Culture Media and culture techniques

1. Culture Media & Culture Techniques
Prof. Dr. Mohammad Minnatul karim
Department of Biotechnology and Genetic Engineering
Islamic University, Kushtia

2.  To study microorganisms we need to isolate
them from their natural habitats.
 By appropriate procedures they have to be
grown separately (isolated) on culture media in
the laboratory and obtained as pure for study.
History
 The earliest media used by Louis Pasteur in
1860.
– Liquid broth made from urine or meat extract
 Liquid medium – diffuse growth
 Solid medium – discrete colonies.

3.  Robert Koch realized the importance of solid
media for the development of pure culture.
– Performed his research on tuberculosis by
growing colonies on potato slices.
 Later he use gelatin as a solidifying agent to
prepare solid media.
– Result not satisfactory because it melts around
30-35oC
– Gelatin is a protein & digested by many bacteria
(Bacteria that capable of producing proteolytic
exoenzyme gelatinase that hydrolyze the protein
of amino acid)

4.  Suggested by Fannie Eilshemius Hesse, wife of
Walther Hesse (who was an assistant to Robert
Koch) that to use agar in culture media.
– Agar melt at 96oC & solidify at 40-45oC
– Not attack by most bacteria
 Julius Richard Petri (Koch’s another assistant)
develop the petri dish in 1887, a easy to use
container for solid culture media.

5. Thus their contribution made possible to isolate
the pure culture of microorganisms and
progress in all area of microbiology

6.  Inoculum : Microorganisms that inserted into
a culture medium (to initiate microbial growth)
or into the body (for immune response).
 Inoculation : Introduction of microbes into
culture medium.
 Culture medium :
Requirement :
Must be sterile
Contain appropriate nutrients
Must be incubated at appropriate temperature
Should be reasonably cheap
Easy to prepare
Should promote a rapid growth

7.  Culture :
 A pure culture contains only one species of
strains
 Mixed culture contains several species of
strains
 Contaminated culture contained unwanted
species of organisms
 Colony : Macroscopically visible
collection of millions of bacteria
originating from a single bacterial
cell on the surface of a solid
media .

8.  An artificial culture media must provide all
the nutritional requirements that
microorganism get from its natural
environment.
 Any culture media must contain:
 Water
 Source of hydrogen and oxygen.
 Essential for the growth of all the
microorganisms.
 Must be free from any chemicals which inhibit
bacterial growth.

9.  Peptone (water soluble)
 Obtained from the protein materials with proteolytic
enzymes.
 Contain a mixture of amino acid, carbohydrates,
mineral salts and polypeptides.
 Sodium choride and other electrolytes
(potassium, iron, calcium, magnesium)
 NaCl2 is essential ingredient of most culture media.
 Sulphates are required as a source of sulphur.
 Phosphates are needed as a source of phosphorus.
 Meat & yeast extract
 Used to enrich media.
 It contains protein degradation products,
carbohydrates, inorganic salts and some growth
factors.

10.  Carbohydrates
 Provide bacteria with carbon and energy
 Help to differentiate the bacteria
 Agar : A gelatinous material derived from cell
wall of red algae, specifically used as a
solidifying agent of culture medium .
Properties of Agar
 Used for preparing solid medium
 2% agar is employed in solid medium
 Obtained from seaweeds.
 No nutritive value
 Not affected by the growth of the bacteria.
 Melts above 95oC & sets at 40-45oC

12. Based on chemical composition :
Simple media / basal media
 Most commonly used in routine laboratory diagnostic
procedure
 Used for primary isolation of microbes.
 Ex: Peptone agar, nutrient agar, nutrient broth
NB consists of peptone, meat extract, NaCl,
NB + 2% agar = Nutrient agar
Synthetic or defined media
– Media prepared from pure chemical substances and
its exact composition is known

13. – Must contain organic compound that serve as a
source of carbon and energy
– Widely used in research
– Ex: peptone water – 1% peptone + 0.5% NaCl in
water
Complex media
– A media that contain some ingredients of unknown
composition.
– Often needed when chemical requirement of
particular microorganisms are unknown.
– Contain peptone, meat extracts and yeast products.
– Ex: Tryptic soy broth, MacConkey agar

