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MicrobiologyLab Report02

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MicrobiologyLab Report02

Uploaded by

giang
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© © All Rights Reserved
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General Microbiology lab | Date 23.03.

2024 | Report 02

ISOLATION OF AEROBIC AND ANAEROBIC


MICROORGANISMS
Student Name Dinh Hang Giang
Student ID 20210286
Class ID 743706

I. PURPOSE & THEORY

In order to study the characteristics of of any species of microorganisms, it is


necessary to obtain a pure culture.

- Pure culture: contains onl the microbial species of interest.


- Mix culture: contains a group of different microbial species.

Isolation: process of separating a microorganism from a mix culture to obtain the


pure culture.

- In the first experiment, the purpose is to isolate aerobic microorganisms


capable of degrading starch, which are able to excret extra cellular enzyme to
degrade starch into maltose.
- In the second experiment, the purpose is to isolate lactic acid bacteria
(facultative anaerobic microorganisms) from a food product using double agar
layer method.

II. ISOLATION OF AMYLASE PRODUCING AEROBIC MICROORGANISMS


1. Materials & Equipment
- Environmental sample: Soy bean paste – heterogeneous sample;
- Isolation medium: Czapek using soluble starch, sodium chloride;
- Micropipettes (1 mL, 100 µL) and their corresponding tip boxes;
- Erlenmeyer flask, Eppendorf tubes, Petri dishes, 50 mL Falcon tubes;
- Glass spreader, vortex mixer, static incubator.

2. Procedure

Medium Preparation

- Prepare Czapek agar medium according to table below and pour into 3 Petri
dishes.

Starch 6 ml – 1% MgSO4 0.1 g


solution solution
NaNO3 0.4 g FeSO4 0.2 ml of 1%
solution
KCl 0.1 g Water 200 mL
KH2PO4 0.2 g Agar-agar 3g
In this experiment, starch solution was used in substitution to saccharose as the
sole carbon source at concentration of 1%. This change occurred in order to create
a selective medium, which favor the separation process of the amylase producing
microorganisms. These microorganisms have the ability excret extra cellular
enzyme to degrade starch into maltose.

Agar is used to inhibit the growth of some anaerobic bacteria contained in the
culture.

- Prepare saline buffer (0.85% sodium chloride solution), aliquot 99mL into an
Erlenmeyer flasj and approximately 40 mL into 50 mL Falcon tube, autoclave at
110oC for 30 minutes.

Sample preparation and dilution

- Prepare saline buffer for dilution: aseptically dispense 900 µL sterile saline
buffer into 5 Eppendorf tubes.
- Measure 1 mL of soy bean paste sample, put into Erlenmeyer flask containing
99 mL sterile saline buffer. Shake well. This is the 10 2-fold solution.
- Aseptically transfer 100 µL diluted sample from the 10 2-fold solution into 900
µL saline buffer. This is the 10 3-fold solution. Repeat the dilution step from the
previously made dilution until achieve a 10 7-fold solution. Vortex well before
each dilution.

Inoculation

- Label the bottom of Petri disk.


- Dispense 100 µL of 105, 106, 107 fold dilutions into the bottom part of three
Petri dishes containing Czapek-starch agar medium.
- Gently spread the inoculum on the surface of the agar medium using a sterile
glass spreader until the surface of the agar medium is relatively dry (feel the
resistance on the surface of the medium).

Incubation and observation

- Incubate the plate at 30oC for 2 days. Remember to turn agar side up to
prevent water drops intefering the medium.
- After two days, observe the growth of mixed culture.
- To further obtain a pure culture, we must separate a colony from the mixed
culture.
3. Result

These observations was made at around 16:00, date 29/03/2024.

Figure 1.1. Dilution concentration 10-5

There observed three different types of microorganisms. One resulted in small dots
and round-shaped, with opaque yellow-ish color. The other is translucent with
round shape, but appeared in larger size. The other is white-opaque with irregular
shape, but appeared in larger size.

There observed a fungal colony.


Figure 1.2. Dilution concentration 10-6

There observed three different types of microorganisms. One resulted in small dots
and round-shaped, with opaque yellow-ish color. The other is transluvrny with
round shape, but appeared in larger size. The other is white-opaque with irregular
shape, but appeared in larger size.

Figure 1.3. Dilution concentration 10-7


There observed three different types of microorganisms. One resulted in small dots
and round-shaped, with opaque yellow-ish color. The other is white-opaque with
round shape, but appeared in larger size. The other is white-opaque with irregular
shape, but appeared in larger size.

4. Discussion and Evaluation

The obtained result was observed after 6 days when it supposed to be 2 days, so
the microorganisms has grown significantly.

