Microbial Growth

SYLVIA T. PRATIWI
Objectives:

 Classify microbes into five groups on the basis of preferred temperature

range.
 Explain the importance of osmotic pressure to microbial growth.

 Provide a use for each of the four elements (C, N, S, P) needed in large
amounts for microbial growth.
 Explain how microbes are classified on the basis of O2 needs.
 Identify ways in which aerobes avoid damage by toxic forms of O2.

 Describe the formation of biofilms and their potential for causing infection.

 Distinguish between chemically defined and complex media.

Objectives:

 Justify the use of each of the following: anaerobic techniques, living host
cells, candle jars, selective, differential, and enrichment media.
 Define colony and CFUs and describe how pure cultures can be isolated
with streak plates.
 Explain how microbes are preserved by deep-freezing and lyophilization.
 Distinguish between binary fission and budding.

 Define generation time and explain the bacterial growth curve.

 Review some direct and indirect methods of measuring bacterial cell

growth.
The Study of Microbial Growth

 Growth takes place on two levels

 Cell synthesizes new cell components and increases in size
 The number of cells in the population increases

 Microbial growth: Increase in cell number, not cell

size!
 The Basis of Population Growth: Binary Fission
 Factors that Influence Growth

 Growth vs. Tolerance

 Many microbes can survive under conditions in which they cannot

grow

 The suffix “-phile” is often used to describe conditions permitting

growth, whereas the term “tolerant” describes conditions in which
the organisms survive, but don’t necessarily grow

 For example, a “thermophilic bacterium” grows under conditions of

elevated temperature, while a “thermotolerant bacterium” survives
elevated temperature, but grows at a lower temperature
Bacterial growth
 Binary fission
 Generation time
 Phases of growth
Binary fission

1. Prokaryote cells grow

by increasing in cell
number (as opposed to
increasing in size).
2. Replication is by binary
fission, the splitting of
one cell into two
3. Therefore, bacterial
populations increase by
a factor of two (double)
every generation time.
The Rate of Population Growth

– Generation or doubling time: The time required for a complete

fission cycle
– Each new fission cycle or generation increases the population by a
factor of 2
– As long as the environment is favorable, the doubling effect continues
at a constant rate
– The length of the generation time- a measure of the growth rate of an
organism
• Average generation time- 30 to 60 minutes under optimum
conditions
• Can be as short as 10 to 12 minutes
– This growth pattern is termed exponential
Generation time

4-9
 The time required to for a population to double (doubling time) in
number.
 Ex. Escherichia coli (E. coli) double every 20 minutes
 Ex. Mycobacterium tuberculosis double every 12 to 24 hours
Principles of Bacterial Growth

 Growth can be calculated

 Nt = N0 x 2n
 (Nt ) number of cells in population

 (N0 ) original number of cells in the population

 (n) number of divisions

 Example
 N0 = 10 cells in original population
 n = 12
 4 hours assuming 20 minute generation time
 Nt = 10 x 212
 Nt = 10 x 4,096
 Nt = 40,960
 Growth in Batch Culture
1. Bacteria growing in batch culture produce a growth curve with up to four
distinct phases.
2. Batch cultures are grown in tubes or flasks and are closed systems where no
fresh nutrients are added or waste products removed.
3. Data from an entire growth period typically produce a curve with a series of
phases
4. Lag phase occurs when bacteria are adjusting to them medium. For example,
with a nutritionally poor medium, several anabolic pathways need to be turned
on, resulting in a lag before active growth begins.
5. In log or exponential phase, the cells are growing as fast as they can, limited
only by growth conditions and genetic potential. During this phase, almost all
cells are alive, they are most nearly identical, and they are most affected by
outside influences like disinfectants.
6. Due to nutrient depletion and/or accumulation of toxic end products,
replication stops and cells enter a stationary phase where there is no net
change in cell number.
7. Death phase occurs when cells can no longer maintain viability and numbers
decrease as a proportion.
Growth in Batch Culture
Mean Generation Time and Growth Rate

 The mean generation time (doubling time) is the amount of time

required for the concentration of cells to double during the log
stage. It is expressed in units of minutes.

