1. Define a culture medium and give its general purpose and importance.

A culture medium, also known as a growth medium, is a liquid or gel that aids in the
development of microorganisms. There are several types of media that may be used to grow various
types of cells. Most microbiological experiments require culture media in order to create clean cultures,
develop and count microbial cells, and nurture and select microorganisms. A medium that is rich in
nutrients and can be used by almost any microorganism to grow. Allows a wide range of microorganisms
to flourish.

Source:

Sandle, T. (2014). Assessment of Culture Media in Pharmaceutical Microbiology. Retrieved 14

September 2021, from https://www.americanpharmaceuticalreview.com/Featured-Articles/163589-
Assessment-of-Culture-Media-in-Pharmaceutical-Microbiology/

2. How would you ensure that the culture media are free from contamination? Give examples of
precautionary measures.

During cell culture, aseptic method or sterile technique is utilized to avoid contamination of
sterile medium and equipment. When working with live cell cultures and the reagents/media that will
be utilized for such cultures, sterile procedure should always be applied. This approach entails the use of
flame to destroy contaminating organisms as well as a general manner of operation that minimizes
contamination of sterile media and equipment.

When dealing with living creature cultures, it's critical to keep the surroundings where cells are
grown and controlled as free of other species as possible. This necessitates limiting the exposure of
sterilized culture medium containers to outside air and using a flame to “re-sterilize” container lids and
rims. This entails passing rims and lids through a Bunsen burners flame to destroy germs that come into
touch with such surfaces.

In general, sterile method is a taught state of being, or mantra, in which every use of sterile
material comes with the proviso of taking every effort to keep it as free of contaminants as feasible for
as long as possible. The Bunsen burner is the sterile technician's best buddy when it comes to destroying
germs with heat.

Source:

Wilder, C. (2012). 8 Tips on Handling Microbial Cultures. Retrieved 14 September 2021, from
https://www.genengnews.com/magazine/8-tips-on-handling-microbial-cultures/

3. Table form. Enumerate and discuss the different classifications of culture media according to
physical state, composition, use, or purpose. Give example of each.

Classification based physical state

Type Description Uses Example

Solid medium Solid in nature due to Used to isolate bacteria Blood agar, chocolate
agar concentration of agar, MacConkey agar
about 1.5-2.0%.
Colonies grow by
 adhering to the
physical surface
Semi-solid medium Soft consistency with Used to find bacterial Stuart's and Amie's
agar concentration of mobility and culture media
0.5% microaerophilic
bacteria
Liquid or broth Solidifying agents are Used in fermentation Nutrient broth
medium absent and hence the tests
medium is liquid in
nature
Classification based on composition

Type Description Uses

Simple or basal media Maintains normal grow of bacterial culture Used to culture and maintain Staphylococc
Enterobacteriaceae

Defined or synthetic Chemical composition used in the medium is Used in studying metabolism and n
media known or defined. requirement of microbes

Complex or semi- Contains some chemicals with unknown Used for faster growth of microbes
synthetic media composition complicated needs.

Classification based on use or purpose

ype Description Uses Example

Supportive or Supports minimal growth of bacteria Used for initial microbial Tryptic so
maintenance isolation
media

Selective and Helps growth of only specific organism by enrichment Used to select and culture Thayer M
enrichment of specific nutrients and inhibits growth of other only specific bacteria and Agar(Ente
media organism by selection. Selection factors included are remove contamination alkaline p
pH, antibiotics, dyes, etc.

Enriched media Enriched with nutrients like egg yolk, serum, blood etc Used in fast bacterial growth Blood aga
which helps fast growth of colonies

Differential or Allows differentiating species in same culture using Differentiation of microbes Blood aga
indicator media dyes or metabolites. using color is easy. white- n
mannitol
 Transport media Contains components that prevents pathogen Used to transport microbe Stuart's a
contamination and favor minimal bacterial growth sample by preventing
contamination and
desiccation

Assay media Contains components that help in biochemical assay  Used in vitamins, amino acids, Antibiotic
and antibiotics assays.

Enumeration Determines bacterial population Used to find bacterial Media for

media population in food products
like milk, water and also soil.

Anaerobic media Produce reduced environment by addition of 1% Specially used to grow Robertson
glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% anaerboes
cysteine, or red hot iron filings for the growth of
anaerobic microbes.

Source:

Culture Media. (2021, January 4). Retrieved September 15, 2021, from
https://bio.libretexts.org/@go/page/9162

MILNE Library. (n.d.). Bacteriological Culture Methods. Retrieved 15 September 2021, from
https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/bacteriological-culture-methods/

4. Table form. Cite 5 examples of culture media commonly used in the bacteriology laboratory and
give its specific purpose.

