MLS 409 Exam 2

What are the general requirements for setting up a urine culture?

• Media: BAP & MAC

• Incubation: 35-37C, ambient air, 18-24 hours

Which type of urine specimen do you use calibrated loops for?

Midstream clean catch, straight & indwelling catheter, nephrostomy/cytoscopy

How do you collect a specimen from an indwelling catheter source?

Collect from sampling port, not the bag

How do you culture a suprapubic aspirate urine specimen?

Plate specimen directly

What is the purpose of doing colony counts on urine specimens?

To distinguish a UTI from contamination

• > 100,000 CFU/mL indicates a UTI

• Isolation of colonies

What does it mean when there are 1-2 organisms vs multiple organisms on a urine plate?

Less organisms = infection, more = contamination

What are common urinary pathogens?

• E. coli (most common!)

• Klebsiella pneumoniae

• Proteus mirabilis

• Enterococcus faecalis

• Staphylococcus saprophyticus (UTIs in young women)

• Pseudomonas aeruginosa

Describe the following characteristics common to the Neisseriaceae:

• Gram stain morphology

• Oxidase production

• GNDC

• Oxidase positive (aerobic)

Describe the specific specimen collection, transport, and processing (including nutritional and atmosphere) requirements of Neisseria gonorrhoeae and Neisseria meningitidis cultures.

• Culture immediately (temperature sensitive, transport at room temp)

• Capnophilic (2-8% CO₂)

N. gonorrhoeae

• Media (enriched): CHOC and modified Thayer-Martin agar

• Susceptible to drying (directly inoculate)

• Inoculate with dacron or rayon swabs (others are inhibitory)

• If delayed, use enriched selective media with transport system

N. meningitidis

• Media: SBA, CHOC, modified Thayer-Martin

What is Thayer-Martin media? (Including its purpose, proper use, significant ingredients, inhibitory/selective properties)

• CHOC agar with added antibiotics

  • Vancomycin, colistin, nystatin, trimethoprim

• Selective for pathogenic Neisseria sp.

  • N. gonorrhoeae: small, gray to tan, translucent, raised

  • N. meningitidis: small, tan, sometimes mucoid, convex

  • N. lactamica (non-pathogenic, but still grows): small, tan, convex

Explain the oxidase test

Purpose: detect cytochrome c oxidase

Positive: purple color change

Negative: no color change

Explain the nitrate reduction test

Purpose: detect reduction of nitrate to nitrite

Positive: red after adding reagent (nitrite), or colorless after zinc (N₂ gas)

Negative: red only after adding zinc

Explain the butyrate esterase test

Purpose: detects butyrate esterase enzyme

Positive: blue color change

Negative: no color change

Explain the beta-lactamase test

Purpose: Detects the penicillinase/beta-lactamase enzyme

Positive: color change

Negative: no color change

Predict the carbohydrate utilization test results & growth on Thayer-Martin for the Neisseria sp and Moraxella catarrhalis

 

	Glucose	Maltose	Lactose	Sucrose	TM
N. gonnorhoeae	Positive	Negative	Negative	Negative	Growth
N. meningitidis	Positive	Positive	Negative	Negative	Growth
N. lactamia	Positive	Positive	Positive	Negative	Growth
M. catarrhalis	Negative	Negative	Negative	Negative	No Growth

What tests can help differentiate between Moraxella and Neisseria sp?

Butyrate esterase

• Neisseria: negative

• Moraxella: positive

Nitrate

• Neisseria: negative

• Moraxella positive

Growth on Thayer-Martin

• Neisseria: Growth

• Moraxella: No Growth

What are the expected test results from a BactiCard for Neisseria sp and Moraxella catarrhalis?

Note: these tests are performed from enriched selective media only

 

BactiCard Performed on isolates from enriched selective media only
	N. gonorrhoeae	N. meningitidis	N. lactamica	M. catarrhalis
IB Pos: teal	Negative	Negative	Negative	Positive
PRO Pos: purp	Positive	Variable	N/A	N/A
GLUT Pos: purp	Negative	Positive	N/A	N/A
BGAL Pos: teal	Negative	Negative	Positive	N/A

Draw the flowchart for GN(D)C

What are some examples of transport media used for Neisseria sp?

