Bacteria

Cells are prokaryotic and amongst the smallest known cells ( length 0.5-20 µm ).

No membrane bound nucleus.Have ribosomes,but no other organelles.DNA present as a long circular molecule.( see handout for bacterial structure)

Habitat

Bacteria are found everywhere.AirWaterSoilOn plants and animals.In plants and animals.

Types of Bacteria

There are three major groups of bacteria based on their shapes; coccus, bacillus, and spirillum.

1. Coccus

Plural is cocci.Spherical bacteriumSingle cells - monococciPairs - diplococciChains - streptococci

2.BacillusPlural is bacilli.A rod - shaped bacterium.Exists as single cells, in

pairs (diplobacilli), and in chains (streptobacilli).

3. SpirillumPlural is spirilli.A spiral - shaped

bacterium.Exist only as single

cells.

Bacterial Structure

Cell Wall - outermost structure of the cell.

Made up of a substance called peptidoglycan ( a long chain of sugars linked to amino acids ).

Penicillin destroys bacteria by interfering with the peptidoglycan molecules.

Bacterial Structure Continued

Cell Membrane - Found beneath the cell wall.

May be folded inward.

Bacterial Structure Continued

Capsule: This is a layer of slime secreted over the cell wall of the bacterium.

The capsule provides protection for the bacterium.

Bacteria that have capsules are said to be encapsulated.

Most pathogenic bacteria are encapsulated.

Bacterial Structure Continued

Flagella - Some bacteria in the bacilli and spirilli categories can move by way of flagella ( whip - like structures that propel the bacteria).

Note: Cocci do not have flagella.

Bacterial Physiology-Nutritional NeedsLike all living things bacteria need food.Bacteria are placed in one of two

categories, depending on how they get their food.

The two categories are: Heterotrophic bacteria and Autotrophic bacteria.

Heterotrophic Bacteria

Heterotrophs must get their food from a source of pre-formed organic matter:

(A) Saprobes- feed on the remains of dead plants and animals.

(B) Parasites - live on or in the organism and cause disease. For example, Mycobacterium tuberculosis.

Autotrophic Bacteria

Autotrophs can make their own food:

Photosynthetic-use a special type of chlorophyll called bacteriochlorophyll.

O2 is not released in bacterial photosynthesis

Chemosynthetic -obtain energy by breaking down inorganic material such as iron or sulfur.

Bacterial Physiology-Reproduction

The growth of bacteria is limited by the following factors:

temperature pH moisture content nutrient availability competition from other organisms

Bacterial Physiology-Reproduction continued

Bacteria reproduce asexually by binary fission. Using this process bacteria reproduce about every 20 minutes.

Bacterial Physiology-Reproduction continuedThe rate of

reproduction can be plotted in a graph called a Growth Curve.

Under ideal conditions one bacterial cell can produce a mass of 2 million Kg in 24 hours.

In reality this never happens. Why?

GRAM STAINING

REAGENTS USED IN GRAM STAIN

1. CRYSTAL VIOLET Primary stain Violet colored, stains all micro-org2. GRAM IODINE Mordant Forms Crystal violet iodine complexes3. DECOLORIZER Acetone + Methanol Removes Crystal violet iodine complex

from thin peptidoglycan layers Dissolves outer layer of Gram negative

org

REAGENTS USED IN GRAM STAIN

4. GRAM SAFRANINE Counter stain Red colored Stains thin walled

Gram neg org Pus cells cytoplasm &

lobes of nuclei also stain red

The Gram Stain ProcedureStep 1 - Prepare a Smear

Watch what happens to the “Bacteria” at each step

“Bacteria”

Suspend some of the material to be stained in a drop of water on a microscope slide, spread the drop to about the size of a nickel.

Allow to air dry. Heat fix by gently warming

The Gram Stain ProcedureStep 2 - Apply the Primary Stain

Flood the Smear with Crystal Violet

Allow to stand for 1 min

Rinse with water to remove excess stain

The Gram Stain ProcedureStep 3 - Apply the Mordant

Flood the Smear with Iodine solution

Allow to stand 2 min

The Gram Stain ProcedureStep 4 - Rinse

Rinse with water to remove excess Iodine

The Gram Stain ProcedureStep 5 - Decolorize

Drip Decolorizer (80% Methanol +20% Acetone) across the slide about 5 sec

The effluent should appear pale or clear

The Gram Stain ProcedureStep 6 - Rinse

Rinse with water to remove excess alcohol

The Gram Stain ProcedureStep 7 - Counterstain

Flood the slide with Safranin solution

Let stand for 2 minutes

The Gram Stain

Step 8 - Rinse, Dry and Observe

Gram-Positive Gram-Negative

Rinse with water to remove excess stain

Blot dry

Observe under Oil Immersion

CELL WALL OF GRAM POS & NEG

CELL WALL OF GRAM POS & NEG

GRAM + & GRAM – BACTERIACell Wall Structures Gram Positive

organismsGram Negative organisms

Inner cytoplasmic membrane

Present Present

Peptidoglycan layer Thick Thin

Teichoic Acid Present Absent

Outer membrane layer Absent Present

Lipid A, LPS , Lipo-protien components

Absent Present

Peri-plasmic space Absent Present

GRAM VARIABLEGram variability Old culturesDecolorize

improperlyDead and alive

bacteriaAutolytic organisms

e.g. Streptococcus pneumoniae