14. Based on physical nature :
Liquid media
– No agar.
– Use for inoculum preparation, specially when the
inoculation is suspected to be low.
– Mixed organisms cannot be separated.
– Properties of bacteria are not visible in liquid media
and presence of more than one type
bacteria.
– Ex: Nutrient broth

15. Semi solid medium
 Contain 0.5% agar.
 Soft consistency.
 Used to demonstrate motility of bacteria.
 Ex: SIM medium (Sulphide Indole Motility
medium)
Solid media
– Nutrient material that contains 2% agar as solidifying
agent (plates, slant, deeps).
– Used to separate & isolate mixed
bacteria.
– Colony morphology, pigmentation,
hemolysis can be observed.
– Ex: Nutrient agar, Blood agar

16. Biphasic media
A culture setup contain both liquid and solid
media in the same bottle
The inoculum added to the liquid and when
subculture are prepared, the bottle is simple tilted
to allow the liquid to flow over solid medium.
Agar, egg yolk and serum is used (serum and
egg yolk are normally liquid, they can be made
solid by coagulate using heat).

17. Based on function :
Supportive media
– They support the growth of many microorganisms.
– Called as general purpose media.
– Ex: Tryptic soy broth, tryptic soy agar.
Enriched media
– When blood, serum, egg yolk and other nutrients are
added to supportive media to encourage
fastidious microbes,
it is called enriched
media.
– Ex: Blood agar,
Chocolate agar.
Blood agar Chocolate agar

18. Selective media and enrichment media
– Selective media are used for the growth of only
selected microorganisms.
– The inhibitory substance is added to a selective media
such as, if any microorganism is resistant to a certain
antibiotic, then that antibiotic can be added to the
medium to prevent other cells, which do not
possess the resistance, from growing.
– Enrichment media similar to selective
media but designed to increase the
numbers of desired organisms to
detectable levels.
– Usually culture is in liquid form which
provides favorable environment and
nutrient requirement.

20. Differential media:
 .Also known as indicator media because these contain
certain indicators (e.g. chemicals or dyes) which
changes in color especially when the definite organism
(i.e. the organism of interest) being cultured.
 Unlike selective media which only encourage the
growth of particular microbes, differential media
differentiate between different groups of bacteria.
 Some culture media can serve as both selective and
differential media.
 Eg: cystein lactose electrolyte deficient (CLED) media,
mannitol salt agar (MSA), MacConkey agar and blood
agar .

21. Figure: MacConkey agar (MAC) is a differential media that help
microbiologists to differentiate lactose fermenting bacteria (e.g. Escherichia
coli) which ferments lactose to produce pink colonies on MAC from non-
lactose fermenting bacteria (e.g. Salmonella spp.) which does not ferment
lactose and thus appear as pale or colorless colonies on the growth medium.

22. Indicator media
 These media contain an indicator which
changes its color when a bacterium grows in
them.
 Eg: Blood agar, Christensen’s urease medium

25. Transport media
– Media used for transporting the samples.
– Delicate organisms may not survive the
time taken for transporting the specimen
without a transport media.
– Such media prevent drying of specimen,
inhibit overgrowth of unwanted bacteria.
– Contain only buffer and salt.
– Lack of carbon, nitrogen and organic
growth factor to prevent microbial
multiplication.
– Eg: Stuart’s media (non nutrient semi
solid media),amies media

26. Anaerobic media
– These media are used to grow those bacteria that
required low oxygen.
– Before use the media must be boiled in water bath to
expel any dissolved oxygen and then sealed with
sterile parafin
– Eg: Thioglycolate medium.
Thioglycolate medium

30.  Nutrient Agar is a general purpose, nutrient
medium used for the cultivation of microbes
 Nutrient agar is popular media because it can
grow a variety of types of bacteria and fungi, and
contains many nutrients needed for the bacterial
growth.

31. Composition of Nutrient Agar
0.5% Peptone
 It is an enzymatic digest of animal protein.
 Peptone is the principal source of organic nitrogen
for the growing bacteria.
0.3% beef extract/yeast extract
 It is the water-soluble substances which aid in
bacterial growth, such as vitamins, carbohydrates,
organic nitrogen compounds and salts.

