Catabolic Taxonomic Tests

Gram + tests

catalase, Durham fermentation tubes, amylase test, spores, hemolysis, DNAase, Mannitol Salt, Bile Esculin

Gram - tests

Durhman fementation tubes, TSI, Nitrate Reduction, Oxidase, Citrate Utilization, Indole, Urea, EMB, SIM

What is required before interpreting the results of differential tests?

growth

Gram + cocci tests

catalase, fermentation, mannitol salt plate, DNAase, hemolysis

Catalase Test

differntiates between staphylococci (+) & streptococci (-); for organisms that respire with oxygen & use catalase to break down H2O2 into water & O2; + shows bubbles when hydrogen peroxide (3%) is added to growth on slide

Durham Fermentation Tubes ingredients

complex medium with fermentable sugar, phenol red, and durham tube

Durham Fermentation Tubes test

fermentation makes acidic products that lower the pH and cause phenol red to turn medium yellow; some gaseous products like CO₂, H₂ are collected in durham tube (also + for fermentation)

Durham Fermentation Tube Results

R/no gas → no fermentation; Y/gas or no gas → fermentation

Mannitol Salt Agar medium

selective, differential, and complex; high concentration of salt (7.5%), mannitol sugar, phenol red, proteose peptone

Mannitol Salt Agar test

selects for Staphylococcus with high saline levels; if ferments mannitol, acidic byproduct causes phenol red to turn agar yellow

Mannitol Salt agar results

pathogenic Staphylococci (ex: aureus) ferment mannitol while non-pathogenic Staphylococci (ex: epidermidis) don’t

DNAase medium

complex w/ Bacto Tryptone & Deoxyribonucleic Acid (DNA)

DNAase test

If DNAase is present, it hydrolyzes surrounding DNA to use as source of carbon & as energy for growth so when add HCl, clear zone around the growth; if DNA is intact & undigested, adding HCl to DNA makes plate cloudy and opaque

Hemolysis

hemolysins lyse red blood cells & degrade hemoglobin; beta-hemolysin, alpha-hemolysin, gamma-hemolysin

Beta-hemolysin

breaks down red blood cells and hemoglobin completely so clear zone around growth

Alpha-hemolysin

partially breaks down red blood cells; leaves greenish color behind

Gamma-hemolysis

no hemolysis occurs

Gram + rod tests

catalase, fermentation, amylase, endospores, hemolysis

Amylase medium

complex medium containing starch and beef extract

Amylase Test

production of amylase hydrolyzes starch into amylose and amylopectin; adding Gram’s iodine turns starch brown-blue color so halo around organism that has amylase

Amylase Test Results

+ → halo around growth; - → no halo

Gram + Rod Lactose Fermentation Test

-; R/no gas

Bile Esculin Medium

complex with esculin, bile salts, and ferric citrate

Bile Esculin Test

differential & selective to identify Enterococcus and Group D Streptococcus species; Enterococcus grows and hydrolyzes esculin to form dextrose & esculetin which then reacts with ferric ions to turn medium to dark brown/black

Bile Esculin Results

dark brown/black → Esculin positive bacteria (Enterococcus); - → no color

Triple Sugar Ion (TSI) medium

complex with cysteine & methionine from peptone, sodium thiosulfate, glucose, sucrose, lactose, phenol red, FeSO₄

How does Triple Sugar Ion (TSI) test for H₂S production?

bacteria that degrade amino acids into H₂S or reduce sodium thiosulfate to H₂S which reacts with Fe³⁺ to make FeS which is a black precipitate

Triple Sugar Ion (TSI) test

detects bacteria that ferment glucose, glucose + lactose/sucrose/both, or don’t; gas may crack agar → positive for slant; acidic byproducts cause phenol red to change medium yellow

Triple Sugar Ion (TSI) results

ferment glucose (R/Y sink initially ferments glucose but runs out & uses fermentation products to respire so slant changes to Y), glucose + lactose/sucrose/both (Y/Y), no fermenters (need growth but R); gas → + fermentation; black percipitate → H₂S

Nitrate Reduction medium

complex with potassium nitrate

Nitrate Reduction Test

tests for ability to use nitrate as terminal electron acceptor during respiration (anaerobic respiration); nitrate reduced to nitrite (red after adding reagent a + b) or nitrate reduced to nitrogen gas (denitrification; no change after reagent a, b, + zinc)

