Eprac 2: Cultivation Techniques- Selective And Differential Media

Question 1

TSB (Tryptone soya broth)

Answer 1

Broth culture

Highly nutritious medium

Recommended for general lab use

Contain tryptone and peptone -> supports growth of many fastidious organisms

Question 2

Kings A and Kings B

Answer 2

Broth cultures

Used to enhance fluorescein and pyocyanin pigments of Pseudomonas

Turns green for pyocyanin production or yellow for fluorescein production

Question 3

Solid media

Answer 3

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

Question 4

Non selective- non differential

Answer 4

General purpose media

Allow growth of many types of bacteria

Question 5

Selective media

Answer 5

Permit growth of desired organism while inhibiting growth of others

Question 6

Differential media

Answer 6

Permit growth of many organisms and contain reagents which allow observer to distinguish between different types

Question 7

Selective/differential media

Answer 7

Both select for desired organisms by inhibiting growth of others

Allow differentiation between types of organism selected

Question 8

Nutrient Agar

Answer 8

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

Question 9

Mueller Hinton agar (MHA)

Answer 9

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

Question 10

Salt agar/TSA

Answer 10

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

Question 11

Rogosa agar

Answer 11

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

Question 12

Cysteine Lactose Electrolyte Deficient agar (CLED)

Answer 12

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

Question 13

Blood agar

Answer 13

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

Question 14

Alpha haemolysis

Answer 14

Partial degradation of rbc

-> greenish zone around colonies

Question 15

Beta haemolysis

Answer 15

Total degradation of rbc

-> clear zone around colonies

Question 16

Mannitol Salt Agar (MSA)

Answer 16

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

Question 17

CHROMagar Candida

Answer 17

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

Question 18

Hektoen Enteric Agar (HE)

Answer 18

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

Question 19

Xylose Lysine Deoxycholate agar (XLD)

Answer 19

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

Question 20

Tinsdale agar

Answer 20

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)

Question 21

MEGA (Membrane Enriched Enterococcus Glucose Azide Agar)

Answer 21

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

Question 22

MESLS (Membrane Enriched Sodium Lauryl Sulphate Agar)

Answer 22

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

Question 23

Monitoring pollution using E.coli

Answer 23

E.coli used as indicator organism for monitoring faecal contamination of water sources

Ideal indicator organisms characteristics:

1. Present when pathogens present
2. Present only when there is danger of pathogens
3. Occur in greater no. than pathogens
4. Survive in environment as long as pathogens
5. Easy to detect
6. 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

Question 24

Agar slopes

Answer 24

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

Question 25

Urea slopes

Answer 25

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