BACT211

WEEK 5: CULTIVATION OF BACTERIA

SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

BACTERIAL CULTIVATION
1.To grow and isolate all bacteria present in a
clinical specimen
2. To determine which of the bacteria that grow are
most likely causing infection and which are
likely contaminants or colonizers (normal
microbiota)
➢ Organisms present on our skin
➢ We have bacteria in our body fighting
infections
3. To obtain sufficient growth of clinically relevant
bacteria to allow identification, characterization,
and susceptibility testing
• Cultivation is the process of growing
microorganisms in culture by taking bacteria from
the infection site (i.e., the in vivo environment) by
some means of specimen collection and growing
them in the artificial environment of the laboratory
(i.e., the in vitro environment)
• Culture media
o Nutritional needs classification: • Solid medium: solidifying agent added to the
fastidious and non-fastidious nutrients and water
• Agarose is the most common solidifying agent
PHASES OF GROWTH MEDIA • Melting at high temperatures (95°C) but
• Two phases: resolidifies after the temperature falls below 50°C.
1. Broth (liquid)
• Stable solid gel referred to as agar
2. Agar (solid)
• Agar plate
• Broth media, nutrients are dissolved in water from
• Pure colony: all bacterial cells within a single
clear to turbid (i.e., cloudy)
colony are the same genus and species, having
• Some broths may also contain a pH indicator, such
identical genetic and phenotypic characteristics
as phenol red, that may change color in the
(i.e., are derived from a single clone)
presence of bacterial metabolites
• In addition to the amount of growth present, the SELECTION OF CULTURE MEDIA
location of growth within the broth, such as the
1. Nutritive media: blood or chocolate agars
thioglycolate broth, which contains a small
➢ Support the growth of a wide range of
amount of agar (making it a semisolid medium),
microorganisms and are considered
provides an indication of the type of organism
nonselective because, theoretically, the
present based on oxygen requirements.
growth of most organisms is supported
• Strict anaerobes will grow at the bottom of the ➢ Nutritive media can also be differential, in
broth tube, whereas aerobes will grow near the that microorganisms can be distinguished on
surface. the basis of certain growth characteristics
o Microaerophilic organisms will grow evident on the medium.
slightly below the surface where oxygen ➢ Blood agar is considered both a nutritive and
concentrations are lower than
differential medium because it differentiates
atmospheric concentrations. organisms based on whether they are alpha
o In addition, facultative anaerobes and (a)-, beta (b)-, or gamma (g)-hemolytic
aerotolerant organisms will grow o Alpha → incomplete/partial
throughout the medium, because they hemolysis (green-brown)
are unaffected by the variation in o Beta → complete hemolysis
oxygen content.
(yellow)
o Gamma → no change
2. Selective media: support the growth of one group
of organisms but not another by adding
antimicrobials, dyes, or alcohol to a particular
medium.
➢ Can also be differential media if, in addition
to their inhibitory activity, they differentiate
between groups of organisms
➢ MacConkey agar, for example, contains the
dye crystal violet, which inhibits gram-
positive organisms.
o No growth of gram (+)

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 BACT211
WEEK 5: CULTIVATION OF BACTERIA
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

