1

KINNAIRD COLLEGE FOR WOMEN UNIVERSITY,

LAHORE

FINAL PROJECT
MANUAL

SUBMITTED BY: Ayesha Masood (F19BSFN051)

SUBMITTED TO: Miss Fasiha Ilyas
COURSE TITLE: Food Microbiology
SECTION: C
SEMESTER: 4
SESSION: 2019-23
MAJOR: Food Science & Human Nutrition
DATE OF SUBMISSION: 8th May, 2021

2
Contents
WHAT IS MICROBIOLOGY? ................................................................................................................. 4
MICROBIOLOGY LAB INSTRUCTIONS ............................................................................................. 5
EQUIPMENTS USED IN MICROBIOLOGY LABORATORY ......................................................... 10
MICROSCOPE AND ITS TYPES .......................................................................................................... 22
PRACTICALS........................................................................................................................................... 30
PRACTICAL#1 ..................................................................................................................................... 31
PRACTICAL#2 ..................................................................................................................................... 33
PRACTICAL#3 ..................................................................................................................................... 36
PRACTICAL#4 ..................................................................................................................................... 40
PRACTICAL#5 ..................................................................................................................................... 44

3
 WHAT IS MICROBIOLOGY?

The term “microbiology” has been derived from Greek word “mikros” meaning small and
“bios” meaning life. Microbiology is the study of all living organisms that are too small to be
visible with the naked eye. This includes bacteria, archaea, viruses, fungi, prions, protozoa and
algae, collectively known as 'microbes'. These microbes play key roles in nutrient cycling,
biodegradation/ bio-deterioration, climate change, food spoilage, the cause and control of
disease, and biotechnology.
Food microbiology is the study of the microorganisms that inhibit, create, or contaminate food.
This includes the study of microorganisms causing food spoilage; pathogens that may cause
disease (especially if food is improperly cooked or stored); microbes used to produce fermented
foods such as cheese, yogurt, bread, beer, and wine; and microbes with other useful roles, such
as producing probiotics.

4
 MICROBIOLOGY LAB INSTRUCTIONS

General Safety Rules

1. Listen to or read instructions carefully before attempting to do anything.
2. Wear safety goggles to protect your eyes from chemicals, heated materials, or things that
might be able to shatter.
3. Notify your teacher if any spills or accidents
occur.
4. After handling chemicals, always wash your
hands with soap and water.
5. During lab work, keep your hands away from
your face.
6. Tie back long hair.
7. Roll up loose sleeves.
8. Know the location of the fire extinguisher, fire
blanket, eyewash station, and first aid kit.
9. Keep your work area uncluttered. Take to the lab
station only what is necessary.
10. It is suggested that you wear glasses rather than contact lenses.
11. Never put anything into your mouth during a lab experiment.
12. Clean up your lab area at the conclusion of the laboratory period.
13. Never “horse around” or play practical jokes in the laboratory.
14. Material Safety Data Sheet (MSDS) should be present in the laboratory (a document that
contains information on the potential hazards (health, fire, reactivity and environmental)
and how to work safely with the chemical product).

Glassware Safety
1. Chipped or cracked glassware should not be
used. Show it to the teacher.
2. Broken glassware should not be disposed of in
a classroom trashcan. There is a special glass
disposal container for it.
3. When pouring liquids into glassware, make
sure the container you are pouring into is
resting on a table at least a hands breadth from
the edge.
4. If a piece of glassware gets broken, do not try to clean it up by yourself. Notify the
teacher.
5. Do not place hot glassware in water. Rapid cooling may make it shatter.

5
Chemical Safety
1. Chipped or cracked glassware should not be
used. Show it to the teacher.
2. Broken glassware should not be disposed of in
a classroom trashcan. There is a special glass
disposal container for it.
3. When pouring liquids into glassware, make
sure the container you are pouring into is
resting on a table at least a hands breadth from
the edge.
4. If a piece of glassware gets broken, do not try
to clean it up by yourself. Notify the teacher.
5. Do not place hot glassware in water. Rapid
cooling may make it shatter.

Heating Safety
1. Chipped or cracked glassware should not be used. Show it to the teacher.
2. Broken glassware should not be disposed of in a classroom
trashcan. There is a special glass disposal container for it.
3. When pouring liquids into glassware, make sure the container
you are pouring into is resting on a table at least a hands breadth
from the edge.
4. If a piece of glassware gets broken, do not try to clean it up by
yourself. Notify the teacher.
5. Do not place hot glassware in water. Rapid cooling may make it shatter.

First Aid
1. Injury: Burns
To Do: Immediately flush with cold water until burning sensation is lessened.
2. Injury: Cuts, bruises
To Do: Do not touch an open wound without safety gloves. Pressing
directly on minor cuts will stop bleeding in a few minutes. Apply
cold compress to bruises to reduce swelling.
3. Injury: The eyes
To Do: Flush eyes immediately with plenty of water for several
minutes. If a foreign object is lodged in the eye, do not allow the eye
to be rubbed.

