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Agar Script

The document outlines a demonstration on preparing nutrient agar and performing inoculation using aseptic techniques. It details the materials required, the preparation and sterilization processes, and various inoculation methods. The importance of proper labeling and storage of bacterial samples is also emphasized to ensure accuracy and integrity in experiments.

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Kevin Yu
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33 views4 pages

Agar Script

The document outlines a demonstration on preparing nutrient agar and performing inoculation using aseptic techniques. It details the materials required, the preparation and sterilization processes, and various inoculation methods. The importance of proper labeling and storage of bacterial samples is also emphasized to ensure accuracy and integrity in experiments.

Uploaded by

Kevin Yu
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Introduction:

1. “ Good morning, my name is Diana Viloria Masangkay, today I will demonstrate how to prepare
nutrient agar and how to perform inoculation using proper aseptic technique”

2. “Our goal is to prepare a sterile growth medium for bacterial culture and ensure contamination-
free inoculation.”

Materials and PPE Explanation:

1. Before we start, I would like to discuss the materials that we are going to use:

a) First is our personal Protective Equipment:

i. Disposable Gloves
ii. Gown
iii. Face Mask
iv. Googles
v. Disposable head cover

This will ensure that we are free from contamination and safe for the procedure that we will do.PPE
refers to a diverse range of specialised gear designed to shield us from various hazards, such as
chemical exposure, physical impacts, harmful particles, and more.

b) Second are the materials that we are going to use during our activity:

i. Nutrient agar powder


1. Nutrient agar is a general-purpose solid medium supporting growth of a wide
range of non-fastidious organisms. It typically contains (mass/volume): 0.5%
peptone – this provides organic nitrogen. 0.3% beef extract/yeast extract – the
water-soluble content of these contribute vitamins, carbohydrates, nitrogen, and
salts.
ii. Distilled water
1. water that has been purified by boiling it into vapor then condensing it back into
liquid in a separate container. Impurities in the original water that do not boil
below or near the boiling point of water remain in the original container.
iii. Measuring tools
1. measures things like distances and angles. Tape measures are made of flexible
material such as cloth or thin metal with specific measurement markings. Steel
rules are made of stiff metal that also has specific measurement markings.
iv. Petri dishes
1. (alternatively known as a Petri plate or cell-culture dish) is a shallow transparent
lidded dish that biologists use to hold growth medium in which cells can be
cultured, originally, cells of bacteria, fungi and small mosses
v. Test tubes
1. a clear glass or plastic container that is much longer than it is wide, commonly
has a U-shaped bottom, and has an open top. Test tubes are used to hold, mix,
and heat chemical experiments. They are used as homes for microorganisms
when people want to culture (grow) them.
vi. Autoclave
1. Autoclaves are also known as steam sterilizers, and are typically used for
healthcare or industrial applications. An autoclave is a machine that uses steam
under pressure to kill harmful bacteria, viruses, fungi, and spores on items that
are placed inside a pressure vessel.
vii. Inoculation loop
1. (also called a smear loop, inoculation wand or microstreaker) is a simple tool
used mainly by microbiologists to pick up and transfer a small sample of
microorganisms called inoculum from a microbial culture
viii. Bunsen burner
1. type of gas burner that is used in many chemistry procedures in a laboratory
setting. It is used to heat substances, to combust substances, and to sterilize
objects on high heat.
ix. Culture sample
1. a test to confirm whether you have a bacterial infection. The test can also
identify what type of bacteria caused the infection, which helps guide treatment
decisions. For a bacteria culture test, a healthcare provider takes a sample of
blood, stool, urine, skin, mucus or spinal fluid.

Nutrient Agar Preparation:

1. We will start the process of preparing the Nutrient Agar.


a) First we need to measure the correct amount of nutrient agar powder to ensure the
accuracy of our experiment.
b) Next is we need to dissolve the nutrient agar powder to distilled water by stirring until fully
mixed and to make sure that no solid particles will remain that may affect the process.
c) And last, pour the solution into sterilization container. Please make sure that we follow the
sterile technique in transferring the mixed solution to avoid discrepancies to the result.

Sterilization Process:

1. For the sterilization process, our prepared solution will be sterilized using autoclave technique.

a) Place the prepared media bottles into a metal tray.


b) Add distilled water until it covers the bottom of the tray; about 1-2 cm deep.
c) Place into the autoclave.
d) Autoclave at 121*C for 15 minutes at 15 psi.
e) Once the cycle is complete, wear heat-resistant gloves to remove the tray and bottles from
the machine.
f) Allow bottles to cool to approximately 60*C.