o Growth of gram (-) PREPARATION OF ARTIFICIAL MEDIA

➢ Columbia agar with colistin and nalidixic
• Media Sterilization
acid (CNA) is a selective medium for gram-
o The timing of autoclave sterilization
positive organisms because the
should start from the moment the
antimicrobials colistin and nalidixic acid
temperature reaches 121°C and usually
inhibit gram-negative organisms
requires a minimum of 15 minutes.
o No growth of gram (-)
▪ 15 psi
o Growth of gram (+)
o Once the sterilization cycle is completed,
3. MacConkey agar, for example, differentiates
molten agar is allowed to cool to
between lactose-fermenting and nonfermenting
approximately 50°C before being
gram-negative rods by the color of the colonial
distributed to individual petri plates
growth (pink or clear, respectively)
(approximately 20 to 25 mL of molten
4. Enriched media contains growth enhancers that
agar per plate).
are added to nonselective agar to allow fastidious
▪ 4-6 inches agar thickness
organisms to flourish. Chocolate agar is an
o If other ingredients are to be added (e.g.,
enriched medium.
supplements such as sheep blood or
5. Enrichment broth is a liquid medium designed to
specific vitamins, nutrients, or
encourage the growth of small numbers of a
antibiotics), they should be incorporated
particular organism while suppressing other flora
when the molten agar has cooled, just
present
before distribution to plates.
➢ Enrichment broths are incubated for a certain
▪ Should be cooled down
period and then must be subcultured to
o Delicate media components that cannot
isolate the particular organism.
withstand steam sterilization by
➢ Lim broth (Todd Hewitt with CAN) is used
autoclaving (e.g., serum, certain
to enhance the growth of group B
carbohydrate solutions, certain
Streptococci
antibiotics, and other heat-labile
6. Supplemental broth media can be used as a
substances) can be sterilized by
supplement to agar plates to detect small numbers
membrane filtration.
of most aerobes, anaerobes, and microaerophiles.
o Passage of solutions through membrane
➢ Thioglycollate broth is an example
filters with pores ranging in size from
➢ In some cases (sterile body fluids, tissues, or
0.2 to 0.45 μm in diameter will not
deep abscesses in a patient receiving
remove viruses but will effectively
antimicrobial therapy), backup broth (also
remove most bacterial and fungal
called supplemental or enrichment broth)
contaminants.
medium is inoculated, along with primary
o Finally, all media, whether purchased or
solid (agar) media, so small numbers of
prepared, must be subjected to stringent
organism present may be detected this allows
quality control before being used in the
detection of anaerobes in aerobic cultures and
diagnostic setting
organisms that may be damaged by either
• Geobacillus stearothermophilus present in
previous or concurrent antimicrobial therapy.
autoclave
o Growth only in supplemental broth
o If positive, all organisms are killed
media
• Spores should be destroyed
➢ Thioglycollate (thio) Broth, Brain-heart
• Do not open autoclave right away
Infusion Broth (BHIB), and Tryptic Soy
Broth are common backup broths
BASIC ASEPTIC TECHNIQUE
PRIMARY PLANTING • Aseptic transfer of a culture from one culture
1. Nonselective agar plate vessel to another is successful only if no
2. Enriched medium for fastidious organisms for contaminating microorganisms are introduced in
normally sterile body fluids or a site in which the process. A transfer may involve the transport
fastidious organisms are expected of organisms from an isolated colony on a plate of
3. Selective and differential medium for enteric solid medium to a broth tube, or inoculating
gram-negative bacilli for most routine bacterial various media (solid or liquid) from a broth culture
cultures for various types of tests
4. Selective medium for gram-positive organisms
for specimens in which mixed gram-positive and GENERAL PROCEDURE
gram-negative bacteria are found 1. Work area disinfection
5. Additional selective media or enrichment ➢ The work area is first treated with a
broths for specific pathogens as needed disinfectant to kill any microorganisms that
6. Broth medium may be used as a supplement with may be present. This step destroys vegetative
specimens from sterile body fluids, tissues, lesions, cells and viruses; endospores, however, are
wounds, and abscesses not destroyed in this brief application of
disinfectant.
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 BACT211
WEEK 5: CULTIVATION OF BACTERIA
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

➢ 10 minutes after
2. Loops and needles
➢ The transport of organisms will be performed
with an inoculating loop or needle. To
sterilize the loop or needle prior to picking up
the organisms, heat must be applied with a
Bunsen burner flame/alcohol lamp flame,
rendering them glowing red-hot.
3. Culture tube flaming
➢ Before inserting the cooled loop or needle
into a tube of culture, the tube cap is removed
and the mouth of the culture tube flamed.
Once the organisms have been removed from
the tube, the tube mouth must be flamed
again before returning the cap to the tube
4. Liquid medium inoculation
➢ If a tube of liquid medium is to be inoculated, INOCULATION OF BACTERIA
the tube mouth must be flamed before • When we try to study the bacterial flora of the
inserting the loop into the tube. To disperse body, soil, water, food, or any other part of our
the organisms on the loop, the loop should be environment, we soon discover that bacteria exist
twisted back and forth in the medium. If an in mixed populations.
inoculating needle is used for stabbing a solid • It is only in very rare situations that they occur as
medium, the needle is inserted deep into the a single species. To be able to study the cultural,
medium. morphological, and physiological characteristics
5. Final flaming of an individual species, it is essential, first of all,
➢ Once the inoculation is completed, the loop that the organism be separated from the other
or needle is removed from the tube, flamed species that are normally found in its habitat; in
as before, and returned to a receptacle. These other words, we must have a pure culture of the
tools should never be placed on the tabletop. microorganism. Several different methods of
The inoculated tube is also flamed before getting a pure culture from a mixed culture are
placing the cap on the tube. available to us.
6. Petri plate inoculation • The two most frequently used methods involve
➢ To inoculate a Petri plate, no heat is applied making a streak plate or a pour plate. Both plate
to the plate and a loop is used for the transfer. techniques involve thinning the organisms so that
When streaking the surface of the medium, the individual species can be selected from the
the cover should be held diagonally over the others
plate bottom to prevent air contamination of
the medium.
7. Final disinfection
➢ When all work is finished, the work area is
treated with disinfectant to ensure that any
microorganisms deposited during any of the
procedures are eliminated.

