Isolation and Cultivation of Microorganisms Culture Media

Module 7 | S-BIOL316 General Microbiology Lecture

● Enrichment medium
Definition of Terms
- Used to increase the number of
microorganisms with unusual physiological
● Pure culture (Axenic culture) characteristics.
- A culture which contains a single species of - With special nutrients (ex: blood).
microorganism. - Ex: blood agar.
- A population of cells arising from a single cell. ● Assay media
● Cultivation - Prescribed composition used for assay of
- Increasing the population of microorganisms by vitamins, AAs, and antibiotics.
providing their nutritional and physical - Used to determine qualitative/quantitative
requirements. production of such a compound by an
● Nutrients organism.
- Extracellular substances which provide the cell
with materials for building protoplasm and for
energy generation. Isolation Techniques
● Culture medium
- Any nutrient material for growth and cultivation 1. Plating
of microorganisms in the laboratory. ● Colony
- A macroscopically visible (surface or
subsurface) growth or cluster of
Uses of Culture Media
microorganisms on a solid medium.
● Streak plating
- For growth and maintenance of microbial cultures.
- To favor the production of particular compounds.
- To study microbial action on some constituents of
the medium.

Types of Culture Media

- According to physical state:

● Liquid (broth)
- No solidifying agent.
● Spread plating
● Semi-solid
- 0.1-0.5% solidifying agent.
● Solid
- 1-5-2.0% solidifying agent.
➔ Solidifying agent: agar or gelatin.
- According to chemical composition:
● Synthetic
- All components are chemically defined.
● Complex
- Not all components are chemically defined. ● Pour plating
- Ex: potato infusion, beef extract, yeast extract.
- According to principle function, purpose, or
application:
● General purpose media
- Can support most or almost all types of
species.
- Ex: nutrient agar (NA).
● Differential media
- Distinguishes one type of bacteria from
another.
- With special reagents like pH indicators or
dyes.
- Ex: eosin methylene blue agar (EMBA).
● Selective medium
- Allows the growth of a specific type of
microorganism only.
- With selective agents (ex: salts, dyes,
antibiotics)
- Ex: bacillus cereus agar (BCA).
Isolation and Cultivation of Microorganisms Culture Media
Module 7 | S-BIOL316 General Microbiology Lecture

2. Enrichment culture
Steps in Preparing Pure Cultures
- Isolation of specific types of microorganisms by a
combination of nutrient and physical conditions.
- Used for the isolation of unusual physiological 1. Isolation
types of microorganisms which are present in 2. Transfer
small numbers and which grow slowly. 3. Verify the purity
4. Make stock cultures

Culture Preservation

- To retain the viability of the stock culture for a long

period of time while maintaining its purity and trait of
being “true-to-type.”
● Methods
1. Periodic transfer to fresh media
3. Serial dilution ○ Considerations
- Used if the desired microorganism is present at a - Time interval of transfers
higher level than any other microorganism. - Proper medium
- Outcome is 10-fold reduction of cells in every - Proper storage temperature
transfer. 2. Overlaying cultures with mineral oil
○ Aim
- Limit the availability of O2.
○ Advantages
- Simple
- Enables one to remove some growth
under the oil and inoculate it in a fresh
medium and still preserve the initial
culture.
○ Disadvantages
- Viability of microorganisms varies with
species.
4. Single-cell isolation technique
3. Freeze-drying (lyophilization)
- Uses a micropipette or a microprobe to physically
○ Employs
pick a single cell and transfer it on an agar
- Temperature = -70° C.
medium.
- Rapid drying in a frozen state.
5. Membrane filter technique
- Dry ice in alcohol.
- For samples with low population.
4. Freezing with liquid nitrogen
- Uses a sterile membrane filter having a pore size
○ Advantages
that retains microorganism.
- Long-term survival.
- Less opportunity for changes in the
characteristics of culture.
- Smallness of storage containers.
○ Considerations
- Temperature = -196° C.
- Cryoprotective agent (glycerol).
- Liquid nitrogen refs.
5. Drying
○ Limitation
- Drying temperature = 45° C.
- For spore- and cyst- formers.

