Clinical Chemistry_Lab

College of Medical Technology

CULTURE & CULTURE MEDIA Classification of Culture Media

Culture According to consistency
- Growth of microorganism in a culture 1. Liquid medium
medium - Does not contain any amount of agar
Culture medium - Allows the growth of aerobes, anaerobes
- Mixture of nutrients such as carbon, and facultative anaerobes
nitrogen, sulfur, phosphorus,hydrogen, Examples: Brain heart infusion (BHI), trypticase soy
oxygen and buffers. broth(TSB) and thioglycollate (liquid medium for
- A liquid, semi-solid or solid-medium can be anaerobes).
utilized to observe the growth patterns of 2. Semi-solid medium
microorganisms as well as their transport - Contains 0.5% to 1% agar.
and storage. - Used to observe bacterial motility and
Agar detect indole and sulfide production.
- Sulfonated polymer is made up of Examples: sulfide indole motility (SIM) medium
D-galactose, 3-6 anhydro-L'Galactose, and
D-glucuronic acid and is usually derived 3. Solid medium
from algae. - Contains 2% to 3% agar.
- Melt if the temperature is 80C to 90C and Examples: Triple sugar iron (TSI) agar, MacConkey
solidify at 40C to 50C (MAC) agar, blood agar plate (BAP) and Chocolate
- Cooling temperature for distribution of agar plate (CAP)
culture medium into petri plates is 55C to
60C According to composition
- Molten agar in the amount of 20mL to 25mL 1. Synthetic or defined medium
should be transferred to sterile plates. - Medium in which all components are
known
Types of Culture - For research purposes as either solid or
1. Pure culture liquid medium
- Composed of only one species - Preferred for the isolation of cyanobacteria
2. Mixed culture and chemoorganotrophs.
- Composed of more than one species Example: BG-11 medium
3. Stock culture 2. Non-synthetic or complex medium
- Composed of several species contained in a - Medium where some of the substance are
separate culture medium (one species per unknown (peptones, meat, and yeast
culture medium) extracts)
- Used for academic and industrial purposes - Useful for isolation of medically significant
bacteria
Examples: Nutrient broth (NB) medium, TSB and
MAC agar
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- Also used as supplement to agar plates to

3. Tissue culture medium detect aerobes, anaerobes and
- Used for obligate intracellular bacteria microaerophiles (thioglycollate)
(Rickettsia and Chlamydia) Examples: alkaline peptone water, selenite F,
Examples: W138, HeLa 229 cells and McCoy cells thioglycollate, tetrathionate, Gram negative (GN)
(used for isolation of Chlamydia) broth and Lim broth.
HeLa 229 cells
- Human cervical tissue cells Alkaline peptone water
McCoy cells and W138 cells - Used at pH 8.5 to promote the growth of
- Fibroblast Vibrio species before inoculation into
*Embryonated eggs for propagation of Rickettsia Thiosulfate-citrate bile salts sucrose (TCBS)
agar
Selenite F
According to Dispensing or distribution method for
- For isolation of Salmonella from feces, urine
medium
and water samples.
Plated media
Thioglycollate
- Distributed into dish or plate
- General support is an enrichment medium
Tube media
that promotes the growth of almost all
- Prepared as either liquid, slant, butt or butt
non-fastidious bacteria.
Examples: Triple sugar iron agar, SIM, Simmons
Tetrathionate
citrate agar (SCA) and lysine iron agar (LIA)
- Selective enrichment broth for the isolation
of Salmonella and Proteus;
According to use - addition of bile salt and thiosulfate into
1. Simple media, general purpose media, and medium suppresses the growth of other
supportive media coliform bacilli
- Routinely used in the laboratory and Gram negative broth
without additional supplements - For isolation of Salmonella and Shigella
- Media that support the growth of most - Used for enrichment and selective medium
non-fastidious bacteria - Contains sodium citrate and sodium
- Composed of meat and soybean extracts deoxycholate which inhibit Gram-positive
Examples: Nutrient Agar, Nutrient Broth, and bacteria
Trypticase soy broth (TSA) Lim broth
- Used as enrichment medium for group B
2. Enrichment media (liquid-tYpe media) streptococci
- Used to propagate growth of certain group
of bacteria from a mixture of organisms 3. Enriched media and non-selective media
- Contain specific nutrients and without - With additional supplements such as blood,
supplements vitamins and yeast extract which are
necessary for growth of fastidious
organisms
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- Solid type media d. Phenylethyl alcohol for gram positive

