Eprac 2: Cultivation Techniques- Selective And Differential Media Flashcards
TSB (Tryptone soya broth)
Broth culture
Highly nutritious medium
Recommended for general lab use
Contain tryptone and peptone -> supports growth of many fastidious organisms
Kings A and Kings B
Broth cultures
Used to enhance fluorescein and pyocyanin pigments of Pseudomonas
Turns green for pyocyanin production or yellow for fluorescein production
Solid media
Allows us to isolate individual organisms from mixture and to make preliminary identification based on their growth characteristics
Allows us to identify bacterial species based on colony morphology
Non selective- non differential
General purpose media
Allow growth of many types of bacteria
Selective media
Permit growth of desired organism while inhibiting growth of others
Differential media
Permit growth of many organisms and contain reagents which allow observer to distinguish between different types
Selective/differential media
Both select for desired organisms by inhibiting growth of others
Allow differentiation between types of organism selected
Nutrient Agar
General purpose medium
Non selective- non differential
Contain peptone and yeast extract
Suitable for cultivation of non-fastidious organisms (organisms that don’t have complex nutritional requirements)
Used for routine examination of water, sewage and food products for microorganisms
Addition of different bio fluids such as horse or sheep blood, serum or egg yolk can make it suitable for cultivation of more fastidious organisms
Mueller Hinton agar (MHA)
Non selective-non differential
Rich medium containing meat infusion, casein hydrolysate, starch and agar
Provides complex nutrients: some bacteria may be unable to produce on their own
Starch binds toxic metabolites produced by bacteria, ensuring growth is not inhibited
Used for antibiotic susceptibility testing
Inclusion of high quality agar ensures large clear zones of inhibition will be evident when sensitive organisms meet active antibiotics
Salt agar/TSA
Selective
Nutritious medium
Contains 7.5% NaCl which is inhibitory to most organisms
Selective for salt tolerant organisms such as Staphylococci
Staphylococcus aureus: grows
Staphylococcus epidermidis: grows
Escheria coli: growth inhibited
Rogosa agar
Selective for isolation and enumeration of Lactobacilli
Isolate Lactobacilli in faeces, saliva, mouth rinses and dairy products
Contains acetate -> lowers pH -> inhibit growth of most bacteria and moulds, but allow lactic bacteria to grow
Low conc of magnesium, manganese and iron ensures optimal growth of Lactobacilli. Glucose used as carbon source
Incubated for 3 days at 37 degrees in candle jar -> prevents evaporation and provides micro-aerophilic conditions (reduced oxygen conc environment) favoured by Lactobacilli. Increased CO2 has stimulating effect on growth
Lactobacillus: grows; Escherichia coli: growth inhibited
Cysteine Lactose Electrolyte Deficient agar (CLED)
Differential nutritious medium with single major carbon source (lactose) and an acid base indicator (bromophenol blue) -> allow detection of lactose fermentation (yellow)
Designed for use in urine bacteriology. Supports growth of all urinary pathogens -> visualisation of colony morphology and diagnostic characteristics
Electrolyte deficient -> prevent swarming of Proteus species
Fermentation of lactose -> yellow colonies Bacteria that decarboxylate L-cystine -> alkaline reaction -> blue colonies
Enterococcus faecalis: yellow opaque colonies
Escheria coli: yellow opaque colonies
Klebsiella spp: yellow to white mucoid colonies
Pseudomonas spp: green colonies, rough periphery
Proteus spp: translucent blue colonies
Blood agar
Enriched differential medium: differentiate alpha and beta haemolytic Streptococci
Used for isolation of fastidious organisms (Neisseria meningitidis, Streptococcus pyogenes, Streptococcus pneumoniae)
Defibrinated horse blood incorporated as source of nutrients and as indicator of haemolysis
Alpha haemolysis
Partial degradation of rbc
-> greenish zone around colonies
Beta haemolysis
Total degradation of rbc
-> clear zone around colonies
Mannitol Salt Agar (MSA)
Selective/differential
Nutritious medium containing 7.5% salt, single major carbon source (mannitol) and an acid base indicator (phenol red) -> detection of mannitol fermentation (turns plate yellow)
High salt conc restricts growth to salt tolerant organisms, while indicator allows differentiation of mannitol fermenters (pink to yellow)
