medical microbiology i - lecture3
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MEDICAL MICROBIOLOGY I
Lecture 3
Culturing of bacteria anaerobically, on
special agar and enriched agar
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Anaerobic Culture
Anaerobic bacteria need special media for
growth because they need low oxygen
content, reduced oxidation-reduction potential
and extra nutrientsand extra nutrients
Media for anaerobes may have to be
supplemented with nutrients like hemin and
vitamin K
Boiling the medium serves to expel any
dissolved oxygen
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Anaerobic Culture
Addition of 1% glucose, 0.1% thioglycollate,
0.1% ascorbic acid, 0.05% cysteine or red hot
iron filings can render a medium reduced
Robertson cooked meat that is commonly Robertson cooked meat that is commonly
used to grow Clostridium species.
Medium contain a 2.5 cm column of bullock
heart meat and 15 mL of nutrient broth
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Anaerobic Culture
Before use the medium must be boiled in
water bath to expel any dissolved oxygen and
then sealed with sterile liquid paraffin
Methylene blue or resazurin is an oxidation- Methylene blue or resazurin is an oxidation-
reduction potential indicator that is
incorporated in the thioglycollate medium
Under reduced condition, methylene blue is
colourless
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Gas-Pak System
Generating an anaerobic atmosphere within an
anaerobic jar
1. Inoculated media are placed in an anaerobic jar
along with a catalyst (consists of a disposable along with a catalyst (consists of a disposable
H2 and CO2 envelope) and an indicator strip
(blue)
2. A Gas-Pak envelope is activated by cutting off a
top corner of the envelope and placed inside
the jar
3. 10 mL of water is added into the jar
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Gas-Pak System
5. The jar is sealed and placed in an aerobic
incubator
6. An anaerobic atmosphere should be attained
within 2 hours after sealing the jar.within 2 hours after sealing the jar.
7. If an anaerobic atmosphere has been
attained, the strip will turn white and
moisture will accumulate on the side of the
jar
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Gas-Pak System
H2 and CO2 are generated in the following
manner
Citric acid and sodium bicarbonate react to
form waterform water
Sodium hydride reacts with water to form
sodium borohydrate and hydrogen
The palladium-coated catalysts reacts with
hydrogen and any remaining oxygen to form
water
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Enriched Media
Addition of extra nutrients in the form of
blood, serum, egg yolk etc. to basal medium
makes them enriched media
Enriched media are used to grow nutritionally Enriched media are used to grow nutritionally
exacting (fastidious) bacteria
Blood agar, chocolate agar, Loefflers serum
slope etc. are few of the enriched media
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Blood Agar
A general purpose medium used to cultivate
nutritionally demanding microorganisms and to
differentiate between those that lyse red blood
cells (haemolytic) and those that do notcells (haemolytic) and those that do not
It is especially useful to distinguish streptococci
based on their haemolytic properties
Bacteria growing on BA produce a wide variety
of products that affect the integrity of red
blood cells contained in the medium
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Blood Agar
Streptococcus pyrogenes produce streptolysin
P and S, both of which are capable of
completely lysing red blood cells
Streptolysin O is oxygen-labile, where as Streptolysin O is oxygen-labile, where as
streptolysin S in oxygen-stable
Some streptococci are able to partially lyse the
red blood cells, resulting in greenish or
brownish discolouration around the colony -
alpha haemolysis
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Blood Agar
Beta haemolysis appears as a clear or
colourless zone surrounding the colony
Species that fail to produce visible effects on
red blood cells are said to be gamma red blood cells are said to be gamma
haemolytic or non-haemolytic
Haemolysis is due to the release of enzymes
by microorganisms
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Blood Agar
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Selective and Enrichment Media
Designed to inhibit unwanted commensal or
contaminating bacteria and help to recover
pathogen from a mixture of bacteria
While selective media are agar based, While selective media are agar based,
enrichment media are liquid in consistency.
Both these media serve the same purpose
Any agar media can be made selective by
addition of certain inhibitory agents that DO
NOT affect the pathogen
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Selective Media
Various approaches to make a medium
selective include:
1. addition of antibiotics
2. addition of dyes2. addition of dyes
3. addition of chemicals
4. alteration of pH
5. combination of some of these
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Enrichment Media
A medium of known composition and specific
conditions of incubation which favours the
growth of a particular type or species of
bacteriumbacterium
Liquid media that also serves to inhibit
commensals in the clinical specimen
Selenite F broth, tetrathionate broth and
alkaline peptone water are used to recover
pathogens from faecal specimens
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Chocolate Agar
A non-selective, enriched growth medium
It is a variant of blood agar
It contains red blood cells which have been lysed
by heating very slowly to 56Cby heating very slowly to 56C
Used for growing fastidious (fussy) respiratory
bacteria such as Haemophilus influenza
These bacteria need growth factors, like NAD
and hematin which are inside red blood cells,
thus a prerequisite to growth is lysis of the red
blood cells
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Chocolate Agar
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Differential Media or Indicator
Media
Certain media are designed in such a way that
different bacteria can be recognized on the
basis of their colony colour
Various approaches include incorporation of Various approaches include incorporation of
dyes, metabolic substrates etc. so that those
bacteria that utilize them appear as differently
coloured colonies
Examples: MacConkey agar, CLED agar, TCBS
agar, XLD agar etc.