32. 1.5% agar
 It is the solidifying agent.
0.5% NaCl
 The presence of sodium chloride in nutrient
agar maintains a salt concentration in the
medium that is similar to the cytoplasm of the
microorganisms.
Distilled water
 Water is essential for the growth of and
reproduction of micro-organisms and also
provides the medium through which various
nutrients can be transported.

33. Use:
 It is frequently used for isolation and purification of
cultures.
 It can also be used as a means for producing the
bacterial lawns needed for antibiotic sensitivity
tests. In actuality, antibiotic sensitivity testing is
typically performed
on media specially
formulated for that
purpose.

35.  Blood Agar (BA) are enriched medium used
to culture those bacteria or microbes that do
not grow easily, such bacteria are called
“fastidious” as they demand a special,
enriched nutritional environment as compared
to other bacteria.
 It is also a differential media in allowing the
detection of hemolysis (destroying the RBC) by
cytolytic toxins secreted by some bacteria,
such as certain strains
of Bacillus, Streptococcus, Enterococcus,
Staphylococcus, and Aerococcus.

36.  Blood agar can be
made selective for
certain pathogens by the
addition of antibiotics,
chemicals or dyes.
Examples includes
crystal violet blood agar
to select Streptococcus
pyogens from throat swabs, and kanamycin or
neomycin blood agar to
select anaerobes from
pus.
Pseudomonas aeruginosa on Sheep Blood
Agar
Artist: Courtney Toth

37. Composition of blood agar
0.5% Peptone
 It is an enzymatic digest of animal protein.
 Peptone is the principal source of organic nitrogen
for the growing bacteria.
0.3% beef extract/yeast extract
 It is the water-soluble substances which aid in
bacterial growth, such as vitamins, carbohydrates,
organic nitrogen compounds and salts.
1.5% agar
 It is the solidifying agent.
0.5% NaCl
 The presence of sodium chloride in nutrient agar
maintains a salt concentration in the medium that is
similar to the cytoplasm of the microorganisms.

38. Distilled water
 Water is essential for the growth of and
reproduction of micro-organisms and also
provides the medium through which various
nutrients can be transported.
5% Sheep Blood
 Sheep RBCs are most sensitive to the
hemolytic toxins released by bacterial cells thus
causing hemolytic zones around the
colonies over the period of time.

39. Uses of Blood Agar
 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)).

40. MacConkey agar (MAC) 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 inoculated with E. Coli.
The pink indicates that it's lactose positive.

41. Principle of MacConkey Agar
 MacConkey agar is used for the isolation of gram-
negative enteric bacteria and the differentiation of
lactose fermenting from lactose non-fermenting
gram-negative bacteria.
 Pancreatic digest of gelatin and peptones (meat
and casein) provide the essential nutrients,
vitamins and nitrogenous factors required for
growth of microorganisms.
 Lactose monohydrate is the fermentable source of
carbohydrate.

42.  The selective action of this medium is attributed
to crystal violet and bile salts, which are inhibitory
to most species of gram-positive bacteria.
 Sodium chloride maintains the osmotic balance in
the medium.
 Neutral red is a pH indicator that turns red at a pH
below 6.8 and is colorless at any pH greater than
6.8.
 Agar is the solidifying agent.
 Distilled water is essential for the growth of and
reproduction of micro-organisms and also
provides the medium through which various
nutrients can be transported.

44. Uses of MacConkey Agar
 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.

45.  Sabouraud Dextrose Agar (SDA) is
used for the isolation, cultivation, and
maintenance of non-pathogenic and
pathogenic species of fungi and yeasts.
 The pH is adjusted to approximately 5.6
in order to enhance the growth of fungi,
especially dermatophytes (fungi that
require keratin for growth) and to slightly
inhibit bacterial growth in clinical
specimens.

46. Principle of SDA
 Peptone (Enzymatic Digest of Casein and
Enzymatic Digest of Animal Tissue) provide the
nitrogen and vitamin source required for
organism growth in SDA.
 Dextrose is added as the energy and carbon
source.
 Agar is the solidifying agent.
 Distilled water is essential for the growth and
also provides the medium through which various
nutrients can be transported.

