Micro I Rotation: Set Ups Quiz

What are the components of xylose lysine deoxycholate (XLD) media?

Lysine, xylose, lactose, sucrose, ferric ammonium citrate.

• Sodium deoxycholate inhibits Gram positive organisms; phenol red as indicator

What bacteria does xylose lysine deoxycholate (XLD) agar select for?

Salmonella and shigella

India ink procedure

1. Place a small drop of specimen (ex. CSF, culture) onto a clean glass slide

2. Add an equal drop of India ink next to or directly onto the specimen and mix gently with a sterile wire or pipette

3. Place a coverslip carefully over the mixture to avoid creating air bubbles and to keep the thin layer

4. Examine the wet mount under a microscope using high-power or oil immersion lenses (40x to 100x)

India ink purpose

To detect the fungus Cryptococcus in clinical fluid (e.g. CSF).

• The presence of a halo indicates a positive result

• For a negative result, no encapsulated yeast cells are observed under the microscope. The background is uniformly dark with no clear halos surrounding yeast structrures

Cryptococcus (purpose of India ink) is a prevalent fungal infection prevalent in..

immunocompromised and HIV patients

Occult Blood Testing

Detects human hemoglobin in fecal samples

OC Auto Fecal Occult Blood Testing

• Detects human hemoglobin in fecal specimens

• Colorectal cancer screening (lower GI bleeding)

• Anti-human HBA0 abs in latex reagent react to hemoglobin in the sample, resulting in latex agglutination → change in optical density is picked up by instrument

• No dietary restrictions required (does not interfere with test)

Hemoccult Sensa Fecal Occult Blood Test

• Detects blood (heme) in stool via oxidation of guaiac by hydrogen peroxide to a blue-colored compound. Heme in stool catalyzes the oxidation of guaiac in test paper. Blue = positive

• Not a tests for colorectal cancer or any specific disease

• NSAIDs, vit C and red meat/ vegetables, menstrual blood can interfere with the result

True/ False: OC Auto Fecal Occult Blood Testing does not have dietary restrictions while Hemoccult Sensa Fecal Occult Blood Testing does.

True

What are general restrictions of fecal occult blood testing?

Samples collected from patients with active bleeding (from hemorrhoids/ fissures/ menstruation/ pregnancy) could result in false positives

Rapid Respiratory Syncytial Virus (RSV) and Influenza testing

• Lateral flow immunoassay detects Flu A, Flu B, or RSV nucleoprotein antigens (note: one kit for each type)

• Nasopharyngeal specimen in viral transport media

• Conjugated abs bind to viral antigens; abs-ag conjugate complex migrates across the test strip to the reaction area, bind to the ab on the membrane and is visualized as a line, read by instrument

Inappropriate specimens for anaerobic culture include:

• Bronchial washings

• Sputum/ tracheal (nasal swab)

• Throat/ oral

• Catheter tip

• Stool/ rectal swab

• Urine, unless invasively obtained (suprapubic,

• Unsterile specimen

What are protocols when rejecting a specimen?

Must fill out a nonconforming report

Gram Stain Procedure

1. Heat fix slide

2. Perform series of staining

   1. Crystal violet: Primary stain which stains Gram positives and negatives purple

   2. Iodine: Fixative/ mordant. Will fix crystal violet to Gram negative bacterial cell walls

   3. Alcohol wash: (Ethanol) removed CV from Gram negative bacteria; does not remove stain from Gram positives.

   4. Safranin: counterstain. Stains Gram negative bacteria pink.

Why do Gram positive bacterial cells stain purple?

The thick peptidoglycan cell wall traps the crystal violet stain and resists decolorization under alcohol.

Why do Gram negative bacterial cells stain pink?

The large lipid content in the cell walls is removed by the alcohol wash along with the CV stain.

When performing urine cultures, what volume of sample does the inoculation loop hold?

1 uL

In a urine culture plate, you count 10 colonies. What is the CFU?

• Inoculation loop volume = 1 uL

• 1 mL = 1000 uL

• 10 colonies / 1 uL x (1000 uL: 1 mL) = 10,000 colonies / mL

Autoclaving

• Primary means of sterilization in microbiology lab (media plates, sterile saline/water, glassware)

What is the protocol for autoclaving?

Double-walled chamber uses steam heat at 121 C for 15 minutes under 115 lbs of atmospheric pressure

Mannitol Salt Agar: Components

Mannitol, phenol red indicator, salt concentration of 7.5% inhibits most bacteria

What type of media is Mannitol Salt Agar?

Selective and differential

• Yellow: S. aureus (ferments mannitol)

• Pink: S. epidermidis (does not ferment mannitol)

Pink colonies on Mannitol Salt Agar indicate

S. epidermidis (does not ferment mannitol)

Yellow colonies on Mannitol Salt Agar indicate

S. aureus (ferments mannitol)

Rapid Urea Medium Helicobacter Pylori

• Used for detection of urease produced by Helicobacter pylori in gastric biopsy specimens

• Gastric biopsy tissue is inoculated into media by provider at time of collection

• Inoculated media is returned to Micro and read by Set Ups tech at 24 hours

• Pink = positive for urease production and assumed positive for H. pylori

What is the positive control for Rapid Urea Medium Helicobacter Pylori (Urease) Test?

Proteus vulgaris or Proteus mirabilis

The media for a rapid urea medium helicobacter pylori (urease) test is pink after 24 hours of inoculation. What are the results?

The test is positive. The patient is positive for Helicobacter pylori.

Criteria for sputum cultures

• Epithelial cells : <10 epithelial cells

• PMNs: >25 PMNs

Quality of specimen must be assessed at 10x (low power)

You count 21 epithelial cells in a sputum specimen slide at 10x. What decision must you make?

• Reject the specimen

• A large amount of epithelial cells indicate contamination with saliva and the culture results cannot be properly interpreted

Sterile sites specimen include..

fluids, tissues, CSF

Normally, sterile sites specimen do not contain PMNs or bacteria. A Wayson stain as a..

• follow up/ confirmation stain for negative smears

• for specimen that show > 3+ PMNs and no bacteria seen

• assists in seeing hidden bacteria by staining DNA material (bacteria as well as cellular DNA) blue

Gram stain grading: 1+

Number of organisms: 0-1

Gram stain grading: 2+

Number of organisms: 2-6

Gram stain grading: 3+

Number of organisms: 7-29

Gram stain grading: 4+

Number of organisms: >30

Cystine Heart Agar (Cystine Glucose Blood Agar): Components

cystine, dextrose/ glucose, blood

Cystine Heart Agar (Cystine Glucose Blood Agar) selects for..

Francisella tularensis

Chocolate Agar: Components

Peptone base, hemin, hemoglobin or IsoVitaleX