Lab Manual DMT 10023

FUNDAMENTAL
OF
DMT 1023
JABATAN TEKNOLOGI MAKANAN
NAMA :
NO PENDAFTARAN :
KELAS :
PENSYARAH :
DMT1023 - FUNDAMENTAL OF MICROBIOLOGY LAB MANUAL
Table of Contents
CONTENT PAGE
1 Laboratory Rules Procedures 2
2 Lab 1: Microbiology Laboratory 4
3 Lab 2: Sterilization Technique of Equipment 6

and Materials
4 Lab 3: Media culture preparation 8
5 Lab 4: Solid media inoculation technique 9
6 Lab 5: Culture transferring technique 11
7 Lab 6: Using Microscope 17
8 Lab 7: Simple Stains 23
9 Lab 8: Negative Stains 24
10 Lab 9: Gram Stain 25
11 Lab 10: Serial dilution 26
12 Lab 11: Determination of microbial counts 27

using pour plate and spread plate methods
FOOD TECHNOLOGY DEPARTMENT

2/32
Laboratory rules and procedures
A. Laboratory safety rules
1. Be always aware of safety precautions

2. Be familiar with every step in the procedure of the experiment
3. Before starting the experiment examine every piece of equipment, materials and
culture.
4. Be always alert regarding the hazards while handling the cultures of microorganism.
5. Remember the better ways to carry out experiments by the right procedure and
safety precaution.
6. Be aware of the safety of your friends and yourself.
7. Be prepare to protect yourself from accidents while in the laboratory.
8. Be calm and act quickly in any emergency.
9. Suggest and carry out any safety precaution if your think it is needed
10. Make sure your know the location of safety equipment, first aid kits and other items
in the microbiology laboratory.
B. Standard operation procedure in the microbiology laboratory
For the safety and convenience of everyone working in the laboratory, it is important that
the following laboratory rules be observed at all times:
1. Place only those materials needed for the day's laboratory exercise on the
benchtops. Purses, coats, extra books, etc., should be placed in the lab bench
storage areas or under the lab benchs in order to avoid damage or contamination.
2. Since some of the microorganisms used in this class are pathogenic or potentially
pathogenic (opportunistic), it is essential to always follow proper aseptic
technique in handling and transferring all organisms
3. No smoking, eating, drinking, or any other hand-to-mouth activity while in the

lab. If you need a short break, wash your hands with disinfectant soap and leave the
room.
4. If you should spill a culture, observe the following procedures:
a. Immediately place the culture tube in the plastic baskets found in the hood
in the back of the room so no one else touches the contaminated tube.
b. Have your partner spray isopropyl alcohol liberally over the spill. Be sure
your Bunsen burner is turned off before you spray any alcohol! After a few
minutes, use paper towels to dry the area.
c. Both you and your partner wash your hands with disinfectant soap.
d. Notify your instructor.
5. Report any cuts, burns, or other injuries to your instructor.
6. Using a wax marker, properly label all inoculated culture tubes or petri plates with
the name or the initials of the microorganism you are growing, your initials or a group
symbol, and any other pertinent information.

3/32
Place all inoculated material only on your assigned incubator shelf, the shelf
corresponding to your lab section. Culture tubes should be stored upright in
plastic beakers, while petri plates should be stacked and incubated upside-
down.
7. After completing an experiment, dispose of all material properly:
a. Remove all wax pencil markings from culture tubes and place the tubes
upright in the plastic baskets found in the disposal hood at the back of the
room.
b. Place petri plates in the plastic bags found in the disposal hood at the back
of the room.
c. Put all used pipettes and swabs in the biowaste disposal containers found
in the front of the room and under the hood.
8. Use caution around the bunsen burners. In a crowded lab it is easy to lean over a
burner and ignite your hair or clothing.
9. Always clean the oil off of the oil immersion lens of the microscope with a piece
of lens paper at the completion of each microscopy lab.
10. Return all equipment, reagents, and other supplies to their proper places at the end
of each lab period.
11. Disinfect the benchtop with isopropyl alcohol before and after each lab period. Be
sure your Bunsen burner is turned off before you spray any alcohol!
12. Always wash your hands with disinfectant soap before leaving the laboratory.
C. General Directions
1. Always familiarize yourself in advance with the exercises to be performed.
2. Disinfect the bench tops with isopropyl alcohol before and after each lab.
3. The first part of each lab period will be used to complete and record the results of
prior experiments. When you come into the lab, always pull out and organize any
culture tubes or petri plates you have in the incubator from previous labs. We
will always go over these results as a class. You may wish to purchase a set of
colored pencils to aid you in recording your results in the lab manual.
4. The latter part of each lab period will be used to begin new experiments. Preliminary
instructions, demonstrations, and any changes in procedure will be given by your
instructor prior to starting each new lab exercise.
5. After completing an experiment, dispose of all laboratory media and contaminated

materials in the designated areas as described above.
6. Wash your hands with disinfectant soap before leaving the lab.

4/32
LAB 1
TOPIC : MICROBIOLOGY LABORATORY
OBJECTIVE : MICROBIOLOGY LABORATORY FAMILIARISE
INTRODUCTION
In this first lab student will be introduce to the microbiology laboratory condition and
environment. You will be able to see and identified the equipment and material in the
microbiology laboratory. Laboratory rules and important equipment that always being use
will be discussed in this first lab. Student must write report individually after each lab so
that each individual must understand every lab exercises had been done.
EQUIPMENT AND MATERIAL
Microbiology laboratory equipment and material should be used under aseptic condition
while doing lab exercises. In working with microorganisms, method of transferring
growing organisms often used, aseptic technique is important to preventing unwanted
microorganisms.
In working with microorganisms we must also have a sterile nutrient-containing medium

in which to grow the organisms. Anything in or on which we grow microorganisms is
termed a medium. A sterile medium is one that is free of all life forms. It is usually
sterilized by heating it to a temperature at which all contaminating microorganisms are
destroyed. There are few medium that used such as Nutrient Agar, Nutrient Broth,
Potato Dextrose Agar and Standard Method Agar. Reagents that always been use such
as crystal violet, safranin, methylene blue and ethyl alcohol.
Autoclave, incubator, oven, dilution bottle, pipette, petri dish, and colony counter are
some example of material that been use often in every lab exercises.
INCUBATOR
This guide is written to reflect the use of a 37 °C incubator. The transformation

experiment can be conducted without the use of an incubator, however, the number of
days required to culture colonies to the optimum size depends on the ambient
temperature. Best results are obtained if starter plate colonies are fresh (24-48 hours
growth) and measure about 1-1.5 mm in diameter. Refrigeration of cultured plates will
significantly lower transformation efficiency. 37 °C (98.6 °F) is the optimum temperature
for growing E. coli and lower temperatures will result in a decreased growth rate. At 28
°C (82 °F) two days incubation is required to obtain optimum size. 21 °C (70 °F) requires
three days incubation of plates to obtain optimum size.

