Bacti - Orientation

Gram Stain is one of the most useful tools in microbiology. It aids in the identification of bacterial organisms by visualizing organisms and cells from a direct specimen.

The presence or absence of organisms and cells aid in empiric therapy of infectious processes and aid in the presumptive diagnosis of pneumococcal, meningococcal or gonococcal infections

Methanol fixing preserves cell morphology (visualizes WBC, RBC, epithelial cells, etc.). Heat fixing lyses the cellular elements.

Differential stain based on dye retention and microscopic arrangement

Components:
Crystal Violet: primary stain, purple.
Gram’s Iodine: mordant.
Acetone-Alcohol: decolorizer.
Safranin: counterstain, pink

Thick peptidoglycan layer allows retention of crystal violet-iodine complex. Appears blue/purple.

Thin peptidoglycan layer surrounded by outer lipopolysaccharide membrane. Appears pink / red.

1. Place a small loopful of distilled water on a clean slide.
2. Touch a sterile inoculating loop to a colony, rub it into the droplet and create even suspension.
3. Let the smear dry at room temperature.
4. Heat-fix.
5. Cool to room temperature before staining

- Flood fixed smears with CV for 1 minute, then decant and rinse with water.
- Flood with gram’s iodine for 1 minute, then decant and rinse with water.
- Decolorize with acetone-alcohol solution, stopping when runoff becomes clear, remove excess decolorizer with water. (Adjust decolorizing time to thickness of smear and type of decolorizer used)
- Counterstain with safranin for 30 seconds to 1 minute, then decant and rinse with water. (Faint organisms may require longer counterstain time. Alternative stains such as carbol fuchsin may be used.)
- Let air dry or blot dry (if blot drying, be careful as to not rub and remove the smear from the slide).

*Stain time can be adjusted. there is no need to actually spend a lot of time or watch the clock as you perform gram stain.

Gram Positive Cocci:
Spheres in irregular clusters, singly, pairs or short chains

Gram Positive Cocci:
spheres in pairs, short or long chains. Typically more elongated than staph

Gram Positive Cocci:
resemble strep, but with lancet shape, capsule may be observed

straight or slightly curved. V, L, Y formations. Pleomorphic, diphtheroid-like (club shaped)

small rounds with rounded ends, but possibly short coccal forms

vary from short plump palisading to long slender rods

large fat in single, pairs or chains. Frequently gram variable. Spores not always visible

Usually impossible to identify based strictly on gram stain

larger and more elongated

may be curved

small curved, faint staining

small pleomorphic or coccobacillary

small, short, almost coccoid

thin long with pointed ends

coccobacillary

Moraxella catarrhalis is true cocci

Gram Negative Cocci:
spheres in single or pairs. Pairs often have “kidney bean” or “coffee bean” shape

Organisms may look atypical in very young, old, dead or degenerating cultures and in specimens collected post-antibiotic therapy.

Must evaluate gram stain quality (thickness, stain time, stain precipitate)

Primary, non-selective

5% sheep blood and trypticase soy agar

Alpha: green around colony.
Beta: complete clearing around colony.
Gamma/Non-hemolytic: no hemolysis around colony

Recovers fastidious organism such as Neisseria gonorrhoeae and Haemophilus species

Same preparation as BAP, but RBCs are lysed releasing hemin (X factor) and NAD (V factor)

with IsoVitalex

Lacks lipids. Lipophilic organisms such as certain species of Corynebacterium (i.e., Corynebacterium jeikeium) will not grow

Bile salt and crystal violet inhibit gram positive flora

Neutral red pH indicator

Lactose fermenters produce organic acid which reduce pH, turning neutral red reddish pink. The neutral red is absorbed by the bacteria which appears bright pink on the agar

Lactose positive (Lactose fermenter, LF). Lactose negative (Non-lactose fermenter, NLF)

Strong lactose fermenters produce enough acid to cause precipitation of bile salts resulting in pink halo surround colonies

The gram positive cocci appear in clusters. The colonies are typically flatter, more opaque and are catalase positive

The gram positive cocci appear in pairs/chains and the colonies are typically raised, more translucent. Catalase negative.

