism. Try to compare each other.
2. Be careful not to disturb (by shaking) the broth cultures, as this may dislodge the surface
growth.
3. Complete the table in the notes and observation part. If you were able to recognize
features other than those indicated, note them on the table.
4. Consult your instructor if you have doubts as to what is the appropriate description of the
culture you are examining. You can actually use terms which you think would be more
descriptive and at the same time others will understand also.
Questions

1. Are the cultural features studied in this exercise equally important in identifying a
bacterial species? If not, which one are the most important?
 Yes, the cultural features studied in this exercise are equally important in
identifying a bacterial species.

2. Which of the following cultural characteristics are subject to mutation?

 The cultural characteristic subjected to mutation are the pigment and the amount
of growth including the size colonies.

Examination of colonial morphology of a pure culture of a bacterial species must include

the following

1. Pigmentation of the colony (ies) or color

2. Surface characteristics
a. Dry or powdery
b. Rough
c. Smooth or glistening
d. Wrinkled
3. Shape of colony
a. Circular or round
b. Irregular
c. Filamentous
d. Granular
e. Rhizoid
f. Punctiform
4. Estimated size of colonies (preferably in mm)
5. Form edge of colony
a. Curled
b. Lobate
c. Filamentous
d. Serrate undulate
e. Entire
6. Forms of elevation
a. Raised
b. Umbonate
c. Flat
d. Convex
e. Pulvinate
f. Subsurface
7. Odor
8. Imparts pigment to agar medium
DIAGRAMS OF BACTERIAL COLONY ONFIGURATION/CHARACTERISTICS
COMMONLY RECOGNIZED

Examination of broth cultures must consider the following

1. Surface
a. Pellicle – thick membrane like growth covering the entire surface of the broth
b. Membranous- thin membrane-like growth covering the entire surface of the broth
c. Flocculent- made of floating adherent masses of bacteria
d. Ring type- growth characteristically dense around the broth surface

2. Subsurface
a. Turbid- cloudy
b. Granular- small particles are seen
c. Flocculent- small masses are seen floating around
d. Flaky- large particles are in suspension

3. Sediment
a. None
b. Granular-small particle
c. Flocculent-small masses
d. Flaky- large particles
e. Viscid

4. Amount of growth
a. None
b. Slight or scanty
c. Moderate
d. Abundant
e. Pigmentation
DIAGRAM OF SELECTED FEATURES OF BROTH CULTURES

Examination of agar slant cultures must consider the following

1. Amount of growth
a. scant or slight
b. Moderate
c. Abundant
d. None

2. Color

3. Opacity
a. Opaque
b. Transparent
c. Translucent (partially transparent)

4. Form
a. Filiform; characterized by uniform of even growth (even) along the lie of inoculation
b. Echinulate- margins of growth exhibit toothed appearance (pointed)
c. Beaded- separate of semiconfluent colonies along the line of inoculation
d. Effuse- growth is thin, veil like, usually spreading
e. Arborescent- branched, tree- like
f. Rhizoid- root like in appearance
DIAGRAMS OF SELECTED FEATURES OF AGAR SLANT CULTURE

NOTES AND OBSERVATION: AGAR PLATE CULTURES

Organism Color Shape Size Edge Elevation Surface Pigment

B. subtilis White Round 4mm Serrate Umbonat Smooth Cream
e
E. coli Cream Round 4mm Undulate Raised Wrinkled Yellow
Mycobacterium
P. vulgaris
P. aeruginosa Cream Round 5mm Entire Umbonat Smooth Cream
e
S. marcencens White Round 3mm Entire Flat Dry Very
light
green
S. aureus Cream Filamentous 4mm serrate Raised dry Cream

AGAR SLANT CULTURES

Organism Growth Color Opacity Form Pigmentation
B. subtilis Abundant Cream Opaque Effuse Cream
 E. coli Scanty Yellowish Translucent Filiform Yellowish
Mycobacterium
P. vulgaris
P. aeruginosa Moderate Cream Opaque Effuse Cream
S. marcencens Scanty Cream Opaque Effuse Cream
S. aureus Abundant Cream Translucent Effuse Cream

BROTH CULTURE
Organism Surface Subsurface Sediment Growth Color
B. subtilis None Translucent Granular None Yellowish
E. coli None Turbid Granular None Yellowish
Mycobacterium
P. vulgaris
P. aeruginosa Flocculent Granular Granular Scanty Cream
S. marcencens Flocculent Translucent Granular Abundant Cream
S. aureus Flocculent Flocculent Flocculent Scanty Yellowish

Notes and sketches

AGAR PLATE CULTURES

Bacillus subtilis Escherichia coli Staphylococcus aureus

Salmonella spp.

AGAR SLANT CULTURES

Bacillus subtilis Escherichia coli Staphylococcus Salmonella spp.

aureus

BROTH CULTURES
Checked by:

EXERCISE NO. 7: PURE CULTURE TECHNIQUE

Bacteria are naturally found in mixed populations in their natural habitat. It is only in
very rare situations that they are found as a single species. They have to be separated as a single
species in order to study their individual morphology and physiological characteristics. The
technique that are included in this exercise are used to cultivate pure cultures of a particular
bacterial species. A pure culture refers to a culture in which each cell is a direct descendant of
the same single cell in a culture comprised of cells of only one species.
There are different methods of getting a pure culture from a mixed bacterial population.
Two most commonly used techniques used in the laboratory are the streak method and pour plat
(or loop dilution). Both methods involved thinning out the organism so that the individual
species can be selected from the others.
After completing the exercise, you are expected to:
1. Be able to isolate individual colonies from mixed cultures by means of the streak-and
pour-plate methods
2. Recognize the advantages and disadvantages of the steak-plate and pour plate methods
PART A. STREAK-plate technique
The steak plate method was originally developed by two bacteriologist, Loeffler and
Gaffky. The streak-plate method or “streaking out” of a culture is a standard way of obtaining a
pure culture in initial isolation, or of checking cultures to detect possible contamination. The
technique involves the spreading of a single loopful of inoculum over a surface of an agar plate
in such a way as to separate the inoculum into a single cells or small clumps in the course of
spreading. After proper incubation, the cells so separated will develop into separate colonies. An
isolated single colony will comprise a population of a single bacterial species. To further purify
this colony, a representative inoculum from a single colony can be picked and streaked again on
a new agar plate.
Materials
2 tubes of nutrient agar (kept at 50C)
2 sterile petri dishes
1 tubes of mixed culture of Serratia marcencens, Staphylococcus aureus and Escherichia
coli

Procedures
1. Before performing this exercise, make sure you have cleaned and disinfected your
laboratory table top.
2. The petri dishes will be provided to you packed in paper. Carefully remove the paper
while avoiding to open it. Label the petri dish with your group number, section and date
on the bottom surface.
3. When you are ready to pour the melted agar on a petri dish ask from the instructor one
tube of melted nutrient agar (request only one at a time). Be sure to have a piece of tissue
paper when you get the tube from the water bath. Place the tube in the test tube rack or
plastic can and taken it to your table top. Let the tube to cool down to 45C (this is the
temperature that the skin can tolerate). BE SURE TO DO THIS CAREFULLT AND
SWIFTLY SO THAT THE AGAR WILL NOT SOLIDIFY BEFORE YOU POUR IT.
4. Using the aseptic techniques, remove the cotton plug with melted agar (45C) and flame
the mouth of the tube.
5. Using the left hand, carefully open the petri dish and pour the melted agar medium into
the bottom of the dish and cover immediately.
6. Put the petri dish on a level surface and let it stand until the agar solidifies.
7. While waiting for the agar to solidify, prepare a gram-stained smear of the culture. Label
the slides properly and set it aside. You can also perform this step after you have
performed the steak-plate and pour-plate techniques. Examine the slide under oil
immersion and record observations.
8. Perform the quadrant streak method on one plate and the radiant streak method on the
second plate. Follow the steps for each method
a. Spread the organism over a small area near the edge of the dish (area).
b. Sterilize the loop and let it cool. Make 5-6 streaks from the area 1 into area 2.
Stay near the edge of the dish.
c. Sterilize the loop again and allow it to cool down.
d. Make 6-7 streaks from area 2 into area 3.
e. Flame the loop again and let it cool down. Make as many streaks from area 3 into
4. The remainder of the medium is streaked out.
 f.
Flame the loop again before putting it down.
g.
Spread the organism over a small area near the edge of the dish. This is area 1.
h.
Flame the loop and allow it to cool down for about 5-10 seconds.
i.
From the edge of area 1 make 7-8 streaks to the opposite side of the dish.
j.
Flame the loop again and let it cool down. Cross streak over the last group of the
streaks.
k. Flame the loop again before putting it down.
9. ALWAYS INCUBATE AGAR PLATES IN AN INVERTED POSITION AT 37C for
18-24 hours unless directed otherwise.

PART B. POUR PLATE METHOD

This technique was originally developed by brilliant bacteriologist, Robert Koch. This
technique consist of (1) cooling melted agar (42-45C), and (2) inoculating the agar medium with
an inoculum just prior to pouring it into a sterile petri dish. Thus, the bacteria are distributed
throughout the agar medium and trapped in position as the medium hardens. The solidified agar
medium restricts the movement of bacteria from one are to another. Bacterial growth occurs both
on the surface and in the depths of the agar medium.
Materials
3 nutrients agar tubes (kept at 50C)
3 sterile petri dishes
Mixed culture used in Part A
Water bath set at 45-50C
Procedure
1. Label the 3 petri dished from 1 to 3. Get 3 agar tubes and label them from 1 to 3.
2. Cool the agar to 45-50C.
3. Inoculate tube 1 with one loopful of the cultures. Mix the organisms in tube 1 by shaking
the tube from side to side or rolling the tube between the palms of the hands. BE
CAREFUL NOT TO WET THE COTTON PLUG IN THE MEDIUM.
4. Inoculate tube 2 with one loopful from tube 1. Return tube 1 to the water bath. Mix tube 2
the same as in step 3.
5. Inoculate tube 3 with one loopful from tube 2. Return tube 2 to the water bath. Mix tube 3
the same way as in step 3.
6. Pour the melted agar from tube 3 on the petri dish 3. Make sure to observe aseptic
technique you followed in Part A.
7. Pour likewise the melted agar from tube 2 to dish 2 and tube 1 to dish 1.
8. After the medium has completely solidified incubate the dishes, inverted, at 37C for 18-
24 hours.

