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Applied Veterinary Bacteriology and Mycology: Introduction Chapter 5: Identification systems used in diagnostic

bacteriology

Applied Veterinary Bacteriology and Mycology:Bacteriological techniques Chapter 5: Identification systems used in diagnostic bacteriology Author: Dr. J.A. Picard

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTSBACTERIOLOGICAL IDENTIFICATION FOR THE CLINICAL LABORATORY........................................2

INTERPRETATION OF GROWTH/COLONY MORPHOLOGY..................................................................2

SECONDARY CULTURES......................................................................................................................... 4

IDENTIFICATION OF BACTERIA...............................................................................................................4

TYPES OF IDENTIFICATION SYSTEMS.................................................................................................17

Biochemical profiles.............................................................................................................................. 17

Gas-liquid chromatography...................................................................................................................18

THE NORMAL FLORA............................................................................................................................. 21

Mouth, nasopharynx............................................................................................................................. 21

OCCURRENCE OF PATHOGENS IN ANIMAL SPECIES.......................................................................22

Laboratory animal infections.................................................................................................................22

Rats and Mice....................................................................................................................................... 22

Guinea Pigs.......................................................................................................................................... 22

Rabbits................................................................................................................................................. 23

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bacteriology

BACTERIOLOGICAL IDENTIFICATION FOR THE CLINICAL LABORATORYAlthough new technologies such as the polymerase chain reaction (PCR), DNA hybridization and the ELISA test are available for the identification of various bacteria, they are often too expensive for routine laboratory use. Thus clinical microbiologists continue to depend on morphological and biochemical criteria e.g. substrate utilization systems for the identification of micro-organisms isolated from clinical specimens.

Identification of bacteria follows primary isolation and colony purification. This is normally based on the following tests:

Colony morphology Gram’s stain (in some cases specialized stains e.g. for mycobacteria). Catalase reaction Oxidase reaction Growth on MacConkey agar Oxidation-fermentation test Motility test Secondary identification tests

INTERPRETATION OF GROWTH/COLONY MORPHOLOGYInterpretation of bacterial growth requires considerable experience. In short, it hinges on the ability of the microbiologist to distinguish between what is significant (abnormal) and what is to be expected from normal tissue. The clinical history, necropsy findings and smear examination must be borne in mind when evaluating the growth.

Examine all the cultures of each specific case. The stereo-microscope can be invaluable at this stage. If a stereo-microscope is not available, a hand lens can be used. This helps to distinguish between different bacterial colonies. A good light source is required to examine the cultures. Circle suspect colonies on the under-surface of the plate. Colonies should be fully described according to shape, size, colony elevation, opacity, the presence of pigments, consistency and odour. Each circled colony should then be sub-cultured to obtain a pure culture for identification.

Below are a few tips:

1. Examples of micro-organisms exhibiting distinctive odours include: Pseudomonas aeruginosa: grape juice odour Proteus spp.: burnt chocolate

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Pasteurella spp.: mouse urine Nocardia & Streptomyces spp: musty basement Clostridium spp.: faecal, putrid Prevotella melaninogenica: acrid

2. It is often preferable to re-incubate very small colonies so as to be able to assess purity.3. Although growth rate is not an indication of pathogenicity, slow growing bacteria are often more

pathogenic than those which grow fast on culture.4. The growth of the suspected pathogen on the various media must be closely compared before a

decision is made as to which colonies should be isolated.5. Some bacterial species, such as some streptococci, clostridia and Bacillus, do not retain their Gram-

positive properties very well and consequently often stain Gram-negative.6. Clostridium perfringens seldom forms spores on artificial media.

Figure 1: Terms used to describe gross colony morphology

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SECONDARY CULTURESAfter 24 hours incubation (or as long as it requires to distinguish the individual colonies clearly), make a subculture from a single colony of the suspect pathogen by touching the colony with a sterile (flamed and cooled) inoculation needle and then inoculating it onto a non-selective agar medium.

When the bacterial growth is very heavy and the colonies too close together to distinguish from each other, a stereo-microscope may be used to advantage. A straight sharp needle may facilitate the isolation of a particular colony.

Only select colonies from selective media, should there be no alternative i.e. the colony is not represented on non-selective media. Inoculate a non-selective agar medium. Isolations from selective solid media into nutritious fluid media should not be attempted, even if the culture appears “pure”. Selective media often inhibits, but may not kill, bacteria.

Make a smear of the colony and stain it using Gram’s method, and any other stains that may be deemed necessary, from the remainder of the same colony.

Incubate agar for at least 18 hours before carrying out the appropriate identification tests. Should there be enough isolated colonies on the initial non-selective agar medium, identification and an antimicrobial sensitivity test can be done from the primary plates. Please note that all tests should be done using a pure culture as using mixed cultures can lead to spurious results.

IDENTIFICATION OF BACTERIAPrimary identification of the majority of bacteria is based on the Gram’s stain. Bacteria are divided into Gram-positive and Gram-negative bacteria. Thereafter they are divided according to colony and microscopic morphology as well as catalase, oxidase and oxidation-fermentation tests.

Based upon these results secondary identification tests are done.