Nitrate Reduction Results

red after reagent a + b → nitrate to nitrite; red after reagent a + b + zinc → unreduced nitrate (negative result); no color change after reagent a + b + zinc → nitrate to N₂

Explain the use of zinc in the nitrate reduction test

chemically reduces nitrate in the medium to nitrate, turning medium to red; added only if reagent a + b don’t turn medium red; if turns red, unreduced nitrate so - result & no nitrate reduction; if no color change, no nitrate in medium so reduced nitrate to nitrogen gas

Oxidase Test

oxidase strip contains chromogenic reducing agent that changes to blue or purple when oxidized by cytochrome C oxidase; add some growth onto the oxidase strip

Oxidase result

red/violet/blue color → positive for cytochrome C oxidase; no change → don’t reduce O₂ with cytochrome C oxidase

Citrate Utilization

only test where absence of growth is negative since no growth means does not use citrate; tests for ability to use citrate as sole carbon source; oxidize citrate to CO₂ that combines with sodium and water to make sodium carbonate, an alkaline product, so Bromothymol blue turns the medium blue

Citrate Utilization Medium

defined with sodium citrate, water, and Bromothymol Blue

Indole Medium

complex with tryptone broth (lots of tryptophan)

Indole Test

tryptophanase deaminates and hydrolyzes tryptophan to make indole, pyruvate, and ammonium; Kovac’s reagent detects indole by reacting with it to make a red ring

Indole Test Results

red ring → indole +; yellow ring → - indole

Sulfur Indole Motility (SIM) Medium

Ferrous ammonium sulfate, sodium thiosulfate, low amoutn of agar, peptone w/ methionine & cysteine, tryptophan

Sulfur Indole Motility (SIM)

uses Kovac’s reagent to test for indole (red ring); motility agar to test for motility; if produces black precipitate, produces H₂S

Urease Medium

complex with phenol red and urea

Urease Test

urease hydrolyzes urea to ammonia and carbon dioxide, making alkaline environment; phenol red turns fuchsia (pink) for alkaline environments

Urease Test results

pink → urease +; no color change → urease -

Eosin Methylene Blue (EMB) medium

selective, differential, and complex with lactose, Eosin Y, Methylene Blue

Eosin Methylene Blue (EMB)

used to isolate fecal coliforms; lactose encourages growth of fecal coliforms; if ferments lactose, Eosin Y and methylene blue make dark purple precipitate & inhibit most Gram + organisms

Eosin Methylene Blue (EMB) results

green metallic sheen → E. coli (vigorous fermenter); mucoid pink → slow/weak fermenters; light grey → - fermentation

You inoculate a nitrate tube and a glucose fermentation tube with bacterium X. A day later the nitrate tube was yellow after the addition of reagent a, b, and zinc. The fermentation tube had growth but no color change. What do these results tell you about the physiology of bacterium X?

positive result for nitrate → N₂ so can denitrification (anaerobic respiration); still grew in fermentation tube so respirers and uses nitrate as terminal electron acceptor

Which identification procedure gives better results with mixed cultures, the gram stain or Indole test? Explain.

gram stain bc identifies the shape & if they are gram + or - and then can use other tests based on these results; indole only determines if it can produce indole/has tryptophanase, but not useful for mixed culture

If you grow an organism in a glucose durham tube in a shaker bath. Would you expect this culture to be positive? Why or why not?

no bc it’s fermentation which does not require oxygen; increases oxygen in tube which can inhibit growth of anaerobes & cause aerotolerant aerobes to use respiration instead of fermentation since that is more efficient

Would you try a nitrate reduction test on an aerobe? Why or why not?

no bc aerobes use oxygen as teh terminal electron acceptor over nitrate bc reduced oxygen has a higher redox potential than nitrate; will get negative response for nitrate reduction

How would you test an organism for sorbitol fermentation? Explain how you would know it’s positive.

Inoculate in a complex medium containing sorbitol and phenol red in a Durham fermentation tube; if it ferments, it will release acidic byproducts which will cause phenol red to turn the medium yellow & may produce gaseous products so gas would be in tube; signifies positive for sorbitol fermentation

You inoculate a glucose Durham tube with bacterium X. A week later you read the results and the medium has not changed color but was turbid. What conclusion(s) can you draw from this result?

does not ferment glucose bc no color change unless ran out of glucose to ferment so used fermentation products as energy causing the medium to change color back to yellow; could be a respirer or an autotroph, using CO₂ as carbon energy or O₂ as terminal electron acceptor