An Assignment on

Microbial Culture Media

Course Title: Bioprocess Technology
Course Code: MBIO-5105
Submitted By Submitted To
Name: Farhana Ferdous Tarin Md. Shaminur Rahman
Roll: MS-210301 Lecturer
Session: 2021-22 Dept. of Microbiology
Dept. of Microbiology Jashore University of Science and
Jashore University of Science and Technology.
Technology.

Submission Date: 30/09/2023

 Microbial Culture Media
Culture media are mediums that provide essential nutrients and minerals to support the growth of
microorganisms in the laboratory.
Microorganisms have varying nature, characteristics, habitat, and even nutritional requirements,
thus it is impossible to culture them with one type of culture media. However, there are also
microorganisms that can’t grow on a culture media at all in any condition – these are called obligate
parasites.
Culturing microorganisms is essential for diagnosing infectious diseases, obtaining antigens,
developing serological assays for vaccines, genetic studies, and identification of microbial species.
Furthermore, it’s also essential for isolating pure cultures, storing culture stock, studying
biochemical reactions, testing microbial contamination, checking antimicrobial agents and
preservatives effect, testing viable count, and testing antibiotic sensitivity.
Culture Media Preparation:
1. Weigh the amount of ingredients powder on weighing machine.
2. Dissolve the ingredients in distilled water.
3. Adjust PH of the medium if needed.
4. Add agar and boiled it to dissolve.
5. Pour the media into flask.
6. Autoclave the media when ingredients fully dissolve.
7. Sterilization is done in autoclave to prevent from contamination, at 121ºC for 15 min at
15lbs.
8. After the autoclave place the media flask in laminar air flow.
9. Sterilize the laminar air flow with 70% alcohol.
10. A bit cools down the media and pours into sterile Petri-plates for solidification.
11. Then sample is ready to spread(spreader) / streak
12. (Inoculation loop) on the medium for identification or isolation of microbes.
13. Sealed the Petri plates with paraffin, label them.
 14. Keep them inverted in incubator at 37ºC for 24hrs.
15. Observe the result next day colonies formation is visible on the media.
Classification and Types of Culture Media
Growing microorganisms in the lab involve mimicking the organisms’ natural habitat or
environment, and this is possible in the laboratory by formulating culture media that meets their
requirements. Therefore, many culture media were developed by scientists according to the
microbial species to be cultured.
The basic media contains a source of carbon & energy, nitrogen source, growth factors, and some
trace elements. Some commonly used media components include peptone, agar, water, casein
hydrolysate, malt extract, meat extract, and yeast extract. In addition, the pH of the medium should
be set accordingly.
However, some additional components or nutrients are added to the media when growing specific
microorganisms.
Defined medium
A defined medium has a known quantity of all ingredients, like carbon source (Glucose or
Glycerol) and nitrogen source (Ammonium salt or Nitrate as inorganic nitrogen). The medium
needs in metabolic, nutritional, and physiological growth experiments.
Undefined medium
This medium has different complex ingredients in unknown quantities, for example- yeast extract,
beef, various salts, and enzymatic protein.
Complex media
This media is other than basal media; it has added ingredients to bring the characteristics of
microorganisms with unique nutrients.
Culture media can be classified in three ways: based on their consistency, nutritional component,
and applications.
A. Classification of culture media based on consistency
1. Solid media: In these media, the agar which is an unbranched long chain of
polysaccharides is added in the concentration of 1.5-2.0%. Most commonly, 1.3% agar is
used to prepare solid media in labs. The agar-containing media solidifies at 37
ºC. Sometimes, in the place of agar, some other inert solidifying agents are used, such as
gellan gum. Solid media are used to grow microorganisms in their full physical form,
prepare bacterial pure cultures, or isolate bacteria to study colony characteristics. The
bacterial growth on solid media varies in appearance as mucoid, round, smooth, rough,
filamentous, irregular, and punctiform. The media is not hydrolyzed by microorganisms
and is free from growth-inhibiting substances. Examples of solid media are blood agar,
nutrient agar, McConkey agar, and chocolate agar.
