Clinical Chemistry_Lab
College of Medical Technology
CULTURE & CULTURE MEDIA
Culture
- Growth of microorganism in a culture
medium
Culture medium
- Mixture of nutrients such as carbon,
nitrogen, sulfur, phosphorus,hydrogen,
oxygen and buffers.
- A liquid, semi-solid or solid-medium can be
utilized to observe the growth patterns of
microorganisms as well as their transport
and storage.
Agar
- Sulfonated polymer is made up of
D-galactose, 3-6 anhydro-L'Galactose, and
D-glucuronic acid and is usually derived
from algae.
- Melt if the temperature is 80C to 90C and
solidify at 40C to 50C
- Cooling temperature for distribution of
culture medium into petri plates is 55C to
60C
- Molten agar in the amount of 20mL to 25mL
should be transferred to sterile plates.
Types of Culture
1. Pure culture
- Composed of only one species
2. Mixed culture
- Composed of more than one species
3. Stock culture
- Composed of several species contained in a
separate culture medium (one species per
culture medium)
- Used for academic and industrial purposes
Classification of Culture Media
According to consistency
1. Liquid medium
- Does not contain any amount of agar
- Allows the growth of aerobes, anaerobes
and facultative anaerobes
Examples: Brain heart infusion (BHI), trypticase soy
broth(TSB) and thioglycollate (liquid medium for
anaerobes).
2. Semi-solid medium
- Contains 0.5% to 1% agar.
- Used to observe bacterial motility and
detect indole and sulfide production.
Examples: sulfide indole motility (SIM) medium
3. Solid medium
- Contains 2% to 3% agar.
Examples: Triple sugar iron (TSI) agar, MacConkey
(MAC) agar, blood agar plate (BAP) and Chocolate
agar plate (CAP)
According to composition
1. Synthetic or defined medium
- Medium in which all components are
known
- For research purposes as either solid or
liquid medium
- Preferred for the isolation of cyanobacteria
and chemoorganotrophs.
Example: BG-11 medium
2. Non-synthetic or complex medium
- Medium where some of the substance are
unknown (peptones, meat, and yeast
extracts)
- Useful for isolation of medically significant
bacteria
Examples: Nutrient broth (NB) medium, TSB and
MAC agar
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3. Tissue culture medium
- Used for obligate intracellular bacteria
(Rickettsia and Chlamydia)
Examples: W138, HeLa 229 cells and McCoy cells
(used for isolation of Chlamydia)
HeLa 229 cells
- Human cervical tissue cells
McCoy cells and W138 cells
- Fibroblast
*Embryonated eggs for propagation of Rickettsia
According to Dispensing or distribution method for
medium
Plated media
- Distributed into dish or plate
Tube media
- Prepared as either liquid, slant, butt or butt
Examples: Triple sugar iron agar, SIM, Simmons
citrate agar (SCA) and lysine iron agar (LIA)
According to use
1. Simple media, general purpose media, and
supportive media
- Routinely used in the laboratory and
without additional supplements
- Media that support the growth of most
non-fastidious bacteria
- Composed of meat and soybean extracts
Examples: Nutrient Agar, Nutrient Broth, and
Trypticase soy broth (TSA)
2. Enrichment media (liquid-tYpe media)
- Used to propagate growth of certain group
of bacteria from a mixture of organisms
- Contain specific nutrients and without
supplements
- Also used as supplement to agar plates to
detect aerobes, anaerobes and
microaerophiles (thioglycollate)
Examples: alkaline peptone water, selenite F,
thioglycollate, tetrathionate, Gram negative (GN)
broth and Lim broth.
Alkaline peptone water
- Used at pH 8.5 to promote the growth of
Vibrio species before inoculation into
Thiosulfate-citrate bile salts sucrose (TCBS)
agar
Selenite F
- For isolation of Salmonella from feces, urine
and water samples.
Thioglycollate
- General support is an enrichment medium
that promotes the growth of almost all
non-fastidious bacteria.