5/32
AUTOCLAVE
A steam autoclave may be used to sterilize media, glassware, waste, instruments, etc.
To accomplish the desired end goal and to protect the user and the environment from
hazardous materials, the autoclave must be used correctly. Additionally, wastes must be
managed in compliance with state and local regulations.
For the autoclave process to be effective in achieving sterilization, sufficient temperature,

time and direct steam contact are essential. Air must be completely removed from the
sterilizer chamber and from the materials to allow steam penetration so that the material
being autoclaved will be at treatment temperature for sufficient time to achieve kill.
Factors that affect air removal include type and quantity of material to be autoclaved,
packaging, load density and configuration, and container type, size, and shape.
The parameters for the sterilization cycle will depend upon the amount and type of
material. Usually 121 C° at 15 psi for a minimum of 30 minutes is recommended.
However, the temperature and cycle time should be determined using a worst case load
and using a biological indicator as verification that sterilization was achieved. A biological
indicator should be used frequently enough to ensure that the sterilization parameters
are effective in treating biohazardous waste
OTHER EQUIPMENT
Other equipment that usually used is Bunsen burner, needle, inoculation loop, distilled
water and thermometer etc.
SAFETY AND CONTAMINATION CONTROL
Always aware of safety precautions while in laboratory. Do prepare and check all
equipment that will be use before beginning any experiment. In case of emergency,
please do not panic but stay calm and inform your lecturer, if necessary. Report must be
done of any kind of accident happens.
Every time, before and after doing any experiments wash your hand with soap and
water. Laboratory bench must be clean using disinfectant before and after doing any
experiment. Do not speak when transferring any culture. Strictly, avoid of having any
foods or drinks or tasting any material in microbiology laboratory. Failure to follow all the
procedures will affect your health. All equipment used in experiment must be sterile
thoroughly. Always ware lab coat and shoes while in microbiology laboratory.
Do not take away any equipment and materials from microbiology laboratory. All
equipment used must be clean before leave the microbiology laboratory. Always make
sure the microbiology laboratory is clean after experiment.
REPORT
Observe the prior equipments and materials in the microbiology laboratory and records
the types, brands, characteristics, functions and operating procedures each of the
equipments and materials. If necessary, do get your reference from any related book as
to complete your laboratory report before summit.
“REMEMBER, SAFETY FIRST”

6/32
LAB 2
TOPIC : STERILIZATION TECHNIQUE OF EQUIPMENT AND MATERIALS
OBJECTIVE : LEARNING STERILIZATION TECHNIQUE
INTRODUCTION
All the experiment should be done in microbiology laboratory. The equipment used and
materials must be in sterilization or aseptic condition.
Definition of sterilization is the act or process, physical or chemical that destroys or

eliminates all viable microbes including resistant bacterial spores from a fluid or a solid.
There is various way of physical procedure:
a) Burning – usually use for needle, inoculation loop, pipette, test tube and conical
flask.
b) Dry heat – using oven which the temperature was 170 -180 C and between 1 – 2
hour defend on the quantity of equipment. Used to sterilize glassware.
c) Steam under pressure – using autoclave with temperature 121 C° at 15 psi for 15 -
30 minutes. Used to sterilize media culture and small equipment.
MATERIALS AND PROCEDURES
A. Burning Method
1. Light up the Bunsen burner, get the blue flame.

2. Burn the inoculation loop until red.
3. Open bottle lid and put the end of hot inoculation loop on the surface of solution.
4. Dip the loop into the solution and close the bottle.
5. Transfer the inoculate into the other bottle.
B. Dry Heat Method
1. Wash pipette and petri dish with soap and dry them.
2. Put the pipette into pipette container.
3. Set the oven to 180C, put pipette container and petri dish that have been
washed into the oven. After 1 hour or more switch off the oven and left the pipette
container and petri dish to be cooled by them.

7/32
C. Steam Under Pressure
1. Twist the control knob for the drain valve on the autoclave to the closed position.
2. Open the door on the autoclave and add distilled water in the bottom of this
opening.
3. Load the materials to be sterilized into the steel basket in the autoclave. (Liquids
in the bottles should be ¾ or less full with lids slightly loosened, clean petri dishes
should be loaded in top side up and contaminated petri dishes get loaded agar
side down.
4. Close the door and lock it up
5. Set the time, temperature and pressure appropriately (121°C, 15-30minutes and
15psi)

8/32
LAB 3
TOPIC : MEDIA CULTURE PREPARATION
OBJECTIVE : PREPARING MEDIA CULTURE
INTRODUCTION
In nature, microorganism can be found everywhere. Because of that, it is difficult to study

microorganism in their pure habitat. Researches have been done in artificial condition in
the laboratory. Media culture is a medium where microorganism grows in purpose and
called as culture. Three types of main medium are liquid, semi solid and solid. Each of
medium has it own characteristic. Those characteristic suitable to one or more condition
or experiment purpose.
A. Preparing Liquid Media
1. Prepare clean and dried bottles, bikers and flasks.

2. Read the instruction on the media bottle before used the media.
3. Calculate how much media must be prepare and how much media powder must
be dilute.
4. Weigh the powder and dilute into distil water.
5. Stir the media solution with glass rod until it dissolved. If necessary, heat the
media solution until dissolve.
6. After all media dissolve, put the liquid media into bottle for 3 full. Then close
4
the bottle.
7. Sterile the media
B. Preparing Solid Media
Slant Agar
1. Continues upper step but put only half media into bottles.
2. Sterile. After that put the bottle slanted in 30 position.
3. Store the media for next experiment.
Plate Agar
1. Continues upper step, used flasks. Close the mouth of the flasks with wool and
aluminum foil.
2. Sterile in autoclave. After autoclave left it to cool but make sure that the media
body does not become solid.
3. Pour into petri dish in 1 volume. Left it to be solid and cool.
3
• Media culture must be used after it is prepare. But for this lab, store the media in
chiller to be used in next lab.