• Jembec

• Transgrow

• Gono-Pack

Describe Neisseria gonorrhoeae, including its clinical significance, diseases caused, and treatment/prevention.

Clinical Significance

• Present in genital, rectal, throat, eye, synovial fluid specimens

• Infects any site containing columnar or transitional epithelial cells

• Primarily spreads through sexual contact

• Always pathogenic

Diseases

• Causes gonorrhea (reported to the health department)

  • Males: acute urethritis, prostatitis, epididymitis, intracellular GNDC

  • Females: can be asymptomatic or cause severe discharge

    • Can lead to cervicitis, leading to pelvic inflammatory disease (PID), sterility, and ectopic pregnancies

• Can also cause: Disseminated gonococcal infection

Treatment/Prevention

• Treated with ceftriaxone or cefixime

• Diseases prevented by screening mothers and providing eye treatments for infants within 2 hours of delivery

Describe Neisseria meningitidis, including its clinical significance, diseases caused, and treatment/prevention.

Clinical Significance

• Source of infection: oral secretions and respiratory droplets, can be found in CSF

• People can be asymptomatic carriers 

• Can enter the bloodstream = septicemia and/or meningitis

• AST not performed 

• Serotypes A, B, C, W-135, and Y cause most cases

Diseases

• Endemic Meningitis (Reported to Health Dept) 

• Risk factors: college-age young adults 

• Symptoms: fever, lethargy, irritability, headache, photophobia, stiff neck, seizures 

  • Septicemia = purpura with petechial rash 

Treatment and Prevention 

• Penicillin for meningitis (AST not performed) 

• 3rd gen cephalosporins for meningococcemia 

• Prevent with vaccines and prophylaxis treatment to close contacts

Describe Neisseria lactamica including its clinical significance, diseases caused, and treatment/prevention.

Clinical Significance: 

• Normal flora of nasopharynx, rarely implicated in disease 

• Sterile sites (may req full identification)

Describe Moraxella catarrhalis including its clinical significance, diseases caused, and treatment/prevention.

Clinical Significance:

• Normal flora of upper respiratory, alimentary, and genitourinary tracts but opportunistic pathogens in children and elderly

• Most beta-lactamase positive  

Diseases

• Otitis media and maxillary sinusitis (3rd common cause) 

• Acute bronchitis & Pneumonia (in immunocompromised)

What is the colonial morphology of Moraxella catarrhalis?

Morphology: hockey-puck, smooth, opaque, gray to white that develops into a pink/salmon color. 

Describe the following characteristics common to the Enterobacterales:

• Gram stain morphology

• Oxidase reaction

• Glucose fermentation

• Nitrate reduction

• Growth on MacConkey agar

• GNR / GNCB

• Oxidase negative (except Plesiomonas)

• Glucose fermenters

• Nitrate reduction positive

• Growth on MAC + HE/XLD agar

Describe MAC agar, including its purpose, proper use, and significant inhibitory/selective properties

• Select for GNR (bile silts and crystal violet inhibit GP)

• Differentiates lactose fermenters/non-fermenters

Describe Hektoen enteric agar (HE), including its purpose, proper use, and significant inhibitory/selective properties

• Selective for GNR (higher concentration of bile salts than MAC)

  • Still inhibits many normal flora Enterics

• Differentiates: lactose and sucrose fermenters; ferric salt producers (H₂S - hydrogen sulfide)

Interpretation

• Lactose &/ sucrose fermenter = yellow/orange colonies

• Lactose &/sucrose non-fermenter = colorless, blue/green

• H₂S production = black precipitate

Describe Salmonella-Shigella agar (SS) including its purpose, proper use, and significant inhibitory/selective properties

• Selective for GNRs (very inhibitory, but other Enterics can grow)

• Differentiates lactose fermenters and H₂S producers

Interpretation

• LF: pink colonies

• NF: colorless colonies

• H₂S: black precipitate

Describe Xylose-lysine-deoxycholate agar (XLD) including its purpose, proper use, and significant inhibitory/selective properties

