ISOLATION OF BACTERIAL PATHOGENS IN PURE CULTURES

(Bacterial Culture media)

Dr Mostafa Mahmoud Ahmed, Ph DConsultant Microbiologist, GDHA, Riyadh, KSA.Associate Prof. of Microbiology & Immunology.

Faculty of Medicine – Ain Shams University

CULTURE MEDIA

Culture media generally provide sources of carbon , energy and nitrogen in the form of available carbohydrates and amino

acids.

Special media provide specific requirements as inorganic salts or particular growth

factors.

Types of Culture Media

- Basic media - Enriched media- Selective media - Enrichement media - Indicator ( Differential ) media- Transport media

I . BASIC MEDIA

- These are simple media used to support the growth of microorganisms that do not have special nutritional requirements.

- They include nutrient broth, peptone water, and nutrient agar.

1 -Nutrient Broth

-Filtrate of cooked fresh minced meat + 1% peptone + 0.5% NaCl.

-Clear yellowish fluid medium.

-Sterilized in autoclave at 121°C for 30 min.

-Base for most culture media.

2 -Peptone water

-1% peptone + 0.5% NaCl dissolved in water

-Clear colourless fluid medium

-Sterilized in autoclave at 121°C for 30 min.

-Base for sugar media -Indole production test

3 -Nutrient agar

-2-3% agar powder dissolved in nutrient broth.

-Yellowish semi-transparent medium ) Plates or tubes as slopes or deep agar.(

-Sterilized in autoclave at 121°C for 30 min. Uses:

-Base of different culture media -Plates : for isolation & identification of bacteria

-Slopes : for preservation of pure cultures -Deep agar : for anaerobic bacteria

Nutrient Agar Plates

II. ENRICHED MEDIA- Prepared by the addition of substances such as blood, serum or egg to a basic medium.

- Used for cultivation of fastidious organisms that cannot grow on simple media and need highly nutritive substances for growth.

-Used for culturing sterile body fluids such as blood or CSF, where the finding of any organisms = infection due to that organism. And also for primary identification of microorganisms e.g. hemolysis on blood

- e.g. blood agar, chocolate agar, Loeffler’s serum .

1 -Blood agar .5-10% sterile defibrinated sheep or human

blood + melted nutrient agar at 55°C.

.Red opaque solid medium .

.N. agar is sterilized in autoclave at 121°C for 30 min . Blood is added under complete aseptic condition at 45-

55 oC.

-Supports the growth of most delicate organisms e.g. Streptococcus pyogenes.

-Identifying bacteria according to their haemolytic action on the red cells.

2 -Chocolate agar

.Prepared as blood agar followed by raising the temperature to l00 °C for 2 min. to rupture

red cells and release nutrients as X and V factors.

.Brown opaque solid medium

.Sterilized as blood agar.

.Used for the isolation of Neisseria meningitides, Haemophilus influenza and Streptococcus pneumonae

Blood Agar Medium

Plate

Chocolate Agar Medium

Plate & Slope

3-Loeffler's serum

.3 parts of sheep or horse serum+ 1 part glucose broth

.Opaque whitish solid medium.

.Serum is sterilized by filtration & glucose broth by Koch's steamer .

.The medium is solidified in hot air inspissator at 75°C for 2 hr for 2 successive days .

Uses : Culture of Corynebacterium diphtheria

III. SELECTIVE MEDIA. Solid media that contain substances (e.g. bile salts or other chemicals, dyes, antibiotics) which

inhibit the growth of one organism to allow the growth of another .

. Used when culturing a specimen from a site having a normal microbial flora to prevent unwanted

contaminants overgrowing a pathogen.

. They include the following media:-

1 -Lowenstein Jensen medium .3 parts beaten eggs + 1 part water+

malachite green.

.Green opaque solid medium

.Sterilized in hot air inspissator at 75 °C for 2hr for 2 successive days.

.Used for Isolation of Mycobacterium tuberculosis

2 -MacLeod's tellurite blood agar

.Blood agar + 0.02-0.04% K tellurite

. .Red opaque solid medium

.Sterilized as blood agar.

.Used for isolation of Corynebacterium diphtheriae from contaminated materials .

3 -Modified Thayer-Martin agar

.Chocolate agar + vancomycin + colistin + nystatin .

.Brown opaque-solid medium.

.Sterilized as Chocolate agar.

.Used for Isolation of Neisseria from non sterile specimens.

