sugar fermentation tsi and litmus milk

1. Sugar Fermentation TSI and Litmus milk Lab 16

3. Phenol Red Broth for Sugar Fermentation Lactose fermenters are organisms that split lactose into glucose and galactose and ferments the monosaccharides (hydrolysis reactions) Glucose fermenters produce pyruvate (glycolysis)and ferment pyruvate to products alchohol and CO2 and H. Phenol Red is an acid indicator Yellow below 6.8, Pink above 7.4, and red from 6.8 to 7.4. Durham tube is and indicator for gas production. Differentiation of Enterobacteraciae from other Gram – rods

4. Tests results Acid Gas - produces yellow bubble in the tube Acid no Gas- produces yellow with no bubble Red Broth control pH 7.3 Degradation of peptone produces alkaline media with NH3 as the end product and will be pink/reddish E.Coli S. Aureus Alcaligenes faecalis

5. BIOCHEMICAL TESTSEx 5-2 Phenol Red Broth for Sugar Fermentation Reading results Yellow color in the phenol red pH indicators shows acid hot pink shows alkaline Gas production will show as a bubble in the durham tube

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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.

14. 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, glucose 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

15. 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

16. 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

17. 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

18. The results Alkaline “blue”reaction- 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

19. Some examples

20. 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

21. 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

22. 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