• Selective for GNRs

  • Sodium desoxycholate inhibits GPC and some GNR

  • Lower concentration of bile salts = better Shigella recovery

• Differential for lactose, sucrose, xylose fermenters; lysine decarboxylation, H₂S producers

Interpretation

• Fermenters: yellow colonies

  • + Xylose fermenters that do not decarboxylate lysine

• Non-fermenters: red

  • Xylose fermenters that decarboxylate lysine, turning pH back to red

• H₂S: black precipitate

Describe Eosin-methylene blue agar (EMB) including its purpose, proper use, and significant inhibitory/selective properties

• Selective for GNRs

• Differential for lactose/sucrose fermenters

Interpretation

• Fermenters: purple, strong = metallic

• Non-fermenters: clear colonies

• E. coli: metallic green sheen

Describe Cefsulodin-irgasan-novobiocin agar (CIN) or Yersinia selective agar including its purpose, proper use, and significant inhibitory/selective properties

• Selective for GNRs; used to recovery primarily Yersinia enterocolitica

  • Ingredients inhibit most organisms found in the stool

• Differentiates mannitol fermenters

Interpretation

• Fermenter: pink

  • Yersinia: bulls eye colonies with pink center and clera periphery

• Nonfermenter: clear

What is the purpose of using Gram-negative broth?

Selectively enriched to promote Salmonella and Shigella sp

Describe Sorbitol MacConkey Agar (SMAC) including its purpose, proper use, and significant inhibitory/selective properties

A specialized MAC agar

• Selects for GNRs

• Differentiates sorbitol fermentation (MAC: lactose fermentation)

Interpretation

• Fermenter: pink

• Nonfermenter: clear

What is the most common etiologic agent of subacute bacterial endocarditis?

Viridans streptococci

Discuss the appropriateness of performing direct Gram stains on blood cultures

• Guides empirical therapy

• Differentiate pathogens from contaminants

• Facilitates AST testing

What organisms are indicative of septicemia/bacteremia?

• S. aureus

• E. coli

• Streptococcus sp

• Enterococcus sp

• Candida sp

What organisms are indicative of skin contamination?

• CoNS

• Bacillus sp

• Micrococcus sp

Explain the indole test

Detects tryptophanase, which hydrolyzes tryptophan to indole

Positive: blue/purple

Explain the lysine and ornithine decarboxylase (LDC, ODC) test

Detects lysine decarboxylase, turning lysine into cadaverine

Detects ornithine decarboxylase, turning ornithine into putrescine and CO₂

• Bacteria first ferments glucose, lowering pH = yellow

• Acidic environment triggers decarboxylation, raising pH = purple

Explain the glucose oxidation-fermentation reaction

Two test tubes = one aerobic and anaerobic

• Fermentative: both tubes turn yellow (can break down glucose with/without oxygen)

• Oxidative: only the aerobic tube turns yellow (needs oxygen)

• Inert/Non-saccharolytic: bacteria cannot use glucose at all

Explain the urease production test

• Detects urease, which breaks down urea into ammonia and CO₂

• Positive: pink agar

Explain the gelatin liquefaction test

• Detects gelatinase, which breaks down gelatin into smaller amino acids, causing the media to lose its ability to solidify at cold temperatures

• Positive: medium remains liquid after chilling

• Negative: medium solidifies upon chilling

Explain the nitrate reduction test

• Detects if bacteria can reduce nitrate to nitrite

• Positive: medium turns red after adding reagent; or medium remains colorless after adding reagent and zinc (N₂ gas)

• Negative: medium remains colorless after adding reagent; or medium turns red after adding zinc

Explain the citrate utilization test

• Detects if bacteria can use citrate as a carbon and energy source

• Positive: blue medium

• Negative: medium remains green

Explain the Voges-Proskauer (VP) test

• Determines if 2,3-butanediol is a produce of glucose fermentation

• Positive: red/purple color near top of tube

• Negative: no color change

Explain the deaminase reaction test (tryptophan, lysine, phenylalanine)

• Determines if bacteria can remove an amine group (NH₂) from an amino acid

• Tryptophan: indole test

• Phenylalanine: positive result is a green color on a slant

• Lysine: positive result is a red color on a slant

Expalin the orthonitropheynl-beta-D-galactopyranoside (ONPG) test

• Detects beta-galactosidase, which is needed to break down lactose

• Positive: liquid medium turns yellow

What are the clinically relevant lactose-fermenting members of the order Enterobacterales?