Staphylococcus aureus: grows and turns plate yellow
Staphylococcus epidermidis: grows; no colour change
Escheria coli: growth inhibited
Streptococcus: growth inhibited
CHROMagar Candida
Selective/differential media for identification of yeasts
Selective medium for isolation and presumptive identification of yeast and filamentous fungi
Facilitates detection of mixed yeast cultures in specimens (differences in morphology and colour)
Yeasts produce different colours on base, surface, centre and edges of colonies, characteristic for each species
Candida albicans: light to medium green colonies
Candida tropicalis: small, smooth, dark to metallic blue colonies
Candida krusei: large, rough, pale pink to mauve with white colony borders
Candida parapsilosis: large, smooth, white to pale pink colonies
Candida guilliermondii: small, smooth, dark purple with white colony borders
Candida stellatoidea: small, smooth, deep greeny-blue colonies
Hektoen Enteric Agar (HE)
Selective-differential for isolation of food poisoning organisms (Salmonella and Shigella from enteric specimens)
Bacteria that use sugars -> turn pH indictor yellow to red (example E.coli)
Peptone metabolism by Salmonella and Shigella-> increase in pH -> blue
Medium contains thiosulfate -> form black precipitate in presence of Hydrogen sulfide
Salmonella produces Hydrogen sulfide: black/brown colonies
Shigella: does not produce Hydrogen sulfide -> blue to green moist colonies
Pseudomonas spp: green to brown colonies
Coliforms: pink to yellow/orange/red colonies
Xylose Lysine Deoxycholate agar (XLD)
Selective/differential for isolation of pathogens from enteric specimens
At neutral pH, indicator phenol red remains pink
Salmonella ferments xylose and decarboxylate lysine to remain red. Production pf hydrogen sulfides -> colonies have black centre
Shigella cannot ferment xylose -> remains red
Coliforms (E.coli) ferment lactose and sucrose -> acidifying medium -> yellow colonies
Pseudomonas spp: pink irregular colonies
Tinsdale agar
Selective/differential for identification of Corynebacterium species (isolation and differentiation Corynebacterium diptheriae from diptheroids)
C. Diphtheriae -> grayish black colonies surrounded by dark halo
C.xerosis -> gray/black colonies with dark brown halo
Diptheroids found in upper respiratory tract don’t form such colonies
Dark brown halo (cystinase activity -> producing H2S which interacts with tellurite salt in agar)
MEGA (Membrane Enriched Enterococcus Glucose Azide Agar)
Example of selective/differential media for water quality
Used in membrane filtration analysis of water samples to select for faecal streptococci
Contains sodium azide at conc inhibitory to coliform organisms and most other Gram -ve, not faecal streptococci
2,3,5-triphenyl tetrazolium chloride (TTC) in medium can be reduced by faecal streptococci-> insoluble red dye formazan
Faecal streptococci: pink, red and maroon colonies
MESLS (Membrane Enriched Sodium Lauryl Sulphate Agar)
Example of selective/differential media for water quality
Select for faecal coliforms
Sodium Lauryl Sulphate -> inhibit growth of most non-coliform bacteria
Contains lactose as sole carbohydrate available for fermentation and phenol red as indicator of acidity
- > lactose fermenting faecal coliforms: yellow
- > non-coliform: pink
Monitoring pollution using E.coli
E.coli used as indicator organism for monitoring faecal contamination of water sources
Ideal indicator organisms characteristics:
- Present when pathogens present
- Present only when there is danger of pathogens
- Occur in greater no. than pathogens
- Survive in environment as long as pathogens
- Easy to detect
- Shouldn’t multiply in water -> distort no.
E.coli meets all of criteria
Limitations of E.coli: doesn’t reflect possible presence of protozoa such as Cryptosporidium, not linked in any way with possible viral contaminants known to cause water borne epidemics such as Enteroviruses
Agar slopes
Slope cultures: in bottles with screw-caps -> reduces evaporation and dehydration. Caps can’t be accidentally knocked off
Sometimes preferred to broth (first sign of contamination is more readily noticed on agar surface)
Great transport medium
Urea slopes
Used for detection of organisms which are able to hydrolyse urea by production of urease
Urease: characteristic of all species of Proteus. Commonly used to differentiate this from other members of Enterobacteriaceae
