Medias & Stains

Types of agar, Gram stain, special stains pg. 140-142

Routine Medias (8)

• Blood Agar (BA, BAP)

• Chocolate Agar

• Phenylethyl Alcohol Agar (PEA)

• Columbia Colistin-Nalidixic Acid (CNA)

• Thayer-Martin Agar

• CAMPY-Blood Agar

• Thioglycolate Broth

• Lowenstein-Jensen Agar, Middlebrook 7H10 Agar, and Petragnani Agar

Purpose of BAP

Most bacteria; determines hemolytic reactions

Purpose of Chocolate Agar

Chocolate Agar - Haemophilus and Neisseria sp.; enriched with Hemoglobin or IsoVitalleX

Purpose of Phenylethyl Alcohol Agar (PEA)

Phenylethyl Alcohol Agar (PEA) - Selects for gram pos cocci and anaerobic gram neg bacilli

Purpose of Columbia Colistin-Nalidixic Acid (CNA)

Selects for gram pos cocci

Purpose of Thayer-Martin Agar

N. gonorrhoeae and N. meningitidis

Purpose of CAMPY-Blood Agar

Campylobacter sp.

Purpose of Thioglycolate Broth

“Back-up” for Anaerobes

Purpose of Lowenstein-Jensen Agar, Middlebrook 7H10 Agar, and Petragnani Agar

Mycobacterium sp.

Anaerobic Media (5)

• Bacteroides Bile Esculin Agar (BBE)

• Kanamycin-Vancomycin Laked Blood (KVLB)

• Cycloserine-Cefoxitin Fructose Agar (CCFA)

• CDC Anaerobic Blood Agar

• Cooked or Chopped Meat Medium

Purpose of Bacteroides Bile Esculin Agar (BBE)

Selects for B. fragilis Group (Black Colonies)

Purpose of Kanamycin-Vancomycin Laked Blood (KVLB)

Bacteroides sp. (Enhances Pigment Production)

Purpose of Cycloserine-Cefoxitin Fructose Agar (CCFA)

C. difficile

Purpose of CDC Anaerobic Blood Agar

Anaerobes (enriched with Hemin, Cystine, and Vitamin K)

Purpose of Cooked or Chopped Meat Medium

Cooked or Chopped Meat Medium - Anaerobes

Special Media (9)

• Bordet-Gengou Agar

• Buffered Charcoal Yeast Extract (BYCE)

• Cystine-Glucose Agar

• Fletcher’s Medium

• Skirrow Agar

• Thiosulfate Citrate-Bile Salts (TCBS)

• Vaginalis Agar (V-Agar) (human blood)

• Cystine-Tellurite Blood (Tinsdale)

• Loeffler’s Medium

Purpose of Bordet-Gengou Agar

B. pertussis

Purpose of Buffered Charcoal Yeast Extract (BYCE)

Legionella sp.

Purpose of Cystine-Glucose Agar

F. tularensis

Purpose of Fletcher’s Medium

Leptospira

Purpose of Skirrow Agar

Helicobacter pylori

Purpose of Thiosulfate Citrate-Bile Salts (TCBS)

Vibrio sp.

Purpose of Vaginalis Agar (V-Agar) (human blood)

Gardnerella vaginalis

Purpose of Cystine-Tellurite Blood (Tinsdale)

C. diphtheriae (Black Colonies)

Purpose of Loeffler’s Medium

C. diphtheriae (Enhances Grouping and Metachromatic Granules)

Select Biochemical Reactions (7)

• Carbohydrate fermentation with phenol red

• Esculin

• Hippurate hydrolysis

• Motility

• 6.5% NaCl Broth

• Phenylalanine or tryptophan deaminase

• Urea

Carbohydrate fermentation with phenol red:

• Uninoculated Color or Negative Reaction

• Positive Reactions

• - Red

• + Yellow

Esculin:

• Uninoculated Color or Negative Reaction

• Positive Reactions

• - Nondescript

• + Black

Hippurate hydrolysis:

• Uninoculated Color or Negative Reaction

• Positive Reactions

• - No change

• + Purple

Motility:

• Uninoculated Color or Negative Reaction

• Positive Reactions

• - Growth along stab line

• + Blurred stab line

6.5% NaCl Broth:

• Uninoculated Color or Negative Reaction

• Positive Reactions

• - Clear

• + Turbid

Phenylalanine or tryptophan deaminase:

• Uninoculated Color or Negative Reaction

• Positive Reactions

• - Nondescript

• + Green surface after 10% FeCl₃

Urea:

• Uninoculated Color or Negative Reaction

• Positive Reactions

• - Nondescript

• + Bright pink

Stains Commonly Used in Microbiology/Mycobacteriology (4)

• Gram stain

• Kinyoun & Ziehl Neilson

• Auramine-Rhodamine

• Calcofluor White

• Primary stain - Crystal violet

• Decolorizer - Alcohol/Acetone

• Counterstain - Safranin

• Results:

  • Pos - Purple

  • Neg - Pink

• Principle:

  • Iodine mordant

  • Methanol or heat fix

  • Violet dye & iodine form complex in cell; washes out of gram neg cells

Gram stain

• Primary stain - Carbol Fucshin

• Decolorizer - Acid Alcohol

• Counterstain - Methylene Blue

• Results:

  • Pos - Pink

  • Neg - Blue

• Principle:

  • Acid fast

  • For Mycobacteria

Kinyoun & Ziehl-Neilson

• Primary stain - Fluorescent stain

• Decolorizer - Acid Alcohol

• Counterstain - Potassium Permanganate

• Results:

  • Pos - Orange Fluoresc.

  • Neg - No Fluoresc.

• Principle:

  • For Mycobacteria

Auramine-Rhodamine

• Primary stain - (—) + 10% KOH

• Decolorizer - N/A

• Counterstain - N/A

• Results:

  • Pos - Bluish-white fluoresc.

  • Neg - No fluoresc.

• Principle:

  • For yeast and fungi

  • KOH to break down debris and mucous

Calcofluor White