4 -Thiosulphate citrate bile sucrose agar) TCBS(

.Alkaline agar + sucrose + thiosulphate + citrate and bromothymol blue indicator.

.Greenish transparent solid medium.

.Sterilized in autoclave at 121°C for 30 min.

.Used for isolation of Vibrio cholerae.

TCBS medium

5 -Deoxycholate citrate agar (DCA)

.Agar + lactose + neutral red indicator + Na deoxycholate and citrate.

.Reddish semi transparent solid medium.

.Sterilized in autoclave at 121°C for 30 min.

.Used for isolation of Shigella and Salmonella.

XLD Media .Agar + lactose + phenol red indicator + ferric

citrate + desoxycholate + xylose + lysine + sucrose + yeast extract.

.Reddish semi transparent solid medium.

.Sterilized by boiling. .Used for isolation of Shigella and Salmonella.

IV. ENRICHMENT MEDIA... Fluid media that contain substances which favour the

growth of wanted organisms on the expense of others. . Usually used as a preliminary step for isolation of

pathogens before subculturing on solid selective media. Examples are:

. Selenite broth for isolation of Salmonella and Shigella species from faeces.

. Tetrathionate broth for isolation of Salmonella from faeces.

. Alkaline peptone water for isolation of Vibrio cholerea.

V. INDICATOR (DIFFERENTIAL) MEDIA

. These are media to which dyes or other substances (Indicators) are added to differentiate microorganisms.

. Indicators change colour when acid is produced following fermentation of a specific carbohydrate e.g. MacConkey's agar medium.

MacConkey's agar medium

. Peptone, agar, lactose, bile salt and neutral red indicator.

. Reddish transparent solid medium

. Sterilized in autoclave 121°C for 30 min.

. Supports the growth of most Gram- negative bacilli,

especially the enterobacteriaceae, but inhibits the growth of

Gram positive organism and some fastidious Gram-

negative bacteria, such as Haemophilus and Neisseria.

MacConkey's agar MacConkey's agar mediummedium

VI. IDENTIFICATION MEDIA These include media to which substrates or chemicals are added to help identify bacteria isolated on primary cultures. i.e. organisms identified must be first isolated in pure culture.

Organisms are mainly identified by a change in the colour of the medium and or the production of gas.

They include peptone water sugars, litmus milk, and gelatin media.

1 -Peptone water sugar media

-Peptone water + 1% tested sugar + 1% Andrade's indicator.

-Durham tube is an inverted tube to visualize gas bubbles produced from sugar fermentation.

. -Yellowish transparent

-Sterilized in Koch's steamer for 20 min on three successive days (tyndallisation).

-Used to test the biochemical activity of bacteria on carbohydrates.

2 -Litmus milk -Steamed fresh milk for 1 hour (cream is

removed) + litmus solution.

-Mauve

-Sterilized in Koch's steamer for 20 min on three successive days (tyndallisation).

-Used to test the saccharolytic activity of bacteria.

-For identification of enterococci )bleaching.(

3 -Gelatin

-10-15% gelatin sheets dissolved in nutrient broth .

-The medium is solid below 24°C. Above this temperature it melts into yellowish fluid.

-Sterilized in Koch's steamer for 20 min on three

successive days (tyndallisation).

-Used to test the proteolytic activity of different organisms.

VII. TRANSPORT MEDIA - Semisolid media that contain ingredients to prevent

the overgrowth of commensals & ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately.

Examples: 1- Cary-Blair medium for preserving enteric

pathogens.2- Amies transport medium for ensuring the

viability of gonococci. 3 -Thioglycollate broth and deep agar for

anaerobic organisms.

CULTURE MEDIA FOR ANAEROBESMedia for anaerobes is the same as media for

aerobes except that: 1. They are richer in organic constituents . 2. Contain reducing agents (cysteine & haemin).

3. Contain a redox indicator .The inoculated media are incubated in anaerobic

environment using anaerobic gas pack .

Robertson's cooked meatAnaerobic enrichment media

-5 gm cooked minced meat to which broth is added .) Anaerobiosis is achieved through

reducing substances in the meat e.g. haemin and glutathione.(

-Sterilized in autoclave at 121°C for 30 min.

Anaerobic GasPak SystemA method for the exclusion of oxygen from a

sealed jar used for incubation of anaerobic cultures in a nonreducing medium .

Methods of isolation of pure culture

The streak-plate method to obtain pure

cultures

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