47. Uses of SDA
 SDA is primarily used for the selective cultivation of
yeasts, molds and aciduric bacteria.
 The medium is often used with antibiotics for the
isolation of pathogenic fungi from material
containing large numbers of other fungi or bacteria.
 This
medium is
also employed
to determine
microbial
contamination
in food,
cosmetics, and
clinical
specimens.

48. Mueller and Hinton developed Mueller
Hinton Agar (MHA) in 1941 for the
isolation of pathogenic Neisseria species.
Nowadays, it is more commonly used for
the routine susceptibility testing of non-
fastidious microorganism by the Kirby-
Bauer disk diffusion technique.
This type is also commonly used for
susceptibility testing of Campylobacter.

49. Principle of MHA
 Beef extract and Acid hydrolysate of
casein provide nitrogen, vitamins, carbon, amino
acids, sulphur and other essential nutrients.
 Starch is added to absorb any toxic metabolites
produced. Starch hydrolysis yields dextrose, which
serves as a source of energy.
 Agar is the solidifying agent.
 Five percent sheep blood and nicotinamide
adenine dinucleotide may also be added when
susceptibility testing is done on Streptococcus
species.
 Distilled water is essential for the growth of and
reproduction of micro-organisms and also provides
the medium through which various nutrients can be
transported.

50. Uses of MHA
 The major use of Mueller Hinton Agar is for
antimicrobial susceptibility testing.
 It has become the standard medium for the
Bauer Kirby method and its performance is
specified by the NCCLS.
 It can be used to cultivate Neisseria
 It is specified in FDA Bacteriological Analytical
Manual for food
 testing, and
procedures
commonly
performed on
aerobic and
facultative
anaerobic
bacteria.

51.  Salmonella Shigella (SS) Agar is
moderately selective and differential
medium for the isolation, cultivation and
differentiation of Salmonella spp. and
some strains of Shigella spp.
 SS Agar is a modification of the
Desoxycholate Citrate Agar.
 It is recommended for testing clinical
specimens and food testing for the
presence of Salmonella spp. and
some Shigella spp

52. Principle of Salmonella Shigella Agar
 The inclusion of Bile salts, sodium
citrate and brilliant green serve to inhibit gram-
positive, coliform organisms and inhibit
swarming Proteus spp., while allowing Salmonella spp.
to grow.
 Beef extract, enzymatic digest of casein,
and enzymatic digest of animal tissue provide
sources of nitrogen, carbon, and vitamins required for
organism growth.
 Lactose is the carbohydrate present in Salmonella
Shigella Agar.
 Thiosulfate and ferric citrate permit detection
of hydrogen sulfide by the production of colonies with
black centers.
 Neutral red turns red in the presence of an acidic pH,
thus showing fermentation has occurred.

53. Uses of Salmonella Shigella Agar
 It is used as a selective and differential medium for
the isolation of Salmonella and
some Shigella species from clinical and non-
clinical specimens.
 This medium is not recommended for the primary
isolation of Shigella.
 It was also developed to aid in the differentiation of
lactose and
non-lactose-fermenters
from clinical specimens,
suspected foods, and
other such samples.

54.  Xylose Lysine Deoxycholate (XLD) agar
is both a selective and differential
medium for the isolation
of Salmonella and Shigella spp from clinical
specimens and food samples.
 The pathogens are differentiated not only
from the non-pathogenic lactose fermenters
but also from many non-pathogens which
do not ferment lactose or sucrose.
 Additionally, the medium was formulated to
increase the frequency of growth of the
more fastidious pathogens, which in other
formulations have often failed to grow due
to the inclusion of excessively toxic
inhibitors.

55. Principle of XLD Agar
 XLD contains yeast extract as a source of nutrients
and vitamins.
 Sodium chloride also supplies essential electrolytes
for transport and osmotic balance.
 It utilizes sodium deoxycholate as the selective agent
and, therefore, is inhibitory to gram-positive micro-
organisms.
 Xylose is incorporated into the medium since it
is fermented by practically all enterics except for
the Shigella and this property enables the differentiation
of Shigella species.
 Lysine decarboxylation in the absence of lactose and
sucrose fermentation causes reversion to an alkaline
condition and the color of the medium changes back to
red.