 Growth rate (min-1) = 1

mean generation time
 Mean generation time can be determined directly from a semilog
plot of bacterial concentration vs time after inoculation
 Lag Phase

• Relatively “flat” period

• Newly inoculated cells require a
period of adjustment, enlargement,
and synthesis
• The cells are not yet multiplying at
their maximum rate
• The population of cells is so sparse
that the sampling misses them
• Length of lag period varies from
one population to another
Exponential Growth (Logarithmic or log)
Phase

 When the growth curve increases

geometrically
 Cells reach the maximum rate of
cell division
 Will continue as long as cells have
adequate nutrients and the
environment is favorable
 The number of cells growing greatly
out number the number of cells
dying.
Stationary Growth Phase

 The population enters a survival

mode in which cells stop growing
or grow slowly
 The rate of cell inhibition or
death balances out the rate of
multiplication
 Depleted nutrients and oxygen
 Excretion of organic acids and
other biochemical pollutants
into the growth medium
 The number of cells growing will
equal the amount of cells
dying.
 Endospores begin to form in this
phase.
Rapidly Declining Phase

 The curve dips downward

 Cells begin to die at an
exponential rate
 The amount of cells dying out
numbers the amount of cells
growing.
 The dead cells become nutrients
for the growing cells.
Death Phase

 The curve continues to dips

downward
 Most cellular activity stops
 Endospores are formed and
released from the parent cells.
Phases of Growth

Basic phases of growth:

1. Lag phase: new growth

medium, period of delay while
cells prepare to divide

2. Log phase (exponential growth

phase): cellular reproduction
most active during this period,
generation time reaches a
constant minimum

3. Stationary phase: state of

equilibrium where number of
cell deaths equals number of
cell divisions
Phases of Growth

Basic phases of growth:

4. Rapidly Declining Phase:

cells die logarithmically,
endospores formed

5. Death phase: number of

deaths exceeds number of
new cells
Potential Importance of the Growth Curve

• Implications in microbial control, infection, food microbiology,

and culture technology

• Growth patterns in microorganisms can account for the

stages of infection

• Understanding the stages of cell growth is crucial for working

with cultures

• In some applications, closed batch culturing is inefficient, and

instead, must use a chemostat or continuous culture system
Graphing Bacterial Growth

• The data from growing bacterial populations are graphed by

plotting the number of cells as a function of time
– If plotted logarithmically- a straight line
– If plotted arithmetically- a constantly curved slope

• To calculate thesize of a population over time: Nf = (Ni)2g

– Nf is the total number of cells in the population at some
point in the growth phase
– Ni is the starting number
– g denotes the generation number
Basic Chemostat System – A continuous
Culture
Lab Chemostat System
 Growth in Continuous Culture

 A “continuous culture” is an
open system in which fresh
media is continuously added to
the culture at a constant rate,
and old broth is removed at the
same rate.
 This method is accomplished in
a device called a chemostat.
 Typically, the concentration of
cells will reach an equilibrium
level that remains constant as
long as the nutrient feed is
maintained.
The Difference
between Batch culture
& continuous Culture

https://pediaa.com/difference-
between-batch-and-continuous-
culture/
Environmental factors

 Temperature
 Oxygen requirement
 pH
 Water availability
 Factors that Influence Growth

 Obligate (strict) vs. facultative

 “Obligate” (or “strict”) means that a given condition is required for growth

 “Facultative”, often applied to sub-optimal conditions, means that the

organism can grow under the condition, but doesn’t require it

 For example, an obligate thermophile requires elevated temperatures for

growth, while a facultative thermophile may grow in either elevated
temperatures or lower temperatures
 Factors that Influence Growth

 Temperature
 Most bacteria grow throughout a range of approximately 20 Celsius
degrees, with the maximum growth rate at a certain “optimum
temperature”
❑ Enzymes, the machinery of the cell, are influenced by external factors and
can be shown to have a range where they function that includes an optimal
value that produces the highest activity.

