Basic Laboratory Techniques for Isolation, Cultivation, and Cultural

Characterization of Microorganisms. Culture Transfer Techniques.

Lab Report #1

Alibek Ysmaiyl
26.09.2016
Lab instructors name: Damira Kanayeva
Introduction to Microbiology with Lab-Lab
Fall semester/2016
Section 1
Introduction
Serratia marcescens is a short rod-shaped and facultative anaerobe bacterium classified as an
opportunistic pathogen. Firstly, the bacterium was discovered by Bartolomeo Bizio in 1819.
Generally, S. marcescens can be found in soil, water, plants and animals. There is an optimal
environment for growth of S. marcescens, namely bacterium growth at 37oC, in pH 5-9 ranges.
S. marcescens is well known due to their red pigmentation called prodigiosin which is produced
at temperature below 30oC [1][Fig.1].

Figure 1. S. marcescens via SEM

Therefore, it is proposed that when observing the results red pigments should be appeared.

For optimal growth of bacteria, the amount of nutrient should be enough. Basically, their nutrient
contains low-molecular-weight substances which are came from the degradation of complex
nutrients. So, culture medium is a solution that contains these nutrients. There are three types of
culture medium such as liquid, semisolid or solid. In this laboratory, nutrient broth which lack of
the solidifying agent and nutrient agar with solidifying agent were used. Moreover, the
concentration of solidifying agent called agar in nutrient agar has to be 1.5 to 1.8%. In addition,
due to the position of solidification of agar this medium can be differentiated into agar slant tube
with an angle and agar deep tube with axial arrangement [2][Fig.2].

Figure 2. Types of nutrient media

One of the most important procedures in microbiology laboratory session is appropriate using of
aseptic techniques. Proper aseptic technique prevents from the contamination of incubated
culture from unknown bacteria. First of all, the surface of table must be cleaned with alcohol.

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 Secondly, all equipment should be sterilized with Bunsen burner prior to and after the
experiment [3].

The main purposes and objectives of this laboratory session were to learn proper use of aseptic
techniques, the transfer of culture form one type medium to another, to develop skills in
uncapping and recapping with fingers around Bunsen burner, and to prepare stock cultures.

Materials and methods

Table 1. Materials and equipment

Cultures Media Equipment

24 hour nutrient broth and Nutrient broth, nutrient agar Bunsen burner, inoculating
nutrient agar slant cultures of slant and nutrient agar deep loop and needle, and marking
Serratia marcescens. tube. pencile.

Procedure:
1. Sterile tubes were labelled with the name of the organism and initials
2. One tube with culture and another with medium were hold on the left hand and separated
to form a V shape
3. On the blue flame of Bunsen burner the inoculating loop was heated until red wire was
reached.
4. After that, tubes were uncapped with little fingers of right hand, and then the necks of
tubes were passed through Bunsen burner to maintain sterilization.
5. Slant-to- broth transfer: the inoculum was obtained from agar slant and transferred into
broth medium with a slight agitation.
6. Broth-to-slant transfer: a loopful of broth was obtained and placed into slant by spreading
in a zigzag motion.
7. Slant-to-agar deep transfer: the inoculum was obtained from agar slant and inserted in a
straight line until the bottom of tube was reached and then withdrawn along the line of
insertion.
8. The necks of tubes were passed through the Bunsen burner and tubes were recapped.
9. The loop was heated in blue flame in order to sterile from remaining bacterium.
10. Finally, all tubes were incubated for 24-48 hours at 25oC.

Results
Table 2.Observations on nutrient medium

Nutrient Broth Nutrient Agar Slant Nutrient Agar Deep

Growth (+) or (-) + + +
Orange-red pigmentation (+) or (-) + + +
The distribution of the growth Pink film at the top Pinkish yellow in Pink distribution In
and slightly at the color condensation at the line of insertion

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 bottom, entire the base of the slant loop of NA deep.
medium remained
transparent yellow

Discussion
Generally, the hypothesis was supported by results, that is, red color pigments were observed in
each medium. However, the insignificant growth of culture in agar slant tube was observed, and
this can be explained by the fact that at the base of slant there were highest amount of oxygen.
Even if Serratia marcescens is an anaerobic bacterium, it can survive in the presence of oxygen,
but with the limited amount of growth. The nutrient broth and nutrient agar deep tube were
positive for growth. In nutrient broth, the culture was distributed mainly at the top and slightly at
the bottom. In nutrient agar deep tube, the pink colored spread was observed along the line of
insertion. The exponential growth in these two tubes can be explained by the fact that the amount
of oxygen was limited, and therefore the high level of growth was observed.

Conclusion
Taking all points into consideration, after the completion of the laboratory session main aseptic
techniques were developed. Moreover, culture transfer techniques, namely broth-to-slant, slant-
to-broth, and slant-to-agar deep transfer techniques were learned. According to results, it could
be seen that the experiment was completed successfully because of proper use of aseptic
techniques.

Answer to review questions

1. Explain why the following steps are essential during subculturing:

a. Flaming the inoculating instrument prior to and after each inoculation.

The importance of flaming of instrument is in order to reach sterility.

b. Holding the test tube caps in the hand as illustrated in Figure 1.1 on page 8.

In order to do not contaminate the caps or tubes because of possible contamination on hands.

c. Cooling the inoculating instrument prior to obtaining the inoculum.

If instrument is not cooled prior to the excess heat can kill the bacteria or fix the samples.

d. Flaming the neck of the tubes immediately after uncapping and before recapping.

In order to do not spread other bacteria on the neck of tube into nutrient, and also to do not
spread incubating bacteria through the neck

2. Describe the purposes of the subculturing procedure.

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The main purposes are to separate the bacteria to make observations on them, and to prepare
stock cultures. Moreover, the above procedure should be done aseptically in order to avoid
contaminations.

3. Explain why a straight inoculating needle is used to inoculate an agar deep tube.

The reason for this is that inoculum has to be pulled out from the bottom of tube through a
straight line by line of insertion.

4. There is a lack of orange-red pigmentation in some of the growth on your agar slant
labeled S. marcescens. Does this necessarily indicate the presence of a contaminant?
Explain.

Actually this could mean that there was wrong inoculation of bacteria or incorrect nutrient for
bacteria growth.

5. Upon observation of the nutrient agar slant culture, you strongly suspect that the culture
is contaminated. Outline the method you would follow to ascertain whether your suspicion
is justified.

To grow the nutrient agar slant culture more in order to observe the growth of unknown bacteria.

References
Figure 1- "Serratia Marcescens." - Stock Image C001/5157. Accessed September 26, 2016.
http://www.sciencephoto.com/media/79559/view.

Figure 2- "EXe." EXe. Accessed September 26, 2016.

https://learning.uonbi.ac.ke/courses/SBT202/scormPackages/path_2/13_culture_methods.html.

[1]. Serratia marcescens. https://microbewiki.kenyon.edu/index.php/serratia_marcescens

(accessed Sep 26, 2016).

[2]. Cappuccino, James G and Natalie Sherman. 2011. Microbiology. San Francisco:
Pearson/Benjamin Cummings. Basic Laboratory Techniques for Isolation, Cultivation, and
Cultural Characterization of Microorganisms.

[3]. Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab
http://vlab.amrita.edu/?sub=3 (accessed Sep 26, 2016).