Biochemical Identification Of Gram-Negative Bacteria

Gram-negative bacilli and coccobacilli [rod-shaped, elongated], non-spore forming, facultatively anaerobic

ENTEROBACTERIACEAE DO NOT produce cytochrome C oxidase except for

FAMILY ENTEROBACTERIACEAE MOTILE at body temperatures except

To ensure isolation of both opportunistic and fastidious pathogens, laboratories must provide appropriate transport media, such as

enterobacteriaceae optimal growth in

Low temperatures (1 degree Celsius to 5 degrees

tolerate high temperatures (45 to 50 degrees Celsius,

General Culture Medi

Selective or Differential Medium:

All Enterobacteriaceae produce similar growth on blood and chocolate agars; colonies

may be mucoid – presence of polysaccharide capsule

“swarm” on blood and chocolate agars

Y. enterocolitica produces

Broth cultures of Y. pestis exhibit a characteristic

5% SBA are pinpoint at 24 hours but exhibit a rough, cauliflower appearance at 48 hours

Best used to characterize gram-negative rods

Lactose fermenters can be differentiated with non- lactose fermenters

Lactose fermenter: appear as

can ferment lactose within 18-24 hours of incubation

can ferment lactose within 48-72 hours of incubation

Rapid Lactose Fermenters:

Late Lactose Fermenters:

even prolonged incubation, they cannot ferment lactose;

Non-lactose fermenter appear

Non-lactose fermente

determines the presence of the cytochrome oxidase system that oxidizes reduced cytochrome with molecular oxygen.

Kovac’s oxidase test uses a

positive result of oxidase test

determines whether an organism has the ability to reduce nitrate to nitrite and reduce nitrite further to nitrogen gas (N2)

After 24 hours of incubation of NITRATE AND NITRITE REDUCTION _________ is added

indicates the presence of nitrite

Ph indicator of MALONATE UTILIZATION

in MALONATE UTILIZATION bacteria able to use ____________ as sole carbon source

in MALONATE UTILIZATION use _________ as a nitrogen source

A positive test result in increased alkalinity from utilization of the ammonium sulfate, changing the indicator from

Gelatinase activity is detected by loss of gelling (liquefaction) of gelatin

proteolytic enzymes that break down gelatin into amino acids

this test is used to differentiate organisms based on the production of deoxyribonuclease

positive result of DNA HYDROLYSIS

negative result of DNA HYDROLYSIS TEST

Slow Urease Producers:

Rapid Urease Producers:

determines whether a microorganism can hydrolyze urea

Urease hydrolyzes urea to form

agar use in urease test

positive result in urease test

negative result in urease test

it determines whether an organism can use sodium citrate as a sole carbon source.

Part of a series referred to as IMViC

Contains ammonium salts as the sole nitrogen sourc

ph indicator of CITRATE UTILIZATION

positive result of citrate utilizatoon

negative result of citrate utilization

use light inoculum

citrate utilization incubate up to what degree of celcuis and up to what.

Presence of cloudiness spreading from the inoculation line

This can be indicated by the presence of a black precipitate

Organisms that possess the enzyme tryptophanase are capable of deaminating tryptophan with the formation of the intermediate degradation products of indole, pyruvic acid, and ammonia

reagent of sim agar

positive result of sim agar

miscellaneous test

Amino acid utilization

deamination of phenylalanine

Determines whether an organism possesses the enzyme that deaminates phenylalanine to phenylpyruvic acid

positive result of PHENYLALANINE DEAMINASE (PAD) TES

H2S Producing of LIA

Deaminase producing

in LIA

Red Slant/Acid Butt (yellow)

in LIA Alkaline Slant (purple)/Acid butt (yellow)

Alkaline Slant (purple)/Alkaline butt (purple)

to differentiate gram-negative bacilli based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S)

medium of LYSINE IRON AGAR

ph indicator of LIA

positive and negative result of Lysine Deaminase (Slant)

positive and negative result of Lysine Decarboxylase (Butt)

method of LIA

GLUCOSE METABOLISM AND ITS METABOLIC PRODUCTS

im VP test Detects the organism’s ability to convert the acid products to

after incubation of VP test what reagent we will add first as a catalyst

after adding a-naphthol in VP text next we add what

poristive resukt of VP test

negative result of VP test

If glucose is metabolized by the mixed acid fermentation pathway, stable acid end products are produced, which results in

methyl red test

reagent of methyl red test

postive result of MR TEST

negative resukt of MR test

MRVP broth → inoculate → incubate for

B-Galactosidase hydrolyzes ONPG, a colorless compound, into galactose and o-nitrophenol, a YELLOW compound. – L

The test can be performed by making a heavy suspension of bacteria in sterile saline and adding commercially prepared

can be observe on the formation of bubbles or splitting of the medium in butt

There are members of the Enterobacteriaceae family that are H2S producers in TSI

H2S production

A/A ((yellow) slant/acid (yellow) butt) –

K/A (Alkaline (red) slant/acid (yellow) butt)

K/K (Alkaline (red) slant/alkaline (red) butt)

Used to determine glucose and lactose or sucrose utilization and H2S production, gas production

Composition: of TSI

ph indicator of TSI

H²s indicator of TSI