ASCP-BOC MLS Exam: Microbiology

List 2 examples of media which are both enriched and selective.

1. Thayer-Martin (T-M) or Martin-Lewis (ML) 2. Campylobacter blood agar (Campy BAP)

Describe the principle of the Kinyoun stain.

Acid fast bacilli (AFB) have thick layers of mycolic acid in their cell wall, which allows them to resist decolorization with strong acids and remain red. Non-acid fast organisms lack mycolic acid, so they are decolorized by strong acids and are stained blue by the counterstain.

Describe the principle of the acridine orange stain in microbiology.

Acridine orange is a fluorescent dye that binds nucleic acid. It is sometimes used to detect microorganisms in direct smears from clinical specimens (e.g. blood cultures, CSF), as it will stain human tissue and microorganisms different colors when buffered at a low pH.

List the 3 steps in staining with acridine orange for microbiology.

1. Fix slide with absolute methanol 2. Stain with acridine orange stain 3. Flush with water and allow to dry

List the 4 steps in staining with KOH and calcofluor white.

1. Add 10-40% KOH 2. Stain with calcofluor white 3. Warm slide slightly 4. Mount with coverslip

List 8 examples of enriched media.

1. Chocolate agar (CHOC) 2. Anaerobe blood agar (ABA) 3. Brain heart infusion (BHI) 4. Thayer-Martin (T-M) or Martin-Lewis (ML) 5. Campylobacter blood agar (Campy BAP) 6. Trypticase soy broth (TSB) 7. GN broth 8. Thioglycollate broth (THIO)

List 7 examples of selective media.

1. Colistin-nalidixic acid agar (CNA) 2. MacConkey agar (MAC) 3. MacConkey agar with sorbitol (MAC-SOR) 4. Hektoen-enteric agar (HE) 5. Thayer-Martin (T-M) or Martin-Lewis (ML) 6. Campylobacter blood agar (Campy BAP) 7. Thiosulfate-citrate-bile salt-sucrose (TCBS)

List the 5 steps in trichrome staining.

1. Fix slide with PVA 2. Stain with trichrome stain 3. Rinse several times with alcohol 4. Stain with xylene 5. Mount with coverslip

List the 3 steps in the Giemsa stain.

1. Fix slide with absolute methanol 2. Stain with Giemsa stain 3. Flush with water and allow to dry

List 4 examples of media which are both selective and differential.

1. MacConkey agar (MAC) 2. MacConkey agar with sorbitol (MAC-SOR) 3. Hecktoen-enteric agar (HE) 4. Thiosulfate-citrate-bile salt-sucrose (TCBS)

List the 3 components of the Giemsa stain.

1. Methylene blue 2. Eosin 3. Azure B

List the 2 primary purposes of sheep blood agar (SBA) media.

1. Primary plating and subcultures 2. Determining hemolytic activity

List the 3 steps in the modified Kinyoun stain.

1. Primary stain = carbolfuchsin 2. Decolorizer = H2SO4 3. Counterstain = methylene blue

List the 3 steps in the Kinyoun stain.

1. Primary stain = carbolfuchsin 2. Decolorizer = HCl 3. Counterstain = methylene blue

List the 4 steps in the Gram stain procedure.

1. Primary stain = crystal violet 2. Mordant = iodine 3. Decolorizer = alcohol 4. Counterstain = safranin

List 3 examples of non-selective media.

1. Sheep blood agar (SBA) 2. Horse blood agar (HBA) 3. Mueller-Hinton (MH)

Describe the role of iodine in the Gram stain.

Binds crystal violet to teichoic acids in thick peptidoglycan layer of GP organisms

In the trichrome stain used for stool samples, protozoan cytoplasm typically appears __________, while nuclear chromatin and ingested cells and bacteria appear __________. Additionally, ingested yeast and mold appear __________ and helminth eggs appear __________. The background is __________.

Blue-green with purple tinge, red or purple-red, green, red, green

Describe the role of alcohol in the Gram stain.

Dissolves lipid outer layer in GN organisms, allowing crystal violet to escape the cell

Calcofluor white permits the visualization of fungal elements by making them __________ and requires the use of __________.

Fluorescent, fluorescent microscopy

Identify the stain often used to stain blood films for blood protozoa (e.g. malaria) and filarial nematodes.

Giemsa

Describe the principle of the Gram stain.

It takes advantage of differences in cell wall composition. GP organisms have a thick peptidoglycan layer with teichoic acids, while GN organisms have a thin peptidoglycan layer with a lipid outer membrane.

Describe the principle of staining with KOH and calcofluor white.

KOH digests debris (e.g. cells, tissue) so that fungal elements are more readily visible. Calcofluor white is a fluorescent dye that binds chitin, which causes fungal elements to fluoresce.

Using a modified Kinyoun stain, acid fast organisms will stain __________, partially acid fast organisms will stain __________, and non-acid fast organisms will stain __________.

Red, red, blue

After a specimen has been stained with acridine orange, human tissue and cells will appear __________ or __________, while bacteria appear __________.

Pale green, yellow, bright orange

Describe the principle of the modified Kinyoun stain.

Partially acid fast organisms have thin layers of mycolic acid in their cell wall, so they are decolorized by the strong acid used in the Kinyoun stain but are not decolorized by the weak acid in the modified Kinyoun stain, so they remain red. This differentiates partially acid fast organisms from truly non-acid fast organisms, which are still decolorized by the weak acid and are stained blue by the counterstain.

Staining a blood smear with Giemsa stain will cause RBCs and platelets to appear __________, the cytoplasm to appear __________, the nuclear chromatin to appear __________, and parasites (e.g. malaria, filarial nematodes) to appear __________.

Pink, pale blue, magenta, dark purple

The most common fixative used with the trichrome stain is:

Polyvinyl alcohol (PVA)

Trichrome staining is used on __________ and __________ stool samples and is used to look for __________.

Preserved, unconcentrated, protozoan cysts and trophs

Using a Gram stain, Gram positive organisms stain __________, while Gram negative organisms stain __________.

Purple-blue, pink-red

Using a Kinyoun stain, acid fast organisms will stain __________, partially acid fast organisms will stain __________, and non-acid fast organisms will stain __________.

Red, blue, blue

Describe why free NAD is not available in SBA plates.

Sheep blood contains NADase, so NAD is unavailable unless RBCs in the agar are lysed to release free NAD from the cells.

Describe the role of crystal violet in the Gram stain.

Stains all organisms

Describe the role of safranin in the Gram stain.

Stains the colorless GN organisms to visualize them (NOTE: also stains GP organisms, but color is masked by crystal violet stain)

The key difference between the Kinyoun stain and the modified Kinyoun stain is that the Kinyoun uses a __________ as the decolorizer, while the modified Kinyoun uses a __________ as the decolorizer.

Strong acid, weak acid

Identify a type of cell that is often mistaken for a protozoan troph or cyst in trichrome stained slides.

WBC