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Figure 2: General procedures for the isolation of bacteria and fungi from clinical specimens

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Figure 3: Primary identification of some Gram-positive bacteria of veterinary importance

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Figure 4: Primary identification of some Gram-negative bacteria of veterinary importance

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Table 1: Selection of commercial biochemical tests for the Gram-negative bacteria, according to microscopic morphology, colony appearance, catalase and oxidase tests

ShapeColony

MorphologyColonies Cat. Oxi. Biochemical tests

Short & long rods Large on BTA + -Enterobacteriaceae, API 20EAPI 10S

Short & long rods Yellow on MacConkey + -EnterobacteriaceaeAPI 20EAPI 10S

Short & long rods Black on XLD + -

EnterobacteriaceaeSalmonellaAPI 20EAPI 10S

Short & long rods (beaded)

Large or fine, MacConkey positive. + D

PseudomonasAPI 20NEMicrobact 12A & 12B

Diplococci Large or fine + DPseudomonasAPI 20NEMicrobact 12A & 12B

Diplococcobacilli Large or fine + DPseudomonasAPI 20NEMicrobact 12A & 12B

Pleomorphic Fine D D

Haemophilus, Pasteurella, ActinobacillusAPI 20NEMicrobact 12A & 12B

Pleomorphic from chickens or ostriches D D

Fowl PasteurellaAPI20NE (identification is not listed, you must use own tables).

Pleomorphic Large and sometimes beta-haemolytic D + Fish bacteria (with salt

broth)

PleomorphicLarge or fine, growth at 25°C. Found in fish, tortoises & snakes.

D D Fish bacteria (with salt broth)

Rods Flat & spreading at 25°C. Found in fish D D Myxobacteria

Pleomorphic, bent or spiral-shaped

Fine. Growth better with added CO2

D D Campylobacter

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Table 2: Selection of biochemical tests for the Gram-positive bacteria, according to microscopic morphology, colony appearance, catalase and oxidase tests

ShapeColony

MorphologyColonies Cat. Oxi. Biochemical tests

Short & long rods, sometimes with spores Usually large + D Bacillus

Short & long rods Fine D DGram-positiveCoryneAPI

Short & long rods, sometimes with spores

AnO2,Beta-haemolytic

- DClostridiumAPI32A

Fine rodsFine alpha-haemolytic and from ostrich stomach.

- D Ostrich bacillus

Pleomorphic Large or fine + DGram-positiveCoryneAPI

Pleomorphic and sometimes branched

Fine white beta-haemolytic - D CAMP test

Cocci or tetradsLarge, usually white, sometimes yellow and beta-haemolytic.

+ D Staphylococcus, Micrococcus

Cocci or coccobacilli in broth can occur as chains)

Fine alpha- or beta-, or gamma-haemolytic - - Streptococcus

Large oval to round cells with budding.

Large dull, sticky colonies. Rarely slimy + D Yeasts

Branched large hyphae, sometimes with fruiting bodies

Large, chalky, fluffy or leathery. D D Fungus

Branched filamentous bacteria, sometimes breaks up into cocci.

Usually grows into the agar. + D

Nocardia, Streptomyces, Actinomyces

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Table 3: Recognition of the different bacterial genera by their Gram’s stain and colony morphology

BacteriumBlood agar MacConkey agar Gram’s stain

General commentColony Haem

olysis Growth LF/NLF Reaction Shape

Salmonella spp.Greyish,round and shiny2-3 mm

- + NLF - R

Pale colonies on MAC agar. No smell (unlike most other members of the Enterobacteriaceae

Non haemolytic E. coli

Greyish, round and shiny2-3 mm

- + LF - R

Non-haemolytic but otherwise similar to the haemolytic strains. Characteristic “coliform” smell.

Yersinia spp.Greyish, round and shiny2-3 mm

- + NLF - R

Serratia marcenscens and S. rubidea

Red/orange convex, round and shiny.2-3mm

- + NLF - R

Produces red pigment (prodigosin). Some strains are white at 37°C (no pigment)

Klebsiella spp.

Grey, mucoid colonies that coalesce.2-4mm

- + LF - RColonies tend to be large, mucoid and light pink on MAC agar. Non-motile.

Enterobacter spp.

Grey, mucoid colonies that coalesce.2-4mm

- + LF - R Very similar to colonies of Klebsiella spp. Motile.

Proteus spp.Grey, swarming growth over the agar.

- + NLF - R

Characteristic swarming on non-selective agar which can be in waves. Turns BTA brown. Very foul smell. Colonies pale and discreet on MAC, but edges may be irregular.

Pseudomonas spp. other thanP. aeruginosa

Grey or yellowish-green, flat and spreading.2.5-4mm

- + NLF - RSome may produce the yellowish-green pigment, pyoverdin.

Corynebacterium renale

Grey-white, round and moist0.5-1 mm

- - + RColonies older than 48 hours become drier.Urease positive

Brucella spp.

Translucent, convex and round0.5 mm

- - - R(C)

Some brucellae require 10% CO2 for growth. Colonies not visible until 2-3 days after incubation. MZN positive.

Campylobacter Small, “dew- - - - R Requires 2-3 days for

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fetusdrop”, round and opaque0.5 mm

curved

growth under reduced oxygen tension. Curved rods, if in pairs they have a "seagull" appearance

Actinobacillus spp.

Grey to translucent, round and shiny0.5-1mm

- V V - R Usually urease positive

Pasteurella multocida

Translucent, smooth, round and shiny1-2 mm

- - - R

Colonies appear pinkish on BTA. Characteristic sweetish smell. Indole positive.

Bordetella bronchiseptica

Small geyish-white and round0.5-2 mm

- + NLF - R

Colonies very small at 24 hours but become much larger later. Unreactive bacterium.

Rhodococcus equi

Salmon pink and mucoid. Colonies coalesce

- - + R(C)The colour becomes more definite with time. Mucoid colonies tend to merge.

