Olive A.

Amparado
Sasha Anne L. Valdez
OUTLINE FOR MODULE 4 Part 1:
I. Module Objectives

II. Define Microbial Growth

III. Factors Affecting Microbial Growth

IV. Define Microbial Nutrition and Nutrients

V. Macronutrients, Micronutrients, and Vitamins

VI. Different types of Culture Media

Module 4: Microbial Growth Requirements Part 1
Module Objectives:

1. Define microbial growth.

2. Identify factors that could affect microbial growth.

3. Define microbial nutrition and classify different nutrients

needed for microbial growth.

4. Identify different types of culture media and their uses.

5. Recognize the significance of knowledge on principles of

microbial nutrition in relation to successful laboratory microbial
cultivation.
 What is Microbial Growth?
increase in the number of cells or microbial
population rather than in the size of
individual cells
• To increase the population of
microorganisms, their nutritional and
physical requirements must be provided.
• is the orderly increase of all the chemical
constituents of the bacteria. Multiplication is
the consequence of growth. Death of
bacteria is the irreversible loss of ability to
reproduce.
https://www.slideshare.net/shababali1/3-bio265-microbial-growth-instructor-dr-
di-bonaventura
Factors Affecting Microbial Growth

• biochemical
factors (nutrition) • physical factors
– macronutrients – pH
– micronutrients – temperature
– vitamins – oxygen concentration
– moisture
– hydrostatic pressure
– osmotic pressure
– radiation
• generation time
 What is Microbial Nutrition?
• supplying cells with chemical tools (nutrients) they need
to make monomers of macromolecules that mainly
comprise microbial cells

What are Nutrients?

• made up of chemical elements

• extracellular substances that provide the microbial cell
with materials to
➢ build protoplasm
➢ generate energy
 E. coli data

https://physics.bgu.ac.il/COURSES/CellPhysics/2017A/FILES/Guy_Alli
s_Divisome_Structure.pdf
 https://water-detective.net/articles/the-unseen-life-of-rivers-
mysterious-aquatic-microorganisms/

“different microorganisms need different sets of nutrients,

which are required in different amounts”

❑ macronutrients – nutrients required in relatively larger

amounts
❑ micronutrients – nutrients required in lesser quantities
 https://www.biologydiscussion.com/microorganisms/nutritional-
C, O, N, H (predominantly required) requirements-of-microorganisms/55070
https://www.biologydiscussion.com/microorganisms/nutritional-requirements-of-microorganisms/55070
https://www.biologydiscussion.com/microorganisms/nutritional-requirements-of-microorganisms/55070
 What is a Culture Medium
• any nutrient material prepared/used for the growth and
cultivation of microorganisms in the laboratory

What are Culture Media for?

1. for the growth and maintenance of microbial cultures

2. to favor the production of particular compounds

3. to study microbial action on some constituents of the medium

https://slideplayer.com/slide/5976886/
 Types of Culture Media
(based on physical state or consistency)
Consistency Composition Purpose/s Examples

1) liquid with no Inoculum Nutrient

solidifying agent preparation; Broth;
fermentation test Lactose Broth
2) semi- with 0.1- 0.5% Motility test Sulfur Indole
solid solidifying agent Motility
(SIM)
Medium
3) solid with 1.5-2.0% Colony morphology Nutrient
solidifying agent observation; Agar; Blood
hemolysis and Agar
pigmentation
characterization
1) Liquid culture medium (broth)
• Difficult to identify all types of microorganisms
• Suitable for isolation of bacteria from blood culturing
and water analysis

https://blog.biomall.in/preparation-of-culture- http://w3.marietta.edu/~spilatrs/biol202/
media/ labresults/lactose_broth.html
2) Semisolid culture medium

https://microbenotes.com/motility-test-
principle-procedure-and-results/

https://catalog.hardydiagnostics.com/
cp_prod/Content/hugo/MotilityTestMe
dia.htm
 • Used to check for motility & also
used as a transport media for fragile
organisms.

