CHEM-2032 AAU | AAiT | CoBME Dec 2019

ADDIS ABABA UNIVERSITY

ADDIS ABABA INSTITUTE OF TECHNOLOGY
CENTER OF BIOMEDICAL ENGINEERING

LABORATORY REPORT #1

COURSE NAME : BIOCHEMISTRY

EXPERIMENT NUMBER : 1

TITLE OF THE EXPERIMENT:

EXPERIMENT 1 – ASEPTIC TECHNIQUE AND CULTURING YEAST CELLS ON

MEDIA

STUDENT NAME ID NO

MICHAEL DEBEBE ATR/7915/11

MIKIAS TESHOME ETR/5400/11

NATNAEL ABEBE ETR/7426/11

SADOR YONAS ATR/4026/11

YARED MINWUYELET ATR/4702/11

LAB INSTRUCTOR : ALENE ADMAS

DATE OF EXPERIMENT: 6, DECEMBER 2019
DATE OF SUBMISSION: 19, DECEMBER 2019

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I.OBJECTIVES

 The objectives of this laboratory experiment were to practice following the aseptic
technique in order to avoid contamination and to isolate yeast and create a pure culture.
 To verify the practices and procedures used in aseptic technique used in an organized
way to avoid contamination and getting our desired result in a very mannered exercise.
 The experiment uses cultured solid and liquid media to observe the introduction of
microorganisms from a visitor colony, culturing them and observing the results

II. INTRODUCTION

AN OVERVIEW OF ASEPTIC TECHNIQUE

Microorganisms are everywhere, and some are beneficial for us while others are harmful. To
protect patients from harmful bacteria and other pathogens during medical procedures, healthcare
providers use aseptic technique.

Aseptic technique is a set of principles and practices used by cell culture workers to reduce the
presence of unwanted microorganisms or other cell lines in their cultures. It means using
practices and procedures to prevent contamination from pathogens. It involves applying the
strictest rules to minimize the risk of infection. Healthcare workers use aseptic technique in
surgery rooms, clinics, laboratories, outpatient care centers, and other health care settings.

What is aseptic technique used for?

Following aseptic technique helps prevent the spread of pathogens that cause infection.

Healthcare professionals commonly use aseptic technique when they’re:

 handling surgery equipment

 carrying out culturing procedures
 helping with a baby’s birth by vaginal delivery
 handling dialysis catheters
 performing dialysis
 inserting a chest tube
 inserting a urinary catheter
 inserting central intravenous (IV) or arterial lines
 inserting other draining devices
 performing various surgical techniques

Aseptic technique types

According to The Joint Commission, there are four chief aspects of the aseptic technique:
barriers, patient equipment and preparation, environmental controls, and contact guidelines. Each
plays an important role in infection prevention during a medical procedure.

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Barriers

Barriers protect the patient from the transfer of pathogens from a healthcare worker, from the
environment, or from both. Some barriers used in aseptic technique include:

 sterile gloves
 sterile gowns
 masks for the patient and healthcare provider
 sterile drapes

Sterile barriers are those that have not touched a contaminated surface. They’re specially
packaged and cleaned items. Healthcare workers put them on or use them in specific ways that
minimize exposure to germs.

Patient and Equipment Preparation

Healthcare providers also use sterile equipment and sterile instruments. To further protect the
patient, they apply cleansing and bacteria-killing preparations to the patient’s skin before a
procedure.

Environmental controls

Maintaining a sterile environment requires keeping doors closed during an operation. Only
necessary health personnel should be at the procedure. The more people present, the more
opportunities for harmful bacteria to cause contamination.

Contact guidelines

Once healthcare providers have on sterile barriers, they should only touch other sterile items.
They should avoid touching nonsterile items at all costs.

AN OVERVIEW OF CULTURING YEAST CELLS ON MEDIA

This lab will introduce you to standard techniques used in microbiology. Very similar techniques
are used to culture yeast and bacteria, although the culture conditions are optimized for each
organism. In this lab, you will learn sterile techniques required for maintaining the integrity of
yeast strains in the lab, as well as methods for culturing cells and estimating cell numbers.