Staphylococcus epidermidis (coagulase negative)

White, shiny, round and convex2-3 mm

- +LFMost

+ C

Colonies are similar to coagulase + staphylococci, but always white and non-haemolytic.

Micrococcus spp.

White, yellow, tan or pink. Round, convex and shiny.2-3 mm

- - + C Colonies are usually pigmented.

S. aureus orS. pseudintermedius

White or yellow, smooth rounsd and shiny2-3 mm

+target

+ LF + C

Human and bovine strains are bright yellow. Hold plate to bright light to see characteristic double haemolysis. Are catalase positive.

Clostridium perfringens

Grey, flat and often irregular edge2-3 mm

+target

- + R

Aerobic with double or target haemolysis. Colonies tend to have irregular edges.

Haemolytic E. coliGrey, smooth and round2-3 mm

+ + LF - R Characteristic “coliform” smell.

Aeromonas hydrophila

Grey, flat, round and shiny2-3 mm

+ + v - RFoul smell, different to that of E. coli. Good growth on MAC. Oxidase positive.

P. aeruginosa

Blue-green, flat, round. Some have a metallic sheen. 2.5-4 mm

+ + NLF - R

Amount of pyocyanin (blue-green pigment) varies between strains. Characteristic fruity-musty smell.

Bacillus spp. Grey, dry, granular with

± v v + R Usually large, dry rhizoid colonies. Motile except for

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irregular edges3-5 mm

spores B. anthracis. B. anthracis non-haemolytic.

C. pseudotuberculosis

Opaque, dry, crumbling0.5-1 mm

V - + RThe cells tend to be less pleomorphic than A. pyogenes.

Trueperella pyogenes

Translucent, pin-point0.5 mm

+hazy

- + R(C)

Hazy haemolysis along streak lines, even before colonies are seen. Very pleomorphic in the Gram stained smear. Catalase negative.

Beta-haemolytic streptococci

Translucent, glistening and round0.5-1 mm

+ - + C

The size of the clear zone of beta-haemolysis varies with species. Catalase negative.

Listeria monocytogenes

White, smooth and round0.5-1 mm

+ - + R(C)

Colonies resemble that of beta-haemolytic streptococci. Young colonies may yield coccoid cells. Catalase positive.

Moraxella bovisWhite, smooth and round0.5-1 mm

+(-)

- - R(C)

Colonies very similar to the above two bacteria. Gram-negative and cells in pairs as rods or fat cocci.

Pasteurella haemolytica

White/grey, smooth and round0.5-1.5 mm

++pin-point

LF - RSome strains are haemolytic only under the colonies.

Enterococcus faecalis


+alpha

+pin-point

LF + C Red pin-point colonies on MAC.

Alpha-haemolytic streptococci


+alpha

- + C

Erysipelothrix rhusiopathiae

White, smooth and round. Some strains rough0.5-1.5 mm

+alpha 48 hours

- + R

Alpha-haemolysis under the colonies only at 48 hours. Rough, dry colonies especially from chronic forms of the disease. Catalase negative.

+ = positive; - = negative; v= variable reaction; LF = lactose fermenter; NLF = non-lactose fermenter; MAC = MacConkey agar; BTA = blood agar; MZN = modified Ziel-Neelsen; C = cocci, R = rodsSize of colonies can be variable. Size is given and described after 48 hours of incubation, as even with fast-growing bacteria the colonies are more characteristic at this stage.

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Table 4: Biochemical tests recommended for the identification of Gram-negative bacteria

Standard tests Haemophylis, Pasteurella, Actinobacillus

Fowl Pasteurellas Pseudomonas

LactoseDulcitolSucroseMaltoseInositolPhenylalanineUreaMalonateCitrateTSI3 ml MRP brothGershman’s

If Salmonella suspected add:SalicinSorbitolLysine & control

3 ml MRP brothUreaGlucoseMannoseTrehaloseMannitolInositolAesculin

BTA with staph streak (CAMP test)

If it cannot be identified with these tests add:OrnithineArabinoseXyloseRhamnoseGalactoseSucroseLactoseMaltoseAdonitolDulcitolSorbitolSalicin

3 ml MRP brothUreaGlucoseMannoseSucroseLactoseMaltoseTrehaloseMannitolInositolSorbitolSalicinAesculinBTA for ONPG & Porphyrin

42°C BTANitrateUreaO/F without oilGlucoseCitrateAesculinGershman’s3 ml MRP brothBrucella glucose with H2SHorse serum

Table 5: Identification of some Gram-negative bacteria

Campylobacter Fish bacteria Myxobacteria

AnO2 BTABTA in Campylobacter gasBTA in candlejar @ 42°CBTA in candle jar at 25°C.TSISodium hippurateNitrateBruc. glucose & H2SSeleniteNutrient broth25°C for motility

Gershman’s0% Sout broth5% Salt broth7% Salt broth3 ml MRP brothMethylredCitrateArginineLysineOrnithineUrea

MaltoseSucroseMannitolTrehaloseGlycerolSorbitolSalicin5% BTA37°C BTA-ONPG0129 BTA discStarch

At 25°C:0% Salt broth2% Salt brothGelatinAesculinGlucoseO & F without oilCasein agarDNAse agarNitrateBruc. gluc. & H2SCitrate

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GelatineGlucoseArabinose

Vibrio agar Gershman’sStarchAnO2 @ 25°CAnO2 Cytophaga agarEggyolkPhosphatase agar

