ii)media lecture

7/30/2019 II)Media Lecture

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Artificial cultivation of bacteria,

culture media

Patrick


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Cultivation

The process of growing microorganisms inculture

involves taking bacteria from the infection site

growing them in artificial environment in thelaboratory


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Introduction

In nature, bacteria exist as mixed populations.

In the laboratory these populations must be

separated so that characteristics of individualspecies may be observed.

A number of basic techniques are used in

microbiology with this end in mind.


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Introduction

First, microorganisms must be removed fromnatural environments and cultured

this requires artificial media and surfaces on

which bacteria may grow this also requires knowledge of nutritional

requirements and environmental requirements

bacteria of interest must be separated from allother bacteria


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Introduction

This requires separation techniques that allowisolation of a pure culture

once a pure culture is achieved, it should bemaintained

This requires that all media and lab supplies besterile

techniques are needed that facilitate working

with pure cultures This requires aseptic technique and techniques

of storage for pure cultures


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Uses of media & culture media provide nutrients for the growth of

microbesCulture;

gives in vitro diagnosis of infectious diseases.

Isolating bacteria from sterile sites of the body

provides an initial step in studying morphology

and its identification.

provides antigens for developing serological

assays or vaccines.


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Uses of media & culture

For genetic studies and manipulations in vitro.

provides a reliable way estimating their

numbers (viable count).

solid media is a convenient way of separating

bacteria in mixtures.


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Classification of media

Based on three factors

consistency,

nutritional component, functional use,


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Based on Consistency

Culture media are

liquid,

semi-solid

or solid and biphasic.


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Liquid media

These are available for use in test-tubes, bottles. They also called broths (e.g nutrient broth).

Here, bacteria grow uniformly producing general

turbidity. Certain aerobic bacteria and those containing fimbriae

(Vibrio & Bacillus) are known to grow as a thin film

called surface pellicle on the surface of undisturbed

broth.

Sometimes the initial turbidity may be followed by

clearing due to autolysis (pneumococci)


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Liquid media

Mostly used when a large number of bacteria areneeded

When the number in sample is suspected to be low.

This is the practical approach in blood cultures.

It can be used to obtain viable count (dilution

methods).

However, properties of bacteria are not visible

presence of more than one type of bacteria can notbe detected.


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Solid media Any liquid medium can be converted to solid media by

adding a solidifying agents.

Agar agar;

is the most commonly used solidifying agent.

Agar, is a galactan obtained from marine algae It melts at 95oC and solidifies at 42oC,

It doesnt contribute any nutrients

it is not hydrolyzed by most bacteria It is free from growth promoting or

growth retarding substances.

it is used at 1-3% to make a solid agar medium.


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Semi-solid agar

Made by reducing the amount of agar to 0.2-0.5% useful in demonstrating bacterial motility

Also for transporting some bacteria

e.g. Stuarts and Amies media

mannitol motility medium.


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Biphasic media

Sometimes, a culture system comprises of both

liquid and solid medium in the same bottle.

This is known as biphasic medium.

The inoculum is added to the liquid medium

subcultures are made, by tilting the bottle

This allows the liquid to flow over the solid

medium This eliminates frequent opening of the culture

bottle to subculture


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Other solidifying agents Besides agar, egg yolk and serum too can be used

to solidify culture media.

While serum and egg yolk are normally liquid,

they can be rendered solid by coagulation using

heat.

Serum containing medium; Loefflers serum slope

egg containing media;

Lowenstein Jensen medium

Dorset egg medium are solidified


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Based on nutritional component

Media can be classified as simple, complex andsynthetic (or defined).

non-fastidious bacteria grow with minimal

requirements

Fastidious bacteria require extra nutrients.

Simple media; peptone water, nutrient agar

Complex media; blood agar, chocolate agar. Synthetic or defined media; Davis & Mingioli

medium are specially prepared media for

research purposes


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Based on functional use

These include

basal media,

enriched media,

selective/enrichment media,

indicator/differential media,

transport media and holding media.