6
7
LAB SAFETY SYMBOLS

8
9
 EQUIPMENTS USED IN MICROBIOLOGY LABORATORY

1. Petri dish
A Petri dish is a shallow transparent lidded dish that biologists use to hold growth medium in
which cells can be cultured, originally, cells of bacteria, fungi and small mosses. The container is
named after its inventor, German bacteriologist Julius Richard Petri.

2. Bunsen burner
A Bunsen burner, named after Robert Bunsen, is a kind of gas burner used as laboratory
equipment; it produces a single open gas flame, and is used for heating, sterilization, and
combustion. The gas can be natural gas or a liquefied petroleum gas, such as propane, butane, or
a mixture.

3. Glassware
Laboratory glassware refers to a variety of equipment used in scientific work, and traditionally
made of glass. Glass can be blown, bent, cut, molded, and formed into many sizes and shapes,

10
and is therefore common in chemistry, biology, and
analytical laboratories. Examples include beakers,
glass jars, test tubes, petri dishes, microscope glass
slides.

4. Pipette
A pipette (sometimes spelled pipet) is a laboratory
tool commonly used in chemistry, biology and
medicine to transport a measured volume of liquid,
often as a media dispenser.

5. Hot air oven

It is used for sterilization of glassware’s, such as test tubes, pipettes and petri dishes. Such dry
sterilization is done only for glassware’s. Liquid substances, such as prepared media and saline
solutions cannot be sterilized in oven, as they lose water due to evaporation. This kind of dry
heat sterilization is recommended when it is undesirable that hot air make contact with the
material to be sterilized. This is true for glass wares – glass petri plates, Pipettes as well as for
substances like oil, powder, etc.

6. Drying oven
For preparation of certain reagents, the glassware’s, after proper cleaning and rinsing with
distilled water, are required to be dried. They are dried inside the drying oven at 100°C till the
glassware’s dry up completely.

11
 7. Autoclave
Autoclave is the nucleus of a microbiology laboratory. It is used not only to sterilize liquid
substances such as prepared media and saline (diluents) solutions, but also to sterilize
glassware’s, when required. Autoclave sterilizes items by heating them with steam to a very high
temperature (121 degrees° C). The advantage of using an autoclave is that it can reach
temperatures higher than boiling water alone so it can kill not only bacteria but also bacterial
spores, which tend to be resistant.

8. Incubator
Incubator is a warm cabinet that the temperature is set to a proper temperature for microbial
growth. A good temperature for most bacteria is about 35ºc and a good temperature for most
fungi is 25°C. Incubator can be of two types:
(a) Microbiological incubator: Profuse growth of microbes is obtained in the laboratory
by growing them at suitable temperatures. As most of the microbes pathogenic to man
grow profusely at body temperature of normal human being (i.e. 37°C), the usual
temperature of incubation is 37°C.
(b) BOD incubator (low temperature incubator): Some microbes are to be grown at lower
temperatures for specific purposes. The BOD low temperature incubator which can

12
 maintain temperatures from 50°C to as low as 2-3°C is used for incubation in such
cases.
The incubator maintains optimal temperature, humidity and other conditions such as the CO2
and oxygen content of the atmosphere inside.

9. Fridge (refrigerator)
It serves as a repository for thermo labile chemicals, solutions, antibiotics, serums and
biochemical reagents at cooler temperatures and even at sub-zero temperatures (at less than 0°C).
Stock cultures of bacteria are also stored in it between sub-culturing periods. It is also used for
the storage of sterilized media, so as to prevent their dehydration.

10.Deep fridge
It is used to store chemicals and preserve samples at very low sub-zero temperatures.

13
 11.Electronic Top-pan Balance:
It is used for weighing large quantities of media and other chemicals, where precise weighing is
not of much importance.

12.Electronic Analytical Balance:

It is used to weigh small quantities of chemicals and samples precisely and quickly.

13. Double-Pan Analytical Balance:

14
It is used to weigh chemicals and samples precisely. Weighing takes more time, for which it is
used in emergency only.

14.Distilled water plant

Water is used in the preparation of media and reagents. If the media are prepared using tap water,
the chemical impurities present in it may interfere with the growth of the microorganisms in the
media. Moreover, the higher is the bacteria content of the media, the longer is the time required
for their sterilization and greater is the chance of survival of some bacteria.
Distilled water, though not bacteria- free, contains less number of bacteria. That is why; it is
preferred in the preparation of microbiological media. It is also used in the preparation of
reagents, because the chemical impurities present in tap water may interfere with the proper
functioning of the reagent chemicals.