EVALUATION OF ISOLATION TECHNIQUES

 1. Examine your streak plate cultures and look for the colonies that are well isolated from
others. Compare the quadrant streak method from the radiant streak method.
2. Note down your observations and make sketches of the different colonies. Compare the
appearance of the colonies with those you observed in Exercise 6. Characterize the
different types of colonies (make sure you have 3) using the recommended terms in
Exercise 6.
3. Examine your pour plate cultures and note how crowded the colonies appear on the first
plate as compared with second and third plates. Look for the presence of the three
different colonies. Indicate also if you observe the presence of surface and subsurface
colonies.
4. Sketch the colonies found in your pour-plate.

NOTES AND OBSERVATION

Streak plate-quadrant streak method

Pour plate technique

QUESTIONS
1. Did you obtain a suitable separation of the bacterial colonies making up the mixed
culture?
Yes, the colonies of bacteria are far apart or are isolated.
2. Which of the two technique was the easiest to perform? Why?
In my opinion quadrant streak is easier to perform because you only need few materials
and a steady hand.
3. Which do you prefer to perform, the quadrant or radiant streak method of streak planting?
Why?
I prefer the quadrant streak method because usually you will be able to get a few visible
individual colonies in just one plate if your streaking skills are good.
4. What is colony picking?
Colony picking is the process of selecting and getting a certain colony of bacteria out of
a
mixture and growing it as a pure culture.
5. Are there any disadvantages to streak-plate method? If none, explain why?
 Storage space. If you need to use usual methods for bacterial isolation your Petri plate
stack will grow significantly.
 Agar must be prepared ahead of time. If you don’t have the time of knowing your
sampling size prior to your lab work, this can be a hassle
 It can be time-consuming. You’ll need to sterilize your loop, streak a quadrant, and repeat
that process several times just for a single plate. If you need a lot of samples this will take
some time
PART C. VERIFICATION OF ISOLATES FROM THE STREAK PLATE.
Materials
Streak plate cultures from Part A
Glass slides
1 set of gram stain
Gram stained bacterial smears prepared in Part A.

Procedure
1. Look closely on the isolated colonies found in your streak-plate cultures (quadrant and radiant
method). Usually the isolated colonies are found on the area where the last streak were
performed.
2. Select the isolated colonies that are different morphologically and mark them with a marking
pen at the bottom surface of the dish. Encircle each colony and designate a number (1-3)
3. Characterize the three different colonies using the term you learned in exercise 6
4. Prepare gram stained smear of each colony. You can pick out the colony by gently touching
the surface of the colony with a sterilized loop.
5. Go back to your gram stained smears from those prepared from colonies picked out from the
streak plates.

SKETCHES AND OBSERVATIONS

Colony No. Colour Shape Size Elev’n Gram stain/
shape
1.Escherichia coli yellow rod 4µm umbonate (-)
2.Staphylococcus cream cocci 4µm raised (+)
aureus
3.

QUESTIONS
1. Compared with the gram stained smear of the mixture. Did your gram stained smears
of the isolated colonies contain the bacterial forms similarly?
Yes, it contained the bacterial forms similarly.

2. Were you able to detect the presence of contamination?

No, we were not able to detect the presence of contaminants.

Checked by:
Reference:
Miller, C. (2007). Book review. Manual Therapy, 12(3). Retrieved November 23, 2018, from
https://www.researchgate.net/publication/257380059_Laboratory_Manual_in_General_Micr
obiology_For_Undergraduate_Students_Short_Version.
EXCERCISE NO.8. BACTERIA MORPHOLOGY
After completing this exercise, the student is expected to
1. Know and be able to recognize the basic shapes and arrangement of bacteria.
2. Be able to describe the basic morphological features of bacteria stained with Gram stain.
Students of Microbiology are expected to be able to recognize the basic morphological features
of bacteria. The use of the Gram stain as an important corollary to this knowledge of bacterial
morphology as used as a tool in the classification of bacteria. Successful identification of a
bacterial species relies heavily on the correct description and interpretation of Gram stain
reaction.
The general morphological shapes of bacteria were first described by Anton van Leeuwenhoek in
the late 1600’s. Aside from these differences in shape, definite patterns in cellular number +/-
and arrangement are known to exist among different bacterial species. The basic morphological
forms of bacteria include:
1. Spherical of cocci form
May be arranged in pairs-diplococci
Chains of 4 or more-streptococci
4 bacteria in square arrangement-tetrad
Irregular group like grape clusters-staphylococcus
8 cocci in cuboidal packet-sarcinae

2. Rod shaped or bacillus form (bacillary)

 May be arranged: singly
In pairs-diplobacilli
In chains- streptobacilli
3. Spiral shaped or coiled
Flexible, wavy with several coils-spirochete
Rigid coils with one or more curves-spirilla
Short, incomplete coils-vibrio,comma shaped
4. Pleomorphic-varied shapes
Materials
Agar slant and broth culture of
Streptococcus spp.
Staphylococcus aureus
Escherichia coli
Bacillus subtilis
Corynebacterium pyogenes
1 set of Gram staining reagents
Glass slides

Procedures
Each student must perform this exercise individually.
1. Prepare gram stained smears of the agar slant and broth cultures of each bacterial smears.
You can place 2-4 smears in one slide. MAKE SURE TO LABEL THEM PROPERLY.
2. CHARACTERIZE THE CELLS YOU OBSERVE UNDER THE OIL IMMERSION
BASED ON THE TERMS FOUND IN THE FIRST PART OF THIS EXERCISE.
There are additional terms used to describe bacillary forms such as
1. Length of rod-short, very short, medium-sized, long very long
2. Thickness of rod-plump, thin or slender, very thin, regular
3. Ends-rounded, straight or flat ends
4. Sides-straight or flat ends.

3. COMPLETE THE TABLE BELOW.

Microorganism Shape Arrangement Gram reaction Sketch of the
cell
Streptococcus cocci Chains Gram positive
sp.
 S. aureus cocci Clusters Gram positive

E. coli bacilli Singly Gram negative

B. subtilis bacilli Chains Gram positive
C. pyogenes Pleomorphic or Singly, short Gram positive
cocci chainis or V
shaped pairs

Checked by:

EXERCISE NO. 9 BACTERIAL ANATOMY

This exercise will enable the student to study the gross anatomy of selected bacteria
limited to those components that may be seen with an ordinary light microscope. When you
perform the gram stain, the component of the bacteria that will be studied are the capsule, spore,
flagella, and granules. A bacterial species may not possess all these components.

PART A: THE CAPSULE

After completing this exercise, the student is expected to

1. Be able to carry out a negative staining
2. Be able to differentiate between bacterial capsule and artifacts

Some bacterial species are surrounded by a pronounced gelatinous or slimy layer called a
capsule. It is believed that all bacteria have some amount of slim layer but most of the time the
layer is not sufficiently thick to be discerned. Some consists of a polysaccharide and others
appear to be water soluble so the students is advised to be very careful not to excessively wash
the smear.

Materials
24-48 cryticase soy agar (BA) and litmus milk culture of
Klebsiella pneumonia
Pasteurella multocida
Bacillus subtilis
Unknown culture

Two methods will be performed in this exercise, Anthony’s method and Negative staining
technique
ANTHONY’S METHOD
1% aqueous solution of crystal violet
20% solution of CuSO4.5H20

Procedure
1. Prepare an air-dried smear from the cultures. CAREFULLY DO NOT FLAME THE
SLIDE.
2. Put enough 1% aqueous solution of crystal violet on the smear and let it stand for 2
minutes.
3. Wash off the stain with 20% copper sulfate.
4. Drain and air dry. Examine under oil immersion. The cells should appear dark blue
and if capsulated, there will be a blue violet colored structure around them.
5. Prepare the sketches of the representative fields under the microscope and note down
the agar plate features of each culture.

NEGATIVE STAINING TECHNIQUE

Indian ink (pelican brand) or nigrosine
Sterile toothpick

This method consists of mixing the organism in a small amount of India ink or nigrosine
and spreading them over the clean slide. India ink or nigrosine do not adhere to the organism, but
they do obliterate the background. When examine under the microscope, if the microorganism is
encapsulated it appear transparent in a darkened background. This technique is useful to
determine the cell shape, size, presence of capsule but reveals nothing about the structure inside
the cells. Since no heat is applied in this technique, the cell size is more precise than with those
stained and heat fixed.
NOTE THE STAIN IS DRAGGED OVER THE SLIDE NOT PUSHED

Procedure
1. Place a loopful of India ink suspension on a clean slide (thoroughly cleaned) and add
a small amount of culture material. Using your dissecting needle (inoculating needle)
thru to dispose the material into the India ink.
2. Place the edge of another slide (with even edges and clean) against the India ink
suspension so that the fluid is drawn along the edge towards the opposite side. Now,
push the slide away from the India ink. If you have not used too much nigrosine or
India ink, the smear will be thick at the starting end and feather thin at the finished
end.
3. Allow the slide to air dry. DO NOT HEAT THE SLIDE.
4. Examine the slide under the oil immersion. Select that part of the smear that shows
cell distinctly with a dark background. The capsule will appear as a clear zone.
5. Draw sketches of the representative fields under the microscope.
 6. Compare the two methods as to the ease of performing the technique and ability to
demonstrate the capsules.
7. Using the sterile toothpick, remove a small amount of material from between your
teeth. Use this material for preparing another negatively stained smear. Draw sketches
of the representative fields under the microscope.

NOTES AND OBSERVATIONS

Anthony’
Organism Color Size Shape Edge Elevation Negative
s
Purple
K. cell with Purple cell
0.5-2
pneumoni White Bacilli Undulate Umbonate light blue with clear
µm.
a stained halo
capsule
Clear halo;
P. 0.3-1.0 Blue
Yellow Bacilli Flat Raised dark
multocida µm. violet
background
White to 0.7-0.8 Blue
B. subtilis Bacilli Undulate Umbonate
cream µm violet
Dental
- - - - - - -
material
QUESTIONS

1. What is the principle of the negative stain?

The principle of the negative stain is to use an acidic dye (e.g nigrosine or India
ink) which will steadily give up a hydrogen ion (proton) and the chromatophore of the
dye becomes negatively charged and will repel the bacteria’s negatively charged cell
surface. The glass slide will stain but the bacterial cells will not and will show up as a
clear spots against a dark background.
2. Relate the colony characteristics to capsule formation as observed microscopically.

Capsule formation give the diffused character of the microorganism in regular

stain appear as a rod or colloid colonies.

