MICRO biochemical tests used to ID bacteria

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Potential biochemical reactions; enzymes

catalase
oxidase
urease
ONPG
nitrate reduction

Potential biochemical reactions; Metablolism of carbs and related products

oxidation fermentation

Potential biochemical reactions; specific break down products of the organism

indole
methyl red
voges proskauer

Potential biochemical reactions; ability to utilize specific substances

citrate

Potential biochemical reactions; metabolism of protein and/or amino acids

gelatin hydrolysis
H2S production

Catalase used for:

organisms that produce the enzyme catalase

Catalase mechanism

detoxifies hydrogen peroxide by breaking it down into water and oxygen

Catalase what is it?

chemically it is a hemoprotein, similar in structure to hemoglobin 2H2O2------> 2H2O + O2

Catalase; present in?

most cytochrom containing aerobic and facultative anaerobic bacteria (except strep)

Catalase enzyme is responsible for?

for protecting bacteria from hydrogen peroxide (H2O2) accumulation, which can occur during aerobic metabolism

Accumulation of H2O2 is?

toxic to the organism

Catalase breaks down?

H2O2 in to H2O and O2

catalase procedure; reagents

3% H2O2 stored in dark brown bottle in fridge
Isolate of organism 18-24 hours old

catalase procedure; test

slide
tube
direct plate
(what to test it on)

catalase procedure; QC

S. aureus- positive
Strep species- negative

catalase positives:

Micrococcus, staph, bacillus, listeria monocytogenes, corynebacterium (except C. pyogenes and C. haemolyticum), moraxella species, enterobacteriaceae, gonococcus and meningococcus, vibriocholerae, pseudomonas/aeromonas/plesiomonas

Catalase negatives

Strep, clostridium, erysipleothrix

Catalase precautions:

avoid testing colonies and or pieces of bld agar
-only fresh isolates should be used 18-24 hrs old
-reagents should be fairly fresh, it is unstable and breaks down when exposed to light
-reagenst should be stored in the refrigerator

Oxidase utilization:

To ID microorganisms that contain enzyme cytochrome oxidase

oxidase is important for what chain?

electron transport chain

Oxidase mechanism

during aerobic respiration- transfers electrons from the electron transport chain to O2 (the final electron acceptor) and reduces it to water

Oxidase also participates in what?

the nitrate metabolic pathway

Oxidase test; artificial electron donors and acceptors are provided:

reagent dye is P-phenylenediamide dihydrochloride acts as artificial electron acceptor substituting the oxygen.

Oxidast test: when elelctron donor is oxidized by cytochrome oxidase it turns DARK PURPLE= positive

in the reduced stage, dye is colorless, but in the presence of cytochrome oxidase, dye is oxidized to indophenol blue

Oxidase reagents

1% kovacs (tetra methyl-p-phenylenediamide dihydrochloride)
-1% gordans and Mcleods (dimethyl-p-phenylenediamide dihydrochloride

Oxidase Methods

moist filter paper
dry disk
direct plate

Oxidase QC

pseudomonas spp- positive
E.coli- negative

Oxidase precautions:

use platinum wire or wooden stick
read within 10 seconds
fresh prepared reagents
store reagents in dark bottles
avoid pigmented colonies and those on macconkey
-avoid colonies growing in glucose medium, it's fermentation will inhibit oxidase enzyme activity

Oxidase positive:

Neisseria species, haemophilus sps, pseudomonas sps (except P maltophilia), aeromonas species, alcaligene, vibrio sps, campylobacter, plesimonas shigelloides, micrococcus sps., moraxella

Oxidase negative

enterobacteriaceae, acinetobacter sps, brucella canis, bordetella parapertussis, francisella tularensis, gardenella vaginalis

Urease Identifies organisms:

capable of hydrolyzing urea using enzyme urease

urease; this break down of urea forms:

weak base (two ammonia molecules)

Urease results in:

alkalinity-increase pH of the media above 8.4

urease broths and agars: stuarts urea broth (ph 6.8)

phenol red, monopotassium phosphate, disodium phosphate, urea, phenol red indicator
distilled water.