15.Ultra-pure water purification system

For precision analytical works, now-a-days, instead of using double- or triple-distilled water,
micro- filtered water is used. In case of distilled water, there is chance that, few volatile
substances present in the water get volatilized during heating of the water and subsequently get
condensed into the distilled water collected

15
 16.Homogenizer
For microbiological analysis, liquid samples are directly used, whereas solid samples have to be
mixed thoroughly with a diluents (usually physiological saline), so as to get a homogenous
suspension of bacteria. This suspension is assumed to contain bacteria homogenously.

17.pH meter
A pH meter is an instrument for determining the pH of liquid media, liquid samples and buffers.
It has a glass pH electrode. When not in use, it should be kept half immersed in water contained
in a small beaker and preferably be covered by a bell jar to avoid dust accumulation in the water
and loss of water through evaporation.

16
 18.Hot plate
Hot plate is used to heat chemicals and reagents. The hot plate is made of an iron plate, which
gets heated by an electric heating element from below. The required degree of heating is
obtained by a regulator.

19.Shaking Water bath

Sometimes, heating at very precise temperatures is required. Such precise temperatures cannot be
obtained in an incubator or oven, in which temperature fluctuates, though slightly. However,
precise temperatures can be maintained in a water bath, which provides a stable temperature.

20.Quebec colony counter

17
In enumeration of bacteria in samples, it is assumed that a single bacterium gives rise to a single
visible colony, when grown on a plate of solidified nutrient medium. Thus, by counting the
number of colonies, the number of bacteria in a sample can be estimated.

21.Electronic colony counter

Electronic colony counter is of two types:
(a) Hand-held electronic colony counter
(b) Table-top electronic colony counter.
Both are used for different purposes.

22.Magnetic stirrer
In the preparation of solutions, certain chemicals require stirring for long time, to be dissolved in
certain solvents. Magnetic stirrer is used to dissolve such substances easily and quickly. A small
teflon-coated magnet, called ‘stirring bar’, is put into a container containing the solvent and the
solute.

18
 23.Sonicator
It is used to rupture cells using high frequency waves.

24.Vortex mixer
It is an instrument used for thorough mixing of liquids in test tubes. It has a rotor, whose speed
can be controlled. On the tip of the rotor is a foam-rubber top. When the bottom of a test tube is
pressed upon this foam-rubber top, the rotor starts rotating, thereby rotating the bottom of the test
tube at high speed.

25.Laminar flow hood

19
It is a chamber used for aseptic transfer of sterilized materials, as well as for inoculation of
microbes. Dust particles floating in the air harbor microbes. These microbe-laden dust particles
may enter into the sterilized media and contaminate them, when they are opened for short
periods of time during inoculation of microbe or transfer from one container to another.

26.Microscope
It is a device that produces a magnified image of objects too small to be seen with the naked eye.
Such objects are thus called “microscopic.” The microscope is widely used in medicine and
biology.

20
21
 MICROSCOPE AND ITS TYPES

Microscope is an optical instrument that uses a lens or a combination of lenses to produce

magnified images of small objects, especially of objects too small to be seen by the unaided eye.
Such objects which cannot be seen by an unaided eye are called microscopic.

History of microscope
In 1665, an English physicist, Robert Hooke looked at a sliver of cork through a microscope lens
and noticed some "pores" or "cells" in it.

Vocabulary for microscope

 Magnification: increase of an object’s apparent size
 Resolution: power to show details clearly
Both are needed to see a clear image.

Parts of a microscope

1. Ocular lens:
Magnifies; where you look through to see the image of your specimen. They are usually 10x or
15x power. Our microscopes have an ocular lens power of 10x.
2. Arm:
It supports the tube and connects it to the base.
3. Stage:
The flat platform where the slides are placed.

22
 4. Coarse adjustment knob
Moves the stage up and down.
5. Fine adjustment knob
Small, round knob on the side of the microscope used to fine-tune the focus of your specimen
after using the coarse adjustment knob
6. Base
The bottom of the microscope used for support
7. Body tube
Connects the eyepiece to the objective lens.
8. Revolving nosepiece
The part that holds two or more objective lenses and can be rotated to easily change power.
9. Objective lens
Adds to the magnification. Usually you will find 3 or 4 objective lenses on a microscope. They
almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most
common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X, 400X and
1000X. The shortest lens is the lowest power, the longest one is the lens with the greatest
power. Lenses are color coded. The high power objective lenses are retractable (i.e.
40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded)
thereby protecting the lens and the slide.
10. Stage clips
Stage clips hold the slides in place. If your microscope has a mechanical stage, you will be able
to move the slide around by turning two knobs. One moves it left and right, the other moves it
up and down.
11. Diaphragm
Controls the amount of light going through the specimen. Many microscopes have a rotating disk
under the stage. This diaphragm has different sized holes and is used to vary the intensity and
size of the cone of light that is projected upward into the slide. There is no set rule regarding
which setting to use for a particular power. Rather, the setting is a function of the transparency of
the specimen, the degree of contrast you desire and the particular objective lens in use.
12. Light
Makes the specimen easier to see.