Banking Microbes

● Culture collections
- Organizations which maintain authentic pure
cultures of microorganisms.
- Provide ‘type’ strains to microbiologists
throughout the world.
- Ex: American Type Culture Collection, National
Collection of Type Cultures, Japanese Type
Culture Collection, Philippine National Collection of
Microorganisms.
Isolation and Cultivation of Microorganisms Culture Media
Module 7 | S-BIOL316 General Microbiology Lecture

■ Limitations
Isolation and Enumeration of Bacteria
1. Dead cells are not distinguished from
living cells.
- The number of microorganisms in a culture, sample, 2. Small cells are difficult to see under
or specimen is measured to assess the levels of the microscope, and some cells are
microbial contamination in raw material or probably missed,
manufactured products. 3. Precision is difficult to achieve,
- Enumeration is also done to evaluate the effects of 4. A phase-contrast microscope is
antimicrobial agents or the decontamination required when the sample is not
processes, stained.
5. The method is not usually suitable for
cell suspensions of low density, but
Methods for Counting Microorganisms
samples can be concentrated by
centrifugation or filtration to increase
Viable counts Total counts Rapid methods sensitivity.
(traditional) (traditional) (indirect viable ○ Turbidity method
counts) - Most common means of estimating the
Pour plate Direct Epifluorescence total number of bacteria present in a
method microscopic often coupled sample.
counting (using with image - Measuring the turbidity using a
Helber or analysis
spectrophotometer or colorimeter and
haemocytometer
counting reading the concentration from a
chambers) calibration plot is a simple means of
standardizing cell suspensions for inocula in
Spread plate Turbidity methods ATP testing
method
antibiotic assays or other tests of
antimicrobial chemicals.
Membrane filter Dry weight Impedance ● Viable count technique
method determination method - Records living cells alone.
MPN Method Nitrogen, protein, Manometric
- One that can divide and form offspring.
or nucleic acid methods - To determine the number of cells in the sample
determinations capable of forming colonies on a suitable agar
medium.
● Total count technique - Often called the plate or colony count.
- A counting procedure enumerating both living and - Used to enumerate bacteria in milk, water, foods,
dead cells. soils, cultures, etc., and the number of bacteria is
○ Direct microscopic count expressed as colony-forming units (CFU) per ml.
- Possible using special slides known as - The usual practice is to count colonies only on
counting chambers, consisting of a ruled plates that have between 30 and 300 colonies.
slide and a coverslip. ➔ Serial dilutions are employed to reach the
- Constructed so that the coverslip, slide, and final desired dilution.
ruled lines delimit a known volume. ➔ Each colony that can be counted is called a
- The number of bacteria in a small known colony-forming unit (CFU).
volume is microscopically counted, and the - By extrapolation, this number can be used
number of bacteria in the larger original to calculate the number of CFUs in the
sample is determined by extrapolation. original sample rather than the number of
- Petroff-Hausser counting chamber. bacteria per milliliter.
■ Formula ○ Formula
- Number of bacteria per cc = average - Number of CFUs per ml of sample =
numbers of bacteria per large number of colonies (30-300 plate) ×
double-lined square × dilution factors dilution factor of the plate counted.
of the large square (1,250,000) × ○ Advantage
dilution factor of any dilutions made - Used routinely and with satisfactory
before placing the sample in the results for estimating bacterial
counting chamber. populations in milk, water, foods, and
■ Advantages many other materials.
1. Rapid, simple, and easy method - Easy to perform and can be adapted
requiring minimum equipment. to measuring population of any
2. Morphology of the bacteria can be magnitude.
observed as they are counted. - A sensitive method (theoretically, a
3. Very dense suspensions can be single viable cell can be detected).
counted if they are diluted - Allows for inspection and
appropriately. identification of the organism
counted.
Isolation and Cultivation of Microorganisms Culture Media
Module 7 | S-BIOL316 General Microbiology Lecture

○ Limitation
- Only living cells develop into a single
colony.
- Clumps or chains of cells develop
into a single colony.
- Colonies develop only from those
organisms for which the cultural
conditions are suitable for growth.

