Mirco Lab

What is an appropriate way to streak?

• The quadrant streak pattern

- dividing the plate into four sections

• T-streak

- divided in three sections

ALL DONE IN A ZIGZAG

Orange

Coarse-focus

purple

Fine-focus

slide- positioning

yellow

red

objective lense

blue

ocular

black

revolving nosepiece

green

slide clip

Appropriate clean-up procedures

• lens paper to remove oil

• dispose slides in sharps (red box)

• wipe down table

Capsule stain protocol

• 1 loopful sheep serum

• 1 drop congo red

• whatever mix we are working one

• smear using second slide

• let dry

• Maneval’s reagent

- 1 min

• Gently rinse with water

Gram Satin protocol

• heat fix

• crystal violet

- 1 min and then rinse with water

• gram’s iodine

- 1 min then rinse

• Decolorize with alcohol, then rinse with water

• Safranin

- 1 min

• Blot dry

Endospore stain protocol

• heat fix

• Malachite green

- 10 min exposure

• rinse

• safranin

- 1 min then rinse with water

Acid-Fast stain protocol

• heat fix

• rinse smear with water

• acid-alcohol decolorize

- 1 min, rinse with water

• counterstain with methylene blue

• 1 min, rinse

• blot dry

Capsule Stain: Positive result:

Clear halo (capsule) surrounding the bacterial cell.

Capsule Stain: Negative result:

No halo present; capsule absent or not visible

Capsule Stain: Type of stain

• Structural stain

• Also uses an indirect (negative) staining technique because the background is stained instead of the capsule.

Capsule stain: Why each step matters

• Background stain creates contrast.

• Air drying preserves the capsule.

• Counterstain colors the cell.

Capsule stain:If mistakes are made

• Heat fixing: Capsule shrinks or disappears (false negative).

• Skipping Congo Red: No dark background.

• Skipping Maneval's stain: Cells difficult to see.

• Overwashing: Capsule may wash away.

Gram stain: Positive result

• Purple cells

• Gram-positive

• Thick peptidoglycan cell wall

Gram stain: Negative result

• Pink/red cells

• Gram-negative

• Thin peptidoglycan with outer membrane

Gram stain:Type of stain

Differential stain

Gram stain: Why each step matter

• Crystal violet stains all cells.

• Iodine locks the stain inside cells.

• Alcohol differentiates Gram-positive from Gram-negative bacteria.

• Safranin makes Gram-negative cells visible.

Gram stain: If mistakes are made

• Forget iodine → Crystal violet washes out → Gram-positive cells may appear Gram-negative.

• Over-decolorize → Gram-positive cells become pink.

• Under-decolorize → Gram-negative cells stay purple.

• Skip safranin → Gram-negative cells appear colorless.

Endospore Stain:Positive result

• Green endospores inside or outside pink vegetative cells.

• Organism is capable of producing endospores.

Endospore Stain: Negative result

• Only pink vegetative cells.

• No spores present.

Endospore Stain: Type of stain

Structural stain

Endospore Stain: Why each step matters

• Heat opens the tough spore coat.

• Water removes stain from regular cells.

• Safranin provides contrast

Endospore Stain:If mistakes are made

• No heat → Spores stay colorless.

• Skip water rinse → Everything stays green.

• Skip safranin → Vegetative cells are difficult to see.

• Too much heat → Distorted cells.

Acid-Fast Stain: Positive result

• Bright red or fuchsia cells.

• Organism contains mycolic acids in the cell wall.

Acid-Fast Stain:Negative result

• Blue cells.

• Lacks mycolic acid-rich cell walls.

Acid-Fast Stain: Type of stain

Differential stain

Acid-Fast Stain:Why each step matters

• Carbolfuchsin stains all cells.

• Heat helps stain penetrate the waxy cell wall (Ziehl-Neelsen method).

• Acid-alcohol differentiates acid-fast from non-acid-fast bacteria.

• Methylene blue stains decolorized cells

Acid-Fast Stain: If mistakes are made

• Too much decolorizer → Acid-fast cells may lose the red stain (false negative).

• Too little decolorizer → Non-acid-fast cells remain red (false positive).

• Skip methylene blue → Non-acid-fast cells appear colorless.

• Insufficient heating (Ziehl-Neelsen) → Poor penetration of carbolfuchsin into acid-fast cells.

Blood Agar (BAP): Type of media

• Differentia

Blood Agar (BAP): Tests for the ability to

produce hemolysins, which lyse (break open) red blood cells.

Blood Agar (BAP): Important ingredient

5% Sheep blood

Blood Agar (BAP): Differential agent

Red blood cells (sheep blood)

Blood Agar (BAP): positive

• (Beta) hemolysis = complete destruction of red blood cells; clear zone around colonies

• (Alpha) hemolysis = greenish or brown discoloration around colonies

Blood Agar (BAP): negative

(Gamma) hemolysis = no hemolysis; agar unchanged

MacConkey Agar (MAC): Type of media

Selective and Differential

MacConkey Agar (MAC): What does it test?

• Growth of Gram-negative bacteria

• Ability to ferment lactose

MacConkey Agar (MAC): Important ingredients

• Bile salts

• Crystal Violet

MacConkey Agar (MAC): Differential agents

• Lactose

• Neutral Red (pH indicator)

MacConkey Agar (MAC): positive

Pink/red colonies = Positive for lactose fermentation

MacConkey Agar (MAC): negative

Colorless and lacks lactose fermentation

Mannitol Salt Agar (MSA): Type of media

Selective and Differential

Mannitol Salt Agar (MSA):What does it test?

1. Salt tolerance

2. Mannitol fermentation

Mannitol Salt Agar (MSA): Important ingredients

7.5% Sodium Chloride (NaCl)

Mannitol Salt Agar (MSA): Differential agents

• Mannitol

• Phenol Red

Mannitol Salt Agar (MSA): postive

Yellow agar = Positive for mannitol fermentation

Mannitol Salt Agar (MSA): negative

Red/pink agar = Negative for mannitol fermentation

Eosin Methylene Blue (EMB): What does it test?

• Growth of Gram-negative bacteria

• Lactose fermentation

Eosin Methylene Blue (EMB):Selective agents

• Eosin Y

• Methylene Blue

Eosin Methylene Blue (EMB): Differential agent

Lactose

Eosin Methylene Blue (EMB): positive

Metallic green sheen = Strong lactose fermenter

Eosin Methylene Blue (EMB): negative

Purple/dark colonies = Weak lactose fermenter

Volvox

Spirogyra

Rhizopous stolonifer

Penicillium roquefortii

Candida albicans

Paramecium caudatum

Amoeba proteus

Plasmodium falciparum

Trypauosoma cruzi

Colony shape

round

Colony shape

irregular

Colony shape

spindle

Colony shape

Filamentous

Colony shape

Rhizoid

Arrangement

streptococcus

Arrangement

spirochete

Arrangement

Vibrio

Arrangement

Streptobacillius

Arrangement

Diplobacillius

Arrangement

Palisades

Arrangement

Diplococci

Arrangement

Tetracoccus

Arrangement

Staphylococci:

Arrangement

Sarcinae