Types of microscope

23
The different types of microscopes are discussed below.
1. Light Microscopes
The most common type of microscope you’re likely to come across, these microscopes rely on
lenses and light to illuminate a specimen for optimal image-gathering. They can be used for
viewing living cells, insects, for performing dissections, or for clinical blood and tissue
assessment.

2. Compound Microscopes
Compound microscopes can be found in schools and labs across the world. They fit on a desktop,
they’re portable, affordable, and easy to use. Their light source comes from the bottom, and light
must pass through the specimen to travel through the microscope’s lenses and make it fully
visible. They are most often used to view objects at a cellular level and can reach magnifications
up to 1000x.

3. Stereoscopic Microscopes
These are common in labs and educational settings, as well. A stereoscopic microscope has a
light source on the top to illuminate the specimen, causing reflection into the microscope lens.
They have weaker magnification than compound microscopes, to make it easier to see opaque,
larger objects up close, at a maximum magnification of about 50x. Dual light paths inside the

24
microscope tube create layered imaging, which provides a 3-dimensional image in the eyepiece,
an improvement over the flat imaging in a compound scope. These are commonly used for
dissection, coin appraisal, gem and mineral study, and entomology. They can also be used for
intricate watch or microchip repair.

4. Confocal Microscopes
Confocal microscopes use lasers to scan a specimen and create high resolution, high
magnification images. Because they provide depth-selection by scanning the specimen, they can
create sectional detail (without physical dissection) that can be used to build a 3D image.
Confocal microscopes are most often used in biomedical sciences to image living cells or
embryos marked by fluorescence. They can typically reach a maximum magnification of 2000x.

5. Electron Microscopes
An electron microscope doesn’t need light to create an image. Instead, it sends accelerated
electrons across or through a specimen to render a digital image. These microscopes have the
highest power and highest resolution available and are used to see detailed structure at the
cellular and macromolecular levels. While this may seem like the answer to all things
microscopy, electron beams destroy samples. This means you can’t use them to view live
specimens.

25
 6. Scanning Electron Microscopes (SEM)
SEM microscopes scan the surface of a specimen in a rectangular pattern to provide information
about topography and composition. The sample is set on a stage inside a vacuum chamber, which
removes any electron-inhibiting air to aid acceleration. Information is then sent to a computer for
interpretation and a digital image. SEMs can reach resolutions of about 10 nanometers, and have
a maximum magnification strength of 30,000x.

7. Transmission Electron Microscopes (TEM)

Unlike the scanning structure of an SEM microscope, a TEM must pass electrons through a thin
specimen to receive information, comparable to the way light must pass through a specimen on a
compound microscope. Rather than reflecting off the specimen’s surface, the TEM’s electrons
pass back and forth through the microscope’s vacuum chamber to build an image. Stronger than
an SEM microscope, a TEM produces high magnification power of up to 1-nanometer
resolution, or about 500,000x.

26
 8. Reflection Electron Microscopes (REM)
These microscopes are used to study the microscopic surface structure and composition of
crystals. A narrow beam of electrons is refracted from the first few atomic layers of the crystal at
high resolution (up to about 1 nanometer). It is combined with spectroscopy (the study of light
dispersal) to form an image.

9. X-Ray Microscopes
Because X-rays can penetrate matter efficiently, they can be used to view the internal structure of
opaque specimens such as rocks, bones, or metals. While lacking the power of an electron
microscope, they don’t require a vacuum tube or accelerated electrons and so can handle any
kind of specimen. X-ray microscopes can reach a resolution of about 20 nanometers.

27
 10. Scanning Probes
SPMs can create nanoscale images at a resolution of less than 1 nanometer. A probe tip about as
wide as a single atom scans across the specimen surface. It detects any deflections in the
specimen and measures them via laser, then sends the information into photodiodes, which
interpret the information into a digital image. These microscopes are used to study objects on a
nanoscale and look inside cells and molecules.

11. Scanning Acoustic

Used to image the internal structures of specimens without causing damage. They can achieve
resolution down to 100 nanometers, are often used to inspect optical or electronic devices.
Specimens are submerged in liquid and subjected to sound waves, which echo back to a
transducer which pixelates the information and creates an image.

12. Fluorescence microscope

Fluorescence microscopy is a type of light microscope that works on the principle of
fluorescence. A substance is said to be fluorescent when it absorbs the energy of invisible shorter
wavelength radiation (such as UV light) and emits longer wavelength radiation of visible light
(such as green or red light).