 2. Semisolid media: This media has 0.2-0.5% agar concentration, and due to the reduced
agar concentration, it appears as a soft, jelly-like substance. It’s mainly used to study the
motility of microorganisms, distinguish between motile and non-motile bacterial strains
(through U-tube and Cragie’s tube), and cultivate microaerophilic bacteria – bacteria on
this media appear as a thick line. Examples of semi-solid media are: Hugh and Leifson’s
oxidation fermentation medium, Stuart’s and Amies media, and Mannitol motility media.
3. Liquid media: These media do not contain any traces of solidifying agents, such as agar
or gelatin, and large growth of bacterial colonies can be observed in the media. Liquid
media are also called broths, they allow for uniform and turbid growth of bacterial strains
when incubated at 37ºC for 24hrs. The media is used for the profuse growth of
microorganisms and fermentation studies. Examples include Tryptic soy broth, phenol red
carbohydrate broth, MR-VP broth, and nutrient broth.
Other than these, there are also biphasic media, which consist of both solid and liquid media. And
sometimes in the place of agar, egg yolk and serum are added to the media as a solidifying agent.
While naturally, these substances are liquid, they are solidified by using heat, and the prepared
media is sterilized using the inspissation technique. Examples are Lowenstein Jensen medium and
Dorset egg medium, which contain egg yolk, and Loeffler’s serum slope, which contain serum.
B. Classification based on the nutritional component
1. Simple media: It’s a general-purpose media that supports the growth of non-fastidious
microbes, and it is primarily used for the isolation of microorganisms. Examples are
nutrient broth, peptone water, and nutrient agar.
2. Complex media: These are media containing nutrients in unknown quantities that are
added to bring about a particular characteristic of a microbial strain. Examples are tryptic
soy broth, blood agar, and nutrient broth.
3. Synthetic media: Synthetic media is a type of chemically defined media and is produced
from pure chemical substances. A defined media refers to a medium having a known
concentration of ingredients, like sugar (glucose or glycerol) and nitrogen source (such as
ammonium salt or nitrate as inorganic nitrogen). It is generally used in scientific research,
and an example is Czapek Dox Medium.
C. Classification of culture media based on application/chemical composition
• Basal media: These are routinely used simple media having carbon and nitrogen sources
that boost the growth of many microorganisms. They are also known as general-purpose
media and are considered non-selective media. The basal media do not require enrichment
sources for the growth of non-fastidious bacteria and are suitable for growing
Staphylococcus and Enterobacteriaceae. They are generally used to isolate microorganisms
in labs or in sub-culturing processes. Examples are nutrient broth, nutrient agar, and
peptone water.
 • Enriched media: This media is prepared by adding additional substances like blood,
serum, or egg yolk in the basal medium. It’s used to grow fastidious microorganisms as
they require additional nutrients and growth-promoting substances.
Examples are chocolate agar, blood agar, and Loeffler’s serum slope. Chocolate media is
used to grow N. gonorrhea while blood agar (which is prepared by adding 5-10% blood by
volume to a blood agar base) is used to identify hemolytic bacteria.
• Selective media: This media allows the growth of certain microbes while inhibiting the
growth of others. It’s an agar-based medium that is used to isolate microorganisms in labs.
Below is a list of common selective media and the bacteria they’re used to culture:
Culture media Inhibiting substances Bacteria
Contains antibiotics; Used for Neisseria
Thayer Martin Agar vancomycin, colistin, and gonorrhoeae
nystatin
Contains bile salts Used for Enterobacteriaceae
MacConkey’s Agar
members
Lowenstein Jensen Medium Addition of malachite green Used for M.tuberculosis
Mannitol Salt Agar Contains 10% NaCl Used to recover S.aureus
Contains 0.0002% crystal Used for Streptococcus
Crystal Violet Blood Agar
violet pyogenes
Thiosulfate citrate bile salts Have elevated pH of about Used for isolating Vibrio
sucrose (TCBS) agar 8.5-8.6 cholerae
Wilson and Blair’s Agar Addition of dye brilliant green Used for recovering S. typhi
Potassium tellurite medium Contains 0.04% Potassium Used to recover C.diphtheriae
tellurite
Pseudosel Agar (cetrimide Contains cetrimide (antiseptic Used to recover Pseudomonas
agar) agent) aeruginosa
Salmonella-Shigella Agar Contains bile salts, brilliant Used for the isolation of
green, and sodium citrate Salmonella.