Example: BAP and CAP bacteria
e. Columbia colistin-nalidixic acid (CNA) agar:
BAP (Sheep's Blood agar) Gram positive bacteria
- Differentiates hemolytic patterns of f. Gram negative broth: Salmonella and
bacteria(streptococci) Shigella
- Alpha(incomplete), beta(complete) and
gamma(no hemolysis) Inhibitory substances
CAP (Chocolate Agar plate) - Added to culture media to isolate desired
- Uses horse blood organism and make a selective medium
- Routinely used for the recovery of a. Gram positive bacteria: crystal/gentian
Haemophilus species violet, basic/ carbol fuchsin and bile salt
b. Gram negative bacteria: potassium tellurite
4. Differential media and sodium azide
- Allow the indicator of metabolic differences c. For swarming bacteria- alcohol and chloral
between groups of bacteria hydrate
- Can detect hemolysis patterns HEA
Examples: MAC, BAP, eosin methylene blue EMB - Contains bile and dyes (acid fuchsin and pH
and Hektoen enteric agar (HEA) indicator blue) that inhibit indigenous
MAC microbiota of the lower GIT (non-enteric
- Differentiates lactose fermenters (pink pathogens)
colonies) from non-lactose fermenters XLD agar
(colorless colonies) - Contains xylose, lysine, sucrose, 0.25%
- Neutral red pH indicator that detects sodium deoxycholate salt and sodium
fermentation of sugar and eventually thiosulfate
production of acid. - Differentiates colonies of Salmonella (red
with black centers) from Shigella (red or
5. Selective media clear colonies without black centers)
- 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: *HEA, XLD and MAC are used for recovery of fecal
a. Gentamicin blood agar: Streptococcus bacteria
b. Bacitracin Chocolate agar: Haemophilus MSA
c. Blood agar plate w/ ampicillin: Aeromonas - Supports growth of Staphylococcus aureus
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TMA In selection of culture media inoculation should

- Selective for Neisseria species start from nonselective to the selective plates
6. Special media
- Used to isolate bacteria with specific growth
requirements Incubation Conditions
Example: Lowenstein Jensen medium and TCBS 1. Aerobes-21% O2 + 0.03% CO2
agar 2. Anaerobes- 0% O2, 5-10% CO2+ 5-10% H2
LJ medium +80-90% N2 (anaerobe jars, bags or chambers)
- Protein-rich medium composed of whole 3. Capnophiles- 5-20% CO2 +15% O2 (CO2
Incubator or bag; 3% CO2 in candle jars)
eggs and malachite green that supports
4. Microaerophiles- 5-10% O2 + 8-10% CO2
growth of mycobacteria and is sterilized by
Ideal incubation temperature: 35C
inspissation and not by autoclaving
Most routine bacterial culture is held at 48-72
TCBS
hours
- Selective for isolation of Vibrio
Aerobic bacteria for 18-24 hrs and Anaerobic
- Sterilization is performed by boiling and
culture requires 48 hrs of incubation.
never by autoclaving
Anaerobes and Broth cultures may be held for 5-7
days
Inoculation of Specimen
Unusual organisms require a special medium or
● Sterile Body fluids -pus, urine and sputum
conditions beyond routine setup
are inoculated directly into selected media
● Specimens that require direct / bedside
inoculqted are blood, genital specimens,
Manner of Reporting (Grading) of Growth on
corneal scrapings, sterile fluids like synovial
and peritoneal fluids and nasopharyngeal
plate
1. 4+ : many, heavy growth;if growth is up to the
swabs for isolation of Bordetella pertussis
4th quadrant
2. 3+ : moderate growth; if growth is up to 3rd
Inoculation Techniques quadrant
Streaking: most common manner of inoculation 3. 2+ : signifies a few or light growth if growth is in
Stabbing of medium the 2nd quadrant
performed usually with group A streptococci to 4. 1+ : rare growth; if growth is in the 1st quadrant
create anaerobiosis and promote sub-surface only.
hemolysis Anaerobic Cultivation
Overlapping inoculation - Use of a special culture medium
used for antimicrobial sensitivity test (disk diffusion incorporated with thioglycollate and
method) cysteine (reducing agent)
Urine specimen - Boiling culture medium to remove (drive
- Inoculated using quantitative isolation off) oxygen
technique with calibrated loop(0.01 mL or - Use of anaerobic chamber system with a
0.001 mL) to deliver specific volume 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 - stage in bacteria that increase

colored logarithmically since cellular production is
3. Growth in nutrient broth most active during this period
● Distribution in the broth - phase where microorganisms utilized in
a. May be turbid (10^6 CFU/mL of broth is physiological and biochemical testing
needed to create turbidity) - target microbial agents
b. Confined on surface of broth as a film 3. Stationary phase/Plateau
(pellicle) or accumulated as sediment - period where there is a balance between
c. May be reported as granular or viscous in cell division and dying organisms even if
appearance viable microorganisms remain constant
● Odor - metabolic activities of surviving cells slow
- Growth may produce putrid, fruity or down and nutrients are limited
aromatic odor. 4. Death or decline phase
4. Growth in gelatin slant - period where cessation of bacterial growth
● Growth along the line of inoculation as the number of dead cells exceeds the
a. Confined within the zone of number of living microorganisms
inoculation streak - stage where loss of nutrients and increase
b. Varying degrees of spreading outside in amount of toxic waste
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.
Reproduction
Transverse Binary fission
Stages of bacterial growth most common asexual reproductive process in
1. Lag phase or period of rejuvenescence which single cell divides into two daughter cells
- period when there is no cell division or after developing a transverse cell wall
abrupt increase in cell number
- start of biosynthesis where there is no Growth measurement
increase in cell mass 1. By cell count- microscopy, electronic
- adjustment phase to a new environment particle counter or colony count
2. Log or exponential phase (Balance growth) 2. By cell mass- nitrogen content and
- microorganisms are actively growing and turbidimetry
dividing 3. By cell activity- biochemical activity