28
 13. Dark field microscope
Dark-field microscopy is a technique that can be used for the observation of living, unstained
cells and microorganisms. In this microscopy, the specimen is brightly illuminated while the
background is dark. It is one type of light microscopes, other being bright-field, phase-contrast,
differential interface contrast, and fluorescence.

14. Phase contrast microscope

Phase-contrast microscopy is basically a specially designed light microscope with all the basic
parts in addition to which an annular phase plate and annular diaphragm are fitted. It is situated
below the condenser. It is made up of a circular disc having a circular annular groove

29
PRACTICALS

30
 PRACTICAL#1
OBSERVATION OF ONION EPIDERMIS CELLS WITH THE
HELP OF A MICROSCOPE
Onion tissue provides excellent cells to study under the microscope. The main cell structures are
easy to see when viewed with the microscope at medium power. Also present in the onion cell, is
a well-developed cell wall and a cell membrane just beneath it.

OBJECTIVE:
To study the structure of the onion epidermal cell, with particular emphasis on the nucleus and
nucleoli.

MATERIAL:
 onion
 microscope
 glass slide
 cover slip
 iodine

SAMPLE:
Onion epidermal cells

PROCEDURE:
1) Get a glass slide and cover slip for yourself and make sure they are both thoroughly
washed and dried.
2) Remove the single layer of epidermal cells from the inner (concave) side of the scale leaf
(The thinner the better).
3) Place the single layer of onion cell epithelium on a glass slide. Make sure that you do not
fold it over or wrinkle it.
4) Place a drop of iodine stain on your onion tissue.
5) Put the cover slip on the stained tissue and gently tap out any air bubbles.
6) Observe the cells under 4x, 10x, and 40x with the diaphragm wide open.

OBSERVATIONS:
The onion cells are long, rectangular in shape.

31
32
 PRACTICAL#2
IDENTIFICATION OF BACTERIA BYGRAM STAINING

OBJECTIVE:
 Smear preparation
 Procedure of gram staining and reading slides
 Quality control for gram stain

PRECAUTIONS:
Wear apron, mask and gloves before performing this practical.

SAMPLE:
Specimen from patient

MATERIAL REQUIRED:
 Specimen from patient  Biosafety hood
 Culture plate with growth  Bunsen burner
 Broth culture  Four primary reagents
 Sterile Pasteur pipette Crystal Violet (primary)
 Smear loop Gram iodine (mordant)
 Clean slides Acetone (decolorizer)
 Glass marker Safranin (secondary stain)
 Discard jar with 1% sodium  Staining rack/ rods
hypochlorite solution
PROCEDURE:
 Preparation of smear
It is important to prepare good quality smear from a good quality specimen.
Material Required:
 Specimen from patient  Discard jar with 1% sodium
 Culture plate with growth hypochlorite solution
 Broth culture  Biosafety hood
 Sterile Pasteur pipette  Bunsen burner
 Smear loop
 Clean slides
 Glass marker

33
The smear can be prepared with patients’ sample, culture plate, or broth culture. The procedures
must be performed under a biosafety hood.
Smear preparation from patient sample:
1) Take a glass slide and label it.
2) With an inoculating loop, place a small portion of sample on the slide and spread to
obtain a smear of medium thickness.
3) Let it air-dry
4) Mark the position of the smear with a glass marker or pen.
5) Heat fix the smear by passing over the flame for 2-3 seconds, taking care not to burn it.
Smear preparation from broth culture
1) Take a tube containing the broth culture and remove the cotton plug.
2) Flame the mouth of the tube and take a loop-ful of culture using a sterile inoculating
loop.
3) Replace the cotton plug
4) Spread the culture on the slide and let it air-dry.
Smear preparation from solid culture media
1) Take a loop-ful of normal saline and smear it on the slide.
2) With an inoculating loop, pick a bacterial colony from culture plate and emulsify onto the
saline while spreading the smear.
3) Let air-dry.

 Gram staining
There are various modifications of gram-staining procedure. The most common method is
discussed below.
Materials Required:
 Four primary reagents
Crystal Violet (primary)
Gram iodine (mordant)
Acetone (decolorizer)
Safranin (secondary stain)
 Staining rack/ rods
Gram-staining procedure
1) Place a slide with heat fixed smear on staining tray.
2) Gently smudge the slide with crystal violet and let it stand for 1 minute.
3) Tilt the slide slightly and rinse with gentle stream of tap water.
4) Gently flood the smear with Gram iodine and let it stand for 1 minute.

