School of Chemical & Life Sciences

Diploma in Food Science & Nutrition

Module Code : CLF105

Module Name : Food Microbiology I
Academic year / Semester : 2016/17 / Semester 1

Name of student : ____________________

Admin no : ____________________

Laboratory Manual

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 MODULE SCHEDULE
Week Practical Remarks

1 - Lab Briefing
- Care and Use of Microscope
2 - Gram stain
- Endospore stain
3 - Biotechniques
- Aseptic Technique 1
4* Video on serial dilution, spread plate technique & streaking
technique
5 - Aseptic Technique 2
- Serial Dilution
- Spread Plate Techquie
6 Revision for Test 1 Practice:
- Aseptic Technique
- Serial Dilution
- Gram Stain
- Streaking
7 Practical Test I & Review
8* Bacterial Culture and Characteristics
9-10 - Break
11 - Common Test
* Literature Review on Foodborne Viruses
12 Examination of foods for the Standard Plate Count Report writing
13 Examination of foods for Yeasts and moulds Report writing
14 Biochemical Tests: Report writing
- Selective and Differential Media;
- Motility, Oxidase & Catalase Tests
15 Effects of Physical Control & Chemical Agent on Microbial Report writing
Growth
16 Anaerobic Culture and Growth
17 Practical Test II & Review
18-20 Study/ Exam
*Self-directed learning

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Laboratory Safety
The smooth and safe operation of a laboratory depends on observing specific safety rules and
regulations, and carefully applying standard chemical-handling and culture-handling
procedures. Students are expected to follow the following rules and regulation in all
laboratories.

General Rules and Regulations

1. Never eat, store food, or smoke in the laboratory. The food samples used in class are not
for consumption.
2. To prevent contamination of your street clothes, always wear a laboratory coat and closed
toe shoes during class. Remove this protective apparel before leaving the laboratory.
3. Keep your books and bag in the cabinet and not on the working bench. Only leave your
practical manual and a pencil on the working bench. Keep your work area organized
because multiple people working in a small space can lead to hazardous mistakes.
4. Report fires or personal injuries such as cuts or burns to your instructor immediately. Your
instructor will show you the location and demonstrate the use of emergency equipment
such as fire extinguishers, fire blankets, and first-aid kits. Know their locations and how
to use them.
5. Always double-check the label of any chemical or microbial culture you use.
6. Keep any and all flammable liquids away from an open flame (Bunsen burner).
7. Do not inhale the fumes of any substance directly. When required to note the odor of a
culture, for example, take a deep breath of fresh air and exhale normally. With your hand
waft the vapors toward your nose and sniff slowly.
8. Do not pipette cultures or chemicals by mouth. Always use a pipette aid or pump. (Your
instructor and a later exercise will demonstrate the proper way to pipette.)
9. Do not use your fingers as a stopper for a tube or a bottle when shaking the tube to mix
its contents.
10. If you get a chemical on your skin, or in your eyes, immediately flood with water at the
emergency eyes bath and shower and call for help.
11. Never remove equipment, media, or microbial cultures from the laboratory.
12. Sanitize work area. Benchtops should be washed down with sanitizer before starting work
and before leaving for the day.
13. Dispose of chemicals, biological materials, cell culture, microbial culture and paper as
indicated by your instructor. Do not put paper or related materials in the sink.
14. Wash your hands thoroughly with detergent and water after handling any cultures, after
removing gloves, and before leaving the laboratory.
15. Whenever you are in doubt about a personal health problem or laboratory procedure, ask
your instructor before you attempt to do anything.

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Laboratory Operating Procedures
1. Each laboratory class will begin promptly at the beginning of the period. At each meeting,
the instructor will give a lecture demonstration that will include general directions
designed to make your work easier and your methods more efficient.
2. You are expected to read the assigned laboratory exercise/manual and carry out the
suggested reading assignments before each class.
3. Store your personal belongings, such as textbooks and bag, in the cabinet specified by your
instructor. Never let them clutter the operating space on the laboratory bench, where they
might become contaminated through exposure to microbial cultures and cell cultures.
4. As you perform your experiments, record data and make sketches and labels in pencil.
Always perform separate steps of an exercise in the sequence given in the manual.
5. Unless your instructor specifies otherwise, each experiment is to be completed within one
laboratory session.
6. Leave all laboratory facilities and equipment in good order at the close of each period.
Place waste paper and contaminated glassware in the receptacles provided. Inform the
instructor of any defects in equipment.
7. Conserve water and gas. Turn off water faucets and gas burners when they are not in use.

