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261 terms

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
Phenylalanine deaminase (PD) information:
Detects ability of organism to produce deaminase enzyme
Phenylalanine deaminase (PD): Deaminase enzyme:
enzyme removes amine group from amino acid phenylalanine
Phenylalanine deaminase (PD): amine group:
is released as free ammonia.
-phenylpyruvic acid is also produced, which is detected by adding 10% ferric chloride to test
Phenylalanine deaminase (PD) is useful for:
differentiating members of genera below from other enterobacteriaceae:
proteus
morganella
providencia
Phenylalanine deaminase (PD): reagents:
Phenylalanine agar/slant (contains nutrients and DL-phenylalanine)
-10% ferric chloride
Phenylalanine deaminase (PD) QC:
Positive- Proteus vulgaris
negative- e.coli
Phenylalanine deaminase (PD); Procedure:
inoculate agar
-incubate at 35C overnight with loose caps
-add 4-5 drops of 20% ferric chloride
-evaluate for color reaction
Phenylalanine deaminase (PD); Interpretation:
POS- dark green color- pyruvic acid was produced by org. and reacted with the ferric chloride
NEG- medium remains straw color- phenylalanine deaminase not produced
Phenylalanine deaminase (PD) positive organisms
Proteus mirabilis
morganella morganii
providencia sps
Phenylalanine deaminase (PD) negative organisms
e.coli
enterbacter sps
citrobacter sps
serratia sps
Decarboxylase; Information
Based on the principle that certain organisms have the ability to remove (hydrolyse) a carboxyl or hydroxyl group from an amino acid to form an amine.
-this removal increases pH
-decarboxylase is the enzyme responsible for this
Decarboxylase; the broth contains:
nutrients
-dextrose or glucose (fermentable carbs)
-pyridoxal (enzyme cofactor for decarboxylase)
pH indicators (bromcresol purple and cresol red)
-one amino acid: arginine, lysine or ornithine
Decarboxylase; principle
under anaerobic conditions, certain orgs can ferment glucose
-pH decreased, detected by change in color to yellow
-reduced pH activates decarboxylase that alter amino acids to alkaline by-products
-formation of alkaline amines increases pH, medium returns to purple/reddish purple
-arginine is hydrolyzed to ornithine and then decaroxylated
-ornithine decarboylation yields putrescine
-lysine decarboxylastion results in cadaverine
Decarboxylase; specification
each decarboxylase produced by an organism is specific to the amino acid on which it acts
-Therefore, we tst the ability to produce
Arginine decarboxylase
lysine decarboxylase
ornithine decarboxylase
Decarboxylase; Media
3 different, but very similar media
Decarboxylase; QC; LYSINE
Positive-Kleb pneumo
Negative-E. cloacae
Decarboxylase; QC; Ornithine
Positive- e. cloacae
Negative- Kleb pneumo
Decarboxylase; QC; Arginine
Positive- e. cloacae
Negative- Kleb. pneumo
Decarboxylase; Procedure
obtain broths of each amino acid
-inoculate each tube
-overlay surface of medium w/sterile mineral oil (creates anaerobic envir, also prevents alkalination at surface of medium)
-incubate 35C overnight
-eval for rxns daily for up to 4 days before reporting as pos or neg.
Decarboxylase; interpretation:
medium will 1st turn yellow if glucose is ferment.