https://www.slideshare.net/HiwrHastear/ty
pes-of-culture-media
 3) Solid culture medium

Agar plate Agar slant

Agar deep

http://outbreaknewstoday.com/venezuela-
serratia-marcescens-kills-11-children-
barquisimeto-hospital-98992/

https://slideplayer.
https://www.alamy.com/stock-
com/slide/782573
photo-agar-slant-tube-cultures-of-
6/
serratia-marcescens-left-and-
kocuria-rhizophila-16369146.html
 Agar
• complex polysaccharide (usually derived from
red algae)
• used as solidifying agent for culture media in
Petri plates, slants, and deeps
• no nutritive value; generally not metabolized by
microbes
• not affected by growth of bacteria
• Liquefies at 100°C
• Solidifies at ~40°C
https://slideplayer.com/slide/5976886/
 Types of Culture Media
(based on chemical composition)

Type Components Examples

1) Synthetic All components are Glucose

or chemically-defined Inorganic Salt
chemically- (precise nutrient Phosphate
defined composition and
amounts)
2) Complex Not all components are Nutrient Agar,
chemically-defined yeast extract
 Synthetic Medium
Complex Medium

http://www.mmc.gov.bd/downloadable%2
0file/Culture%20media%20by%20Shya
mal.pdf
https://laboratoryinfo.com/nutrient-agar/
1) Synthetic or Defined Medium
• prepared from pure chemicals and exact
chemical composition (kinds and amounts) is
known

• uses: important for genetic and specific or

precise studies
• disadvantages:
– preparation is time-consuming
– microorganisms grow relatively slow
– prepared only for microorganisms with known
nutritional requirements
 https://www.slideshare.net/HiwrHastear/type
s-of-culture-media

https://www.amazon.in/DUB
OS-BROTH-500GM-
DEHYDRATED-
CULTURE/dp/B0793MN9K9
 https://www.amazon.in/DUB
OS-BROTH-500GM-
Ingredients (Gms / Litre)
Tryptone 0.500
DEHYDRATED-
CULTURE/dp/B0793MN9K9

L-Asparagine 2.000
Polysorbate 80 (Tween 80) 0.200
Potassium dihydrogen phosphate 1.000
Disodium hydrogen phosphate 2.500
Ferric ammonium citrate 0.050
Magnesium sulphate 0.010
Calcium chloride anhydrous 0.0005
• for Culture of Zinc sulphate 0.0001
M. tuberculosis from
Sputum Copper sulphate 0.0001
Final pH ( at 25°C) 6.6±0.2
**Formula adjusted, standardized to suit
performance parameters

https://himedialabs.com/TD/M067.pdf
2) Complex Medium
• chemically undefined medium; precise
chemical composition (kinds) unknown
• contains certain similar ingredients but
chemical composition varies slightly from
batch to batch
• made from extracts from natural materials
(beef, blood, casein, yeast, soybeans)

• use: routine purposes

• advantages:
– easy to prepare
– support rapid growth of most microorganisms
https://slideplayer.com/slide/5976886/
 Types of Culture Media
(based on principal purpose, function, or application)

Types Characteristic Examples

general Can support most Nutrient Agar,

purpose or almost all Tryptic Soy
types of species Agar, Brain
Heart Infusion
Agar
https://digitalworldbiology.com/archive/how
differential Can distinguish -well-do-you-know-your-bacteria

visually one type

of bacterium
from another Eosin
Methylene
selective Allows growth of
Blue, Mannitol
a specific type of
Salt Agar
microorganism
only https://microbeonline.com/macc
onkey-agar-mac-composition-
preparation-uses-and-colony-
characteristics/
1) Supportive/ General
Purpose

e.g.: Nutrient Broth (NB),

Nutrient Agar (NA)
https://microbiologyinfo.com/nutrient-
agar-composition-preparation-and-uses/