56.  To prevent similar reversion by lysine positive
coliforms, lactose and sucrose are added to produce
acid in excess.
 Degradation of xylose, lactose and
sucrose generates acid causes phenol red indicator
to change its color in the medium from red to yellow.
 To add to the differentiating ability of the formulation,
an H2S indicator system, consisting of sodium
thiosulfate and ferric ammonium citrate, is included
for the visualization of hydrogen sulfide produced
under alkaline conditions causes colonies to develop
black centers. This reaction is inhibited by the acid
conditions that accompany carbohydrate
fermentation. The non pathogenic H2S producers do
not decarboxylate lysine.

57. Uses of XLD Agar
 XLD Agar is a selective differential medium for
the isolation of Gram-negative enteric pathogens
from fecal specimens and other clinical material.
 It is especially suitable for the isolation
of Shigella and Salmonella species.
 Microbiological
testing of
foods, water
and dairy
products.

58.  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.
 It encourages the growth of a group of certain
bacteria while inhibiting the growth of others.
 It is a selective medium prepared for the isolation
of presumptive pathogenic staphylococci.
Principle of Mannitol Salt Agar
 Mannitol Salt Agar contains peptones and beef
extract, which supply nitrogen, vitamins,
minerals and amino acids essential for growth.

59.  The 7.5% concentration of sodium chloride results in the
partial or complete inhibition of bacterial organisms other than
staphylococci.It also supplies essential electrolytes for
transport and osmotic balance.
 Mannitol is the fermentable carbohydrate, fermentation of
which leads to acid production, detected by phenol
red indicator, aids in the differentiation of staphylococcal
species. Coagulase positive staphylococci
(e.g., Staphylococcus aureus) produce yellow colonies and a
surrounding yellow medium while coagulase negative
staphylococci produce red colonies and no color change of
the phenol red indicator.
 Agar is the solidifying agent.
 Addition of 5% v/v Egg Yolk Emulsion enables the detection
of lipase activity of staphylococci along with mannitol
fermentation. The salt clears the egg yolk emulsion and lipase
production is detected as yellow opaque zone around the
colonies.

60. Uses of Mannitol Salt Agar
 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.
 It is also used in the bacteriological examination of
swimming pool
water, spas and
drinking water
using membrane
filtration

61.  Thiosulfate-Citrate-Bile Salts-Sucrose
 (TCBS) Agar is used for the selective isolation of
cholera vibrios and Vibrio parahaemolyticus from a
variety of clinical and nonclinical specimens.
 Vibrio species are most widely recognized for their
role in human intestinal infections, and cholera
and V. parahaemolyticus diarrhea are important
worldwide.
 All pathogenic Vibrio spp., except Vibrio hollisae, will
grow on TCBS Agar.

62. Principle of TCBS Agar
 Thiosulfate and sodium citrate, as well as the alkalinity of the
medium, considerably inhibit the growth of Enterobacteria.
 Ox bile and sodium cholate slow the growth of enterococci and
inhibit the development of Gram-positive bacteria. The acidification
of the medium resulting from the fermentation
of sucrose by Vibrio makes bromthymol blue turn yellow.
 Bromthymol Blue and Thymol Blue are pH indicators.
 Using thiosulfate as a sulfur source, the production of hydrogen
sulfide is visualized in the presence of ferric citrate.
 Yeast extract and peptone provides the nitrogen, vitamins,
and amino acids in TCBS Agar.
 Sodium chloride provide optimum growth and metabolic
activity of halophilic Vibrio spp.
 Agar is a Solidifying agent.
 An increased pH is used to enhance growth of Vibrio cholerae,
because this organism is sensitive to acid environments.

63. Uses of TCBS Agar
 TCBS Agar is used for the isolation of Vibrio
cholerae and other enteropathologic Vibrio (in
particular Vibrio parahaemolyticus) in fish,
seafood and biological samples of animal
origin.
 TCBS agar has also been used to control
outbreaks of the crown-of-thorns seastar
(Acanthaster planci).

65. Microbial culture methods
 A microbiological culture, or microbial
culture, is a method of multiplying microbial
organisms by letting them reproduce in
predetermined culture medium under
controlled laboratory conditions.
 Culture methods employed depend on the
purpose for which they are intended.

