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Scierep - Scientific report

Scientific report

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Bukidnon State University

Academic year: 2021/2022

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Central Mindanao University

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Microbial Culture Media Preparation, Sterilization Procedures

and Aseptic Technique

Exercise No. 3

A Laboratory Scientific Report

Presented to

Aimae Jean B. Lunsayan

Department of Biology

College of Arts and Sciences

Central Mindanao University

In Partial Fulfillment

of the Requirements for the subject

GENERAL MICROBIOLOGY, LABORATORY

Georgina Mae C. Balanay

Janna Victoria L. Adlao

Myzel Jane L. Aninao

Necka Earl D. Amolo

Arghie R. Agsinao

October 2022

Table of Contents

Introduction..........................................................................................................................................

Introduction..........................................................................................................................................
Methodology........................................................................................................................................
Results...............................................................................................................................................
Discussions........................................................................................................................................
Conclusion.........................................................................................................................................
References........................................................................................................................................

Methodology........................................................................................................................................

I. Aseptic Techniques

Apply disinfectant/antiseptic such as 70% isopropyl or ethyl alcohol.

Wash hands with soap and water and dry with a paper towel.

Disinfect the work area with alcohol or thin bleach before and after work is finished.

Flame the inoculating loop before and after transfer of bacteria from one container to another. Never lay an unflamed inoculating loop on the bench top.

Do not lay the tube plugs on the bench top while bacteria are removed from or transferred to the container. Plug should remain under your control throughout the transfer.

Flame the mouth of glass containers or the lid of the plate before and after removing/transferring bacteria from them or when plating media.

Do not talk while plating the media,

performing culture transfer, etc.

Students with long hair should keep it tied to prevent it from touching the surface of the bench top or the plate.

Work quickly and efficiently to minimize exposure time of the culture to the environment.

Pressure Cooker/Sterilizer

Preparation of glassware for sterilization.

Place the plate at the center of the paper in landscape position and fold both edges over the plate.

Wrap each pair in an 8x11 inches piece of short bond paper.

To Prepare Petri dish for Sterilization:

Dry glassware at room temperature or in a drying oven set at 50’C.

Wash glassware with detergent and water. Liquid detergent are preferred.

Wrap pipettes in 2’s using 2 pieces of paper. Put it on top of the other allowing for a minimum of 30% overlap lengthwise.

Push a loose cotton plug into a larger tip using a piece of wire.

To prepare pipettes for sterilization:

Place all wrapped paper plates in an autoclavable transparent cellophane and tie with a rubber bond.

Holding the paper, press both sides of the upper paper opening with your pointing fingers and fold over the plate. Do the same to the lower opening.

Operation procedures for Pressure-Cooker type sterilizer:

Place wrapped tubes and flask in an autoclavable cellophane and tie with a rubberband.

Individually, wrap the mouth of flasks with paper and tie with a rubber band.

Wrap the anterior region of bundled tubes (max. of 10 tubes) with paper and tie it with rubber band.

Allow to dry and put on appropriate fitting cotton plugs.

Wash and clean them with liquid detergents.

Transfer techniques:

Pour enough tap water to about 2inches deep into the cooker.

Put in place the metal stand and the inner chamber (wire basket) of the cooker.

Arrange materials to be sterilized inside the inner chamber, leaving ample space for steam circulation. Place large and heavy materials first at the bottom of the cooker.

Put the cover and rotate it until its mark coincides with that of the first container.

Remove contents and dry wrapped materials in the oven or at room temperature.

Turn off the heat. Wait for the pressure to go down to zero. Open the stopcock to release remaining pressure. Loosen the screw and rotate the cover. Lift the cover away from you.

Turn to medium heating when the pressure reaches 15lbs. Maintain this pressure for 15mins. Extend the period at 15lbs to 30mins if you are discarding cultures.

Make sure that the stopcock is open. This will produce the hissing sound when the cooker is vented.

Vent for about 5minutes to remove air from the inside.

Turn on the stove to maximum heating. Or you can pre-heat the pressure-cooker.

Lock opposite pairs of the screw at the same time and with equal tightness, again to prevent release of steam.

Hold both tubes in the palm of your left hand (if you are right-handed) and beat the loop to redness with your right hand (if you are right-handed).
Obtain two broth tubes, one with bacterial growth and the other sterile.
Flame the mouth of the tubes briefly to kill any air-borne microbes that may contaminate the top of the tube during the transfer.
Unplug both tubes by placing one cotton plug between your pointing and middle finger and the other between the ring and small finger, with the palm of the hand facing up.
Place the loop in the sterile broth tube, and mix in the broth gently for a few seconds.
Carefully place the loop into the tube with bacterial growth, mix briefly, and carefully withdraw the knop without touching the rim of the tube.
While withdrawing the loop, tap it gently on the inside of the tube well below the lip.
After the loop is withdrawn, flame the tubes again and put back the cotton plugs.
Heat the loop to redness and place it on the test tube rack.