Pour Plate Technique

- A method of melted agar inoculation followed by

● Rapid methods
Petri dish inoculation.
- Enumerate viable microorganisms, usually
- The known volume (0.1-1.0 mL) of culture is pipetted
bacteria and yeasts, within a matter of hours
into a sterile Petri plate; melted agar medium is then
and eliminate traditional methods 24-48 hour
added and mixed well by gently swirling the plate on
incubation periods.
the tabletop.
- Employ various means for indirect detection of
- After solidification of the gel, the plate is inverted
living cells.
and incubated at 37° C for 24-48 hours.
- Fast, readily automated, and eliminate the
- Useful for quantifying microorganisms that grow in a
need for numerous Petri dishes and incubators
solid medium.
but may require the purchase of expensive
- Because it embeds colonies in agar, it can supply a
equipment.
sufficiently oxygen-deficient environment that can
○ Epifluorescent technique
allow the growth and quantification of
- Uses fluorescent dyes that either exhibit
microaerophiles.
different colors in living and dead cells or
appear colorless outside the cell but
Spread Plate Technique become fluorescent when absorbed and
subjected to cellular metabolism.
○ ATP testing
- A volume of an appropriately diluted culture, usually
- Living cells generate ATP that can readily
no greater than 0.1 mL, is spread over the surface of
be detected by enzyme assays.
an agar plate using a sterile glass spreader.
- Light emission can be measured and
- The plate is then incubated until the colonies appear,
related to bacterial concentration.
and the number of colonies is counted.
○ Impedance technique
- Cultures are never exposed to 45° C.
- Resistance, capacitance, or impedance
● Membrane filter method
of a culture medium changes due to
- A large known volume of sample is passed
bacterial or yeast growth and
through the membrane, and then the
metabolism, and these electrical
membrane is placed, without inversion, on the
properties vary in proportion to cell
agar surface,
concentration.
- Nutrients diffuse through the membrane and
○ Manometric technique
allow retained cells to grow into colonies.
- Appropriate for monitoring the growth of
- Detects lower concentrations of
organisms that consume or produce
microorganisms than other methods, but it
significant quantities of gas during their
can’t be used to test microbial loads of viscous
metabolism, producing CO2 from
samples.
fermentation.
● Most probable number (MPN) method
- May be used when the anticipated count is
relatively low, from <1 to 100 microorganisms Maintenance and Preservation of Organisms
per mL.
- Involves inoculating multiple tubes of culture
- Once a microorganism has been isolated and grown
medium, usually 3 or 5, with 3 different volumes
in pure culture, it becomes necessary to maintain the
of the sample (0.1 mL and 0.001 mL).
viability and purity of the microorganisms by keeping
- Commonly used in the water, food, and dairy
the pure culture free from contamination.
industries.
- The pure cultures are transferred periodically onto or
into a fresh medium (subculturing) to allow
continuous growth and viability of microorganisms.
➔ Becomes difficult to maintain a large number of
pure cultures successfully for a long time.
➔ There is a risk of genetic changes as well as
contamination.
Isolation and Cultivation of Microorganisms Culture Media
Module 7 | S-BIOL316 General Microbiology Lecture

