MICROBIAL GROWTH

LEARNING OUTCOMES
• Explain the physical and chemical requirements of growth
• Distinguish between the various types of culture media
and explain how they affect bacterial growth
• Explain the methods on microorganism preservations
• Describe the growth patterns of microbes
• Discuss the methods of measuring cell growth
• Explain on biofilms
 PHYSICAL REQUIREMENTS
• The temperature requirements for microbial growth can be categorized into
physical requirements or chemical requirements.
• Among the physical requirements that are crucial in the growth of microbes:
• Temperature
• pH
• Osmotic pressure
• Temperature:
• Each bacteria have
• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature
 PHYSICAL REQUIREMENTS
• Temperature:
• Microorganisms can be classified into FIVE groups
• Psychrophiles (1 mark) can grow well at 0°C, have optimal growth at 15°C
or lower, and usually will not grow above 20°C (1 mark)
• Psychrotrophs (facultative psychrophiles) (1 mark) can also grow at 0°C,
but have growth optima between 20°C and 30°C, and growth maxima at
about 35°C (1 mark)
• Mesophiles (1 mark) have growth optima of 20 to 45°C, minima of 15 to
20°C, and maxima of about 45°C or lower (1 mark)
• Thermophiles (1 mark) have growth optima of 55 to 65°C, and minima
around 45°C (1 mark)
• Hyperthermophiles (1 mark) have growth optima of 80 to 110°C and
minima around 55°C (1 mark)
PHYSICAL REQUIREMENTS

Taken from Brock Biology of Microorganism 11/e

 PHYSICAL REQUIREMENTS
• pH
• Most bacteria grow best at pH near neutral (6.5-7.5), however there are
exceptions
• Acidophiles – highly tolerant of low pH
• Neutrophiles – near neutral
• Alkaliphiles – highly tolerant of high pH

preserve food
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 PHYSICAL REQUIREMENTS
• Osmotic pressure
• When a bacterial call is placed in a hypertonic solution, the cellular water
content will pass out through the plasma membrane and cause plasmolysis
(shrinkage of cell’s cytoplasm)
• salting can help preserve food – salted fish
• Sweetened condense milk
• Extreme halophiles – adapted to salty environment that they require it for
growth  aka obligate halophiles
• Facultative halophiles  do not require high salt conc, but can tolerate up to
2% salt (which most bacteria cannot)

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 CHEMICAL REQUIREMENTS
• Carbon
• Carbon is needed because it is the backbone structure of any organic molecules
• It is the second most important requirement for growth after water
• Chemoautotrophs and photoautotrophs derive their carbon from carbon dioxide
• Chemoheterotroph get their carbon from their energy source
• Nitrogen, sulfur and phosphorus
• Nitrogen and sulfur is needed for amino acids  backbone of protein
• Nitrogen and phosphorus is needed to make nucleotides  DNA molecule
• ATP  energy storage

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 CHEMICAL REQUIREMENTS
• Nitrogen, sulfur and phosphorus
• Most organisms are not able to use molecular nitrogen (N2) directly
• Some microbe have the ability to fix the molecular nitrogen into ammonium
 nitrites which is readily used by most organism
• Trace elements
• Element that are needed in small amount
• Minerals  iron, copper, zinc (usually co-factors for enzymatic reaction)
• Oxygen
• Different microbe have different requirements for oxygen
• Microbe can be classified based on their oxygen requirements
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 CHEMICAL REQUIREMENTS
• Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be
detoxified.
• Three main enzymes break down those toxic byproducts: superoxide dismutase, peroxidase, and
catalase.
• Each one catalyzes a different reaction.
• Reactions of type seen in Reaction 1 are catalyzed by peroxidases.
(1) X−(2H+)+H2O2→oxidized-X+2H2O
• In these reactions, an electron donor (reduced compound; e.g., reduced nicotinamide adenine
dinucleotide [NADH]) oxidizes hydrogen peroxide, or other peroxides, to water. The enzymes
play an important role by limiting the damage caused by peroxidation of membrane lipids.
• Reaction 2 is mediated by the enzyme superoxide dismutase (SOD) and breaks down the
powerful superoxide anions generated by aerobic metabolism:
(2) 2O2-+2H+→H2O2+O2
• The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.
(3) 2H2O2→2H2O+O2
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 CULTURE MEDIA
• Culture  microbes growing in or on
culture media
• Culture media is the food used to grow
and control microbes.
• Culture medium or growth medium is a
liquid (broth) or gel (agar) designed to
support the growth of microorganisms.
• It has to be sterile  only the intended
microbe will grow
• There are different types of media
suitable for growing different types of
cells.
• Chemically defined  know the
exact composition of the media
• Complex  contain extract and/or
digest of yeast, meat or plant
 CULTURE MEDIA
• Types of culture media:
• Enrichment media
• Defined media which has been added with specifically required
substances such as blood, serum or egg
• Helps in promoting the growth of targeted microbe which in normal
circumstances may be difficult to grow
• Selective media
• selects the growth of a specific microbe while inhibiting growth of others
• by adding certain salt or dyes
• Differential media
• Differentiate a specific bacteria from others
 COMMONLY USED CULTURE MEDIA