9/32
LAB 4
TOPIC : SOLID MEDIA INOCULATION TECHNIQUE
OBJECTIVE : LEARNING SOLID MEDIA INOCULATION TECHNIQUE
INTRODUCTION
Agar
Solid or semisolid; liquefies at 100°C and solidifies at 40°C.
Agar plates – These are petri dishes that contain agar for growing microorganisms.
They have a large surface area and are useful for isolating and studying
microorganisms.
After they are inoculated, petri dishes are incubated in an inverted position. This
prevents condensation from dripping from the cover onto the agar.
Agar slants – useful for maintaining cultures. It is a solid media in a test tube with a
slanted surface on which to culture the microorganism.
Agar deep tubes – useful for studying gas requirements of microbes. It is a solid media
in a test tube which does not have a slanted surface. These are typically inoculated by
stabbing the media with a sterile needle.
Microorganisms grow on the surface of agar plates and slants. A needle is used to
inoculate a deep tube by pushing cells underneath the agar.
Bacterial cells on the agar or in the broth will reproduce rapidly if other environmental
conditions such as temperature are favorable.
A single cell on the agar will shortly produce a colony of cells that is easily visible to the
naked eye. Such a colony is a pure culture because it is a single species.
A. Streak Plating (Tube to Plate Transfer)

1. Loosen the tube cap, but do not remove it.
2. Hold tube in the palm of the left hand, with the side of the tube resting against the
insides of the fingers and using the thumb to hold them in place.
3. Hold the loop between the thumb and index finger of the right hand. Insert the loop
into the upper portion of the flame at about a 60o angle. Flame the loop until it is red-
hot, remove from the flame and let it cool. Briefly count to ten.
(The loop is flamed to kill any organisms on it and cooled so it does not kill cells in
the culture.)
4. Using the ring and little fingers of the right hand, remove the cap from the tube
containing the culture and flame the lip of the tube, passing it through the flame only
once.
(The tube is flamed to kill organisms around the lip, but it is flamed only briefly so the
tube does not get too hot.)

10/32
5. Insert the cooled loop without touching the sides of the tube and just immerse the
loop in the top portion of the culture. This is usually sufficient to obtain the proper
number of cells for transfer. It may be necessary to mix the culture tube first to
suspend the cells.
6. Withdraw the loop, flame the lip of the tube and replace the cap by bringing the cap
to the tube.
(The culture is capped before continuing with the manipulation to protect the original
culture. The cap is brought to the tube, rather than vice-versa, to minimize the
possibility of contamination by microorganisms in the air.)
7. Using the left hand, lift the lid of the plate to approximately a 45o angle.
8. Insert the loop to touch the surface of the agar at the furthest point from the open
side.
9. Starting from the top (where you touch the surface of the agar with the loop) streak
back and forth about one-quarter of the way down the plate. Make streaks very close
together. When finished flame the loop.
10. Rotate the plate slightly counterclockwise. Draw the sterile loop through the first
series of streaks once and streak about half of the open area without touching the
first series of streaks again. Do not flame the loop, but go directly to step 11.
11. Rotate the plate counterclockwise. Without touching the second section, finish
streaking the plate. When you are finished streaking, flame the loop before setting it
aside.
B. Tube to Tube Transfer (using a loop)
1. Loosen the tube caps, but do not remove them.

2. Hold both tubes in the palm of the left hand, with the sides of the tubes resting
against the insides of the fingers and using the thumb to hold them in place. The
tube containing material to be transferred should be the nearest the little finger.
3. Hold the loop between the thumb and the index finger of the right hand. Flame the
loop as described in the above procedure.
4. Using the ring and little finger of the right hand, remove the cap from the tube
containing the culture and flame the lip of the tube, passing it through the flame only
once.
5. Insert the cooled loop and retrieve a portion of the culture as stated before.
6. Withdraw the loop, flame the lip of the tube and replace the cap by bringing the cap
to the tube.
7. Remove the cap of the fresh tube, flame the lip, insert the loop into the slant agar and
streak gently. Withdraw the loop, flame the lip of the tube and replace the cap.
Flame the loop.
8. Tighten the caps on the two tubes and replace then in a rack or carton.

11/32
LAB 5
TOPIC : CULTURE TRANSFERING TECHNIQUE
OBJECTIVE: UNDERSTANDING CULTURE TRANSFERING TECHNIQUE
INTRODUCTION
Microorganisms are often transferred from one medium to another with a wire loop or
needle. Before the loop or needle is used to remove a sample of microorganisms, it must
first be sterilized.
Subculturing refers to transferring microorganisms from one container with medium to

another.
Wire loops are used to transfer microorganisms from liquid media to liquid or solid
media. It made from platinum or nichrome.
Needles are used for transferring microorganisms to deep tubes.
Pipettes are used to transfer liquids. A mechanical device must be used with pipettes to
create a vacuum.
A. Transferring using inoculation loop
1. Remove the culture tube stopper or cap with one (do not set it down) and flame
the mouth of the tube to surface sterilize the mouth. The heated tube surface will
generate a thermal current that prevents contamination of the culture.
2. Without setting any of the culture materials on the bench, place the sterile
inoculation loop in the culture.
3. Replace cap on the culture tube with the active microbes and put it in the test
tube rack.
4. Without setting the loop down, pick-up a sterile fresh culture tube with media with
one hand, and remove the cap with the other hand.
5. Flame the mouth of the clean culture tube.
6. Place the inoculation loop containing the microbes in the fresh media and swirl
the loop in the loop in the media to ensure even dispersal in the media.
7. If using a solid media slant tube, follow steps 1-5 and then zig-zag the inoculation
loop across the slanted surface of the solid media in the tube.
8. Flame the mouth of the newly inoculated culture tube and replace the cap.
9. Place the culture tube in test tube rack.
10. Repeat until all of the sterile tubes have been inoculated. Use a fresh disposable
culture loop for each tube or flame the metal loop after each tube has been
inoculated.
11. Incubate the culture at the recommended temperature (check with your supplier
for growth requirements). If using environmental samples, incubation at room
temperature will avoid the accidental culture of human pathogens.
12. Dispose of all culture materials in a biohazard bag and sterilize all old cultures
before pouring out cultures and washing culture tubes. Disposable culture dishes
should be melted in an autoclave or pressure cooker prior to disposal.