Table 6: Biochemical tests recommended for the isolation of Gram-positive bacteria

Gram-positive Staphylococcus Streptococcus BacillusActinomyces/

Corynebacterium

O & F (x2)NitrateGlucoseUreaAesculinHorse serumBrucella glucose & H2SSodium HippurateAnaerobic BTAStarchDNAseCaseinIf identification not possible with these tests, then add:OrnithineLysineArginineMannitolSalicinAdonitolArabinoseRaffinoseRhamnoseXyloseTrehaloseAesculinSorbitol

NitrateGlucoseMaltoseO & FDNAse

For identification of coagulase negative staphs:ThioglycollateMethylredXyloseArabinoseRaffinoseSalicinSucroseMaltoseMannitolMannoseTrehaloselactoseGalactoseNitrateArginineUrea45°C BTAPhosphatase agarDNAse agarBTA & nalidixic acid diskanaerobic BTA

LactoseMannitolRaffinoseSalicinSorbitolTrehaloseInulinAesculinMethylene blueSodium hippurate45°C BTACAMP testStreptex

If identification not possible with these tests, then add:ArabinoseFructoseGalactoseXyloseArginineGlycerolDolcitolLactose milkBruc. glucose & H2SAdonitolStarch agarPhosphatase agar

Egg yolkAnaerobic BTACitrate3ml MRP brothNitrateGlucoseHorse serum

XyloseGalactoseMannoseLactoseSucroseTrehaloseRaffinoseSalicinPhosphatase agarInositol

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Table 7: Identification of some Gram-positive bacteria

Micrococcus Listeria Anthrax Nocardia Megabacteria

Anaerobic BTAGlucoseGlycerolAesculinNitrateUrea

GalactoseLactoseMannitolRhamnoseSucroseXylosePhosphatase agarCAMP test with Rhodococcus equi & S. aureus.

SalicinMethylin blue milkGelatinLactose milkPenicillin disk3 ml MRP brothGershman’sGamma-phage

45°C BTACasein agar

Brucella glucose slant & H2SGalactoseRhamnoseLactoseSucroseRaffinoseSalicinMannitolAesculinAnaerobic BTAMAC

Table 8: Summary of selected, commonly used biochemical tests for the identification of bacteria

Test Medium Incubation Product tested for and reagent used

Result

Negative Positive

Citrate utilization

Simmons citrate. Inoculate solid agar slant.

Up to 7 days at 37°C

Ability to use citrate as the sole carbon source

Green pH 6.9(E. coli)

Blue(Salmonella)

Decarboxylase (lysine and ornithine) and dehydrolase (arginine tests)

Broth base & 0,1% glucose with:0.5% L-arginine or0.5% L-lysine or0.5% L-ornithineORLysine iron agarMIO medium (ornithine)

Up to 4 days at 37°C

Arginine and ornithine to putrescine. Lysine to cadaverine. Products are alkaline. Bromocresol purple used as the indicator.

Yellow (acid), glucose only attacked.(Proteus)

Purple (alkaline)(Salmonella)

Gelatin liquefaction

Stab inoculation of nutrient gelatin.

22°C for 30 days or 37°C for 14 days

Proteolytic activity (gelatinases) and gelatin liquefied.

No liquefaction Liquefaction (not solid at 4°C)

Charcoal gelatin discs, placed in a broth

37°C for 14 days

As above. Charcoal particles are released when the gelatin is liquefied

No change Free charcoal particles.

X-ray film method. Small strip in heavy inoculum of bacteria in trypticase soy broth

37°C for 48 hours

As above.Gelatin layer on X-ray film strip

No change

(Enterobacter spp.)

Removal of gelatin layer leaving blue plastic film.(Serratia marcescens)

Hippurate Sodium hippurate 24 hours at Centrifuge test. Add 0.2ml No precipitate Permanent

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hydrolysis 10g in 1 litre of brain heart infusion broth.

37°C with an uninoculated control

ferric chloride reagent to 0.8ml of the supernatant

(A. equuli, S. pyogenes)

precipitate(A. ligniersii,S. agalactiae)

Hydrogen sulphide

Iron salts in media e.g. TSI and SIM (least sensitive method)

16 hours at 37°C

Hydrogen sulphide gas production

No change(E. coli)

Blackening of medium (Salmonella)

Lead acetate paper strip (most sensitive method). Strip suspended over trypticase soy broth or serum glucose agar slants (Brucella)

35°C for up to 7 days. Change lead acetate strip daily.

Hydrogen sulphide gas production

No change to lead acetate strip

Blackening of the lead acetate strip.(Brucella spp.)

Indole test Tryptone water 1-2 days at 30°C

Trytophan splits to Indole. Add 0,5ml Kovac’s reagent to medium and shake. Read in 1 minute.

Reagent layer: yellow(Salmonella)

Reagent layer: deep red(E. coli)

SIM medium in tubes As above

Kovac’s reagent (0,2ml) to tube. Stand for 10 minutes

Oxalic acid test paper suspended over medium

No change in reagent colour

No change in test strip

Reagent dark red

Pink colour at lower end of paper

Spot test for indole

Use bacterial colonies on either blood or nutrient agar incubated 24-48 hours at 37°C

Filter paper saturated with Kovac’s reagent. Rub colony over filter paper with a glass rod.

No reaction (P. haemolytica)

Blue colour on streak within 30 seconds (P. multocida)

Malonate utilization

Malonate broth (0,3% sodium malonate)

24 hours at 37°C

Utilization of malonate as a sole carbon source. Bromothymol blue indicator.