• supporting growth of a wide range of non-

fastidious organisms.
• It typically contains (mass/volume): 0.5%
Peptone - this provides organic nitrogen. 0.3%
beef extract/yeast extract - the water-soluble
content of these contribute vitamins,
carbohydrates, nitrogen, and salts.
https://pediaa.com/diffe
rence-between-
selective-and-
differential-media/
2) Selective Medium
• allows the growth of a specific type of
microorganisms only and suppresses the growth of
others

• contains selective agents/additives/toxic chemicals

(salts, dyes, antibiotics, and other inhibitors)
– sodium azide, potassium tellurite, thallium acetate (0.1-0.5
g/L), crystal violet (2 mg/L), penicillin (5-50 units/mL)
• extreme pH value or unusual carbon source to favor
growth of a particular organism

• i.e. Thayer-Martin agar (Neisseria gonorrhoeae);

Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar;
NA with penicillin (Gram-negative bacteria)
• Selective media allow certain types of
organisms to grow, and inhibit the growth of
other organisms. The selectivity is
accomplished in several ways.

• organisms that can utilize a given sugar are

easily screened by making that sugar the only
carbon source in the medium.

Tellurite agar - is used to select for Gram + organisms

Nutrient agar - supplemented with penicillin can be

used to select for Gram - organisms.
https://www.slideserve.com/hoshi/thayer-martin-agar
What component/s of
Thayer Martin Agar
make/s it selective to
Neisseria gonorrhoeae?
 https://en.wikipedia.org/wiki/File:Neisseria_
gonorrhoeae_01.png

Comparison of two culture media types used to grow

Neisseria gonorrhoeae bacteria.

Known as overgrowth, the non-selective chocolate agar medium on the left, due to its
composition, allowed for the growth of organismal colonies other than those
of Neisseria gonorrhoeae, while the selective Thayer-Martin medium on the right,
containing antimicrobials that inhibit the growth of organisms other than N.
gonorrhoeae, shows no overgrowth, but is positive for N. gonorrhoeae bacteria.
https://www.eolabs.com/product/pp0270- https://www.slideshare.net/DanaSinzianaBreharCi/vib
tcbs-medium/ rio-campylobacter-helicobacterpseudomonas-
46210466

Thiosulfate-citrate-bile salts-sucrose agar,

or TCBS agar, is a type of highly selective agar culture
plate that is used for the isolation of V. cholerae and V.
parahaemolyticus as well as other Vibrio species.
 https://microbiologyinfo.com/thiosulfate-citrate-bile-salts-sucrose-tcbs-agar-
composition-principle-uses-preparation-and-colony-morphology/

Contains high concentrations of sodium

thiosulfate and sodium citrate to inhibit the growth
of Enterobacteriaceae.

Inhibition of Gram + bacteria is achieved by the

incorporation of ox gall (a naturally occurring substance
containing a mixture of bile salts) and sodium cholate (a
pure bile salt).
 https://www.researchgate.net/figure/Cultural-characteristic-of-Vibrio-
nereis-A-and-Vibrio-harveyi-B-on-TCBS-agar_fig1_308707480

Sodium thiosulfate also serves as a sulfur source and its presence, in

combination with ferric citrate, allows for the easy detection
of hydrogen sulfide production.

Saccharose (sucrose) is included as a fermentable carbohydrate for

metabolism by Vibrio species.

The alkaline pH of the medium enhances the recovery of V.

cholerae and inhibits the growth of others. Thymol
blue and bromothymol blue are included as indicators of pH changes.
What component/s of
Thiosulfate Citrate Bile
Salts Sucrose (TCBS)
Agar make/s it
selective to Vibrio
species?
https://www.slideshare.net/NAMDEOSHIND
E15/culture-media-165080927
3) Differential Medium
• can distinguish or differentiate visually one type
of bacterium from another

• identifies microorganisms by the appearance of

their colonies and exploits the ability of a
particular microorganism to change the appearance
of the medium

• with special reagents like pH indicators and dyes

• i.e. blood agar (Streptococcus species); Mac-

Conkey agar (E. coli and lactose fermenters)
Blood agar contains general nutrients and 5% sheep blood.
It is useful for cultivating fastidious organisms and for
determining the hemolytic capabilities of an organism.

