Bacterial culture media بكتريا عملي

Bacterial Culture Medium

By

Dr. Nabil El Aila

Assistant Professor of Molecular Microbiology

Medical Technology Department

Al -Aqsa University

Culture and Medium

• Culture is the term given to microorganisms that are cultivated in the lab for the purpose of identifying and studying them.

• Medium is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth.

- Nutrients (agar, pH indicators, proteins and

carbohydrates) used to grow organisms

outside of their natural habitats

Dr. Nabil El Aila General Microbiology

Basic requirements of culture media

• Nutrients

- Energy source

- Carbon source

- Nitrogen source

• Mineral salts – Sulphate, phosphates, chlorides &

carbonates of K, Mg & Ca.

• A suitable pH – 7.2 – 7.4

• Accessory growth factors

- Tryptophan for Salmonella typhi

- X & V factors for H. influenzae


Agar - Agar

• Agar is obtained from Sea weeds

• Agar contain long chain polysaccharides. Inorganic salts and

protein like substance

• Used as solidifying agent for culture media in Petri plates,

slants, and deeps

• Generally not metabolized by microbes

• Liquefies at 98

C and Solidifies ~42

C


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Culture medium

– is the mixture of various nutrients that is

suitable for the growth of microorganisms.

Types of Culture Media

based on the function and chemical components

based on the physical state


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Based on the physical state

• Liquid medium:

– Without agar.

– for the proliferation of bacteria.

• Solid medium:

– 1.5-2.5% agar.

– for the isolation and identification of bacteria

– e.g., slant, Petri dishes/plates.

• Semisolid medium:

– 0.3-0.5% agar.

– for the observation of bacterial motility and preservation of bacteria.


ii. Growth of bacteria in culture medium

i. Liquid medium or Broth

1) Homogeneous turbidity

2) Surface

3) Bottom

ii. Solid agar medium

Colony and mossy

1) Smooth colony

2) Rough colony

3) Mucoid colony

iii. Semi-solid agar medium


Blood culture – ‘Liquid Medium’

Semisolid medium

The organism in the tube on the left in the

figure is motile and the organism in the tube on

the right is nonmotile.


Types of culture media

• Basic media

• Enriched media

• Selective and enrichment media

• Enrichment media

• Differential media or indicator media

• Transport media

• Anaerobic media

Dr. Nabil El Aila

General Microbiology


• Basic media are basically simple media that supports most

non-fastidious bacteria.

Peptone water, nutrient broth and nutrient agar considered

basic medium.


Peptone

• Peptone contain partially digested proteins

• Proteases

• Polypeptides

• Aminoacids

• Inorganic salts

Phosphates

Potassium and Magnesium

Riboflavin

Meat exract called as Lab lemco

Carbohydrate media

• Peptone water – 100 ml,

Desired sugar 1 gm% and

Andrade's indicator –

0.005% soln(1ml)

• Dissolve the desired

carbohydrate in peptone

water and steam for 30 min

or sterilize by filtration.

• Use: To test the fermenting

ability of an organism


Nutrient Agar

• Contain 2% agar added

to Nutrient agar

commonly used

• Concentration can be

increased to 6% to

prevent swarming

• Can be reduced to 0’5%

Pigment producing Staphylococci


• Basic media

• Enriched media




• Transport media

• Anaerobic media

Dr. Nabil El Aila


• 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 exacting

(fastidious) bacteria.

- Blood agar, chocolate agar, Loeffler’s serum slope etc are

few of the enriched media.


Enriched Medium

• To culture medium Blood serum or egg are added to medium

• eg Blood agar

• Chocolate agar

• Egg



Different types of hemolysis on

Blood Agar


Other Enrichments – Chocolate Agar

• Several organic

materials are added to

the basic constituents of

the Medium such as

Blood, yeast, yeast

extract etc


Chocolate agar


• Basic media

• Enriched media




• Transport media

• Anaerobic media

• Selective and enrichment media are designed to inhibit unwanted

commensal or contaminating bacteria and help to recover pathogen

from a mixture of bacteria.

- 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 don’t affect the pathogen.

- Various approaches to make a medium selective include

addition of antibiotics, dyes, chemicals, alteration of pH or

a combination of these.