3. What functions are attributed to the capsule?

Capsule protects the bacteria from phagocytosis due to the presence of high
molecular weight polysaccharides which makes the bacteria very slippery. And due to
the capsule containing water it also protects the bacteria from desiccation. These help
the bacteria t adhere to surfaces and makes the bacteria resistant to complement
invasiveness.

4. Can the presence of the capsule on the pathogen always be correlated with virulence?
Explain your answer.
 Yes, the presence of the capsule of the pathogen is always correlated with
virulence. The capsule enhances the ability of the bacteria to cause a disease via
protection from phagocytosis, which enables the bacteria to perform its action without
being affected by macrophages when inside the host body. These are the first to be
known as virulence determinants of microorganisms (bacteria). Capsule is the most
phenotypically expressed virulence factor.

5. Are all capsule demonstrated in stained smears? Why?

No, some staining techniques can’t properly demonstrate capsule. Heat fixing
procedures that is required in other staining techniques can cause the capsule of
bacteria to shrink due to water it contained.

PART B: THE ENDOSPORE

After completing this exercise the student is expected to:

1. Be able to carry out a standard procedure for the demonstration of bacterial endospores
2. Be able to detect the presence of bacterial endospores in a culture

Endospores (or simply called spores) are actually cell forms that are produced by two
species of bacteria belonging principally to the genera Bacillus and Clostridia. The spores are
called endospores since they are formed within the bacterial cell, one endospore per cell. The
endospores are most resistant of the resistant cell forms known. Spore forming bacteria like
Bacillus and Clostridial species are widespread in nature and are very important agents of decay
and some may be potential pathogens by virtue of their capacity to form extremely powerful
toxins.
Ordinary dyes used in staining bacteria like methylene blues are unable to stain the
endospores because the dyes do not penetrate the spores out. Once stained, however, special
techniques of spore staining, the dye is retained by the spore and cannot be removed easily.
There are occasions when one perform the Gram staining techniques spores can be seen in the
smear.

Two methods of spore staining will be used in this exercise

1. Hansen’s method
2. Schaffer-Fulton method

Materials
48 hour cultures (nutrient agar slant) of Bacillus subtilis and Escherichia coli
Gram staining set

Hansen’s method
Staining set composed of
Carbol fuchsin
 5% acetic acid
Methylene blue

Schaffer-Fulton method
Staining set composed of
5% aqueous malachite green
0.5% aqueous safranin
Procedure
1. Prepare bacterial smears of B. subtilis and E. coli in one slide side by side. Prepare one
for each staining method. Fix the smears with gentle heat.
2. Prepare also the wet mounts of each organism on a slide and examine with the high dry
objective. Note that the spores are more refractile than the vegetative cells. Sketch what
you observe.

HANSEN’S METHOD
1. Stain the fixed smears with steaming carbol fuschin for 5 minutes
2. Decolorize with 5% acetic acid until the field is light pink for about 15 seconds
3. Rinse gently with tap water
4. Counterstain with methylene blue for 2 minutes
5. Rinse with tap water, drain, air dry, and examine under oil immersion
6. The spores will stain pink to red while vegetative cell will be blue
7. Make sketches of the representative fields under the microscope

SCHAFFER-FULTON METHOD
1. Put enough malachite green (cover whole surface) on the smear and allow the dye to
stand for one minute
2. Cover the surface with a slide-sized piece of tissue paper
3. Het to steaming with a flame and continue heating for 5 minutes. Do not allow the
stain to dry while steaming by adding enough stain when drying is noticed along the
edge of the slide
4. Let the slide cool down and wash it gently with tap water.
5. Put enough safranin on the smear and let it stand for one minute
6. Rinse with tap water, drain, and air dry. Examine under oil immersion lens.
7. The endospore will stain green and the cytoplasm is pink.
8. Draw sketches of the representative fields under the microscope.
9. Examine your smears carefully and take note of the size and appearance of the spores
and vegetative cells. Distinguish between free spores and spores still inside the
vegetative cells. Note the location of the spores within the cells. Note also their shape
and how they affect the shape of the cell.

PREPARE GRAM STAINED SMEARS OF EACH ORGANISM. Compare your gram stained
smears with those prepared using the two methods. Note their differences.

NOTES AND OBSERVATIONS

 Microorganism Sch-Fulton method Hansen’s method Gram stain
B. subtilis Green/ + Pink/+ Blue/+
E. coli Pink/- Blue/- Red/-

QUESTIONS:
1. What are the two functions of endospore?
The endospores primary function is to ensure the survival of the bacterium
through stressful periods in the environment. Another function is to enable the bacteria to
lie dormant for extended periods of time until the environment are favorable.

2. Briefly sketch the general feature of a bacterial spore cycle.

PART C. GRANULES

The reserve materials may accumulate in bacterial cells in the form of insoluble granules
under certain growth conditions. These granules may vary in size, chemical constituents and
amount. The type of granules is dependent on the kind of bacterium involved and its growth
conditions. Bacillus species when grown in carbohydrate rich media with low levels of nitrogen
accumulates reserves as large granules of polymerized betahydroxybutyric acid (or poly-beta-
hydroxybutyric acid) and these stains with sudan black. On the other hand, there are granules
composed of polymerized meta-phosphate and are sometimes called volutin. There are certain
members of the genus Corynebacterium that contains these granules.
Metachromatic granules stain with methylene blue, appearing as dark blue granules in light blue
cell.

QUESTIONS:
1. What are the functions of these granules?

They are used by the bacteria as storage for its nutrients. These nutrients are used
by the bacteria as energy-source. Polysaccharide granules are the storage form of glucose
while polyphosphate granules are the storage form for inorganic phosphates. Poly-β-
hydroxybutyrate granules are the reserve carbon and energy source.
 2. Will you be able to determine the nutritional condition of the bacteria grown in a
specified medium? Explain your answer in terms of examination of smear stained with
Loeffler’s methylene blue solution.

Yes, it is possible to determine the nutritional condition of the bacteria grown in a

specified medium through the appearance of the granules. They are formed when there is
abundance if nutrients in the medium. Its absence indicates low nutrients in the
environment or medium. Metachromasia is observed when polymerized polyphosphates
(granules) are accumulated in high concentration inside the cell.

PART D. FLAGELLA

There are certain bacterial species that can move by the aid of certain appendages. The
most common appendage in bacteria is the flagellum. It is a filament-like structure found on the
surface of a bacterium are really too small to be seen with a light microscope. While it is difficult
to observe they can be seen when enough dye and mordant are made to form around them, thus
increasing their diameter. Flagella attaining is quite difficult to perform even in the hands of a
specialist, and so it will not be attempted as a class exercise.

Although it is difficult to stain the flagella, there are ways of determining whether a
bacterial species is able to move (motile). Motility is a property of many bacteria and confers an
important biological property essential in its identification. There are two ways of indirectly
showing motility of an organism, namely, (1) hanging drop preparation and (b) growth patterns
in motility media, like the SIM or sulphide-iron-motility medium and the TTC
(triphenyltetrazollum chloride) medium.

After completing this exercise, the student is expected to:

1. Able to prepare and examine microscopically hanging drop specimens.
2. Be able to distinguish between motility in Brownian movement.
3. The covers to be able to distinguish the flagella from stained smear.
4. To be able to know how to properly inoculate and interpret changes in media used to
differentiate motile bacteria.

Materials
 Hollow ground slide, cover slip
 Petrolatum
 Log phase of Proteus vulgaris, Staphylococcus aureus, Escherichia coli, and Bacillus
subtilis in nutrient broth
 4 tubes of SIM
 Inoculating loop and needle

HANGING DROP PREPARATION

 1. Clean thoroughly a hollow ground slide by washing it with soap and water first. Blot
it dry carefully with tissue paper and then wipe it with lens paper. Do the same thing
with a cover slip.
2. With the use of a match stick, apply a film of petrolatum on the 4 edges of the
coverslip on a piece of tissue paper with a greased side.
3. Place a loop full of a broth culture on the centre of the coverslip on a piece of the
coverslip. DO NOT SPREAD THE DROP.
4. Place the hollow ground slide in the depression side down, over the coverslip so that
the drop is in the centre of the hollowed-out area and the petrolatum adheres to the
slide. Turn the preparation right side up. The drop must hang from the cover slip in
the concavity without touching the bottom or side of the depression.
5. Examine the preparation first under low power objective lens to locate the edge of the
drop. Focus just inside the edge and change to high dry lens for more detail. DO NOT
FOCUS DOWNWARD. Rather, lower the body tube until the tip of the low power
objective lens is almost touching the coverslip. Then raise the tube slowly and bring
the field into focus. Adjusting the light as necessary. NEVER ATTEMPT TO SET
THE OIL IMMERSION LENS IN HANGING DROP PREPARATIONS.
6. Do not confuse Brownian movement, brought about by the molecular bombardment,
with true motility, characterized by definite movement and considerable distance
covered relative to the size of the bacterium.
7. Examine each culture using the hanging drop method and determine which one is
motile.

8. Note your observation.

INOCULATION OF SIM MEDIUM

1. Observing aseptic techniques, inoculate each of the microorganism into each tube of
SIM with a single stab of the inoculating wire. Limit the stab to half the depth of the
semi-solid agar.
2. Incubate the culture at 37°C.
3. During the next laboratory period, examine the cultures for the diffuse growth
emanating from the stab line. Describe the appearance of the growth. Indicate
whether the culture is motile or non-motile on the table.
4. Correlate these results with those derived by the hanging drop.
SKETCHES

NOTES AND OBSERVATIONS

Hanging drop: SIM medium: Motile/

MICROORGANISM
motile/nonmotile nonmotile
P. vulgaris Motile Motile
E. coli Motile Motile
B. subtilis Motile Motile
S. areus Nonmotile Nonmotile

QUESTIONS
1. Differentiate flagella from pili.

Flagella are long, thin appendages free at one end and attaches to the cell at the
other end. They extend from the interior of the cell body. Flagella are mostly composed
of flagellin protein. On the other hand, pili are short, thick, straight, hair-like surface
appendages. They act as the receptors of some viruses. Pili originate from the plasma
membrane and are mainly composed of pilin protein.

2. Aside from using the flagella for movement, what are the other functions or use are
associated with them?

Aside from locomotion function of the flagella, it also acts on contributing on

bacterial virulence. Flagella are important as adhesive organelles. They are used to sense
chemicals and change in temperature since they are sensitive to these.