12/32
B. Transferring using pipette
This technique is used when doing sample dilution or liquid analysis such as water or
milk. Pipetting by mouth is not an acceptable laboratory practice. Fluids are drawn up
into pipettes using pipette fillers. There are several options. A simple rubber bulb is
suitable for a 1 ml pipette. For 10 ml pipettes, use a triple valve rubber bulb, a hand
operated pump or an electronic pipette filler.
Pasteur pipettes are glass pipettes used to transfer fluids from one place to another.
They are not graduated and are therefore not used to measure volumes. Like graduated
pipettes they should be plugged with cotton wool and sterilized before use.
1. Sterilized all equipment and material used.

2. All aseptic technique must be practices.
3. Take out 1 ml sterile pipette from aluminum container.
4. Flame the end of pipette to sterilize the pipette.
5. Sucked up 1.0 ml inoculums from culture tube or bottle.
6. Transfer the 1.0 ml inoculums to new culture tube or bottle containing 9.0 ml distil
water.
7. Pipette had been used for transfers must not been use and place it in pipette jar
containing disinfectant solution.
8. Label the new culture tube or bottle and stored it in chiller.

13/32
MICROSCOPE
Introduction
A good microscope is an essential tool for any microbiology laboratory. There are
many kinds of microscopes, but the type most useful in diagnostic work is the compound
light microscope. By means of a series of lenses and a source of bright light, it magnifies
and illuminates minute objects such as bacteria and other microorganisms that would
otherwise be invisible to the eye. This type of microscope will be used throughout your
laboratory course. As you gain experience using it, you will realize how precise it is and
how valuable for studying microorganisms present in clinical specimens and in cultures.
Even though you may not need to use a microscope in your planned profession, a
firsthand knowledge of how to use it can stand you in good stead. In addition, your
laboratory experience with it will give you a lasting concept of the existence of living
forms too small to be seen with normal vision. Armed with such a view of “invisible”
microorganisms, you should be better able to understand their role in transmission of
infection.
The following are important parts of the compound microscope and their function:
1. Look at the microscope you have and compare it to the figure 6.0. Note that its
working parts are set into a sturdy frame consisting of a base for support and an arm
for carrying it. (Note: when lifting and carrying the microscope, always use both
hands; one to grasp the arm firmly, the other to support the base. Never lift it by the
part that holds the lenses.
2. Observe that a flat platform, or stage as it is called, extends between the upper lens
system and the lower set of devices for providing light. The stage has a hole in the
center that permits light from below to pass upward into the lenses above. The object
to be viewed is positioned on the stage over this opening so that it is brightly
illuminated from below. Our microscopes are equipped with a mechanical stage,
once the specimen is placed into position it no longer needs adjustment by directly
touching it.
3. The light source is at the base. Most microscopes have a built in illuminator.
4. Light from the bulb is directed upward through the condenser, placed under the
center opening in the stage. The condenser contains lenses that collect and
concentrate the light, directing it upward through any object on the stage. It also has
a shutter, or iris diaphragm, which can be used to adjust the amount of light
admitted. A lever is provided on the condenser for operating the diaphragm.
5. Above the stage, attached to the arm, a tube holds the magnifying lenses through
which the object is viewed. The lower end of the tube is fitted with a rotating
nosepiece holding three or four objective lenses. As the nosepiece is rotated, any
chosen objective can be brought into position above the stage opening. The upper
end of the tube holds the ocular lens, or eyepiece.
6. The rotating nosepiece can be raised or lowered by means of coarse and fine
adjustment knobs. These are located below the stage. They are placed in tandem
with the smaller fine adjustment extending from the larger coarse wheel. The coarse
adjustment knob is used to bring the objective down into position over any object on
the stage, while looking at it from the side to avoid striking the object and thus
damaging the expensive objective lens. The fine adjustment knob moves the tube
to such a slight degree that it cannot be observed from the side. It is used when one
is viewing the object through the lenses to make the small adjustments necessary for
a sharp, clear image. The adjustment knobs should always be turned slowly and
gently, with one’s attention on the relative positions of objective and object. Avoid
bringing the objective down with the fine adjustment while viewing, because this
unperceived motion may force the lens against the object. Bring the lens safely down
14/32
first with the coarse knob, then, while looking through the ocular, turn the fine knob to
raise the lens until you have a clear view of the subject. Rotating the fine adjustment
too far in either direction may cause it to jam. If this should happen, never attempt to
force it. To avoid jamming, gently locate the two extremes to which the fine knob can
be turned, then bring it back to the middle of its span and keep it within one turn of
this central position.
7. The total magnification achieved with the microscope depends on the combination of
the ocular and objective lens used. Look at the ocular lens on your microscope. You
will see that it is marked 10X, meaning that it magnifies 10 times. Now look at the
three objective lenses on the nosepiece. The short one is the low-power objective.
Its metal shaft bears a 10X mark, indicating that it gives tenfold magnification. When
an object is viewed with the10X objective combined with the 10X ocular, it is
magnified 10 times 10, or 100X. Each of the other objectives carries a different
magnification, with the oil immersion lens being the most powerful at 100X itself.
Note the higher the magnification used, the more intense the light must be, but the
density of the object also determines the amount of illumination needed. For
example, more light is needed to view stained than unstained preparations.
8. The focal length of an objective is directly proportional to the diameter of its lens. You
can see this by comparing your three objectives when positioned as close to the
stage as the coarse adjustment permits.
9. Take a piece of clean, soft lens paper and brush it lightly over the ocular and
objective lenses and the top of the condenser. With subdued light coming through,
look into the microscope. If you see specks of dust, rotate the ocular in its socket to
see whether the dirt moves. If it does, it is on the ocular and should be wiped off
more carefully. It you cannot solve the problem, call the instructor. Never wipe the
lenses with anything but clean, dry lens paper.
10. Because students in other laboratory sections may also use your microscope, you
should examine the microscope carefully at the beginning of each laboratory session
and clean it completely when finished.
MICROSCOPE PART
EYEPIECE:
◼ Magnifies material being viewed by 10X.
◼ The part of the microscope you look into.
◼ Sometimes contains a pointer that can be seen as you look into the
eyepiece.
◼ May also be called the ocular.
NOSE PIECE:
◼ Part of microscope to which the objectives are attached.
◼ Rotates to allow for the changing of objectives to increase or decrease
magnification.
ARM:
◼ Part of microscope to which the nosepiece is attached.
◼ A secure part of the microscope to hold on to when the microscope is
being carried.
◼ Sometimes includes a cord hanger for power cord storage.