- Examples of selective media like Salmonella shigella agar,

XLD agar, Heckton Enteric agar, Mannitol salt agar, Lowenstein

Jensen’s medium

• Enrichment media are 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 fecal specimens.

Salmonella Shigella agar

- For isolation and differential medium for pathogenic Gram-negative bacilli in particular, Salmonella and Shigella. Inhibitor for Coliforms.


For isolation of Salmonella and Shigella species from clinical samples

XLD agar (Xylose Lysine

Deoxycholate)

For isolation of Salmonella and Shigella species from clinical samples

Hecktoen Enteric agar


Lowenstein Jensen Medium - cultivation of

Mycobacterium tuberculosis

Lowenstein-Jensen’s medium

• Mineral salt soln - 600ml Malachite green soln - 20ml (2gm% in D.water) Beaten egg - 1000ml (20-22 eggs)

• Mix the above

• Distribute in Mc Cartney bottles

• Sterilize by Inspissation

Use: To cultivate Mycobacteria


• For selective isolation of pathogenic staphylococci

• Mannitol fermentation by pathogenic staphylococci is

indicated by a yellow halo surrounding the colonies.

•Sodium chloride is the inhibitor agent.

•Phenol red is the PH indicator

Mannitol Salt Agar ( MSA )

Enrichment Medium

• If the sample contain more

than one type of bacteria,

undesired bacteria grwoth

can be reduced or

eliminated.

• The desired organism is

facilitated to grow

• Eg Tetrathionate broth

• Selenite F broth


• Basic media

• Enriched media




• Transport media

• Anaerobic media

Dr. Nabil El Aila


• 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 dyes,

metabolic substrates etc, so that those bacteria that

utilize them appear as differently coloured colonies.

- Such media are called differential media or indicator

media.

- Examples: MacConkey’s agar, CLED agar, TCBS

agar etc.


MacConkey agar

• MacConkey agar is useful medium for cultivation of enterobacteria

• It contains a bile salt to inhibit non intestinal bacteria

• Lactose in combination with Neutral red distinguish the lactose fermenting from the non lactose fermenting Salmonella and Dysentery group

Differential Medium

Mac Conkey's agar

• Bringing out different

characters of bacteria their

atypical characters

• Mac Conkey’s medium

Contain peptone, Lactose

Agar, Neutral red and

taurocholate and show

growth of Lactose

fermenters as pink colored

colonies


Lactose fermenting and Non lactose

fermenting

TCBS medium

(Thiosulphate Citrate bile salts sucrose agar)

For cultivation of Vibrio cholera


Cystine-Lactose-Electrolyte-Deficient Agar (CLED agar)

For isolation and differentiation of urinary pathogens on the basis

of lactose fermentation



• Basic media

• Enriched media




• Transport media

• Anaerobic media

Dr. Nabil El Aila


• 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 (desiccation) of specimen, maintain the

pathogen to commensal ratio and inhibit overgrowth of unwanted

bacteria.

- Some of these media (Stuart’s & Amie’s) are semi-solid in

consistency.

- Addition of charcoal serves to neutralize inhibitory factors.

- Cary Blair medium and Venkatraman Ramakrishnan medium are

used to transport feces from suspected cholera patients.

- Sach’s buffered glycerol saline is used to transport feces from

patients suspected to be suffering from bacillary dysentery.

Transport Medium

• Stuart’s medium contain

reducing agents to

prevent oxidation.

• Charcoal to neutralize

certain bacterial

inhibitors to Gonococci,



• Basic media

• Enriched media




• Transport media

• Anaerobic media

Dr. Nabil El Aila


• Anaerobic media: Anaerobic bacteria need special media for

growth because they need low oxygen content, reduced

oxidation –reduction potential and 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.

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.

• Methylene blue or resazurin is an oxidation-reduction potential

indicator that is incorporated in the thioglycollate medium.

Under reduced condition, methylene blue is colorless.