For routine culture, scientists usually use rich media that supply all the nutrients that cells
need to grow. The individual components of rich media are often undefined. For example, yeast
are commonly grown in a medium known as YPD, which is simple and inexpensive to prepare.
The “Y” in YPD refers to a yeast extract, which contains the water-soluble compounds generated
when yeast are forced to self-digest.The “P” refers to peptone, a mixture of
peptides and amino acids prepared by digesting animal protein with proteases. The “D” refers to
dextrose, or glucose, which is the favored carbon source of yeast.

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Yeast can be grown in liquid cultures or on the surface of plates containing solid media.
Agar is usually used to solidify liquid growth media when preparing plates. Strains are typically
maintained on agar plates for routine use. Cells grow in colonies on plates. The cells in a colony
are genetically very similar, if not identical, because they are derived from the same progenitor
cell. Most yeast strains can be stored on plates in the refrigerator for several months with
minimal loss of viability.

Yeast growth phases

When yeast are grown in liquid medium, the culture follows a well-established pattern
for microbial growth. (Bacteria follow this same general pattern, although they divide much
more rapidly.) Cultures are usually started by inoculating media with a small number of cells. A
lag phase follows the inoculation, during which cells become acclimated to the new environment
and begin to condition the media with their own metabolites. Lag phase is followed by an
exponential, or log phase, when the number of cells increases exponentially.

Preparation Instructions

 Prepare the Potato Dextrose Agar and Potato Dextrose Broth

 Initialize your digital balance to measure the amount of PDA and PDB
 Set up test tube racks with five test tubes per group.
 Set up petri dishes and slants with two petri dishes and two slants per group.
 Set up the flasks with two flasks per group.
 Set up your pipettes with it’s respective diametrical tips
 Prepare 200 ml of distilled water that will be poured to the two flasks of PDA and
PDB with 100 ml each.
 Dictate the usage of autoclave sterilizer , laminar flow and incubator

Pre Lab Definitions

 Culture: To grow living organisms in a prepared mixture of nutrients (media).

 Media: Substance containing nutrients needed for cell growth.
 Agar: Jelly to which food has been added for the growth of microbes.
 Sterile/Aseptic Technique: Laboratory procedures used in handling cultures,
media and equipment that prevent contamination.
 Colony: Group of bacteria.
 Inoculate: To introduce a microbe to an environment where it can grow.
 A balance: is used to weigh chemicals. The chemicals are always in some form of
container and never placed directly on the balance. It is important not to move a
balance because they have been calibrated for the exact position they are in.
 Petri Dish: A round, shallow dish used to grow yeast.
 Slant culture: A leaned holder used to grow yeast
 Graduated cylinders: This is a primary measuring tool for the volume of a liquid.

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 Pipette: They are all for measuring an exact volume of liquid and placing it into
another container.
 Spatulas and scoopulas: are for scooping solid chemicals. They are typically used
to scoop a chemical out of its original container onto a weigh boat so that it can be
weighed on a balance.
 Bunsen burner: an ideal flame for heating purposes.
 Inoculation loop: Used to pick up and transfer a small sample (inoculum) from a
culture of microorganisms to petridishes or slants.

III. MATERIALS AND METHODS

Table 1: Materials used

Material used Quantity per group Purpose

Safety Gloves 1 pair per person As the name indicated it is used to

prevent any contamination of
chemicals with experimenter.

Balance 1 Is used to weigh chemicals such as

PDA and PDB.

Spatulas and scoopulas 2 To scoop solid chemicals

PDA(agar) and PDB (broth) from
their package into balance.

Test Tubes(10 ml) 5 To store distilled water and to be

as a site of the mixture of “Tejj”
until it becomes dilute, Used to
mix, heat, or store substances.

Test tube rack 1 Used to hold test tubes.

Erlenmeyer Flask(250ml) 2 This allows easy mixing and

swirling of the the raw materials
without too much risk of
spilling.In our case, one flask used
for mixing of the distilled water
and PDA(agar).The second flask
for mixing of the distilled water
and PDB(Broth).

Aluminum foil 2 To prevent entrance of

microorganism into the yeast
source solution

Measuring cylinder 1 To measure water volume

Graduate Cylinder 1 It is the primary measuring tool

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for the volume of a liquid.

Pipettes 3 Measures an exact volume

of liquid and places it into
another container.They are
of different sizes with
different tip ranges.

Petri dishes 2 A locale where PDA and PDB

placed.