• Nutrient agar/broth
• General purpose complex media use for
cultivating bacteria
• Supports wide range of non-fastidious
bacteria
• Contain 0.5% peptone (nitrogen source),
0.3% beef/yeast extract (vitamin,
carbohydrates, nitrogen and salt ), 0.5%
NaCl

https://microbiologyinfo.com/nutrient-agar-composition-
preparation-and-uses/
 COMMONLY USED CULTURE MEDIA
• Blood agar – for microbes that require blood & chocolate agar – red
blood cells have been lysed
• Both are considered enrichment media – contain blood or lysed red
blood cells
• Helps in promoting the growth of targeted microbe which in normal
circumstances may be difficult to grow

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 COMMONLY USED CULTURE MEDIA
• Blood agar – especially useful in determining specific bacteria based on their ability to
break down blood
• Differentiation properties
• Alpha () hemolysis – partial hemolysis
• Beta () hemolysis – full hemolysis
• Gamma () hemolysis – no hemolysis

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 COMMONLY USED CULTURE MEDIA
• Mannitol salt agar  contains high conc. of NaCl that selects for
microbe that can withstand osmotic pressure
• Also contain mannitol and phenol red (pH indicator)  microbe
that can ferment mannitol and produce acid  yellow
(differential properties)

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 COMMONLY USED CULTURE MEDIA
• Salmonella-Shigella agar  selects for growth of Salmonella and
Shigella
• Selective media  Bile Salts, Sodium Citrate and Brilliant Green serve to
inhibit Gram-positive & coliform
• Differential media
• Thiosulfate and Ferric Citrate permit detection of hydrogen sulfide by the
production of colonies with black centers.
• Lactose fermenters  pink colonies

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 COMMONLY USED CULTURE MEDIA
• Thiosulfate Citrate bile salts sucrose (TCBS) agar
• Contain selective agents (sodium thiosulphate, sodium citrate)
providing an alkaline pH to inhibit Gram-positive organisms and
suppress coliforms
• Ox Bile: The bile salts inhibit growth of Gram-positive
microorganisms
• Ferric citrate: Sodium Thiosulfate is also a sulfur source, and acts
with Ferric Citrate as an indicator to detect hydrogen sulfide
production.
• Differentiation:
• V. cholerae large and slightly flattened, yellow colonies with
opaque centers and translucent peripheries.
• V. parahaemolyticus  green to blue-green colonies as it does
not ferment sucrose.
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 ANAEROBIC CULTURE METHOD
Because 21% of our air is composed of oxygen, culturing anaerobic
microbes require special methods.

1. Anaerobic chamber
• Especially of strict anaerobes
2. Anaerobic jar
• For not-so-strict anaerobes
3. Candle jar
• For bacteria requiring high conc. of carbon dioxide and low
oxygen
4. Oxyrase addition to culture media  remove all oxygen in
media
ANAEROBIC CHAMBER

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 ANAEROBIC JAR

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CANDLE JAR

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 PURE CULTURE
• Contain only one species or strain.
• Most patient specimens and environmental samples
contain several different kinds of bacteria
• Streak-plate method is commonly used
• Colony formation: A population of cells arising from a
single cell or spore or from a group of attached cells (also
referred to as CFU).
• Only ~1% of all bacteria can be successfully cultured
• Aseptic technique critical!
 OBTAINING PURE CULTURE

MICROBIOLOGY - AN
INTRODUCTION, by
Tortora, Funke, and Case,
10th edition
 GROWTH OF BACTERIAL CULTURES
• Bacteria grow by binary fission in an exponential growth pattern
• Most yeast grow by budding
• Generation time is referred to as the time required for cell to divide
(also known as doubling time)
• Ranges from 20 min (E. coli) to > 24h (M. tuberculosis)

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 GROWTH OF BACTERIAL CULTURES
• Calculating bacterial cells by no of generation MICROBIOLOGY - AN INTRODUCTION
Tortora, Funke, and Case, 10th edition
GROWTH OF BACTERIAL CULTURES

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GROWTH OF BACTERIAL CULTURES
Arithmetic vs. Exponential Plotting

MICROBIOLOGY - AN INTRODUCTION
Tortora, Funke, and Case, 10th edition
MEASUREMENT OF BACTERIAL GROWTH

• Measurement of bacterial growth can be done in two ways:

• Direct method
• Viable cell count  plate count
• Filtration
• MPN – most probable number
• Direct microscopic count
• Indirect method
• Optical density reading
• Metabolic activity
• Dry weight
Viable cell counts: Plate counts: Serial dilutions put on
plates CFUs (colony forming unit)

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MICROBIOLOGY - AN INTRODUCTION
Tortora, Funke, and Case, 10th edition
 Most Probable Number – detect coliforms in water

• Principle
• Water to be tested is diluted serially and inoculated in lactose broth
• Coliforms, if present in water, utilize the lactose present in the medium to
produce acid and gas.
• The presence of acid is indicated by color change of the medium and the
presence of gas is detected as gas bubbles collected in the inverted Durham
tube present in the medium.
• The number of total coliforms is determined by counting the number of tubes
giving positive reaction (i.e both color change and gas production) and
comparing the pattern of positive results (the number of tubes showing
growth at each dilution) with standard statistical tables.
• MPN test is performed in 3 steps
• Presumptive test
• Confirmatory test
• Completed test
 Most Probable Number – detect coliforms in water

• What are coliforms??

