CFB 20303LMPRACTICAL

MALAYSIAN INSTITUTE OF CHEMICAL AND

BIOENGINEERING TECHNOLOGY.

FOOD MICROBIOLOGY (CFB 20303)

UNIKL MICET
DOC. NO. : JAN 12
FDTLM2033 LAB MANUAL (REV. NO. 1)

PRACTICAL 1 : PREPARATION OF CULTURE MEDIA AND ASEPTIC

TECHNIQUE IN MICROBIOLOGY

OBJECTIVES
1. After completing this laboratory, student should be able to prepare
microbiological media on their own.
2. Student will be exposed to media preparation and aseptic technique involve
during media preparation.

KEYWORDS
Culture media, Aseptic Technique

INTRODUCTION

There is a wide variety of media which are used in microbiology, but the procedures
used in their preparation are generally the same. They include weighing out the
dehydrated media, dissolving it in dH2O, sterilizing it (usually by autoclaving),
pouring the plates, preincubation to check for contamination and to dry out the
plates, and storage.

Agar is particularly suited as a solidifying agent because it will not melt until the
medium is heated to near boiling(95°C-100°C), but will remain melted until cooled
to around 42°C. It is not degraded by the vast majority of bacteria, and therefore
maintains its structure during bacterial growth, and because it is not a nutrient for
bacteria, it also allows strict control of growth factors.

Because medium boils up during decompression from autoclaving, we will

autoclave only 70 mL of medium in a 100 liter bottle. You will need to calculate the
amount of powder needed per 70 mL of medium since the directions on reagent
bottles are for 1 full liter.

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Aseptic technique is the name given to the procedures used by microbiologists to

prevent microbial contamination of themselves, which may result in infection,
contamination of the environment they are working in (e.g. fomites), and contamination
of the specimen they are working on, which is especially important when a pure culture is
desired. It is used whenever specimens are to be transferred between media, for example,
when subculturing.

2. MATERIALS AND EQUIPMENT

100 mL beaker or weighing dry media
Balance
thermometer, -10°C to 110°C
stirring hot plate
100 mL media bottle with cap
Bunsen burner
autoclave
incubator
dehydrated media
70 mL dH2O
6-8 sterile petri dishes
Lysol

3. PROCEDURES

Agar Preparation Methods

A. Weighing the dry media

1. Tare out the 100 mL beaker, use counterweights if necessary, and record tare
weight.
2. Sum the desired mass of the dehydrated medium + tare weight, set balance to read
the sum.
3. Add dry reagent with care to beaker with a clean spatula. After each addition,
gently tap the edge of beaker with spatula to judge progress. When close to the
desired weight, tilt and roll the reagent bottle, tap its neck to sprinkle in the final
amount until equal swings are achieved. Do not remove any excess back to the

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reagent bottle. Replace the cover immediately on the reagent bottle (it is
hygroscopic). Record the actual amount added.
4. If there are more than single substances in your medium, repeat steps 2 & 3 for
other dry materials. (Use each new apparent weight as the new tare, repeat the
sequence of steps.) Check again to be sure all reagents are securely capped.
5. Add dH20 to the beaker with stirring, break up clumps of powder, to the desired
volume (70 mL here).

B. Dissolving the media

1. Stirring with a magnetic bar, heat to boiling with hot plate: do not allow to boil
over, nor to burn on bottom.
2. The media should clarify near boiling (95-100 C).
3. Pour into a 100 mL bottle. Cap loosely, label bottle with name of medium and
your group's name, place in autoclave.

C. Autoclaving the media

(After all student groups have placed their loosely capped bottles in autoclave)
Place dissolved, loosely capped media in the autoclave.

1. Set for slow exhaust, autoclave the medium at 1 psi. pressure for 15 minutes (for
most media) at 121 °C. This takes at least 45 minutes – 1 hour.
2. Remove from autoclave (CAUTION: HOT STEAM), allow to cool to 50-60°C
(feels hot, but possible to hold).

Pouring the plates

1. Open a sterile package of petri dishes preserving bag for later storage.
2. Mark the sides of the dishes according to code lines to indicate the type of media
they contain (or label the petri dishes on bottom in small letters: plate type, date.)
3. On a sterile field, each student pours at least four plates using sterile technique as
demonstrated. (Position stacks of four or five sterile plates near edge of desk,
remove and hold cap with little finger, flame lip of bottle, fill bottom plate first, at
least half full, repeat, holding lid over plate as you pour.)
4. Flame the lip of the bottle whenever the lid is replaced. The last student pours all
of the rest of the medium.
5. Rinse bottle immediately after pouring last plate before the remnant solidifies.
6. When plates have solidified, invert, place in 37°C incubator for 48 hours to check
for sterility and to dry out excess moisture. Store in labeled plastic bag at 4C°.
Pre-warm before using.