No change (most Salmonella spp.)

Growth and a deep blue colour(S. arizonae)

Methyl red (MR) test

Glucose phosphate peptone water (5 ml) MR-VP broth

2 days at 37°C or 3-5 days at 30°C

Small molecular weight acids such as formic and acetic.Add 5 drops of MR reagent to medium

Yellowish (E. cloacae)

Red (acid)(E. coli)

Nitrate reduction

Nitrate broth (0,1% KNO3: 5ml)

24 hours at 37°C (rarely up to 5 days)

Nitrate (NO3)

Nitrite (NO2)

Nitrogen gas (N2)Add 5 drops of reagents A*

and B#. Shake and wait 1-2 minutes.

Colourless. Add a pinch of zinc dust.

NO3 converted to NO2

(negative: red)

Red (NO3 to NO2)

Colourless (positive)

KNO3 (40%) on filter paper. Place on

37°C and examine at 4

Nitrate reduction No reaction or very narrow

Wide zone of browning of

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blood agar and stab inoculate test bacterium 20mm from paper strip. Use a heavy inoculum and E. coli as a positive control

and 24 hourszone of browning around medium

medium between colony and strip (E. coli)

ONPG test

Peptone water & 0,15% o-nitrophenoly-beta-D-galactopyranoside

24 hours at 37°C

The enzyme beta-galactosidase. Identifies potential lactose fermenters

Colourless (Salmonella spp.)

Yellow(S. arizonae)

Phenylalanine deaminase test

Phenylalanine medium (BBL) 0,2% DL-phenylalanine slant agar)

Inoculate heavily. 35°C for 4 or 18-24 hours

Phenyl-pyruvic acid formed.Add 4-5 drops of aqueous ferric chloride. Rotate and read in 1-5 minutes.

Yellowish

Green reaction in slant. (Proteus, Morganella and Providencia spp.)

Phosphatase test

Nutrient agar & 0,01% phenolpthalein diphosphate

18-24 hours at 37°C

Sufficient phosphatase to split to phenol-phtalehin diphosphate.Hold colonies on the agar over an open bottle of ammonia

Unchanged colonies (coagulase negative staphylococci)

Colonies bright pink (coagulase positive staphylococci)

Urease tests

Christensen media: either an agar slant or broth base containing 2% urea

Up to 24 hours at 37°C

Urease: splits urea with formation of ammonia (alkaline).Phenol red indicator

Yellow (Salmonella spp.)

Red(Proteus spp.)

Spot test. Moisten filter paper with a few drops of 10% urea agar base concentrate and rub some culture onto the filter paper with a glass rod.

As above No changePink or red streak within 2 minutes

Voges Proskauer (VP) test

5ml glucose phosphate peptone water. (MR-VP broth)

3-5 days at 30°C

Acetoin derived from glucose.3ml of 5 & alphanaphthol in absolute ethyl alcohol and then 1ml of 40% KOH. Shake and leave for 5 minutes.

Colourless (E. coli)

Red(most Enterobacter spp.)

TYPES OF IDENTIFICATION SYSTEMSBiochemical profiles

In the diagnostic laboratory, biochemical profiles are most commonly used to identify a micro-organism, either by using a manual or automated system. Biochemical profiles are determined by the reactions of individual organisms with each of the substrates in the system. Most bacteria can be identified to species

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level by these tests, allowing a small laboratory to identify a wide range of bacteria. Systems used rely on the following indicators:

pH-based reactions (many 15- to 24-hour identification systems). As a general rule, carbohydrate utilization by micro-organisms results in acid production, while protein utilization or the release of nitrogen-containing products results in an alkaline pH.

Enzyme profiles (many 4-hour systems). Enzyme profile tests are usually based on pre-formed enzymes and require minimal microbial growth. When a colourless chromogen (colour) or fluorogen (fluoresces in U-V light) is hydrolyzed by an appropriate enzyme, the chromogen or fluorogen is released resulting in a visible reaction.

Carbon-source utilization. This method measures metabolic activity. Tetrazolium-labelled carbon sources are colourless until electrons activated by metabolic activity are transferred to the dye, creating a purple colour.

Visual detection of growth (yeast identification systems). Assimilation assays depend on the ability of micro-organisms to grow in the presence of a substrate. Visual detection of growth is a positive result.

Gas-liquid chromatography

Chromatography has been used to separate various bacterial components such as cellular fatty acids, in order to isolate, characterize and identify bacteria. As this is a technically difficult technique, requiring specialized instrumentation, and limited to the identification of a small group of bacteria, it has only been used in reference laboratories and for research purposes. This technique has been most commonly used to identify obligate anaerobes, by identifying short chain fatty acids that are produced by glucose fermentation. The mycolic acids of Nocardia spp., Rhodococcus spp., Corynebacterium spp. and mycobacteria can be analysed in this fashion.

Recently, these methods have been standardized and automated (Microbial identification systems, Newark, Del.), making them more easily available. They allow objective analysis, less labour input and relatively low cost per sample. They are also able to identify both asaccharolytic organisms and those with enzymic profiles that are not distinctive. Disadvantages are the large capital expense required for initial equipment purchase, non-acceptance of alternative identification strategies, and the size of the library of known organisms that is required. As these tests are developed further, they may in the future become more accessible to diagnostic laboratories.

Immunoassays

Immunoassays are commonly used in diagnostic laboratories to both identify the micro-organism and to detect antibodies. Tests that are most commonly used to identify micro-organisms include agglutination tests e.g. Streptex, serotyping of E. coli and Salmonella sp., fluorescent antibody tests i.e. for identification of histiotoxic clostridia and precipitation tests e.g. identification of galactan produced by Mycoplasma mycoides subsp. mycoides.