66. The indications for culture are:
 To isolate bacteria in pure cultures.
 To demonstrate their properties.
 To obtain sufficient growth for the preparation
of antigens and for other tests.
 For bacteriophage & bacteriocin susceptibility.
 To determine sensitivity to antibiotics.
 To estimate viable counts.
 Maintain stock cultures.

67. Culture methods include:
 Streak culture
 Lawn culture
 Stroke culture
 Stab culture
 Pour plate method
 Liquid culture
 Anaerobic culture methods

68. STREAK CULTURE
 Streak culture is used to obtain isolated colonies from
an inoculum by creating areas of increasing dilution on
a single plate.
 Platinum wire or Nichrome wire is used.
 One loopful of the specimen is transferred onto the
surface of a well dried plate.
 Spread over a small area at the periphery.
 The inoculum is then distributed thinly over the plate by
streaking it with a loop in a series of parallel lines in
different segments of the plate.
 On incubation, separated colonies are obtained over
the last series of streaks.

71. LAWN CULTURE
 Provides a uniform surface growth of the
bacterium.
 Uses
– For bacteriophage typing.
– Antibiotic sensitivity testing.
– In the preparation of bacterial antigens and
vaccines.
 Lawn cultures are prepared by flooding the
surface of the plate with a liquid suspension of
the bacterium.

72. Antibiotic sensitivity
testing

73. STROKE CULTURE
 Stroke culture is made in tubes
containing agar slope / slant.
 Uses
–Provide a pure growth of bacteria
–For biochemical testing
–Other diagnostic tests

75. STAB CULTURE
 Prepared by puncturing a suitable medium
such as gelatin or nutrient agar with a
long, straight, charged wire.
 Uses
–Demonstration of gelatin liquefaction.
–Use to determine oxygen requirements
of bacterial cells.
–Use of short term storage or shipment
of culture.

77. Gelatin liquefaction Oxidation – Fermentation
medium

78. POUR PLATE CULTURE
 Agar medium is melted (15 ml) and cooled to
45oC.
 1 ml of the inoculum is added to the molten agar.
 Mix well and pour to a sterile petri dish.
 Allow it to set.
 Incubate at 37oC, colonies will be distributed
throughout the depth of the medium.
 Uses
– Gives an estimate of the viable bacterial count in a
suspension.
– For the quantitative urine cultures.

80. LIQUID CULTURES
 Liquid cultures are inoculated by touching with a
charged loop or by adding the inoculum with
pipettes or syringes.
 Uses
– Blood culture
– Sterility tests
– Continuous culture methods
 Disadvantage
– It does not provide a pure culture from mixed
inoculation.

81. Blood culture bottles

83. Production of vacuum:
 Incubate the cultures in a vacuum desiccators.
 Displacement of oxygen with hydrogen,
nitrogen, helium or CO2 .
 Ex: candle jar

85. Chemical method:
 Alkaline pyrogallol absorbs oxygen
 Chromium and sulphuric acid
McIntosh-Fildes’anaerobic jar :
 Consists of a metal jar or glass jar with a metal
lid which can be clamped air tight.
 The lid has 2 tubes-gas inlet and gas outlet
 The lid has two terminals-connected to electrical
supply.
 Under the lid-small grooved porcelain
spool, wrapped with a layer of
palladinised asbestos.

87. Acknowledgment :
 Tortora G.J., Funke B.R.,Case C.L.(Tenth edition) Microbiology an
introduction
 http://www.labtestsguide.com/differential-culture
 https://microbiologyinfo.com/category/culture-media/
 https://slideplayer.com/slide/13763489/
 https://slideplayer.com/slide/12757858/
 https://www.brainkart.com/article/Types-of-Culture-Media_17850/
 “Culturing Media.” Boundless Microbiology
 https://www.stevenson.edu/academics/undergraduate-
programs/biology/blog-news-events/microbiology-students-create-
agar-art
 https://www.facebook.com/PetriDishPicasso/photos/blood-agar-
seen-here-is-a-useful-growth-medium-to-select-for-and-
differentiate-s/502991140105423/
 https://m.facebook.com/asmfan/photos/a.10155021120285200.107
3741837.62453295199/10155021225215200/