Report and Discussion

You are required to report the observations made and discuss the difficulties if any, faced
during your work.
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EXPERIMENT l (B) :ASEPTIC TECHNIQUES IN MICROBIOLOGY

Pre-lab exercise: Discuss the definition and importance of aseptic techniques in

microbiology. Aseptic techniques are one of the ways for prevention of contamination.

A. Laboratory design
Good illumination and easily cleaned surfaces are important requirements in all
microbiological laboratory. Cleanliness and aseptic conditions must be maintained at all
times in the laboratory. Microbiological laboratory must be situated on the upper floor
of a building to avoid contamination from the dusty air.

B. Apparatus and equipment used in the laboratory

Glasswares, and inoculating devices must be sterilized before use to avoid
contamination. Test tubes, Erienmeyer flasks, media flasks, Petri dishes, pipettes tips
and Durham tubes must be sterilized before use. Inoculating devices such as pipettes and
tips, glass rods, straight wires and wire loops made of platinum, chromium or nickel are
recommended to enable heating during sterilization by burning.

C. Disposable presterilized equipments

Usually plastic apparatus such as Petri dishes, vials, culture tubes or test tubes can be
purchased insterile conditions.

Glassware/apparatus and chemicals for 2 students.

1. A bottle of Lysol
2. 2 bottles of sterile molten nutrient agar
3. 2 flasks containing nutrient broth
4. 2 sterile Petri dishes and 2 non-sterile Petri dishes
5. Bunsen burner (to be placed in laminar flow cabinet)
6. An inoculating loop
7. A bottle of ethanol at 75% concentration
8. Sterile pipettes and tips
9. A bottle of sterile distilled water
10. A laminar flow cabinet
11. An incubator shaker

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Experimental Procedures

1. One student will work in the laminar flow cabinet while another student will work
at the bench in the laboratory. Results obtained by the two students are compared
2. Each student will performed the procedures as shown in Table 1.1, one taking care
of the aseptic conditions while the other without considering the aseptic conditions

Reports
1. Describe and discuss the observation on the results obtained.
2. Based on the results, describe the procedures that are considered aseptic in ensuring the
aseptic conditions for microbiological work. How do these procedures affect the
results?

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Table 1.1 Procedures for the experiments
Procedures with aseptic techniques
1. Wipe the table top of the laminar
flow cabinet with either alcohol or
Lysol
2. Light the Bunsen burner
3. Arrange the required
apparatusclose to the Bunsen burner
and allow for 10 min before starting
the experiment. Place the inoculating
loop in the alcohol solution.
4. Aseptically pour the molten agar
into an empty Petri dish and close the
lid quickly and allow the agar to
harden.
5. Using the pipette pick up the sterile
tips and aseptically dispense 0.1 ml of
distilled water and transfer it into the
nutrient broth
6. Flame the wire loop until it is red
hot and cool it down in the air. Dip the
loop into the distilled water and
aseptically streak an S-shape on the
surface of the agar which has harden.
7. Close the lid of the Petri dish
quickly.
8. Incubate the agar plates in an
incubator and the nutrient broth in the
incubator shaker for 24 h at 37°C.
9. Observe for any growth of
microorganisms in the agar and broth.
10. Wipe the tabletop after completing
the work and remove all the
apparatus/glassware.
CFB 20303LMPRACTICAL
Procedure without aseptic
techniques
1. Work is performed on the bench in
the laboratory.
2. Arrange the required apparatus on
the table.
3. Pour the molten agar into the Petri
dishes by allowing the agar to flow
across the dish.
4. Close the lid of Petri dish and allow
it to harden.
5. Using the pipette, pick up the tips
and dispense 0.1 ml of distilled
water and transfer it to the nutrient
broth
6. Similarly dip the loop into
the distilled water and streak an S--
shape on the surface of the agar which
has harden.
7. Close the lid of the Petri dish
quickly
8. Incubate the agar plates in the
incubator plates and the nutrient broth
in the incubator shaker for 24h at
37°C.
9. Observe for any growth of the
microorganisms in the agar and in the
broth.
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REFERENCES

1. Harrigan, W.F., Laboratory Methods in Food Microbiology, Academic Press,

1998.
2. Tortorello, M.L. and Gendel, S.M., Food Microbiology & Analytical Methods,
Marcel Dekker, 1999

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