Basic technique of microbiology

1. Basic handling of microbiology
By- Shreya Yadav
190101032
Biochemical Engineering

2. Experiments
 Laboratory Equipments- autoclave, microscope, laminar air flow (LAF), incubator, weighing balance, micropipette, etc.
 Washing/ Cleaning
 Micropipette caliberation
 Media Preparation
o Nutrient Broth (NB) preparation
o NB agar preparation
o Preparation of E.Coli culture
o Yeast Peptone Dextrose(YPD) preparation
o YPD agar preparation
o Preparation of S.Cerevisae
 Methylene blue degradation
 Cross-linking of polymers

3. Autoclave
An autoclave is an equipment that provides a
physical method of sterilization by killing bacteria,
viruses, and even spores present in the material put
inside of the vessel using steam under pressure. It
works on the principle of moist heat sterilization
where steam under pressure is used to sterilize the
material present inside the chamber. It is run at a
temperature of 121° C for at least 30 minutes and
uses saturated steam under at least 15 psi of
pressure.

4. Laminar air flow (LAF)
It is an equipment that is generally used in microbiology
laboratories. It consists of a chamber with an air blower
attached to its rear side that allows the flow of air with a
uniform velocity in straight lines that are parallel to each
other. The main purpose of a laminar flow cabinet/hood is
to form a contaminant-free work environment. It makes use
of a filter pad and a special filter system known as a high-
efficiency particulate air filter or HEPA filter. It works on the
principle of laminar flow of air

5. Incubator
An incubator, in microbiology, is an insulated and enclosed
device that provides an optimal condition of temperature,
humidity, and other environmental conditions required for
the growth of organisms. The usual temperature to be
maintained is 35–37°C for bacteria.

6. Micropipette
Micropipette is a standard but essential instrument in the
laboratory utilized to precisely and accurately transfer
volumes of liquid within small microliter volume.

7. Media preparation
• Nutrient broth (NB) preparation:
1. Suspend 2.6 g of nutrient broth in 200 ml of distilled water.
2. Stir it for fully dissolving all components in water.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the nutrient broth has been autoclaved, allow it to cool.
• Nutrient broth (NB) Agar preparation:
1. Suspend 2.6 g of nutrient broth and 5 g of agar powder in 200 ml of distilled water.
2. Stir this mixture for fully dissolving all components.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the NB agar has been autoclaved, allow it to cool but not let it solidify.
5. Pour NB agar medium into each plate and leave plates on the sterile surface until the agar get solidify.
6. Replace the lid of each Petri plate and store the plates in a refrigerator.

8. Culture preparation (of E.Coli)
1. For 10 minutes we on the UV light in LAF.
2. Take out the culture sample (E.Coli) from freezer (20˚C) and
thaw it so that it come into liquid state.
3. After 10 minutes we off the UV light and on the lamp light
and mains then opened the shutter of LAF and washed
hands with IP solution.
4. Took the media (49 ml) and poured the culture (1ml) into
media for culture solution. Then we took 1 ml of it and pour
it into fresh 49 ml media.
5. In this way we did serial dilution of stock culture up to 5
times.
6. Then we pour 1 ml of culture from 5th dilution into a 49 ml
media, making it 50 ml culture.
7. Then sealed the culture with wax tape and put prepared
culture into orbital shaker and left there for 24hrs.
8. Off the lamp light and mains.

9. Media preparation
YPD preparation (per 100 ml):
1. Took yeast extract, dextrose & peptone in above given quantity and dissolve it into 100 ml of distilled water.
2. Then shake it properly.
3. Put this media into autoclave for 15-25 minutes at 121˚C.
4. Once the media is autoclaved allow it to cool at room temperature.
5. Take it to LAF for performing experiment.
Components Quantity
Yeast extract 1000.5 mg
Peptone 2001.6 mg
Dextrose 2000.8 mg

10. Media preparation (YPD Agar media)
1. Took yeast extract, dextrose & peptone in above given quantity
and dissolve it into 100 ml of distilled water.
2. Weigh and put 2.5 gm of agar into it.
3. Then shake it properly.
4. Put this media into autoclave for 15-25 minutes at 121˚C.
5. Once the media is autoclaved allow it to cool but not to solidify.
6. Pour this YPD agar media into each petriplate and leave the
plates on the sterile surface until the agar has solidified.
7. Replace the lid of each Petri dish and invert it then store the
plates in a refrigerator
Quantity
Yeast extract 1.049 g
Peptone 2.097 g
Dextrose 2.5059 g
Agar 2.503 g

11. Culture preparation (of S.Cerevisae)
1. For 10 minutes we on the UV light in LAF.
2. Took 5 mg of yeast powder and pour it into 0.1 ml of RO water in eppendorf of 1.5 ml size.
3. Then vortex it in order to prepare culture.
4. Then pour prepared culture into 100 ml of media.
5. Put this culture on shaker for 48 hrs.

12. Methylene blue degradation
Photocatalytic degradation oxidizes complex organic
compounds into small molecular inorganic
substances, such as carbon dioxide and water, under
light. The reaction is thorough and causes no
secondary pollution. Methylene blue is used not only
to dye paper and office supplies but also to tone up
silk colors.
Procedure:
1. We took the 5 ml stock solution of methylene
blue.
2. Then we made 1500 µl solution by adding 8.4 ml
water and 600 µl of methylene blue along with
18 mg of nanoparticle.
3. Then we put the solution into shaker for 1 hour.
4. Then we took two falcon tubes.
5. In first one we poured 1500 µl of solution and
1350 µl of water along with 150 µl of solution A.
6. And in second one we poured 1500 µl of
solution and 1200 µl of water along with 150 µl
of solution A and 150 µl of solution B.

13. Cross-linking of polymer (gelatin) with transglutaminase (TGA).
Cross-link is a bond or a short sequence of bonds
that links one polymer chain to another. These links
may take the form of covalent bonds or ionic bonds
and the polymers can be either synthetic polymers
or natural polymers (such as proteins). Here it
refers to the linking of proteins together to check
for protein–protein interactions and other creative
cross-linking.
Procedure:
1. First took the polymer (milk powder) and
weighed it for three concentration of set-A;
C₁=800.0 mg, C₂=1200.6 mg, C₃=1500.8 mg)
2. Then prepare the stock solution of sample by
dissolving polymer into 10 ml of RO water.
3. Prepare the stock solution of
transglutaminase (10.2 mg/ml) in water.
4. Pour the TGA in sample solution such that
1600 µl in C₁, 2400 µl in C₂ and 300 µl in C₃.
5. Now wait and observe the gel time.
Result: Gel was observed after 45 minutes.

14. Thank You