Wk 4 Flashcards
Bacteria where their needs are complex and
relatively complex, and exceptional media components
must be used for growth
Fastidious
– Bacteria where their needs are relatively
basic and straightforward
Nonfastidious
is an aqueous solution to which all the
necessary nutrients required for growth of micro-
organisms have been added
culture medium is an aqueous solution to which all the
necessary nutrients required for growth of micro-
organisms have been added
Example of liquid media
Pectin water and nutrient broth
(prepared by adding 0.2-0.5% agar
to the liquid medium.
Semi-solid media
disadvantage and advantage of liquid medium
Disadvatage:
- Growths of bacteria do not exhibit special
characteristic appearances and hence, are difficult
to identify
- Isolated colonies can’t be separated from a liquid
medium
o Advantage:
- Different micro-organisms give distinct appearances
in the form of colonies which are very useful in
identification
- Indispensable for the isolation of pure cultures
(microorganisms are easily separated and grown as
a pure line, thus helping an identification
support the growth of
micro-organisms that do not have special
nutritional needs
- Basic or basal media
Mainly used maintaining stock cultures of control
strains of bacteria and for subculturing of
bacteria from selective media
- Basic or basal media
n inhibitory substances added
to them which inhibit the growth of unwanted
bacteria and only promote the growth of
pathogenic or disease-causing bacteria
- Selective media
These are used for sites which have a normal
microbial flora to prevent the growth ofunwanted
bacteria overcrowding the pathogenic bacteria
- Selective media
o Ex. DCA medium for Dysentery bacilli
- Selective media
These media have certain substances or
indicators added to them which help to
differentiate between bacteria by a color change
Indicator or differential media
o Ex. MacConkey media
Indicator or differential media
Media used to transport specimens from the
patient’s bedside or health centers to a
microbiology laboratory
- Transport media
media used in transport media
Usually semi-solid media which promote the
growth of certain delicate organisms which might
usually not survive the time taken for
transportation
Prevent the pathogenic bacteria to be overgrown
by commensal bacteria
- Transport media
Ex. Cary-Blair’s medium u
- Transport media
used for transportation
and preservation of enteric bacteria
Cary-Blair’s medium
- Separate room should be dedicated for
o Preparation of culture media
o Pouring of culture media
o Sterilization of culture media
In the preparation of complex culture media, what media should be used
In the preparation of complex culture media, ready-made
standardized dehydrated media can be used to ensure
good performance and reproducibility
how many media is suggested for a
basic microbiology laboratory
minimum of five dehydrated media
`tend to absorb moisture from the air
and are sensitive to heat, light and extreme changes in
temperature
Dehydrated media
o Follow the storage conditions as indicated for dehydrated media
- Below 25°C in a dry area
- Away from direct sunlight, autoclaves, hot-air
ovens, other heat sources - Use the stock in lot/batch number order (product
with earlier batch number should be opened first) - Note on the label the container is first opened
- Order the medium in an appropriate size o
container - A large container which is opened several times
will spoil the medium overtime
o It is advisable to prepare
one week’s requirement only
how to prepare media and volume
Prepare the medium in a container which is about twice
the final volume of the medium needed so that the dry
powdered medium can be adequately mixed
Dry culture media powder which does not dissolve
might not be sterilized in the autoclave and can be a
source of contamination
Media containing agar should be heated first to
completely dissolve the agar before autoclaving
Most culture media will require final sterilization in an
autoclave at 121°C for 20 minutes
Do not adjust the ____of dehydrated media prior to
sterilization
ph
A liquid medium can be tested by
dipping a narrow
range pH paper into a sample of the medium at room
temperature and comparing the color of the paper
against the pH color chart provided
agar medium can be tested by
laying a narrow
range pH paper on its surface after it has solidified
used to make blood agar and chocolate
agar, etc
Nutrient agar
mostly used as an indicator medium
for gram negative bacteria
MacConkey agar
Sensitivity test agar (Mueller-Hinton agar) –
for
antimicrobial sensitivity testing
selective medium for growth
of enterobacteria like Salmonella and Shigella
Deoxycholate citrate agar
Over-heating of the media should be avoided as i
may cause the formation of toxic products due to
chemo-oxidation
it is preferred to work within an
inoculation hood or cabinet
filled with a filtered air supply
heat the mouth of the container from which the
medium is being poured in a flame for making
a slant or a
slope
To reduce condensation of water on the lids, the media
should be at what temp
cooled down to 52°C before pouring
ayer of moisture is undesirable and should be removed
by drying the set plates in a drying cabinet at what temp
60°C for 15-30 mins
Screw-capped bottles of nutrient broth and agar can be
stored for
6 months at low ambient temperatures (12-16°C).
Agar plates should be stored at __________
to avoid loss of moisture.
Agar plates should be stored at 2-8°C in sealed containers
to avoid loss of moisture.