❑ The range of enzyme activity determines the range for growth of specific
bacteria, analogously leading to a value for optimal growth rate.

❑ In the case of temperature, bacteria are divided into categories based on

the temperature range where they can grow and the temperature that
provides optimal growth.
Temperature

 Psychrophile
 0o to 18o C
 Psychrotroph
 20°C to 30°C
 Important in food spoilage
 Mesophile
 25°C to 45°C
 More common
 Disease causing
 Thermophiles
 45°C to 70°C
 Common in hot springs and hot water heaters
 Hyperthermophiles
 70°C to 110°C
 Live at very high temperatures, high enough where water threatens to
 Factors that Influence Growth

 Oxygen concentration
 Strict aerobes: Require oxygen for growth (~20%)
 Strict anaerobes: Grow in the absence of oxygen; cannot grow in the
presence of oxygen
 Facultative anaerobes: Grow best in the presence of oxygen, but are able
to grow (at reduced rates) in the absence of oxygen
 Aerotolerant anaerobes: Can grow equally well in the presence or
absence of oxygen
 Microaerophiles: Require reduced concentrations of oxygen (~2 – 10%)
for growth
•Oxygen is a very reactive molecule and can affect cells in several ways. The effect of oxygen
is often determined using thioglycollate broth, a special medium that contains a reducing
agent (thioglycollate) that removes oxygen so that a gradient occurs within the tube.

•Obligately aerobic bacteria can obtain energy only through aerobic respiration and have to
have oxygen available. Thus, they will grow only at the surface of thioglycollate broth.

•Obligately anaerobic bacteria die in the presence of oxygen and can only grow at the bottom
of thioglycollate broth. Some anaerobes are so sensitive to oxygen that even thioglycollate
broth is not anoxic enough to provide suitable anaerobic conditions.
 4-35
• Microaerophiles require oxygen for growth but the 20% in air is too toxic. As a
result, they grow near the top but beneath the surface of thioglycollate broth
where the oxygen concentration is typically 4 – 10%.

• Facultative anaerobes can use oxygen for aerobic respiration but can switch to
fermentative metabolism in the absence of oxygen. As a result, they will grow
throughout thioglycollate broth. (Heavier growth at top.)

• Aerotolerant anaerobes are anaerobic bacteria that can grow in the presence of
air.
 Factors that Influence Growth

 pH
❑ Neutrophiles grow best around
neutral pH (7)
❑ Acidophiles grow best at pH < 7
❑ Alkophiles grow best at pH > 7
❑ Acidotolerant grow best at pH 7
but can also grow at lower pH
❑ Alkotolerant grow best at pH 7
but can also grow at higher pH
 Water Activity

❑ Liquid water is essential for life.

❑ Aqueous solutions actually have different amounts of water available,
depending on how many solutes are dissolved in it.
❑ Water activity (aw) can be decreased by the addition of any soluble
molecule although salt (NaCl) and sugars are probably the most
common.
Water Activity

 Microbes that require a high water activity (near or at 1) are termed

nonhalophiles. (Halophile = salt-loving)
 Some bacteria require salt to grow and are called halophiles. If a
very high concentration of salt is required (around saturation), the
organisms are termed extreme halophiles.
 A nonhalophile that can grows best with almost no salt but can still
grow with low levels of salt (~ 7%) is called halotolerant.
 In general, fungi are more tolerant of low water activity. (That’s why
your jelly is more likely to get contaminated by fungi than bacteria.)
 Factors that Influence Growth

 Salt concentration
 Halophiles require elevated
salt concentrations to grow;
often require 0.2 M ionic
strength or greater and may
some may grow at 1 M or
greater; example,
Halobacterium
 Osmotolerant (halotolerant)
organisms grow over a wide
range of salt concentrations or
ionic strengths; for example,
Staphylococcus aureus
Microbial Nutrition
Cell metabolism
Nutritional Categories of
Microorganisms

 Microorganisms are often grouped according to the sources of energy

they use:

