Key Techniques and Tests in Microbiology

Function of the condenser in microscopy

Focuses light through the specimen; important during wet mount preparation.

Steps of Gram stain and their purpose

1. Crystal violet stains all cells purple 2. Iodine binds dye to cells 3. Alcohol removes dye from Gram-negative cells 4. Safranin stains Gram-negative cells pink

Selective vs Differential media

Selective: supports growth of specific organisms (e.g., MSA). Differential: shows visible differences in colonies (e.g., MAC for lactose fermentation).

Catalase test

Detects presence of catalase enzyme. Staphylococcus = positive (bubbles), Streptococcus = negative.

Acid-fast stain significance

Used to detect Mycobacterium spp. which retain carbolfuchsin due to waxy mycolic acid in their cell walls.

Distinguishing S. pyogenes and S. agalactiae

S. pyogenes is bacitracin sensitive (Taxo A), S. agalactiae is resistant.

Prokaryotic vs Eukaryotic movement

Prokaryotes: flagella, smaller (0.1-5 µm). Eukaryotes: cilia, pseudopods, larger (10-100 µm).

Parfocal definition

Microscope remains in focus when switching between objectives, requiring only fine adjustment.

E. coli indicators on media

Hot pink colonies on MacConkey agar; green metallic sheen on EMB; indole positive (red ring with Kovac's).

Proteus mirabilis characteristics

Gram-negative, non-lactose fermenter, swarming motility on BAP, urea positive.

Function of trypticase soy agar (TSA)

Basic nutritive media used for growth of both Gram-positive and Gram-negative bacteria.

Role of chocolate agar

Enriched media with cooked blood, supports growth of fastidious organisms like Neisseria spp.

MacConkey agar usage

Selective for Gram-negative; differential based on lactose fermentation.

Purpose of acid-fast staining

Differentiates organisms like Mycobacterium based on resistance to decolorization by acid alcohol.

Blood agar hemolysis types

Alpha: green zone (partial), Beta: clear zone (complete), Gamma: no change (none).

Steps in acid-fast stain

1. Carbolfuchsin stain 2. Acid-alcohol decolorizer 3. Methylene blue counterstain.

Kirby-Bauer test principle

Tests antibiotic sensitivity by measuring zones of inhibition around antibiotic disks on agar.

Throat culture swab technique

Swab posterior pharynx, avoid tongue and saliva; streak on blood agar and incubate with CO₂.

Function of bile-esculin agar

Selective for Enterococcus faecalis; black precipitate indicates positive esculin hydrolysis.

Purpose of mannitol salt agar (MSA)

Selective for Staphylococcus (high salt); differential based on mannitol fermentation (yellow = positive).

Staphylococcus for catalase

positive (bubbles)

Streptococcus

what bacteria has a negative catalase test?

What is the primary stain used in the Gram stain?

Crystal violet

What color are Gram-positive bacteria after Gram staining?

Purple

What color are Gram-negative bacteria after Gram staining?

Pink or red

What does the catalase test detect?

Presence of the enzyme catalase

What result indicates a positive catalase test?

Bubbling when hydrogen peroxide is added

What organism is catalase positive and forms clusters?

Staphylococcus spp.

What media is selective for Gram-negative bacteria?

MacConkey agar

What media is selective for Gram-positive bacteria?

Mannitol Salt Agar (MSA)

What is the function of MacConkey agar?

Selects for Gram-negative and differentiates lactose fermenters

What does a pink colony on MacConkey agar indicate?

Lactose fermentation

What is the appearance of E. coli on EMB agar?

Green metallic sheen

What is the purpose of the acid-fast stain?

To identify Mycobacterium spp.

What does a positive indole test look like?

Red ring after adding Kovac's reagent

What test differentiates Enterococcus faecalis?

Bile esculin test (black precipitate = positive)

What test is used in the Kirby-Bauer method?

Antibiotic susceptibility test

What are the three hemolysis types seen on blood agar?

Alpha (green), Beta (clear), Gamma (no change)

What organism causes swarming on blood agar?

Proteus mirabilis

What shape and Gram reaction is Staphylococcus aureus?

Gram-positive cocci in clusters

What stain is used in a simple stain?

Methylene blue

What does a simple stain show?

Cell shape, size, and arrangement