Some bacteria produce exoenzymes that lyse red blood cells

and degrade hemoglobin; these are called hemolysins.

Bacteria can produce different types of hemolysins.

https://www.amazon.com/Hardy-Diagnostics-A40- https://www.studyblue.com/notes/note/n/blood-
Cefoperazone-Campylobacter/dp/B01M4OW6EJ agar/deck/6258465
Beta-hemolysin breaks down the red blood cells and hemoglobin
completely. This leaves a clear zone around the bacterial growth. Such
results are referred to as β-hemolysis (beta hemolysis).

Alpha-hemolysin partially breaks down the red blood cells and leaves a
greenish color behind. This is referred to as α-hemolysis (alpha
hemolysis). The greenish color is caused by the presence of biliverdin,
which is a by-product of the breakdown of hemoglobin.

If the organism does not produce hemolysins and does not break down
the blood cells, no clearing will occur. This is called γ-hemolysis
(gamma hemolysis).

https://microbenotes.com/
hemolysis-of-streptococci/
https://www.slideshare.net/drabbasnaseem/staining-culture-medias
https://slideplayer.com/slide/10384388/
https://www.cdc.gov/groupbstrep/lab/index.html
https://mltexpo.blogspo
t.com/2018/04/maccon
key-agar-principles- https://www.amazon.com/Hardy-
composition.html Diagnostics-G36-Macconkey-
Sorbitol/dp/B01MG8W432

https://slideplayer.com/slide/8282469/
https://www.slideshare.net/doctorrao/enterobacteriaceae-basics
https://www.slideshare.net/doctorrao/enterobacteriaceae-basics
https://slideplayer.com/slide/11433811/
https://slideplayer.com/slide/11433785/
https://microbiologyinfo.com/macconkey-agar-composition-principle-uses-
preparation-and-colony-morphology/
https://in.pinterest.co
m/pin/4083498911932
20631/
What component/s of
MacConkey Agar
(MCA) enable/s it to
differentiate different
groups of
microorganisms?

How do these
components make MCA
a differential medium?
https://www.slideshare.net/NomanCh1/isol
ation-and-identification-of-salmonella-ecoli
Which of the culture
media previously
discussed can be
classified under more
than one type?
 There are culture media that can be
categorized under more than one
type. Examples include Mac Conkey
Agar (MAC), Mannitol Salt Agar
(MSA) and Eosin Methylene Blue
(EMB) Agar. What type of culture
media are these two? What makes
them such type?

https://www.dreamstime.com/ferment-non-ferment-
mannitol-salt-agar-staphylococcus-mannitol-salt-agar- https://www.youtube.com/watch?v=DYDD
image110190660 wE-HhCM
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.

distinguishing pathogenic microbes in a short period of time. It contains a high

concentration (about 7.5%-10%) of salt (NaCl), making it selective for Gram
positive bacteria (Staphylococcus and Micrococcaceae) since this level of
salt is inhibitory to most other bacteria.

also a differential medium for

mannitol-fermenting
staphylococci, containing
carbohydrate mannitol and the
indicator phenol red, a pH
indicator for detecting acid
produced by mannitol-fermenting
staphylococci.

https://laboratoryinfo.com/mannitol-salt-agar/
If an organism can ferment mannitol, an acidic byproduct is formed that causes
the phenol red in the agar to turn yellow. It is used for the selective isolation of
presumptive pathogenic (pp) Staphylococcus species.

