Bacteria §Cells are prokaryotic and amongst the smallest known cells ( length 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. §Air §Water §Soil §On 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 bacterium §Single cells - monococci §Pairs - diplococci §Chains - streptococci

2.Bacillus §Plural is bacilli. §A rod - shaped bacterium. §Exists as single cells, in pairs (diplobacilli), and in chains (streptobacilli).

3. Spirillum §Plural 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 Needs §Like 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. §O 2 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: è temperature è pH è moisture content è nutrient availability è 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 continued §The 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-org 2.GRAM IODINE §Mordant §Forms Crystal violet iodine complexes 3.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 Procedure Step 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 Procedure Step 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 Procedure Step 3 - Apply the Mordant Flood the Smear with Iodine solution Allow to stand 2 min

The Gram Stain Procedure Step 4 - Rinse Rinse with water to remove excess Iodine

The Gram Stain Procedure Step 5 - Decolorize Drip Decolorizer (80% Methanol +20% Acetone) across the slide about 5 sec The effluent should appear pale or clear

The Gram Stain Procedure Step 6 - Rinse Rinse with water to remove excess alcohol

The Gram Stain Procedure Step 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

GRAM + & GRAM – BACTERIA Cell Wall StructuresGram Positive organisms Gram Negative organisms Inner cytoplasmic membrane Present Peptidoglycan layerThickThin Teichoic AcidPresentAbsent Outer membrane layerAbsentPresent Lipid A, LPS, Lipo-protien components AbsentPresent Peri-plasmic spaceAbsentPresent

GRAM VARIABLE §Gram variability §Old cultures §Decolorize improperly §Dead and alive bacteria §Autolytic organisms e.g. Streptococcus pneumoniae