METHODS IN MICROBIOLOGY

 Methods in microbiology are those techniques which are used to study microbial
populations.
 Terms
i) Culture -A population of microorganisms that is grown under
defined conditions
ii) Pure culture – A culture of a single microorganism.
iii) Isolation – Separation of a particular microorganism from a mixed
population. The process of getting a pure culture of a
microorganism from a mixed population.
iv) Cultivation – Growing microbes in the lab under artificial
environments or conditions.
v) Culture medium/media - Any material prepared for the growth of
bacteria in a laboratory.

Methods of isolation of microorganisms

Petriplate methods
Involve culturing of microorganisms in petri dishes on suitable growth media and
incubating at suitable temperatures of growth.
a) Streak plate method
 An inoculating loop is used to pick and introduce a small amount of culture on
the surface of a growth medium.
 This is followed by incubation at suitable temperatures for growth to occur.
 The streaking is done on the surface of solidified medium.
 The streaked plates show the heaviest growth in the first sections and less
growth on the subsequent sections.

Further reading: https://microbeonline.com/streak-plate-method-principle-

purpose-procedure-results/

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 b) Spread plate method
 A small amount of sample is poured onto the surface of solid medium and
spread evenly by swirling the plate back and forth before incubation.

c) Pour plate method

 The sample to be cultured for growth is poured into an empty petridish
 Melted/molten medium (about 50oC) is poured on-top of the sample.
 The plate is swirled back and forth to mix well before incubation.
 Assignment 2: Learn to differentiate the three petri plate methods plus
advantages and disadvantages of each method

DILUTION METHODS
 Involves making dilution series (a series of dilutions), plating and incubating.

 A serial dilution is a series of sequential dilutions used to reduce a dense culture

of cells to a less concentrated one.

 Method:

 A set of serial dilutions is made

 A sample of each dilution is cultured by the pour plate

technique

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  Colonies grow within and on the agar medium

 The colonies can be enumerated to estimate the number of

viable cells in the initial sample

 To get the number of bacterial cells in the initial sample, the number of colonies
counted on the plate is multiplied by the reciprocal of the dilution factor.

CFUs = No. of colonies on agar plate x reciprocal of dilution factor

 For example if 32 colonies are counted on a plate of 1:10,000; then the number of
bacterial cells per ml of the initial sample is 32 x 10,000 = 32,0000

CULTIVATION OF MICROORGANISMS
Requirements/Essentials for growing microorganisms
a) Growth Nutrients
 Laboratory growth media must contain all the nutrients necessary for the
synthesis of new organisms.
 Carbon – required by most organisms
 Nitrogen – Can be in the form of NO3- or NH4+. Is a component of amino acids
 Sulphur - Is provided as sulphates. Is a constituent of proteins and RNA.
 Assignment 3: Read about the nutrients required by microorganisms

b) Growth factors
 Are organic compounds which microbial cells must contain in order to grow but
which they are unable to synthesize.

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  Include amino acids or vitamins which cannot be synthesized by the
microorganism being cultivated

c)Oxygen requirements
 Anaerobic microorganisms are cultivated in tightly sealed tubes.
 Anaerobic bacteria do not grow in the presence of oxygen.
 They do not use oxygen for growth and metabolism but obtain their energy from
fermentation reactions.
 Anaerobic bacteria are killed by oxygen or toxic oxygen radicals.
 Oxygen must be provided if the microorganism is aerobic.
 Aeration can be done by bubbling air and shaking the growth tube so that they
get enough oxygen for respiration.
o Bacteria may be classified into four groups on oxygen requirement
 Aerobes
o They cannot grow without oxygen
o Require oxygen for cellular respiration
o e.g. Mycobacterium tuberculosis.
 Facultative anaerobes
o These grow under both aerobic and anaerobic
conditions.
o e.g. Enterobacteriaceae.
 Anaerobes
o They only grow in absence of free oxygen
o In the presence of oxygen they are killed
o e.g. Clostridium, Bacteroides
 Microaerophiles
o grow best in oxygen less than that present in the air
o e.g. Campylobacter.
 Aerotolerant
o Do not use oxygen for respiration but are not affected
by the presence of oxygen.
o They tolerate oxygen
d) pH requirements

 Like temperature, pH also plays a role in determining the ability of bacteria to

grow or thrive in particular environments.
 Most commonly bacteria grow optimally within a narrow range of pH between
6.7 and 7.5.
o Acidophiles

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  prefer acidic conditions.
 exampleThiobacillusferrooxidans can survive at pH 1.
o Alkalinophiles
 Example Vibrio choleracholera, can thrive at a pH as high as 9.0.
o Neutrophiles
 Buffers are added to culture media to increase acidity or alkalinity based on the
pH requirements of the microbe being cultivated.

