Colony characteristics of micrococcus luteus

Ex 3-7: The Gram Stain

Pages 195-202

Today we will do 3 things:

Observe the 2 streak plates and assess the broth culture and slants from Tuesday for growth patterns

Gram stain some reference organisms

Observe our environmental plates on dissecting scope for Colony Morphology (Ex. 2-2)

Tools to characterize and identify microorganisms

Microscopic morphology
Colony morphology
Culture characteristics
Biochemical activities
Each of these is determined by an organism’s genes, and will be consistent for a bacterial species or isolate
Culture Characteristics in Broth

Surface:
Ring, pellicle, none

Sediment:
present, absent

Broth Clarity:
Uniform turbidity, flocculent *, clear

Amount of growth:
0 to +3 (none/slight/moderate/abundant)

*

Growth Patterns in Broth

pellicle*growth on surface
flocculent (clumps)
uniform turbidity
sediment
1

4

3

2

*

*

*

*

Serratia marcescens. Gram negative, short bacilli that produce mucoid colonies which have entire margins and umbonate elevation. Note that there are both red and white colonies present on this plate. Some strains of S. marcescens produce the red pigment prodigiosin in response to incubation at 30o C, but do not do so at 37o C. This is an example of temperature-regulated phenotypic expression.

Broth: turbid appearance of the broth and the red color present in both the sediment and pellicle.

Slant: white to red color, smooth, moist growth

Micrococcus luteus. Circular, pinhead colonies which are convex with entire margins. This gram positive coccus produces a bright yellow, non-diffusable pigment.

Broth: produced granular and pellicle growth

Slant: abundant, opaque, grayish white, smooth, flat, slimy, and moist.
Escherichia coli. This gram negative, coccobacillus forms shiny, low convex, mucoid colonies which have entire margins and are slightly raised. Older colonies often have a darker center.

Broth: turbid growth with a deposit which disperse upon shaking.

Slant: off-white in color and showed a spreading growth pattern: usually it appeared as a cloudy film over the whole surface of the slant

Staphylococcus saprophyticus A Gram-positive, cocci forms slightly convex colonies that are opaque, smooth, circular, gray-white, white to cream colonies.

Broth: uniform turbidity with a fine to slight mucoid deposit.

Slant:

*

Culture Characteristics on a Slant

Slants are useful for keeping stock cultures of bacteria
Growth occurs on slant
The butt* allows for diffusion of nutrients to the organisms and diffusion of waste products away from organisms
Observe pattern of growth on the slant:
Note any other unique/obvious characteristics – e.g., color
*

Purpose:
To determine appearance of growth on various media
This is consistent for an organism and is useful to characterize and identify organisms
Genetically determined, yet environmentally influenced
Compare organisms to see if they’re the same or different
Can tell us about their properties, such as the conditions/media at which they grow
3) Colony Morphology/Culture Characteristics

Colony Morphology/Culture Characteristics

7 Characteristics of Colony Morphology:

Shape
Margin
Elevation
Size
Color
Texture
Optical Properties
Shape
Elevation
Margin
Colony Characteristics (p. 64)

Size – measure in mm (or a comment)
Color – describe the color (specifically)
Texture – typically refers to the colony surface or its texture when probed with a loop
Optical Properties – how it transmits light (opaque, translucent, transparent)
Dissecting scopes are useful to observe colonies

Colony Characteristics

Summary: Characteristics to observe

On a plate

(as an isolated colony)

Colony Morphology

Shape
Margin
Elevation
Size
Color
Texture
Optical properties
On a slant

Growth on slants

Pattern of growth

Anything else that’s obvious or unique

In a broth

Growth in Broth

Surface

Sediment

Broth

Amount of growth

Streak Plate

Last time you created a streak plate from a mixed broth culture (Namely, E. coli, M. luteus, and Serratia marcescens)

A good streak plate:

Is appropriately labeled
Has an appropriate pattern
for streak lines
Uses the entire plate
Has well-isolated colonies
Is free of contamination
Did you obtain individual colonies?

Observe & critique your streak plate

To get more well-isolated colonies:

Use a small amount of initial bacterial inoculum (in first quadrant).
Remember to flame loop after each quadrant.
Don’t go back into previous quadrant so much (2 or 3 pass throughs).
Keep streak lines moving in forward direction.
Use entire plate.
Types of Stains used by Microbiologists

Simple Stains – a single dye is used to add contrast, so microbes can be viewed microscopically
Positive stain
Negative stain
Differential Stains
Gram stain
Acid-fast stain
Special Structure Stains
Capsule stain
Spore stain
Flagellar stain
X

Differential stains detect differences between organisms
The Gram stain differentiates bacteria into two groups: gram-positive and gram-negative, based on differences in cell wall structure.
Gram-positive bacteria tend to be killed more easily by antibiotics like penicillin.
Gram-negative bacteria can be more resistant to antibiotics.
Differential Stains: Gram Stain

Will react in a crystal violet dye and will stain dark purple or violet.

Cell wall is unique because it is composed of several peptidoglycan layers (multilayered) making it thicker.

Presence of techoic acids

Lacks an LPS (lipopolysachharide) content, periplasmic space and outer membrane making this group low in both lipoprotein and lipid composition.

Gram Positive

Normally does not react to a crystal violet dye, stains pinkish red [decolorized in accepting safranin (a counterstain)]

Thinner peptidoglycan cell wall layer because it is just made of one single layer as opposed to gram positive’s multi-layered wall

Lacks teichoic acids, but has a high content of LPS

Has both a periplasmic space and an outer membrane

Has a high amount of lipoprotein content as well as lipids.
Gram Negative

Gram Stain
Start with an air-dried, heat-fixed smear.

primary stain mordant decolorizer counterstain

Crystal violet-iodine complexes form in cell
Gram-positive bacteria
Alcohol dehydrates & condenses thick peptidoglycan layer
Crystal violet-iodine complexes do not leave
Gram-negative bacteria
Alcohol dissolves outer membrane (LPS) and leaves holes in thin peptidoglycan layer
Crystal violet-iodine washes out
Gram Stain Mechanism

Differential Stains: Gram Stain

For every Gram stain, observe & record:

Gram reaction

cell shape

cell arrangement

size (if you can)

Gram Stain – Common Problems

Smears are too thick
Decolorizing too little or too much
Old cells and dead cells tend to stain Gram negative
Because the cell wall is not intact
Should always use fresh cultures
Demonstration of a Gram stain

Air-dried, heat-fixed smear
Remember: small amount of organism spread out in a small drop of water
Gram stain procedure – p 200
Decolorization is the most crucial step
Until run-off is clear

page 200

Expectations: Each student should:

Observe & critique your streak plates

Prepare & Gram stain 1 slide

Slide: Three smears on slide:
Gram Pos., Mixed, and Gram Neg.

Gram stain procedure – p 200
Observe with oil immersion objective (100 X)
Observe simple stained slides from last lab and colony morphology
S. Sapro

Or M. luteus

S. marcescens

Or E. coli

Mixed

coccus bacillus spirillum

Bacterial Shape

Singly
Pairs: diplococci, diplobacilli
Clusters: staphylococci
Chains: streptococci, streptobacilli
Arrangement

For Next Lab:

Read:

Ex 3.9 Capsule Stain
Ex 3.10 Endospore Stain
Ex 3.12 Flagella Stain