urease broths and agars; Christiansens urea agar (ph 6.8)

peptone, glucose, sodium chloride, monopotassium phosphate, urea, phenol red indicator, agar, distilled water

urease; procedure:

innoculate broth or streak plate with organism in question
-incubate 18-24 hours at 35C

urease; QC

proteus species-stron positive
klebsiella sps-weak positive
E. coli- negative

Urease interpretation; rapid hydrolyzers show

positive results in 1-2 hours. Proteus species

Urease interpretation; Less active species can require:

3 or more days. Enterobacteriaceae

Urease interpretation; Stuarts broth

red color thru out broth- positive
yellow color thru out broth- negative

urease interpretation; Christensens agar

red thru out agar- positive
red color initially on slant portion only, then gradually entire tube- positive (slow urea splitter)
-yellow or straw color thru out agar- negative

Urease alternate methods:

reagent impregnated urease strips (2 hours)
-urease tablets
-urea discs
-ewings urea broth

urease precautions

both broth and agar depend on demonstration of alkalinity
-protein hydrolysis can result in alkalinity
-false positives may be detected

urease positives

Kleb pneumo, proteus, bacillus lentus, enterobacter, cryptococcus, heliobacter pylori, bordetella bronchoseptica, actinobacilllus species

urease negatives

escherichia, providencia, salmonella typhi, edwardsiella, aeromonas

B-galactosidase (ONPG) information

a disaccharide compound composed of glucose and galactose connected thru an O2 linkage known as galactoside bond

B-galactosidase (ONPG); lactose fermentation depends upon

two enzymes:
-B-galactoside permease
-B-galactosidase

B-galactoside permease:

permits transport of B-galactoside (such as lactose) in to the bacterial cell wall

B-galactosidase:

Cleaves the B-galactoside bond after it enters in to the bacterial cell liberating glucose and galactose

ONPG

O-Nitrophenyl-B-D-galactopyranoside

ONPG test is used to:

determine the presence or absence of the enzyme B-galactosidase in an organism

ONPG; true lactose non-fermenters:

do not posess either of the enzymes

ONPG; Late lactose fermenters:

do not have permease, but do posess B-galactosidase

B-galactosidase reagents/materials required

discrete isolated bacterial colony growing on solid nonselective medium
-ONPG broth or commercially prepared ONPG discs
-0.5 mL for each test being performed
-Toluene

B-galactosidase QC;

E. coli- positive
Proteus species- negative

B-galactosidase procedure

loop full of bacterial growth to 0.5 mL of saline, producing a heavy suspension
-add 1 drop toluene to suspension & mix for a few seconds
-add equal volume of ONPG solution
-incubate 35-37C up to 24 hours
-evaluate presence or absence of color rxn between 4 and 24 hours

B-galactosidase interpretation

yellow-presence of enzyme-lact. ferm.-positive
colorless-absence of enzyme-lact nonfer- neg.

B-galactosidase precautions

don't eval color rxn beyond 24 hours
-discard reagent if discolored
-avoid testing orgs grown on media containing glucose as it inhibits B-galactosidase
-organisms with yellow pigment should not be tested

B-galactosidase positive

E. coli, kleb pneumo, enterobacter species, citrobacter sps.,neisseria lactamica

B-galactosidase

morganella morganii, neisseria gonorrhoeae, proteus vulgaris, proteus mirabilis, providencia rettgeri

Nitrate reduction information

nitrate may be reduced to multiple compounds by two processes:
-anaerobic respiration
-denitrification

-anaerobic respiration

does not use O2 as final electron acceptor, but instead uses non-O2 electron acceptors such as nitrate

Denitrification

If nitrate used as elelctron acceptor then bacteria performs this step to produce nitrogen gas

Nitrate reduction; This test determines the ability of the organism to reduce:

nitrate NO3 to nitrite NO2 using the enzyme nitrate reductase

Nitrate reduction; also tests the ability of the organism to perform

nitrification on nitrate and nitrite to produce molecular nitrogen

Nitrate reduction; reagents

nitrate reduction broth- has nutrients, potassium nitrate
-sulfanilic acid
-alpha- napthylamine
-zinc dust

