microbiology bacterial culture media

1. Bacterial
Culture
Media
Presented By: Group 4, Pharm-D batch: IV

2. 01
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Table of contents
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Introduction (M.Umar) Tryptic Soy Agar
( Syeda Faiza Pirzada)
Eosin Methylene Blue
Agar (Saniya Ilyas)
Sheep Blood Agar
(Rizwan and Waseem)
Macckonkey Agar
(Noman and Usama)
Mannitol Agar (Sarah
Bibi)
Hemolytic Reactions
(M.Shahid)
Thayer Martin Agar
(Usman Afzal)

3. What is Bacterial Culture?
• Bacterial culture is a method, that allows the multiplication of
bacterial cells in or on a culture medium under controlled
laboratory conditions.

4. What is Culture Media?
• Culture medium or growth medium is a liquid or
gel designed to support the growth of
microorganisms.
Culture media contain nutrients, energy sources, growth-promoting
factors, minerals, metals, buffer salts, and gelling agents.

5. Frequently Used Terms
Agar:
A gelatinous material derived from algae, specifically used as a culture medium
of bacteria.
Fastidious Bacteria:
Fastidious bacteria require special nutritional supplements and conditions to
grow.
Non-Fastidious Bacteria:
Bacteria which do not need such special nutritional supplements or conditions.

6. Types of Mediums for Bacterial Culture:
Basal media / General-Purpose Media:
Basal media also called general-purpose media are basically simple media that support the
growth of most non-fastidious bacteria. Peptone water, nutrient broth, and nutrient
agar (NA) are considered basal media. These media are generally used for the primary
isolation of microorganisms.
Enriched Media:
Addition of extra nutrients in the form of blood, serum, egg yolk, etc. to the basal medium
makes an enriched medium. Enriched media are used to grow fastidious bacteria.

7. Selective Media:
Selective medium is designed to suppress the growth of some microorganisms
while allowing the growth of others.
Differential Media:
Differential media contain compounds that allow groups of microorganisms to be
visually distinguished by the appearance of the colony or the surrounding media,
usually on the basis of some biochemical difference between the two groups.

8. ● Mueller–Hinton agar containing 5% chocolate sheep blood and antibiotics
is known as Thayer–Martin agar (or Thayer–Martin medium, or VPN agar).
Because the medium hinders the development of most other
microorganisms.
● it is primarily used for growing and isolating pathogenic Neisseria bacteria,
such as Neisseria gonorrhoeae and Neisseria meningitidis. When cultivating
Neisseria meningitidis, a simple chocolate agar is utilised to start with a
generally sterile bodily fluid (blood or CSF). Thayer–Martin agar was first
created in 1964, and a more refined version was published in 1966.
Thayer martin agar:
*USMAN AFZAL*

9. ● It generally comprises the antibiotics listed below, which make up the VPN
acronym.
● Vancomycin, which is able to kill most Gram-positive organisms, although
some Gram-positive organisms such as Lactobacillus and Pediococcus are
intrinsically resistant.
● Polymyxin, commonly known as colistin, is an antibiotic that is used to kill
most Gram-negative bacteria except Neisseria meningitidis. However,
certain Gram-negative bacteria, such as Legionella, are resistant to it.
● Nystatin is a fungicide that can destroy the majority of fungus.
● Trimethoprim prevents Proteus spp. from swarming.
Component:

10. ● A comparison of two types of culture medium for
growing Neisseria gonorrhoeae bacteria.
● Note that the non-selective chocolate agar medium
on the left allowed for the growth of organismal
colonies other than those of Neisseria gonorrhoeae
due to its composition, whereas the selective Thayer–
Martin medium on the right, which contains
antimicrobials that inhibit the growth of organisms
other than N. gonorrhoeae, shows no overgrowth but
is positive for N. gonorrhoeae bacteria,
Testing for Neisseria gonorrhoeae:

11. ● Eosin Methylene Blue (EMB) agar is a differential microbiological
medium, which slightly inhibits the growth of Gram-positive
bacteria and provides a color indicator distinguishing between
organisms that ferment lactose (e.g., E. coli) and those that do not
(e.g., Salmonella, Shigella).
● The medium is important in medical laboratories to distinguish
gram-negative pathogenic microbes in a short period of time.
Eosin-methylene blue agar
*SANIYA ILYAS*

12. Composition of EMB agar
Ingredients Gms/liter
Peptic digest of animal tissue 10.000
Dipotassium phosphate 2.000
Lactose 5.000
Sucrose 5.000
Eosin – Y 0.400
Methylene blue 0.065
Agar 13.500

13. ● EMB agar is characterized by the presence of a combination of the
two dyes eosin and methylene blue in the ratio of 6:1.
● Peptic digest of animal tissue serves as a source of carbon, nitrogen,
and other essential growth nutrients.
● Lactose and sucrose are the sources of energy by being fermentable
carbohydrates.
Principle of EMB agar

14. ● EMB agar is useful in differentiating gram positive and gram-
negative bacteria.
● It helps in the isolation and differentiation of enteric bacilli and
gram-negative bacilli.
● It differentiates microorganisms in the colon-typhoid-dysentery
group.
● It also helps in the isolation and differentiation of lactose
fermenting and non-lactose fermenting enteric bacilli.
Uses

15. Result interpretation on EMB agar
Organisms Growth
Escherichia coli
Blue-black bull’s eye; may have a green
metallic sheen
Pseudomonas aeruginosa Colorless
Enterobacter aerogenes Good growth; pink, without sheen
Klebsiella pneumoniae Pink, mucoid colonies

