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

• GNCB

• 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 (chromogenic substrates)?

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

• Not resistant 

• 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: pink

• 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 contamination?

• CoNS

• Bacillus sp

• Micrococcus sp