• Escherichia coli

• Citrobacter freundii

• Klebsiella pneumoniae

• Klebsiella oxytoca

• Enterobacter cloacae

• Klebsiella (Enterobacter) aerogenes

What are the clinically relevant species that are non-lactose fermenting members of the order Enterobacterales and also LDA/TDA positive and ONPG negative?

• Proteus vulgaris

• Proteus mirabilis

• Morganella morganii

What are the clinically relevant species that are non-lactose fermenting members of the order Enterobacterales and also LDA/TDA negative?

• Shigella sonnei

• Shigella species

• Salmonella species

• Salmonella typhi

• Serratia species

• Citrobacter freundii

• Citrobacter koseri

• Yersinia entercolitica

What are the general characteristics of Enterobacterales?

• GNRs

• Glucose fermenters

• Oxidase negative

• Nitrate reduction positive

Identify the key test results of LDC, LDA/TDA, H2S, ODC, Motility, Indole, and ONPG to differentiate:

• Salmonella sp

• Shigella sp

• Shigella sonnei

 Lactose Nonfermenters; LDA/TDA negative

Organism	LDC	LDA/TDA	H2S	ODC	Motility	Indole	ONPG
Salmonella sp	Pos	Neg	Pos	Pos	Pos	Neg	Neg
Shigella sp	Neg	Neg	Neg	Neg	Neg	-/+	Neg
Shigella sonnei	Neg	Neg	Neg	Pos	Neg	Neg	Pos

Identify the key test results of ODC, LDC, Indole, Citrate, VP, Motility, and H₂S to differentiate:

• Escherichia coli

• Klebsiella pneumoniae

• Klebsiella oxytoca

• Klebsiella (Enterobacter) aerogenes

• Enterobacter cloacae

 Lactose Fermenters, ONPG positive

Organism	ODC	LDC	Indole	Citrate	VP	Motility	H2S
E. coli	Pos/Neg	Pos/Neg	Pos	Neg	Neg	Pos/Neg	Neg
K. pneumoniae	Neg	Pos	Neg	Pos	Pos	Neg	Neg
K. oxytoca	Neg	Pos	Pos	Pos	Pos	Neg	Neg
K. (E.) aerogenes	Pos	Pos	Neg	Pos	Pos	Pos	Neg
E. cloacae	Pos	Neg	Neg	Pos	Pos	Pos	Neg

Identify the key test results of Urease, H₂S, ODC, Indole, LDA/TDA to differentiate:

• Proteus mirabilis

• Proteus vulgaris

 

Organism	Urease	H2S	ODC	Indole	LDA/TDA
Proteus mirabilis	++	+	+	-	+
Proteus vulgaris	++	+	-	+	+

Draw out the abbreviated ID scheme for E. coli

Draw out the abbreviated ID scheme for Proteus sp

What does Serratia marcescens look like on SBA and MAC?

SBA: creamy, gray white

MAC: translucent/pale

Produces a red pigment (prodigiosin) at room temperature

What does Yersinia enterocolitica look like on CIN/Yersinia agar?

• Smooth colonies with a bulls-eye appearance

What is the only lactose fermenting GNR that also produces H₂S?

Citrobacter freundii

Define the following terms:

a. Capsular antigen (K)

b. Somatic antigen (O)

c. Flagellar antigen (H)

d. Vi antigen

• Capsular antigen (K): surrounds bacterial cell outside the O antigen, protection from phagocytosis and complement

• Somatic antigen (O): heat-stable polysaccharide portion of the lipopolysaccharide (LPS) in the outer membrane of GN bacteria

• Flagellar antigen (H): heat-labile protein (flagellin) that makes up flagelal

• Vi antigen: surface capsular polysaccharide antigen that overlies O antigen, inhibiting phagocytosis

T/F: AST is routinely performed on suspected pathogenic Enterobacterales isolates except Salmonella (except typhoidal isolates) and Shigella

True

Biochemical ID of Shigella sp should be confirmed with what?