Important human pathogen that can cause a variety of infections ranging from skin infections (cellulitis, abscess, carbuncles) to systemic illnesses (bacteremia, endocarditis, meningitis, osteomyelitis)

Staphylococcal Scalded Skin Syndrome: exfoliation of the skin in neonates and young children

Toxic Shock Syndrome via TSST-1 (assoc. tampons)

Stimulate T-cells and induce TNF and cytokine interleukin-1

Food poisoning via enterotoxin

Nosocomial pathogen of hospitalized patients. Methicillin-resistant SA (MRSA). Vancomycin-intermediate and –resistant (VISA, VRSA)

Colonizes the nares (screens in-patient, surgical patients for MRSA by culture or PCR)

Gram Stain: GPCCl - Gram-Positive Cocci in Clusters
Dense, large, smooth, butyrous colonies. Typically, golden/yellow. Beta-hemolytic

Catalase (+)
Slide coagulase (+)
Tube coagulase (+)

Laboratories may opt for Staphaurex™ Latex Agglutination test (detects Protein A) as substitute for slide coagulase test

Primarily causes acute UTI in young, sexually active females.

Cystitis, urethritis, pyelonephritis

S. saprophyticus bacteremia seen as complication of UTI

Urethritis, catheter-associated UTI, prostatitis in elderly men

Possible agent of non-gonococcal urethritis in males

Gram Stain: GPCCl
Large, smooth colonies, usually white. Non-hemolytic

Catalase (+).
Slide and tube coagulase (-)
Novobiocin Resistant (zone of inhibition <=16 mm)

high salt concentration inhibit most gram negative and some gram positive

organism that ferments mannitol produce yellow colonies

Staphylococci (+) vs Streptococci/Enterococci (-)

Can be used to differentiate among gram positive rods and some fastidious organisms

Streak colony onto slide and place a drop of hydrogen peroxide on it
- Positive: Rapid production of bubbles(effervescence)
- Negative: No or weak bubbling
- Catalase present in RBC, avoid carryover

2H₂O₂ -- CATALASE --> 2H₂O + O₂

Clotting of coagulase plasma (rabbit plasma and EDTA) by enzyme coagulase
S. aureus (+)
Other staph (-)*

Slide coagulase detects bound coagulase (clumping factor)

Tube coagulase detects extracellular free coagulase

- Emulsify a portion of colony (using loop or wooden stick) into rabbit plasma
- Macroscopic clumping in 10 seconds is positive

Negative slide coagulase must be confirmed by tube coagulase method

- Emulsify several colonies (using loop or wooden stick) into 0.5 mL rabbit plasma
- Incubate at 35-37oC
- Observe at 1-4 hours for positive result, if negative incubate at room temperature overnight

Staphylokinase producing SA can revert result to negative

Using a swab, prepare 0.5 McF suspension of bacteria to be tested in 2mL 0.85% saline.

Using a new swab, soak it in the suspension and wring out excess solution

Streak the swab soaked with 0.5 McF suspension onto Mueller Hinton agar plate, creating a lawn (streaks should be tight with no gaps in between)

Drop a novobiocin disk on to the surface of the streaked agar

Incubate in ambient air at 35–37°C overnight

Measure the diameter zone size:
Resistant: <=16 mm.
Susceptible: >16 mm

Both Staphylococci and Micrococci are gram positive cocci in clusters. Micrococci tend to be larger in size, form tetrads, may be yellow pigmented

Gram positive cocci in pairs/chains. May appear rod-like from agar plate media

Hemolysis on Sheep Blood Agar is key to identifying Streptococci

Alpha: Partial/incomplete hemolysis, greening around the colony.
- S. pneumoniae and S. viridans

Beta: Complete lysis of RBC, clearing zone around colony.
- S. pyogenes (GAS/BSA) and S. agalactiae (GBS/BSB)

Gamma: Non-hemolytic, no hemolysis around colony.
- Enterococcus faecalis

Oxygen labile
Antigenic
Stab agar to enhance hemolysis

Oxygen stable
Non-antigenic

Normal upper respiratory flora

Otitis media and bacteremia in infants and children

Community-acquired bacterial pneumonia

Common cause of meningitis (vaccine available)

Sinusitis, conjunctivitis, arthritis, endocarditis

Gram positive diplococci, lancet-shaped

alpha, glistening, gray; mucoid in strains has capsule

umbilicated (depressed center) due to autolytic enzyme

Catalase (-).
Optochin (P Disk) - S.
Bile (10% sodium deoxycholate) soluble

respiratory, gastrointestinal, urogenital, skin

Common cause of subacute bacterial endocarditis (heart valves), abscesses, bacteremia (subsequent to dental procedures)

Gram Stain: GPCPrch, sometimes elongated and rod-like

minute to small, gray, domed, smooth or matte, alpha or gamma hemolytic

Catalase (-).
Optochin R.
Bile insoluble.
Bile-esculin (+/-).
6.5% NaCl (-).
PYR (-).
LAP (+)

