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Aseptic Technique Lab Report

The document discusses aseptic technique and the transfer of microbes in a laboratory setting. It describes how pure cultures of microorganisms are isolated and maintained through aseptic transfers between different media like broth, slants, and agar plates. The objective is to learn proper aseptic technique to avoid contamination and allow microbial growth to be observed. Results showed turbidity in broths and bacterial growth patterns on slanted and deep agar corresponding to the different microbe and media. Maintaining sterile technique through practices like flame sterilization of loops and proper handling is crucial for pure cultures.

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548 views11 pages

Aseptic Technique Lab Report

The document discusses aseptic technique and the transfer of microbes in a laboratory setting. It describes how pure cultures of microorganisms are isolated and maintained through aseptic transfers between different media like broth, slants, and agar plates. The objective is to learn proper aseptic technique to avoid contamination and allow microbial growth to be observed. Results showed turbidity in broths and bacterial growth patterns on slanted and deep agar corresponding to the different microbe and media. Maintaining sterile technique through practices like flame sterilization of loops and proper handling is crucial for pure cultures.

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nasuhaazmi234
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LAB TOPIC: ASEPTIC TECHNIQUE AND TRANSFER OF

MICROBES

1.0 INTRODUCTION

In nature, microorganisms almost always exist as mixed populations of many widely


differing types. However, before most properties and characteristics of a particular organism can
be determined, the organism must first be isolated in pure culture. A pure culture is one that
contains a single strain of microorganisms. For example, a pure culture of the bacterium
Escherichia coli contains only Escherichia coli cells of a particular strain - no other living
microorganisms are present. The term strain refers to a population of cells all descended from a
single cell. The isolation and maintenance of pure cultures are some of the most important
procedures in microbiology and one with which all students must be familiar.

Like all other organisms, microorganisms require nutrients and a favorable environment
to grow and multiply. Microbes are generally grown in a culture medium that contains essential
nutrients and provides a suitable environment (e.g. proper pH). Microbes are essentially grown in
a liquid medium, broth, or on a solid medium, agar.

Since most laboratory studies are made with pure cultures, it is necessary to sterilize
culture media, that is, completely eliminate all living organisms. This medium must be
maintained in a sterile condition, free from living organisms, until inoculated with a pure culture.

To grow a microbial culture in a sterilized medium, a number of the cells, the inoculum,
are transferred, inoculated, into or onto the medium with special precautions to maintain the
purity of the culture. The procedures used in the microbiology laboratory to prevent
contamination of pure cultures are commonly referred to as the aseptic technique.
2.0 OBJECTIVE

In this experiment, we need to acquire knowledge of aseptic techniques in the field of


microbiology. Additionally, we need to avoid the contamination of cultures by undesirable
microbes in the laboratory. After, a subculture transfers cultures from one media by inoculating
into another media. To inhibit lab microbes from being distributed in the environment and
contaminating the investigator is also one of the objectives.

For isolation and purification of microbes, the objectives are to learn how to make sterile
microbiological agar plate media and pour agar plates. Besides, to learned how to perform the
aseptic technique to inoculate and grow a pure culture in agar plates.
3.0 MATERIALS AND METHODS
Procedure
3.1 Transfer the inoculum (broth) into broth.

3.2 Transfer the inoculum (broth) into slant agar.


3.3 Transfer the inoculum (slant) into the broth.

3.4 Transfer the inoculum (slant ) into the slant.


3.5 Transfer the inoculum (slant ) into deep agar.

3.6 Streaking technique


4.0 RESULTS

Results Observation

Turbidity appears to be present in


nutrient broth from slant culture
to nutrient broth as well as from
broth culture to nutrient broth.
This shows the growth of E. Coli
in both nutrition broths.

a) Nutrient Broth
i) Nutrient Broth from Broth Culture to Nutrient Broth
ii) Nutrient Broth from Slant Culture to Nutrient Broth

b) Nutrient Slant Agar The slanted nutrient agar in both


slanted test tubes has pinnacles
on top of it.
This indicates that E. Coli has
grown on both slanted nutrient
agar.

i) Slant Agar from Broth Culture to Slant Agar


ii) Slant Agar from Slant Culture to Slant Agar
c) Nutrient Deep Agar The deep agar test tubes have
pellicles, sediment at the bottom
of the test tubes and a mass of
organisms appears on top.

i) Deep Agar from Broth to Deep Agar

a) Result of streaking E.Coli on b) Result of spreading E. coli on


Nutrient Agar MacConkey Agar
5.0 DISCUSSIONS

The process of transferring bacteria to the medium that has been prepared must be done
carefully to avoid contamination. we need to change the tip loop size accordingly. immerse the
loop in the solution containing bacteria then touch the wall of the test tube so that the bacteria
content is not excessive.

The bacteria (E.coli) that have been included in the media by stabbing the needle into the
center of the agar in the tube will be allowed at room temperature were then incubated for 24
hours. The higher temperature of the lab may speed up the rate of growth cultures, but it also
causes dehydration of the media, and the bacteria will slowly die after 24 hours (Ahern, 2019).
The non-motile bacteria will remain near the inoculation zone, while motile bacteria will
spread and visibly blur the media. This bacteria tend to float at the surface and produce a surface
membrane called a pellicile and also forming sediment at the bottom of test tube. Motile bacteria
move with structures called flagella. Their movement will create a uniform cloudiness (turbidity)
in the broth. Semi-solid medium differs from solid agar in that it contains less agar and thus
allows motile bacteria to move through it. In this stab, we can observe that the growth of bacteria
appears around the line of inoculation in the deep after 24 hours. The bacteria spread from the
stab line creates a pinkish feathering effect.