Checked by:
EXERCISE NO. 10 PHYSIOLOGIC CHARACTERISTICS OF BACTERIA

So far, you have learned the basic morphological and cultural characteristics of bacteria
that are used as the bases for the identification of bacteria. These two characteristics are not
enough to make a definitive identification of a particular bacteria species. The species
determination need also the definition of the biochemical (or physiological) characteristics of the
unknown. Although the bacterium seems to be very simple microorganism being single-celled, a
completely physiological study would be difficult. In this course, we will be limiting the study to
those physiological characteristics basically used for bacterial identification. The knowledge
learned in this exercise is very essential to the study of pathogenic bacteria that you will meet in
the second (advanced microbiology) semester. Just like learning how to be proficient in
performing the basic aseptic handling, staining, and cultivation techniques in the earlier part of
the course, you will be expected to:
1. learn about some of the physiological characteristics of bacteria
2. know how to accumulate information about an organism being identified
3. be able to know the principles behind the tests
4. be able to perform and interpret tests for selected aspects of metabolism
 There are many and diverse biochemical reactions that make up the synthetic and
energetic metabolism of bacteria. The detailed discussion on bacterial metabolism will be
discussed in the lecture part. Most of the concepts that will be taken in the laboratory part will be
a recapitulation of whatever had been covered in the lecture. Most of the tests that you will
encounter in this exercise are actually detection of the by-products or end-products of the action
of enzymes on substrate provided in the growth medium. The major substrate that will be used in
these tests are carbohydrates and proteins.

Part A. Carbohydrate Metabolism

Materials:
24-hour nutrient broth culture of: Bacillus subtilis Escherichia coli
Proteus vulgaris
Fermentation media containing phenol res as indicator & Durham tube:
Glucose or dextrose broth
Lactose broth
Sucrose broth
Reminders about inoculation of media:
1. Remember to observe aseptic techniques
2. Label each tube of medium with the name of the medium, name of the organism
inoculated with and the group number and section
3. Before inoculating the medium, observe carefully the appearance and color of the media.
Be sure to acquaint yourself with the different media in their uninoculated appearance so
that you will be able to appreciate the change(s) that will take place after incubation

Procedure:

1. inoculate one set of fermentation media (dextrose, lactose, and sucrose broths) with the
organism using sterile inoculation loop
2. incubate all tubes at 37˚ until the nest lab session
3. observe that reactions that will develop in the fermentation tubes. Record your findings in
the Notes and Observation Section by indicating the following:
A for aid production = medium turns yellow form pink
Alk for alkaline reaction = medium turned darker pink / red color due to
protein breakdown w/o utilization of sugar
G for gas production as indicated by presence of bubble in the Durnham
tube
AG for both acid and gas production
Ng for no growth as indicated by absence of turbidity in the medium

NOTES AND OBSERVATION

Glucose Lactose Sucrose
 Uninoculated

Bacillus subtilis A K A
Escherichia coli A (+) G (+) A (+)
Proteus vulgaris A A A

QUESTIONS:

1. What information can you derive from the reactions of the different bacteria on the
fermentation media? Explain your answers.
 The reaction of the different bacteria on the fermentation media shows the age of
media used and the age of the organism because it indicates the appearance of the
fermented culture.
2. Name two precautions you must consider before reading the reaction on the fermentation
media?
 Two precautions that must be considered are; a) age of organism and b) age of
media before reading the reactions on the fermentation media.
3. Differentiate fermentation from respiration?
 Fermentation is the anaerobic conversion of foodstuff to particular products while
respiration refers to the energy yielding metabolic process that does not involve
oxidation or electron transport of oxygen or other electron receptor.

You have learned from the above exercise that fermentation media with the Durnham tube will
indicate the ability of microorganism to ferment a sugar. This technique will not indicate what
fermentation products are produces. Specifically using glucose broth, a glucose fermenter
produces a large quantity of organic acids (mixed acid fermentation) or produces 2,3 butanediol
instead. The methyl red (MR) and Voges-Proskauer (VP) test re used instead to make this
differentiation.

METHYL RED – VOGES-PROSKAUER (MRVP) TESTS

After performing this test, the students is expected to:

1. know the basic principle behind the MRVP tests

2. know the indication of these test
3. know how to perform the MRVP test
4. know how to interpret the MRVP test results

MIXED ACID FERMENTATION. That re certain bacteria that ferment glucose to produce large
amount of lactic, acetic, succinic, formic acid, carbon dioxide, hydrogen and ethanol.
Enterobacteria such as those belonging to genera Escherichia, Salmonella and Proteus are mixed
acidic fermenter. It is the accumulation of these acids in the medium that lowers the pH to 5.0
and less. So if the reagent methyl red (which is actually an indicator) turns red. These bacteria
are generally producers of gas because they produce the enzyme formic hydrogenylase which
splits formic acid into equal parts of carbon dioxide and hydrogen. This enzyme accounts for the
low concentration of formic acid in the medium.

BUTANEDIOL FERMENTATION. Members of the Enterobacter and Serratia and other

bacteria produce large amount of 2,3 butanediol and ethanol instead of a mixture of acids from
glucose. These end-products are responsible for less lowering the pH in the medium with the
result they give negative reaction the in the MR test (yellow in color instead of red). This 2,3
butanediol cannot be detected but its precursor, acetylmethyl carbine, can be easily detected
using Barritt’s reagent composed of alphanapthol and OH. When this reagent is added to 48 hour
culture in MRVP medium and allowed to stand for some times, the medium change to pink to
red in the presence of acetylmethyl carbinol (or acetoin). The acetoin and 2,3 butanediol are
present together, the test is considered valid although it is an indirect method for detecting2,3
butanediol production. This test is called Voger-Proskauer test.

Materials:
Nutrient agar slant culture of Escherichia coli
Nutrient agar slant culture of Enterobacter aerogenes
4 tubes of MRVP medium
MRVP reagent

Procedure:

1. Inoculate two tubes of MRVP medium with each organism

2. Incubate at 37˚C for 48 hours
3. To test for mixed acid fermentation (MR test) add 3 to 4 drops of each methyl red to each
tube. The medium especially the top part will become red immediately. This is the
positive reaction.
4. To perform the VP test, add 10 to 20 drops of the VP reagent to each tube. Shake the tube
and let them stand for about 1 hour (remove cotton plug). A positive VP reaction id pink
to red.
5. Record your results and observation. Be sure to call your instructor to help you determine
if you have the positive and negative reaction.

NOTES AND OBSERVATION

MR test VP test
Escherichia coli Red (+) Yellow (-)
Enterobacter aerogenes Yellow (-) Red (+)

QUESTIONS:
 1. What is the key intermediate substance in the metabolism of carbohydrate by bacteria?
 The key intermediate substance in the metabolism of carbohydrate by bacteria is
the pyruvate.
2. Glucose is the main carbohydrate compound that serves as a carbon source of bacteria.
Enumerate four different pathways wherein glucose is converted to the key intermediate
substance (answer to # 1)?
 The four different pathways wherein the glucose is converted to the key
intermediate substance includes:
o Interberg-Drekens or Hexose Monophosphate (HMP) pathway
o Embden-Meyerhof-Parnas (EMP) pathway
o Enter-Doudroff (ED) pathway
o Phosphoketolase (PK) pathway

3. Why is it essential that cultures must be grown in MVRP medium for at least 48 hours
before testing?
 It is essential that cultures must be grown in MRVP medium for at least 48 hours
before testing in order for the bacteria to ferment the available carbohydrate in the
media.
4. Why is it necessary to remove the cotton plug in the VP medium after adding the VP
reagent?
 It is necessary to remove the cotton plug in the VP medium after adding the VP
reagent to maintain the aeration and to expose the medium to atmospheric
oxygen. It enhances the VP reaction.

TRIPLE SUGAR IRON AGAR REACTION (TSI)

After completing this exercise, the student is expected to:

1. know the principle behind the TSI medium

2. know how to inoculate properly the TSI medium
3. know how to read and interpret the TSI reactions

The TSI medium contains three types of sugar (dextrose, lactose and sucrose) ferrous substrate,
incorporated with basal medium to sustain bacterial growth. This medium will indicate if the
inoculum can utilize these three sugars and iron. Since this medium contain a pH indicator like
the fermentation medium you encountered previously, production can be detected by change of
color of the medium. Utilization of the ferrous substrate is indicated by the production of black
color in the medium. The black color is due to the hydrogen sulphide. In order to appreciate the
color change in this medium, the student is advised to note carefully the color of the
uninoculated medium.

Materials:
Nutrient agar slant culture of Escherichia coli, Proteus Vulgaris and
Pseudomonas
Aeruginosa
 Demonstration of Salmonella inoculated in TSI slant

Procedure:

1. Observe the appearance of the TSI agar slant and note them down.
2. Using the inoculating needle, inoculate the TSI agar slant by stabbing the center of the
slant through the butt. Your instructor will demonstrate to the class how to do this
technique. BSURE TO MAKE A CLEAN STAB INTO THE MEDIUM AND AVIND
MOVING YOUR NEEDLE SIDEWISE WHEN INSIDE THE AGAR. After stabbing
the butt, withdraw the needle upon reaching the surface of the slant streak it. This
technique is call the stab and streak method.
3. Seal the tube aseptically and label each tube properly with the name of the organism etc.
4. Incubate the tube at 37˚C and read your culture within 18 hours and NO MORE THAN
THIS TIME PERIOD. The time of reading the reactions is critical because of color
changes after this time will be misleading and inaccurate.
5. Read the result or note down your observations on the changes that took place in each
tube by the following table of reactions and interpretation accompanying this exercise.
The dorsal surface of the surface of the medium is called slant while the medium beneath
it is called the butt. The reaction noted in the slant is written down as the numerator while
the reaction (so in the butt are written as the denominator, e.g. A/AG meaning acid
slant/acid butt with the gas production.