Optochin and bile solubility helps differentiates from Streptococcus pneumoniae

Bile esculin and 6.5% NaCl helps different from other alpha-hemolyticus Streptococci (including Enterococci)

Differentiates Streptococcus pneumoniae from other alpha hemolytic Streptococci

Optochin = ethylhydrocupreine hydrochloride (5.0 ug) interferes with ATPase and production of ATP

zone of inhibition ≥ 14mm in diameter

Equivocal: any ZOI <14mm should be confirmed by bile solubility test

Activates autolysin produced by S. pneumoniae which enhances lytic reaction.

S. pneumoniae (+/soluble).
Other alpha-hemolytic streptococci (-/insoluble)

Add drop of 2% sodium deoxycholate into well-isolated colonies on SBA

Incubate with lid up at 35°C for 30 min

Positive: colony disintegrates, imprint may remain
Negative: intact colonies

Streptococcal pharyngitis

Tonsillitis, sinusitis

Usually self-limiting, but if treatment is necessary penicillin is still the treatment of choice

Implicated in other superficial cutaneous infections: impetigo (red skin lesions), erysipelas (soft tissues), cellulitis (spreading inflammation)

Group A streptococcus toxic shock-like syndrome

Isolation of S. pyogenes from non-respiratory site is a significant finding and is reported to the clinician (implicated in necrotizing fasciitis)

carditis, polyarthritis, subcutaneous nodules, chorea (neurologic – muscular spasms, weakness)

inflammation of the glomeruli leads to hypertension, hematuria, proteinuria

Complications mostly thought to be through formation of antistreptococcal antibodies that cross-react with antigens on various organs

Gram Stain: GPCPrCh

Colony morphology: Grayish white, transparent to translucent, matte or glossy small to pinpoint colonies with large zone of beta hemolysis

Catalase (-)
Lancefield Group A
PYR (+)
Bacitracin (A Disk, Taxo A) - susceptible
SXT (Trimethoprim/sulfamethoxazole) - resistant
Bile-esculin (-)
6.5% Nacl (-)
LAP (+)

Produces Streptolysin S (oxygen-stable) and Streptolysin O (oxygen-labile)

Normal flora of gastrointestinal tract

Female may carry GBS as vaginal flora. Can cause sepsis and meningitis in neonates

Postpartum fever, osteomyelitis, wound infections, endocarditis

Gram Stain: GPCPrCh
Colony morphology: larger colonies than Grp A, translucent to opaque; flat, glossy, narrow zone of BH, some strains are gamma.

Biochemical:
Catalase (-)
Lancefield Group B
PYR (-)
Bacitracin (R)
SXT (R)
Bile-esculin (-)
6.5% NaCl (+)
LAP (+)
CAMP (+)
Hippurate (+)

Distinguish beta-hemolytic streptococcus

antigen is extracted from organism using nitrous acid extraction procedure or physical grinding and react with group-specific antisera

Lancefield antigen: A, B, C, F, G

Group F is now associated with Streptococcus anginosus group (small colony beta streptococcus)

Antibody from latex particles reacts and agglutinates with streptococcal antigens extracted from bacterial cell wall.

Limitations: organisms that share similar antigenicity may cross-react, e.g.:

pneumoniae with Strep C latex
Listeria monocytogenes with Strep B/G
Streptococcus porcinus types as group B
Viridans group Streptococcus maybe havemore than 1 demonstrable group antigen
A, C, G Streptococci must be differentiatedfrom minute A, C, F, G Streptococcus anginosus

Extraction Method Procedure
Extraction:
Add 2 drops each of Reagent 1 and 2 to test tube
Pick 1-4 beta colonies (more maybe used for younger/smaller colonies) with wooden stick and mix into the reagent
Add 4 drops of Reagent 3 and mix by tapping the tube

Testing:
Add 1 drop of your extracted bacterial antigen to 1 drop of selected strep grouping latex on a PathoDX reaction card
Mix with applicator stick
Observe for agglutination

Direct Method Procedure:
Rub colonies onto PathoDx reaction card (mechanically releases the antigen on the cell wall)
Add selected strep grouping latex and mix as per previous procedure
Non-specific clumping may be observed due to increased amount of organism used

Pyrrolidonyl aminopeptidase enzyme hydrolyzes L-pyrrolidonyl-β-naphthylamide to β-naphthylamide