Staphylococcus aureus produces yellow colonies with yellow zones, whereas

other coagulase-negative staphylococci produce small pink or red colonies with
no colour change to the medium

https://www.bioscience.com.pk/topics/microbiology/item
/232-mannitol-salt-agar-test
https://www.slideshare.net/doctorrao/skills-in-medical-
microbiology
https://www.pinterest.ph/pin/201043570844601151/
https://www.youtube.com/watch?v=5_nWidnxfQ8
https://slideplayer.com/slide/10804039/
https://slideplayer.com/slide/7585533/
 Types Characteristics Examples

4) enrichment Used to increase the number of Cellulose agar, Petroleum

microorganisms with unusual broth, Blood agar
physiological characteristics;
contains special nutrients for
microorganisms of interest

5) assay Used for assay of vitamins, fermentation media, TSI

amino acids, antibiotics; may be agar, Vitamin B12 assay
used for qualitative or medium
quantitative production of a
compound by a microorganism
4) Enrichment Medium
• used to increase the number of microorganisms with
unusual physiological characteristics (i.e. pathogens from a
mixed culture)

• contains special nutrient(s) for the microbe of interest

(i.e. cellulose, petroleum, blood) and inhibitory substances
to suppress unwanted microorganisms

• i.e. Cellulose Agar (for cellulolytic microorganisms);

Petroleum broth (for petroleum-utilizers); Blood Agar (for
hemolytic bacteria), Selenite F broth (for Salmonella and
Shigella), Alkaline peptone water (for Vibrio cholerae)
https://laboratoryinfo.com/chocolate-agar/
5) Assay Medium
• used for the assay of vitamins, amino acids,
antibiotics, etc.; used for qualitative or
quantitative production of such a compound by
a microorganism

• of prescribed composition

• i.e. fermentation media, Triple Sugar Iron

Agar, Media for Antibiotic Sensitivity Testing,
Vitamin B12 Assay Medium
 Sugar Medium
• medium containing any fermentable substances (i.e.
glucose, arabinose, lactose, starch)

• consists of 1% of the sugar in peptone water

• contain a small tube (Durham tube) for the detection

of gas by the bacteria

http://faculty.fiu.edu/~gantarm/Ex8WaterBact1.pdf
https://slideplayer.com/slide/7623494/
https://www.slideshare.net/Millatlaboratory/biochemical-tests-58986607
https://www.slideserve.com/salena/microbiology-unknown-lab
https://slideplayer.com/slide/17423304/
 Antibiotic Sensitivity Testing

https://www.researchgate.net/figure/Antibiotic-sensitivity-pattern-
https://www.himediastore.com/b12-assay- of-Clostridium-perfringens-in-Mueller-Hinton-
medium-using-l-leichmannii-9879 agar_fig2_317340043
Other Types of
Culture Media
 Transport Medium
• medium used for transporting
samples (prevent microbial
proliferation; maintain viability
of microorganisms)

• i.e. Stuart’s medium (non nutrient

soft agar gel containing a reducing
agent); buffered glycerol saline (for
enteric bacilli)

https://www.slideshare.net/doctorr
ao/culture-media3
 Quality Control

Appearance
White to light blue
coloured
homogeneous
free flowing
powder

https://www.slideshare.net/doctorr
ao/culture-media3
Transport Medium Stuart is recommended for the preservation and
transportation of Neisseria species and other fastidious organisms from the
clinic to laboratory.
This medium is a chemically defined, semisolid, non-nutrient medium which
prevent microbial proliferation. Because of this composition the medium ensures
that microorganisms present are able to survive for a sufficiently long period of
time. The medium provides an adequate degree of anaerobiosis which can be
monitored by means of the redox indicator methylene blue.
 Maintain viability of enteric
pathogens while transporting
stool specimens to the
laboratory with Buffered
Glycerol Saline.

http://www.himedialabs.com/intl/en/products/Microbio
logy/Dehydrated-Culture-Media-General-Animal-
based-Media-Bacterial/Buffered-Glycerol-Saline-
Base-M204
 Biochemical Reaction Medium

• medium used to provide additional information for

the identification of the bacterium

• i.e. Triple sugar iron agar (sugar fermentation);