The pH of the prepared medium in its final form at an
ambient temperature of _______should lie within the range
given on the product labe
The pH of the prepared medium in its final form at an
ambient temperature of 25°C should lie within the range
given on the product labe
STERILITY
A few plates, tubes or bottles prepared from each lot or
batch should be incubated for _________________
A few plates, tubes or bottles prepared from each lot or
batch should be incubated for 2-5 days at 35-30°C and 50-
55°C
- For a lot of 100 or less units, _______ should be tested
- For a lot of 100 or less units, 3-5% units should be tested
For a larger lot, _________ should be tested
for sterility
For a larger lot, 10 random plated or tubes should be tested
for sterility
For testing new lots or batches of media,
inoculate old and
new lots in together and compare the performance of the
two lots side by sid
To test whether the medium prepared supports the growth
of bacteria,
inoculate a few units with appropriate stock
cultures or fresh isolate
Chiefly used as the basal medium for carbohydrate
fermentation tests
- Peptone water
o Used to test the formation of indole
- Peptone water
Usually supplied as golden granular powder with a low
moisture content and gives a pH between 5 and 7 in a
1% solution
- Peptone water
o It is hygroscopic
- Peptone water
peptone waterph
gives a pH between 5 and 7 in a
1% solution
- If you are preparing peptone water, the recipe is
o Peptone, 10g
o NaCl, 5g
o Water, 1L
o Dissolve the ingredients in warm water and adjust the
pH to 7.4-7.5
o Filter and distribute as required
o Autoclave at 121°C for 15 minutes
o Reconstitute the dehydrated nutrient broth
according to manufacturer’s instructions
o Add 2% agar to the solution
o Heat to completely dissolve the agar
- Nutrient agar
part from being an enriched medium, it also is used
as an indicator medium to study the haemolysis of
bacteria like Staphylococcus aureus and
Streptococcus pyogenes
- Blood agar
Blood used for the preparation of blood agarshould
be as fresh and stored at what temp
Blood used for the preparation of blood agarshould
be as fresh and stored at 2-8°C
RECOMMENDED BLOOD FOR BA IS
Defibrinated blood is recommended for use rather
than blood containing an anticoagulant
CONCENTRATION OF BLOOD FOR BA
o Concentrations can vary from 5-50% although10%
WHAT TEMP FOR INCUBATING BA
o Incubate at 35°C
At 100ml blood to 1L of sterile agar base, agar willtend
to s
agar willtend
to settle at the bottom of the flask, so all flasks should
be swirled to facilitate proper mixing of agar and
aeration of blood. Pour into plates after
Prepared from blood agar by heating and contains
certain extra nutrients required for growth of fastidious organisms like Neisseria and Pneumococcus
- Chocolate agar
how o cool choco agar
Cool it in a water bath at 75°C at 10% sterile blood
he heat causes red cells to lyse and release the
nutrients
chocolate agar
medium turns chocolate brown within—————
Then pour into plates.
10 minutes.
o Used for the cultivation of enterobacteria
- McConkey Agar
Contains bile salts which inhibit the growth of grampositive bacteria and promotes growth of only gramnegative bacteria
- McConkey Agar
dye of macconkey
Contains a dye called neutral red which gives a pink
color to colonies of lactose-fermenting bacteria
autoclaving of macconkey
Heat in autoclave with free steam (100°C) for 1 hour
and then 115°C for 15 minutes.
solation of bacteria in pure culture from clinical
specimens
Streak Culture
inoocluation loop type
Platinum, Nichrome wire or plastic disposal wire loop is
used
- Provides a uniform surface growth of the bacterium.
Lawn Culture
Used for for bacteriophage typing or antibiotic sensitivity
testing
Lawn Culture
Also used in the preparation of bacterial antigens and
vaccines
Lawn Culture
Lawn cultures are prepared by
flooding the surface of the
plate with a liquid suspension of the bacterium
pour plate melting req
- Agar medium is melted (15 ml) and cooled to 45oC.
ml of inoculum added for pour plate method
- 1 ml of the inoculum is added to the molten agar.
pour plate method incubation
- Plates are incubated at 37oC
It gives an estimate of the viable bacterial count in a
suspension.
Pour Plate Method
- Used for quantitative urine cultures.
Pour Plate Method
- Sample is pipetted onto the surface of an agar.
What method
Spread Plate Method
are inoculated by touching with a charged
loop or by adding the inoculum with pipettes or syringes.
Liquid cultures
Used for Blood culture, Sterility tests or for continuous
culture methods
Liquid Culture
Disadvantage: It does not provide a pure culture from
mixed inocula
Liquid Culture
Done in Kirby-Bauer Disk Diffusion method of Antibiotic
Sensitivity
Lawn Culture Technique
Lawn Culture Technique agar used
Lawn Culture Technique
Streak perpendicular
lawn culture