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Molecular methods

At present, DNA probes and nucleic acid amplification techniques are most useful for the characterisation of micro-organisms for which culture and serologic methods are difficult, expensive or unavailable. Amplified and non-amplified DNA probes are used for the identification of micro-organisms in samples (in situ hybridization); for culture confirmation of slow-growing bacteria such as the mycobacteria and pathogenic dimorphic fungi; and the identification of enterotoxin producing E. coli. Amplification of microbial DNA is the most sensitive technique, making it suitable for the identification of micro-organisms in samples. As these techniques are expensive, detect both viable and non-viable micro-organisms and are not available for all micro-organisms, they should only replace conventional techniques where they would lead to a quicker patient isolation, a better choice of patient therapy, and a decrease in time when the patient is infectious.

Rapid manual and automated methods

Commercial systems both automated and in kit form, are available for the identification of commonly isolated species. These tests have enabled laboratories to more rapidly report results which are often more accurate. They also are less labour-intensive as no complex media preparation is required. These kits/systems, however, do not replace a good microbiologist, who is able to determine the possibility of a false result e.g. Bacillus species often resembles Gram-negative bacteria and may be misidentified by an automated system as members of the family Enterobacteriacae. Errors in commercial systems include:

Human error: wrong isolate, misidentified module. System inadequacies: insufficient data in database, inability to interpret atypical reactions. Incorrect identifications. Results for a given test may not be equally valid among all identification systems e.g.

Christensen’s urea agar is more sensitive in the detection of urease than that of the API-system. Batch variation. Laboratory practice variation.

Table 9: Summary of selected identification systems

System Manufacturer Organisms identified Storage temp. (°)

No. of tests Incubation Automa

ted

ANI BioMérieux Vitek Anaerobes 2 - 8 28 4 h: aerobic Yes

API 20A BioMérieux Vitek Anaerobes 2 - 8 21 24 h: anaerobic No

API 20C BioMérieux Vitek Yeasts 2 - 8 20 24 – 48h No

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API 20E BioMérieux Vitek

Enterobacterieae & nonfermenting Gram-negative bacteria

2 - 8 21 4 – 24h No

API 20 Strep BioMérieux Vitek Streptococci & enterococci 2 - 8 20 4h: aerobic No

API An-IDENT BioMérieux Vitek Anaerobes 2 - 8 21 24 h No

API Corne BioMérieux Vitek Corynebacteria 2 - 8 20 24 – 48h No

API NFT (Rapid NFT)

BioMérieux Vitek

Gram-negative non- Enterobacterieae 2 - 8 20 2 – 4 h No

API Rapid 20E BioMérieux Vitek Enterobacterieae 2 - 8 21 4 h No

API Staph-IDENT

BioMérieux Vitek Staphylococci & micrococci 2 - 8 10 No

API Staph (STAPH-Trac)

BioMérieux Vitek Staphylococci & micrococci 2 - 8 20 24 h No

Bacterial Identification Panel

AlamarEnterobacterieae & nonfermenting Gram-negative bacteria

RT 26 18 – 20h Reader only

Crystal E/NF BDMSEnterobacterieae & nonfermenting Gram-negative bacteria

2 - 8 30 18 – 20 h No

Crystal Rapid Stool/Enteric BDMS Gram-negative stool

pathogens 2 - 8 30 18 – 20 h No

Enterotube II BDMS Enterobacteriea 2 - 8 15 18 – 24 h No

EPS (Enteric Pathogen Screen)

BioMérieux Vitek

Edwardsiella, Salmonella, Shigella & Yersinia 2 - 8 10 4 – 8h No

ES MicroPlate Biolog Aerobic gram-negative bacteria 2 - 8 95 4 – 24 h No

Fox Dual GNI Micro-Media Systems


-20 - 40 33 4 – 24 h Yes

GM Microplate Biolog Aerobic Gram-negative bacteria 2 - 8 95 4 – 13 h Reader

only

GNI BioMérieux Vitek


2 - 8 29 4 – 24 h Yes

GP Microplate Biolog Most Gram-positive cocci & bacilli 2 - 8 95 4 – 15 h Reader

only

GPI BioMérieux Vitek Gram-positive cocci & bacilli 2 – 8 - 20 29 16 – 20 h Yes

ID Tri-Panel Difco/Pasco Gram-negative & Gram-positive bacteria 30 Reader

only

MicroID Organon Teknika Enterobacteriea 2 - 8 15 No

Minitek BDMS Anaerobes, Enterobacterieae, Gram-

2 - 25 4 – 21 (dependi

Enterobacteriacceae & Neisseria

No

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positive bacteria, Neisseria spp. nonfermenters & yeasts.

ng on need)

spp. For up to 72 h for yeasts

THE NORMAL FLORAIt is important that the veterinary microbiologist is familiar with the kinds of organisms encountered normally in and on animals. Such knowledge is necessary in the interpretation of the results of microbiological examinations. The so-called normal flora consists of the wide variety of bacteria and fungi that live in or on the normal animal without producing disease. Included in this flora are many potential pathogens and opportunistic organisms. The term normal flora is a convenient concept, but it should be kept in mind that the kinds and numbers of bacteria present vary greatly under different circumstances. The intestinal flora of the young animal differs markedly from that of the older animal. The flora is also influenced by geographic location, nutrition, and climate. The technical procedures are biased to recover pathogenic organisms and thus frequently give a distorted idea of the kinds of numbers of bacteria present. The normal flora of the domestic animals has not been studied in as detailed a fashion as that of human beings. What little information that is available, and firsthand experience in the diagnostic laboratory, indicate a considerable similarity between the normal flora of humans and that of domestic animals. Some of the bacteria that can be expected to occur normally in and on domestic animals are tabulated below.

