(MMI) Lab Module 3: Plates and Plating Techniques

Students will apply aseptic, pure culture, and selective isolation techniques to cultivate, enrich, and isolate microorganisms from mixed cultures

Isolation

•refers to a single, pure culture (colony/CFU) of an organism obtained from a mixed culture

Colony Forming Unit (CFU)

•a group of cells originating from a single cell or small group of cells

Four-Quadrant Streak (GOLD STANDARD FOR ISOLATION)

•used for high cell density or mixed samples (a loop is used)

•is “diluted” quadrant by quadrant until isolates are formed

•Qualitative Streak Method

Zig-Zag Streak

•used for low cell density or pure samples (a swab is used)

•sample is not diluted

•Quantitative Streak Method

Colony

•a visible mass of cells

•Isolated colonies in either the 3rd or 4th quadrants or both

•Good techniques for plating should include:

•Good use of plate space

•No unnecessary intersections or overlapping of quadrants

•No cuts or gouges into the agar

•Decreasing bacterial density with each subsequent quadrant

Factors Influencing Colony Morphology

•Temperature

•Incubation Time

•Nutrient Conditions

(Morphology is read from the 3^rd or 4^th quadrant)

COLONY MORPHOLOGY CATEGORIES

S S P O T M E

Size

Shape/Whole Colony

Pigment

Optical

Texture

Margin

Elevation

Size

: the physical size of an individual colony

• punctiform (<1mm), small, medium, large, etc.

Shape/Whole Colony

: the physical shape of an individual colony

• round, irregular, filamentous, rhizoid, etc.

Pigment

: color produced by the bacterium

• yellow, red, green, etc.

Optical

Opacity of the colony

• transparent, translucent, opaque

Texture

The physical “feel” of a colony

• dry, wet, mucoid, etc.

Margin

the physical appearance of the edge of a colony

smooth/entire, irregular, lobate, etc.

Elevation

how tall/short the colony is in relation to the agar level

• convex, umbonate, flat, raise, etc.

What is a great instrument to use for magnifying colonies up close (morphology) ?

Stereoscope

Morphology Example (SSPOTME)

Size: punctiform

Shape: round

Pigment: yellow

Optical: opaque

Texture: moist

Margin: smooth/entire

Elevation: convex

Differential Medium (Experiment 3-3)

to tell the difference between, or differentiate, different organisms

•Differentiation based on hemolysis

Hemolysis

•Lysing, or breaking down, of red blood cells

• Either complete or partial lysing of cells

• Carried out by an enzyme known as hemolysin (an exotoxin)

Make an observation, what type of hemolysis is this?

No change around growth

Organism does not hemolyze RBC’s

Gamma or γ - hemolysis

Make an observation, what type of hemolysis is this?

Greenish discoloration around growth

Organism partially hemolyzes RBC’s

Alpha or α - hemolysis

Make an observation, what type of hemolysis is this?

Clearing around growth

Organism hemolyzes RBC’s completely

Beta or β - hemolysis

Significance of Hemolysis in Microbiology

•Indicates pathogenicity of an organism

• Pathogenicity: ability of an organism to cause disease

• Many organisms are beta-hemolytic

Streptococcus spp. are…

are frequent producers of hemolysins. There are known as Streptolysins, toxic proteins produced by Group A Streptococcus that destroy red blood cells

Streptolysin O (Oxygen Sensitive)

AEROBIC ENVIRONMENT (O2)	ANAEROBIC ENVIRONMENT (NO O2)
No activity	Maximum activity

Streptolysin S (Oxygen Stable)

AEROBIC ENVIRONMENT (O2)	ANAEROBIC ENVIRONMENT (NO O2)
Minimum Activity	Maximum activity

Streptolysins have better activity in _________ _________.

anaerobic environments

• This is why we stab the blood agar plat

• electrical tape

What kind of hemolysis and streptolysin is being demonstrated?

1. We can see in this image that the streptolysin appears to work the same at the surface of the agar as it does inside the stab. This would be an example of Streptolysin S

What kind of hemolysis and streptolysin is being demonstrated?

2. Here we can see that the alpha-hemolysis is more apparent in the stab than it is on the surface of the plate. This tells us that this is Streptolysin O

What kind of hemolysis and streptolysin is being demonstrated?

3. And here we can see that there is no hemolysis which means that no streptolysin is present.

Capable organisms (Beta Hemolysis (Complete Lysis))

• Streptococcus pyogenes (Group A Strep / GAS)

• Streptococcus agalactiae (Group B Strep / GBS)

• Staphylococcus aureus

Capable organisms (Alpha Hemolysis (Incomplete/partial Lysis))

• Streptococcus pneumoniae

• Viridans Group Streptococci

Organisms that are NOT capable of Hemolysis (Gamma Hemolysis)

1. Enterococcus faecalis & Enterococcus faecium

2. Staphylococcus epidermidis

3. Staphylococcus saprophyticus