Bacterial Culture and Media 

What is a Culture ?

Culture is the process of cultivation of the micro-organisms in the laboratory for the purpose of identifying & studying them. Bacterial Culture and Media 

What is a Media ?

Media are the artificial food for microorganisms, which are used for the cultivation of microorganisms in the laboratory under appropriate biochemical & biophysical environment.

What is a Colony?

Visible mass of bacteria produced by multiplication of single bacterial cell on a solid media. About 108 – 109 bacteria form visible colony.

What is a Pure culture?

When only one species of micro organism grow in a media.

What is a Mixed culture?

When more than one species of micro organism grow in a media.

Inoculation:

The process of introducing a micro organism or suspension of micro organism into a culture medium.

Subculture:

Inoculation of a second medium from a previous culture.

Colony forming unit:

An individual cell which is able to clone itself into an entire colony of identical cells.

Colonial morphology:

Cultural characteristics of a bacterium on an agar plate.

Incubation:

A process of maintaining a bacterial culture at a particular temperature for a set length of time in order to measure bacterial growth. Optimum temp for incubation: 370C .Instrument used for incubation is called incubator.

Purposes / Need for Bacterial Culture and media

• Isolate bacteria in pure cultures.
• Demonstrate their properties.
• Obtain sufficient growth for preparation ofantigens & for other tests.
• Typing bacterial isolates.
• Antibiotic sensitivity.
• Estimate viable counts.
• Maintain stock cultures.

Factors that must be controlled during growth of Bacterial Culture in a Media

• Nutrients
• PH
• Temperature
• Aeration
• Ionic strength of media
• Salt concentration

Classification of  Bacterial Culture and media

• Based on the consistency:

Liquid — Peptone water, Nutrient broth
Semisolid — MIU (motility indole urease media)
Solid — Blood agar, nutrient agar

• Based on Oxygen requirement:

— Aerobic media
— Anaerobic media

Based on the constituents/chemical composition Bacterial Culture and Media 

A. Routine laboratory media: Bacterial Culture and Media

• Simple/Basal media
• Complex/special media:
• Enriched media
• Differential/indicator media
• Enrichment media
• Selective media
• Sugar media
• Transport media
• Storage media

B. Defined synthetic media Bacterial Culture and Media 

Basic constituent of media

 Agar or agar- agar
 Peptone – mixture of partially digestedproteins
 Yeast or meat extract
 NaCl

Solidifying agents

• Agar, gelatin, serum (Loeffler serum slope), egg (Lowenstein Jensen media), potato etc.
Agar: agar is a polysaccharide extract obtained from sea weed. Most commonly used solidifying agent.
• Agar used in different concentration for different types of media-

•  1% – 2% for solid media
•  0.5% for semisolid media
•  No agar in liquid media

Agar is ideal solidifying agent because:

• Bacteriologically inert
• Melting point : 98°C, Solidifying point : 42°C
• Remain solid at all incubation temperature
• No nutritive value
• Transparent – so colony morphology better demonstrated
• No growth promoting & growth inhibitory substances
• Not destroyed at high temperature, so can be sterilized by autoclave.

Liquid media

• Usually known as broth
• Used for profuse growth
• Cannot separate mixed organisms.
• Example:
– Peptone water(1% peptone +0.5%Nacl + water), Nutrient broth ( peptone water + 1% meat extract), Glucose broth, trypticase soya broth, brain heart infusion broth etc.

Advantages:

• Liquid media are most commonly used for profuse growth where organisms are likely to be few e.g. blood culture.
• Large specimen can be tested
• Liquid media may also be used for biochemical testing
• Used as transport media

Disadvantages:

• Isolation not possible
• Identification not possible
• Can not separate mixed organisms

Semi solid media

• This form of culture medium is prepared by adding a small amount of agar (0.4–0.5%) to a liquid medium. Semi-solid media are used mainly as transport media, and for motility and biochemical tests.
• Example: MIU media.

Solid media

• Media are solidified by incorporating a solidifying agent such as agar.
• Examples:
– Nutrient agar (nutrient broth + 1% to 2% Agar)
– Blood agar media
– Chocolate agar media
– MacConkey’s agar media etc.

Advantages:

• Identification of bacteria by studying colony character
• Mixed bacteria can be separated
• Isolation of bacteria as pure culture
• Easy to inoculate
• Easy to pick up for sub culture
• Antibiotic sensitivity testing

Simple media

• Simple media- consists of only basic necessities
 Also called Basal medium
 Most commonly used in routine labs.
e.g. Nutrient broth, nutrient agar, peptone water.
• These are simple media such as nutrient agar and nutrient broth that will support the growth of microorganisms that do not have special nutritional requirements.