 Phototrophs use light as an energy source

 Photosynthesis
 Chemotrophs use chemicals as energy sources
 Chemoorganotroph : obtains energy from the oxidation of
reduced organic com- pounds
 Chemolithotroph : obtain energy from the oxidation of reduced
inorganic compounds such as sulfide, ammonia and hydrogen,
and use carbon dioxide as carbon source.
 NUTRITIONAL REQUIREMENTS OF
BACTERIA

1. MACRO-NUTRIENTS OR MACRO-ELEMENTS
2. OTHER MACRO-NUTRIENTS
3. MICRO-NUTIENTS OR TRACE EEMENTS
4. ORGANIC GROWTH FACTORS
5. LIGHT
6. WATER

1. THE MACRO-NUTRIENTS OR MACRO-ELEMENTS:

o carbon, hydrogen, oxygen, nitrogen, phosphorus and sulphur.

o They are essential elements because they required in large amounts.
o They contain 95% of dry weight of the microbial cells.
THE REMAINING FOUR MACRO-
NUTRIENTS:
They exist in cell cations & a play variety of role.

NUTRIENTS FUNCTIONS
Micronutrients

 Elements needed in trace quantities

 Manganese, Zinc, Cobalt, Copper, Nickel
 Part of Enzymes
 From tap water
 Growth factors
 Organic compounds
 Vitamins
 ORGANIC GROWTH FACTORS

Organic compounds that are essential cell compounds or precursors of such

components are called ‘Growth factors’.

The major classes of growth factors are:

1. AMINO ACIDS: Needed for protein synthesis.
2. PURINE & PYRIMIDINE: Helps in nucleic acid synthesis.

 VITAMINS: small organic molecules that usually make up all or part of enzyme,
co-factor & are needed in only very small amounts to sustain growth, also
known as bacterial vitamins.
 Some bacteria’s synthesis their own vitamins & while other need to take them
from outside.
 This varies from bacteria to bacteria.
 CULTURE MEDIA

 A Nutrient material prepared for the growth of micro-organisms in a lab. is

called a ‘culture medium’.
 When microbes are introduces into a culture medium to initiate growth,
they are called an ‘inoculum’.
 The microbes that grow & multiply in or on a culture medium , are referred
to as ‘culture’.
 Culture media can be solidifying by the addition of agar, a complex
polysaccharide from red algae.
CULTURE MEDIA

 Used to grow bacteria

 Can be used to:

❖ Enrich the numbers of bacteria.

❖ Select for certain bacteria and suppress others.

❖ Differentiate among different kinds of bacteria.

Composition of culture media

 Provide similar environmental and nutritional conditions that

exist in its natural habitat

 A culture medium contains water, a source of carbon & energy,

source of nitrogen, trace elements and some growth factors
BASIC REQUIREMENTS OF CULTURE MEDIA

 NUTRIENTS :

❑ Energy source
❑ Carbon source
❑ Nitrogen source

 MINERAL SALTS :

❑ Sulphates, phosphates, chlorides and carbonates of K, Mg and Ca

❑ A suitable pH- 7.2- 7.4
 GROWTH FACTORS
ARGININE E.COLI

GLUTATHIONE GONOCOCCI
CHOLESTEROL MYCOPLASMA

ARYL SULPHATE, AMIDE ATYPICAL MYCOBACTERIA

GLYCEROL MYCOPLASMA HOMINIS

SULFONAMIDES RIKETTSIA

TRYPTOPHAN SALMONELLA TYPHI

L-CYSTEINE LISTERIA MONOCYTOGENS

SODIUM CHLORIDE VIBRIO PARAHAEMOLYTICUS

FACTOR X & V H.INFLUENZAE

Culture media classification

 Consistency
 Nutritional component
 Functional use
CLASSIFICATION
BASED ON BASED ON BASED ON
PHYSICAL PRESENCE OF NUTRITIONAL
STATE MOLECULAR FACTORS
OXYGEN AND
REDUCING
SUBSTANCES