C. Broth-to-Slant Transfer

Culture Media and their Preparation

A. Familiarize some commonly used culture media.

Repeat steps 2 to 5 as you did for the broth-to-broth transfer.

Obtain a broth tube with bacterial growth and a sterile agar slant.

Heat the loop to redness and place it on the test tube rack.

Touch the loop to the lower part of the sterile agar slant, and draw it gently, running up the surface of the slant once in a zigzag manner while still touching the agar.

Carefully place the loop in the broth tube, mix briefly, and carefully remove the loop without touching the rim of the tube.

Withdraw the loop Flame the tubes again and put back the cotton plugs.

Pull out all available dehydrated culture media in the shelf

Understand their respective uses

Read the respective instructions on how to prepare them

Classify them based on their physical state, composition and functional type

Use dry spoon in scooping media and tightly close media containers immediately after opening.

C. Preparation of nutrient broth (NB)

Open the plate with the thumb and pointing finger of the left hand, with the rest of the fingers supporting the bottom of the plate. Maneuver the fingers to prevent burning cither from the flame or from the hot media.

Flame first the entire lid of the plate (using the left hand) and the mouth of the flask (using the right hand and with the cotton plug placed in between the pointing and middle fingers).

Leave undisturbed for several minutes to solidify. Put the plug back in the flask.

Using the right hand, pour approximately 15-20ml NA from the flask. Work near the flame.

Close the plate and place it gently on the tabletop.

Dispense 8-10 ml dissolved NB into a test tube. Plug with cotton. Prepare 3 NB tubes, bundle them in a beaker and place the beaker in an autoclavable cellophane. Sterilize.

Weigh computed amount and place in a 100-ml flask containing 30 ml dH O. Stir to dissolve.

Compute for the number of grams needed to prepare 30 ml of the medium.

Results

Figure 1. Nutrient gar with bacterial growth incubated for about three (3) days. Aseptic techniques reduce the possibility of contamination of cultures by environmental microorganisms or contamination of the environment by the microorganisms being handled.

Conclusion

Overall, this experiment demonstrated the significance and value of

aseptic procedures, sterilizing procedures, and culture media.

Microorganism cultivation frequently results in microbial contamination,

especially when not done aseptically. It was expected that the results would

be tainted if this were the case. The primary goal of this analysis is to avoid

this outcome, not only to avoid contaminated culture but also to avoid some

infections that could endanger lab personnel. Regardless of how carefully

and aseptically the processes are carried out, if the tools are not sterilized

and by doing the methodology improperly, the results will be contaminated

and unnecessary outcomes. As a result, this overall demonstrates how

training workers or students, laboratory layout, cleaning, and maintenance,

as well as awareness of the importance of culture media, and adhering the

experiment’s methodology are important factors in preventing contamination

in cell culture laboratories.

References

Finlay, B. B., & Falkow, S. (1997, June). Common themes in microbial

pathogenicity revisited. Microbiology and Molecular Biology Reviews,

61 (2), 136–169. doi/10.1128/mmbr.61.2.136-169.

IntroductionDisinfection & Sterilization Guidelines | Guidelines Library |

Infection Control | CDC. (n.).

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on?fbclid=IwAR3ReAH475YSGWNWurWKugGkjv-

Y5YcCIFfDy0gJNd8HSJCj3yUnvf29gkI

NCBI - WWW Error Blocked Diagnostic. (n.). Retrieved October 19, 2022,

from ncbi.nlm.nih/pmc/articles/PMC7123386/?

fbclid=IwAR1i1W9HWPp5-

WWLeFJKd72Z415w5U8jwolzjmhdGWSb6FAQCrXPbsFJ6vw

NIH Human Microbiome Project defines normal bacterial makeup of the.

(2015, August 31). National Institutes of Health (NIH).

nih/news-events/news-releases/nih-human-

microbiome-project-defines-normal-bacterial-makeup-body

Singh, A. (2022, March 27). How to Prepare Culture Media and Preserve

Cultures. Conduct Science. conductscience/how-to-

prepare-culture-media-and-preserve-cultures/?

fbclid=IwAR0XApFnr9jY_oyUjql1a0U_WjcSlbos0mA_E765d89oinLiV

B82WkC-dmw

Sterilization, Disinfection, and Decontamination | Office of Lab Safety | The

George Washington University. (n.).

labsafety.gwu/sterilization-disinfection-and-

decontamination?

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