Wk 4 Flashcards by astrum glen

Bacteria where their needs are complex and
relatively complex, and exceptional media components
must be used for growth

Fastidious

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– Bacteria where their needs are relatively
basic and straightforward

Nonfastidious

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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

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Example of liquid media

Pectin water and nutrient broth

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(prepared by adding 0.2-0.5% agar
to the liquid medium.

Semi-solid media

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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

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support the growth of
micro-organisms that do not have special
nutritional needs

Basic or basal media

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Mainly used maintaining stock cultures of control
strains of bacteria and for subculturing of
bacteria from selective media

Basic or basal media

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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

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These are used for sites which have a normal
microbial flora to prevent the growth ofunwanted
bacteria overcrowding the pathogenic bacteria

Selective media

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o Ex. DCA medium for Dysentery bacilli

Selective media

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These media have certain substances or
indicators added to them which help to
differentiate between bacteria by a color change

Indicator or differential media

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o Ex. MacConkey media

Indicator or differential media

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Media used to transport specimens from the
patient’s bedside or health centers to a
microbiology laboratory

Transport media

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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

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Prevent the pathogenic bacteria to be overgrown
by commensal bacteria

Transport media

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Ex. Cary-Blair’s medium u

Transport media

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used for transportation
and preservation of enteric bacteria

Cary-Blair’s medium

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Separate room should be dedicated for

o Preparation of culture media
o Pouring of culture media
o Sterilization of culture media

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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

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how many media is suggested for a
basic microbiology laboratory

minimum of five dehydrated media

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`tend to absorb moisture from the air
and are sensitive to heat, light and extreme changes in
temperature

Dehydrated media

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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

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o It is advisable to prepare

one week’s requirement only

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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

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

Wk 4 Flashcards

(87 cards)