Serogrouping

Which Enterics are commonly responsible for GI infections?

• Salmonella sp

• Shigella sp

• E. coli

• Yersinia enterocolitica

Which Enterics are commonly responsible for opportunistic/nosocomial infections?

• K. pneumoniae = destructive pneumonia

• Enterobacter cloacae

• Serratia sp

• Citrobacter sp

• Proteus sp = implicated frequently in UTIs

• Morganella morganni

Which Enterics are commonly responsible for UTIs?

• E. coli (#1 etiologic agent of UTI)

• Proteus sp

Which Enterics are commonly responsible for typhoid fever?

Salmonella typhi

• The most pathogenic of the Salmonella sp

• Rose spots appear

• Constipation followed by bloody diarrhea

Which Enterics are commonly responsible for neonatal meningitis?

E. coli

Which Enterics are commonly responsible for hemolytic uremic syndrome (HUS)?

E. coli

Which Enterics are commonly responsible for respiratory infections?

K. pneumoniae

Which Enterics are commonly responsible for wound infections?

• Preotus

• E. coli

• K. pneumoniae

Which Enterics are always pathogens?

• Shigella sp

• Salmonella sp

• Yersinia

• E. coli O157:H7

Describe the specific specimen processing requirements for Haemophilus species, including nutritional requirements, atmosphere requirements, and length of incubation.

Haemophilus sp are fastidious organisms that are capnophilic, facultative anaerobes that require V (NAD) and X (Hemin) growth factors

• Avoid drying and extreme temperatures, culture immediately

• CHOC

• CO₂

• Moist environment

• Incubate for 24-48 hours

Describe the characteristic Gram stain and colonial morphology of Haemophilus species.

• GNCB

• Tannish

• Translucent

• Mousy odor

• H. ducreyi: small, grey, yellow/tan, hockey-puck

What tests/characteristics are used to identify Haemophilus sp?

• X and V requirements

  • Disk test, watch for growth (contains either X, V, or XV)

  • Growth around either X/V/XY = requires that factor

  • Growth around all disks: does not require any factor

• Porphyrin test (ALA test)

  • Determines if organism can synthesize X factor from ALA

  • Positive: red/orange fluorescence after reagent/UV light, does not require X factor, organism produces porphyrin

• Hemolysis on horse blood agar

  • Detects organism’s ability to produce hemolysins that lyse horse RBCs

  • Helps differentiate between hemo/nonhemolytic species

• Satellitism

  • Demonstrates dependence on V factor (NAD)

  • S. aureus inoculated, lyses RBCs, releasing X favor and secretes V factor, which allows Haemophilus to grow

Describe the difference in test results between H. influenzae and H. parainfluenzae (X/Y test; Porphyrin; Horse blood; Satellitism)

	Influenzae	Parainfluenzae
X/Y Test (Growth)	XV	V & XV
Porphyrin	Negative	Positive
Horse Blood	No	No
Satellitism	Positive	Positive

Describe the function and clinical significance of the capsule as a virulence factor for Haemophilus influenzae.

H. influenzae’s polysaccharide capsule is its most significant virulence factor (6 antigen types a-f, based on capsular polysaccharides). Helps prevent phagocytosis.

• Encapsulated strains, especially Hib cause severe invasive infections, especially in unvaccinated children younger than 5

  • Vaccines are available

• Un-encapsulated species can still cause disease

  • Localized infections, risk factors for elderly and immunocompromised

What kind of diseases do H. influenzae cause?

• Respiratory infections

• Meningitis

• Otitis media

• Conjunctivitis (ssp. aegypticus)

What disease does H. ducreyi cause?

Chancroid/Soft Chancre

Should you perform AST on Haemophilus sp?

Yes (ish)

• Traditionally, ampicillin was the drug of choice, but resistance has been seen

• Beta-lactamase should be performed

• Other AST can be done

What are the GNR glucose non-fermenters?