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Laboratory Manual
How to Use the Laboratory Manual
1. This laboratory manual has been designed to help you to observe, learn, remember, and
apply fundamental concepts, principles, and techniques of microbiology and cell biology.
Think of it as an extension of your textbook. An organized use of the manual will provide
considerable benefits. Undoubtedly, you will develop your own method of using the
manual; however, here are some suggestions that may help you to gain the most from your
efforts and time.
2. Before starting a specific exercise, read the introduction to the section. It will provide
important background information and give you a general overview of the exercises in the
section and their relationship to one another. Use the stated objectives as indications of
what you should know and the skills you should acquire upon completion of the exercise.
3. Examine the items listed in the Materials section. This procedure will acquaint you with
the microorganisms and items to be used in carrying out specific procedures. Check each
item as it is obtained. Refer to this section if you are ever in doubt about which
microorganisms or materials are to be used.
4. Next, read the Procedure section and look at any figures specified. Underlining steps
of a procedure and all the important information can be helpful in locating specific steps
of a procedure and can save time. Place a check at each step after it is completed. This
practice will help you to quickly locate your place in the procedure. Certain exercises
contain step-by-step illustrations of procedures to show the proper handling of equipment
and cultures.
5. Then read the questions in the Questions sections. This practice will help you to
determine the types of information you will need to obtain from the exercise.
6. Now you are ready to perform the procedure. If you have gone through the earlier steps of
this section, you should not find the exercise difficult. It is always easier and less
frustrating to carry out an exercise if you know what is expected and what is to be done
before starting.
7. Following the procedure, examine your preparations and complete your laboratory report
with Results/Observations, discussion and Questions sections.
8. Finally, review the main portions of each exercise to reinforce your understanding of the
concepts, principles, terminology, and techniques presented.

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Format for Report Writing
Name : xxxxx
Class : FSxxxx
Date : / /
Module name : Food Microbiology I
Exp No : 4

Title (refer to experiment)

Objective(s) (refer to experiment)

Introduction (Summary of theory, approximately 8-12 lines. Maximum 12 lines)

Materials and methods (Summary of procedure)

Results & Discussion (Results/Drawings/Tables/Discussion)

References

IMPORTANT TIPS:

TIPS:
1. When drawing bacteria or specimen under microscope always:
a. Draw bacteria or specimen within a circle
b. State total magnification
c. Name of microorganisms. Escherichia coli or Escherichia coli
d. Provide shape (rod, spherical etc) , arrangement (single, clusters), colour
(blue, green etc) if stained

2. Submit your report at the next practical session or as advised by your instructor.

3. Late reports will be penalized.

4. Copying is strictly prohibited. Plagiarism is a serious offence. If you are caught

COPYING DIRECTLY from internet/books/other source/practical manual without proper
referencing, you will be penalized severely.

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Experiment No 1: Care and Use of Microscope

PART I: CARE & USE OF MICROSCOPE

OBJECTIVES

1. Identify the parts of the microscope, and understand their functions.

2. Demonstrate the proper method of focusing, changing objectives, carrying the
microscope, and cleaning the microscope.
3. Calculate the total magnification of any ocular and objective combination.
4. Define and understand the following terms and concepts: resolving, working distance,
depth of field, and magnification.
5. Observe specimens with the oil immersion objective lens.

THEORY

The compound microscope is a precision instrument. It is essentially a system of

accurately ground lenses arranged to give sharp, clear, magnified images of minute objects.
Magnification that may be obtained with different lens systems ranges from approximately 100
to 2,000 times (X) the diameter of the specimen being observed.

While magnification is an important microscope property, the limiting factor that

determines the usefulness of a microscope is its resolving power. The resolving power of an
optical system is its ability to distinguish fine detail by producing separate images of small
parts of a specimen that are only a short distance apart. In practice, the limit of resolution
obtainable with a compound light microscope is about 0.2 pm, or roughly 1,000 times the
resolving power of the unaided human eye.

The quality of the image obtained depends on the degree of contrast between the specimen
and its surroundings and at times between parts of a specimen. Such contrast is due in part to
differences in light absorption and various techniques that affect absorption used to improve
contrast. For example, the use of chemical stains results in colour differences. Reducing the
intensity of the light illuminating a specimen makes it easier to distinguish cells that have a
refractive index similar to how the light illuminating a specimen makes it easier to distinguish
cells that have a refractive index similar to the fluid in which they are suspended. In this
exercise, the wet-mount technique is used to emphasize certain properties of the light
microscope and to introduce the features of living and nonliving cells, respectively.

Use and Maintenance of Microscope

Microscopes vary greatly from laboratory to laboratory. Your instructor will demonstrate and
explain the general construction and manipulation of the particular instrument you will use.
There is, however, a set of basic rules that should always be observed for all instruments to
ensure maximum efficiency and proper maintenance.