Enriched media

• The media to which additional growth factors have been added.
• Enriched media are required for the growth of organisms with exacting growth requirements such as H. influenzae, Neisseria species, and some Streptococcus species.
• Basic media can be enriched by adding whole or lyzed blood, serum, egg, peptones, yeast extract, vitamins and other growth factors.
• Examples: Blood agar media, chocolate agar media, loeffler serum slope etc.

Blood agar media

Nutrient agar + 5 to 10% defibrinated sheep blood
• Blood added to sterile melted nutrient agar at 450 to 500 C but gently avoiding froth formation.
• Good for culturing many bacterial species including Gram positive & Gram negative species.
• Human blood not used because of presence of inhibitory substances.
• Blood agar also an indicator media because haemolysis can be seen.

• 3 types of haemolysis-

•  β-haemolysis: complete haemolysis. Clear zone around the colony. e.g. Steptococcus pyogenes.
•  α-haemolysis: greenish zone around the colony by forming biliverdin due to incomplete haemolysis. e.g. S. pneumonia.
• γ-haemolysis: no haemolysis. e.g. Enterococcus faecalis.

Chocolate agar media

• Heated blood agar
• Ingredients are essentially same as blood agar (lysed blood)
• Blood added to sterile melted nutrient agar at 750c and allow to remain at 750c after gently mixing till it is chocolate brown in colour.
• Heating of blood causes lysis of red cells, so nutrients (factor V & factor X) come out. So, chocolate agar media is more enriched than blood agar media.
Use: To culture fastidious organisms like H. influenza, Neisseria, S. pneumoniae.

Selective media

• A culture medium to which certain substances are added which enhance the growth of the wanted/ particular pathogenic organisms and suppress the unwanted/other bacteria.
• Examples:
– Thiosulphate citrate bile sucrose (TCBS) agar media for Vibrio cholera
– MacConkey’s agar media selective for enterobacteriaceae
– Tellurite blood agar for C. diphtheria
– Lowenstein Jensen media for Mycobacterium tuberculosis
• These are solid media which contain substances (e.g. bile salts or other chemicals, dyes, antibiotics) which inhibit the growth of one organism to allow the growth of another to be more clearly demonstrated.

Lowenstein Jensen media

• It is selective media for M. tuberculosis due to presence of –
Malachite green- inhibit the growth of other organisms
Glycerol which enhance the growth of M. tuberculosis but inhibitory to M. bovis
• It is also enriched media due to egg (egg also act as solidifying agent).

Differential/indicator media

These are the media to which dyes or other substances are added to distinguish between different groups of bacteria on the basis of their biological characteristics; Causes observable change in medium when biochemical reaction occurs.
• Indicator may be blood, neutral red, phenol red, bromothymol blue etc.
• Many differential media distinguish between bacteria by incorporating an indicator which changes colour when acid is produced following fermentation of a specific carbohydrate e.g. MacConkey agar.
• Change in the colour of indicator with the growth of bacteria.
Examples:
• Blood agar media
• MacConkey,s agar media
• TCBS agar media
• Salmonella-Shigella (SS) agar media etc.

MacConkey’s agar media.

• Selective media as well as indicator or differential media.
• Selective for enterobacteriaceae due to presence of bile salt which inhibit other non enteric bacteria & crystal violet inhibit Gram positive bacteria.
• Differentiate lactose fermenters (E. coli, Klebsiella) from non-lactose fermenters (Salmonella,Shigella) by changing colour.
• Contains lactose as substrate and Neutral red as an indicator
• Neutral red is a P H indicator that turns pink at acidic PH & is colourless at alkaline PH

• When lactose fermenting bacteria grow on this media they ferment lactose of the media & produce acid which reduce the PH of the media, reduction of PH cause changing of the colour of the indicator to pink and thus media & colonies turn pink.
• When non lactose fermenting bacteria grow on this media they do not ferment lactose of the media so no change of PH & no change of the colour of the indicator. So, colonies will be pale or colourless.