RECORDING AND INTERPRETATION OF TRIPLE SUGAR IRON REACTION

APPEARANCE RECORDED AS
INTERPRETATION (S)
Acid slant/acid butt A/A glucose, sucrose, and/or lactose
fermented
Acid slant/acid butt, gas A/AG; A/A glucose, sucrose and/or
lactose fermented with gas/prod’n
Alkaline slant/acid butt Alk/A only glucose fermented
Alkaline slant/acid butt, gas Alk/AG; Alk/A only glucose fermented with
gas
prod’n
Alkaline slant/acid butt, gas blackening Alk/AG, H2S only glucose fermented with
gas and
hydrogen sulphide prod’n
Alkaline slant/acid butt, blackening Alk/A, H2S only glucose fermented with
hydrogen
sulfide produced
Acid slant/acid butt blackening A/A, H2S all three sugars fermented but
no gas
prod’n hydrogen sulphide produced
Alkaline slant/alkaline butt Alk/Alk no sugar fermented but
organism grow
on medium, proteolysis
No change/no change NC/NC organism cannot grow in this
medium
Alkaline slant only/no change alk/NC uses peptone aerobically

Yellow color of the medium = acid production

Alkaline color medium = alkaline color due to peptone degradation
Gas production (hydrogen and carbon dioxide) is indicated by presence or cracks,
bubbles in the
medium, indention of medium
Blackening of medium = hydrogen sulphide production (black precipitate which is
product of
hydrogen sulphide gas plus ferric ammonium citrate)

PRECAUTION TO BE OBSERVED WHEN READING TSI REACTIONS

1. TSI reactions must be read and interpreted within 18 hours of incubation period. Of read
earlier, a false positive result of acid/acid may occur, or the carbohydrate fermented may
not have yet produced enough acid change the pH indication. A TSI tube read after 24
hours may give a false positive result of alkaline/alkaline due to utilization of peptones,
resulting in an alkaline pH.
2. An organism may produce so much hydrogen sulphide that the acidity produced in the
butt is completely masked. However, if 2S is produced in the butt is completely, an acid
condition does exist in the butt even not observable and should be recorded as such.

Results and Observation

1. Write down the reactions you observed on the TSI slants

Organism Butt Slant Gas prod’n H2S prod’n Recorded as

E. coli A/K A/K + - A/AG or K/K
P. vulgaris A/A A/K + + K/K
P. aeroginosa A/K A/K - - K/K
Salmonella A/K A/K + + K/K

Questions:

1. Explain the basis of the results obtainable with the TSI slant.
 
The pH indicator is phenol red, the ferrol sulfate of ferric ammonium citrate with
sodium thiosulfate detects hydrogen sulfide production. The medium is red if
uninoculated, when an alkaline reaction occurs yellow, it is an acid reaction.
2. Can mixed culture be used to inoculate a TSI slant? Explain your answer.
 No, because different organisms have different reaction thus if mixed culture is
used to inoculate a TSI plant, there would be different or misleading result.

Part D. Protein Metabolism

In the previous exercise you studied the use of carbohydrates by microorganism. These
substances are the prime sources of energy and carbon skeletons for the synthesis of substances
of the microorganism. Another important substance utilized by the microorganism is the protein.
Proteins are the nitrogen-containing compounds that involved in the enzymatic and structural
activities of the microorganisms. This part of the metabolism will consider the several reactions
of the microorganisms such as protein degradation, urease activitiy and citrate reduction. The
ability of microorganism to degrade an intact protein is facilitated by the use of enzymes.

After completing this exercise the student is expected to be able to perform and interpret test for
selected aspects of protein metabolism.

Materials:

1. 24 hr nutrient broth cultures of Esherichia coli, Proteus vulgaris, Pseudomonas

aeruginosa, Staphyloccocus aureus.
2. One set of media containing: 1 nutrient gelatin, 1 tryptone broth, 1 nitrate broth and 1
urea broth.
3. Reagents for each test will be provided after incubation of the media(next class period)

Method:

1. Each group will be assigned a particular microorganism to inoculate one set of media.
Make sure to observe and note down the results of other groups for comparison of results.
2. Inoculate one set of media containing the nutrient gelatin, tryptone broth, nitrate broth,
and urea broth. With assigned culture, use the inoculating needle to stab the nutrient
gelatin medium. Use the inoculating loop to inoculate the broth media.
3. Incubate the media at 37˚C for 24 hours.
4. After incubation, determine the results of the test as follows:
a. Gelatin hydrolysis
i. Place the gelatin tube into a beaker with iced water/refrigerator (4-
10˚C) for 15-30 minutes.
ii. Gelatinase activity is indicated when the medium remains liquid
after refrigeration.

b. Indole test
i. Add 5 drops of Kovac’s reagent to each tube of tryptone broth
 ii. The development of the red color is positive reaction.

c. Nitrate reductase activity

i. Add 1 drop of sulfanilic acid (solution A) and 1 drop of dimethyl-
alpha-naphthylamine (solution B) to the nitrate broth.
ii. Nitrate reduction is indicated by a red/brown color.

d. Urease Activity
i. The development of the red color in the urea broth is a positive
reaction.

5. Record all your observations and results on the following table:

Organism Gelatinase Trytophanase Urease Nitrate Citrate

Reductase
E. coli + + - - -
P. vulgaris + + - - variable
P. + - - - -
aeroginosa
S. aureus + - - - -

Questions:

1. How can the nitrogen metabolism tests be used in the classification of the bacteria
selected in this exercise?
 Nitrogen metabolism tests can be used in the classification of bacteria, if there
will be specific changes that the media or indicator induces the test that will
specifically show the type of bacteria.
2. Discuss the virulence of bacterial pathogens on the basis of proteinase activity.
a. The bacterial pathogen that secretes proteinase has the ability to breakdown
protein substances that are available in the chord and connecting it to its
metabolic needs.

Litmus Milk Test

Milk is composed of materials that most bacteria require for growth. The addition of an
indicator, litmus, provides the investigator a means of determining an organism’s acid or alkali
production and oxidation and reduction activities. The characteristic reactions observe with
litmus milk are:

1. acid and acid curd formation

2. alkaline condition
3. rennin curd production
4. peptinozation
5. reduction
 6. gas formation

The production of acid by an organism is a function of its ability to utilize lactose, one of the
sugars most abundantly present in the milk acid production is demonstrated when litmus medium
changes from blue to red. If the organism is able to produce considerable amount of acid, this
may cause insoluble complex of calcium and csein to from resulting in a curd. An organism that
cannot attack lactose might utilize the milk protein as a source of nitrogen and carbon, and an
alkaline reaction might result. This is indicated in intensification of blue color in the litmus
medium. Of the amount of acid or alkali produces is low, no apparent change in the medium
maybe observed. However a rennin curd may develop even in low acid or alkali production in
the organism contains enzyme that can produce retracts and yields grayish liquids known as
whey. Either type of peptinozation (or solubilisation). Peptonisation is characterized by reduction
of curd size and the formation of a brownish liquid. This reaction can be observed easily when it
occurs alongside the curd. Reduction of the litmus by oxidation-reduction activities of bacteria
can be shown by the litmus color. This is quite apparent when the pink acid curd begins to turn
white. The change develop the bottom of the tube and moves upward. Another readily apparent
characteristic is gas production.

Materials:

1. 24 hour nutrient broth of; Escherichia coli, Proteus vulgaris, Bacillus subtilis
2. Litmus milk tubes, one control tube uninoculated.

Methods:

1. Inoculate the litmus milk with your assigned organism using inoculating loop/sterile
pipette
2. Incubate all tubes at 37˚C for 24 hours
3. Observe each culture using control litmus and record your results and observation on the
table;

Organism Acid Alk No Curd Peptonisation Reduction Gas

change
E. coli + - + Curd - - +
P. - - + Curd - - -
vulgaris
 B. + - - curd - - -
subtilis

Questions

1. Why does reduction usually begin at the bottom of the tube?

 Reduction usually begin at the bottom of the tube because the concentration of the
organism is larger at the bottom and also the concentration of available nutrient is at
the bottom of the tube.
2. What is the differential tube media?
 Differential media refers to the culture media that distinguished groups of organism
based on their differences of biological characteristic.

3. Described the two test beside from litmus milk that permits observation of several
characteristics of an organism.
 Triple Sugar Iron Agar Slant Reaction uses three different sugar and an indicator
incorporated in a slant. The change in the color of the slant indicates other changes, as
such the type of bacteria present.
 TSI also shows the presence of certain acid in inoculated medium.

Checked by

Exercise 11
ENUMERATION OF BACTERIA
One of the standard microbiological technique required in the study of bacteria is enumeration of
bacteria. Enumeration of bacteria is the determination of the number that are present in a given
unit of volume or area. Enumeration of bacteria is used also as a biological parameter of bacterial
growth. The quantification of bacteria is used also as a means of estimating the quality of water,
food, and other components of the environment. The method of choice of bacterial enumeration
depends on various factors such as kind of material to be examined, availability of equipment,
technical skills, etc.
A. QUANTITATIVE PLATING METHOD (Standard Plate Count)
This method involves the determination of the viable bacterial cells that are present in a known
amount of material. Basically, this method involves the serial dilution of the known amount of
material and known volume of this diluted material mixed into an appropriate liquefiable solid
medium. Each colony develops on the plate during subsequent incubation is considered to arise
from a single cell present in the original inoculum. Only dilutions with countable colonies are
selected.
Materials:
24 hour culture of E. coli
2 water blanks containing 99.0 ml
3 water blanks containing 9.0 ml
6 sterile Petri dishes
6 sterile 1.0 ml pipette
1 sterile 5.0 ml pipette
6 tubes containing melted nutrient agar for pour plating

Methods:
1. Study and follow the dilution and plating protocol to obtain the dilutions of 1/100,000,
1/1,000,000, 1/10,000,000.
2. Make sure to clean and disinfect thoroughly your bench top before proceeding to perform the
exercise.
3. Label the water blanks and the petri dishes with the corresponding dilution that will be made.
Only dilution of 10-5, 10-6, 10-7, will be plated.
4. When using pipette make sure to observe the following:
a) Hold the pipette with the index finger over the upper end
b) Handle the pipette aseptically and must not be laid over the bench top but placed
in a suitable container.
c) Follow the sequence of the use of the pipette.
5. Make sure to mix well the tube containing the diluent and the inoculum before taking out any
sample and subsequent plating.