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 BACT211
WEEK 5: CULTIVATION OF BACTERIA
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

• Involves detecting the presence of a gene

• The presence of a specific gene or a particular
nucleic acid sequence unique to the organism is
interpreted as a definitive identification of the
organism
• The genotypic approach is highly specific and
often very sensitive.
• Specificity refers to the percentage of patients
without disease that will test negative for the
presence of the organism. Sensitivity indicates the
percentage of patients in whom the organism is
present who actually test positive
• Phenotypic criteria are based on observable
physical or metabolic characteristics of bacteria—
that is, identification is through analysis of gene
products rather than through the genes themselves.
• Most of the phenotypic characterizations used in
diagnostic bacteriology are based on tests that
establish a bacterial isolate’s morphology and
metabolic capabilities.
• The most commonly used phenotypic criteria
include the following:
o Microscopic morphology and staining
characteristics
o Macroscopic (colony) morphology,
including odor and pigmentation
o Environmental requirements for growth
o Resistance or susceptibility to
antimicrobial agents
o Nutritional requirements and metabolic
capabilities
o Biochemical reactions including
enzymatic reactions or chemical
profiles

MACROSCOPIC (COLONY) MORPHOLOGY

• Evaluation of colony morphology includes
considering colony size, shape, odor, color
IMPORTANT NOTES (pigment), surface appearance, and any changes
• The identification of a bacterial isolate requires that colony growth produces in the surrounding
analysis of information gathered from laboratory agar medium (e.g., hemolysis of blood in blood
tests that provide characteristic profiles of bacteria. agar plates).
• Identification scheme or workup of the organism • A characteristic odor can support an identification
o Identification schemes can be classified of an organism such as Pseudomonas aeruginosa,
into one of two categories: which is described as having a fruity or grapelike
1. Those based on genotypic smell. (Note: Smelling plates in a clinical setting
characteristics of bacteria and can be dangerous and is strongly discouraged.)
2. Those based on phenotypic • Although these characteristics usually are not
characteristics. sufficient for establishing a final or definitive
• Certain schemes rely on both genotypic and identification, the information gained provides
phenotypic characteristics preliminary information necessary for
• In addition, some tests, such as the Gram stain, are determining what identification procedures should
an integral part of many schemes used for follow.
identifying a wide variety of bacteria, whereas • However, it is unwise to place too much
other tests may only be used in the identification confidence on colony morphology alone for
scheme for a single species, such as the preliminary identification of isolates.
fluorescent antibody test for identification of • Microorganisms often grow as colonies whose
Legionella pneumophila. appearance is not that different from many other
• Genotypic identification methods involve the species, especially if the colonies are relatively
characterization of some portion of a bacterium’s young (i.e., less than 14 hours old).
genome using molecular methods for DNA or
RNA analysis.
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 BACT211
WEEK 5: CULTIVATION OF BACTERIA
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

• Therefore, unless colony morphology is

distinctive or unless growth occurs on a particular
selective medium,

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 BACT211
WEEK 5: CULTIVATION METHOD
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

AKILLA DUANNE HABER|1

 BACT211
WEEK 5: CULTIVATION METHOD
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

AKILLA DUANNE HABER|2

 BACT211
WEEK 5: CULTIVATION METHOD
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

AKILLA DUANNE HABER|3

 BACT211
WEEK 5: CULTIVATION METHOD
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

AKILLA DUANNE HABER|4

 BACT211
WEEK 5: CULTIVATION METHOD
SECOND SEMESTER | SY 2023-2024 | PROFESSOR MARK JOHN R. RAVINA, RMT, DTA

AKILLA DUANNE HABER|5