Culture media Description Purpose

Nutrient Agar Solid medium that contains about 2% Used for maintenance and storage of bacterial colonies
of agar

Blood Agar Contains about 5% of sheep or horse Used as differential media to find beta hemolysis microbe
blood hemolysis microbes like Viridans streptococci (green zone

MacConkey Agar Contains agar, peptones, sodium Selective medium that favors growth of enterobactericea
chloride, bile salt, lactose and neutral
red

Mueller Hinton Contains beef extract, casein Used in antimicrobial drug sensitivity tests and used to m
Agar hydrolysate, agar and starch

Lowenstein- Contains  egg, malachite green and selective media that helps culture Mycobacterium tuberc
 Jensen Medium glycerol

Sources:

Culture Media. (2021, January 4). Retrieved September 15, 2021, from
https://bio.libretexts.org/@go/page/9162

MILNE Library. (n.d.). Bacteriological Culture Methods. Retrieved 15 September 2021, from
https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/bacteriological-culture-methods/

5. What are the proper ways of storing culture media if they are not in use?

Preparation culture medium has a wide range of shelf-life recommendations. At low ambient
temperatures (12-l6°C), screw-capped bottles containing nutritional broth and agar can be kept for six
months. All material must be kept out of direct sunlight. To prevent moisture loss, keep agar plates at 2-
8°C in airtight containers. REMEMBER NOT TO FROST.

 Because fresh material is superior than stored media, avoid storing it for lengthy periods of
time. Some extremely labile beta-lactam selective agents have extremely limited active
lifetimes, therefore media containing them should be utilized within a few days of manufacture.
 Establishing shelf-lives for all produced media and date-stamping the containers or holders
accordingly is acceptable laboratory practice.
 Poor bacteriological performance is frequently caused by moisture loss from agar plates. As a
sterility check, do not preincubate all plates overnight. Only clearly damp plates need to be
dried prior to inoculation.
 Ascertain that all of the plates are incubated in a humidified atmosphere.
 Before inoculation, inspect the prepared medium. Look for symptoms of contamination, such as
uneven filling or bubbles on the agar's surface, color changes, haemolysis, and dehydration signs
including shrinkage, breaking, and volume loss. Any plates or tubes that are faulty should be
discarded.

Source:

The CABRI Consortium. (2013). Laboratory procedures for microorganisms. Retrieved 14

September 2021, from http://www.cabri.org/guidelines/micro-organisms/M203Ap1.html

6. Are there culture media that can have more than one purpose? If so, give 3 examples and
explain why.

Yes.

 Blood agar: differential media , it differentiate alpha/partial (green-brownish) and

beta/complete hemolysis (transperent coloney).
 Mc Conley agar: differential and selective media, inhibit growth of gram positive
bacteria and allow gram negative bacteria to grow and form pink coloney of lactose
fermenting bacteria( eg: E. coli)
 Chocolate agar: enriched media , allow the growth of fastidious pathogenic bacteria.

Source:
 Sanders E. R. (2012). Aseptic laboratory techniques: plating methods. Journal of visualized
experiments : JoVE, (63), e3064. https://doi.org/10.3791/3064

7. What would happen to plates poured with agar that is too hot? Could it still be used?

If plates are poured with agar that is too hot, then the agar would kill the bacteria. Agar is the
support medium for bacterial and fungal culture, as nutrients, antibiotics, salts and various growth
enhancers. It is a dried hydrophilic, colloidal extracted from various species of red algae. Mainly used in
cultures for bacteria and other microorganism. Too hot agar would kill the bacteria as the bacteria
would get denatured.

Source:

Sanders E. R. (2012). Aseptic laboratory techniques: plating methods. Journal of visualized

experiments : JoVE, (63), e3064. https://doi.org/10.3791/3064

8. What would happen to plates poured with agar that is too cool? Could it still be used?

The agar plate need not to be too hot or too cold. It is autoclaved and heated at 55°C to melt
and then cold to 48°C prior to pouring. After pouring it is allowed to set at room temperature and after
cooling plate is placed in fridge at 4°C. If the agar is too cool, the medium may be lumpy once solidified.
Too cold agar becomes clumpy on solidification and this can't be used

Source:

Sanders E. R. (2012). Aseptic laboratory techniques: plating methods. Journal of visualized

experiments : JoVE, (63), e3064. https://doi.org/10.3791/3064

Madigan M, Martinko J, eds. (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall.
ISBN 0-13-144329-1.