DILUTION/PLATING PROTOCOL
Culture Dilution Pipette Plate #

1.0 ml 1 -
-2
99 ml water blank 1/100(10 ) 2 -
1 ml
99 ml water blank 1/10,000(10-4) 3 -
1 ml
9.0 ml water blank 1/100,000(10-5) 4 1
1 ml
1 ml
9.0 ml water blank 1/1,000,000(10-6) 5 2
1 ml
1ml
9.0 ml water blank 1/10,000,000(10-7) 6 3
1 ml

6. Using a sterile pipette 1.0 ml, withdraw 1.0 ml from E.coli broth culture and transfer to a 99.0
ml sterile water blank. Discard the pipette (# 1). Mixed the dilution blank well by shaking the
blank for at least 20 times (circle of 8 movement). Do this step every time you add a dilution
blank and withdrawn a sample from it.
Take a second sterile 1.0 pipette and, after mixing the dilution blank well, remove a 1 ml
sample to the second 99 ml sterile blank. Discard the pipette #2.
Using a third pipette transfer 1 ml of the sample from the mixed dilution blank and transfer it
to the third sterile dilution blank (99 ml). Mix well and discard pipette #3.
Complete the dilution scheme by following the protocol using the appropriate pipette to add
1.0 ml samples to duplicate. Petri dishes as well as making dilutions. Make sure that each
dilution blank is mixed well before sampling and that only those pipettes for a given dilution
are used for introducing sample into petri dishes.
7. After doing the dilutions and transfer to the petri dishes, do the pour plating method (as you
have learned from previous exercise). Let the agar cool down and solidify. Invert the plates
and incubate at 37oc. examine your plate on next lab period.
8. Lay out plates on the table in order of dilution and compare them. SELECT THE PLATE
THAT HAS NO FEWER THAN 30 COLONIES NOR MORE THAN 300 COLONIES FOR
YOUR COUNT. Plates with less than 30 or more than 300 colonies are statistically unreliable.
If your last plate contains more than 300 colonies, record your data as TNC or too numerous to
count.
9. Using a Quebec colony counter, count the colonies making sure you do not count the same
colony twice. Count every colony, regardless of how small or insignificant.
10. Get the average of the two counts per dilution. Calculate the number of organism per ml
of the culture by multiplying the number of colonies by dilution factor.

For example: if you counted 200 colonies on the 1/1,000,000 dilution plate,
200 X 1,000,000 = 200,000,000 bacteria per ml or this can be
expressed also as 2 X 108 bacteria/ml
*Use only two significant figure. For example, if your average count was 253, use
250.
11. Record your result on the results and observation.
RESULT AND OBSERVATIONS:
Dilution 1/100,000 1/1,000,000 1/10,000,000
Count

Calculation of number of cells/ml:

B. TURBIDIMETRIC ANALYSIS
The standard plate count is the most reliable in determining bacterial concentration of a given
volume of sample because only viable cells are counted. However, this method is limited to a
few samples at a time because of the obvious necessity of preparing a large number of media
plus extra labor involved. In case a large quantities of sample have to be examined, the use of
another method is recommended. Once method is indirect method using the turbidity of
suspension. The extent to which light is scattered is proportional to the concentration of bacteria.
A spectrophotometer is used to measured turbidity as a function of light lost in passage through
the uniformly suspended culture compared to a control medium. The intensity of the light after
passing the culture is measured by the defection of the galvanometer of the spectrophotometer.
The turbidity readings must be standardized against some other estimate numbers such as plate
count or direct count to arrive at a standard curve which then may be used repeatedly to relate
turbidity to number with the same culture system. One disadvantage of this method is that both
dead and live cells are counted.
This exercise will be demonstrated in class but the method used will be given in this exercise.
Principle behind the use of spectrophotometer
A culture of bacteria acts as a colloidal suspension which will intercept light as it passes through.
Within certain limits the amount of light that is absorbed is directly proportional to the
concentration of the cells. A spectrophotometer has a photocell that can measure the amount of
light that passes through the culture. A light source in the instrument transmit a beam of white
light through the two lenses and an entrance slit into diffraction grating which disperses the light
into the horizontal beams of pf all colors of the spectrum. The spectrum of light falls on a dark
screen with a slit cut in it. Only that portion of a spectrum which happen to fall on the slit goes
through the sample. It will be a monochromatic beam of light. By turning a wavelength control
knob on the instrument. The light that get through the culture that activates a photo-tube, which
in turn registers percent transmittance on the galvanometer. The higher the percent transmittance,
the fewer are the cells in suspension. Before the turbidity can determined, the instrument has to
be calibrated with a tube of sterile nutrient broth to establish 100% transmittance. After it is
calibrated, turbidity, in terms of transmittance, is read by inserting a cuvette of the culture into
the sample holder of the instrument
To illustrate the direct proportional relationship between the concentration of the bacterial cells
and the absorbance of light you will measure the percent transmittance of various dilutions of the
culture you used in previous exercise. These value will be converted to optical density and
plotted in a graph.
Materials:
4 hour broth culture of E. coli
Spectrophotometer
5 ml pipette
Bottle of sterile nutrient broth (20)
Method:
1. Calibrate the spectrophotometer according to the instruction of the manufacturer.
2. In handling the cuvettes, keep the following points in mind:
3. Rinse the cuvette several times with distilled water to clean it before using it.
a. Keep the lower part of the cuvette spotlessly clean by keeping it free of
liquids, smudges and finger prints. Wipe with lint free tissue only.
b. Insert the cuvette into the sample holder with its index line registered with the
index line on the holder.
c. After the cuvette is seated, line up the index lines exactly
d. Handle these cuvettes with great care. THEY ARE VERY EXPENSIVE.

4. Label the cuvette near the top with 1:1, 1:2, 1:4, 1:8, 1:16
5. With a 5 ml pipette, dispense 3 ml of sterile nutrient broth into tubes 1:2, 1:4, 1:8, and
1:16.
6. Mix the contents in the 1:2 tube by drawing the mixture up and put into the pipette and
discharging into the tube 3 times.
7. Transfer 3 ml from the 1:2 tube to the 1:4 tube, mix 3 times and go on to the other tubes
in similar manner. Tube 1:16 will have 6 ml of diluted organism
8. Measure the percent transmittance values of these 5 tubes by inserting each one into the
sample holder and closing the lid. Record the readings on the Result and Observations.
9. Convert the percent transmittance values to optical (OD) using the formula:
OD=2 –log of percent transmittance
10. Records the OD values in the table and plot the values on a graph.

RESULTS AND OBSERVATION

Questions:
1. What is the breed method of enumerating bacteria?
 Direct/Viable
 A direct/viable method involves a standard plate count, in which repeated
dilutions of a sample are counted to calculate the count in the original sample.
 Indirect/Viable
Indirect/viable methods such as MPN (most probable number) involve making
a statistical inference about the microbe count based on patterns of growth.
 Direct/Total
The microbes are counted with the aid of fluorescent stains and dyes, which
make the microbes visible with the aid of a fluorescent microscope.
 Indirect/Total
Spectroscopy is a form of indirect/total enumeration, which involves
estimating the amount of microbes based on the amount of light passed through
the culture by a spectrophotometer.

2. What is Macfarland nephelometer? How is it prepared?

 Macfarland nephelometer- an instrument for estimating the number of bacteria in
suspensions used for calculating the opsonic index and for vaccines.
 Macfarland turbidity standards are prepared by mixing various volumes of 1%
sulfuric acid and 1% barium chloride to obtain solutions with specific optical
densities. 0.5 McFarland turbidity standard provides an optical density
comparable to the density of a bacterial suspension 1.5x 10^8 colony forming
units (CFU/ml)
Checked by:
 Exercise 12
Effects of Environment on Microorganism
Like any other living cells or entities, microorganism interact with the environment for its
growth and survival. There are many factors present in the environment that interact with.
Manipulation of these factors may be beneficial to a scientist in studying the factors that affects
microbial growth and survival. One of the benefits of knowing how to manipulate these factors is
the ability to control microbial populations of one particular group over another. There are many
factors in the environment that affects microbial population but this exercise will deal only with
three of them: pH, temperature and oxygen.
A. Effect of pH in microorganism
After completing this exercise, you are expected to:
1. Be able to recognize the influence of hydrogen-ion concentration on microbial growth.
2. Know the pH range in which microbial growth can occur.
The hydrogen ion concentration (usually designated as pH) influence most biological activities
of microorganisms. The optimal pH for microorganism, and the tolerance to departure from
optimal, vary considerably. Most microorganism grow best in pH near neutrality. Few
microorganisms grow below 4.0. Each organism has its own optimum pH for growth.
Comparatively speaking, the fungi are less sensitive to pH changes in the environment than
bacteria.
Materials:
18-hour nutrient broth cultures of: (screw cap)
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
Staphylococcus aureus
Nutrient broth (5ml each) adjusted to pH 5, 6, 7, 8, 9
Method:
1. Label each nutrient broth with the corresponding pH and culture assigned per group.
2. Shake well the culture tube and then carefully get on loopful aseptically and transfer to a
nutrient broth.
3. Incubate the tube at 37 C and examine them during the next laboratory period.
4. Make observations on the growth of each tube and record them. Estimate the growth by the
turbidity of the medium according to the following scale:
 No growth - or 0 Fair growth 2+/++
Slight growth + or 1+ Good growth 3+/+++