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Basal media

These are basically simple media

they support growth of a broad range of

organisms

They usually have complex constituents Constituents of such media are generally defined

e.g. peptone water, nutrient broth and nutrient

agar are considered basal medium


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Enriched media

Addition of extra nutrients in the form of blood,serum, egg yolk etc, to basal medium

Enriched media are used to grow fastidious

bacteriaExamples;

Blood agar,

chocolate agar, Loefflers serum slope etc


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Selective media

These are designed to inhibit unwanted

organisms or contaminating bacteria

this helps to recover pathogen from a mixture of

bacteria.

selective media are agar based

addition of certain inhibitory agents to agar

media makes it selective

These should not affect the pathogen in choice

Examples; antibiotics, dyes, chemicals, alteration

of pH or a combination of these.


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Enrichment media

These are liquid media that also serves to

inhibit commensals in the clinical specimen.

Examples;

Selenite F broth, tetrathionate broth

alkaline peptone water

These are used to recover pathogens fromfecal specimens.


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Differential or indicator media Certain media are designed to different bacteria

on the basis of their colony colour.

by incorporating dyes, metabolic substrates etc,

bacteria that utilize them appear as differently

coloured colonies

Such media are called differential media or

indicator media

Examples: MacConkeys agar, CLED agar, TCBS

agar, XLD agar etc


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Transport media Clinical specimens must be transported to the

laboratory immediately after collection

to prevent overgrowth of contaminating

organisms or commensals.

This can be achieved by using transport media.

Such media prevent drying of specimen,

They also maintain the pathogens they inhibit overgrowth of unwanted bacteria.


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Transport media

Some of these media (Stuarts & Amies) are

semi-solid in consistency

Addition of charcoal serves to neutralize

inhibitory factors

Cary Blair medium is used to transport samples

from cholera suspects

Sachs buffered glycerol saline is used to

transport samples of bacillary dysentery suspects


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Anaerobic media

Anaerobic bacteria need special media ( no oxygen) and

extra nutrients

They need to be supplemented with nutrients like

hemin and vitamin K

Boiling the medium expels any dissolved oxygen

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 used to grow

Clostridium spps

It contains heart meat and nutrient broth.


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Anaerobic media

Before use the medium must be boiled in water

bath to expel any dissolved oxygen and then

sealed

Methylene blue or resazurin is an oxidation-

reduction potential indicator

it is incorporated in the thioglycollate medium

Under reduced condition, methylene blue is

colourless


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Use of physical separation procedures

Streak plate tech

For achieving single colony characteristics

streak plate technique

pour plate;

spread plate;
http://en.wikipedia.org/wiki/File:Agar_plate_with_colonies.jpg


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Growth conditions

Bacteria may be isolated from a variety ofenvironments

For cultivation of bacteria in the lab, the

conditions of the environments must bemimicked

Refer to previous lecture on growth

requirements


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Bacterial requirements Water

Oxygen

Carbon dioxide

Temperature

PH Light

Nutrients

Growth factors Inorganic salts

Osmotic effect


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Salt conditions Halophiles; these bacteria require relatively high

concentrations of salt for growth (10-20%);

Halophiles must possess special membranes and

enzyme

Some organisms are salt tolerant. These do notrequire salt for growth but can grow in its presence

An example is Staphylococcus aureus. which is found

on skin, which often has a high salt concentration(10% NaCl).


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Preparation and preservation

Heat stable ingredients Care must be taken to adjust the pH of the

medium before autoclaving.

Dehydrated media are commercially available These must be reconstituted as per

manufacturers recommendation.

Most culture media are sterilized by autoclaving.


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Heat labile ingredients

like glucose, antibiotics, urea, serum, blood are not

autoclaved.

These are filtered and added separately after the

medium is autoclaved.

a representation from each lot is tested forperformance and contamination before use.

Once prepared, media may be held at 4-5oC in the

refrigerator for 1-2 weeks.

Certain liquid media in screw capped bottles or tubes

or cotton plugged can be held at room temperature for

weeks.


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Thank you

ii)media lecture

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