Nitrate reduction; procedure:innoculate the nitrate broth

-if the org has the ability, it'll reduce NO3 to NO2
-the NO2 in medium wil produce nitrous acid

Nitrate reduction; procedure:Add sulfanilic acid & alpha-napthylamine

-if nitrous acid is present, it'll react with the sulfanilic acid to produce diazotized sulfanilic acid
-the diazotized sulfanilic acid then reacts w/ the alpha-napthylamine to form a red colored compound

Nitrate reduction; procedure:No red color means?

the organism was unable to reduce the nitrates, or that it was able to denitrify the nitrate to produce ammonia or molecular nitrogen

Nitrate reduction; procedure: requires?

an additional step, adding a small amt of powdered zinc
** great care must be taken, powdered zinc is hazardous

Nitrate reduction; Post zinc addition; red color= unreduced nitrate present= NEGATIVE result

-addition of zinc reduced nitrate to nitrite
-nitrite in the medium formed nitrous acid
-nitrous acid reacted with sulfanilic acid
-diazotized sulfanilic acid was produced and reacted with alpha-napthyamine creating red complex

Nitrate reduction; Post zinc addition; NO COLOR production is a POSITIVE result

no nitrate to reduce
-no nitrite present in medium
-denitrification took place
-ammonia or molecular nitrogen were formed

Nitrate reduction positives:

E. coli, Enterobacter aerogenes, salmonella typhimurium, pseudomonas stutzeri, yersinia enterocolitica

Nitrate reduction negatives

acinetobacter caloceticus
acinetobacter baumannii
clostridium bifermentans
alcaligenes piechaudii

Oxidation fermentation information

certain gram neg. bacilli metabolize glucose by fermentation or aerobic respiration

Oxidation fermentation information; during anaerobic process of ferementation

pyruvate is converted to a variety of mixed acids depending on type of fermentation
-the high concentration of acid produced during fermentation is detected

Oxidation fermentation information; during aerobic respiration:

glucose metabolized producing small amts of weak acids
-increased concentration of glucose enhances production of weak acids that are detected

Oxidation fermentation; This test is used to determine if gram neg. bacilli;

-have the ability to metabolize carbohydrate oxidatively
-have the ability to metabolize carbs by fermentation
-are nonsacchrolytic and therefore have no ability to use the carb in the media

Oxidation fermentation; reagents

of basal medium: peptone, NACL, glucose, bromthymol blue

Oxidation fermentation (OF); procedure:

2 tubes OF media innoculated for each test org.
-inoculated by stabbing halfway down the tube
-overlay 1 tube w/ 1cm of mineral oil- prevents diffusion of O2 in to the medium and creates anaerobic environment
-Incubate 35C for 48 hours- slow growing bacteria may take 3-4 days before results can be observed.

Oxidation fermentation; Interpretation; fermentive results:

acid production changes pH indicator from green to yellow
-acid production in both tubes- yellow thru out
FERMENTORS= E.COLI, vibrio cholerae

Oxidation fermentation; Oxidative results:

no color change in oil covered tube
-acid product., yellow color thru part or all (dependant on length of incubation)
oxidizers: Pseudo aeruginosa, bacillus subtilis

Oxidation fermentation; Negative results

no color change in oil covered tube
-no color change in open tube, or even increase in pH which causes color change at top from green to blue

INDOLE information

determines organism ability to split tryptophan to form the compound inodole

INDOLE was originally used to:

distinguish E.coli from enterobacter aerogenes.

Numerous variations have been created and used over the years

INDOLE; Theory:

tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that express tryptophanase enzyme.