- Replaced by some modern methods that do not 4. Close tube with screw-cap
need frequent subculturing. or cork. Dip cap or cork
● Short-term storage into molten paraffin wax
○ Periodic transfer to fresh media to seal.
- Stains can be maintained by periodically ■ Cooked-meat medium (anaerobes)
preparing a fresh culture from the previous - Used for the preservation of anaerobic
stock. bacteria.
- The temperature and the medium chosen 1. Inoculate tubes of cooked meat
should support a slow rather than a rapid medium with the isolate.
rate of growth so that the time interval 2. Incubate overnight at 35° C.
between transfers can be as long as 3. Close tube with screw-cap or cork.
possible. 4. Store at room temperature.
- Has the disadvantage of failing to prevent Transfer every two months.
changes in the characteristics of a strain ● Long-term storage
due to the development of variants and - Permits intervals of months or even years
mutants. between subcultures.
○ Refrigeration - Isolates may be stored indefinitely if they are
- Pure cultures can be successfully stored at maintained frozen at -70° C or below; these
-0.4° C in refrigerators or cold rooms. temperatures can be achieved in an “ultralow
- Applied for a short duration because the freezer” (-70° C) or a liquid nitrogen freezer
metabolic activities of the microorganisms (-196° C).
are significantly slowed down but not ○ Freezing at -70° C
stopped. - Frozen, non-fastidious organisms should
- Their growth continues slowly, nutrients are be thawed, reisolated, and refrozen
utilized, and waste products are released every 5 years; fastidious organisms
into the medium. should be thawed, reisolated, and
■ Preservation in glycerol at -20° C refrozen every 3 years.
1. Grow a pure culture on an - Acid-fast bacilli (AFB) may also be frozen
appropriate solid medium. at -70° C in 7H9 broth with glycerol.
2. When the culture is fully developed, - Viruses may be stored indefinitely at -70°
scrape it off with a loop. C in a solution containing a
3. Suspend small clumps of the culture cryoprotectant, such as 10% dimethyl
in sterile neutral glycerol. sulfoxide (DMSO) or fetal bovine serum.
4. Distribute in quantities of 1-2 mL in ○ Cryopreservation
screw-capped tubes or vials. - Helps the survival of pure cultures for
5. Store at -20° C. avoid repeated long storage times.
freezing and thawing. Transfer after - The microorganisms of culture are
12-18 months. rapidly frozen in liquid nitrogen at -196° C
■ Stab cultures in the presence of stabilizing agents such
- At room temperature are used for as glycerol or dimethyl sulfoxide that
non-fastidious organisms only. prevent cell damage due to the
1. Prepare tubes with a deep butt of formation of ice crystals and promote
carbohydrate-free agar. Tryptic soy cell survival.
agar is recommended. - This method has been successful with
2. Stab the organism into the agar. many species that cannot be preserved
3. Incubate overnight at 35° C. by lyophilization and most species can
4. Close tube with screw-cap or cork. remain viable under these conditions for
Dip cap or cork into molten paraffin 10-30 years without undergoing a
wax to seal. change in their characteristics.
5. Store at room temperature, transfer ○ Lyophilization (Freeze-drying)
after one year. - A process where water and other
A. Stab culture in cystine solvents are removed from a frozen
trypticase agar (CTA) method product via sublimation.
- Recommended for the ➔ Occurs when a frozen liquid goes
preservation of Neisseria directly to a gaseous state without
and streptococci. entering a liquid phase.
1. Prepare tubes of cystine - Results in a stable, readily rehydrated
trypticase agar. product.
2. Stab the organism into 1. Pre-freezing the product in laboratory
the medium. freezer to form a frozen structure.
3. Incubate overnight at 35° 2. Primary drying to remove most water.
C. 3. Secondary drying to remove bound
water.
Isolation and Cultivation of Microorganisms Culture Media
Module 7 | S-BIOL316 General Microbiology Lecture

- Recommended to use slow rates of

cooling, as this will result in the formation
of vertical ice crystal structures.
- Freeze-dried products are hygroscopic
and must be protected from moisture
during storage.
- Results in microbes go into dormant
state and retain viability for years.
- Most frequently used technique by
culture collection centers.
■ Advantages
1. Only minimal storage space is
required; hundreds of lyophilized
cultures can be stored in a small
area.
2. Small vials can be sent
conveniently through the mail to
other microbiology laboratories
when packaged in a special sealed
mailing container.
3. Lyophilized cultures can be revived
by opening the vials, adding the
liquid medium, and transferring the
rehydrated culture to a suitable
growth medium.
○ Paraffin Method
- Preservation of organisms by overlaying
culture medium with mineral oil.
- Sterile liquid paraffin is poured over the
slant of the culture and stored upright at
room temperature.
- The layer of paraffin ensures anaerobic
conditions and prevents dehydration of the
medium.
- Helps microorganisms or pure culture to
remain dormant; the culture can be
preserved from months to years.
- Advantage is we can remove some of the
growth under the oil with a transfer needle,
inoculate a fresh medium, and still preserve
the original culture.