Mouth, nasopharynx

Micrococci (aerobic and anaerobic, pigmented and nonpigmented); Staphylococcus spp.; haemolytic and non-haemolytic streptococci; Veillonella and other Gram-negative cocci; coliforms and Pasteurella spp.; diphtheroids; pneumococci; yeasts, including Candida albicans; Haemophilus spp.

Jejunum, ileum

Only a small number of bacteria are present in this portion of the intestinal tract of animals.

Large intestine

Enteroccus spp.; E. coli; Klebsiella; Enterobacter; Pseudomonas spp.; Proteus spp.; staphylococci; Cl. perfringens, Cl. septicum, and other clostridia; Gram-negative anaerobes; spirochetes; lactobacilli.

Trachea, bronchi, lungs

Few, if any, bacteria and fungi reside in these structures; possibly very low numbers of Pasteurella spp. may be present.

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Vulva, prepuce

Vulva: diptheroids; micrococci; coliforms and Proteus spp.; enterococci; yeasts; Gram-negative anaerobes.

Prepuce: the same kinds of organisms. A bull may be a carrier of C. foetus venerealis and a stallion of certain serovars of Klebsiella pneumoniae and Taylorella equigenitalium.

Vagina

The numbers and kinds of bacteria vary with the reproductive cycle and age. The cervix and anterior vagina of the healthy mare possesses few bacteria. Some of the organisms recovered from the vagina are coliforms and Proteus spp.; certain salmonellas and klebsiellas; diphtheroids and lactobacilli; mycoplasmas; yeasts and fungi.

Skin

Animals, by virtue of their habits and environment, frequently possess a large and varied bacterial and fungal flora on their hair and skin. Stapylococcus epidermidis and S. aureus occur commonly, as do other micrococci. Of the many other organisms isolated, it is not known which make up the resident flora and which are “transients”.

Milk

Micrococci, staphylococci, non-haemolytic streptococci, mycoplasmas and diphtheroids including Corynebacterium bovis are frequently shed from the apparently normal mammary gland.

OCCURRENCE OF PATHOGENS IN ANIMAL SPECIESIn Table 20, the organisms most frequently associated with infection in various organs and systems of the more important animal species are listed.

Laboratory animal infections

Rats and Mice

Salmonella spp.; pyogenic streptococci; Bacillus piliformis; Pasteurella pneumotropica, P. multocida; Corynebacterium kutscheri; Bordetella bronchiseptica; Streptobacillus moniliformis; Streptococcus pneumoniae; mycoplasmas; Yersinia pseudotuberculosis.

Guinea Pigs

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Salmonella spp.; Bordetella bronchiseptica; Streptococcus pneumoniae, pyogenic streptococci; Klebsiella pneumoniae; Yersinia pseudotuberculosis; Streptobacillus moniliformis.

Rabbits

Salmonella spp.; Pasteurella multocida; Bordetella bronchiseptica; Yersinia pseudotuberculosis, Y. enterocolitica; pyogenic streptococci; Haemophilus spp.; Clostridium piliforme, Fusobacterium necrophorum; Treponema cuniculi.

Table 10: Potentially pathogenic bacteria of different organ systems in different animals

Bovine Ovine Porcine

The respiratory system

P. multocida,M. haemolytica; A. pyogenes;B. bronchiseptica; H. somni; Actino. actinoides (rare); mycoplasmas., including M. mycoides mycoides SC.

M. haemolytica,P. trehalosiP. multocida;C. pseudotuberculosisT (A). pyogenes; mycoplasmas;chlamydia

P. multocida,M. haemolytica;T (A). pyogenes;H. parasuis, A.suis, A. pleuropneumoniae M. hypneumoniaM. hyorhinis

Mastitis

S. aureus, S. epidermidis; Micrococcus spp.; Strep. qalactiae, Strep. dysqalactiae, Strep. uberis; C. bovis, A.pyogenes; E. coli; Ps.aeruginosa; M. haemolytica, P. multocida; Klebsiella spp.; other Gram-negative organisms; Mycoplasma bovis, yeasts

S. aureus; M. haemolytica, P. multocida; S. agalactiae, S. dysgalactiae, Strep. uberis; A. pyogenes;C. pseudotuberculosis; Histophilus ovis, mycobacteria; mycoplasmas.

Streptococci;S. aureus;F. necrophorum; Actinomyces bovis;A. lignieressii;A. pyogenes, mycobacteria; coliforms.

The gastro-intestinal tract

Escherichia coli; Salmonellas;Cl. perfringens types B and C;M. paratuberculosis.

E. coli; Cl. perfringens type A (red gut), type B (lamb dysentery), type C (struck) type D (pulpy kidney), type E;M. paratuberculosis; Salmonella

E. coli;Salmonella (especially S. choleraesuis);B. hyodysenteriae;B. pilisicoliLawsonia intracellularis;Cl. perfringens type C.

Genital infections. Streptococci, staphylococci, enteric bacteria and Pseudomonas aeruginosa are commonly associated with genital infection in all species

Campylobacter fetus subspp. fetus and intestinalis; Brucella abortus; chlamydia;M. bovigenitalium;L. monocytogenes;T. pyogenes.