1. Always carefully remove the microscope from its place of storage by holding it firmly by
the arm with one hand and supporting it at the base with the other. Keep the instrument in
an upright position.

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2. Place the microscope at least 6 inches (about 15 cm) from the edge of your laboratory table.
3. Do not tamper with any parts of the instrument. If the microscope does not seem to be
functioning, notify the instructor immediately. Since students in other laboratory sections
may use the same instrument, any part previously damaged and not reported could be
charged to you.
4. Do not touch the lenses with your fingers. Perspiration contains fatty acids and other
substances that can damage the lenses. Always use lens paper for cleaning the optical
system. Never use paper towels or cloths. These materials will scratch the delicate lens
surfaces.
5. Wipe the lenses of the microscope before and after use.
6. Use a clean, soft, linen cloth to remove dirt, dust, or other materials from parts other than
the lenses.
7. Do not allow liquids, particularly acids and alcohol, to come in contact with any part of the
microscope.
8. Always use a cover slip when examining objects of organisms mounted in water o or other
fluids.
9. Always raise the body tube sufficiently before either placing a slide on, or removing a slide
from, the microscope stage.
10. Remove immersion oil from the microscope with lens paper only.
11. Before putting the microscope away, always rotate the low-power objective into working
position (i.e., in line with the body tube), open the diaphragm, and raise the condenser to
its higher fixed position.
12. When disconnecting the light source, do not pull on the wire; instead, grasp the plug
firmly to remove it from the socket.
13. If a cover is provided for the microscope, be certain that it covers the entire instrument.

Microscope Parts and Principles of Microscopy

The ordinary compound microscope consists of a series of optical lenses, mechanical

adjustment parts, and supportive structures for various components. The optical lenses include
the ocular or eyepiece; usually three objectives with different magnifying powers; and the
substage condenser. The coarse-, fine-, and condenser-adjustment knobs together with the iris-
diaphragm lever are the major mechanical parts concerned with the operation of the instrument.
The various components of the scope are held in position by, or contained within, such
supportive structures as the base, arm, pillar body tube (barrel), and revolving nosepiece.

To serve its purpose, a microscope must provide adequate magnifying power so that the
finest details of a specimen are separated enough to be visible to the eye. The magnifying power
of microscope lenses usually is indicated by markings on objectives and eyepieces e.g. l0X,
40X, 100X, designated as low power, high-dry, and oil immersion, respectively. Some
microscopes will have a fourth objective for rapid scanning of microscopic fields that is only
4X. The total magnification is obtained by multiplying the magnification of the objective with
that of the eyepiece.

While magnification is an important microscope property, the limiting factor that

determines the usefulness of the light microscope is its resolving power. The resolving power
of an optical system can be defined as the ability to show clearly two points that are close
together as separate entities. This feature is largely determined by the wavelength of the light
source and the angular aperture (opening) of the lens system being used. Resolution is also
affected by the refractive index of the medium through which light passes before entering the
microscope objective. (The path of light rays are bent or refracted from a straight path as they
pass through different media or substances. The refractive index refers to the ratio of the speed

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of light in air to its speed in another medium.)
The relationship of these factors is expressed in the following combined formula:

Numerical aperture (NA) =  sin 

where  represents the refractive index of the medium through which light passes before
entering the objective lens and sin  is the trigonometric sine of one-half the angle formed by
light rays (in the shape of a cone) coming from the condenser and passing through the
specimen. Values for NA are engraved on the barrel of objectives and specify the maximum
resolution obtainable with the proper use of the appropriate accessories.

The objective lenses serve not only to magnify but also to focus the light rays from a
specimen in order to form a real image within the body tube. This resulting image is further
magnified by the ocular lens system of the microscope. The total magnification obtained is
determined by multiplying the magnification of the objective used by the magnifying power of
the ocular lens.

The individual objectives of a microscope consist of a combination of convex and concave

lenses. These objectives are contained in a revolving nosepiece that is used to rotate the desired
objective into place. The distance from the specimen and a point roughly midway between the
component lenses of the objective is known as the focal length. It is important in determining
the distance required for focusing. The distance between an objective and a specimen is the
working distance. This distance decreases as the magnification is increased; more light is
needed for better and more accurate viewing of a specimen.