Addition of color developer para-dimethylaminocinnamaldehyde complex with free naphthylamide to produce red color

Moisten PYR disk with small amount of water
Rub small amount of colony to be tested onto the disk
Wait 2 minutes
Add a drop of color developer on to the disk

Positive – pink color. Negative – no color

Differentiates
1. β-hemolytic Grp A streptococci (S) from other β-hemolytic streptococci (R). 2. Staphylococcus (R) from Micrococcus (S)

Antibiotic: bacitracin (0.04 units). Inhibits synthesis of bacterial cell walls

Streak lawn on BAP with 2–3 colonies of isolate. Place bacitracin disk on surface of agar

Incubate plate for 18–24h at 35–37°C in:
- streptococci: 5–10% CO₂.
- staphylococci: ambient air

Positive (Susceptible): zone of inhibition >7–10mm

Enterococcus species can cause UTI, bacteremia, endocarditis, intrabdominal and pelvic infections, wound and soft-tissue infections, neonatal sepsis.

Gram Stain: GPCPrCh

Colony morphology:
- Small, cream, or white, round, convex, smooth, entire
- Typically gamma but can be beta or alpha.

Biochemical:
Catalase (-) some have peroxidase that slowly catalyzes H2O2 – not truly positive
Bile-esculin (+)
6.5% NaCl (+)
PYR (+)
Lancefield group D (not typed)
LAP (+)

Differentiates enterococci (+) from non-enterococci (–).

Heart infusion broth containing 6.5% NaCl, small amount of glucose, and pH indicator bromocresol purple.

Inoculate from 18–24h culture into 6.5% NaCl broth at 35–37°C in ambient air.

Positive: visible turbidity in broth, with or without colorchange from purple to yellow

Negative: no turbidity and no color change

Presumptive ID of enterococci and organisms in the Streptococcus bovis group.

Ability to grow in the presence of 4% bile and hydrolyze esculin to esculetin (Esculetin reacts with Fe3+ to form dark brown / black precipitate).

Inoculate onto the surface of a slant and incubate at 35–37°C in ambient air.

Positive: black slant.
Negative: no color change

Most coryneform bacteria are considered part of normal flora (skin, urogenital, respiratory)

Pleomorphic, diphtheroid, non-spore-forming gram positive rod (PLGPR)
Palisading/picket fence/chinese letters/forming V, Y, L arrangement

Varies depending on the species. Catalase strongly positive.

Can be reported as Corynebacterium species, unless further speciation is needed.

Bacteremia and sepsis, valve endocarditis, meningitis, pneumonia, and soft-tissue infections.

Nosocomial infections and infections associated with prosthetic device implants.

Corynebacterium jeikeium.

May appear coccobacillary instead of a pleomorphic gram-positive rod.

Pinpoint colonies; lipophilic and grows poorly on chocolate agar due to absence of lipids.

CAT (+)
MOT (-)
URE (-), NIT (-), Glucose (+)

Multidrug resistant but remains susceptible to vancomycin.

All members have the following characteristics:
Ferments glucose
Oxidase negative (exception of Plesiomonas)
Nitrate (+)
Growth on Mac

Escherichia coli.

Most species are normal GI tract flora except Salmonella, Shigella and Yersinia

May cause UTIs, gastroenteritis, septicemia, food poisoning, wound infections, pneumonia, and meningitis.

Salmonella, Citrobacter freundii, Proteus (except P. penneri, which is variable).

Motility (-): Shigella, Klebsiella, Yersinia (35oC)

Klebsiella, Enterobacter, Serratia.

Serratia and Proteus.

Salmonella (70%), Citrobacter (65%), Enterobacter cloacae.

Shigella, Salmonella typhi, Yersinia, Providencia (except P. alcalifaciens).

-Carbohydrate utilization (glucose, lactose, sucrose)
-Gas production (CO2)
-H2S production
-KIA (Kligler Iron Agar) is essentially same as TSI, except does not contain sucrose

Sugar: Lactose, Sucrose, Glucose in 10:10:1
pH indicator: Phenol Red
H2S indicator: Ferrous sulfate (sodium thiosulfate as source of sulfur) complex with H2S to form black precipitate

Uninoculated tube is red

Stab the butt once and then streak the slant. incubate tube at 35-37°C in ambient air for 18-24 h with loosen cap

Record result as slant / butt
Yellow color = Acid (A)
Pink / red color = Alkaline (K)
Formation of black precipitate = H2S positive (*H2S forms under acidic condition only)
Bubbles in the media = gas produced (Denotes with “G”)

Yellow slant.