Brucella ovis,B. melitensis;C. fetus subsp. intestinalis;L. monocytogenes; Histophilus ovis (Haemophilus somnus), Pasteurella spp., A. seminis;C. pseudotuberculosis; Chlamydophila. abortus

Brucella suis; Escherichia coli, mycobacteria;Ps. aeruginosa;T. pyogenes,multocida;pyogenic streptococci

Abscesses, ulcers A. pyogenes, A. bovis; D. congolensis; E. rhusiopathiae; S.

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and infections of the skinStreptococci and staphylococci are the most common causes.

Actinobacillus lignieresii,Sporothrix schenckii;D. congolensis.T. verrucosum

C. pseudotubercolosis, Corynebacterium (?) sp. (Bolo disease); Pseudomonas spp. (fleece rot).

hyicus;Group E streptococci (jowl abscesses); S. porcinus;T. pyogenes. M. avium

The central nervous system E. coli; Listeria monocytogenes;

Histophilus somni; streptococci;P. multocida, Pasteurella strains EF4 group; C. psittaci; Staphylococcus aureus.

E. coli;L. monocytogenes;Staph. aureus;P. haemolytica.

E. coli;L. monocytogenes;P. multocida; streptococci

The eyes Moraxella bovis; Branhamella ovis; chlamydia.

Moraxella ovis; Moraxella spp.; mycoplasma; chlamydia.

Joints

E. coli; pyogenic streptococci; Salmonella;Actinomyces pyogenes; Staphylococcus aureus; chlamydia;mycoplasmas; Haemophilus somnus.

E. coli; pyogenic and fecal streptococci; E.rhusiopathiae; Haemophilus agni; chlamydia;Strep. dysgalactiae Mycoplasma agalactiae;A. pyogenes;F. necrophorum; Histophilus ovis.

E. rhusiopathiae;A. pyogenes; various pyogenic streptococci; mycoplasmas; Staph. aureus; Haemophilus parasuis; Brucella suis; E. coli; Actinobacillus suis.

Respiratory system

Bordetella bronchiseptica;P. multocida; Klebsiella,Str. canis; Nocardia asteroides; mycoplasmas; Cryptococcus neoformans; Blastomyces dermatitidis; Actinomyces viscosus

P. multocida; Nocardia asteroides; Bord. bronchiseptica; chlamydophila; Cryptococcus neoformans.

Strep. equi, Str. equisimilis;Str. zooepidemicusStrep. pneumoniaeR. equi (foals);A. equuli (foals);P. multocida; Ps. mallei;Klebsiella;B. bronchiseptica;Mycoplasma felisC. neoformans; Asperqillus.

A. paragallinarumP. multocida,G. anatis, P. gallinarum;O. rhinotracheale ; various mycoplasmas; A. fumigatus.

GIT

Salmonella; possibly other enteric bacteria;Staph. intermedius (puppies); Borrelia canis; Spirillum spp.; Campylobacter.

Salmonella, Candida albicans (kittens).

SalmonellaClostridium perfringensClostridium sordelli

Genital tract

Brucella canis, other brucella species (rare); Klebsiella; Enterobacter; Proteus spp.; Candida albicans;P. aeruginosa; mycoplasmas.

Salmonella; R. equi (foals);A. equuli (foals).The mare’s cervixS. aureus; various streptococci; Klebsiella; P. aeruginosa; various fungi; R. equi; Candida albicans; Enterobacter; E. coli; A. equuli; T. equigenitalis

The skin S. intermedius; pyogenic

Pasteurella multocida,

Actinobacillus equuli; Klebsiella; Rhodococcus equi; Salmonella

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streptococci; Nocardia asteroides; Blastomyces dermatitidis; S. schenkii; Actinomyces viscosus; fungal dermatophytes.P. aeruginosa; Candida albicans

N. asteroides; fungal dermatophytes.

abortus-equi; Candida albicans; pyogenic streptococci; E. coli;P. aeruginosa

Canine otitis externa

S. aureus;P. aeruginosa; various streptococci;C. albicans; Proteus spp.Malassezia pachydermatis;C. perfringens.

C. pseudotuberculosis (chest abscesses); Histoplasma farciminosum; Sporothrix schenckii; fungal dermatophytes

CNS C. neoformans C. neoformans

L. monocytogenes; Strep. equi;Staph. aureus. Clostridium tetani

The eyes

Staph. aureus; Staph. epidermidis;P. aeruginosa; Cl. perfringens; Candida albicans; Cryptococcus neoformans, chlamydia; mycoplasmas; Moraxella spp.,

Staph. aureus; Staph. epidermidis; Pseudomonas aeruginosa; Clostridium perfringens; Candida albicans; chlamydia; mycoplasmas; Moraxella spp., and Neisseria flavus.

Strep. equi;Strep. equisimilis; Staph. aureus.

Joints

Staphylococcus aureus; Pseudomonas aeruginosa; various streptococci;Candida albicans;Malassezia pachydermatis; Proteus spp.; Clostridium perfringens.

A. equuli;Staph. aureus; pyogenic and faecal streptococci; E. coli; Rhodococcus equi; Klebsiella; Salmonella

Urinary infections

Proteus (usually mirabilis);P. aeruginosa; enterococci; E. coli; Enterobacter; S. aureus; pyogenic and fecal streptococci; Corynebacterium pilosum (rare), C. cystitidis.

E. coliC. pilosum,C. cystitidis.

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