15/32
OBJECTIVES LENSE:
◼ Magnify material being examined.
◼ Scanning – magnifies 4X.
◼ 10X – magnifies 10X.
◼ High dry – magnifies 40X.
◼ Oil immersion – magnifies 100X.
(Total magnification = magnifying power of eyepiece times the magnifying
power of the objective in use.)
STAGE:
◼ Platform on which microscope slide rests.
MECHANICAL STAGE:
◼ Used for adjusting the position of the slide for viewing.
SLIDE HOLDER:
◼ Secures slide to the mechanical stage.
MECHANICAL STAGE CONTROL KNOBS:

◼ Move the slide.
COARSE ADJUSTMENT KNOB:

◼ Coarse focusing.
FINE ADJUSTMENT KNOB:

◼ Precise focusing.
CONDENSER
◼ Concentrates light & directs it through opening in microscope stage.
IRIS DIAPHRAGM:
◼ Regulates the amount of light passing through the substage condenser.
◼ Increases resolving power of the microscope.
IRIS DIAPHRAGM LEVER:

◼ Adjusts the iris diaphragm to increase or decrease microscope resolution.
CONDENSER CONTROL KNOB:

◼ Raises & lowers condenser.
ILLUMINATOR
◼ Light source.
ILLUMINATOR CONTROL:
◼ Controls the intensity of light.
◼ Serves as the on/off switch for some microscopes.
BASE:
◼ Provides support for microscope.

16/32
LAB 6
TOPIC : USING MICROSCOPE
OBJECTIVE : HANDLE THE MICROSCOPE PROPERLY, IDENTIFY ITS PARTS, AND

FOCUS ON A SLIDE USING ALL THREE POWERS
Activity
Pre-Activity Discussion
1. Show the compound microscope.

2. What is the microscope used for?
3. Define terms: magnification, total magnification, and resolution.
4. Discuss “Care and Handling of the Microscope” with the class.
5. Explain guidelines for making drawing on lab reports; students must:
• Only use pencil to erase and shade areas for drawings
• Identify area viewed through the microscope with a circle
• Draw specimens to scale (i.e. show if specimen takes the whole field of view)
• Label drawings with the specimen name and magnification
• Make drawings large enough to view details; label significant details
• Write all labels outside of the circle
6. Discuss the importance of making a wet mount.
7. Ask students to identify things used to make wet mounts.
B. Student Activity
1. Cut out the letter "e" from the newspaper and place it on the slide face up and then
add a drop of water to the slide.
2. Place the cover slip on top of the "e" and drop of water at a 45-degree angle and
lower.
3. Place the slide on the stage and view in low power (4X) and center the "e" in your
field of view.
4. Move the slide to the left, right, up and then down.
5. View the specimen in high power (10X and 40X). Use the fine adjustment ONLY to
focus.

17/32
MICROSCOPE
Adjustable eyepiece
Nose piece
Eyepiece
Objectives Lense
Mechanical Stage
Slide Holder
Main Power
Illuminator
Stage Control
Y-axis Mechanical
Stage Control Knob
Condenser X-axis Mechanical
Stage Control Knob
Arm
Iris Diaphragm
Illuminator Coarse Knob

Filter
Fine Knob
Base
Illuminator

18/32
The Letter “e”
Specimen: Letter “e”

Magnification: Total magnification:



19/32
Questions
1. How does the letter “e” as seen through the microscope differ from the way an “e”
normally appears?
2. When you move the slide to the left, in what direction does the letter “e” appear to
move?
3. When you move the slide the right, in what direction does the letter “e” appear to
move?
4. When you move the letter “e” up, in what direction does the letter “e” appear to
move?
5. When you move the letter “e” down, which direction does the letter “e” move?
6. How does the ink appear under the microscope compared to normal view?
7. Why does a specimen placed under the microscope have to be thin?

20/32
SPECIAL STAINS
Introduction
Bacteriological stains are dyes that make bacterial cells more visible by
increasing the contrast between cell and background. The dyes are usually organic
(carbon containing) molecules of complex, but known structure. All dyes selectively
absorb light of certain wavelengths and thus have a color. Many dyes useful for staining
bacterial cells also specifically bind (attach) to the surface of bacterial cells. This
category of dye is referred to as positive stains. Crystal violet and safranin, which are
used in the Gram stain, are both positive stains. With positive stains the cell picks up the
color. The background remains transparent. Some dyes do not bind to the surface of
bacterial cells. These dyes, called negative stains, color the background. With
negative staining bacterial cells appear white.
Stains help to provide information about cell morphology – size, shape, and
arrangement. Stains can also provide more detailed information about such cell
properties as the presence of a cell wall, of carbohydrate, lipid, and phosphate nutritional
reserves, of flagella, and of nucleoids, which contain DNA, the genetic material. Perhaps
the most important use of stains in microbiology is to help in the identification of bacteria.
Some staining procedures produce different results when applied to different kinds of
bacteria. In this course we will use the Gram stain procedure which distinguishes two
important classes of bacteria, the Gram positive (G+) and the Gram negative (G-).
Because it distinguishes between these two classes, the Gram stain is a differential
stain.
The systematic use of bacterial stains began in the 1870’s. In 1884 the Danish
physician Christian Gram reported a method for staining using the dye crystal violet. He
observed that organic solvents (e.g. ethanol) could more readily remove the crystal violet
from some bacteria (G-) than from others (G+). This phenomenon of decolorization is
what makes the Gram stain differential. The Gram stain has remained an important
technique because its result is taxonomically significant. That is, closely related types of
bacteria usually all have the same Gram reaction. The Gram stain is thus an indicator
for a fundamental division among bacteria. There are other general differences between
G+ and G- bacteria. Some of them are given in the table below.
Gram Positive Bacteria Gram Negative Bacteria

Thick cell wall (peptidoglycan) little lipid in Thin cell wall (peptidoglycan) much lipid in
cell envelop (1-4%) no outer membrane a cell outer membrane(11-22%)
surrounding the cell wall
More sensitive to the antibiotic penicillin Less sensitive to penicillin
Growth inhibited by basic dyes such as Little growth inhibition compared to G+’s
crystal violet
Multiple growth factors requirements in Few growth factor requirements in most
some species (vitamins, amino acids) species
Examples of Gram positive bacteria include Staphylococcus, Streptococcus, and