Selective + enriched media

• Lowenstein Jensen media (M. tuberculosis)
• Loeffler serum media (C. diphtheria)
• Thayer Martin media (Neisseria)
• Modified New York city media (Neisseria)

Selective + indicator media

• MacConkey,s agar media
• TCBS agar
• Mannitol salt agar media (S. aureus)

Enriched + indicator media

• Blood agar media

Enriched + selective + indicator media

• Tellurite blood agar media (C. diphtheria)

Enrichment media

 For mixed cultures or materials containing more than one bacterium.
 Contains substances which stimulates wanted bacteria & inhibits unwanted bacteria. e.g. Tetrathionate broth

Transport media

• Usually semi solid or liquid media
• Are used in case of delicate organisms whenever there is a delay in the transportation of the specimen to the lab
• To Maintain viability of pathogenic bacteria & to prevent the multiplication of non-pathogenic bacteria
• Examples:
– Stuart’s medium (Gonococci)
– Amies transport media (Gonococci)
– Cary-Blair’s medium (V. cholerae or Campylobacter jejuni)

• These are mostly semisolid media that contain ingredients to prevent the overgrowth of commensals and ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately after collection.

Indication of blood culture

• Septicaemia
• Infective endocarditis
• Enteric fever
• Osteomyelitis
• Meningitis
• Pneumonia
• Brucellosis
• Leptospirosis
• PUO

Blood culture system

• Conventional method
• Lytic centrifugation method
• Automated method

• BACTEC System
• BacT/ALERT system

Blood culture media

• Brain-heart infusion broth
• Tryptica soya broth
• Glucose broth

Possible pathogens isolated from blood culture and Media

Gram negative

Ecoli, Klebsiella, Pseudomonas, Enterobacter, Salmonella typhi, Acinetobacter, Brucella, H. influenza, Bacteroides etc.

Gram positive

Enterococcus, Staph. aureus, Streptococcus pyogenes, Viridans streptococcus, S. pneumonia, Clostridium spp etc.
Fungi Candida, Aspergillus, Cryptococcus, H. capsulatum etc.

Automated blood culture

Advantages:
• Early growth of the organism. So early detection within 2-6 hours (Only give positive indication)
• Chances of contamination is low, as media prepared commercially and sterilized.
• Also useful for culturing the blood of the patients who are taking antibiotic because media contain antibiotic neutralizing substances.
• Large number of sample can be dealt at a time.
• Rate of false positivity and false negativity is very low.

Disadvantages:
• Expensive
• Power supply should be continuous.
• Highly technical hand is required.

Antibiotic sensitivity testing (AST)

Media used for antibiotic sensitivity testing
(AST):

• Meuller Hinton agar media (ideal media for AST)
• Nutrient agar media

Methods of antibiotic sensitivity testing:

• Disc diffusion method
• Dilution method (minimum inhibitory
concentration- MIC)

Anaerobic culture

• Needed for culture of obligate anaerobes
Anaerobic culture is done by-
• Anaerobic culture media
• Culture in anaerobic atmosphere

Anaerobic atmosphere can be achieved by

1. Displacement of O2 with other gases.
2. Absorption of O2 by chemical or biological means
3. Reduction of O2

Name of anaerobic culture system
• Anaerobic jar technique (by evacuation – replacement procedure)
• Gas Pak (Anaerobic jar technique by disposable gas generator)
• Anaerobic glove boxes
• Anaerobic disposable plastic bags

Anaerobic media

Using reducing agents that deplete oxygen like 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid. e.g. Thioglycollate broth, Robertson’s cooked meat medium.

Robertson’s Cooked Meat Medium:

• Broth medium for cultivation & preservation of anaerobic organisms
• Fat free minced cooked meat in broth, with a layer of sterile vaseline over it.
• SH group & unsaturated fatty acid remove the oxygen.
• Meat digested by proteolytic organisms & turn black.

Candle jar

• The burning candle within a closed jar consumes oxygen.
• Releases carbon dioxide until it extinguishes itself
• Rarely produces complete anaerobiosis.
• Suitable for growing microaerophilic and capnophilic bacteria.

Sterilizing bacterial culture media and culture

The following methodsare used to sterilize culture media:
• – Autoclaving
• – Steaming at 1000C (Tyndallization)
• – Filtration

• Autoclaving: The majority of culture media are sterilized by autoclave.
• Heat-labile substances like serum, sugar, urea, gelatin solutions must be sterilized by steaming or tyndallization.
• Egg containing media (Lowenstein-Jensen’s medium), Loeffler’s serum slope by inspissation

Filtration

• It is used mainly to sterilize additives that are heat-sensitive and cannot be autoclaved, or less stable substances that need to be added to a sterile medium immediately before it is used.
• Examples include serum and solutions containing urea and certain carbohydrates.

Bacteria that can not be cultured in artificial Bacterial Culture and Media :

• Rickettsiae species
• Chlamydiae species
• Mycobacterium leprae
• Treponema pallidum

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