Tetrathionate
- Selective enrichment broth for the isolation
of Salmonella and Proteus;
- addition of bile salt and thiosulfate into
medium suppresses the growth of other
coliform bacilli
Gram negative broth
- For isolation of Salmonella and Shigella
- Used for enrichment and selective medium
- Contains sodium citrate and sodium
deoxycholate which inhibit Gram-positive
bacteria
Lim broth
- Used as enrichment medium for group B
streptococci
3. Enriched media and non-selective media
- With additional supplements such as blood,
vitamins and yeast extract which are
necessary for growth of fastidious
organisms
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- Solid type media
Example: BAP and CAP
BAP (Sheep's Blood agar)
- Differentiates hemolytic patterns of
bacteria(streptococci)
- Alpha(incomplete), beta(complete) and
gamma(no hemolysis)
CAP (Chocolate Agar plate)
- Uses horse blood
- Routinely used for the recovery of
Haemophilus species
4. Differential media
- Allow the indicator of metabolic differences
between groups of bacteria
- Can detect hemolysis patterns
Examples: MAC, BAP, eosin methylene blue EMB
and Hektoen enteric agar (HEA)
MAC
- Differentiates lactose fermenters (pink
colonies) from non-lactose fermenters
(colorless colonies)
- Neutral red pH indicator that detects
fermentation of sugar and eventually
production of acid.
5. Selective media
- Incorporated with antibiotics, dyes or
chemicals to inhibit the growth of other
organisms while promoting the growth of
the desired organism
Example: HEA, MAC, xylose lysine deoxycholate
(XLD) agar, bismuth sulfite agar (BSA), mannitol salt
agar (MSA) and Thayer-Martin agar (TMA)
Other selective media:
a. Gentamicin blood agar: Streptococcus
b. Bacitracin Chocolate agar: Haemophilus
c. Blood agar plate w/ ampicillin: Aeromonas
d. Phenylethyl alcohol for gram positive
bacteria
e. Columbia colistin-nalidixic acid (CNA) agar:
Gram positive bacteria
f. Gram negative broth: Salmonella and
Shigella
Inhibitory substances
- Added to culture media to isolate desired
organism and make a selective medium
a. Gram positive bacteria: crystal/gentian
violet, basic/ carbol fuchsin and bile salt
b. Gram negative bacteria: potassium tellurite
and sodium azide
c. For swarming bacteria- alcohol and chloral
hydrate
HEA
- Contains bile and dyes (acid fuchsin and pH
indicator blue) that inhibit indigenous
microbiota of the lower GIT (non-enteric
pathogens)
XLD agar
- Contains xylose, lysine, sucrose, 0.25%
sodium deoxycholate salt and sodium
thiosulfate
- Differentiates colonies of Salmonella (red
with black centers) from Shigella (red or
clear colonies without black centers)
*HEA, XLD and MAC are used for recovery of fecal
bacteria
MSA
- Supports growth of Staphylococcus aureus
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TMA
- Selective for Neisseria species
6. Special media
- Used to isolate bacteria with specific growth
requirements
Example: Lowenstein Jensen medium and TCBS
agar
LJ medium
- Protein-rich medium composed of whole
eggs and malachite green that supports
growth of mycobacteria and is sterilized by
inspissation and not by autoclaving
TCBS
- Selective for isolation of Vibrio
- Sterilization is performed by boiling and
never by autoclaving
Inoculation of Specimen
● Sterile Body fluids -pus, urine and sputum
are inoculated directly into selected media
● Specimens that require direct / bedside
inoculqted are blood, genital specimens,
corneal scrapings, sterile fluids like synovial
and peritoneal fluids and nasopharyngeal
swabs for isolation of Bordetella pertussis
Inoculation Techniques
Streaking: most common manner of inoculation
Stabbing of medium
performed usually with group A streptococci to
create anaerobiosis and promote sub-surface
hemolysis
Overlapping inoculation
used for antimicrobial sensitivity test (disk diffusion
method)
Urine specimen
- Inoculated using quantitative isolation
technique with calibrated loop(0.01 mL or
0.001 mL) to deliver specific volume
In selection of culture media inoculation should
start from nonselective to the selective plates
Incubation Conditions
1. Aerobes-21% O2 + 0.03% CO2
2. Anaerobes- 0% O2, 5-10% CO2+ 5-10% H2
+80-90% N2 (anaerobe jars, bags or chambers)
3. Capnophiles- 5-20% CO2 +15% O2 (CO2
Incubator or bag; 3% CO2 in candle jars)
4. Microaerophiles- 5-10% O2 + 8-10% CO2
Ideal incubation temperature: 35C
Most routine bacterial culture is held at 48-72
hours
Aerobic bacteria for 18-24 hrs and Anaerobic
culture requires 48 hrs of incubation.
Anaerobes and Broth cultures may be held for 5-7
days
Unusual organisms require a special medium or
conditions beyond routine setup
Manner of Reporting (Grading) of Growth on
plate
1. 4+ : many, heavy growth;if growth is up to the
4th quadrant
2. 3+ : moderate growth; if growth is up to 3rd
quadrant
3. 2+ : signifies a few or light growth if growth is in
the 2nd quadrant
4. 1+ : rare growth; if growth is in the 1st quadrant
only.