Results and observation

Microorganism PH of medium Growth pH for optimal
growth
Escherichia coli 5 + pH of 7
6 +
7 +++
8 ++
9 +
Pseudomonas 5 + pH of 7
aeruginosa 6 ++
7 +++
8 ++
9 +
Bacillus subtilis 5 + pH of 8
6 +
7 ++
8 +++
9 ++
Staphylococcus 5 ++ pH 4.2 to 9.3
aureus 6 ++
7 ++
8 ++
9 ++
Questions
1. What microbial activities does pH affect?
Very low or very high pH values will prevent microbial growth, therefore the microbial
activity that the pH affect is the growth of the microbes.
2. What affect does pH have on toxin production?
Toxin production is decreased as the pH is decreased. The effect of pH on toxin production
is directly proportional.
3. Why do high-acid food require less heat treatment for preservation in canning than low-
acid foods?
High-acid food require less heat treatment for preservation in canning because there is no
fear of Clostridium botullinum growth. To be safe, such foods only needs to reach
pasteurization temperature. These pasteurization temperatures are sufficient to kill all
microorganisms except for bacterial spores.
B. Effect of Temperature on Microorganisms
Temperature influence the rate of growth, rate of death, length of growth phases, total quantity
of cells, morphology and many other features of the growth of microorganisms. Below the
optimum temperature, the rate of growth is decreased, a few degrees above the optimum, cells
are killed. Microorganism that grows best at 25 to 35 C are mesophiles. Those microorganisms
that have an optimum temperature growth range between 50 to 60 C are called thermophiles.
Animal pathogens generally have an optimum temperature for growth of slightly above most
saprophytic mesophiles. The optimum temperature for growth for some bacteria may be altered
by continual cultivation at different temperature.
After completing this exercise, you are expected to:
1. Be able to explain resistance to heat of different bacteria.
2. Be able to outline and explain in experiment to isolate spore-forming bacteria from natural
materials.
Materials:
18 Hour nutrient broth culture of:
Escherichia coli Bacillus subtilis
Serratia marcescens Pseudomonas aeruginosa
5 nutrient agar plates
Water bath set at 80 C
Method:
1. Using your marking pen, divide each petri dish containing the nutrient agar into 4
quadrants. Label each quadrant with the organisms you will inoculate it with and the temperature
it will be incubated at. You can make codes for the culture and the temperature. The incubation
temperatures that will be used are 4 C, room temperature, 37 and 55 C.
2. On each assigned quadrant streak a loopful from the culture. To save time and effort, you
can streak each quadrant of each plate with assigned microorganism. For example, if you
assigned the code I to E. coli, streak all the quadrants labeled as I on the 4 plates. Proceed doing
the same procedure for the rest of the cultures.
3. After you have finished the inoculation of the plates, incubate them at the assigned places
for 4 C, room temperature, 37 (incubator) and 55 C.
4. On the fifth plate, streak the cultures subjected to 80 for 10 minutes in a water bath.
Incubate the inoculated plate at 37 C.
5. On the next lab period, examine the plates and compare the amount of growth. Record any
gross features such as color or colonial size that appears to vary with temperature.
Result and observation
Amount of growth/colonial features
Microorganism 4C Room 37 C 55 C
temperature
E. coli +++
Serratia
marcescens
Bacillus subtilis
P. aeruginosa
Indicate the amount of growth the same way you used in the previous exercise. Note down the
colonial features at each cultivation temperature.
Results of growing cultures after exposing to 80 C for 10 minutes:
E. coli
Bacillus subtilis
Serratia marcescens
P. aeruginosa
Questions
1. Give two reasons for heat resistance of bacteria.
Heat resistance of bacteria is due to the presence of bacterial spores. Concentration of DPA and
metal ions, the size of the spore core, and the protoplast-to-sporoplast ratiocoli the factors
affecting the heat resistance of bacteria.
2. How did Louis Pasteur produce a living vaccine against Bacillus anthracis infection?
Louis Pasteur made anthrax bacteria avirulent by growing them at an unusually high
temperature. These attenuated organisms were then used as a vaccine.
C. Effect of Oxygen on Microorganism
Respiration in all organism is necessary for the energy. A growing bacterial cell requires
energy for the many types of chemical reactions essential for growth, cell division, movement,
and other biological activities. Generally speaking, respiration is concerned with the oxidation of
metabolites. The release of energy in these oxidation reactions involves the transfer of hydrogen
from one substance to another. The metabolite which gives up the hydrogen is said to be
oxidized. The hydrogen acceptor, usually oxygen is said to be reduced in the process. These
oxidation-reduction reactions occur stepwise with the gradual release of energy.
Obligate aerobes grow only in the presence of dissolved oxygen and uses the oxygen in their
respiration processes. Many bacteria and fungi are obligate aerobes. On the other hand, obligate
anaerobes are those that either do not need oxygen in their respiration processes or are inhibited
by traces of oxygen. Many bacteria and few fungi (mostly yeast_ can grow aerobically or
anaerobically and can shift respiration accordingly. These microorganisms are facultative
anaerobes.
After completing this exercise, you will be expected to:
1. Be able to distinguish aerobes, facultative anaerobes, and obligate anaerobes.
2. Be able to perform shake culture, paraffin plug technique and OF test.
Materials:
Broth culture of Clostridium sporogenes, E. coli, Pseudomonas aeruginosa
Shake cultures: 3 nutrient agar deeps (with screw cap) kept at 50 degrees
Paraffin plug technique: 3 tubes of brain-heart-infusion broth (previously boiled) class before,
tubes of melted paraffin
Thioglycolate Medium: 3 tubes of thioglycolate medium
O-F test: 6 sterile O-F medium, sterile paraffin oil
Method:
1. Before proceeding in performing this exercise be sure to familiarize yourself with the
different media that will be used. Take not specially the following: color and consistency.
LABEL ALL TUBES PROPERLY BEFORE PROCEEDING WITH THE EXERCISE.
2. Make sure to read the instruction first before the doing the exercise.
SHAKE CULTURE TECHNIQUE
1. Cool the nutrient agar deeps to about 45 C by carefully rotating them under cold running
water.
2. Inoculate each nutrient agar deep with one culture. Label properly.
3. Shake the tube gently by striking the tube with your fingers.
4. Cool the tubes rapidly under cold running water/put in a beaker with iced-water.
5. Incubate at 37 C until next laboratory period.
PARAFFIN PLUG TECHNIQUE
1. Inoculate each tube of brain heart infusion broth with one culture. Label the tube properly.
2. Remove the cotton plug/screw cap from the tube, tilt the tube and place the melted paraffin
on top of the medium using a sterile Pasteur pipette. The paraffin layer must at least 1/4 inch in
thickness. Replace the cotton plug/screw cap.
3. Incubate the tube at 37 C until the next laboratory period.
O-F Test
 Microorganism Open tube Closed tube Classification
Clostridium Alkaline (Green) Alkaline (Green) Anaerobe
sporogenes Non saccharolytic
(glucose not
metabolized)
Pseudomonas Acid (Yellow) Alkaline (Green) Oxidative
aeruginosa
E. coli Acid (Yellow) Acid (Yellow) Fermentative
Questions
1. What is the reason for heating the beef heart infusion broth before inoculation?
The reason for heating the beef heart infusion broth before inoculation, is to coagulate some
of the proteins, also for best results.
2. Differentiate:
Aerobe from anaerobe
Aerobe is a microorganism that grows in the presence of air or requires oxygen for
growth. While Anaerobe is a microorganism that grows without the presence of air, or requires
oxygen-free conditions.
Facultative from microaerophilic
Facultative is having the capacity to live under more than one specific set of
environmental conditions, as a bacterium that can live with or without the presence of
oxygen. While Microaerophilic is an aerobic bacterium that requires oxygen, but less
than is present in the air, and grows best under modified atmospheric conditions.
USE OF THIOGLYCOLATE MEDIUM
1. Inoculate each tube of the thioglycolate medium with the culture provided.
2. Incubate the tubes at 37 C until the next lab period.
OBSERVE THE REGIONS OF GROWTH IN EACH TUBE USING THE THREE
TECHNIQUES. RECORD YOUR OBSERVATIONS.
O-F TEST
1. Inoculate two rubes of the O-F medium with one culture
2. Label each tube properly
3. For each pair of O-F tubes, put sterile paraffin on top of one O-F medium just like you did
with the paraffin plug technique. Leave the other tube as is.
4. Incubate all tubes at 37 C until the next lab period.
5. Note the growth on the tubes and the color changes.
 If there is a change in color from blue-green to yellow in both tubes, the organism is both
oxidative and fermentative.
If the color change to yellow in the tube with the paraffin only and not in the other one, the
organism is fermentative only.
If there is color change to yellow in the tube w/o paraffin only and not in the other one, the
organism is oxidative only.
RESULTS AND OBSERVATIONS
Shake cultures: Illustrate the appearance of growth

C. sporogenes Pseudomonas aeruginosa Escherichia coli

Paraffin plug technique/Thioglycollate medium

Growth C. sporogenes Pseudomonas Escherichia coli
aeruginosa
Present
Absent

Checked by:
 Exercise 13 MICROBIAL CONTROL

Part A. Bacteriostatic activity on Dyes

After completing this exercise you are expected to:

1. Be able to recognize the inhibitory action of crystal violet.

2. Understand the selective and inhibitory nature of certain media.

Crystal violet has long been used therapeutically for moniliasis, or thrush, a fungus disease of the
mouth. At low concentration, crystal violet has been found to selectively bacteriostatic for gram
positive bacteria but not for gram negative. This effect can be explained in terms of differences
in the cell walls of these two groups of bacteria. Crystal violet interferes with the synthesis of
Staphylococcus aureus, both types of bacteria are prevented from growing.

Materials:

18 hour nutrient broth of Enterobacter aerogenes and Staphylococcus aureus

Mac Conkey agar plate
Nutrient agar plate

Method:

1. Using a marker pen, divide the bottom of the plate into two equal areas and label each
one with the name of the culture to be inoculated. Do those on both Mac Conkey and
nutrient agar plates.

2. Streak each culture on their designated sectors of the plates.

3. Incubate the plates at 37 until the next lab period.

4. Observe and record your findings

RESULTS AND OBSERVATION:

Sketch your results below:

Mac Conkey Nutrient Plate

Questions:

Differentiate:
1. Bacteriostatic from bactericidal
 Bacteriostatic stop bacteria from growing while bactericidal kill bacteria directly.

2. Selective from differential media

 Selective media allow certain types of organisms to grow, and inhibit the growth
of other organisms. Differential media are used to differentiate closely related
organisms or groups of organisms.

Part B. Antibiotic sensitivity Test

1. Be able to perform an antibiotic-sensitivity test on a bacterial culture.

2. Be able to distinguish the relative resistance and sensitivity of bacterial cultures to

selected antibiotics.

Antibiotics (anti: against; bios: life) are metabolic by-products of one microorganism that is able
to kill other microorganisms. The treatments of bacterial infections often relies heavily on the
use of bacterial infections often relies heavily on the use of some antibiotic as a
chemotherapeutic agent. The choice of the appropriate antibiotic is guided by clinical diagnosis
as to the probable nature of the infectious agent, and by the antibiotic testing of the etiologic
agent, once isolated and identified.

The knowledge of the antibacterial spectra of the various antibiotics is useful in selecting a
suitable chemotherapeutic agent. For example, penicillin is generally effective against Gram
positive organisms while streptomycin is usually effective against gram negative bacteria.
However, certain genera of bacteria frequently develop resistance to antibiotics resulting in a
need for antibiotic susceptibility testing.

The most common procedure for antibiotic sensitivity is the paper disc method. One of the most
commonly used techniques is Kirby-Bauer (1966). The interpretations of the sensitivity patterns
are made by comparing the sizes of zone of inhibition to a standard.
It is important to know that the result of the antibiotic sensitivity method is only meaningful if
the procedure is standardized. Therefore, the disc potency of the antibiotic, concentration of the
inoculum, and type of media used must be constant. Since the various chemotherapeutic agents
have different diffusion rates in agar, the zone size produced by one antibiotic cannot be
compared with that produced by another but only with the standard.

