exercise 34 KTTK: Citrate Utilization test (Micro. lab)

Citrate utilization test designed to

differentiate members of Enterobacteriaceae which are all facultative anaerobes

what the citrate utilization test indicates about the organism

• Indicates whether organism can utilize citrate as their sole carbon source and perform citrate fermentation
• Also indicates whether can use ammonium phosphate as sole nitrogen source
• Simmons citrate agar is defined medium (amount and source of ingredients carefully controlled) with pH starting around 6..9

what enzyme an organism has to have in order to perform citrate fermentation and utilize citrate

Bacteria must possess enzymes citrate permease and citrate lyase

Citrate lyase breaks citrate into

xaloacetate and acetate

Citrate permease

(oxaloacetate decarboxylase Na+ pump) transports oxaloacetate into cell where citrate fermentation performed

Know what it means when it is said that Simmons citrate agar is a
defined medium

(usually poured in a slant format) where sodium citrate is the only carbon source in the medium and ammonium phosphate serves as an inorganic nitrogen source.

Know what else organisms that grow on Simmons citrate agar must
also be able to survive with in addition to sodium citrate as their
only carbon source

ammonium phosphate to ammonia and ammonium hydroxide which alkalinize agar which is observed using bromothymol blue dye

Know why the Simmons citrate agar turns from green to blue when
an organism can utilize the sodium citrate (What does this change
indicate?)

dye appears green at pH 6.9 (the media’s original preparation pH) and blue at a pH level of pH 7.6 (the media’s alkalinized pH if NH₃ and NH₄OH were made).

green color to blue means the bacteria are actively growing and indicates a positive citrate test result

blue above pH of

7.6

green at pH between

6.0 and 7.6

yellow below pH of

6.0

Results of citrate utilization test

• Color change of agar to blue indicates positive test
• No color change or growth indicates negative test
• No color change with growth is usually considered positive test

(application) the citrate utilization test is used

• Used to determine ability of organism to use citrate as sole source of carbon
• Component of IMViC battery of tests (Indole, Methyl red, Voges-Proskauer, and Citrate) used to distinguish between Enterobacteriaceae family members and differentiate them from other Gram-negative rods

Citrate Utilization Test Results

media appear green and there is no growth on the slant’s surface, then the test result is negative; the bacterium cannot use citrate as its sole carbon source.

blue media coloration and the presence of growth indicate a positive test result; the bacterium can use citrate as its sole carbon source.

Citrate Utilization Test Results and Interpretation

Citrate Utilization Test Results

control: No color change and no growth. (-). Citrate is not utilized (organism lacks one or more of the enzymes needed to use citrate as its sole carbon source).

E. coli: No color change and no growth. (-). Citrate is not utilized (organism lacks one or more of the enzymes needed to use citrate as its sole carbon source).

E. aerogenes: Media appears blue (even if only a portion of the media turned blue) and growth is evident. (+). Citrate is utilized (organism has the enzymes needed to use citrate as its sole carbon source).