Bacillus. Gram negatives include Escherichia, Enterobacter, Proteus, Pseudomonas,
and Alcaligenes. It is important to keep in mind that the differences listed in the table are
21/32
differences of degree rather than absolute differences. For example, thinner vs. thicker.
The differential response of G+ and G- cells to the staining procedure is likewise one of
degree, G- cells decolorize (give up the crystal violet stain) faster than G+ cells. If
decolorization is carried out for too long a time, G+ cells will appear G-. There are two
lessons in this. The first is that the Gram stain procedure must be performed carefully
and consistently to give a meaningful result. The second is that even if the procedure is
done properly, some bacterial cells will give atypical results. The most common problem
of this type occurs when an old culture (say, a few days old) is stained. As G + cells age
the Gram reaction can change to G-. So only recent (say, overnight) cultures should be
used for doing the Gram stain. Old cultures often give a Gram variable reaction. Some
bacteria are always Gram variable (e.g. Neisseria) while others stain poorly (e.g.
spirochetes, Mycobacterium).
The Gram stain procedure consists of four steps:

1. Primary staining by crystal violet.
2. Complexing of crystal violet by iodine (mordant).
3. Decolorization by alcohol.
4. Counterstaining by safranin.
The actual mechanisms by which most positive stains bind to cells are unknown.
Crystal violet (step 1), which carries a positive charge, evidently binds to a negatively
charged molecule (or molecules), probably in the cell envelop. Other factors beside the
attraction of a positive charge for a negative charge may also be involved in the binding
of stains to cells.
Iodine must be added (step 2) to form an insoluble complex with crystal violet.
Otherwise even G+ cells will decolorize. The term mordant comes from the textile
industry and refers to a compound, which fixes a dye in a textile fiber.
The key step is the decolorization (step 3). The G+ bacteria with their thick
peptidoglycan layers and with relatively little lipid decolorize slowly whereas the G-
bacteria decolorize rapidly perhaps because the ethanol dissolves their outer membrane
lipid allowing the crystal violet-iodine complex to be released or because their thin
peptidoglycan layer cannot trap the complex.
The purpose of the counterstain (step 4) with the safranin is to make the G - cells
visible in the microscope. Safranin does not stain as intensely as crystal violet and so
has no effect on the appearance of the G+ cells, which are purple.
Another stain that has been used for years is the indirect or negative stain. The
advantage of this stain is that it is the simplest and often-quickest means of discovering
cell shape and possibly refractile inclusions and endospores. It also does not distort
bacteria, which may happen with Gram stains, since cells sometimes, shrink as a result
of heat fixation and alcohol wash. Preparations can easily be made with natural
specimens or cultures by mixing them with a drop of 7% nigrosin solution and making a
thin film. The preparations reveal bacteria unstained, standing out brightly against a
darker background. Negative stains should not be used to measure cells lengths or
widths because some microbes may have capsules or slime layers which do not stain.
In addition, some cells may be partially collapsed. Furthermore, at pH 7.0, nigrosin is
negatively charged, just like the bacterial cell surface. Therefore, when the stain dries,
the bacteria appear larger than life, even if no capsule exists.

22/32
Members of the bacterial genus Mycobacterium contain large amounts of lipid (fatty)
substances within their cell walls. These fatty waxes resist staining by ordinary methods.
Since this genus contains species that cause important human diseases (the agent of
tuberculosis), the diagnostic laboratory must use special stains to reveal them in clinical
specimens or cultures.
When these organisms are stained with a basic dye, such as carbolfuchsin, applied
with heat or a wetting agent (a detergent that decreases the surface tension of fats), the
stain can penetrate the lipid cell wall and reach the cell cytoplasm. Once the cytoplasm
is stained it resists decolorization, even with harsh agents such as acid-alcohol, which
cannot dissolve and penetrate beneath the mycobacterial lipid wall. Under these
conditions of staining, the mycobacteria are said to be acid-fast. Other bacteria whose
cell walls do not contain high concentrations of lipid are readily decolorized by acid-
alcohol after sitting in carbolfuchsin and are said to be non-acid-fast. One medically
important genus, Nocardia, contains species that are partially acid-fast. They resist
decolorization with a weak (1%) sulfuric acid solution, but lose the carbolfuchsin dye
when treated with acid-alcohol. In the acid-fast technique, a counterstain is used to
demonstrate whether or not the fuchsin has been decolorized within the cells and the
second stain taken up.
The original technique for applying carbolfuchsin with heat is called the Ziehl-Neelsen
stain, for the two workers who developed it in the late 1800s. The later modification of
the technique that employs a wetting agent rather than heat to ensure stain penetration
is known as the Kinyoun stain.

23/32
LAB 7
TOPIC : SIMPLE STAINS
OBJECTIVE : UNDERSTAND THE DIFFERENT CHARACTERISTICS OF BACTERIA

AND THUS THE DIFFERENTIATION IN STAINING
INTRODUCTION
Uses only one reagent which provides contrast between the background and the heat-
fixed bacterium itself. The bacterium takes up stain and becomes colored, while the
background remains unstained. Simple stains are typically used on bacterial smears
which have been heat-fixed and thus contain non-living microbes
Materials: 24-hour cultures

Methylene blue
Safranin
Carbolfuschin
Crystal violet
Toothpicks
1. Slides for microscopic smears must always be sparkling clean. They can be cleaned
in xylene and wiped clean with a soft towel.
2. Take three clean slides and with your marking pencil make a circle (about 1.5 cm in
diameter) in the center.
3. Turn the slides over so that the unmarked side is up (when the slides are to be
stained, wax pencil markings should always be placed on the underside so that the
wax will not smear or wash off, or run into the smear itself).
4. Place a loopful of water in the ringed area of the slide. Use your inoculating loop. Mix
a small amount of bacteria in the water and spread it out.
5. Allow the smear to air dry. You should be able to see a thin white film. If not, add
another loopful of water and more bacteria as in step 4.
6. Heat-fix the smear by passing the slide rapidly through the Bunsen burner flame
three times.
7. Place the slides on a staining rack and flood them with safranin. Leave the stain on
for three minutes.
8. Wash each slide gently with distilled water, drain off excess water, blot (do not rub)
with bibulous paper, and let the slides dry completely in air.
9. Prepare two more slides as in steps 1, 2, and 3. Place a loopful of distilled water in
the ringed area on each slide.
10. With the flat end of a toothpick scrape some material from the surface of your teeth
and around the gums. Emulsify the material in the drop of water on one slide. Repeat
the procedure on the other slide.
11. Allow both slides to dry in air, then heat-fix them. Stain one with methylene blue,
carbolfuschin and crystal violet for three minutes; the other with safranin, also for
three minutes.
12. Wash, drain, and dry the slides as in step 8.
13. Examine all slides, with all three microscope objectives. Make a table with the
following information on a separate sheet of paper: Organism or sample, stain used,
color, shape, cell grouping, and appearance (drawing).