-no color change wil occur if glucose is not fermented
-if decarboxylase rxn occurs, pH increases, medium will gradually turn purple/reddish purple
Decarboxylase; precautions
make sure to check the labels of the tubes as the broth on all three is very similar
Decarboxylase; Arginine
positive- E. cloacae
Negative-Panotea agglomerans, E. aerogenes
Decarboxylase; Lysine:
POS- E.coli, salmonella, e. Cloacae, E. aerogenes, Klebsiella
NEG- Citrobacter, Morganella, Providencia
Decarboxylase; Ornithine:
POS- E. cloacae, E. aerogenes
NEG- Pantoea, agglomerans, Klebsiella, Citrobacter
Motility information
Considered a non-biochemical test
Motility; Used to:
determine if the organism is motile
-Organisms must posess flagella to be motile
Motility; two methods:
Hanging drop
semisolid media (most common)
Motility; is often included as part of:
multitest media to differentiate enterbacteriaceae
-MIL- Motility indole lysine
-MIO-motility indole ornithine
-SIM-sulfide indole motility
Motility; Reagents/Material(semisolid media)
-Motility medium- Beef extract, casein, sodium chloride, agar
-inoculating needle
Motility; QC:
motile- e.coli
non-motile-Klebsiella pneumoniae
Motility; procedure (semisolid media)
sterile inoc. needle used to find isolated colony
-stab medium within 1 cm of bottom of tube
-ensure needle remains in same line it entered as it is removed from medium
-incubate 35C for 18-24 hours
-Evaluate results
Motility; Interpretation:
POS- motile org. will spread out into medium from site of inoc.
NEG- non-motile orgs. remain at the site of inoc.
Motility; reagents/materials (hanging drop)
tryptic soy broth or brain heart infusion broth
Motility; QC:
same as semisolid media
Motility; Procedure (hanging drop)
inoculate isolated colony to broth
-incubate 35C for 18-24 hours
-place drop of broth on clean microscope slide, coverslip
-observe microscopically for motility
Motility; interpretation (hanging drop)
POS-individ. bacteria cells moving in random directions, changing positions w/respect to each other, often darting across the field
NEG- no movement, or movement in the same directions as all other cells or debris
Motility; Positive/motile organisms:
pseudomonas sps.
proteus sps
campylobacter jejuni
helicobacter pylori
Motility; Negative/motile organisms
Kleb pneumo
Shigell sonnei
Acinetobacter sps
Multiple Test medias:
KIA-Kligers Iron Agar
LIA-Lysine Iron agar
TSI-triple sugar iron agar
SIM-sulfide indole medium
Litmus milk
KIA; Information; contents:
medium contains lg amt of lactose, sm amt of glucose, pH indicator and iron
KIA information; evaluates:
organisms ability to ferment glucose and lactose to acid and acid plus gas end products
KIA information; allows for:
ID of sulfur reducers which binds to iron to form a black precipitate
-Glucose fermentation will create acidic by products that will turn the phenol red indicator in the media yellow.
KIA; reagents/materials:
Kliger's iron agar
-inoculating needle
KIA; QC:
e.coli
proteus mirabilis
pseudomonas aeruginosa
salmonella typhimurium
shigella flexneri
KIA; Procedure;
sterile inoc. needle to select isolated colony
-stab medium to 1cm of bottom of tube
-needle remains in the same line it entered as removed
-loosen caps to allow exchange of air
-incubate 35C for 18-24 hrs
-eval for acid production on slant/but, gas production and H2S production
KIA; Limitations:
H2S producing orgs may produce a black precipitate to such a large degree that the rxn in the butt is completely masked. If H2S is produced, dextrose is feremented even if it isnot observed
-inoculation with mixed culter will cause erroneous results
KIA; interpretation: Lactose fermentation:
at surface of slant (aerobic) only ferm of carbs present in high concentrations yield more acidic products than can be oxidized to neutrality
POS- yellow slant
NEG- red slant
KIA interpretation; Glucose fermentation:
in the largely anaerobic butt of tube, even fermentation of the trace concentration of glucose yields enough acid to change pH
Glucose POS- result in yellow butt
Glucose NEG- no color change, red butt
Fermentation of lactose in butt will obviously change the pH too, but this does not confuse the interpretation since all lactose positive orgs are also glucose positive
-H2S obsuring rxn, is assumed to be glucose positive
KIA interpretation H2S production:
-lower portions of the tube will turn black, due to formation of iron sulfide
-don't confuse a pigment associated with colony growth on the slant with black ferric sulfide in the butt.