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MacConkey Agar
Grow Gram-negative bacteria and stain them
for lactose fermentation
It contains bile salts, crystal violet dye, neutral
red dye, lactose and peptonered dye, lactose and peptone
The bile salt will inhibit most Gram negative
bacteria, EXCEPT Enterococcus and some
species of Staphylococcus e.g. S. aureus
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MacConkey Agar
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MacConkey Agar
Crystal violet dye will inhibit certain Gram
positive bacteria
The addition of neutral red dye stains the
microbes fermenting lactosemicrobes fermenting lactose
Acting as a visual pH indicator, the agar
distinguishes those Gram negative bacteria
that can ferment the sugar lactose (Lac+) from
those that cannot (Lac-)
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MacConkey Agar
By utilizing the lactose available in the
medium, Lac+ bacteria such as Escherichia coli,
Enterobacter and Klebsiella will produce acid
This acid will lower the pH of the agar below This acid will lower the pH of the agar below
6.8 and results in the appearance of red / pink
colonies
Non-lactose fermenting bacteria such as
Salmonella, Proteus species and Shigella
cannot utilise lactose and will use peptone
instead.
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MacConkey Agar
This forms ammonia, which raises the pH of
the agar and leads to the formation of white /
colourless colonies
Some organisms ferment lactose slowly or Some organisms ferment lactose slowly or
weakly and are sometimes put in their own
category.
These include Serratia and Citrobacter
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Eosin Methylene Blue (EMB) Agar
A selective stain for Gram negative bacteria
It is a blend of two stains: eosin and
methylene blue (6:1)
Inhibits the growth of Gram positive bacteria Inhibits the growth of Gram positive bacteria
Provides a colour indicator distinguishing
between organisms that ferment lactose (e.g.
E. coli) and those that do not (e.g. Salmonella,
Shigella)
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Eosin Methylene Blue (EMB) Agar
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Eosin Methylene Blue (EMB) Agar
Lactose fermentation produces acids which
lower the pH
This encourages dye absorption by the
colonies, display nucleated colonies which colonies, display nucleated colonies which
are now coloured purple-black in the centers
Lactose non-fermenters may increase the pH
by deamination of proteins
This ensures that the dye is not absorbed
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Eosin Methylene Blue (EMB)
On EMB if E. coli is grown it will give a
distinctive metallic green sheen (due to the
metachromatic properties of the dyes, E. coli
movement using flagella, and strong acid end-movement using flagella, and strong acid end-
products of fermentation)
Some species of Citrobacter and Enterobacter
will also react this way to EMB
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Mannitol Salt Agar (MSA)
It contains a high concentration (7.5 - 10%)
of salt (NaCl) making it selective for
staphylococci and Micrococcaeae
Since this level of NaCl is inhibitory to most Since this level of NaCl is inhibitory to most
other bacteria, it is also a differential medium
This medium contains mannitol and the
indicator phenol red
It is a transparent, reddish pink agar
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Mannitol Salt Agar (MSA)
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Mannitol Salt Agar (MSA)
Coagulase positive staphylococci produce
yellow colonies with yellow zones (on the
medium itself)
Coagulase negative staphylococci produce Coagulase negative staphylococci produce
small pink or red colonies with no colour
change to the medium
The medium is used for the selective isolation
of presumptive pathogen (pp) staphylococci
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Thayer-Martin Agar
A Mueller-Hinton agar with 5% chocolate blood
(lysed blood) and antibiotics
It is used for culturing and primarily isolating
pathogenic Neisseria bacteria including N. pathogenic Neisseria bacteria including N.
gonorrhoeae and N. meningitidis as the medium
inhibits the growth of most other
microorganisms
When growing N. meningitidis, usually will start
be inoculating a normally sterile body fluid
(blood or CSF) so a plain chocolate agar is used
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Thayer-Martin Agar
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Mueller-Hinton Agar
Mueller-Hinton agar is a microbiological
growth medium that is commonly used for
antibiotic susceptibility testing.
It is also used to isolate and maintain Neisseria It is also used to isolate and maintain Neisseria
and Moraxella species
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Mueller Hinton Agar
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Bile Esculin Agar (BEA)
Selective differential agar used to isolate and
identify members of the genus Enterococcus,
also known as group D streptococci
Used primarily to differentiate Enterococcus Used primarily to differentiate Enterococcus
from Streptococcus
Members of Enterococcus are capable of
growing in the presence of 4% bile (oxgall)
and hydrolyzing esculin to glucose and
esculetin.
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Bile Esculin Agar
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Bile Esculin Agar (BEA)
Esculetin combines with ferric ions to produce
a black complex.
Certain bacteria are able to hydrolyze esculin.
