JINNAH UNIVERSITY FOR
WOMEN
AEMON NASIR
Department Of Food Science and Technology
Bs - I, 1st Semester 2020
Section: “A”
General Food Microbiology
Course Code: FST/1021
Enrolment NO: JUW07450
Lab REPORT
Teacher Name: Miss AQSA AJAZ
INDEX
SNO. EXPERIMENT PG#
1 TO UNDERSATAND THE FOOD MICROBIOLOGY LABORTARY SAFTEY 1
RULES
2 TO LEARN THE OPERATION OF AUTOCLAVING 2
3 TO EXAMINE DIFFERENT MICROORGANISMS UNDER MICROSCOPE 3–4
4 TO DEMONSTRATE THE OPERATION OF DIGITAL COLONY COUNTER 5
5 TO IDENTIFY GRAM NEGATIVE OR GRAM POSITIVE BACTERIA IN 6
FOOD
6 TO PERFORM SIMPLE STAINING 7
7 TO PERFORM GRAM STAINING AND COMPARISON OF 8
MORPHOLOGICAL SHAPES AND ARRANGEMENT OF BACTERIAL
CELL
8 TO STUDY THE PREPARATION OF NUTRIENT MEDIA, ITS POURING
AND PLATING AND DISPOSAL AFTER USAGE 9
9 TO ENUMERATE HE TOTAL PLATE COUNT BY POUR PLATE
TECHNIQUE IN FOOD SAMPLE 10
10 TO ENUMERATE HE TOTAL PLATE COUNT BY SPREAD PLATE 11
TECHNIQUE IN FOOD SAMPLE
EXPERIMENT # 1
OBEJECTIVE:
To understand the food microbiology laboratory safety rules.
THEORY:
Scientific and educational laboratories can be dangerous places, so staying safe in the lab is
a paramount concern. Never assume that a person has experience with all chemical,
hardware, and lab safety equipment should simply because they are an expert in a related
field. All worn and damaged equipment’s should be replaced immediately. As soon as
equipment or an important lab feature show signs of wear, the manager should replace that
item immediately.
Following are the safety rules with respect to microbiology.
Never work alone in the laboratory without permission and prior knowledge of the
instructor.
Do not engage in rowdy, playful, or unprofessional activities in the laboratory.
Work surfaces must be disinfected at the beginning and at the end of every
laboratory period.
All students must wash their hands at the beginning and at the end of every
laboratory.
No eating or drinking is permitted in the laboratory.
Lab benches are to be kept free of extraneous item while conducting experiments.
This includes personal items s such as backpacks, cell phones, and unnecessary
books.
Students must wear closed-toe shoes that cover the top of the foot, and appropriate
clothing, at all time in the laboratory.
Keep hands away from your face, eyes and mouth when working with chemicals or
microorganisms. This includes not applying cosmetics, not adjusting contact lenses,
and not biting your finger nails.
If any chemical or other agents splash into your eyes, immediately go to the nearest
sink and flush your eyes with water.
Take special precaution when working with open flames. Loose hair, clothing,
dangling jewelry, and nearby paper must be secured. Keep container of alcohol,
acetone, or other flammable liquids at a safe distance from flames.
Report accidents, spills, breakages, or injuries to the instructor. No matter how trivial
they appear. Do not remove cultures, reagents, or other materials from the
laboratory unless specific permission from the instructor has been granted.
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EXPERIMENT # 2
OBEJECTIVE:
To learn the operation of autoclaving.
THEORY: Same as lab manual.
PROCEDURE: Same as lab manual.
DISCUSSION:
German ‘ROBERT KOCH’ and French microbiologist ‘CHARLES CHAMBER’ introduced
autoclave after that autoclaving is automatically produced. Autoclave is an instrumental
device used for two purposes. First is sterilization by steam and the second one is
contamination of infectious bio waste. Autoclaving is the process by the help of autoclave,
it is a two principle mechanism temperature or pressure (moist heat) main motive achieved
by sterilization.
Standard temperature is 121.5 (autoclaving).
It is a standard operating process.
The pressure of autoclaving is 15 psi (pound square inch).
Is the microbial weight is light then it takes 15 minutes and if the microbial weight is
heavy then it takes 30 minutes almost.
There must be some precautions, the two main precautions are:
Don’t use aluminum foil because aluminum foil provides barrier (use bloating paper
or cotton).
There should be some space or gap to let the steam vent out otherwise it will get
burst on heating.
CONCLUSION:
In this experiment, we studied about Autoclaving its importance, working principle and SOP.
This is an instrument used for decontamination of infectious bio or laboratory waste and the
sterilization of laboratory glassware, media, and reagent so there will be fewer chances of
microbial growth. Autoclave is used to sterilize culture media, instruments etc, they are also
used on large industrial scale.
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EXPERIMENT # 3
OBEJECTIVE:
To examine different microorganisms under microscope.
THEORY: Same as manual.