• Enrichment media: It’s a liquid medium, used to increase the relative concentration of
certain microbes before culturing them on a solid medium plate. It’s used as a broth
medium and inhibits the growth of commensal species of microorganisms (those who live
in close association with each other) in the clinical specimen.
It’s also used in isolating fecal and soil microorganisms. Examples are selenite F broth
which is used to isolate Salmonella typhi from a fecal sample, tetrathionate broth, and
alkaline peptone water.
• Differential or indicator media: It contains certain indicators like dyes or metabolic
substrates in the medium composition which gives different colors to colonies of different
microbial species when they utilize or react with these components.
It allows the growth of more than one microorganism, however, the bacterial colonies are
 differentiated based on their color when a chemical change occurs in the indicator, such as
neutral red, phenol red, methylene blue. Examples are:
• Blood agar: In blood agar, three types of blood cell lysis or hemolysis are
observed: alpha, beta, and gamma hemolysis. It allows the growth of many
microorganisms, however, their ability to lyse blood cells differs, and this helps to
distinguish the bacterial colonies. For example, S. pyogenes completely lyse blood
cells (beta hemolysis), thus causing total clearing of the media around its
colonies. S. pneumoniae partially lyse red blood cells, resulting in a greenish-
colored medium, while gamma hemolytic microorganisms like Enterococcus
faecalis, Staphylococcus saprophyticus, and Staphylococcus epidermidis, can’t
lyse red blood cells, thus causing no color change in the medium.
• Mannitol salts agar: The fermentation of mannitol by Staphylococcus
aureus causes the media to change to yellow, however, coagulase-negative
staphylococci that can’t cause fermentation to appear in pink.
• MacConkey agar: It differentiates the gram-negative bacteria based on their
lactose metabolism. The lactose fermenting bacteria, such as Escherichia coli,
Klebsiella spp, Citrobacter, and Enterobacter forms pink-red colonies, while
lactose non-fermenters, like Salmonella, Shigella, Proteus, Providencia,
Pseudomonas, and Morganella form pale or colorless colonies.
• Thiosulfate citrate bile salts sucrose (TCBS) agar: The media contain sucrose,
which is utilized by ferment microbes and helps to distinguish them from non-
ferment microorganisms. Based on this characteristic, different colored bacterial
colonies are formed on the media that help to identify and distinguish them from
each other. For example, V. cholerae ferment the sucrose and form slightly
flattened yellow colonies having opaque centers and translucent peripheries.
Whereas, V. parahaemolyticus can’t ferment the sucrose and forms green to blue-
green colonies.
• Xylose-lysine-desoxycholate (XLD) agar: Xylose-lysine-desoxycholate agar is a
selective and differential medium used for the isolation and differentiation of
enteric pathogens from clinical specimens. This medium is more supportive of
fastidious enteric organisms such as Shigella. For Salmonella, which contains the
lysine enzyme, this reaction reverts the pH to an alkaline state and the colony
appears to be transparent or red with a black center. A number of other similar
media for isolation of enteric pathogens exist, including xylose-galactosidase
medium, which is more specific for Aeromonas spp.
• Transport media: Transport media are useful for clinical specimens which are required
to be transferred immediately to labs to maintain the viability of potential pathogens and
to prevent overgrowth of commensals or contaminating microorganisms. Some of them are
semi-solid in consistency, and examples include:
 • Sach’s buffered glycerol saline: It’s used to transport feces from patients
suspected to be suffering from bacillary dysentery.
• Cary Blair transport and Venkatraman Ramakrishnan media: Fecal samples
collected from suspected cholera patients are transported using these media.
• Pike’s medium: A throat specimen containing Streptococci is transported using
this medium.
• Anaerobic media: This media is for anaerobic bacteria which require low oxygen levels,
extra nutrients, and reduced oxidation-reduction potential. It is supplemented with hemin
and vitamin K nutrients and oxygen is removed by boiling it in a water bath and sealing it
with paraffin film. Examples are: Thioglycollate broth and Robertson Cooked Meat (RCM)
medium which is commonly used to grow Clostridium spp.
• Storage media: It’s used to store microorganisms for a longer period, examples are chalk
cooked meat broth and egg saline medium.
Types of special purpose culture media
1. Assay media
The media assay vitamins, amino acids, and antibiotics. Example- Antibiotic sensitivity test the
media used is Muller-Hinton agar has 1.7% agar for better diffusion of antibiotics. It also contains
starch, which absorbs toxins released by bacteria. In this media plate Zone of inhibition is seen
around antibiotics.