LIQUID MEDIA AEROBIC MEDIA SIMPLE MEDIA

SEMISOLID MEDIA ANAEROBIC MEDIA COMPLEX MEDIA

SOLID MEDIA SYNTHETIC MEDIA

SPECIAL MEDIA
 SPECIAL MEDIA

A. ENRICHED MEDIA
B. ENRICHMENT MEDIA
C. SELECTIVE MEDIA
D. DIFFERENTIAL MEDIA
E. INDICATOR MEDIA
F. TRANSPORT MEDIA
G. SUGAR MEDIA
 Microbiological Media: Consistency

 Liquid (broth) vs. semisolid media

 Liquid medium
 Components are dissolved in water and sterilized
 Semisolid medium
 A medium to which has been added a gelling agent
 Agar (most commonly used)
 Gelatin
 Silica gel (used when a non-organic gelling agent is required)
Microbiological Media:
Nutritional component

 Simple: nutrients agar, peptone water: can support most non-fastidious bacteria
 Defined
 Prepared with precise amounts of chemicals
 Known composition
 e.g. minimal media used in bacterial genetics experiments
 Complex
 Exact composition unknown
 Often consist of plant or animal extracts, such as soybean meal, milk protein,
etc.
 Include most routine laboratory media, e.g., tryptic soy broth
Microbiological Media: Functional use /
application
 Basal media = simple media

 Selective media
 Contain agents that inhibit the growth of certain bacteria while
permitting the growth of others
 Frequently used to isolate specific organisms from a large population of
contaminants
 Any agar media can be made selective by addition of certain inhibitory
agents that don’t affect the pathogen.
 To make a medium selective include addition of antibiotics, dyes,
chemicals, alteration of pH or a combination of these
 SIMPLE MEDIA
Simple or basal media are culture media which contain the minimum
adequate nutrition for non fastidious organisms
Example:- Nutrient broth/agar
Peptone water
Composition:-
Lab-Lemco -10gm
peptone-10gm
NaCl- 5gm
Distilled water-1000ml
- When 2-3% agar is added ,then we have it as nutrient agar.
- For solid media – agar concentration is 0.2-0.4%
Uses:-
1. This is basis of most of the media used in the study at common
pathogenic bacteria.
2. It is used for subcultures of certain organisms.
SEMI-SOLID MEDIA

 Reducing the amount of agar to 0.2-0.5% renders a medium

semi-solid, useful in demonstrating bacterial motility and
separating motile from non-motile strains. Certain transport
media such as Stuart’s and Amies media are semi-solid in
consistency. Hugh & Leifson’s oxidation fermentation test
medium as well as mannitol motility medium are also semi-solid.

Motility of V. anguillarum strains in semi-solid medium.

 BIPHASIC MEDIA

 Sometimes, a culture system comprises of

both liquid and solid medium in the same
bottle. This is known as biphasic medium
(Castaneda system for blood culture). The
inoculum is added to the liquid medium and
when subcultures are to be made, the bottle
is simply tilted to allow the liquid to flow over
the solid medium. This obviates the need for
frequent opening of the culture bottle to
subculture.
PEPTONE WATER

 TYPE : Basic liquid media

 APPEARANCE : clear, colorless, watery, usually in test tube
 Composition :

PEPTONE 10 g
SODIUM CHLORIDE, NaCl 5g
WATER 1 litre
USES OF PEPTONE WATER

➢ The media is used chiefly as the basis for carbohydrate

fermentation media.

➢ Nutrient broths may contain a small amount of sugar

derived from meat and it is essential that the basal
medium to which various carbohydrates are added
for fermentation tests should be free from natural
sugars.
✓ It is also used to test the formation of indole.
✓ Culture of organisms for demonstration of motility
COMPLEX MEDIA
Complex media have added ingredients for bringing out certain properties
for bringing out certain properties or providing special nutrients
required for growth of the bacterium in question.
SYNTHETIC MEDIA
✓ These are prepared from pure chemicals and the exact compositions of
medium is very well known.
Example :- Dubo’s medium

SEMIDEFINED MEDIA
✓ In these media the exact chemical composition of the constituents is
not known because substances like meat and peptone are used.
✓ Most of the culture media used for routine diagnostic work are
semidefined culture media.
SPECIAL MEDIUM
 ENRICHED MEDIA