PROCEDURE: Same as manual.
OBSERVATION:
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DISCUSSION:
ELECTRON MICROSCOPE: A microscope that uses a beam of accelerated electrons and s a
source of illumination.
OPTICAL MICROSCOPE: This microscope is often refers as the light microscope, commonly
uses visible light as system of lenses to magnify small objects.
SCANNING ELECTRON MICROSCOPE: It is as type of electron microscope that produces and
images of a sample by scanning the surface with a focused beam of electrons.
Amoeba is a unicellular eukaryotic organism, belongs to a protozoa family. It does
not have definite shape and move by the means of pseudopodia.
Paramecium is a free-living organism. It usually lives in the stagnant water of pools,
lakes, ditches, ponds, freshwater etc. Its outer body is covered by the tiny hair-like
structures called cilia.
Euglena is a unicellular organism with flagella. These flagella are long whip-like tails
used for movement. They do not have a cell wall, instead they have a thick outer
covering, known as a pellicle that is composed of protein and gives them both
strength and flexibility.
CONCLUSION:
In this experiment, we studied about the morphology of amoeba, euglena and paramecium
and observed them under a microscope. They have different locomotory organs through
which they can move from one place to another. Amoeba has pseudopodia, paramecium
has cilia and euglena has flagella through which they show their movement.
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EXPERIMENT # 4
OBEJECTIVE:
To demonstrate the operation of digital colony counter.
THEORY: Same as manual.
PROCEDURE: Same as manual.
OBSERVATION:
OBS. FOOD PRODUCT COUNTED COLONIES
NO
SAMPLE Citrus Orange (Fungal) 14
A
SAMPLE Milk (Bacterial) 9
B
RESULT:
Sample A consist of 14 colonies.
Sample B consist of 9 colonies.
DISCUSSION:
Digital colony counter is an electronic device used to count the number of colonies of
bacteria or fungi. Some components of digital counter are:
1) Glass surface
2) Light source
3) Magnifying glass
4) Auto-marker.
The glass surface is used to place the plate or samples on it. Light source is used to clearly
see the colonies in light. Magnifying glass is used to focus the plate to see colonies. Auto-
marker is used to touch the sample plate and count colonies digitally.
Sample A (FUNGUS) Consists of some yellow, cotton-like, fluffy colonies and some greenish-
black spots which are round in shape. They are present in bundles. They are at some
distance from each other.
Sample B (BACTERIAL) Consist of some red, smooth spots. They are rounded in shape and
of different size. They are at some distance from each other.
CONCLUSION:
There are two samples citrus orange juice as fungi and milk as bacteria. In citrus if the pH is
low it is fungal. If dots can be seen it is bacteria and cotton like can be seen for fungal.
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EXPERIMENT # 5
OBEJECTIVE:
To identify gram negative or gram positive bacteria in food.
THEORY: Same as lab manual.
PROCEDURE: Same as lab manual.
OBSERVATION:
Organism name: e coli / staph. Aureus
Gram reaction: G+ / G-
Shape: rod / round.
RESULT OR DISCUSSION: GRAM- NEGATIVE
In this experiment, we studied about two major groups of bacteria i.e., gram-positive and
gram-negative. Both of them are morphologically different from each other.
We can see the reddish pink and rod shaped bacteria under the microscope named
E-coli.
It is gram negative bacteria G- .
It has thin cell wall and contains pink color due to stain safranin.
Due to thin cell wall they don’t retain gram stain (crystal violet) and can be
decolorized when washed with the solution of absolute and acetone.
They are 90- 95% pathogenic and can spoil food.
Resistant to antibiotics harmful to humans as they are the reasons to cause diseases.
.
CONCLUSION:
Under the microscope, we observe gram-negative. It is identified on the basis of shape, cell
wall and color. They are rod shaped, have thin cell wall and reddish pink in color.
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EXPERIMENT # 6
OBEJECTIVE:
To perform simple staining.
THEORY: Same as lab manual.
PROCEDURE: Same as lab manual.
OBSERVATION:
RESULT OR DISCUSSION:
Staining is a technique by using color of the identification of bacteria. In this we study the
morphology of bacteria by the method of simple staining.
After simple staining we observe slide under microscope at the magnification power
of ×4.
Under microscope the bacteria which found was purple in color due to the
absorption of crystal violet (stain) and retain it due to thick cell wall.
They were few in number and rounded and of different sizes scattered all over the
slide.
By studying the morphology we can say that this is gram positive bacteria.
CONCLUSION
In this experiment, we studied about simple staining. Stains are the solution that contain a
solute called a chromospheres dissolved in a solvent. Chromospheres are the color
possessing portion of the solution and are therefore responsible for the stains color. In this
we identified the bacteria by its morphology. After staining we observed slide under
microscope at magnification power x4.
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EXPERIMENT # 7
OBEJECTIVE:
To perform gram staining and comparison of morphological shapes and arrangement of
bacterial cell.
