Food Microbiology (CFD 20203) Unikl Lab Manual Micet: Malaysian Institute of Chemical and Bioengineering Technology
Food Microbiology (CFD 20203) Unikl Lab Manual Micet: Malaysian Institute of Chemical and Bioengineering Technology
Food Microbiology (CFD 20203) Unikl Lab Manual Micet: Malaysian Institute of Chemical and Bioengineering Technology
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.
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3. PROCEDURES
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).
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.
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).
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.
You are required to report the observations made and discuss the difficulties if any, faced
during your work.
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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.
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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4. Aseptically pour the molten agar 4. Close the lid of Petri dish and allow
into an empty Petri dish and close the it to harden.
lid quickly and allow the agar to
harden. 5. Using the pipette, pick up the tips
and dispense 0.1 ml of distilled
5. Using the pipette pick up the sterile water and transfer it to the nutrient
tips and aseptically dispense 0.1 ml of broth
distilled water and transfer it into the
nutrient broth 6. Similarly dip the loop into
the distilled water and streak an S--
6. Flame the wire loop until it is red shape on the surface of the agar which
hot and cool it down in the air. Dip the has harden.
loop into the distilled water and
aseptically streak an S-shape on the 7. Close the lid of the Petri dish
surface of the agar which has harden. quickly
7. Close the lid of the Petri dish 8. Incubate the agar plates in the
quickly. incubator plates and the nutrient broth
in the incubator shaker for 24h at
8. Incubate the agar plates in an 37°C.
incubator and the nutrient broth in the
incubator shaker for 24 h at 37°C. 9. Observe for any growth of the
microorganisms in the agar and in the
9. Observe for any growth of broth.
microorganisms in the agar and broth.
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REFERENCES
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