❖ When basal medium is added with some nutrients such as blood,

serum or egg is called enriched media.
❖ They are used to grow bacteria which are more exacting in their
nutritional needs.
Examples:-
Dorset’s Egg Medium.
✓ It is a creamy coloured opaque
slope kept in screw copped bottle
✓ Selective medium for isolation
of Mycobacterium tuberculosis.
✓ Composition: Hen’s egg, Nutrient broth
 BLOOD AGAR: Contains
mammalian blood (usually sheep
or horse), typically at a
concentration of 5–10%. BAP are
enriched, differential media used
to isolate fastidious organisms
and detect hemolytic activity
 CHOCOLATE AGAR: A non-
selective, enriched growth
medium. containing red blood
cells that have been lysed by
slowly heating to 80 °C.
Chocolate agar is used for
growing fastidious bacteria,
such as Haemophilus
influenzae.
Cotd.

 Enrichment media: liquid media that also serves to inhibit

commensal in the clinical specimen.
 Selenite F broth and alkaline peptone water are used to
recover pathogens from fecal specimens.
 SELECTIVE MEDIA
❖ It is a medium in which certain substances are present which inhibit all
other bacteria except the desired bacteria.
❖ It encourages the growth of particular species from a mixed inoculum.

Example:- TCBS
-It is light green translucent medium kept in petridish
-It is selective medium for Vibrio cholera
-Principle:-
Bile salt inhibit the growth of normal
commensals (unwanted bacteria).

✓ Vibrio chloerae produce acid by fermentation

of sucrose which acts on bromothymol blue
(indicator) producing yellow colonies.
Salmonella Shigella Agar Salmonella-Shigella agar plate
with DCA (SS)
Selective media: Thayer Martin Medium

 Selective for Neisseria

gonorrhoeae
 5% chocolate sheep blood and
antibiotics (VCN inhibitor):
vancomycin, colistin, nystatin,
trimethoprim)
Selective media: Lowenstein-Jensen (LJ)
medium

 For Mycobacterium species

 incorporated congo red and
malachite green to inhibit
unwanted bacteria.
Selective media: EMB agar

 Selective for gram-negative

bacteria.
 The dye methylene blue in the
medium inhibits the growth of
gram-positive bacteria
 small amounts of this dye
effectively inhibit the growth of
most Gram-positive bacteria
Microbiological Media: Functional use /
application
 Differential media
Certain media are designed in such a way that different bacteria can be
recognized on the basis of their colony colour

 Contain indicators that react differently with different organisms (for

example, producing colonies with different colors) →Various approaches
include incorporation of dyes, metabolic substrates etc, so that those
bacteria that utilize them appear as differently coloured colonies.

 Used in identifying specific organisms

 E.g. MacConkey’s agar, CLED (Cystine lactose electrolyte deficient) agar,
TCBS (Thiosulfate-citrate-bile salts-sucrose) agar, XLD agar, etc
 MacConkey Agar

 Contains bile salts (to inhibit most

Gram-positive bacteria), crystal violet
dye (which also inhibits certain Gram-
positive bacteria)
 Using neutral red pH indicator →
distinguishes Gram-negative bacteria
that can ferment the sugar lactose
(Lac+) from those that cannot (Lac-)
fermentation.
 Lac+ bacteria: E. coli, Enterobacter,
Klebsiella will produce acid, lowers the
pH of the medium → pink colonies.
 Lac- bacteria:
Salmonella, Proteus species, Yersinia, P.
aeruginosa, Shigella utilize peptone,
forms ammonia → raises the pH of
the agar, → forms white/colorless
colonies.
CLED
(Cystine Lactose Electrolyte Deficient
medium)
 It is a valuable non-inhibitory growth medium used in the
isolation and differentiation of urinary organisms.