In the first sample (dilution 10-5), there observed only few large colonies and
many small colonies that are not fully separated. This was from the

In the sample with 10-6 dilution concentration, the colony density was sparse and
separated; therefore it is the most suitable one for isolating microorganisms.

In the sample with 10-7 dilution concentrations, there observed only small
colonies, because the dilution was too high. Apparently, the same type of
microorganisms show up.

There was fungi in 10-5 and 10-7 Petri disk. Since it did not occur in 2 the 10 -6
sample, the fungi might be contaminated from the environment, during the culture
preparation or the innoculation process.

The colonies grow on the agar surface, using carbon source mainly is starch, and
are aerobic heterotrophic microorganisms, capable of biosynthesizing amylase.

III. ISOLATION OF ANAEROBIC MICROORGANISMS


1. Materials & Equipment
- Environmental sample: Pickle juice - homogenous;
- Isolation medium: MRS, sodium chloride, agar-agar;
- Micropipettes (1 mL, 100 µL) and their corresponding tip boxes;
- Erlenmeyer flask, Eppendorf tubes, Petri dishes, 50 mL Falcon tubes;
- Glass spreader, vortex mixer, static incubator.

2. Procedure

Medium Preparation

- Prepare MRS agar medium according to table below and pour into 3 Petri
dishes.

Peptone 10 g Ammonium 2g
citrate
Meat extract 10 g K2HPO4 2g
Yeast extract 5g MgSO4 0.1 g
Glucose 20 g MnSO4 0.05 g
Polysorbate 80 1g Water 1L
Sodium acetate 5g Agar 20 g

MRS is a slightly selective medium for LAB with pH ranges around 6.2. It contains
selective agents (bile salts, antibiotics,…) inhibiting the growth of competing
bacteria such as several gram-positive bacteria.

- Prepare 2% agar solution. Autoclave at 110oC for 30 minutes, keep molten


agar solution in 50oC waterbath until used.

The agar solution is used as a second agar layer in order to protect the inoculum
from oxygen in the atmosphere (LAB is anaerobic microorganisms). It also helps
encapsulating the bacteria within the medium, preventing the bacteria from
diffusing too far way from their original location during incubation.

- Prepare saline buffer (0.85% sodium chloride solution), aliquot approximately


40 mL into a 50 mL Falcon tube, autoclave at 110 oC for 30 minutes.

Sample preparation and dilution

- Prepare saline buffer for dilution: aseptically dispense 900 µL sterile saline
buffer into 5 Eppendorf tubes.
- Measure 1 mL of pickle juice sample, put into Eppendorf tube containing 900
µL sterile saline buffer. Vortex well. This is the 10-fold solution.
- Aseptically transfer 100 µL diluted sample from the 10-fold solution into 900 µL
saline buffer. This is the 10 2-fold solution. Repeat the dilution step from the
previously made dilution until achieve a 10 5-fold solution. Vortex well before
each dilution.

Inoculation

- Label the bottom of Petri disk.


- Dispense 100 µL of 103, 104, 105 fold dilutions into the bottom part of three
Petri dishes containing Czapek-starch agar medium.
- Gently spread the inoculum on the surface of the agar medium using a sterile
glass spreader until the surface of the agar medium is relatively dry (feel the
resistance on the surface of the medium).
- Cover the surface of the inoculated Petri dishes with molten agar to protect the
inoculum from oxygen in the atmosphere. Keep the plate still until molten agar
is solidified.
Incubation and observation

- Incubate the plate at 30oC for 2 days.


- After two days, observe the growth of mixed culture.
- To further obtain a pure culture, we must separate a colony from the mixed
culture.

3. Result

Figure 2.1. Dilution concentration 10-3

There observed two different types of microorganisms. One resulted in dots with
round-shaped, opaque color. There is a large fungal colony.
Figure 2.2. Dilution concentration 10-4

There observed two different types of microorganisms. One resulted in dots round-
shaped, with opaque color. The other is translucent with irregular shape, appeared
in larger size.

Figure 2.3. Dilution concentration 10-5

There observed two different types of microorganisms. One resulted in dots round-
shaped, with opaque color. The other is translucent with irregular shape, appeared
in larger size.
4. Discussion and Evaluation

The under-layer of agar in sample dilution 10 -4 and 10-5 was broken during
inoculum process, because of the lack of skill. Therefore, there was
microorganisms contaminated from other environment. In the sample 10 -3, the
agar was not broken but there obseved large colony of fungi. This may occurred
during inoculation process, the sterile was not ensured.

Meanwhile, it is able to observe the density of colonies decrease from lower to


higher dilution concentrations.

In the last sameple (figure 2.3), the molten agar did not fill up the whole surface,
yet there did not observe any growth of bacteria outside.

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