A plate containing esculin will fluoresce a pale A plate containing esculin will fluoresce a pale
blue under UV radiation
Some bacteria can hydrolise this, leading to
UV dark colonies as opposed to UV light ones.
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Thiosulphate Citrate Bile Salt Sucrose
(TCBS) Agar
Selective isolation medium for pathogenic
Vibrio sp.
Most Enterobacteriaceae other than Vibrio sp.
are suppressed for at least 24hrare suppressed for at least 24hr
Bile salts inhibit Gram-positive organisms
Sodium thiosulphate serves as a source of
sulphur, which in combination with ferric
citrate, detects hydrogen sulphide production
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Thiosulphate Citrate Bile Salt
Sucrose (TCBS) Agar
When sucrose is fermented, it produces acid
which changes the pH
This is indicated by bromothymol blue and
thymol bluethymol blue
The medium is alkaline which enhances the
recovery of Vibrio cholerae
Saccharose (sucrose) is included as a
fermentable carbohydrate for the metabolism
of vibrios
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Thiosulphate Citrate Bile Salt
Sucrose (TCBS) Agar
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Cysteine Lactose Electrolyte Deficient
Medium (CLED) Agar
A valuable non-inhibitory growth medium
used in the isolation and differentiation of
urinary organisms
Being electrolyte deficient, it prevents the Being electrolyte deficient, it prevents the
swarming of Proteus sp.
Cysteine promotes the formation of cysteine-
dependent dwarf colonies
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Cysteine Lactose Electrolyte Deficient
Medium (CLED) Agar
Lactose fermenters produce yellow colonies
on CLED agar; non-lactose fermenters appear
blue
It has a pH of approximately 7.3 It has a pH of approximately 7.3
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Hektoen Enteric Agar (HEK / HE / HEA)
Selective and differential agar primarily used to
recover Salmonella and Shigella from patient
specimens
HEA contains indicators of lactose fermentation HEA contains indicators of lactose fermentation
and H2S production; as well as inhibitors to
prevent the growth of Gram positive bacteria
The pattern of lactose fermentation and H2S
production aids in the identification of the
organism subbed to the plate
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Hektoen Enteric Agar (HEK / HE / HEA)
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Luria-Bertani or Lysogeny Broth (LB)
LB is a nutritionally rich medium and is
primarily used for the growth of bacteria
Ingredients used to promote growth, include:
Peptides and casein peptones Peptides and casein peptones
Vitamins (including B vitamins)
Trace elements (e.g. nitrogen, sulfur, magnesium)
Minerals
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Nutrient Agar / Broth
A microbiological growth medium commonly
used for the routine cultivation of non-
fastidious bacteria
It is useful because it remains solid even at It is useful because it remains solid even at
relatively high temperatures
Also, bacteria grown in nutrient agar grows on
the surface, and is clearly visible as small
colonies
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Nutrient Agar / Broth
Ingredients:
0.5% peptone
0.3% beef extract
1.5% agar 1.5% agar
pH adjusted to neutral at
25C
Liquid media / broth are
used for growth of pure
culture
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Nutrient Agar / Broth
In nutrient broth, the bacteria grows in the
liquid, and is seen as a soupy substance - NOT
as clearly distinguishable clumps
When bacteria are grown in broth, they may When bacteria are grown in broth, they may
exhibit patterns of growth ranging from a
sediment at the bottom of the tube, turbid
growth throughout the tube, or a pellicle
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Nutrient Agar / Broth
Pellicle formation is sometimes due to:
affinity for oxygen
but may also be the result of hydrophobic
compounds present in the cell wall compounds present in the cell wall
or the general formation of dry, light colonies
If an organism produces and releases soluble
pigments, these will spread into the broth and
then change its colour
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Slant Agar
Growth of bacterial culture on agar slants and
in broths can provide us with useful
information concerning:
motility motility
pigmentation
oxygen requirement
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Slant Agar
Bacterial growth on slants depends on the
motility of the microorganism and ranges
from:
even (following the line of the original streak) even (following the line of the original streak)
irregular (slight spreading from the original line)
spreading (the organisms cover the entire surface
of the slant)
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Slant Agar
Non-motile bacteria such as cocci usually
produce even slants when viewed after 24 - 48
hours of incubation
Slightly motile to motile species produce Slightly motile to motile species produce
irregular to spreading growth
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Trypticase Soy Agar
The medium contains enzymatic digests of
casein and soybean meal
This provides amino acids and other
nitrogenous substances making it a nutritious nitrogenous substances making it a nutritious
medium for a variety of microorganisms
Dextrose is the energy source
Sodium chloride maintains the osmotic
equilibrium, while dipotassium phosphate acts
as buffer to maintain pH
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Cetrimide Agar
Selective isolation of the Gram negative
bacteria Pseudomonas aeruginosa
Cetrimide is a selective agent against alternate
microbial floramicrobial flora
It also enhances the production of
Pseudomonas pigments such as pyocannin and
fluorescein, which show a characteristic blue-
green and yellow-green colour, respectively