KIA interpretation; Gas formation:
butt will show bubbles and cracks
LIA information:
differentiation of microbes base on lysine decarboxylation or deanimation and H2S production.
LIA information: media contains:
lysine, glucose, peptones, bromcresol purple (pH indicator), sodium thiosulfate, ferric ammonium citrate
LIA; Lysine deamination:
is an aerobic process which occurs on the slant of the media
-ammonia is produced, reacting w/ferric ammonium citrate, producing a dark red color on slant
LIA; Lysine decarboxylation is:
an anaerobic process which occurs in the but of the media
-amine end-product reacting with pH indicator giving a purple color in the butt of the tube
LIA; reagents/material:
LIA media
inoculating needle
LIA; QC:
citrobacter freundii
e.coli
proteus mirabilis
salmonella typhimurium
LIA; Procedure:
inoculate medium by stabbing base of the tube butt and streaking slant with a needle
-loosely cap to ensure aerobic conditions
-incubate 35C for 18-48 hours
-examine at 18-24 and at 48 hours evaluate:
growth
color changes in tube butt and slant
evidence of H2S production
LIA; interpretation; lysine decarboxylation
POS = purple (alk) butt
NEG = yellow (acid) butt, purple slant
LIA interpretation; Lysine deamination
POS= red slant
NEG= Purple slant
LIA interpretation; H2S:
POS= blackened medium @ apex of slant (proteus & providencia sps produce a red slant over a yellow butt)
NEG= no black discolorization
LIA limitations; Salmonella paratyphi A
unline other salmonella sps doesn't produce lysine decarboxylase, resulting in an alkaline slant and an acid butt
LIA limitations; H2S producing proteus sps.
don't blacken the medium. It is suggested that LIA be used in conjunction w/triple sugar iron agar on other media to confirm differentiation
LIA limitations; the rxn of Morganella morganii:
may be variable after 23 hours incubation and may require longer incubation
TSI; is a differential medium that contains:
lactose, sucrose, glucose peptone, ferrous sulfate (H2S indicator), phenol red (pH), sodium thiosulfate.
TSI; Determines
carb fermentation
gas production
H2S production
TSI fermentation of carbs into acid end-products will result in:
yellow color in slant and or butt as the acids react with phenol red
TSI production of gas (primarily CO2) during fermentation can be determined by:
observing tube for bubbles or cracks
TSI production of hydrogen sulfide utilized sodium thiosulfate as a substrate, producing H2S, which reacts with:
ferrous sulfate causing a black precipitate
TSI reagent/materials
TSI agar
inoculating needle
TSI QC
e.coli
salmonella typhi
pseudomonas aeruginosa
TSI procedure
inoc. TSI by stabbing thru center of medium to bottom of tube
-streak the surface of the agar slant
-loosely cap tubes for aerobic conditions
-incubate 35C for 18-24 hours
TSI procedure; examine and evaluate for:
color change of slant and tube butt
gas production
H2S production
TSI interpretation: Red/Yellow
K/A glucose fermentation only, peptone catabolized
TSI interpretation; Yellow/Yellow:
A/A glucose and lactose and/or sucrose ferm.
TSI interpretation; Red/Red:
K/K no ferm. peptone catabolized
TSI interpretation; Red/no color change
K/NC no ferm. peptone used aerobically
TSI interpretation; Yellow/yellow w/bubble
A/A,G glucose ferm only; gas produced
TSI interpretation; Red/yellow w/bubbles
K/A,G glucose fermentation only, gas produced
TSI interpretation; red/yellow w/bubbles and black precipitate
K/A,G,H2S glucose fermentation only; gas produced, H2S produced
TSI interpretation; red/yellow w/black prec.