Anaerobic Medium

• Robertson’s cooked

meat medium

• Thiglyclolate liquid

medium


Anaerobic Jar (GasPak)

Clamp with clamp screw

Lid with O-ring gasket

Catalyst pellets in reaction chamber

Flash arrester to prevent explosion

GasPak disposable anaerobic indicator strip

Culture plates

GasPak disposable hydrogen and carbon dioxide generator envelope

O O

O H H

H H

Oxygen in jar (O2)

Water (H2O)

Hydrogen gas (H2)

+ 2

2 Platinum catalyst


Other/ungrouped media


Mueller Hinton Agar • It is a microbiological growth medium that support the growth

of most microorganism.

• It is commonly used for antibiotic susceptibility testing

• typically contains (w/v):

- 30.0% beef infusion

- 1.75% casein hydrolysate

- 0.15% starch

- 1.7% agar

• pH adjusted to neutral at 25

C.

• Five percent sheep blood may also be added when

susceptibility testing is done on Streptococcus species or the

sensitivity can be done on Blood agar.

http://en.wikipedia.org/wiki/Percentage_solution

http://en.wikipedia.org/wiki/Casein

http://en.wikipedia.org/wiki/Hydrolysis

http://en.wikipedia.org/wiki/Starch

http://en.wikipedia.org/wiki/Agar

http://en.wikipedia.org/wiki/Celsius

http://en.wikipedia.org/wiki/Celsius

http://en.wikipedia.org/wiki/Streptococcus

Muller Hinton Agar for Antibiotic

Testing


Antibiotic Testing on Blood Agar

Medium


Triple Sugar Iron Agar (TSI):

• It is used for identification of a Gram- negative bacilli of the

enteric group.

• It contains glucose (0.1% ), Lactose (1%), sucrose(1%).

And peptone (2%) as nutritional sources.

• Sodium Thiosulfate serves as the electron receptor for

reduction of sulfur and production of H2S.

• Detects fermentation of sucrose, lactose, glucose, as well as

production of hydrogen sulfide and /or gas .

• Phenol red is the PH indicator; ferric ammonium citrate is

H2S indicator.

Dr. Nabil El Aila


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Triple sugar iron slant


The lysine iron agar slant or LIA slant test is used to distinguish bacteria which

are able to decarboxylate lysine and/or produce hydrogen sulfide from those that

cannot. This test is particularly useful for distinguishing different Gram-negative

bacilli—especially among the Enterobacteriaceae

Lysine Iron Agar (LIA):

http://en.wikipedia.org/wiki/Carboxylic_acid

http://en.wikipedia.org/wiki/Lysine

http://en.wikipedia.org/wiki/Hydrogen_sulfide

http://en.wikipedia.org/wiki/Gram-negative_bacteria



http://en.wikipedia.org/wiki/Bacillus_(shape)

http://en.wikipedia.org/wiki/Enterobacteriaceae

Simmons' Citrate Agar is a defined, enrichment medium that tests for an

organism's ability to use citrate as a sole carbon source and ammonium ions as the

sole nitrogen source.

Simmons' citrate agar


Urea agar base

Urea Agar Base (Christensen Agar Base). for the detection of urease producing organisms.

Sabouraud's Dextrose agar commonly

used Fungal Isolation Medium

Sabouraud's Dextrose Agar

Dextrose - 4 gm%

Neopeptone - 1 gm%

Agar - 1.5 gm%

Distilled water - 100 ml

• Dissolve the ingredients by heating in a water bath, cool and adjust pH to 5.4

• Autoclave and dispense 20 ml amount in test tubes

Use: For the cultivation of Fungi


Sterilization of culture media

• Media are sterilized in the autoclave at 1210 c for 15

min under 15 lb of Pressure

• Heat-labile substances like serum & sugar solutions

must be sterilized by free-steam or filtration

• Egg containing media –-- Lowenstein-Jensen’s

medium, Loeffler's serum slope by inspissation

• Discarded culture plates are to be sterilized by

autoclaving prior to washing


Storage of culture media

• Prepared media in individual

screw capped bottles can be

stored for weeks at room temp

• Poured plates deteriorate

quickly and often contaminated,

hence cold storage is necessary

• For smaller labs domestic

refrigerators & for larger labs

insulated cold room(4-5oc)

• Deep freeze refrigerators for

preservation of sera, antibiotics

& amino acids (-10 to - 400c)


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Bacterial culture media بكتريا عملي