Practical food microbiology Lab (1)

Microbiology Laboratory Safety Rules

1- All materials and clothes other than those needed for the laboratory are to be kept
away from the work area.

2. A lab coat or other protective clothing must be worn during lab. The lab clothing is not
to be worn outside of the laboratory.

3. Clean the lab table before and after lab with the disinfectant solution provided.

4. Wash hands before leaving lab.

5. Any item contaminated with bacteria or body fluids must be disposed of properly.
Disposable items are to be placed in the BIOHAZARD container. Reusable items are to
be placed in the designated area for autoclaving prior to cleaning. Sharps are to be
disposed of in the appropriate container.

6. Reusable items should have all tape and marks removed by the student before being
autoclaved.

7. Because organisms used in this class are potentially pathogenic, aseptic technique
must be observed at all times. NO eating, drinking, application of cosmetics or
smoking is allowed.

8. Labels should be of the self-adhesive type to avoid the temptation of moistening

gummed labels with the tongue. Don’t place the end of the pencils in the mouth whilst
working.

9. Cultures should never be pipetted by mouth, but with rubber teats or bulbs used in
conjunction with the pipette when pipetting samples or dilutions of samples which
may contain dangerous pathogens or toxins.

10. Used pipettes must be placed in pipette jars containing disinfectant solution.

11. Inoculating needles and loops must be sterilized before and after use, by heating in
Bunsen flame until red-hot along the entire length of the wire.

12. Test tube cultures should always be kept in test tube racks. Never lay the test tubes
on the bench top.

13. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be
provided on request.

14. Long hair should be tied back while in lab.

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15. All accidents, cuts, and any damaged glassware or equipment should be reported to
the lab instructor immediately.

16. Microscopes and other instruments are to be cared for as directed by the instructor.

17. Doors and windows are to be kept closed at all times.

Mr.\ Hamada A. A. Abu-Bakr

Supervisor of food technology (413) (practical)
(Practical Food microbiology)

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Aseptic Technique

When working with microorganisms it is desirable to work with a pure culture. A

pure culture is composed of only one kind of microorganism. Occasionally a mixed
culture is used. In a mixed culture there are two or more organisms that have distinct
characteristics and can be separated easily. In either situation the organisms can be
identified. When unwanted organisms are introduced into the culture they are known as
contaminants.

Aseptic technique is a method that prevents the introduction of unwanted organisms into
an environment. When changing wound dressings aseptic technique is used to prevent
possible infection. When working with microbial cultures aseptic technique is used to
prevent introducing additional organisms into the culture.

Microorganisms are everywhere in the environment. May be found on surfaces and

floating in air currents. They may fall from objects suspended over a culture or swim in
fluids. Aseptic technique prevents environmental organisms from entering a culture.

Aseptic technique procedures:

• Doors and windows are kept closed in the laboratory to prevent air currents which
may cause microorganisms from surfaces to become airborne. Once these
microbes are airborne they are more likely to get into cultures.

• Transfer loops and needles are sterilized before and after use to prevent
introduction of unwanted organisms.

• Agar plates are held in a manner that minimizes the exposure of the surface to the
environment.

• When removing lids from tubes, lids are held in the hand and not placed on the
countertop during the transfer of materials from one tube to another.

• Bunsen burner flam must be kept beside the working area to sterilize the air.

These techniques are the basis of laboratory aseptic technique.

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Sterilization methods
The sterilization of media, lab tools, cultures, containers and instruments is one of the
essential methods in microbiological labs. This method is one of the essential aseptic
technique procedures.