SIM Medium (Indole test); Citrate utilization;
Christensens urease medium (Urease test)
TRIPLE SUGAR IRON AGAR (TSI)

• It is a composite media used to study different

properties of a bacterium – sugar fermentation, gas
production and H2S production.
• In addition to peptone, yeast extract & agar, it
contains 3 sugars – Glucose, Lactose, Sucrose.
• The Iron salt – Ferric citrate indicates H2S
production.
• Phenol red is the indicator.
• It is an orange red medium with a slant and a butt.
• pH of the medium – 7.4
TSI REACTIONS:
Yellow – Acid
Pink - Alkaline

Yellow slant / Yellow butt (A/A) – Lactose

fermenters.
• Pink slant / Yellow butt (K/A) – Non lactose
fermenters
• Pink slant / no colour change (K/K) – Non
fermenters
• Black colour – H2S production.
• Gas bubbles or crack in the medium – gas
production. https://germsandwo
• LF – E.coli, Klebsiella rms.wordpress.com
• NLF – Salmonella, Shigella /2013/03/07/triple-
sugar-iron-tsi-
• H2S - Proteus fermentation/
https://fankhauserblog.
wordpress.com/1987/0
8/03/triple-sugar-iron-
agar-and-its-use-and-
interpretation/
INDOLE TEST
• Used to detect indole production by
the organism.
• They produce indole from tryptophan
present in peptone water.
• After overnight incubation, a few
drops of indole reagent (Kovac’s
reagent) is added.
• Positive test is indicated by a pink ring
– Positive indole test – pink ring
– Negative indole test - yellow ring
• Indole positive – E.coli
• Indole negative – Klebsiella, Salmonella

https://www.youtube.com/watch
?v=m27wzevB9bA
https://slideplayer.com/slide/8698531/
https://slideplayer.com/slide/8692510/
https://micro
benotes.co
m/motility-
test-
principle-
procedure-
and-results/

https://micro
biologyinfo.c
om/sulphur-
reduction-
test/
CITRATE UTILIZATION TEST
• Done in Simmon’s Citrate medium.
• To detect the ability of certain
bacteria to utilize citrate as the
sole source of carbon.
• Contains Sodium citrate and
bromothymol blue as the indicator.
• If citrate is utilized, alkali is
produced which turns the medium to
blue.
– Citrate positive – blue color
– Citrate negative – green color
• Positive – Klebsiella
• Negative – E.coli https://www.onlinebiologynotes.co
m/citrate-utilization-test-objective-
principle-procedure-and-result/
 UREASE TEST
• Done in Christensen’s urease
medium.
• This test is used to detect
organisms that produce urease.
• Urease produced by the
organisms split urea into ammonia
and CO2.
– Urease positive – pink color
– Urease negative – yellow color
• Positive – Proteus, Klebsiella
• Negative – E.coli, Salmonella https://microbiologyinfo.co
m/urease-test-principle-
media-procedure-and-
result/
Aerobic Media and Anaerobic Media
(Based on Oxygen Requirement)

https://www.slideshare.net/Nimish
aK4/cultivation-of-bacteria-ppt-
nimisha
Anaerobic media
• These media are used to grow anaerobic organisms.
• e.g: Robertson’s cooked meat medium, Thioglycolate
medium.

https://microbenotes.com/cultural-characteristics-
of-clostridium-perfringens/
https://www.slideshare.net/drimtiyzwanii/clostridium-
https://www.slides
hare.net/sasipras
ad/culture-
methods
References:
Madigan, M.T., Martinko, J.M, and Parker,
J. (2000). Brock biology of
microorganisms. (9th ed.) New Jersey,
USA: Prentice Hall International, Inc.

Pommerville, J.C. (2004). Alcamo’s

fundamentals of microbiology. (7th ed.)
Massachusetts, USA: Jones and
Bartlett Publishers
University of the Philippines-Los Baños