24/32
LAB 8
TOPIC : NEGATIVE STAIN

INTRODUCTION
Uses a single reagent to provide contrast between the background and the living
bacterium. Thus, the background is “stained”, while bacterium does not take up any
stain. Negative stains are typically used when observing live bacteria is desired.
Materials: 24 hour culture

7% nigrosin
toothpicks
Perform the following using the bacterial culture and a scraping from your teeth.
1. Place one small drop of 7% nigrosin stain in the center of a coverslip.

2. With a toothpick, gently scrape your teeth and gums and mix the material with the
nigrosin. You should also, on a separate coverslip, take a small section of the
bacterial colony and mix.
3. Place another coverslip, rotated at 45o, over the nigrosin and slide the two coverslips
apart to from a thin film on each.
4. Turn the top coverslip over and let both films dry completely.
5. Invert each coverslip on a slide.
6. Find a very thin area of the film and observe the bacteria under oil immersion. Most
of the slide will show large cracks from the dried stain, so find an area that has no
cracks and observe the clear bacteria against a faint gray background.
7. Record your findings.

25/32
LAB 9
TOPIC : GRAM STAIN

INTRODUCTION
Is a differential stain which distinguishes bacteria based on cell wall properties. Gram-
positive organisms have a thick layer of peptidoglycan, whereas Gram-negative
organisms have a thin layer of peptidoglycan, plus an additional outer membrane that is
absent in Gram-positive organisms.
Materials: 24 hour culture

Gram stain reagents: 1% aqueous crystal violet
Lugol’s iodine
95% ethanol
2.5% safranin
Perform the following using both bacterial cultures.
1. Using your inoculating loop aseptically remove a small portion of a colony.

2. Place the cells on a slide. Add a loopful of water and smear the cells over an area
about the size of a quarter. Note that it is possible to do two or three different smears
on one slide.
3. Allow the smear to air dry. Do not heat the slide to speed up drying. Heat may cause
boiling and destruction of the cells. Have patience. When the smear is dry it should
appear as a haze on the slide.
4. Once the smear is dry, pass the back of the slide through the flame 3 or 4 times to fix
the cells to the slide. Test the warmth of the slide after each pass by touching it to the
back of your wrist. When the cells are properly fixed, the slide will feel uncomfortably
hot, but will not be intolerable against your skin.
5. Cover the smear with a lot of crystal violet. Stain for 1 minute. Pour off crystal violet
and rinse gently with tap water.
6. Cover the smear with iodine for a few seconds. Pour off and rinse gently with water.
7. Tilt the slide and allow drops of ethanol to flow over the smear. The crystal violet will
wash off the smear. Stop as soon as the wash is colorless. It will take only a few
seconds (about 10 or fewer) to complete this decolorization step. It is important not
to overdecolorize or G+’s will lose the violet stain. Some practice is necessary to
develop a feel for the right extent of decolorization. Rinse with water.
8. Cover the smear with safranin for a few seconds. Pour off and rinse gently with
water. Blot carefully using bibulous paper.
9. Observe the stained bacterial cells under high dry and oil immersion objectives.
Record you observations.

26/32
LAB 10
TOPIC : SERIAL DILUTION
OBJECTIVE : UNDERSTANDING HOW AND WHY SERIAL DILUTIONS ARE USED
INTRODUCTION
What is a serial dilution? This is a method of diluting bacteria to obtain an estimate of

their numbers or to be able to isolate single colonies more reliably. Better accuracy is
obtained with very large dilutions if the total dilution is made out of a series of smaller
dilutions rather than one large dilution. Example: The dilution of 1 mL of cells into 9 mL
of medium is a 1:10 dilution -- 1/(1+9) = 1/10. One mL of this dilution is then transferred
into 9 mL of fresh medium -- another 1:10 dilution -- but this was made with the first
dilution. The total dilution is 1/10 x 1/10 = 1/100 or 1:100.
Figure 10.0: I/10 Dilution
1. Add 9 ml distil water into 6 bottles each. Sterilize it into autoclave. Then let it cool
down.
2. Using a sterilize pipette, remove 1 ml sample into a bottle. Cap the bottle and
shake it up. Label the bottle with 1/10 dilution or 10 -1, sample name, date and your
name.
3. Continues upper step, but using 1/10 bottle and sucked up 1.0 ml into 1/100 bottle
using a fresh pipette. Then continues upper step follow figure 10.0.
4. After complete the dilution, the following analyze can be done.

27/32
LAB 11
TOPIC : DETERMINATION OF MICROBIAL COUNTS USING POUR PLATE AND

SPREAD PLATE METHODS
OBJECTIVE : TO IDENTIFY THE NUMBER OF VIABLE MICRO-ORGANISMS

USING POUR PLATE AND SPREAD PLATE METHODS
INTRODUCTION
a) SPREAD PLATE METHOD
The purpose of the spread-plate technique is to grow and isolate colonies of bacteria.
The spread plate technique is also used for the enumeration of aerobic microorganisms
from the given sample. This can be done by serial diluting the samples, placing 0.1ml of
the diluted sample in the middle of an agar plate and spreading the sample over the
surface with a help of an L-rod. After the incubation the colonies can be counted.
b) POUR PLATE METHOD
A pour plate is an alternative method for using agar plates to obtain isolated colonies.
Pour plates are used when it is necessary to know the number of organisms present per
unit volume of specimen or other sample. In this technique, the sample is appropriately
diluted and a small aliquot transferred to an agar plate.
1. pipette 1.0ml (sterilized)

2. petri dishes (sterilized)
3. sterilized medium agar and peptone water
4. Bunsen burner
5. Incubator
6. Colony counter
7. Dilution bottle

28/32
Pour plate method (Figure 1.1)
A. Preparation of serial dilution
1. Prepare the dilution bottles from 10-1 to 10-6.

2. Use a 24hours cultures as starter (1ml for liquid culture or one loopful for colony
culture) and transfer into first dilution bottle (10-1)
3. Pipette 1 ml into a 9 ml dilution blanks to gives a 1 in 100 dilution (10-2). Make
sure the pipette does not touch the diluent. Discard the pipette.
4. Shake the 10 ml suspension vigorously 25 times. Pipette 1 ml into 9 ml blank to
give a 10-3.
5. Continue the dilution process to get 10-6.
B. Inoculation and pouring of plates
1. Pipette 1 ml of the sample from each dilution into petri dishes.