2. In some instance, if the autoclave machine is not available, we can use boiling technique as an
alternative.

Pouring and Solidification:

Prepping the workspace:

1. To keep as sterile of an environment as possible to avoid contaminating the media and plates, don
a lab coat and gloves, and use a disinfecting agent or wipes to wipe down all surfaces.
2. This includes tabletops and edges, gloves, scissors, permanent markers, etc.
3. Make sure to clean your gloves if they have touched another surface that is not disinfected (eg.if
you touched face, arm, chair, etc.).
4. If available, use Bunsen burners and carefully pour near the open flame to better prevent airborne
contaminants.
5. Once the area is disinfected, bring out the sterile Petri dishes. Keep the sterile dishes closed.
6. Stand a bag up vertically.
7. To conserve the plate bag to reuse for storage, ignore any “Tear Here” markings, and snip a small
corner off the top of the bag. Insert half of the scissors into this opening and cut along the crease of
the bag.
8. Flip the entire stack of plates upside down, with the cut opening at the very bottom of the stack.
9. Gently apply a small amount of pressure downward while simultaneously rolling the bag up.
10. Fold or roll up the empty bag and put aside to use for re-bagging.
Striping the plates

1. To quickly differentiate between plates that have a similar appearance, a stripe code can be used. A
combination of different colored permanent markers can signify different additives to a plate. For
example, a green stripe may mean ampicillin, a type of antibiotic, was added to the media in that
plate. Striping codes are specific to an individual lab, so always double check the code key.
2. To stripe a plate, the top AND bottom must be labeled with the code.
3. Take the marker with the color in the key, and with the unbagged stack of plates, apply a gentle
amount of pressure with your hand onto the top of the stack.
4. Draw a line straight down from the top edge to the base of each plate in the stack.
5. If done quickly, you may need to go back in and redraw the line on the bottom base.
6. If the stripe code has another line or color, repeat the process by adding another line.
7. The spacing of the second line should be within 1 cm from the first line.
8. Repeat the process until the stripe code is complete.

Setting out the plates


1. Begin to unstack the plates. Make sure the plate tops and bottoms do not separate.
2. Place individual plates around the edge of the table (not in stacks), to create a line or chain of
plates.

Pouring the Plates


1. Once the media has cooled to 60*C, the liquid solution is ready to be poured.
2. At this time, an antibiotic (ex: ampicillin) may be added to the media and gently swirled or stirred to
mix. Note: do not add the antibiotic if the liquid is hotter than 60 o C, as the antibiotic would be
denatured.
3. Uncap the media bottle and hold the bottle in your dominant hand. Note: once the bottle is
opened, do not talk. Talking will allow bacteria from your mouth to become airborne and may
contaminate the media.
4. The cap can be held in the same hand (between fingers) as your bottle or can be placed on a
disinfected surface.
5. Grab a plate with your other hand and slide it towards the edge of the table, while keeping it
closed.
6. Once the plate is at the edge, open the lid as if there is an imaginary hinge at one end; so the plate
opens like a clamshell.
7. Pour the media into the bottom of the plate until it just covers the surface. Do not over fill.
8. Close the lid and allow to cool. The media will be solid.
9. Leave the plates out for a day if possible so the condensation will evaporate from the plate. You
may place the plates in a 37*C for 24-48 hours.
10. Stack plates with the same type of media and slide the plastic sleeve over the top.
11. Flip the stack over and seal the plastic sleeve with masking tape.
12. Label the tape with the type of media, date produced, and name of individual that produced the
stack.
13. Store sealed stacks in the refrigerator until use.

Inoculation Process:

1. For the inoculation process, I would like to describe the different inoculation techniques:
a) Streak Plate
i. A streak plate involves the progressive dilution of an inoculum of bacteria or yeast
over the surface of solidified agar medium in a Petri dish. The result is that some of
the colonies on the plate grow well separated from each other. The aim of the
procedure is to obtain single isolated pure colonies.
b) Spread Plate
i. The spread plate method is a technique to plate a liquid sample containing bacteria so
that the bacteria are easy to count and isolate. A successful spread plate will have a
countable number of isolated bacterial colonies evenly distributed on the plate.
c) Pour Plate
i. This method involves mixing a liquid sample with molten agar and then pouring the
mixture into a petri dish to solidify. The primary purpose of pour plating is to count
the number of colony-forming units (CFUs) in a sample, providing a quantitative
measure of microbial concentration.

2. For the process of using the inoculation loop:


a) Flame sterilize the inoculation loop before and after use to prevent cross-contamination
b) Lightly streak the sample onto the agar surface in a zigzag motion

Labelling and Storage:

1. The importance of proper specimen labeling and identification cannot be overstated, and its
significance lies in several areas, including ensuring accurate date and time, correct sample ID and
initials will be essential in reducing errors and potential harm, and maintaining the integrity of the
trademark specimen.

2. Bacterial samples are critical for research, diagnostic, and teaching purposes. Although there are
many ways to store bacteria, the ideal method is a function of bacterial compatibility, experimental
purpose, and cell viability. Proper storage and incubation conditions can be settled at 37*C for 24-48
hours for most of the bacterial cultures.

To summarize today's activity, we are able to know how to prepare including the materials and
methods that we are going to use, the process of sterilization and inoculation of nutrient agar while
maintaining and following the standards using aseptic technique.

And that ends my discussion for today, for any questions and query, you may raise your hands and I
will be willing to answer it at my very best. Thank you.

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