Precautions are really important in order to prevent contamination of the specific


microorganism we are working with. Firstly, wipe the table using alcohol so that can reduce the
bacteria and germs on the table. After that, properly adjust the bunsen burner. We use the blue
flame when using bunsen burner to burn the loop. Next, when transferring the microorganisms
avoid producing aerosols that we can breathe in and droplets that can land on. We spent a lot of
time in lab transferring organisms from one tube to another, or to slides or to plates. Therefore,
we make it as soon as possible and securely.There are more opportunities for the organism to
become contaminated when it is longer exposed to the air. Lastly, handle biohazard spills and
dispose of biohazard materials properly.

6.0 QUESTIONS

1. Describe the growth of the given microorganism in broth


The broth's nutrients are consumed by the bacteria as they develop, which causes the
solution in the test tube to become more turbid or cloudy. This demonstrates that E. Coli
has effectively entered the liquid nutritional broth medium.The growth of the
microorganisms over a specific period can be tracked by keeping an eye on variations in
turbidity and noticing any visible changes in the broth.
2. Describe the growth of the given microorganism in a slant
Bacteria grow on a solid media as colonies. Bacteria are included via an inoculation loop
by streaking the inoculum at the surface of the slant agar. So, the bacteria only grow in the
streak area. The morphology for slant agar is bacteria performed in a beaded pattern which
is separate or semi-connected colonies in white colour. The bacterial morphology is
punctiform.

3. Describe the growth of the given in-stab


Stab cultures are formed by solid agar in a test tube. Bacteria are included via an inoculation
needle being stabbed into the center of the agar. This technique is usually used for short-
term storage of the bacteria. The non-motile bacteria grow in the punctured area and appear
around the stab line only. The morphology for stab agar is papillate.

4. Describe the growth of the microorganism in an agar plate


The microorganism that grows in an agar plate is Bacteria. Bacteria grow in collections of
cells called colonies. Each colony develops from a single or a small number of bacteria.
Different forms, edges, heights, and colors can be found in colonies. Microbiologists can
use colony traits as one piece of evidence to identify unknown bacteria.

7.0 CONCLUSION

This experiment was conducted to study how to make an aseptic technique and transfer
microbes. Generally, the aseptic technique means using practices and procedures to prevent
contamination from pathogens. It involves applying the strictest rules to minimize the risk of
infection. This experiment is between two test tubes by sterilizing the inoculating loop with passing
it at an angle through the flame of a gas burner for a few seconds to ensure that there is no bacteria
entering the test tube mouth and contaminating the medium. The medium used came from previous
experiments that are crucial for observing the development of microorganisms as we are aware
that contamination by microorganisms can change the outcomes of our experiment. All of the
cultivated plates were handled using the proper aseptic procedure. When it comes to applying this
aseptic technique, typically patients will be checked through physical touch, which has the
potential to create cross-contamination and transmit germs, hand hygiene treatments have been
utilized widely to limit the transmission of pathogens.

8.0 REFERENCES

1. Ahern, Holly. “Bacteriological Culture Methods.” MILNE Library, Open SUNY


Textbooks, 2019, milnepublishing.geneseo.edu/suny-microbiology-
lab/chapter/bacteriological-culture-methods/.
2. Caprette, D. (2017b). Agar slant tubes. Rice.edu.
https://www.ruf.rice.edu/~bioslabs/BIOC318/agar_slants.asp
3. Garg, N., & Garg, K. L. (2010). Laboratory Manual of Food Microbiology. K G Mukerji.
4. Kalra, S., & Reddy, S. (2020). Aseptic Barriers Allow A Clean Contact For Contaminated
Stethoscope Diaphragms, (Vol. 4) 21-33.
5. “9.2: Introduction.” Biology LibreTexts, 24 Sept. 2020,
bio.libretexts.org/Courses/College_of_the_Canyons/Bio_221Lab%3A_Introduction_to_
Microbiology_(Burke)/09%3A_Bacterial_Growth_Patterns-
_Building_your_Stock_Cultures_and_Observing_Culture_Characteristics/9.02%3A_Intr
oduction

6. “Use of Liquid Nutrient Broth Media for Growing Bacteria - Page 2.”
Www.scienceprofonline.com, www.scienceprofonline.com/microbiology/use-of-liquid-
nutrient-broth-media-for-growing-bacteria-2.html
7. Yousef, A. E., Carlstrom, C., & Yousef, A. (2002). Food Microbiology: A Laboratory
Manual (1st ed.). Wiley-Interscience.
8. Siddiquee, S. (2017). The Basic Concept of Microbiology. Practical Handbook of the

Biology and Molecular Diversity of Trichoderma Species from Tropical Regions,

PMC7123386, 1–15. https://doi.org/10.1007/978-3-319-64946-7_1


9. Royal Society of Biology. (2011, October). Aseptic techniques. Practicalbiology.org.

https://practicalbiology.org/standard-techniques/aseptic-techniques

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