34
 5) Rinse with tap water. The smear appears as a purple circle.
6) De-colorization is the next and the most important step which uses acetone. Tilt the glass
slide slightly and apply acetone drop-by-drop. Do not decolorize for more than 2-3
seconds otherwise whole smear will be washed off.
7) Gently flood the slide with safranin. Cover the smear and let it stand for 30 seconds.
8) Wash with tap water.
9) Let it air-dry and blot gently.
10) View using a light microscope under oil emulsion.

 Quality control for gram stain

Gram positive – S.aureus ATCC strain
Gram negative – E.coli ATCC strain
These are used to prepare the smear adjacent to the test smear

OBSERVATIONS:
 Bacteria retaining the positive strain is stained purple is gram positive.
 Bacteria that takes up pink color is gram negative.

COMMON ERRORS:
 Wrong labelling
 Too thin or thick smear
 Inadequate fixing
 Stain deposits
 Over de-colorization

35
 PRACTICAL#3
SERIAL DILUTIONS – LIVE BACTERIAL CELL COUNTS

OBJECTIVE:
To find out the number of bacteria in a live culture.
What are serial dilutions used for in microbiology?
To dilute a bacterial culture, so that when you spread a sample onto an agar plate containing
nutrients, only 30-300 bacteria will grow and produce colonies. These colonies are referred to as
Colony Forming Units (CFUs). Each CFU represents a single bacterial cell that multiplied into
millions of cells piled up onto each other until we can see a single colony of bacterial cells.
To calculate the number of live bacteria in a 10ml original solution taken in a test tube:
The answer can be # of bacteria per ml (# bacterial cells/ml)
If the total volume of culture is known, the answer can be total number of bacteria in the tube:
Total number of bacteria in the tube = (number of bacteria/ml) x 10ml

SAMPLE:
Broth culture containing live bacteria

MATERIAL REQUIRED:
 Test tubes containing broth culture
 Original culture tube
 Glass plates
 Incubator

PROCEDURE:
Prepare a series of diluted bacterial cultures in broth
1. Set up a series of seven tubes (label them tube 2-7) including the original culture tube
(tube 1).
2. Add some diluent, which is usually a broth culture in which bacteria can grow. Vary the
volume of diluent in each tube.
3. Take 1.0ml of original culture and add it to tube#2 (which is containing 9.0ml of diluent).
The original bacterial solution added to the tube will be diluted and a 10ml diluted
solution is formed.
4. To calculate dilution, we compare the present tube (tube#2) to the previous tube (tube#1).
5. We calculate dilution by:

36
 Volume added / (volume added + initial volume) or
Volume added / total volume.
In this case the dilution value is 1/10.
6. Final dilution factor compares final tube with tube 1 (the original tube). Final dilution
factor in this case is 1/10.
7. Take 0.1ml of solution from tube#2 and add it to the tube#3. The dilution calculated for
this tube is 1/10. The final dilution factor for this tube is calculated by multiplying the
value of dilution of tube#2 and tube#3. Its value is 1/100.
8. For the third dilution, take 0.5ml solution from tube#3 and add it to tube#4. Tube#4
initially contains 9.5ml of solution.
9. The dilution calculated for this tube is 1/20. The final dilution factor is obtained by
multiplying the dilution value for tube#2, tube#3 and tube#4 which equals 1/2000.
10. Transfer 2.0ml of solution from tube#4 to tube#5. The dilution in this case is 1/5 and the
dilution factor, obtained by multiplying dilution values of the previous tubes, is
calculated to be 1/10000.
11. Take 2.0ml of solution from tube#5 and add it to tube#6, which itself contains 2.0ml. The
dilution is ½ and the dilution factor calculated is 1/20000, calculated by the same
procedure as followed for other tubes.
12. Take 1.0ml of sample from tube#6 and add it to tube#7. The dilution is 1/3 and final
dilution factor is 1/60000.

Spread a sample of each diluted culture on nutrient-rich agar plate

1. Prepare plates for tube#2-7. Take 0.1ml of solution from these tubes and spread it onto
the plate.
2. Incubate the plates to allow the bacteria to replicate and form CFUs.
(A single bacteria divides to form 30-300 CFUs on each plate. If there are more than 300
CFUs on each plate, the colonies tend to run into each other and error is likely to occur. If
the value is less than 30 CFUs the numbers are too low and errors are likely to occur.)
3. The following diagram shows these plates which have been incubated for 18 hours.

37
 The least number of colonies are formed on the plate#7 and the highest number of
colonies are found in plate#2 and plate#3.
Plate#7 = 6 CFUs
Plate#6 = 14 CFUs
Plate#5 = 91 CFUs
Plate#4 = TMTC (Too Many To Count)
Plate#3 = TMTC
Plate#2 = TMTC
4. Plate#5 is taken to calculate the number of bacteria in the original culture.

Based on the number of bacteria (CFUs) represented on the plates, calculate the number of
bacteria present in the original culture tube.