d) Temperature requirements
 Minimum
o The least temperature at which a particular species will grow
 Maximum
o the highest temperature at which they will grow.
 Optimum
o The temperature at which their growth is optimal.
o Mesophiles – moderate temperatures 20 -45oC
o Psychrophiles,
 which prefer cold temperatures,
 0-15°C.
 psychrotrophs.
o Thermophiles
 thrive in very hot environments,
 many having an optimum growth temperature between (50°C)
and (60°C).
o Extreme thermophiles/hyperthermophiles
 grow at temperatures above (91°C).
 Temperature of growth is controlled in the incubators

Example of each group of microbes depending on their growth properties…Prescott’s

principles of Microbiology…page 139

e) Light requirements
 Some microbes like photosynthetic bacteria and algae need light.
 Light may affect temperature so a water bath is used to cool the culture to the
desired temperature.
 Day light /natural light– uncontinuous, uncontrolled illumination
 lamp light/artificial light – provides continuous illumination

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GROWTH MEDIA

 A culture medium is any material used for the growth of bacteria in a laboratory.
 Microbes that grow and multiply in or on a culture medium are known as
microbial cultures.

Classification of media based on nature

1. Solid media
 Is used for growth in petri dishes.
 Solid media is used for the isolation of bacteria as pure culture
 Advantages of using solid media
 Bacteria may be identified by studying the colony characteristics
o Size, elevation, color,borders,texture
 Mixed bacteria can be separated.
 Solidifying agents particularly agar are used to solidify the growth media.
 Agar is a polysaccharide that is extracted from a seaweed (marine red algae)
 Agar is an ideal solidifying agent as it is:
 Biologically inert, i.e. no influence on bacterial growth. It is not
metabolized by bacteria but serves to simply hold/gel the nutrients
in an aqueous growth medium.
 It gels/solidifies and remains solid at 40°C and melts at 100oC
 It is transparent.

 Selective media

 Selects one kind of microorganism out of a large population.
 Contains ingredients (salts & dyes) that inhibit growth of other microbes
while allowing growth of the desired organisms.
 NB: The use of selective media can be done together with modified pH,
temperature and other conditions so that the growth conditions become
more selective for desired microorganism.

 EMB agar
o Contains dyes (eosin and methylene blue) that are toxic to
Gram +ve bacteria.
o Thereby inhibits growth of Gram +ve bacteria while
allowing Gram –ve bacteria to grow.

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 o Also contains bile salt that is toxic for Gram –ve bacteria
other than coliforms.
 Brilliant green agar
o inhibits Gram +ve and most Gram +ve bacteria and is
used to isolate Salmonella species.

 YM agar (yeast and mold)

o Has a low pH deterring bacterial growth.
o Is a medium modified with yeast extract and peptone.
o Used in the cultivation of yeasts and molds.

 MacConkey agar
o Selective for Gram –ve bacteria that ferment lactose.
o Has crystal violet dye and bile salts that inhibit the
growth of Gram +ve organisms.
o It has the neutral red dye (pH indicator) for selection of
lactose fermenters (turns pink if the microbes ferment
lactose)

 Mannitol salt agar

o Selective for Gram +ve bacteria.
o Contains a high concentration of salt (7.5-10% NaCl) for
selection of microbes that can tolerate high salt
concentrations
o Selective for Staphylococcus aureus

 Xylose Lysine Deoxycholate (XLD)

o Selective for Salmonella and Shigella.
o Is red (has phenol red dye) and changes to yellow when
pH is lowered.
o Salmonella can ferment xylose and produce acid thereby
appearing as yellow colonies on XLD medium.
o Shigella does not metabolize xylose and remain as red
colonies on XLD medium.

 Differential media/Indicator media

 Contain dyes for differentiation of required microbes among mixed
cultures.

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  Differential media are used to distinguish among different
organisms growing on the same medium.
 Blood agar
o Contains 5-10% bovine (sheep/horse) blood
o Used to identify/differentiate organisms that lyse red
blood cells e.gStreptococcus pyogenes
o Certain bacteria when grown in blood agar produce
haemolysis around their colonies while others do not.
o If bacteria growing on the medium are capable of
haemolysis, the medium changes colour (becomes
transparent).
Assignment: types of hemolysis exhibited by bacteria on blood agar

 MacConkey agar – differential for lactose fermenters

 Mannitol Salt Agar

o Differential for mannitol fermenters such as Staphylococcus
aureus.
o It contains mannitol and phenol red indicator for detection
of acids produced by mannitol – fermenting staphylococci.
o Fermentation of mannitol produces acidic byproducts that
cause the phenol red indicator to turn yellow.
o S. aureus produces yellow colonies while other staphylococci (non
mannitol-fermenter) produce pink colonies (no colour change in
the medium).