 Being electrolyte deficient, it prevents the swarming of Proteus

species
Description: Lactose & non
lactose fermenters on CLED
medium.
 XLD Agar

 Xylose lysine deoxycholate agar,

 For isolation (recovery)
of Salmonella and Shigella species
from clinical samples and from food
 Salmonella species: red colonies,
some with black centers. The agar
itself will turn red due to the
presence of Salmonella type colonies.
 Shigella species: red colonies.
 Coliforms: yellow to orange colonies.
 Pseudomonas aeruginosa: pink, flat,
rough colonies. This type of colony
can be easily mistaken for Salmonella
due to the color similarities.
Fungal media

 Dermatophyte test medium

(DTM)
 Contains cycloheximide to
inhibit saprotrophic growth,
antibiotic to
inhibit bacterial growth, and
phenol red a pH indicator
useful in distinguishing
a dermatophyte fungus
INDICATOR MEDIUM
❖ These media contain an indicator which changes colour when bacteria grow on
them.

❖ Example:- Wilson and Blair medium

✓ For isolation of Salmonella typhi and S. paratyphi

✓ They appear as black colonies

✓ Principle:- The black colour of colonies is due to the ability of these organisms
to reduce bismuth sulphite to sulphide in the presence of glucose coliforms are
inhibited by brilliant green and bismuth sullphite
Cotd.

 Transport media : temporary storage of specimens being

transported to the laboratory for cultivation.
 maintain the viability of all organisms in the specimen without
altering their concentration.
 contain only buffers and salt.
 lack of carbon, nitrogen, and organic growth factors so as to
prevent microbial multiplication.
 transport media used in the isolation of anaerobes must be free
of molecular oxygen.

 Cary Blair medium for campylobacter species

 Alkaline peptone water medium for v. cholerae.
Cotd.

 Anaerobic media
 Media for anaerobes may have to be supplemented with
nutrients like hemin and vitamin K
 Boiling the medium serves to expel any dissolved oxygen

 Example: Thioglycollate medium, Robertsons Cooked Meat

Medium
 Growth in Batch Culture

 A “batch culture” is a closed system in broth medium in which no

additional nutrient is added after inoculation of the broth.
 Typically, a batch culture passes through four distinct stages:
 Lag stage
 Logarithmic (exponential) growth
 Stationary stage
 Death stage
 Measurement of
Microbial Growth

 Microscopic cell counts

 Calibrated “Petroff-Hausser
counting chamber,” similar to
hemacytometer, can be used
 Generally very difficult for
bacteria since cells tend to
move in and out of counting
field
 Can be useful for organisms
that can’t be cultured
 Special stains (e.g. serological
stains or stains for viable cells)
can be used for specific
purposes
 Cotd.

 Serial dilution and colony

counting
 Also know as “viable cell
counts”
 Concentrated samples
are diluted by serial
dilution
 The diluted samples can
be either plated by
spread plating or by pour
plating
Cotd.

 Serial dilution (cont.)

 Diluted samples are spread onto media in petri dishes and incubated
 Colonies are counted. The concentration of bacteria in the original
sample is calculated (from plates with 25 – 250 colonies, from the
FDA Bacteriological Analytical Manual).
 A simple calculation, with a single plate falling into the statistically
valid range, is given below:

CFU # colonies counted

in original sample =
ml (dilution factor)(volume plated, in ml)
 Cotd.
● Membrane filtration
– Used for samples with low microbial concentration
– A measured volume (usually 1 to 100 ml) of sample is filtered
through a membrane filter (typically with a 0.45 μm pore size)
– The filter is placed on a nutrient agar medium and incubated
– Colonies grow on the filter and can be counted
Cotd.
 Turbidity
 Based on the diffraction or “scattering” of light by bacteria in a broth
culture
 Light scattering is measured as optical absorbance in a
spectrophotometer
 Optical absorbance is directly proportional to the concentration of
bacteria in the suspension
Cotd.

 Mass determination
 Cells are removed from a broth culture by centrifugation and weighed
to determine the “wet mass.”
 The cells can be dried out and weighed to determine the “dry mass.”
 Measurement of enzymatic activity or other cell components
Ref.

 http://www.microbiologyinfo.com/list-of-culture-media-used-
in-microbiology-with-their-uses/