1. Tube#5 has 91 CFUs from 0.1ml solution which was placed in tube#5.
91 CFUs / 0.1ml
910 CFUs / 1 ml
However, we need to calculate the number of CFUs / ml in Tube#1.
2. Based on earlier calculations, we know that tube#5 only has 1/10000 bacteria (per ml)
that tube 1 has. In other words, tube#5 has 10000 times less bacteria than tube#1.
Therefore, if tube#5 is given 10000 times more bacteria (per ml) then tube#5 will have
the same amount of bacteria (per ml) as tube#1.
(91 CFU / 0.1ml) x 10000 (in Tube#5)
= 910000 / 0.1ml (in tube#1)
or 9100000 / 1ml
3. The CFUs in tube#1 is 9100000 CFUs / 1ml. since the original solution was a 10ml
solution, the number of CFUs per 10ml is 91000000 CFUs.
(9100000 CFUs / 1ml) x 10ml
= 91000000 CFUs
The CFU value represents the population of live bacteria.

38
OBSERVATIONS
Plate#5 was chosen to calculate the number of bacteria in the original culture. By using some
calculations as shown above, the number of bacteria in the original culture were found to be
91000000 CFUs.

39
 PRACTICAL#4
PLATE POURING METHOD

OBJECTIVE:
Plasmids can contain one or more antibiotic resistance genes, which confer antibiotic resistance
to the bacteria that carry them. The presence of an antibiotic resistance gene on a plasmid allows
researchers to artificially separate bacteria that contain the plasmid from bacteria that do not (i.e.
growing the bacteria in the presence of the antibiotic).
Luria broth (LB) is a nutrient-rich media that is commonly used in laboratories to culture
bacteria. The addition of agar to LB creates a gel on which bacteria can grow because they are
unable to digest the agar but can obtain nutrition from the LB within. When an antibiotic is
added to this gel, it allows only bacteria with resistance to that antibiotic to be selected - usually
due to a plasmid carrying the antibiotic resistance gene.
LB agar plates are commonly used to culture individual bacterial colonies from freezer stock or
selecting individual from bacterial mixtures.

SAMPLE:
Lactobacillus

MATERIAL:
 Autoclave
 Water bath
 Autoclave tape
 37 g pre-mixed powder consisting of:
5 g peptone
10.0 g peptone from casein
10.0 g sodium chloride
12.0 g agar-agar
 1 L Sterile H2O
 Sterile plates of your desired size (in this case, 60 mm x 15 mm plates which can hold 5-
10 mL of agar)
 Autoclavable flasks
 Sterile pipettes
 Ice bucket to hold antibiotic
 Antibiotic at 1000 x concentration disolved in the appropriate liquid solvent.

PROCEDURE:
Preparing agar mix

40
 1. Measure 37g of pre-mixed LB-agar powder per L of molten agar you’d like to make.
Since we require only 220ml of molten agar, we take 8.14g of agar powder.
2. Transfer the LB-agar powder, which is measured, out into an appropriately sized bottle
for autoclaving. Transfer the sterile water (in our case 220 mL) to the same bottle and
swirl to form a medium/agar colloid.
3. Cover the opening of the bottle with its cap (but do not make an air-tight seal!) and tape
the bottle with autoclave tape. The autoclave darkens during the autoclave process if the
sample has spent at least 10 min at 121 ℃. Use lab tape to label the bottle with your
initials, the date, and the bottle contents.
Autoclaving
4. Place the gel mix in the autoclave and run on a setting that gets the sample to at least 121
℃ under 20 psi for at least 30 min. The high pressure will prevent your gel mix from
boiling over at high temperature, and high temperature sterilizes material.
Bench setup
5. While your samples are sterilizing in the autoclave, you should prepare your plate
pouring station:
Find an empty section of lab bench with a working flame.
Spray down the bench with a 70% ethanol solution and wipe down with a paper towel.
Count out the appropriate number of plates and stack them on your lab bench.
Label the plates with the date and the medium they will contain including the identity of
the antibiotic.
Position the flame just to the side of where plates will be poured – leaving room for bottle
of molten gel mix, a tube rack containing the appropriate antibiotics, and a section for
active pouring.
6. Prepare your antibiotics. Prior to adding your antibiotic to the molten gel mix, you should
create a 1000x stock solution.
Setting the water bath
7. Prepare a water bath at 60 ℃ with sufficient water to submerge ~75% of the bottle
containing your molten gel mixture. 60℃ is a good temperature because the molten agar
will remain liquid at this temperature but most antibiotics will not break down at this
temperature.
8. Retrieve the molten agar mix from the autoclave. Once the autoclave cycle is complete,
open the door to the autoclave leaving it open that way for ~10 min. This will allow any
steam to escape from the autoclave and will cool gel-mix slightly. When removing
anything from the autoclave, use thermally insulated gloves.
Cooling the agar
9. Partially submerge your molten gel-mix in the 60 ℃ water bath. Leave the molten gel-
mix in the water bath for at least 5 min. Do not let any of the water bath water touch the
neck or top of the bottle as this water is not likely to be sterile. Cooled agar should be