Yellow butt.

Lysine decarboxylation, lysine deamination, and H₂S production.

Lysine, dextrose, peptone, ferric ammonium citrate, and sodium thiosulfate.

Bromocresol purple.

Record result as slant / butt
- Yellow color = Acid (A)
- Purple color = Alkaline (K)
- Red color slant = Red (R)
- Formation of black precipitate = H2S positive
*H2S cannot be detected in LIA if lysine decarboxylase is negative (an acid production in the butt may suppress H2S formation)

Lysine decarboxylation produces cadaverine turns the butt alkaline again (returns to purple) - Alkaline Butt (Purple) = LDC +

Lysine deamination produce α-ketocarboxylic acid, which reacts with iron to form a red slant - Red Slant = LDA (+)

Enzyme urease hydrolyze urea to ammonia producing pink color change

Phenol Red.

less buffered, more suitable for organism that produce less urease such as Klebsiella, Enterobacter, Brucella

Detecting high urease–producing organisms, such as Proteus (heavily buffered and virtually selective).

Ornithine decarboxylase
Motility - Turbidity from the stab line
Indole production - Red color after addition of Kovac’s Reagent

Bromocresol purple.

It turns the medium yellow (acidic).

It produces putrescine, turning the medium purple (alkaline).

By growth extending outward from the line of inoculation.

If the organism has enzyme tryptophanase, it will produce indole from tryptophan. Addition of Kovacs Reagent (p-dimethylaminobenzaldehyde) to the surface (3-4 drops) produces red color band on the media

Whether an organism can utilize ammonium dihydrogen phosphate and sodium citrate as the sole nitrogen and carbon source.

Alkaline products are produced, turning the slant blue.

Bromothymol blue.

Distinguish which metabolic pathway is used to break down dextrose / glucose

Methyl Red: mixed acid pathway producing acidic end products (lactic, acetic, formic acid)

Voges-Proskauer: butylene glycol pathway producing neutral end product (acetoin)

Organism that utilizes one pathway will not use the other

Inoculate MR-VP broth and incubate at 35–37°C in ambient air, then split broth into aliquots for separate MR and VP tests.

Mixed acid fermentation.

Methyl red indicator (1-2 drops per 1 mL broth)
at pH 4.4: MR = red (+)
at pH6.2: MR = yellow (-)

Negative or inconclusive.

Methyl red will stay red in thePresence of acid end products

Detects neutral end-products of glucose digestion via butylene-glycol pathway

Alpha-naphthol and potassium hydroxide (KOH).

1.cAdd 0.6mL (6 drops) of alpha-naphthol and 0.3mL (3 drops) of KOH to 0.5 mL of MRVP broth
2.cShake well after addition of each reagent
3. Observe for 5 min

Interpretation
Positive: red (acetoin production)
Negative: yellow

Tryptophanase, which hydrolyzes tryptophan to form indole.

Tryptophan → indolepyruvic acid (intermediate) → indole + pyruvate + ammonium.

p-dimethylaminobenzaldehyde (p-DMAB), isoamyl alcohol, and hydrochloric acid.

A red-colored compound forms at the surface.

Tube method using Kovac’s reagent is more sensitive than the spot indole test.

Tube method using Ehrlich’s reagent (requires extraction with xylene).

p-dimethylaminocinnamaldehyde (DMACA).

Indole combines with DMACA to form blue-green compound

Saturate filter paper with spot indole reagent
- use wooden applicator stick to rub portion of org on paper
- observe for a color change; most turn blue w/in 30 sec
** Cannot be performed on mixed culture (diffusion cause false positive)

Older biochemical grouping/mnemonic used to differentiate Enterobacterales.

Indole, Methyl Red, Voges-Proskauer, Citrate.
Example:
E. coli: ++--
(Indole positive, MR positive, VP negative, citrate negative)
Klebsiella pneumonia: --++
Proteus mirabilis: -+-+
Citrobacter freundii: -+-+
Enterobacter aergoenes: --++

Other biochemical tests are necessary to fully differentiate bacteria species.

Identifies Enterobacterales and other non-fastidious gram-negative rods (can detect some non-glucose fermenters).

Inoculate 1 colony into 5 mL of 0.85% saline and incubate at 35°C in O₂ for 18–24 hours.

TDA(Tryptophan Deaminase): 10% ferric chloride (forms a red complex with indolepyruvic acid).
IND: Kovac’s reagent (1 drop).
VP: Alpha-naphthol + 40% KOH (1 drop each).

