GRAM NEGATIVE VS GRAM POSITIVE Physical differences clinical

GRAM NEGATIVE VS. GRAM POSITIVE Physical differences & clinical signifigance.

Gram- positive bacteria Figure 4. 8 b

Gram-negative bacteria Figure 4. 8 a

Gram-Positive cell walls • Teichoic acids: • Lipoteichoic acid links to plasma membrane • Wall teichoic acid links to peptidoglycan • May regulate movement of cations • Polysaccharides provide antigenic variation

Gram Negative: Outer Membrane (OM) • A bilayer membrane found in gram-negative bacteria • Forms the outermost layer of the cell wall; is attached to the peptidoglycan by a continuous layer of lipoprotein molecules • Proteins called porins form channels through the OM (transports some proteins) • OM has surface antigens and receptors • Does prevent some molecules from entering (e. g. some antibiotics), but generally isn’t that selective

Gram-Negative Outer Membrane Figure 4. 13 c

Damage to Cell Walls • Lysozyme digests disaccharide in peptidoglycan. • Penicillin inhibits peptide bridges in peptidoglycan. (effective on Gram-positive cells) • .

Bacterial Membranes

Gram-Positive Cell Wall • Thicker layers of peptidoglycan • 2 -ring basal body • Teichoic acid • Disrupted by lysozyme • Penicillin sensitive • More penetrable Gram-Negative Cell Wall § Thinner layer of peptidoglycan § 4 -ring basal body § No teichoic acid; porin proteins § Endotoxin § Tetracycline sensitive § Less penetrable Figure 4. 13 b–c

Exotoxins vs. Endotoxins EXOTOXIN ANIMATION ENDOTOXIN ANIMATION

Types of staining techniques: Simple Staining: use of a single stain Differential staining: Use of 2 contrasting stains Separation into groups: gram stain acid stain Visualizatio n of various structures: flagella, capsule, spore or nuclear stain

The Gram Stain (a) Gram-Positive (b) Gram-Negative Table 4. 1

Techniques of Light Microscopy • Wet mounts: A drop of medium containing organisms is placed on slide and used to view living microorganisms • Smears: Microorganisms are spread onto the surface of a glass slide and used to view destroyed organisms • Heat fixation: destroys the organisms, causes organism to adhere to slide, and alters organism to accept stains (dyes)

Step 1: applying primary stain Outline of Gram Stain procedure Step 2: apply mordant Step 3: apply decolorizing agent Step 4: apply counterstain animation

Gram Positive and Gram Negative Cells

Gram Stain Color of Gram-positive cells Color of Gram-negative cells Primary stain: Crystal violet Purple Mordant: Iodine Purple Decolorizing agent: Alcohol-acetone Purple Colorless Counterstain: Safranin Purple Red

Micrograph of Gram-Stained Bacteria Figure 3. 12 b

The Gram Stain Mechanism • Crystal violet-iodine crystals form in cell • Gram-positive • Alcohol dehydrates peptidoglycan • CV-I crystals do not leave • Gram-negative • Alcohol dissolves outer membrane and leaves holes in peptidoglycan • CV-I washes out

Distinguishing Bacteria by Cell Walls • Gram-positive Bacteria have a relatively thick layer of peptidoglycan (60 -90%) • Gram-negative Bacteria have a more complex cell wall with a thin layer of peptidoglycan (10 -20%) • Acid-fast Bacteria is thick, like that of gram- positive bacteria, but has much less peptidoglycan and about 60% lipid (mycolic acid)… Mycobacteria (TB and leprosy)

Acid-Fast Bacteria • Found in bacteria that belong to the genus, Mycobacterium sp. (e. g. tuberculosis, leprosy) • Cell wall is mainly composed of lipid, makes them very hardy. Also has slow diffusion = slow growth rate for many (eg: 20 days for M. leprae) • Lipid component is mycolic acid • Acid-fast bacteria stain gram-positive

Acid-Fast Stain Color of Acid-fast Color of Non–Acid-fast Primary stain: Carbolfuchsin Red Decolorizing agent: Acid-alcohol Red Colorless Counterstain: Methylene blue Red Blue

Acid-Fast Bacteria Figure 3. 13

Q&A • Acid-fast staining of a patient’s sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What color would bacterial cells appear if the patient has tuberculosis?

• Why doesn’t a negative stain color a cell? 3 -7 • Why is fixing necessary for most staining procedures? 3 -8 • Why is the Gram stain so useful? 3 -9

Normal Microbiota • Bacteria were once classified as plants, giving rise to use of the term flora for microbes • This term has been replaced by microbiota • Microbes normally present in and on the human body are called normal microbiota

Normal Microbiota on Human Tongue Figure 1. 7

Normal Microbiota • Normal microbiota prevent growth of pathogens • Normal microbiota produce growth factors such as folic acid and vitamin K • Resistance is the ability of the body to ward off disease • Resistance factors include skin, stomach acid, and antimicrobial chemicals

Biofilms • Microbes attach to solid surfaces and grow into masses • They will grow on rocks, pipes, teeth, and medical implants

Biofilms Figure 1. 8