Industrial training on microbiologist basic handling

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Table of content
1. Introduction
2. Laboratory equipments
3. Washing and Cleaning
4. Preparation of Nutrients broth and Nutrients agar
5. Preparation of YPD broth and YPD agar
6. Culture plating methods
7. Methyle blue degradation
8. Protein cross-linking method

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Introduction
We will explore the central role of microorganisms in nature and in our daily lives. In this process of
discovery, we will become adept with standard microbiological techniques that will allow us to
investigate the structure and physiology of microorganism.
The various sub-discipline of microbiology including bacterial genetics, Bacteriology and applied
microbiology will be introduced..
Therefore, biochemical engineering is one of the major areas in biotechnology important to its
commercialization.

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Autoclave

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Autoclave Principle/Working
The autoclave works on the principle of moist
heat sterilization where steam under pressure is
used to stenilize the material present inside the
chamber.
This principle is employed in an autoclave where
the water boils at 121°C at the pressure of 15 psi
or 775 mm of Hg.
When this steam comes in contact with the
surface, it kills the microbes
Once the sterilization phase is completed, the
pressure is released from the inside of the
chamber through the whistle.

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Laminar air flow
A laminar air flow is an equipment that is generally used in microbiology laboratories. It consists of a
chamber with and air blower attached to its rear side that allows the flow of air with a uniform velocity in
straignt lines tnat are parallel to each other. I ne main purpose or a laminar tlow cabinetnood is to Torm 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.
Precautions for LAF
The laminar flow cabinet should be sterilized with the UV light before and after the operation.
The Uv light and airflow should not be used at the same time.
No operations should be carried out when the UV light is switched on.
The operator should be dressed in lab coats and long gloves.
The working bench, glass shield, and other components present inside the cabinet should be sterilized
before
and after the completion of work.

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Micropipette
Micropipette is a standard but essential instrument in the laboratory utilized to precisely and accurately
transfer
volumes of liquid within small microliter volume.
Components of a micropipette
Micropipettes can be found in various shapes and sizes. There are a few elements that are common to
all
micropipettes. This includes the plunger, digital display and tip cone, as well as the tip ejector, and the
grippy.
Certain micropipettes come with a calibration tool as well as the stand for micropipettes as an option.
Plunger
Tip Ejector
Volume Display
Tip Cone

8. Preparation of media
The media used in the laboratory have to be chosen to suit the nutritional requirements of the species of
organism to be grown. Isolation from a mixture can sometimes be facilitated by the use of media designed
for a special Purpose
NB preparation
. Suspended 2.6 g of nutrient broth in 200 ml of distilled water.
. Stirred it for fully dissolving all components in water.
. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
. Once the nutrient broth has been autoclaved, allow it to cool
NB (nutrient broth) Agar preparation
. Suspended 2.6 g of nutrient broth and 5gof agar powder in 200 ml of distilled water.
. Stirred this mixture for fully dissolving all components.
. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
. Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
. Pour nutrient agar into each plate and leave plates on the sterile surface until the agar has solidified.
. Replace the lid of each Petri dish and store the plates in a refrigerator

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Prepartion of Media
Nutrient Broth
Basically, the nutrient broth is the nutrient agar that lack of the solid at room temperature and are
usually used
to maintain the stocks of microorganisms. In general, they are used to grow fastidious organisms.
Also, you can
enrich your nutrient broth with blood, serum, sugars… etc for special purposes.
Preparation of Nutrient Broth
Add 1.3gm of nutrient broth powder in 100ml of distilled water
Mix and dissolve them completely.
Pour them into the final containers (conical flask)
Sterilize by autoclaving at 121°C for 15 minutes.
Storage condition and shelf life for nutrient broth
You have to store the dehydrated medium at 10-30”C and use before the expiry date on the label.
Once you
prepare the nutrient broth medium, store them at below 25°C.

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Preparation of Nutrient Broth agar
Preparation of Nutrient Broth Agar
Suspend 2.5 gm of nutrient agar powder and 1.3 gm of nutrient broth 100ml of distilled water.
Mix and dissolve them completely.
Sterilize by autoclaving at 121°C for 15 minutes.
Pour the liquid into the petri dish and wait for the medium to solidify. Be sure that you are preparing
the agar in
the clean environment to prevent any contamination.
Once the agar solidifies, the agar is ready to use.
Storage condition and shelf life for nutrient broth
You have to store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Once you
prepare the nutrient agar in the petri dish, store them at 2-8’C.

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Preparation of E.Coli Culture
For 10 minutes we on the UV light in LAF
Took out the culture sample (E. Col) from freezer (20 c) and churned it so that it come into liquid state.
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.
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
In this way we did serial dilution of stock cuture up to 5 times
Then we pour 1 ml of culture from 5th dilution into a 49 ml media, making it 50 ml culture.
Then sealed the culture with wax tape and put prepared culture in orbital shaker and left there for 24hrs.

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Preparation of YPD agar
YPD agar preparation:
YPD media preparation (per 100 ml)Components
Yeast extract 1.049 g
2. Dextrose 2.097 g
3. Peptone 2.032
4. Agar 2.500g
5. procedure
1.Weighed and put 2.5 gm of agar into it.
2.Then vortex it properly.
3.Put this media in autoclave for 15-25 minutes at 121’C.
4.Once the media is autoclaved allow it to cool.
5.Pour this YPD agar media into each petri plates and leave the plates on the sterile surface until the agar
has solidifie
6.Replace the lid of each Petri dish and invert it then store the plates in a refrigerator

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Methylene blue dergradation
Theory
Photocatalytic degradation oxidizes complex organic compounds into small molecular inorganic
substances, such as carbon dioxide and water, under light.
Methylene blue is an oxidation reaction as it oxidizes complex organic compounds into small molecular
inorganic substances, such as carbon dioxide and water, under, light.
Photocatalytic reaction
The rate of reaction can be altered with the help of catalyst and the reaction where catalyst is light are known
as photocatalytic reaction.
Types of photocatalytic reaction:
Homogeneous photocatalytic reaction:Heterogeneous catalysis has the catalyst in a different phase from the
reactants
Heterogeneous photocatalytic reaction:Heterogeneous catalysis has the catalyst in a different phase from the
reactants. Relation between photocatalytic reaction and methylene blue degradation: Methylene blue
degradation is a photocatalytic reaction.

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Procedure
We took the 5 ml stock solution of methylene blue.
Then we made 1500 ul solution by adding 8.4 ml water and 600 ul of
methylene blue along with 18 mg of nanoparticle.
Then we put the solution into shaker for 1 hour.
Then we took two falcon tubes.
In first one we poured 1500 ul of solution and 1350 ul of water along
with 150 l of solution A.
And in second one we poured 1500 ul of solution and 1200 pl of water
along with 150 l of solution A and 150 ul of solution.

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Experiment (Cross-linking)
Cross-linking of polymer (gelatin) with TGA.
Objective-The objective of this experiment is to cross-link a polymer and observe the
changes in the physical properties as a result of this crOSs-linking.
Procedure
First took the polymer (milk powder) and weighed it for three concentrations of set-A;
C,=800.0 mg, C2=1200.6 mg, C=1500.8 mg)
Then prepare the stock solution of sample by dissolving polymer into 10 ml of RO water.
Prepare the stock solution of transglutaminase (10.2 mg/ml) in water.
Pour the TGA in sample solution such that 1600 ul in Cq, 2400 ul in C2 and 300 pl in C3
Now wait and observe the gel time.
Result:
The experiment gets failed after observing it after 10 minutes.
The experiment gets failed after observing it after 25 minutes.
Gel was observed after 45 minutes.

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