• Pseudomonas aeruginosa

• Stenotrophomonas maltophilia

• Acinetobacter baumannii

• Burkholderia cepacia

Fill in this table for Glucose NF GNRs

	Oxidase	42C	ADH	ODC	LDC	Gelatin	Polymyxin B
P. aeruginosa
S. maltophilia
B. cepacia

 

	Oxidase	42C	ADH	ODC	LDC	Gelatin	Polymyxin B
P. aeruginosa	+	Growth	+	-	-	+/-	Susceptible
S. maltophilia	-	No	-	-	+	+	Susceptible
B. cepacia	+/-	+/-	-	+/-	+	+/-	Resistant

What are the expected results of a glucose oxidation-fermentation test for glucose non-fermenting GNRs?

• Aerobic tube turns yellow

• Anaerobic tube remains green

Describe the following characteristics for P. aeruginosa

• Colony morphology

• Gram morphology

• Glucose utilization

• Oxidase

• Pigment

• Growth at 42C

• Colony: rough, spreading, may have green sheen and be beta-hemolytic

• GS: thin GNR

• Glucose oxidizer

• Oxidase positive

• Growth at 42C

Describe key characteristics/tests of S. maltophilia

• Strong maltose oxidizer

• Opportunistic

• Oxidase negative

• Glucose “O” or “I”

• BAP: pale yellow to lavender pigment

• ADH: negative

• ODC: negative

• LDC: positive

• DNase: positive

• Polymyxin B: sensitive

Describe key characteristics/tests of P. aeruginosa

• Glucose oxidizer

• Strict aerobe

• Pigments: pyoverdin/cyanin/rubin/mealnin

• Beta-hemolytic

• Metallic sheen/mucoid

• Non LF

• Grape smell

• Oxidase: positive

• Indole: negative

• Always perform AST

Draw the ID scheme for GNF GNRs

How do GNF GNRs cause disease?

• Predominant opportunistic - affecting immunocompromised or hospitalized patients

• Survive well in moist environments, form biofilms, have antimicrobial resistance

• Other VFs: pili, endo/exotoxins, proteolytic enzymes, extracellular capsule

What kind of diseases can P. aeruginosa cause?

• Opportunistic infections

• Wounds

• Respiratory

• Otitis externa

• Corneal ulcers

• UTI

• Cystic fibrosis

• Burns

• Osteomyelitis

What kind of diseases can S. maltophilia cause?

• Respiratory

• Wound

• Opportunistic

When do you reject a respiratory specimen for culture using a direct specimen Gram stain?

When there are more than 25 epithelial cells/10X field

Describe the procedures for setting up a culture on a sputum specimen, including the types of media used and incubation requirements:

• Direct specimen gram stain done first before culture set up

• Media: BAP, CHOC, MAC

• Sample areas that are purulent or bloody

• Streak for isolation

• BAP & CHOC - CO2; MAC - ambient

• Temp: 35C

• Time: 18-24 overnight, minimum 48

Describe the procedures for setting up a culture on a throat specimen, including the types of media used and incubation requirements:

• Direct gram stain first

• Throat swabs done to find Group A Strep only

• Media: strep selective blood agar

• Use swab to make primary streak, use loop to streak for isolation

• Make cuts in primary streak or incubate anaerobically to allow detection of streptolysin O

• Temp: 35C

• Incubate in anaerobic conditions

• Time: overnight 18-24 hours, minimum 48

Evaluate the normal flora of the following respiratory sites for culture interpretation:

a. Upper respiratory tract

b. Lower respiratory tract

Upper

• Viridans strep

• CoagN Staph

• Nonpathogenic Neisseria

• Diphtheroids

• Anaerobes

• Yeast

Lower

• Normally sterile

Evaluate pathogens commonly found in the following respiratory sites for culture interpretation:

a. Upper respiratory tract

b. Lower respiratory tract

Clinical group	Organisms
Community-acquired pneumonia	Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis
Hospital-acquired/opportunistic	Klebsiella pneumoniae, E. coli, Enterobacter spp., Serratia marcescens, Staphylococcus aureus
Hospital-acquired/opportunistic nonfermenters	Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp.
Other respiratory pathogen	Streptococcus pyogenes
Cystic fibrosis-associated	Burkholderia cepacia complex

Haemophilus species appear on Gram stain as:

GNCB