 Enriched media
 Contain nutrients that enhance the growth of certain
microorganisms that can utilize the components in the media.
 Used to encourage the growth of particular microorganisms in a
mixed culture.
 Contain nutrients required to support the growth of
microorganisms present in particular environments.
 Particularly employed for fastidious microbes
 Blood agar
o Nutritionally rich in whole blood and supplements
the basic nutrients
 Chocolate agar/heated blood agar

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 o Enriched with heat–treated blood which turns brown
and gives it a brown colour.
o Heating the blood inactivates growth inhibitors
o Used in isolation of Haemophilusinfluenzae and
Neisseria meningitides
2. Broth/ Liquid media
 Used for growth in test tubes, bottles or flasks.
 Do not contain agar.
 Broth is good for growing large batches of organisms.
 Most organisms grow uniformly producing turbidity that can be
quantified
 Commonly used in monitoring cell mass during growth
 E.g NB and Trypticase Soy Broth
 Bacteria growing in liquid media can exhibit three types of growth
o Sediment – at the bottom of tube
o Turbid growth – throughout the tube
o Pellicle – thick growth at the top of tube
 May be due to affinity for oxygen

Disadvantages of liquid media

 Bacterial colonies do not form in broths
o A colony is a collection of bacteria that have arisen from the
division of a single parent cell.
 It’s not easy to identify contamination
 Colony morphology cannot be assessed.

3. Semi-solid media
 Such media are soft and are useful in demonstrating bacterial motility and in
separating motile from non-motile strains.
 Contain little quantities of agar to make them viscous but not solid

Classification based on components

1. Chemically defined/synthetic media

 The exact chemical composition is known.

 Usually composed of biochemical/synthetic components.
 Also called minimal medium

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  Provide only the exact nutrients needed by an organism to
grow.
 Their use requires the user to know the exact nutritional requirements of the
organisms being cultivated.
2. Undefined/complex/basal media
 The exact chemical composition is not known.
 Composed of materials of biological origin such as blood, milk, yeast, beef
extracts the chemical composition of which is not determined.
 The extracts are normally included to provide vitamins and minerals.
 Provide a range of growth factors that may be required by any given
organism
 Useful in:
o Cultivating unknown bacteria or fastidious microbes
 Bacteria with complex nutritional requirements.
o Cultivating heterotrophic microorganisms such as pathogens.
 Assignment: Find out examples of defined and undefined media

Other types of media

1. Reducing/anaerobic media
 Anaerobic bacteria need this special type of media for growth.
 Media for growth of anaerobes should be supplemented with nutrients that
chemically remove molecular oxygen (O2) that might interfere with their
growth
 Contain reducing agents that react with molecular oxygen and reduce it to
water
 Example: Thioglycolate broth
o Contains small amounts of agar making it viscous but still liquid
o Commonly used for testing an organisms requirement for oxygen
o After all the O2 has been removed from the medium, fresh O 2 can only
enter at the top of the tube where the medium contacts air
o A redox dye/indicator-resazurin is included
It is pink when oxidized

o Colourless when reduced

o Helps to differentiate oxic and anoxic zones
 Obligate aerobes
o grow only at the top of the tubes
o only up to where oxygen can penetrate

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  Facultative organisms
o grow throughout the tube but grow best near the
top
o growth is better at the top because they prefer to
respire if oxygen is present
 Microaerophiles
o grow close to the top but not right at the top
o Grow away from oxic zone but close enough to
receive little amounts of oxygen
 Obligate anaerobes
o grow only at the bottom of the tube where O2 does
not penetrate
o Are sensitive to oxygen and grow away from the
top of tube
 Aerotolerant organisms
o Grow uniformly throughout the tube
o Growth may not be better near the surface because
the organisms can only ferment

2. Transport media
 Used to transport specimens from sampling sites to laboratories
 Used for samples that must be transported to the laboratory immediately
after collection to prevent overgrowth of contaminating organisms.
 Transport media should fulfill the following criteria:

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  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.

Examples of transport media include:

 Thioglycolate broth for strict anaerobes.
 Stuart medium
 Cary Blair medium

3. Storage media
 Media used for storing/preserving bacteria for long periods of time.
o Examples: Egg saline medium.
Assignment:
 Give a detailed account on how aerobes and anaerobes are cultured under
artificial laboratory conditions.
o Describe the types of media that can be used
o Aeration techniques for aerobes
o Method of exclusion of oxygen for anaerobes

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