Anaerobic Cultivation
- Use of a special culture medium
incorporated with thioglycollate and
cysteine (reducing agent)
- Boiling culture medium to remove (drive
off) oxygen
- Use of anaerobic chamber system with a
vacuum pump and nitrogen gas to remove
residual oxygen
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- Use of a gas-pak jar containing a palladium

catalyst
- Small volumes: use plastic bags or pouches
containing calcium carbonate and catalyst Cultural Characteristics
Manner of Reporting
Components of Anaerobic Chamber
1. Agar-plate colonies
1. Nitrogen gas
● Size
- Used as filler for remaining percentage of
a. Colonies range from small (pinpoint)
anaerobic atmosphere
to large while others appear mucoid
2. Palladium pellets
or slimy
- Used to remove residual oxygen from the
b. Pseudomonas and Proteus spread
chamber by combining with hydrogen to
across the entire agar surface
form water
● Margin or edge
3. Silica gel (desiccant)
a. Observed to be evenly circular or
- Used to absorb the water that is formed
with irregularities
when hydrogen combines with free oxygen
b. Rounded projections, irregular
in the presence of catalyst
notches or rootlike projections are
4. Methylene blue or reazurin
also observed
- An oxygen reduction indicator that becomes
c. Elevation-colonies may be flat or
colorless in absence of oxygen
raised
Anaerobic incubator
● Chromogenesis
- Components are desiccant, oxygen free
a. Colonies may be pigmented or
indicator, catalyst and anaerobic source of
colored but not all bacterial species
gas (nitrogen, hydrogen and carbon dioxide)
b. Some bacteria retain pigment with
- Ideal anaerobic system is anaerobic
cell while other bacteria color the
chamber
medium
- Isolation and susceptibility test is done in
c. Pigment production is best observed
the chamber
from solid media
- CO2 content is low an anaerobic incubator
2. Growth on agar slant
is increased to 10% the oxygen is lowered at
● Amount
18%
- Scanty, moderate or abundant
- Ideal temperature to promote growth of
● Margin or edge
anaerobes is at 37 C for 48 hrs
- Evenly circular or with irregularities
Gas-pak Jar
● Consistency of mass growth
- Takes 30-45 minutes to obtain an anaerobic
- Colonies may appear butyrous or
environment
butterlike, viscous or stingy, dry and
- Water is added to the gas-pak envelope,
brittle
CO2 and hydrogen are produced.
● Chromogenesis
Takes several hours for methylene blue indicator to
change from blue to colorless
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- Colonies may be pigmented or
colored
3. Growth in nutrient broth
● Distribution in the broth
a. May be turbid (10^6 CFU/mL of broth is
needed to create turbidity)
b. Confined on surface of broth as a film
(pellicle) or accumulated as sediment
c. May be reported as granular or viscous in
appearance
● Odor
- Growth may produce putrid, fruity or
aromatic odor.
4. Growth in gelatin slant
● Growth along the line of inoculation
a. Confined within the zone of
inoculation streak
b. Varying degrees of spreading outside
inoculation streak
● Liquefaction of gelatin starts evenly from
top or various liquefied funnel like designs
may occur
Bacterial Growth Curve
Generation time of bacteria can be as brief as 20
minutes for fast-growing bacteria like Escherichia
coli or as long as 24 hrs for slow growing bacteria
such as Mycobacterium tuberculosis.
Stages of bacterial growth
1. Lag phase or period of rejuvenescence
- period when there is no cell division or
abrupt increase in cell number
- start of biosynthesis where there is no
increase in cell mass
- adjustment phase to a new environment
2. Log or exponential phase (Balance growth)
- microorganisms are actively growing and
dividing
- stage in bacteria that increase
logarithmically since cellular production is
most active during this period
- phase where microorganisms utilized in
physiological and biochemical testing
- target microbial agents
3. Stationary phase/Plateau
- period where there is a balance between
cell division and dying organisms even if
viable microorganisms remain constant
- metabolic activities of surviving cells slow
down and nutrients are limited
4. Death or decline phase
- period where cessation of bacterial growth
as the number of dead cells exceeds the
number of living microorganisms
- stage where loss of nutrients and increase
in amount of toxic waste
Reproduction
Transverse Binary fission
most common asexual reproductive process in
which single cell divides into two daughter cells
after developing a transverse cell wall
Growth measurement
1. By cell count- microscopy, electronic
particle counter or colony count
2. By cell mass- nitrogen content and
turbidimetry
3. By cell activity- biochemical activity