2. Minimal media
Principal of minimal media
Minimal media is a defined medium with different compositions depending on microorganisms
cultured. It contains a carbon source like sugar/succinate and inorganic salts like magnesium,
nitrogen, sulfur, phosphorus. Carbon is a source of energy; magnesium and ammonium salts are
the sources of ions for metabolism stimulation. Phosphate is a buffering agent.
The growth comparison of microbe culture and mutant forms- Minimal media and supplementary-
minimal media- allow the differentiation of wild-type and mutant cells.
Use- The selection of recombinants, for the growth of wild-type microorganisms.
3. Fermentation media
The media is for optimum microorganisms. Fermentation media produce high yields of the
product; media provide energy and nutrients for growth, and medium gives the substrate for the
synthesis of products in the fermentation.
Fermentation media contains major and minor components-
Major components – Carbon and nitrogen for energy.
Minor components- This contains inorganic salts, growth factors, vitamins, buffer, anti-foaming
agents, dissolved oxygen, gases, growth inhibitors, enzymes.
The nutrients in fermentation media depend on the organism and type of fermentation process.
Two uses-
Growth media
It has low nutrients and creates raw material for further fermentation.
Fermentation media
It has high nutrients and creates end products.
Example- The yeast requires 1% carbon, but the fermentation of alcohol, demands 12-13% carbon
in the medium.
Role of fermentation media
The media has a high level of nutrients, microorganisms, and optimum conditions. During the
incubation period under optimum conditions, microorganisms undergo metabolism. Fermentation
organisms become hyperactive due to nutrients being in high quantities and, the result is nutrients
getting consumed, media partially degraded.
The waste effluent is the output product. The death of cells occurs if substrate-level reaches the
inhibitory concentration and excess substrate causes them to inhibit vital enzymes. Excess
substrate increases osmotic pressure and disturbs enzymatic activity in cells. Microbes release
excess substrate as partially digested fermentation media and convert it into the insoluble inert
compound as reserve food, which is harmless to cells. Example- yeast extract, beef extract,
BMGY.
Application of culture media
• To culture microbes.
• To identify the cause of infection.
• To identify characteristics of microorganisms.
• To isolate pure culture.
• To store the culture stock.
• To observe biochemical reactions.
• To test microbial contamination in any sample.
• To check antimicrobial agents and preservatives effect.
• To observe microbe colony type, its color, shape, cause.
• To differentiate between different colonies.
 • To create antigens for laboratory use.
• To estimate viable count.
• To test antibiotic sensitivity.
Limitations of culture media
• Risk of cross-contamination.
• High skill required for optimal results.
• Increased drying out of media can occur.
Conclusion
Culture media is a source of nutrients and growth factors required for the growth of
microorganisms and even plants in laboratory conditions. Every organism has different nutritional
requirements based on its habitat or living conditions. Therefore, a single formulation of culture
media can’t be used to grow all organisms in labs.
Many types of culture media have been developed by scientists to grow selective or desired
microorganisms. These are classified based on their nutrient composition, consistency, and
application or use in life science laboratories.
Culture media serve several purposes in labs like isolating specific strains of microorganisms,
identifying disease-causing pathogens, preparing pure culture of a microbial species,
distinguishing bacterial species, and studying their responses to certain antibiotics.
Thus, before deciding which culture media to use, it is critical to determine the purpose of your
study and in some cases the type of microorganism you want to study. This narrows down your
choices and helps you to choose which media is best for your experiment, without wasting your
time and effort.

References:
1. Tankeshwar Acharya (2021). Bacterial Culture Media: Classification, Types, Uses.
Retrieved from https://microbeonline.com/types-of-bacteriological-culture-medium/.
2. Fatima Aiman (2022). Microbial Culture Media- Definition, Types, Examples, Uses.
Retrieved from https://microbenotes.com/types-of-culture-media/#application-of-culture-
media.
3. Rao Sridhar. Bacterial Culture Media. Retrieved
from https://www.microrao.com/micronotes/pg/culture_media.pdf.
4. Aryal Sagar (2022). Salmonella Shigella (SS) Agar- Composition, Principle, Preparation,
Results, Uses. Retrieved from https://microbenotes.com/salmonella-shigella-ss-agar/.