• rod-shaped Gram-negative non-spore forming and motile or non-motile
bacteria which can ferment lactose with the production of acid and gas when
incubated at 35–37°C
• They are a commonly used indicator of sanitary quality of foods and water
• Coliforms can be found in the aquatic environment, in soil and on vegetation; they are
universally present in large numbers in the feces of warm-blooded animals.
• While coliforms themselves are not normally causes of serious illness, they are easy
to culture, and their presence is used to indicate that other pathogenic organisms of
fecal origin may be present.

• MPN test is performed in 3 steps

• Presumptive test
• Confirmatory test
• Completed test
Most Probable Number – Presumptive test

Double strength Single strength Single strength

lactose broth lactose broth lactose broth
50 coliforms in 100 ml of sample…
 Most Probable Number – Confirmatory test
• Some microorganisms other than coliforms also produce acid and gas from lactose
fermentation.
• In order to confirm the presence of coliform, confirmatory test is done.
• From each of the fermentation tubes with positive results transfer one loopful of
medium to:
• 3 ml lactose-broth or brilliant green lactose fermentation tube,
• to an agar slant and
• 3 ml tryptone water.
• Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas
formation after 24 ± 2 hours. If no gas production is seen, further incubate up
to maximum of 48 ±3 hours to check gas production.
• The agar slants should be incubated at 37°C for 24± 2 hours and Gram-stained
preparations made from the slants should be examined microscopically.
• The formation of gas in lactose broth and the demonstration of Gram negative,
non-spore-forming bacilli in the corresponding agar indicates the presence of a
member of the coliform group in the sample examined.
• The absence of gas formation in lactose broth or the failure to demonstrate
Gram-negative, non-spore-forming bacilli in the corresponding agar slant
constitutes a negative test (absence of coliforms in the tested sample).
 Most Probable Number – Completed test

• Since some of the positive results from the confirmatory test may be false, it is
desirable to do completed tests.
• For this inoculum from each positive tube of the confirmatory test is streaked on a
plate of EMB agar.
• In this process, a loopful of sample from each positive brilliant green lactose
tubes is streaked onto selective medium like Eosin Methylene Blue agar or Endo’s
medium.
• One plate each is incubated at 37°C and another at 44.5± 0.2°C for 24 hours.
• High temperature incubation (44.5 ±0.2) is for detection of thermotolerant E.coli.
• Following incubation, all plates are examined for presence of typical colonies.
• Coliforms produce colonies with greenish metallic sheen which differentiates
it from non-coliform colonies (show no sheen).
• Presence of typical colonies on high temperature (44.5 ±0.2) indicate
presence of thermotolerant E.coli.
 Most Probable Number

• Advantages of MPN :
• Ease of interpretation, either by observation or gas emission
• Sample toxins are diluted
• Effective method of analyzing highly turbid samples such as sediments, sludge,
mud, etc. that cannot be analysed by membrane filtration.

• Disadvantages of MPN:
• It takes a long time to get the results
• Results are not very accurate
• Requires more hardware (glassware) and media
• Probability of false positives
Direct Microscopic Count

MICROBIOLOGY - AN INTRODUCTION
Tortora, Funke, and Case, 10th edition
 Spectrophotometry to measure turbidity

• Does not differentiate between

dead cells or living cells
• Only accurate to a certain reading

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 PRESERVING BACTERIAL CULTURES

• Deep-freezing: Rapid cooling of pure culture in suspension liquid to

–50°to –95°C. Good for several years.

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 PRESERVING BACTERIAL CULTURES

• Lyophilization: (freeze-drying): Frozen (–54° to –72°C) and

dehydrated in a vacuum. Good for many years.

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 PRESERVING BACTERIAL CULTURES

• Cryopreservation: freezing in liquid nitrogen (-196C)

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 BIOFILMS
• Microbial communities form slime or hydrogels
• Starts via attachment of planctonic bacterium to surface structure.
• Bacteria communicate by chemicals via quorum sensing
• Sheltered from harmful factors (disinfectants etc.)
• Cause of most nosocomial infections
• Clinical Focus: Delayed Bloodstream Infection Following Catheterization
 LEARNING OUTCOMES
• Explain the physical and chemical requirements of growth
• Distinguish between the various types of culture media
and explain how they affect bacterial growth
• Explain the methods on microorganism preservations
• Describe the growth patterns of microbes
• Discuss the methods of measuring cell growth
• Explain on biofilms