41
 warm to the touch; as a rule of thumb, if the molten agar cannot be taken out of the water
bath wearing only lab gloves, it’s not likely cool enough to add antibiotic to.
Pouring
10. Light the flame at the plate pouring station and dilute your antibiotic into your ~60 ℃
molten gel mix using sterile technique.
11. Swirl the agar bottle to ensure even distribution of the antibiotic throughout the agar.
12. Open one plate at a time next to the flame and begin pouring. Measure the desired
amount of agar with a pipette for the first plate to get a good idea of what that volume
looks like in your particular plate.
13. For the remainder of the plates, pour directly from the bottle.
Be sure to swirl the plates after pouring to remove bubbles and ensure an even
distribution of agar over the bottom of the plate. Cap each plate after pouring and stack as
it is poured.
If the agar partially solidifies in the bottle while pouring, stop pouring and re-make the
gel-mix. If plates are made without any antibiotic, alternatively re-liquefy the agar by
running it through the autoclave again or by microwaving (if using a microwave, beware
of over-boiling!).
Solidification and storage
14. Leave the plates out on the bench to solidify.
It takes roughly 30 min for our plates to solidify at room temperature, however leave
them out at room temperature overnight to allow them to dry. After overnight drying,
place the plates in a plastic bag with an absorbent material to reduce condensation. The
plates are then stored at 4 ℃ until use.
Testing
15. Once your plates have solidified and dried, test them to make sure the antibiotic functions
properly:
Take out two plates.
On the first plate, streak out a strain that is known to be resistant to the antibiotic.
On the second plate, streak out a strain that’s not resistant to the antibiotic.
Incubate both plates overnight at the appropriate growth temperature and check for
growth.

OBSERVATIONS:
Different cultures other than agar were used to grow bacterial colonies.
 A sheep blood plate was used in which bacteria were grown on blood collected from a
wound. Creamy-looking hemolytic colonies of bacteria were seen.
 Bacteria grown on chocolate. Golden colored Staphylococcus aureus grew.

42
43
 PRACTICAL#5
STREAK PLATING METHOD

OBJECTIVE:
Streaking is a method that isolates a pure strain from a species of bacteria. A sample is taken
from a colony and a microbiological culture is grown on the new plate in order for the organism
to be identified properly.
The purpose of streaking is:
 To isolate colonies of an organism (usually bacteria) on an agar plate. This is useful for
separating species in a mixed culture (to purify/isolate a specific strain from
contaminants) or studying an organism's colony morphology.
 To classify the organism: biochemical tests for bacterial identification are only applicable
on pure cultures.

SAMPLE:
Mixed bacterial culture

MATERIAL REQUIRED:
 Mixed culture of bacteria
 Sterile petri dish with appropriate bacterial media (such as trypticase soy agar, nutrient
agar)
 Inoculating loop (usually nichrome, a nickel-chromium alloy, or platinum; it may also be
a single-use disposable plastic loop, which would be discarded between sectors rather than
resterilized)
 Bunsen burner
1) Marking pen

PROCEDURE:
1) Place the inverted plate close to the Bunsen burner to reduce the chance of contamination
2) Turn on the Bunsen burner
3) Loosen the cap of the bottle containing the inoculum
4) Heat the wire of the inoculating loop from the Bunsen burner
5) Lift the bottle and pass its neck near Bunsen burner 2-3 times to sterilize it.
6) Put the loop inside the suspension without touching the sides.
7) Again sterilize the neck of the bottle before placing the cap.
8) Partially lift the lid of the petri dish
9) Rub the loop containing the bacteria on the top area making a circle
10) Reflame the loop; cool slightly

44
 11) Use the loop to streak the bacteria in 3-4 lines from the streaked circle
12) Reflame the loop, allow to cool slightly
13) From the area you just streaked, streak more 3-4 lines
14) Reflame the loop, allow to cool slightly
15) Streak another 3-4 lines
16) Reflame the loop
17) Streak zig zag lines in the last part of agar dish, from outside to center
18) Do not streak again previous area
19) Sterilize the loop
20) Put down the Bunsen burner
21) Put the petri dish, ready for incubation

OBSERVATIONS:
The streaked plate is incubated for 24 hours at 37°C. Carefully examine the colonies that have
emerged in the plate. The general appearance of all colonies should be the same. If there are
several types of colony, streak each one separately on a separate plate to obtain a pure culture.

45
THANK
YOU!

46
47