Slant tubes 2 It is similar to petridish in it’s

purpose but differs in it’s shape
and configuration.

Vortex 1 Whirling mass

Autoclave sterilizer 1 To sterilize all materials (except

yeast source)before went to
laminar flow

Bunsen burner 1 To sterilize the

Laminar flow 1 Prevents any microorganism

contamination and coagulates the
medias.

Inoculation loop: 1 Used to pick up and transfer a

small sample (inoculum) from a
culture of microorganisms to
petridishes or slants

Incubator 1 To put the final (smeared) yeast to

see the result after 24 hour.

Table 2 : Reagents used

Reagents Quantity for one experiment Purpose

Potato Dextrose Agar (PDA) 3.9 gram for 200 ml water (To Used as a liquid medium
the whole group)

Potato Dextrose Broth(PDB) 4.8 gram for 200 ml water () Used as a solid medium

Distilled water 200 ml Used as a solvent for PDA and

PDB solutes

Yeast source(mead) or “Tejj” 1 ml Act as a stock substance

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Alcohol Not defined To clean our hand during

experiment

PROCEDURE : The steps performed are stated below

 First thing we needed to do was to prepare a media: By measuring them using digital
balance, we prepared two types of media:

1. Solid media (Potato Dextrose Agar) containing 3.9 grams of agar and

2.Liquid media(Potato Dextrose Broth)of 4.8 grams which doesn’t contain any agar.

 Then measuring distilled water of 100 milliliters using spatula for 3.9 grams of Potato
dextrose media and 200 milliliters for 4.8 grams of broth (meaning we needed only 2.4
grams for the 100 ml addition of water per one group).

 Then we added an additional 2% of the total mass of PDA and PDB for solidifying agent
(agar) which does not affect the solution.

 Then we used a pipette to take 9 ml samples of our distilled water and added them into
the five test tubes.

 To add the combined media and water in the flask into the autoclave, we covered it with
aluminum to avoid contamination and addition of other microbes which would alter the
overall result of our experiment.

 Then we put the whole equipments (test tubes, test tube racks, petri dishes, slants,
Erlenmeyer flasks and etc ) in to autoclave sterilizer for approximately 15 minutes with
temperature range of 740c-1210c.

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 After it’s been sterilized, we added the sterilized equipments in to laminar flow.In the
laminar flow we pour an approximate 25ml PDA and PDB solutions in to petri dishes
and slants.

 After the 30 minutes inside the laminar flow, we took a stock sample of “tejj” (1
milliliters) and add it in to the test tubes.Remember now the test tubes are filled with
10ml of solution.

 How we did that was, after we added the 1 milliliters of ”tejj” on the first test tube, we
used a vortex to mix it, then using a pipette, we took a sample of 1ml from the current
test tube and added it to the second test tube, the again using the vortex we mix the
second test tube, take a sample from it and added the sample we took to the third test tube
and repeat this process until we reach the 5th test tube. After this process we take samples
from the third and fourth test tubes.

 We used a Bunsen burner to sterilize the inoculation loop, used for swirling the stock
sample to the petri dishes.Swirl your disposable loop founf in test tubes 3 and 4. Use the
disposable loop to spread the solution on the surface of the agar- being careful not to
press too hard!

Figure 1 : Recommended streak pattren

 And similarly we slowly take very small samples and spread them in the slant.

 And finally we placed the mixed form into the incubator and the cells will incubate or
grow at 3000c for 24 hours.

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IV. RESULTS

The result of the experiment can be illustrated in visual mode as follows :

Figure 2 : Yeast colony

Figure 3 : Yeast colony 2

V. CONCLUSION

Aseptic techniques prevent contamination of bacterial cultures and environment in the lab. After
the incubation for 24 hours, bacteria started to colonize on the agar plate as shown above. A

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colony is a population of cells which arise from a single cell growing on a solid medium. We
gained experience in streaking for isolated colonies by streaking bacteria.

VI. REFERENCES

 MedlinePlus - Search Results for: bacteria culture

 Sherman F (2002) Getting started with yeast. Meth Enzymol 350: 3-41
 Aseptic Technique: Uses, Benefits, and Complications
 https://www.healthline.com/health/aseptic-technique
 Aseptic Technique - an overview | ScienceDirect Topics

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