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MATERIALS

1. Compound light microscope

2. 1 set of glass slide and cover slip
3. Len paper
4. Small pieces of newspaper cut-outs (with letter ‘e’)
5. Scissors
6. Immersion oil
7. Prepared slides of Escherichia coli (E. coli)

PROCEDURE

(A) Microscope Parts

1. Place the microscope approximately 6 inches (15 cm) from the edge of the laboratory
table with the microscope arm facing you.
2. Examine the microscope and, with the aid of the diagrams provided, locate the
components listed below that are applicable to your instrument:

a. Ocular
b. Body tube (or barrel)
c. Revolving nosepiece
d. Low-power objective
e. High-dry objective
f. Oil-immersion lens
g. Base
h. Condenser
i. Iris-diaphragm lever
j. Coarse-adjustment knob
k. Fine-adjustment knob
1. Condenser-adjustment knob
m. Mechanical stage

(B) Use of Microscope

1. Wipe all lenses with lens paper.

2. Move the low-power objective into position under the body tube. You will feel a click
when it is correctly in place.
3. Connect your light source and turn it on.
4. Place the letter e slide on the mechanical stage and secure it with the slide holder. The
finger control lever for the slide holder is generally located at the rear left-hand corner
of the mechanical stage. Forward pressure on the lever will cause the slide holder to
open, and the slide may be inserted into the holding area. The bottom of the slide should
rest on the microscope stage and not on the mechanical stage apparatus. Release the
finger control lever to allow the slide holder to close and to hold the slide within the
mechanical stage.
5. Using the control knobs on the mechanical stage, move the slide until the specimen on
the slide is over the centre of the condenser opening in the stage. One control knob
moves the slide left to right and the other moves it front to back.
6. Use the coarse-adjustment knob to lower the low-power objective to approximately
one-quarter inch above the cover slip.
7. Use the condenser-adjustment knob to raise the condenser as far as it will go.
8. While looking into the ocular, move the iris-diaphragm lever back and forth. Note the
change in illumination. The intensity or brightness of illumination is controlled by
adjusting the iris diaphragm.

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 9. Locate your specimen. Focus upward, that is, move the objectives upward with the fine-
adjustment knob until the specimen comes into clear view. In a microscope with a fixed
body tube, the objectives remain in a fixed position. Focus is controlled by moving the
stage downward until the specimen comes into view. If you have difficulty in locating
your specimen, look for a region with a colour or intensity different from the other
portions of the slide.
10. In order to change objectives (switch from low power to high power), do not raise the
body tube; simply turn the nosepiece to bring the desired objective into place. The
specimen will generally stay in focus with any objective, but a slight turn of the fine-
adjustment knob may be necessary. Most microscopes are parfocal, that is, once the
specimen is in focus, it will remain so when the objective lens is changed.
11. When looking through the microscope, keep both eyes open at all times. Keeping both
eyes open prevents eye strain and enables you to observe and draw microscopic views
of specimens without moving your head.

(C) Observation of Prepared Slides

1. Newspaper letter e
i) Examine the letter e as it appears on the slide to the unaided eye in a normal
reading position. Make a sketch of the letter in the Results and Discussion
section of your report.
ii) Focus and examine the preparation under the low- and high-power
objectives following the steps listed in Section (B).
iii) Slowly move the slide first side to side and then forward (up) and backward
(down) with the mechanical stage control knobs. Compare this view of the
orientation of the e with the view with the unaided eye.
iv) Sketch representative microscopic fields.
v) On your sketch of the e as observed under low power, draw a circle around
the portion of the letter seen under high power.

2. E.coli

i) Place the slide on the microscope sample stage. Locate the smear and try to
focus using the low-power lens.
ii) Search for stained area. Use high-power now and try to focus.
iii) Swing the high-dry objective aside.
iv) Place a drop of immersion oil on the preparation and move the high-power
(oil-immersion) lens into position. The lens should touch the oil.
v) Focus upward with the fine-adjustment knob until the specimen comes into
view. Adjust the knob slowly and carefully so that the lens will not break
the slide.
vi) Sketch your observation (shape, size, colour?).

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QUESTIONS
(Hint: Some of the answers can be found in the theory section)

1. What is the magnification of the following objectives without an ocular?

i) Low power
ii) High-dry power

2. As the magnification used is increased, is more or less illumination needed? Explain

your answer.

3. What factors affect the resolving power?

4. Complete the following table by giving the function(s) of each part listed.

Microscope Parts Function(s)

Ocular
Objective
Condenser
Iris-diaphragm
Coarse-adjustment knob
Mechanical stage
Microscope base

5. Sketch representative microscopic field of newspaper ‘e’ and state the magnification
used. On your sketch of the “e” observed under low power, draw a circle around the
portion of the letter seen under high power.

6. Sketch your observation of the E.coli (shape, size, colour?) observed using the oil-
immersion lens.

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