Cell biology and genetic KD/HNDBDS/05/13

MEDIA PREPARATION

Student Name - K.D.R.G.M.Weerasingha

Student Id - KD/HNDBDS/05/13
Lecture in Charge - Mr. N.Roshan
Batch Number - 05
Cell biology and genetic KD/HNDBDS/05/13

Date - 12/03/2020

Title - Media Preparation

Objectives - To study how to prepare media and how to sterilize it.

Introduction –

There is a substance prepare at the laboratory which contains all the nutrients and physical
conditions for microorganism growth is called culture media.
In the natural residence microorganisms observation is a difficult thing because we use
growing techniques before observing microbes.

The requirements for microbial growth

The requirements for microbial growth can be divided into two main categories as physical
and chemical,

Physical requirements

 Correct Temperature range

 Correct pH range
 Correct osmotic pressure

Chemical requirements

 sources of carbon
 nitrogen
 sulfur
 phosphorus
 oxygen
 trace elements
 organic growth factors

These requirements should be contained in the culture media. These Physical requirements
ranges and nutrients percentages are depending on the type of microbiome we hope to grow.
Cell biology and genetic KD/HNDBDS/05/13

The culture media should be free from other microorganisms therefore we use Sterile
Conditions. The commonly use Sterile Conditions is autoclave sterilization. Put the media for
15 minutes in the autoclave at 15 pounds of pressure and 121 °C For this sterilization.

Culture media classification.

Classification based on the physical state

1. Solid media –

Agar is the most commonly used solidifying agent. it is a golden yellow powder,
prepared from seaweeds this can be use growth of bacteria. Agar percentage in solid
media is 1.5 – 2 % of total weight.

Figure 1
e.g. - nutrient agar

2. Semi-solid media –

Such media are soft they are useful in demonstrating bacterial motility and separating
motile from non-motile strains. These Media contain 0.5% or less agar.

Figure 2
Cell biology and genetic KD/HNDBDS/05/13

3. Liquid media –

Sometimes broth can be used as media. These Media do not contain agar

Figure 3
e.g. – nutrient broth

Classification based on chemical contents

1. Synthetic/defined media -

Specially prepared media form pure chemical substance for research and it has a well-
known composition.
e.g. – peptone water

2. Complex media -

The media has ingredients that exact components are difficult to estimate.
e.g. – blood agar

Classification based on function

1. General-purpose media -

These Media use for the growth of all microorganisms.

Cell biology and genetic KD/HNDBDS/05/13
Figure 4

e.g. – nutrient agar, nutrient broth

2. Enriched media -

Solid media-rich in nutritional substances, that improve the growth of

microorganisms.

Figure 5
e.g. – blood agar

3. Selective media –

These Media allow the growth of specialized microorganisms. Prevents others'

growth.

Figure 6
e.g. – McConkey's agar, TCBS agar

4. Enrichment media –
Cell biology and genetic KD/HNDBDS/05/13
Liquid media increase the concentration of pathogenic microorganisms. Enrichment
media are liquid media that also serves to inhibit commensals in the clinical specimen.
e.g. – selenite f broth

Figure 7
5. Differential agar –

These can be used to differentiate between different microorganisms due to colors

resulting from metabolic reactions.
e.g. – mannitol salt agar

Figure 8

6. Transport media –

The media is used to preserve a clinical sample until the processing.

e.g. – Cary Blair transport medium

7. Anaerobic media –

Used for anaerobic bacteria growing.

e.g. – Robertson cooked meet media

8. Assay media –

Used for the assay of vitamins, amino acids, and antibiotics.

Cell biology and genetic KD/HNDBDS/05/13
e.g. – antibiotics assay media

Materials –

Equipment –
 Analytical balance
 Wire gauze
 Bunsen burner
 pH meter
 weight boat
 autoclave
 tripod stand
 spatula
Glassware –
 petri dish
 graduated cylinder
 conical flask
Consumables –
 Distilled water
 Nutrient agar

Methodology –

First, the nutrient agar bottle was taken, and read the label.

Figure 9

It was notified 28g of powder is wanted to make 1L of media, there for 5.6g of powder was
wanted to make 200 mL media.

Next 5.6 g of nutrient agar was measured by using an analytical balance.

Cell biology and genetic KD/HNDBDS/05/13

Figure 10

After that 200 mL of distilled water was measured by using a graduating cylinder. After it
was transferred to the conical flask.

Agar powder was added to the conical flask and abject the pH 7.4.

The wire mesh was placed on the temp and place the cone flask on it and heat it with a
Bunsen burner.

Figure 11

This solution was heated till all the agar was fully dissolved.

After that, autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
Cell biology and genetic KD/HNDBDS/05/13

Figure 12

The Agar mixture was taken out and let cool down.

Figure 13

Finally, the mixture added to the petri dish, and when seems to be solidify put them in the
refrigerator until use.

Observation –

Figure 14

The color of agar plates will be light amber and they are free from microorganisms.
Cell biology and genetic KD/HNDBDS/05/13

Discussion -

To get the readings more accurate, agar was transferred to the weight boat by a spatula and
while the readings were taken the glass of analytical balance was closed to minimize the
affection of wind.

Some faults can occur because of incorrect pH, incomplete solution, the incorrect weighting
of ingredients or incorrect volume of water to dissolve the media, overheating through
prolonged sterilization, incomplete solution of the medium, poor quality water.

Media should be stored in a cool place to prevent evaporation.

Conclusion –

From this practical can be got the basic ideas about culture media classification. At this
practical simple media was made. That was nutrient agar media, finally, sterilize it and stored
it correctly. After doing this practice can be learned how to prepare a culture media correctly.

References -

Tortora, J., and Funke, R. and Case, L. (2019). Microbiology an introduction. Thirteenth
edition. Boston: Pearson.

Al-tameemi, h., (2019). Medical microbiology laboratory (culture media classification).

[online] Available from :< https://www.slideshare.net/HusseinAltameemi2/medical-
microbiology-laboratory-culture-media-classification > [accessed 29 march 2020].