K/A, H2S glucose ferm only, H2S produced
TSI interpretation; Yellow/yellow w/black precipitate
A/A H2S glucose and lactose and/or sucrose ferm, H2S production
TSI interpretation; no change/no change
NC/NC no fermentation
SIM; combination differential medium:
sulfur reduction
indole production
motility
SIM; useful in differentiating:
enteric organisms
SIM; medium contains:
nutrients (peptone-amino acids, including tryptophan)
-iron
-sodium thiosulfate
SIM; sulfur and motility test results should be determined:
prior to performing indole test
SIM; reagents:
SIM agar
inoculating needle
SIM; QC:
Kleb. pneumo
E.coli
Proteus mirabilis
SIM; Procedure
sterile needle used to select isolated colony
-stab medium within 1 cm of bottom of tube
-needle remains in same line upon removal
-incubate 35C for 18-24 hours
-evaluate results- sulfur reduction and motility prior to indole
-add kovac's reagent for indole test, evaluate
SIM interpretation; Motility
POS-haziness that spreads from stab line
NEG-lack of haziness or absence of thickening of stab line
SIM interpretation; Sulfur reduction
POS-black discoloration of media
NEG-No discoloration of media
SIM interpretation; Indole
POS- red color development after addition of Kovac's reagent
NEG-No color development after addition of kovac's reagent
LITMUS MILK; Tests the:
ability of an organism to metabolize milk
LITMUS MILK; Principle:
milk is complex nutritional source that contains proteins (mainly casein) in an aqueous solution of lactose and minerals
LITMUS MILK; Principle; Bacterial enzymes alter:
the media and may bring about various changes
LITMUS MILK; Litmus is added to the medium to detect:
pH changes that may occur as a result of these enzymatic reactions
LITMUS MILK; coag/curdling of milk may occur due to:
fermentation of lactose
LITMUS MILK; fermentation of lactose is evidenced by:
the litmus turning pink as a result of acid production
LITMUS MILK; If sufficient acid is produced:
casein in the milk is coagulated, solidifying the milk.
LITMUS MILK; with some organisms:
the curd shrinks and whey is formed at the surface
LITMUS MILK; some bacteria hydrolyze casein, causing:
the milk to become straw colored and resemble turbid serum
LITMUS MILK; some organisms reduce litmus, in which case:
the medium becomes colorless in the bottom of the tube
LITMUS MILK: Reagents:
litmus milk broth
inoculating loop
LITMUS MILK; QC:
E.coli
Proteus vulgaris
Lactobacillus acidophilus
Enterococcus faecalis
Clostridium sporogenes
Moraxella oslonesis
LITMUS MILK; Procedure:
inoculate broth
incubate 35C for 24-48 hours
evaluate for rxns
LITMUS MILK; limitations:
litmus milk is a complex medium that can produce a diversity of results. due to quite uniqe rxns, it is advised to confirm test results with other methods
-clot formation is simple recorded as "clot" and connot clearly differentiate between clot and curd formation
LITMUS MILK; interpretation; No Change
no carb fermentation, no change in litmus indicator
LITMUS MILK; interpretation; fermentation of lactose
production of acid (pink color) including stormy fermentation (strong evolution of gas) by some strains of clostridium
LITMUS MILK; interpretation; action of proteolytic enzymes on lactoalbumin
production of ammonia or basic amines resulting in alkaline reaction (blue color)
LITMUS MILK; interpretation; coag of casein as evidenced by
formation of curd or clot. If the casein is converted to paracasein by the enzyme rennin, a clear watery liquid called "whey" is produced at the top of a thoroughly coagulated tube
LITMUS MILK; interpretation; peptonization due to the digestion of the milk protein is:
observed by clearing of medium and dissolution of the clot
LITMUS MILK; interpretation; reduction of the litmus in the depths of the tube due to:
the action of reductase enzymes with the resultant removal of oxygen to form decolorized leucolitmus compounds.