The microbial activity of an antibiotic can be determined by determining the smallest amount of
the agent is called the minimal inhibitory concentration (MIC). The exercise you will perform is
the Kirby-Bauer method using the agar-diffusion. This method involves the use of petri dish
containing suitable medium (Mueller Hinton) is the heavily inoculated with the test
microorganism whose antibiotic sensitivity is to be determined. Filer paper discs containing
defined concentrations of specific antibiotics are available commercially. The disc can be placed
individually on the agar surface using sterile forceps or by an automatic disc dispenser. The
preparation incubated for a definite period of time, during which antibiotics diffuse from the disc
into the agar. At some particular distance from each disc, the MIC for the antibiotic is reached.
The MIC’s are recognized by the presence of growth-inhibition (clear) zone surrounding the
various antibiotic disc used. The diameters of such zones can be measured with a ruler. The
results constitute an antibiogram.

Materials:

2-5 hour broth culture of E.coli in 4ml of trypticase soy of Mueller Hinton broth
2-5 hour culture of S. aureus in 4ml trypticase soy broth or Mueller Hinton broth
2 Mueller Hinton agar plates
2 sterile swabs
Pair of forceps
1-4 ml density standard (prepared by adding 0.5 ml of 15 Ba C12 to 99.5 ml of 0.36 NH2SO4)
Antibiotic discs:
Penicillin G 10 units
Ampicillin 10ug
Neomycin 30ug
Streptomycin 10ug
Tetracyclin 30ug

Method:
1. Compare the densities of the broth culture with that of the BaC12 standard. If
necessary dilute with sterile saline to a density visually equivalent to the standard.

2. Swab one plate with each of the broth culture provided. Your instructor will
demonstrate in the class the technique.
 3. Allow the surface of the plates to dry. Using the sterile forceps (sterilize by dipping
the forceps in alcohol and flame it over the Bunsen burner), place the discs at regular
intervals in the surface of the plates. Gently press the discs on the agar to ensure
contact.

4. Invert the plates and incubate at 37 until the next lab period.

5. On the second lab period, measure and record the MIC’s sizes in mm.

6. Interpret the result by comparing the zone sizes to those in the table that will be
provided in class.

Results and Observations

Zone size in Millimetres

Organism Penicillin Ampicillin Coke Zonrox

E. coli
S. aureus

Part C. Disinfectant use test

After completing this exercise you are expected to:

1. Be able to perform a disinfectant-use test with clinical instrument, selected

disinfectants and bacteria

2. Be able to interpret laboratory data obtained with a disinfectant-use test.

The effectiveness of disinfectants needs to be evaluated. The effectiveness of disinfectant is

actually a determination of the killing power of a given disinfectant against a specific
microorganism. It used to be before that the official method of testing disinfectants involved
comparison with phenol. The determination of the value called phenol coefficient was however
limited to testing phenol like compounds that exert bacteriostatic effects, and are not neutralized
by the subculture media used. Today there are many disinfectants that cannot be evaluated with
this test. As an alternative, a more suitable test for non-phenolic disinfectant was developed. This
is the use-dilution method. This method makes use of small glass rods which test organism are
dried for 30 minutes. The seeded rods are then exposed to the test solutions at 20°C for 1, 5, 10
and 30 minutes, rinsed with water neutralizing solution and transferred to tubes of media. After
incubation at 37°C, for 48 hours, the tubes are examined for growth.
In this exercise, you will follow the modified procedure of the use-dilution method to compare
the effectiveness of different disinfectants on two kinds of bacteria: a spore former, Bacillus
megaterium, and non-spore former, Staphylococcus aureus. Instead of the glass rod you will use
your dissecting/surgical instruments.

Materials:

Disinfectant solutions A, B, C, D and E placed in a beaker.

2 tubes of sterile water (5-10ml)
2 tubes of nutrient broth (5-10ml)
Nutrient broth cultures of S. aureus and Bacillus megaterium.

Sterile swab
2 sterile petri dishes
2 nutrient agar plates
Beaker containing disinfectant solution for disposal

Method

1. Select a surgical instrument that you will use in this exercise

2. Wipe a portion of each instrument with a swab wetted with the bacterial cultures assigned
to your group. Put the instrument on top of the inside of the petri dish. Let the instrument
air dry and put the swab to its beaker for disposal.
3. Select a disinfectant solution and immerse the instrument for 20 minutes
4. Rinse the instrument with distilled water and make sure to collect the rinse water on a
sterile petri dish.
5. Using a sterile cotton swab, wipe the rinsed instrument, making sure to take specimens
from all areas that might harbour microorganism.
6. Inoculate properly labelled nutrient agar plate with the swab using the streak method.
7. Taking another sterile cotton swab, dip into the rinse water and streak in on another
nutrient agar plate.
8. Incubate the two plates at 37°C until the next lab period.
9. Examine the plates and determine whether or not the instrument was sterilized and
whether or not the distilled water showed evidence of contamination. Record your
findings in the result and observation section.
10. Gram stain at least two colonial types that grow in the plates. Record the gram stain
reaction and morphology of the organism.

Results and Observations

Disinfectant Instrument Observation Gram reaction

Questions

1. Which bacteria survived the disinfectant? Why?

2. Based upon your results, should the distilled water have been contaminated? If it
wasn’t, why? What is the significance of distilled water contamination?

Checked By:
EXCERCISE NO.14 INTRODUCTION TO MYCOLOGY
After completing this exercise you are expected to:
1. Be able to recognize basic shapes and arrangement of fungi.
2. Be able to recognize the macroscopic and microscopic features of common molds and
yeast, with particular reference to asexual reproductive structures.

The study of fungi (molds and yeast) is called mycology. This field of microbiology deals with
examination of the morphological components and determination of their respective function.
Fungi are characterized by vegetative structures that elongate into branching filaments called
hyphae. These structures originate from specialized reproductive cells called spores, and may or
may not divide into sections by cross walls (Singular, spetum, plural, seta). Those hyphae
containing cross walls are called septae, while those without are nonseptate.
The continua branching and intertwining of fungal filaments results in the formation of visible
structure called mycelium that develops on surface is called the reproductive or aerial mycelium,
while the portion that penetrates the surface is called the vegetative mycelium. Some fungal
species have specialized structures that penetrates the medium to obtain food, they are called
rhizoids because of their characteristic root like appearance.
Spores arise from the aerial mycelia. These may develop asexually or sexually. The development
of these spores and their morphological features are used in the classification of fungi. Spores
arise in a variety ways. Some are produced in swollen structures called sporagia, and the spores
are consequently called sporangiospores. Others arise in the club shaped structure called the
basidia, and these spores are called basidiospores. Other spores grow from conidiophores and are
called conidia. Some fungi give rise to thallospores which develop from hyphae.
Yeast are another group of single celled organism that lack chlorophyll and exhibit characteristic
shapes ranging from spherical to ellipsoidal forms. They are larger than bacteria and so even at
low power they can be demonstrated. The usual mode of reproduction is by budding. Some do
not bud and separate from the mother and so the daughter cells do not separate, forming a pseudo
mycelium. On colonial morphology, yeast are usually buttery in consistency. There are fungi that
can assume both mycelia or yeast form. These are called dimorphic fungi.
This experiment will only be limited to the demonstration of the basic morphological features of
representative fungi. A more detailed study will be performed in the higher microbiology course.
A GUIDE IN THE STUDY OF A FUNGUS CULTURE (Haley and Gallaway, 1978)
A. Gross examination
1. Type of media used
2. Age of culture
3. Topography of colony-flat, with radial grooves
4. Pigment
A. Surface
B. Reverse
5. Texture of colony
A. Granular(gritty)
B. Powdery(soft)
C. Suede(velvety)
D. Glabrous(smooth)
E. Wooly(cottony)
6. Size of colony
A. Covers agar surface
B. Agar surface partially covered
B. Microscopic examination
1. Hyphae
a. Size in diameter
b. Pigmented 9 colorless, brightly colored, dematiaceous
c. Septated or nonseptated
2. Conidiophore
a. Microconidia(size and shape)
b.Macroconidia(size and shape)
3. Conidiophore
a.Simple
b.Complex
 4. Presence of chlamydospores, arthrospores, blastospores
5. Presence of asci and ascospores
6. Other significant features
USING THE ABOVE GUIDE, DESCRIBE THE DIFFERENT FUNGAL CULTURES THAT
WILL BE GIVEN IN THE CLASS

Aspergillus niger
Gross characteristics
1. Type of media used – Potato dextrose agar (PDA)
2. Age of culture – 7 days
3. Topograhy of colony – Globose, with radial grooves
4. Pigment surface – Black reverse Pale yellow or Pale-gray
5. Texture of colony- Woolly (cottony)
6. Size of colony - 40 - 50 mm
Microscopic characteristics
1. Hyphae
a. Size in diameter - 2.5- 8.0 µm
b. Pigment – Colorless
c. Septated or nonseptated - Septated
2. Conidiophore
a.Simple or complex - Simple
3. Conidia
a. Macroconidia- Globose, very rough, 4.0-5.0 µm
(Raper & Fennell, 1965; Samson, 1979).
4. Other features – Rough walled conidia, radiate and biserate conidia

Penicillium
Gross characteristics
1. Type of media used – Malt Extract Agar (MEA)
 2. Age of culture – 7 days
3. Topography of colony -
4. Pigment of colony – Greenish-blue reverse Red
5. Texture of colony – Velvety and Powdery
6. Size of colony – Covers agar surface
Microscopic characteristics
1. Hyphae
a.Size in diameter - 3 µm
b. Pigment - Colorless
c.Septated or nonseptated - Septated
2. Conidiophore: Simple or complex – Simple
3. Conidia: microconidia/macroconidia – Long dry chains, globose
4. Other features
Rhizopus
Gross characteristics
1. Type of media used - Potato dextrose agar (PDA)
2. Age of culture – 4 days
3. Topography of colony - Filamentous, branching hyphae that generally lack cross-
walls
4. Pigment surface- White becoming gray-brown on surface reverse Pale white
5. Texture of colony – Woolly (Cottony)
6. Size of colony – 5-8 mm

Microscopic characteristics
1. Hyphae
a.Size in diameter - 6-15 µm
b. Pigment- Brown
c.Septated or non-septated – Non-septated
2. Conidiophore- Simple
 3. Conidia - Globose, 175 µm with a flattened base, greyish black, powdery in
appearance
4. Other features: Presence of stolons and pigmented rhizoids, the formation of
sporangiophores.

Saccharomyces
Gross characteristics
1. Type of media used – Maltose Extract Agar (MEA)
2. Age of culture -
3. Topography of colony – flat
4. Pigment surface - reverse
5. Texture of colony - Smooth
6. Size of colony -
Microscopic appearance:
Single celled/Multiple celled – Single celled
Shape of cell – Ellipsoidal to elongated

Other features:

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