2. Pour 10-15 ml of molten medium (45ºC) to each plate. Duplicate for each dilution.
3. Mix the medium and inoculum by 5 to-and-fro movements, followed by 5 circular
clockwise movements, followed by 5 to-and-fro movements, followed by 5 circular
anti-clockwise movements. Allow the plates until the medium has set.
4. Incubate all the plates at 37ºC for 24-48 hours (or at the temperature and for the
period specified for the organism to be estimated).
5. Count all the colonies.
C. Plate election and colony counting
1. Choose the unspread plate. Plate is considering spread out if more than half area
of plate covered with the spread colony.
2. Spread colony will be counted as a one colony.
3. Choose the plate with the colony that spread uniformly.
4. Count the plate that contains 25-250 colonies.
5. Count all the colonies on the surface plate including pinpoint colony.

29/32
Spread plate method (Figure 1.2)
A. Preparation of serial dilution
1. Prepare the dilution bottles from 10-1 to 10-6.

2. Use a 24hours cultures as starter (1ml for liquid culture or one loopful for colony
culture) and transfer into first dilution bottle (10-1)
3. Pipette 1 ml into a 9 ml dilution blanks to gives a 1 in 100 dilutions (10-2). Make
sure the pipette does not touch the diluent. Discard the pipette.
4. Shake the 10 ml suspension vigorously 25 times. Pipette 1 ml into 9 ml blank to
give a 10-3.
5. Continue the dilution process to get 10-6.
B. Inoculation and pouring of plates
1. Pour 10-15ml of molten medium (45°C) to each plate. Duplicate for each dilution.
Allow the plates until the medium has set.
2. Pipette 0.1ml of the sample from each dilution into petri dishes. Discard the
pipette
3. Spread the inoculums gently and evenly over the surfaces of the plates with a
hockey stick. Discard the hockey stick. (Note: Care should be taken not to break
the surface of the medium as this could result in irregular counts).
4. Continue until the necessary number of dilutions has been plated and spread.
5. Allow the plates to stand until the inoculums on each has been completely
absorbed, which should be between 15minutes of spreading.
6. Incubate all the plates at 37°C for 24-48 hours.
7. Count all the colonies.
C. Plate election and colony counting
1. Choose the unspread plate. Plate is considering spread out if more than half area
of plate covered with the spread colony.
2. Spread colony will be counted as a one colony.
3. Choose the plate with the colony that spread uniformly.
4. Count the plate that contains 25-250 colonies.
CALCULATION AND REPORT
1. Refer to guidelines to compute and record colony counts

30/32
POUR PLATE METHOD Figure 1.1
i) Transfer inoculums into peptone water
9.0ml
ii) Perform serial dilution
1.0ml
1.0ml
iv) Perform 8 movement

ii) lncubate (37°C/ 24-48 hr)
iii) Count: colonies on plate containing 25-250 colonies and multiply the number
of the colonies by the dilution

31/32
SPREAD PLATE METHOD Figure 1.2
iv)Transfer inoculums into peptone water
9.0ml
ii) Perform serial dilution
0.1 ml
0.1 ml
v) Spread using glass spreader/ hockey stick

vi) lncubate (37°C/ 24-48 hr)
vii) Count: colonies on plate containing 25-250 colonies and multiply the number
of the colonies by the dilution
32/32

Lab Manual DMT 10023

Uploaded by

Copyright:

Available Formats

You might also like

Lab Manual DMT 10023

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Lab Manual DMT 10023

Uploaded by

Copyright:

Available Formats

FUNDAMENTAL

1 Laboratory Rules Procedures 2

2 Lab 1: Microbiology Laboratory 4

3 Lab 2: Sterilization Technique of Equipment 6

5 Lab 4: Solid media inoculation technique 9

6 Lab 5: Culture transferring technique 11

7 Lab 6: Using Microscope 17

8 Lab 7: Simple Stains 23

9 Lab 8: Negative Stains 24

10 Lab 9: Gram Stain 25

11 Lab 10: Serial dilution 26

12 Lab 11: Determination of microbial counts 27

FOOD TECHNOLOGY DEPARTMENT

Laboratory rules and procedures

A. Laboratory safety rules

1. Be always aware of safety precautions

B. Standard operation procedure in the microbiology laboratory

3. No smoking, eating, drinking, or any other hand-to-mouth activity while in the

4. If you should spill a culture, observe the following procedures:

d. Notify your instructor.

5. Report any cuts, burns, or other injuries to your instructor.

FOOD TECHNOLOGY DEPARTMENT

7. After completing an experiment, dispose of all material properly:

1. Always familiarize yourself in advance with the exercises to be performed.

5. After completing an experiment, dispose of all laboratory media and contaminated

FOOD TECHNOLOGY DEPARTMENT

TOPIC : MICROBIOLOGY LABORATORY

OBJECTIVE : MICROBIOLOGY LABORATORY FAMILIARISE

EQUIPMENT AND MATERIAL

In working with microorganisms we must also have a sterile nutrient-containing medium

This guide is written to reflect the use of a 37 °C incubator. The transformation

FOOD TECHNOLOGY DEPARTMENT

For the autoclave process to be effective in achieving sterilization, sufficient temperature,

SAFETY AND CONTAMINATION CONTROL

“REMEMBER, SAFETY FIRST”

TOPIC : STERILIZATION TECHNIQUE OF EQUIPMENT AND MATERIALS

OBJECTIVE : LEARNING STERILIZATION TECHNIQUE

Definition of sterilization is the act or process, physical or chemical that destroys or

MATERIALS AND PROCEDURES

1. Light up the Bunsen burner, get the blue flame.

B. Dry Heat Method

FOOD TECHNOLOGY DEPARTMENT

C. Steam Under Pressure

FOOD TECHNOLOGY DEPARTMENT

TOPIC : MEDIA CULTURE PREPARATION

OBJECTIVE : PREPARING MEDIA CULTURE

In nature, microorganism can be found everywhere. Because of that, it is difficult to study

MATERIALS AND PROCEDURES

A. Preparing Liquid Media

1. Prepare clean and dried bottles, bikers and flasks.

B. Preparing Solid Media

FOOD TECHNOLOGY DEPARTMENT

TOPIC : SOLID MEDIA INOCULATION TECHNIQUE

OBJECTIVE : LEARNING SOLID MEDIA INOCULATION TECHNIQUE

Solid or semisolid; liquefies at 100°C and solidifies at 40°C.

MATERIALS AND PROCEDURES

A. Streak Plating (Tube to Plate Transfer)