To prepare LB medium, inoculate and maintain (spread plate,

streak plate, pour plate & serial dilution methods) E. coli cultures
LB media
• In order for bacteria to be successfully cultured, they must be grown in the appropriate media. LB, also known as
Lysogeny broth, is a nutrient rich broth that is a standard for culturing Escherichia coli, as it allows for quick growth
and high yields.
• Therefore, the proper preparation of LB will be crucial to maintaining our bacterial cultures.
• Furthermore, addition of agar to LB broth creates a gel for bacteria to grow upon, and is therefore used for plating
bacterial cultures on petri dishes.
• Different components in the LB media have different role. Yeast extract powder provide nitrogen source, vitamin and
growth factor; sodium chloride maintains balanced osmotic pressure; agar is the coagulant of medium.

LB media Composition: Tryptone (1%), yeast extract (0.5%), NaCl (0.5%), agar (1%), NaOH (pH 7-7.5)
Materials
Reagents
• 5g Bacto-tryptone
• 2.5g yeast extract
• 5g NaCl
• 7.5g agar (Only necessary if making LB agar plates)
• 500mL of dH2O (distilled water)
Equipment
• 1L Pyrex bottle
• 1L graduated cylinder
• Filter paper and spatula
• Stack of sterile plates
• Bunsen burner/ethanol burner
• 70% EtOH wash bottle
Making the LB broth
This part can be carried out at a regular lab bench.
• Obtain a clean 1L pyrex bottle
• Obtain a graduated cylinder with 500mL of dH2O and add to the bottle. Record the amount added.
• Using filter paper, separately measure out 5g of NaCl, 5g of Tryptone, and 2.5g of yeast extract on a scale and add them to
the bottle. Swirl the bottle in a circular motion to mix. Remember to re-calibrate your scales in between measurements.
• If you are making LB agar plates, weigh and add 7.5g of agar and swirl to mix. Record the amount added.
• Note the contents do not necessarily need to be completely in solution before autoclaving.
• Autoclave it at 121 degree C for 15 minutes at 15 psi pressure
• After inoculation with a bacterial suspension, incubated at 37 degree C for 24-48 hrs on a shaker.
• In case of LB agar plates, petri plates sealed with parafilm, incubated at 37 degree .C for 24-48 hrs in an inverted position.
• The bacterial culture or plates stored at 4 degree C for further use
Pouring the plates (for LB agar)
While pouring the plates, it is crucial to maintain a sterile environment.
• Sterilize the workspace with 70% EtOH before depositing your materials. Light the Bunsen burner.
• Obtain a stack/roll of empty plates. The plates should still be in their plastic sleeve/wrapping, as they should be sterile.
Don’t throw out the wrapping as it can be used to store the plates. It is essential that you minimize any chance of
contaminating the plates. Make sure that you open the package at the top and expose the plates as minimally as possible.
• Once you take the plates out, store them upside down on your lab bench. Label the plates
• Allow the LB media to cool before pouring. The LB will start to settle at ~30°C.
• If you are preparing selective media, add antibiotic to the mixture. Swirl the flask in a circular motion to mix.
• Take an empty plate and open it slightly. You do not need to open it all the way to pour the agar.
• Pour agar until 2/3 of the plate has been covered, or approximately half of the plate has been filled when viewed from the
side. Pour the agar slowly to prevent the formation of bubbles. Swirl the plate in a circular motion to distribute the media
evenly on the plate.
Serial Dilution

• In microbiology, serial dilution is commonly used to determine the number of microorganisms in a sample with an unknown
concentration. An example would be having a bacterial culture and needing to estimate how many bacteria are present. If
you were to count them individually without using serial dilution, it would be almost impossible due to the extremely high
numbers. So instead, a small portion of the culture is diluted until the concentration is at a more manageable level, this
allows for more accurate and manageable counting. The primary purpose of serial dilutions in microbiology is to
systematically reduce the concentration of bacteria, viruses or other microorganisms to a reasonable level which allows for
precise counting and analysis.

• Serial dilutions work by using a fixed dilution factor at each step. The dilution factor is the ratio by which the solution
concentration is being reduced at each step. The key benefit being that scientists can work with lower concentrations whilst
still maintaining a proportional relationship between original and diluted solutions. The diluted volumes are then used for
spread, pour or streak plate methods. The extent of the dilution is determined by estimating the concentration of cells within
the sample. Common dilution methods are 2-fold or 10-fold dilutions. With a 10-fold dilution, 1 ml of sample is mixed with
9 ml of diluent. The total dilution factor is then calculated by multiplying the dilution factors of each step in the series.
Procedure
• Prepare a set of ten test tube with 20 mL capacity and label them T1- T-10.
• Pipette 9 mL of diluent (water or saline) into each of the 10 test tubes (These 10 tubes can be autoclaved if they are
not previously autoclaved).
• Now take the sample in flask and shake it vigorously.
• Pipette out 1 mL of sample solution from flask and transfer into test tube labeled T1.
• Take 1 mL of sample from test tube T1 and add it to test tube T2. Mix well.
• Take 1 mL of sample from test tube T2 and add it to test tube T3. Mix well.
• Repeat the step 5 and 6 until transfer into tube T10.
• The sample present in the tube T1 to T10 can be transferred into nutrient media for the quantitative analysis of
bacterial population. This serial dilution preparation can also be used for the purification of single colony from the
mixed microbial population.
Limitations of Serial Dilution

• It is important that a serial dilution is conducted with precision and accuracy, as small errors during the dilution steps
could lead to substantial errors in the counting.