Sterilization
methods

Physical Mechanical
methods Chemical methods
methods

Sterilization
Heat sterilization Radiation Disinfectants filters

Heat sterilization

Wet heat

Red heat in the

Bunsen flame

Alcoholic flaming

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1- PHYSICAL METHODS:-
A- Heat sterilization:-

1-Dry heat sterilization:

• Hot air oven
™ Temperature (160-180o c).
™ Sterilization time (2-3 h.)
™ Uses (to sterilize the glassware such as test tubes, Petri dishes,
flasks, and pipettes which are packed in stainless steel
containers before sterilization . it is also used to sterilize the
mineral oils and any other material which can tolerate the dry
conditions with out destruction).
™ Lethal effect of this method on microorganisms(the dry hot air
can make dehydration of microbial cells and oxidation of the
internal organelles of the microbial cells)

2- Wet heat sterilization:

• Autoclave:
™ Temperature (121oC). Under pressure of (15 Ib\in2).
™ Sterilization time (15-20 men.)
™ Uses(to sterilize culture media, diluents, gloves, lab coat and
any other materials which are not tolerate the dry condition)
™ Lethal effect of this method on microorganisms (like dry heat
but the wet heat is more efficient for killing the microbial cells.
That is because it has the raped ability to permeate the
microbial cell wall and good ability to make denaturation of the
cell protoplasm.

3- Red heat in the Bunsen flame:

™ Uses ( to sterilize inoculating wires, loops and metal

instruments that are not damaged by heat
™ Lethal effect for microorganisms (the flame makes burning of
microbial cells found on the surface of wires and loops)

4- Alcoholic flaming(flaming after dipping in ethanol)

™ In which we dip the tool which we want to sterilize in ethanol

then expose it to Bunsen flame
™ Uses(this method is used to sterilize the scalpels, spatulas, etc.,
with the instruments not being heated to red heat)

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B- RADIATION:-
• Using UV, X-RAY, γ-RAY
™ Uses (to sterilize plastic tools like plastic Petri dishes and
plastic pipettes).
™ Lethal effect for microorganisms (affect cells DNA and
enzymatic systems of the microbial cells).

2-CHEMICAL METHODS
Chemical disinfectants are used mainly for disinfecting the skin, floors, buildings,
apparatus, and for articles that cannot be heated effectively without damage.

Examples of common disinfectants:-

1-Ethyl alcohol:

™ Concentration (50-70%).
™ Uses (disinfection of hands and any region of the body).
™ Lethal effect on microorganisms (make dehydration of bacterial cells and
coagulation of cell proteins).
™ Why the concentration 50-70%............? Because the higher concentration
than 70% can make raped dehydration of microbial cell wall which prevent
the penetration of alcohol through the cell wall. So, prevent the coagulation
effect.

2- PHENOL:

™ Concentration (2-5%).
™ Uses (to sterilize floors and the working area)
™ Lethal effect(coagulation of cell proteins)

3-Mercueric chloride:
™ Concentration (0.1%).
™ Uses (to sterilize floors and the working area).
™ Lethal effect (mercuric ions can react with SH groups of the amino acids
involved in enzymatic proteins of the microbial cells).

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3-MECHANICAL METHODS:
™ USES (to sterilize the enzymatic and antibiotic preparations which can affect
by heat used in the previous methods).
™ Examples ( the cellulose membranes filters and asbestos filters)
™ Mechanism of sterilization (these filters have pore size lower than the
diameters of microbial cells. So, when the liquid is passed through these
filters the microbial cells are detained on one side of filter).

Cotton wool plugs

The reasons of using Cotton wool plugs in test tubes and pipettes:-

1- To prevent microorganisms from passing in or out and contaminating either the

culture
or the environment.
2- The necessity of air movements in and gaseous products out of cultures.
(In the case of aerobic microorganisms)

How can cotton plugs prevent microorganisms from passing in or out the
culture:-
• The gaps between the cotton wool fibers are even wide enough for micro-
organisms to pass through. However, this does not happen because micro-
organisms (negatively charged) are “filtered” out by being attracted to and
adsorbed on the oppositely charged cotton wool.

Note:-
The cotton wool must remain dry because this filtration property is lost if the cotton
wool becomes moist – hence the use of nonabsorbent cotton wool. For use in test
tubes a plug should be properly made to ensure that it can be held comfortably
without being dropped and its shape and form are retained while being removed from
and returned to a test tube several times.

Laboratory Procedure

• Each student has to make number of cotton plugs as the instructor

will explain.