INDOLE: QC

positive- E.coli
negative- Kleb pneumo

INDOLE; reagents; tube method

tryptophane broth
ether or xylene (for ehrlichs method)
kovac's reagent or Ehrlich's reagent

INDOLE; reagents; spot method

trypticase soy agar or sheep blood agar culture
filter paper
kovac's reagent

INDOLE; procedure; Tube;

inoculate tryptophane broth
incubate at 35C for 24-48 hours
-kovac's-add kovacs reagent
-erhlich's-add ether or xylene,mix well, let stand,add erhlichs reagent

INDOLE; procedure; Spot

saturate filter paper with kovac's
smear colonies on dampened filter paper
if positive, rxn will occur within 1-3 minutes

Indole; interpretation, Tube:

Pos-wine colored ring after adding approp. reagent
NEG- no color change after adding appropriate reagent

Indole; interpretation, spot

pos- red/pink color development
neg- no color development

Indole precautions;

medium containing glucose shouldn't be used.

INDOLE positive organisms

E.coli,
proteus vulgaris
kleb. oxytoca
citrobacter diversus
edwarsiella tarda
morganella morganii

Indole negative organisms

salmonella sps.
kleb. pneumo
proteus mirabilis
enterobacter aerogenes
yersinia pestis
serratia marcescens

Methyl red info

tests ability of bacteria to produce and maintain stable acid end products (lactate, acetate,formate) from glucose fermentation and to over come the buffering capacity of the system

Methyl red is a

qualitative test for acid production

Methyl red (MR) biochemistry

methyl red- ph indicator with range of 4.4 and 6
-ph @ which MR detects acid is considerably lower than ph of other indicators
-to produce a color change, the test organism must produce a large quantity of acid from substrate being used.

Voges-proskauer (VP) is used to;

determine if glucose can be converted to acetoin-specifically testing butylene glycol pathway

Voges-proskauer (VP) reagents added react with:

acetoin in the presence of creatine (catalyst) and diacetyl (color reactant)

Voges-proskauer (VP) tests are performed:

in conjunction with methyl red

Voges-proskauer (VP) organisms will:

be positive for one test or the other, usually not both.

Both MR and VP used for the identification of:

enterobacteriaceae

Methyl red / Voges proskauer reagents

Media- contains glucose and peptone
MR- methyl red is ph indicator
VP-5% alpha naphthol-color intensifier
-40% potassium hydroxide-oxidizing agent

VP testing can be performed using alternative methods:

impregnated strip, however it's rare

Methyl red / Voges proskauer; QC MR

positive- e.coli
negative-enterobacter aerogenes

Methyl red / Voges proskauer; QC VP

positive-enterobacter aerogenes
Negative-e.coli

Methyl red / Voges proskauer; procedure

inoc. MR/VP broth
incubate 35C 24-48 hrs
broth split to perform each test

Methyl red / Voges proskauer; procedure MR

add methyl red
evaluate for color rxn immediately

Methyl red / Voges proskauer; procedure; VP

add alpha napthol (aka barritts reagent A)
-add potassium hydroxide (barritts reagent B)
-mix by gentle shaking to expose medium to atmosphere oxygen
-allow to sit undisturbd for 10-15 minutes
-evaluate for color reaction

Methyl red / Voges proskauer; Interpretation MR

positive- distinct red color ph 4.4
negative-yellow color ph 6

Methyl red / Voges proskauer; interpretation; VP

pos- red color development
neg- yellow color

Methyl red / Voges proskauer; precautions

excess KOH may mask a weak VP positive rxn.

Methyl red positive organisms

e.coli
yersinia sps
listeria monocytogenes
kleb. ozaenae
salmonella sps.
proteus mirabilis

Methyl red negative organisms

ebterobacter aerogenes
kleb. pneumo
kleb. oxytoca
pseudomonas aeroginosa

Voges proskauer; positive organisms

kleb pneumo
enterobacter sps
yersinia enterocolitica
listeria monocytogenes
kleb oxytoca

Voges proskauer; negative organisms

e.coli
micrococcus sps
kleb ozaenae
shigella sps
salmonella sps
proteus mirabilis

Citrate information

screens the bacterial isolate for the ability to utilize citrae as its carbon source
-also looks for ability to use inorganic ammonium salts as its only nitrogen source