• A small mistake such as a pipetting error or miscalculation can change the result by huge margins. This is because as the
method goes on, you could essentially be accumulating the error, therefore this can be seen as a downside to serial
dilutions as human error can never be ruled out.

• There is also a risk of contamination because there is a transfer between each tube.

• However, by addressing these sources of possible error, it can allow you to take the necessary precautions to ensure your
results are as accurate as possible when using a serial dilution.
Spread plate method

Spread plate method is based on spreading a specific volume of diluted liquid specimen over the surface of a solid agar
medium. The principle of the method is to grow distinct colonies after incubation. Only 0.1 ml of the sample is usually
used. The spreading can be done either by a bent glass rod, glass beads or cotton swabs
 Procedure
Materials 1. Preparation: Prepare all the required materials, sterilize the
workspace, put on the required PPE, and bring samples and
• Liquid sample or suspension of solid samples
media to room temperature.
• Pre-solidified solid culture media plates (Petri
2. Sample preparation: Use serial dilution for the liquid sample
dish)
to reduce microbial load. If you are using a solid sample,
• Sterile test tubes and distilled media
dissolve it in a sterile solvent and then dilute.
• Micropipette
3. Labeling: Write down the name of the sample along with the
• Spreaders (bent glass rod, glass beads, or cotton
dilution factor and any other necessary information in the
swabs)
bottom edge of the Petri dish.
• Ethanol (70%) and Bunsen burner
4. Spreading the sample: Dispense 0.1 ml of the sample using a
for sterilization
micropipette in the center of the agar medium. Spread the
• Personal protective equipment (PPE)
sample evenly using either the sterilized glass rod
• Other laboratory facilities, such as laminar
5. Incubation: Allow the sample to be absorbed by agar medium
flow hood
and incubate the plate in an inverted position under optimal
conditions.
Observations
• After the incubation, examine the plates.
• Each colony represents one viable microbial cell
• If the colony morphologies are identical, it suggests that the sample contained one type of microbial species.
• On the other hand, if the colony morphologies vary, it is likely that the sample contained a mix of microbial species
Streak Plate Method
Streaking is a technique used in microbiology for the isolation of single colonies of microorganisms, either from a mixed
species or from the same species. This technique is mostly applicable to bacteria but is also used for some yeasts.
Objectives of Streak Plate Method
1. To obtain a pure culture of bacteria from a mixed culture
2. To obtain well-isolated colonies

The streak plate method is based on dilution during the process of mechanical spreading of inoculum over the surface of
solidified culture media in order to obtain well-isolated colonies of the sample at the terminal streaks.
Microbial cultures are of two types:
[Link] Culture: Two or more species of microorganism or bacteria are present in mixed culture.
[Link] culture: Single species of microorganism or bacteria is present in Pure culture. In nature, bacteria exist in mixed
populations. Our aim is to isolate pure culture containing single bacteria from mixed culture of many bacteria. The streak
plate method is widely used to isolate pure culture.
Quadrant Streaking
It is the most commonly used and the most preferred method where four equal-sized sections of the agar plate are streaked. It
is also referred to as the “four-quadrant streak” or “four sectors” or “four-way streak” method.
In this method, each plate is divided into four equal sectors and each adjacent sector is streaked sequentially. The sector which
is streaked first is called the first sector or the first quadrant, and it has the highest concentration of inoculum. Gradually the
second, third, and fourth quadrants will have diluted inoculum. By the time the fourth quadrant is streaked, the inoculum is
highly diluted giving rise to isolated colonies following the incubation.
Materials Required: The following apparatus is required for the streak plate technique.
1. Bunsen Burner
2. Nichrome Wire Loop
3. Sterile Nutrient Agar plates
4. Mixed Culture of bacteria
The general procedure of the streak plate method can be summarized as:
1. Arrange all the requirements, put on the PPE, sterilize the work surface, and allow all the samples and media to come to
room temperature if were refrigerated.
2. If the sample is very concentrated then dilution can be helpful to get the isolated colonies. (But it is not compulsory as
the sample will be diluted during the streaking process.)
3. Sterilize the inoculating loop by flaming and allow it to cool. Pick a small portion of the isolated colony. (if the sample is
in the suspension, then take a loopful of the sample)
Precautions
• Ensure all equipment and media are sterilized to prevent contamination.
• Handle cultures aseptically to maintain sterility.
• Incubate plates in an inverted position to prevent condensation on the agar surface