Culture Media - Final [PDF]

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CULTURE MEDIA (Principle, Purpose & Interpretation)



Ma.Elizabeth P. Bonghanoy



MacConkey Agar



•Purpose - It is both selective and differential medium designed to isolate and differentiate enteric based on their ability to ferment lactose. - It differentiates microorganisms in the colon-typhoid-dysentery group.



MacConkey Agar



Inhibitor: crystal violet and bile salts CHO: Lactose pH indicator: Neutral Red



MacConkey Agar



MacConkey Agar • Interpretation ✓Lactose fermenting strains – They grow red/pink and surrounded by acid precipitated bile. ✓Non-fermenting lactose strains – The results are colorless and transparent.



Eosin Methylene Blue Agar



• Purpose - Both a selective and differential medium used to isolate fecal coliforms.



Eosin Methylene Blue Agar



Inhibitor: Eosin Y and methylene blue CHO: Lactose/Sucrose pH Indicator: Eosin Y and Methylene blue



Eosin Methylene Blue Agar



Eosin Methylene Blue Agar



• Interpretation ✓Lactose Fermenting: pink (slow fermenters) – purple complex ✓Non Lactose Fermenting: Colorless



Eosin Methylene Blue Agar



Rapid fermentation of lactose & production of strong acids. - The metallic green sheen is due to the acid production from the amide bonding between the eosin and methylene blue



E. coli: Green Metallic Sheen



Eosin Methylene Blue Agar



Klebsiella has capsular antigens (K antigens), leading to different serogroups12. These well developed polysaccharide capsules give the colonies their characteristic mucoid appearance. The pink color is attributed to its slow fermenting property. Klebsiella: Pink mucoid colonies



Eosin Methylene Blue Agar



Some strains with a K antigen possess a capsule which attributes to the mucoid shape. The dark center is due to precipitation of methylene blue.



Enterobacter: Pink colonies with dark center. “FISH EYE” colony



Thiosulfate Citrate Bile Salts Sucrose Agar • Purpose - Used for the selective isolation of Vibrio cholerae and other enteropathogenic vibrios. - Used to control outbreaks of the crown-ofthorns seastar (Acanthaster planci).



Thiosulfate Citrate Bile Salts Sucrose Agar



Inhibitor: Ox bile and sodium cholate CHO: Sucrose pH Indicator: Bromthymol blue and Thymol blue



Thiosulfate Citrate Bile Salts Sucrose Agar



Thiosulfate Citrate Bile Salts Sucrose Agar • Interpretation



Salmonella and Shigella Agar • Purpose - Used as a selective and differential medium for the isolation of Salmonella and some Shigella species from clinical and non-clinical specimens. - It was also developed to aid in the differentiation of lactose and non-lactosefermenters from clinical specimens, suspected foods, and other such samples



Salmonella and Shigella Agar



Inhibitor: bile salts, brilliant green and sodium citrate CHO: Lactose pH Indicator: neutral red H2S indicator: (Fe+NH4 Citrate)



Salmonella and Shigella Agar



or Sodium Thiosulfate (not metabolized)



Salmonella and Shigella Agar • Interpretation Non Fermenting ✓Salmonella: H2S (+), colorless with black center ✓Shigella: H2S (-), Colorless, without black center



Salmonella and Shigella Agar • Interpretation Fermenting ✓Coliform bacteria: H2S (-), pink color ✓Enterobacter and Klebsiella: larger than E. coli, mucoid, pale, opaque cream to pink



Hektoen Enteric Agar • Purpose • is a selective as well as differential media for the isolation and differentiation of enteric pathogens from clinical specimens.



Hektoen Enteric Agar



Inhibitor: bile salts and acid fuchsin CHO: Lactose/Sucrose/ Salicin pH Indicator: Bromthymol blue H2S indicator: (Fe+NH4 Citrate)



Hektoen Enteric Agar



or Sodium Thiosulfate (not metabolized)



Hektoen Enteric Agar • Interpretation ✓Salmonella colonies: blue-green, with hydrogen sulfide gas. ✓Shigella appear greener than Salmonella, with the color fading to the periphery of the colony.



Hektoen Enteric Agar • Interpretation ✓Rapid lactose fermenters (such as E. coli): moderately inhibited, produce bright-orange to salmon pink colonies. ✓Proteus strains: small transparent and more glistening or watery in appearance



Hektoen Enteric Agar



Thioglycollate Broth • Purpose - Used to find out the growth characteristics of various bacteria based on their oxygen requirements. - Differentiation of obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant organisms.



Thioglycollate Broth • Purpose - Recommended for the cultivation of aerobic, microaerophilic, and anaerobic microorganisms. - Recommended for sterility testing of antibiotics, biologicals, and foods and for determining the phenol coefficient and sporicidal effect of disinfectants.



Thioglycollate Broth PRINCIPLE CHO: Glucose Inhibitor: Bile Salts Growth factors: Dextrose, pancreatic digest of casein, yeast extract, L-cystine Reducing agents: Thioglycolate, cystine, and sodium sulfite



Thioglycollate Broth



PRINCIPLE - Low concentration of agar prevents downward diffusion of oxygen, allowing for the growth of anaerobic organisms toward the bottom of the tube



- L-cystine and sodium thioglycollate allows Clostridium and other strict anaerobes to grow in this medium even under aerobic conditions.



Thioglycollate broth • Interpretation ✓Gram-negative, facultatively anaerobic bacilli generally produce diffuse, even growth throughout the broth. ✓Gram positive cocci frequently grow as discrete “puffballs”.



Thioglycollate broth • Interpretation ✓Strict aerobic bacteria such as Pseudomonas spp., tend to grow toward the surface of the broth ✓Strictly anaerobic bacteria grow at the bottom of the broth.



Thioglycollate broth



Tryptone Soy broth • Purpose - For use as a general growth medium for the isolation and cultivation of microorganisms. - Supports the growth of non-fastidious as well as moderately fastidious microorganisms



Tryptone Soy broth • Purpose - Recommended for use in the cultivation, storage, maintenance, and transportation of pure cultures of microorganisms - Not used for the isolation of pathogens from clinical specimens but may be used for maintaining or subculturing bacterial strains (e.g., Enterobacteriaceae and Staphylococci). After supplementation with blood (e.g. 5% sheep blood)



Tryptone Soy broth • Principle CHO: Glucose Amino acids and other complex nitrogenous substances: Enzymatic digests of casein and soybean Osmotic balance: Sodium chloride Buffering agent: Dipotassium hydrogen phosphate



Tryptone Soy broth • Principle - The addition of a small amount of agar renders the broth suitable for the cultivation of obligatory anaerobes, such as Clostridium species.



Tryptone Soy Broth • Interpretation ✓After the incubation period, the broth should appear turbid, an indication of organism growth. ✓In the case of TSB with phenol red, a red to yellow color change also accompanies turbidity, an indication of acid production from dextrose utilization.