Differentiates late lactose fermenters from non–lactose fermenters.

must be able to transport carbohydrate (via β-galactoside permease) and hydrolyze lactose to glucose and galactose (via β-galactosidase)

ONPG can enter the cell without permease.

ONPG → galactose + o-nitrophenol (yellow).

Presence of β-galactosidase.

Shigella sonnei.

Yes, ONPG is included in the API 20E panel.

O (somatic) antigen – cell wall, heat stable;
K (envelop) antigen – capsular, heat labile;
H (flagellar) antigen – heat labile;
Vi antigen – capsular antigen of Salmonella typhi, heat labile.

Salmonella and Shigella species require additional serological identification after full identification by biochemical methods

Wellcolex Salmonella reacted with two test reagents consisting of a mixture of suspensions of red, blue and green latex particles, each of which is coated with antibody specific for different Salmonella serogroups (A, B, C, D, E ,G) based on O antigen as well as Vi antigen

98% of human Salmonella isolates.

They can further test the H antigen for epidemiological purposes.

Each serological group of Shigella corresponds to a specific specie
Shigella dysenteriae (A)
Shigella flexneri (B)
Shigella boydii (C)
Shigella sonnei (D)

Non-glucose fermenting organism produces K/NC (no change) reaction on TSI

Pseudomonas aeruginosa (1st), Acinetobacter baumannii (2nd), and Stenotrophomonas maltophilia (3rd).

Once confirmed as a non-glucose fermenter by TSI, identification by API20NE

Pseudomonas aeruginosa

Metallic sheen and grape / corn tortilla odor

Oxidase positive, acetamide positive, room temperature motility positive

If metallic sheen present, a positive oxidase test is sufficient

If no metallic sheen present, confirm ID with a positive oxidase and acetamide test

Tetramethyl-p-phenylenediamine dihydrochloride

Cytochrome oxidase, which oxidizes the substrate to indophenol (dark purple end product)

- Moisten filter paper with 1% tetramethyl-p-phenylenediamine dihydrochloride
- Use platinum wire or wooden stick to rub small portion of colony (< 24h) into filter paper
- Nickel-based alloy wires containing chromium and iron (nichrome) – may cause false positive results due to oxidation products from flaming
- Observe for color change to deep blue/purple in 10 sec
- Don’t perform the test on selective / glucose containing media (fermentation products inhibit oxidase = false negative

Enzymatic hydrolysis of acetamide with production of NH₃.

Phenol Red or Bromothymol Blue.

Green to blue with Bromothymol Blue, or dark pink to red on the slant with Phenol Red.

Moist natural and hospital environments; multidrug-resistant strains may be recovered in hospitals.

Gram-negative coccobacilli (almost cocci in some cases).

Smooth, opaque, raised, creamy, and smaller than Enterobacterales; lactose-negative but colonies may show a purplish hue that can be mistaken as lactose positive.

Oxidase negative, room-temperature motility negative, full ID using API 20NE.

It can be found in moist natural and hospital environments.

Common colonizer of non-sterile sites (e.g., respiratory tract).

Oxidizes maltose faster than glucose; oxidase negative; room temperature motility positive; full ID using API 20NE.

Intrinsically resistant to many antibiotics; Trimethoprim/sulfmethoxazole is treatment of choice

Identification of non-fastidious, non-enteric gram negative rods (e.g., Pseudomonas, Acinetobacter, Aeromonas, Vibrios, etc.).

8 conventional tests and 12 assimilation tests (bacteria grow if they can utilize substrate).

0.5 McFarland suspension in 2 mL 0.85% NaCl (18–24 hours old growth).

Requires API AUX Medium for assimilation tests; perform separate oxidase test; see next page for NO₃ test; for TRP test add 1 drop of James reagent.

Determines if bacteria reduce nitrate to nitrite.

NO₃ (nitrate) -- nitrate reductase → NO₂ (nitrite).

All Enterobacteriaceae are nitrate positive.

The reagents are sulfanilic acid and N,N-dimethyl-alpha-naphthylamine; sulfanilic acid reacts with nitrite to form a diazonium salt, which then combines with alpha-naphthylamine to produce a red azo dye.

Red color change indicates nitrate positive.

If no color change: either no reduction or reduction of nitrate further to N2
Add zinc: If NO3 still present, Zn will reduce it and form red color
Red after Zn = Nitrate negative
No color change after Zn = Nitrate positive