16. ● Mannitol salt agar or MSA is a commonly used selective and differential
growth medium in microbiology. It encourages the growth of a group of
certain bacteria while inhibiting the growth of others.
CHARACTERISTICS:
 Mannitol Salt Agar (MSA) is a selective and differential medium. The high
concentration of salt (7.5%) selects for members of the genus
Staphylococcus, since they can tolerate high saline levels. Organisms from
other genera may grow, but they typically grow very weakly.
MANNITOL SALT AGAR
*SARAH BIBI*

17. ACTIVE INGREDIENTS:
Ingredients Gms/Litre
Proteose peptone 10.0
Sodium chloride 75.0
D- mannitol 10.0
Beef extract 1.0
Phenol red 0.025
Agar 15.0

18. ● It is used for the selective isolation and differentiation
of Staphylococcus aureus from clinical samples.
● It is also used for the enumeration of staphylococci in
food and dairy products. This medium is also included in
the Bacteriological Analytical Manual for cosmetics
testing.
USES:

19. ● Mannitol Salt Agar (MSA) is used as a selective
and differential medium for the isolation and
identification of Staphylococcus aureus from
clinical and non-clinical specimens.
INSTRUCTIONS:
● Suspend 111 g of the powder in 1 liter of
distilled or deionized water. Mix well. Heat to
boil for 1 minute shaking frequently until
completely dissolved. Sterilize in autoclave at
121°C for 15 minutes.
RESULT:

20. ● (TSA), called Soybean-Casein Digest Agar Medium by the United States
Pharmacopeia is a general-purpose nonselective growth medium that
supports the growth of most Gram bacteria. -negative and non-fastidious
Gram-positive as well as many yeasts and moulds.
● This medium is recommended for use in the cultivation, storage,
maintenance and transport of pure cultures of microorganisms (not
intended for use in the diagnosis of disease or other conditions in humans
TRYPTIC SOY AGAR
*SYEDA FAIZA PIRZADA*

21. ● Suspend the components, dehydrated powder, in water (40 grams in 1000 ml of
purified/distilled water). The medium is boiled for a few seconds until the ingredients
are completely dissolved. Mix well and finally sterilize by autoclaving at 121°C for 15
minutes.
● Cool to 45-50°C and dispense aseptically into sterile Petri dishes. Date the medium
and assign it a lot number.
● Trypticase soy agar is prepared with casein and soy peptones: the combination of
casein and soy peptones makes the medium nutritious by providing organic nitrogen
in the form of amino acids and polypeptides. Sodium chloride maintains osmotic
balance. Agar is the solidifying agent.
● Formula may be adjusted and/or supplemented as required to meet performance
specifications.
Composition of TSA agar

22. ● Tryptic soy agar (TSA) is mainly used as an initial growth medium
for the purposes of observing colony morphology, developing a
pure culture, and achieving sufficient growth for further
biochemical testing and culture storage. TSA slants are being used
to store and ship bacterial cultures.
USES OF TSA

23. ● MacConkey agar (MAC) was the first solid differential media to be
formulated which was developed at 20th century by Alfred Theodore
MacConkey. MacConkey agar is a selective and differential media used for
the isolation and differentiation of non-fastidious gram-negative rods,
particularly members of the family Enterobacteriaceae and the
genus Pseudomonas.
MAcCONKEY AGAR
*NOMAN SHAH and USAMA*

24. ● Suspend 49.53 grams of dehydrated medium in 1000 ml purified/distilled
water.
● Heat to boiling to dissolve the medium completely.
● Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
● Cool to 45-50°C.
● Mix well before pouring into sterile Petri plates.
Preparation of MacConkey Agar

25. ● MacConkey agar is used for the isolation of gram-negative enteric bacteria.
● It is used in the differentiation of lactose fermenting from lactose non-
fermenting gram-negative bacteria.
● It is used for the isolation of coliforms and intestinal pathogens in water,
dairy products and biological specimens.
Uses of MacConkey Agar

26. ● Sheep Blood Agar Base was developed to meet the demand for an
especially nutritious blood agar base which would permit the maximum
recovery of organisms without interfering with their hemolytic reactions
when used with sheep blood.
SHEEP BLOOD AGAR
*RIZWAN and WASEEM*

27. ● Suspend 28 g of nutrient agar powder in 1 litre of distilled water.
● Heat this mixture while stirring to fully dissolve 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.
● When the agar has cooled to 45-50 °C, Add 5% (vol/vol) sterile defibrinated
blood that has been warmed to room temperature and mix gently but well.
● Avoid Air bubbles.
● Dispense into sterile plates while liquid.
Preparation of Blood Agar

28. ● Blood Agar is a general purpose enriched medium often used to
grow fastidious organisms
● To differentiate bacteria based on their hemolytic properties (β-
hemolysis, α-hemolysis and γ-hemolysis (or non-hemolytic)).
Uses of Blood Agar

29. *MUHAMMAD SHAHID*

30.  Alpha-haemolysis (α-homolysis) is a partial or “green” homolysis associated with
reduction of red cell haemoglobin. Alpha homolysis is caused by hydrogen peroxide
produced by the bacterium, oxidizing haemoglobin to green methaemoglobin.
Example
Streptococcus pneumoniae

31. • Beta-haemolysis (β-haemolysis), sometimes called complete haemolysis, is a complete
lysis of red cells in the media around and under the colonies: the area appears lightened
(yellow) and transparent. Streptolysin, an exotoxin, is the enzyme produced by the
bacteria which causes the complete lysis of red blood cells
Example
Streptococcus pyogenes

32.  Gamma haemolysis is a lack of haemolysis in the area around a bacterial colony. A blood
agar plate displaying gamma haemolysis actually appears brownish. This is a normal
reaction of the blood to the growth conditions used (37° C in the presence of carbon
dioxide).
Example
Staphylococcus epidermidis

33. Any Question !