THEORY: Same as lab manual.
PROCEDURE: Same as lab manual.
OBSERVATION: GRAM STAINING FIGURE.
RESULT OR DISCUSSION:
Gram staining is the method of coloring the bacteria to determine the gram positive or gram
negative bacteria on the basis of color, shape and physical or chemical composition of the
cell wall.
We observe slide under microscope with the magnification power of ×100.
They were also of varying sizes and were present single off in the form of colony.
Under microscope, both type of bacteria were present i.e. gram positive and gram
negative. Some of them are purple and rounded while some are pink and rod shaped
scattered all over the slide.
GRAM POSITIVE: The bacteria which were rounded and retain purple color even after
adding ethanol doesn’t decolorized are gram positive.
GRAM NEGATIVE: The bacteria which were of rod shaped and don’t retain purple color and
decolorized by adding alcohol. Then by adding safranin they absorb and retain pink color 6
gram negative.
CONCLUSION:
In this experiment both type of bacteria is present gram-positive and gram negative. To
determine the gram positive and gram negative bacteria on the basis of shape, colour and
physical and chemical composition of cell wall, gram staining is the technique used for
identifying bacteria.
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EXPERIMENT # 8
OBEJECTIVE:
To study the preparation of nutrient media, its pouring and plating and disposal after
usage.
THEORY: Same as lab manual.
PROCEDURE: Same as lab manual.
DISCUSSION:
A substance, either solid or liquid, used for the cultivation, isolation, identification or storage
of microorganisms is called nutrient medium. Agar is a polymer made up of subunit of sugar
galacgose. It is used for culturing bacteria. Different microorganism need selective agar
because all microorganisms have different mode of nutrition and survival. For example:
Nutrient Agar for the growth of non fastidious organisms use
Mac Conley Agar to differentiate between Gram Positive and Gram Negative
EMB Agar to isolate fecal content.
For culturing bacteria we make agar plate. There is some step of making agar plate like first
dissolving certain amount of nutrient agar in lukewarm water to form a gel then sterilize and
pour it in Petri dish and dispose after it used.
CONCLUSION:
In this experiment, we studied that how Nutrient Agar is being prepared. It is useful to know
which microorganism is suitable for specific agar for obtaining the proper results. By knowing
this we will easily able to differentiate and handle properly and get satisfactory result.
9
EXPERIMENT # 9
OBEJECTIVE:
To enumerate he total plate count by pour plate technique in food sample
THEORY: Same as lab manual.
PROCEDURE: Same as lab manual.
CALCULATION:
CFU/ml (or g-1) = No. of counts / dilution factor * volume plated
DISCUSSION:
Pour plate technique is the microbial method to enumerate some viable cells
present in a sample. The specialty of pour plate method is that in this a known
volume of a sample is mixed with agar first and then pour into the plate.
In this the temperature should not be very high or very low because at high
temperature it killed bacteria and at low temperature it resists the growth which
results in process producing no visible growth.
Microorganisms which can be enumerated are aerobic, anaerobic and facultative.
This technique is used to perform viable plate counts, in which the total number of
colony-forming units within the agar and on the surface of the agar on a single plate
is enumerated.
CONCLUSION:
In this experiment, we use pour plate technique. It is used for desirable microorganisms
such as lactic acid bacteria in fermented foods; we further studied, pour plate is necessary
to conduct a microbiological examination of food to determine its quality.
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EXPERIMENT # 10
OBEJECTIVE:
To enumerate he total plate count by spread plate technique in food sample.
THEORY: Same as lab manual.
PROCEDURE: Same as lab manual.
CALCULATION:
CFU/ml (or g-1) = No. of counts / dilution factor * volume plated
DICUSSION:
There are two methods to enumerate the total plate count i.e.:
1. Spread plate technique
2. Pour plate technique
DEFINITION: Pour plate is a microbial technique that uses to enumerate number of viable
cells in a sample as spread plate is another technique uses to enumerate bacteria grown on
the surface of the media.
SAMPLE ADDING: In pour plate sample is added on to the solidified medium surface while
in spread plate sample is mixed with molten agar and pored into a plate.
ACCURACY: Pour plate accuracy is high and the accuracy in spread plate is low.
MEDIUM USED: Liquid molten agar media are necessary for pour plate, solidifies agar plates
are necessary to perform spread plates.
GROWTH OF MICROBES: Aerobes, anaerobes and facultative anaerobes can be enumerated
by pour plate method and only aerobes can be enumerated in spread plate.
USE OF A GLASS SPREADER: In pour plate glass spreader is not used while in spread plate
glass spreader is used to spread the sample evenly on the surface.
CONCLUSION:
In this experiment spread plate technique is used.
In pour plate 1ml volume of a sample is mixed with molten agar whereas in spread plate
0.1ml of sample is added on solidified medium surface. The spread plate is more reliable
and accuracy is higher as compared to pour plate because it is stable at room temperature
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