Citrate: positive diagnostic test relies on

generation of alkaline by-products of citrate metabolism

Citrate; there is a subsequent.....

increase of ph causing color change of ph indicator (bromthymol blue) from green to blue

Citrate; is used to speciate

members of Enterobacteriaceae

Citrate; reagents

Simmon's citrate media

Citrate; QC:

positive; kleb pneumo
negative; E.coli

Citrate procedure:

inocul. slant medium
replace cap; but loosely (citrate utiliz needs H20)
Incubate 35C 18-48 hrs. some orgs may require 7 days

Citrate interpretation Positive

visible growth on slant surface
media will be intense prussian blue color
by-products of citrate catabolism increases ph to cause color change

citrate interpretation negative

scant to no growth on slant surface
no color change of media
negative will be almost indishtinguishable from uninoculated slant.

Citrate Positive organisms

kleb pneumo
salmonella typhiumurium
serratia marcescens
providencia rettgeri
proteus mirabilis (variable)

Citrate negative organisms

E.coli
proteus mirabilis (variable)
morganella morganii
yersinia enterocolitica
shigella dysenteriae

IMViC defined

I-indole
M-methyl red
V-voges-proskauer
C- Citrate

IMViC Historically used for the:

ID of bacteria of bacteria in Enteroacteriacae group
-IMViC rxns still utilized in calculation of ID
-Board of registry exam questions

Gelatin Hydrolysis information

determines ability of organism to produce proteolytic enzymes that liquify gelatin
-Gelatin (yes, related to Jello)-protein derived from connective tissue

Gelatin Hydrolysis; Enzyme required:

Gelatinase
-allows org. to break gelatin down to sm. pieces for ease of use
-when gelatin broken down, no longer able to solidify, remains liquid even if refridgerated

Gelatin Hydrolysis: Short or long test?

lengthy

Gelatin Hydrolysis; Alternative method:

use of x-ray film, psot incubation, loss of gel coating, flim is clear/bluish

Gelatin Hydrolysis; Materials required

gelatin agar- has 12% gelatin
incubator
refrigerator

Gelatin Hydrolysis; QC:

positive; proteus vulgaris
negative; enterobacter aerogenes

Gelatin Hydrolysis; procedure; Stab method:

organism introduced thru stabbing medium
-heavy innoculum required
-incubat 35C for at least 48 hours
-place in refrigerator 30 minutes
evalutate status of media for liquidity
-intact gelatin should be reincubated for 2 wks
-evaluate media status
-NOTE don't disturb or invert tubes during incubation steps

Gelatin Hydrolysis; Interpretation:

Positive- discernable liquifacation will occur at the top of the tube even after refrigeration- GELATINASE produced

Negative-14 day incubation- solid media- no liquid detected- NO Gelatinase produced

Gelatin Hydrolysis; Positive;

proteus vulgaris
serratia marcescens
pseudomonas fluorescens
stenotrophomonas maltophilia

Gelatin Hydrolysis; Negative

enterobacter aerogenes
salmonella typhimurium

H2S production; Information:

H2Sh is produced when amino acids containing sulfur (cysteine or methionine) are metabolized by microorganisms
-once produced, it combines wih ferrous ammoniam sulfate
-insoluable black ferrous sulfide precip is formed
-media may also be used to determine motility thru use of SIM (sulfide indole medium)
-commonly included in TSI (triple sugar iron) tube testing

H2S production; Reagents

SIM agar (semi-solid tube media)

H2S production;: QC

Positive- proteus mirabilis
Negative- Kleb pneumo

H2S production; Procedure:

use inoc. needle, introduce org by stabbing medium straight down the middle to about 1/4 of depth
-incubate at 35C overnight
-evaluate for H2S production

H2S production; Interpretation

Positive-any blackening of the medium
Negative- no black discolorization

H2S production; alternative methods

use of lead acetate

H2S production; positive organisms

proteus mirabilis
salmonella typhimurium
citrobacter species

H2S production; negative organisms

providencia rettgeri
morganella morganii
serratia marcescens
kleb pneumo
e.coli

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