Scribd
Upload
64 views3 pages

Practical 4 Preparation of Culture Media

Uploaded by

ahmed mumtaz
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
64 views3 pages

Practical 4 Preparation of Culture Media

Uploaded by

ahmed mumtaz
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
You are on page 1/ 3

Subject name: Physiology 1

Coarse Code: PHY.915


Instructor name: Miss Hina Gull
UIDNS (Allied Health Sciences)
The University of Lahore

Practical 4: Protocol for the ‘’Preparation of Culture Media’’


Apparatus:

Graduated cylinder, 1 L

Flask, 1 L

Glass stirring rod

Spatula

Weigh boats

Bacto peptone,

Agar powder

pH paper 6.5-10

HCl, 1N 25 ml/dropper

bottle 1/table NaOH,

1N 25 ml/dropper bottle 1/table Test tube rack 2/table

Labeling tape

Heatproof gloves

Autoclave RESERVE Water baths, 48 °C

Petri plates

Cheesecloth square or cotton plug


PROCEDURE:
1. Wipe down lab bench carefully with Disinfectant to help prevent contamination of your media.

2. Measure approximately 250 ml of distilled water (located in 60°C water bath) in a 1 L graduated
cylinder and pour into a 1 L flask.

3. Weigh out 1.5 g beef extract and 2.5 g peptone and add into the flask. Wash your spatula between
bottles and wipe dry. DO NOT return excess material that is weighed out to the container - discard.
Use approximately 100 ml of the water to rinse any powder stuck to the side of the flask down into
the mixture.

4. Stir over gentle heat from a bunsen burner to dissolve completely.

5. Pour the mixture into the 1 L graduated cylinder and add warm water to the 500 ml mark. Pour
back into the flask.

6. Check the pH of the medium and adjust to pH 7.0, if necessary, using the HCl and/or NaOH.
Adding the agar in the next step will not appreciably change the pH.

7. Using a 10 ml pipette, dispense 10 ml of the mixture into each test tube. Make 10 tubes and place
in a test tube rack.

8. Add 6.0 g of agar to the flask and label it NA. Heat to just boiling for 1-2 minutes while stirring
constantly. The agar will not dissolve unless it is boiled; the solution will become completely clear
when it has dissolved. Allow agar to cool until there is no danger of you being burned and then
dispense into the tubes using a 10 ml pipette. Make ten 10 ml tubes.

9. Close the flask with a Styrofoam plug covered with cheesecloth and tape it on top of the flask. Use
another piece of tape to go around the neck of the flask and pass over the first tape. Cap all the
tubes with Morton closures. These should be pushed down completely or else they will be forced off
during the autoclave's exhaust cycle. They are still self venting when pushed down all the way.

10. Keep one tube of each type in a drawer until next period to demonstrate the need for
sterilization. Continue to observe growth for one more period.

11. Autoclave the flask and the tubes for 15 minutes at 121 °C and 15 lb/in2 pressure at the slow
exhaust mode. Watch your instructor for the use of the autoclave.

12. After removing the media from the autoclave, allow the broth tubes to cool, and store for later
use. Place the flask in the 48°C water bath. Quickly lay the tubes of NA on the slant racks on the
center table so that the medium forms a long slant and a short butt and allow them to cool and
solidify. Do not allow the agar to reach the top of the tube. Allow them to cool completely before
returning to the rack. Store for later use. Label rack.

13. Lay your petri dishes on the bench. The cover should be on top. Light your bunsen burner, then
remove the NA flask from the water bath. Carefully wipe the bottom dry to prevent the dripping
water from contaminating the plates.

14. Remove the tapes and cotton plug from the flask. Carefully flame the neck of the flask, open the
plate cover about half way and fill the plate about 1/2 full. The plates have a full line on the side; fill
to that or slightly above. Put in a little too much rather than too little. If there is not enough medium
in the plate, it will dry up in the incubator.
15. Flame the neck of the flask between each plate. Each student must pour at least two plates.
IMMEDIATELY rinse the excess agar out of the flask with hot tap water and place on the discard cart.
Allow plates to solidify completely, which will take 15 minutes. Then invert, label and incubate at 37
°C overnight to dry off excess moisture and check for contamination.

16. Clean all glassware and leave on paper towels beside sink.

Sterile Conditions & Autoclaving:


The media upon which microorganisms are grown must be sterile or free from all other forms of
microbes. The usual method for sterilization of culture media is by means of the autoclave in which
steam under pressure is the sterilizing agent. Autoclave sterilization for 15 minutes at 15 pounds of
pressure and at 121 °C is recommended for quantities of liquid media up to one liter (1 L). These
settings are called the standard autoclaving conditions.

Storage of Media:
Media should always be stored in a cool moist atmosphere to prevent evaporation, preferably in
screw-capped tubes or bottles.

You might also like

pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy