TSI and Litmus milk

1
TSI and Litmus milk

• Lab 21

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TSI- Triple Sugar Iron

• Differentiate Enterobacteriaceae and distinguish
  between G- bacillus (Pseudomonas, Alcaligenes)
• Media contains 3 carbs, beef extract, yeast,
  peptone (source of C and N), sodium thiosulfate
  (reduction of sulfur), pH indicator phenol red.
• Differentiate bacteria on the basis of glucose,
  lactose, sucrose fermentation, sulfur reduction
• E. coli
• Salmonella typhimurium
• Pseudomnas aeruginosa

3
The results

• Glucose only acid produced lowers pH. Turns
  media yellow. Glucose used up quickly, bacteria
  begins to break down Amino Acids and NH3
  produced. The pH begins to rise and reversion
  results, where the media reverts to red.
• Red slant and Yellow butt, glucose only
• Enterobacteriaceae

4
The results

• Lactose and Sucrose fermentation reactions
  produce acid, raises the pH and turns entire
  media yellow. The increased lactose and sucrose
  in the media does not allow for reversion and
  remains yellow.
• Gas fissures may form to indicate gas formation
  and may lift media.
• Enterobacteriaceae

5
The results

• H2S production- result of thiosulfate reduction
  or breakdown of cystein in the peptone. Black
  precipitate in the butt.
• Fermentation reaction indication of acid
  production by one or more sugars. Glucose and
  lactose yellow, glucose only red.
• H2S indication of Enterobacteriaceae

6
The results

• Catabolize Peptone and Amino Acids but do not
  ferment will alkalize the medium, increasing the
  pH. Turn the slant red.
• Aerobic and anaerobic both slant and butt red
• Obligate aerobe slant only red.
• Compare to control tube- changes could be subtle.
  
• Not Enterobacteriaceae

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BIOCHEMICAL TESTSEx 5-21 Triple Sugar Iron
Agar (Hydrogen Sulfide Production)

• Reading results
• Black color on the bottom (butt) of the tube
  shows H2S production
• Yellow color shows acid production - an
  all-yellow tube shows lactose fermentation

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BIOCHEMICAL TESTSEx 5-21 Triple Sugar Iron
Agar (Hydrogen Sulfide Production)

• Reading results
• Black color on the bottom (butt) of the tube
  shows H2S production
• Hot Pink color shows alkaline (protein breakdown)
  a part yellow / part pink tube shows glucose
  fermentation

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Some Examples

• E. coli ferments lactose and or sucrose, acid
  slant (yellow) and butt and gas formation
• Salmonella typhimurium ferments glucose but does
  not ferment lactose and/ or sucrose. See alkaline
  slant and acid butt. H2S formation from breakdown
  of cystein in peptone, and maybe gas formation.
• Pseudomonas does not ferment glucose, and is
  strict aerobe. The slant will be alkaline , only
  red.

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From left to right

• Uninoculated control
• Red slant and red butt, no black color no
  fermentation of glucose, sucrose or lactose. No
  Hydrogen sulfide produced
• Red slant and black butt no lactose or sucrose
  fermentation, H2S has been produced
• Red slant with yellow butt no lactose or sucrose
  fermentation, lactose is fermented, no H2S has
  been produced
• Yellow slant, yellow butt and black coloration
  Lactose, sucrose and glucose fermented, and H2S
  has been produced
• Yellow slant, yellow butt and lifting and/or
  cracking of media, no black coloration Lactose,
  sucrose and glucose fermented, H2S has not been
  produced but gas has been produced
• Yellow slant, yellow butt and no lifting and/or
  cracking of media, no black coloration Lactose,
  sucrose and glucose fermented, H2S has not been
  produced nor has gas been produced

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Litmus Milk

• Contains skim milk, pH indicator azolitmin
• pink 4.5 blue 8.3 (purple in between)
• Clostridium differentiation based on casein
  coagulation
• Enterobacteriaceae reduce litmus (pink result)
• Used to cultivate lactic acid bacteria
• E. coli
• Pseudomonas aeruginosa
• Proteus vulgaris

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4 results

• Lactose fermentation fermentation of lactose
  acidifies the medium and turns the pH indicator
  pink, pH decreases
• Reduction of litmus- fermentation- acid
  accumulates forms an acid clot. Acid clots
  solidify the medium and can appear pink or white
  with a pink band at the top

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The results

• Casein coagulation- bacteria produce proteolytic
  enzymes (rennin pepsin, or chymotrypsin) that
  coagulate casein and produce a curd. A curd
  differs from an acid clot that it will not
  dissolve in alkaline conditions. See retract from
  the sides, and form whey (straw colored)
• Casein hydrolysis- bacteria produce enzymes that
  digest both acid clots and curds, leaving a
  reaction with clear to brownish fluid

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The results

• Alkaline bluereaction- indication of partial
  digestion of casein (production of NH3 pH
  increases)
• Some examples
• E. Coli acid reaction forms pink color
• Enterococcus faecalis forms acid clot
• Clostridium forms fermentation acid with gas
  fissures in the clot
• Some Clostridium forms proteolytic enzymes that
  coagulate casein

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Some examples
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BIOCHEMICAL TESTSEx 5-23 Litmus Milk Medium

• Reading results
• Compare your tubes to a control tube - pink color
  means acid production, purplish-white means
  reduction, blue is alkaline and clearing shows
  digestion

ACID
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BIOCHEMICAL TESTSEx 5-23 Litmus Milk Medium

• Reading results
• Compare your tubes to a control tube - pink color
  means acid production, purplish-white means
  reduction, blue is alkaline and clearing shows
  digestion

REDUCTION WITH CURD
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BIOCHEMICAL TESTSEx 5-23 Litmus Milk Medium

• Reading results
• Compare your tubes to a control tube - pink color
  means acid production, purplish-white means
  reduction, blue is alkaline and clearing shows
  digestion

DIGESTION